Kan Kanamycin

L B Luria-Bertani

Lol

HSQC Heteronuclear single quantum correlation IA 2-Iodoacetamide IM Inner membrane IMP Inner membrane protein IPTG Isopropyl-β-D-thiogalactopyranoside

Keywords: Type II secretion system, OutC, OutD, Secretin, GST pull-down, Cysteine scanning, β-strand addition

NMR Nuclear magnetic resonance NTD N-terminal domain OM Outer membrane OMP Outer membrane protein PCR Polymerase chain reaction PDZ Post synaptic density 95, Disc large protein, Zo-1 PG Peptidoglycan layer PGA Polygalacturonate PMF Proton motive force PMSF Phenylmethyl-

First of all, I would like to thank the members of thesis examination committee, Dr. Ina Attree, Dr. Olivera Francetic, Dr. Nicolas Bayan and Prof. Philippe Lejeune for acting as reviewers and examiners of this thesis.

I wish to express my deep gratitude to my supervisor Dr. Vladimir Shevchik for giving me the opportunity to realize this thesis in France. His ideas and tremendous support had a major influence on this thesis. He encouraged and supported me with much kindness over the past three and half years. By working with him, I learned a lot of scientific and technical knowledge and I am convinced that this knowledge will help me in the future.

Dr. Nicole Cotte-Pattat, director of the UMR5240, is acknowledged for welcoming me in her laboratory. I wish to extend a special thanks to Dr. Guy Condemine for his academic guidance and corrections of my manuscript while to Dr. William Nasser and Dr. Sylvie Reverchon for their continuous encouragements.

I would like to thank to Dr. Mathilde Lallemand, Géraldine Effantin and Camille Villard for their guidence of my experiments, especially Mathilde, she also gave me many helps in everyday life.

I wish to express my appreciation to Véronique Utzinger, Yvette Alfaro and Jean-Michel Prost and Isabelle Goncalves. Without their help of preparing the materials, the experimental work of this thesis could not be carried out on time.

I also wish to express my appreciation to my officemates, Camille pineau, Arnaud Rondelet and Natalia Guschinskaya, who provided useful advices in this thesis and gave many helps, especially for the help on French language. I also had the pleasure of becoming friends and colleagues with Suzan Hassan, Ouafa Zghidi-Abouzid, Sana Boukerzaza-Cmaraovi, Xuejiao Jiang, Elodie Herault and Julien Wawrzyniak during this period.

Last but not least, I would never forget the support and encouragement from my husband, Chao, as well as other members of my family, who constantly listened carefully to all of my problems, who shared my sorrow, and who motivated me in all circumstances. This Ph.D. thesis has been supported by China Scholarship Council (CSC -UT/INSA program). The support is gratefully acknowledged. . V. 1: Plasmids employed in this chapter. Table . V. 2: Primers used in this chapter.

List of figures and tables

General introduction

Results-Chapter VI

Résume v

Résumé

Le système de sécrétion de type II (T2SS) est largement exploité par les bactéries à Gram négatif pour sécréter divers facteurs de virulence depuis le périplasme vers le milieu extracellulaire. La bactérie phytopathogène Dickeya dadanti (ex. Erwinia chrysanthemi) utilise ce système, appelé Out, pour la sécrétion de pectinases responsable de la maladie de la pourriture molle chez de nombreuses plantes. Les deux composants essentiels du système Out, la protéine de membrane interne OutC et la sécrétine OutD, formant un pore dans la membrane externe, sont impliqués dans la spécificité de sécrétion. L'interaction entre OutC et OutD pourrait assurer l'intégrité structurelle et fonctionnelle du système de sécrétion en reliant les deux membranes. Nous avons entrepris une étude structure-fonction de ces deux composants afin d'identifier et caractériser leurs sites d'interaction et de mieux comprendre leurs rôles. Nous avons appliqué une approche intégrative impliquant une analyse in vivo par cystéine-scanning et pontage disulfure, une analyse in vitro par GST Pour étudier le mécanisme d'adressage et d'assemblage de la sécrétine OutD dans la membrane externe, nous avons exploité les interactions entre OutD et deux composants auxiliaires du T2SS, la protéine de la membrane interne OutB et la lipoprotéine de la membrane externe OutS. Nous avons montré une interaction directe entre le domaine périplasmique d'OutB et le domaine N0 d'OutD. Une analyse structure-fonction du complexe OutS-OutD a révélé que la pilotine OutS interagit fortement avec 18 résidus à l'extrémité C-terminale de la sécrétine, entraînant la structuration sous forme hélicoïdale de cette région initialement non structurée.

Ce travail nous permet de mieux comprendre le mécanisme d'assemblage et de fonctionnement du système de sécrétion de type II.

Mots clés: Système de sécrétion de type II, OutC, OutD, Sécrétine, GST pull-down, Cysteinescanning, Addition des brins β.

General introduction

Secretion of various functional proteins outside of bacterial cells is an essential function directly involved in bacterial survival and pathogenesis. The secreted substrates fulfil various functions in different vital processes such as acquisition of nutrients, motility, intercellular communication and many others. Gram-negative bacteria evolved several specialized secretion systems to release these substrates into the medium or translocate them into the target host cell.

Up to date, at least six distinct secretion systems (from type I to type VI) have been identified in Gram negative bacteria, which are recognizable by the characteristic of components forming the secretion machine. Pathogenic bacteria often use these systems to secrete harmful toxins, adhesins, degradative enzymes, or specialized effectors to colonize and survive within eukaryotic hosts, causing acute or chronic infections, subverting the host cell response and escaping the immune system. Studying bacterial secretion offers the possibility to design specifically drugs that can block these machineries and thus attenuate the virulence of pathogenic bacteria.

Our model organism to study the type II secretion system (T2SS) is a plant pathogenic bacterium Dickeya dadantii 3937. This bacterium is responsible for important economic losses since it causes diseases in many plants by secreting several cell wall degrading enzymes through the T2SS named Out (Perombelon and Kelman, 1980;[START_REF] Toth | Dickeya species: an emerging problem for potato production in Europe[END_REF].

I Dickeya dadantii (ex. Erwina chrysanthemi), a phytopathogenic bacterium I.1 Taxonomy

The phytopathogenic bacterium Erwinia chrysanthemi belongs to the Enterobacteriaceae family. Members of this family are Gram-negative bacilli, γ-proteobacteria, facultative anaerobes, nonsporing and with peritrichous flagella. The genus Erwinia was first established in 1917 to encompass all members of the Enterobacteriaceae that cause disease on plants, including both pectinolytic (e.g., Erwina carotovora and Erwina chrysanthemi) and non-pectinolytic (Erwinia amylovora) species. Later, [START_REF] Waldee | Comparative studies of some peritrichous phytopathogenic bacteria[END_REF] proposed moving the pectinolytic Erwinia to a new genus, Pectobacterium. However, this classification had not been widely accepted until 1998 when new insights from 16S rRNA analysis led to resurrect the genus Pectobacterium for all pectinolytic Erwinia [START_REF] Hauben | Phylogenetic position of phytopathogens within the Enterobacteriaceae[END_REF]. Initially, the species Erwina chrysanthemi was assigned to the genus as a pathogen of chrysanthemum [START_REF] Burkholder | A bacterial blight of chrysanthemums[END_REF]. Similar bacteria were subsequently isolated from soft rots and wilts of numerous diseased plant species and were finally gathered into a single species, Erwinia chrysanthemi (Skerman et al., 1980). So, Erwina chrysanthemi (Pectobacterium chrysanthemi) is a complex of different bacteria. For convenience, it was subdivided into six pathovars, namely pv. chrysanthemi, pv. dianthicola, pv. dieffenbachiae, pv. parthenii, pv. zeae and pv. paradisiaca based on host specificity [START_REF] Young | A proposed nomenclature and classification for plant-pathogenic bacteria[END_REF]Lelliott and Dickey, 1984). On the basis of 16S rRNA sequence analysis, E. chrysanthemi pv. paradisiaca has been renamed Brenneria paradisiaca, whereas the others remained cluster with members of the genus Pectobacterium, namely P. chrysanthemi [START_REF] Hauben | Phylogenetic position of phytopathogens within the Enterobacteriaceae[END_REF].

However, more recently, Erwina chrysanthemi complex has been reclassified as a novel genus, Dickeya, by using phenotypic characteristics, DNA-DNA hybridization, serology and 16S rRNA gene sequence analysis [START_REF] Samson | Transfer of Pectobacterium chrysanthemi (Burkholder et al. 1953) Brenner et al. 1973 and Brenneria paradisiaca to the genus Dickeya gen. nov. as Dickeya chrysanthemi comb. nov. and Dickeya paradisiaca comb. nov. and delineation of four novel species, Dickeya dadantii sp. nov., Dickeya dianthicola sp. nov., Dickeya dieffenbachiae sp. nov. and Dickeya zeae sp. nov[END_REF]. Six genomic species were delineated within the novel genus: Dickeya chrysanthemi, Dickeya dadantii, Dickeya dianthicola, Dickeya dieffenbachiae, Dickeya paradisiaca and Dickeya zeae. The strain Erwinia chrysanthemi 3937 that we used to study the mechanism of pathogenicity was reclassified as Dickeya dadantii.

Despite of the new classification, Erwina chrysanthemi remains to be used in literature and in many data banks (protein and DNA) in parallel. observed in many temperate countries (including Europe) [START_REF] Toth | Dickeya species: an emerging problem for potato production in Europe[END_REF]. D. dadantii also causes disease on certain crops and other plants in temperate regions [START_REF] Toth | Soft rot erwiniae: from genes to genomes[END_REF][START_REF] Toth | Dickeya species: an emerging problem for potato production in Europe[END_REF]. D. dadantii causes general tissue maceration, termed soft rot disease (Fig. 1). This symptom mainly results from the degradation of middle lamella of the plant cell wall caused by a set of enzymes secreted by the bacteria.

I.2 Hosts and disease symptoms

I.3 The pathogenic mechanisms of D. dadantii

The pathogenesis of D. dadantii is a multifactorial process. It encompasses the interaction between host, pathogen and the environment leading to latent and active infections. When the bacterium enters the plant, environmental conditions including free water, oxygen availability and temperature are essential for optimal disease development. Free water may allow bacterial cells to move more easily through plant tissue. Low oxygen availability may create a microaerobic or anaerobic environment within the plant, which has little effect on the ability of bacteria pathogen, but has a major effect on limiting oxygen-dependent defenses of the plant [START_REF] Bolwell | Mechanisms for the generation of reactive oxygen species in plant defence-a broad perspective[END_REF]. Temperature is involved in a tight thermal regulation on the production of cell wall degrading enzymes [START_REF] Nguyen | Temperature-dependant production of carotovoricin Er and pectin lyase in phytopathogenic Erwinia carotovora subsp. carotovora Er[END_REF].

The bacterial cell surface structures, e.g., lipopolysaccharides (LPS), exopolysaccharides (EPS), enterobacterial common antigen (ECA) and flagella, are involved in bacterial motility and adhesion, which are necessary for successful invasion and infection of plants.

Soluble forms of iron, essential for all forms of life, are not readily available in plant or animal tissues. Under iron-limiting conditions, D. dadantii is able to express two high-affinity iron acquisition systems, e.g., chrysobactin and achromobactin that allow it to compete with plant for iron absorption [START_REF] Franza | Erwinia chrysanthemi requires a second iron transport route dependent of the siderophore achromobactin for extracellular growth and plant infection[END_REF].

The interaction between the plant and the bacterial pathogen is dynamic and implies signal exchange between the interacting organisms. On one hand, D. dadantii could produce many signal factors that trigger the host defenses, e.g., oligogalacturonides (OGAs) derived from enzymatic breakdown of pectin [START_REF] Norman | Oligogalacturonide-mediated induction of gene involved in jasmonic acid synthesis in response to the cell-wall-degrading enzymes of the plant pathogen Erwinia carotovora[END_REF], pathogen-associated molecular patterns (PAMPs) such as flagellin [START_REF] Zipfel | Bacterial disease resistance in Arabidopsis through flagellin perception[END_REF], and HrpN secreted by the type Ⅲ secretion system [START_REF] Yang | hrp genes of Erwinia chrysanthemi 3937 are important virulence factors[END_REF]. On the other hand, it can translocate into plant cells proteins that interfere with the resistance process. In fact, D. dadantii could inject a pathogenicity factor DspE directly into plant cells through the type Ⅲ secretion system (T3SS) which interferes with the phosphorylation cascades and suppresses the activation of plant defense genes [START_REF] Debroy | A family of conserved bacterial effectors inhibits salicylic acid-mediated basal immunity and promotes disease necrosis in plants[END_REF][START_REF] Peng | Population behavior analysis of dspE and pelD regulation in Erwinia chrysanthemi 3937[END_REF].

However, among all the pathogenicity factors of D. dadantii, the main factor is the secretion of various enzymes, such as pectinases, cellulases and proteases into the extracellular environment by different secretion systems. T1SS is a specific ABC transporter PrtDEF which allows secretion of four metallo-proteases [START_REF] Delepelaire | Protein secretion in gram-negative bacteria. The extracellular metalloprotease B from Erwinia chrysanthemi contains a C-terminal secretion signal analogous to that of Escherichia coli alpha-hemolysin[END_REF]) that appear to have a relatively minor role in pathogenicity, whereas T2SS is essential for pathogenicity [START_REF] Toth | Soft rot erwiniae: from genes to genomes[END_REF]. The D. dadantii possesses two independent T2SS, Out and Stt [START_REF] Bouley | The PDZ domain of OutC and the N-terminal region of OutD determine the secretion specificity of the type II out pathway of Erwinia chrysanthemi[END_REF][START_REF] Ferrandez | Novel mechanism of outer membrane targeting of proteins in Gram-negative bacteria[END_REF]. At least 10 pectinases and one cellulase are secreted by the Out system [START_REF] Kazemi-Pour | The secretome of the plant pathogenic bacterium Erwinia chrysanthemi[END_REF]. These secreted enzymes degrade the plant cell wall components and cause soft-rot disease. More recently genome sequences of D. dadantii 3937 revealed existence of genes and gene clusters coding for two type IV secretion system (T4SS), Type V and Type VI secretion systems (T5SS and T6SS) [START_REF] Glasner | Genome sequence of the plant-pathogenic bacterium Dickeya dadantii 3937[END_REF].

I.3.1 The plant cell wall

The plant cell wall is a complex macromolecular structure (Fig. 2A) that surrounds and protects the cell. It is a specific characteristic of plant essential to their survival. The cell wall is composed of polysaccharides, proteins, aromatic and aliphatic compounds. The polysaccharides are classified into three types: cellulose, hemicellulose and pectin [START_REF] Caffall | The structure, function and biosynthesis of plant cell wall pectic polysaccharides[END_REF].

The pectins, which are most abundant in the plant primary cell walls and the middle lamella, are group of polysaccharides that are rich in galacturonic acid (GalA) [START_REF] Willats | Pectin: cell biology and prospects for functional analysis[END_REF]. The pectic polysaccharides include homogalacturonan (HGA), rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) (Fig. 2B). These three polysaccharide domains could be covalently linked to form a pectic network throughout the primary cell wall matrix and middle lamella. HGA is a linear homopolymer of (1→4)-α-linked-D-galacturonic acid and is thought to contain 100-200 GalA residues. RG-I consists of as many as 100 repeats of the disaccharide (1→2)-α-L-rhamnose-(1→4)-α-D-galacturonic acid. RG-II has a backbone of around 9 GalA residues that are (1→4)-α-linked, and the side chains contain different sugars (mainly rhamnose, arabinose, galactose and glucuronic acid) [START_REF] Willats | Pectin: cell biology and prospects for functional analysis[END_REF]. Both RG-I and RG-II are thought to attach to HGA (Fig. 2B). Natural pectins present high percentages of methylesterification on the carboxyl group of galacturonate residues (from 40 to 65%). The cellulose microfibrils are linked via hemicellulosic tethers to form the cellulose-hemicellulose network, which is embedded in the pectin matrix (Fig. 2A). Cellulose is a polysaccharide consisting of a linear chain of several hundreds to over ten thousands β (1→4) linked D-glucose units. In contrast to cellulose, hemicellulose consists of several sugars, in addition to glucose, especially xylose, it also includes mannose, galactose, rhamnose and arabinose. It consists of shorter chains of about 200 sugar units. rhamnogalacturonan II; GalA: galacturonic acid; Rha: rhamnose; Gal: galactose; Ara: arabinose [START_REF] Willats | Pectin: cell biology and prospects for functional analysis[END_REF].

I.3.2 Pectinases, the major pathogenicity factor of D. dadantii

D. dadantii produces diverse pectinases that play an important role in the maceration characteristic of soft-rot disease. Pectinases are classified into two groups by their site of action: the pectin esterases, responsible for the liberation of esterified group of the polymer, and depolymerases, responsible for the main chain cleavage [START_REF] Robert-Baudouy | Pectic enzymes of Erwinia chrysanthemi, regulation and role in pathogenesis[END_REF].

I.3.2.1 The pectin esterases

HGA constitutes the main chain of pectin which consists of galacturonate residues, and some of them are modified by methyl and/or acetyl esterification (Fig. 3). The pectin esterases remove these modifications, and thus facilitate the further degradation of the polysaccharidic chain by depolymerases. D. dadantii 3937 produces two types of esterases: pectin methylesterases (Pem)

and pectin acetylesterases (Pae). The methylesterases cleave the methyl groups of the pectin and release polygalacturonic acid (PGA) and methanol. PGA is a substrate more easily accessible to depolymerases and commercially available pectin derivative (used for bacterial growth and enzymatic assays). In D. dadantii 3937, the metylesterase activity is mainly due to PemA secreted by the T2SS Out [START_REF] Fries | Molecular basis of the activity of the phytopathogen pectin methylesterase[END_REF] while a second isoenzyme PemB is a cell-linked outer membrane lipoprotein [START_REF] Shevchik | Characterization of pectin methylesterase B, an outer membrane lipoprotein of Erwinia chrysanthemi 3937[END_REF]. The acetylesterases hydrolyze O-acetyl groups in C2 and C3 of the HGA and release PGA and acetic acid. There are two acetylesterases identified in D. dadantii 3937: PaeY secreted by the T2SS Out, and PaeX which is mostly found in the periplasmic space [START_REF] Shevchik | Identification of a bacterial pectin acetyl esterase in Erwinia chrysanthemi 3937[END_REF]2003).

I.3.2.2 The depolymerases

According to their mechanism of cleavage of the glycosidic bonds (β-elimination or hydrolysis), depolymerases are classified into two types: lyases (pectate lyases and pectin lyases)

and hydrolyases (polygalacturonases). The preferential substrate of pectin lyases (Pnl) is high methoxyl pectins while the preferential substrate of pectate lyases (Pels) is low methoxyl pectins (PGA). The pectate lyases cleave internal glycosidic bonds in PGA via β-elimination to yield oligogalacturonates that are 4, 5-unsaturated at the nonreducing end. Polygalacturonases cleave the glycosidic bond of PGA by hydrolysis and generate saturated oligogalacturonates. The depolymerases can also differ in the random or terminal mode of attack of the polymer, endo or exo, respectively. Endo-acting enzymes would degrade the extracellular highly polymerized forms of PGA present within the plant cell wall. Exo-acting enzymes would be most efficient at producing small oligogalacturonides for intracellular transport from pectic fragments that accumulate in the periplasm.

There are 15 various pectinases identified in D. dadantii 3937: 10 pectate lyases (PelA, PelB, PelC, PelD, PelE, PelI, PelL, PelW, PelX and PelZ) [START_REF] Robert-Baudouy | Pectic enzymes of Erwinia chrysanthemi, regulation and role in pathogenesis[END_REF], 4

polygalacturonases (PehN, PehV, PehW and PehX) [START_REF] Nasser | Analysis of three clustered polygalacturonase genes in Erwinia chrysanthemi 3937 revealed an anti-repressor function for the PecS regulator[END_REF][START_REF] Hugouvieux-Cotte-Pattat | PehN, a polygalacturonase homologue with a low hydrolase activity, is coregulated with the other Erwinia chrysanthemi polygalacturonases[END_REF] and one rhamnogalacturonate lyase RhiE [START_REF] Laatu | Rhamnogalacturonate lyase RhiE is secreted by the out system in Erwinia chrysanthemi[END_REF].

Among them, the endo-pectate lyases (PelA, PelB, PelC, PelD, PelE and PelI) are the major pectinolytic enzymes produced by D. dadantii and they play a crucial role in soft rot disease.

Beyond the pectinases, two cellulases, CelY and Cel5 (ex. EGZ or CelZ) are also involved in the degradation of plant cell walls by cleaving the cellulose [START_REF] Py | Mutagenesis of cellulase EGZ for studying the general protein secretory pathway in Erwinia chrysanthemi[END_REF][START_REF] Zhou | Synergistic hydrolysis of carboxymethyl cellulose and acid-swollen cellulose by two endoglucanases (CelZ and CelY) from Erwinia chrysanthemi[END_REF]. The Cel5 is an extracelluar enzyme secreted by the T2SS Out [START_REF] Py | Mutagenesis of cellulase EGZ for studying the general protein secretory pathway in Erwinia chrysanthemi[END_REF] and represents the majority of endoglucanase activity. The CelY is located in the periplasm and is less abundant [START_REF] Boyer | Characterization of a new endoglucanase from Erwinia chrysanthemi[END_REF].

The sequence of the strain D. dadantii 3937 reveals the existence of other pectinolytic enzymes: a pectate lyase PelN (Hassan et al., in preparation), a polygalacturonase PehK [START_REF] Okinaka | Microarray profiling of Erwinia chrysanthemi 3937 genes that are regulated during plant infection[END_REF], two pectin lyases PnlH and PnlG homologue of E. carotovora, and two feruloyl esterases FaeD and FaeT [START_REF] Hassan | Identification of two feruloyl esterases in Dickeya dadantii 3937 and induction of the major feruloyl esterase and of pectate lyases by ferulic acid[END_REF].

I.3.3 Enzymatic degradation of plant cell wall

The process of pectin degradation begins in the extracellular environment, then continues in the periplasm, and culminates in the cytoplasm of the bacterial cell (Fig. 3).

I.3.3.1 Extracellular pectin degradation

At least ten pectinases (PaeY, PemA, PelA, PelB, PelC, PelD, PelE, PelI, PelL and PelZ) are secreted by T2SS Out [START_REF] Kazemi-Pour | The secretome of the plant pathogenic bacterium Erwinia chrysanthemi[END_REF] and involved in extracellular degradation of pectin.

Pectin acetylesterase PaeY [START_REF] Shevchik | Identification of a bacterial pectin acetyl esterase in Erwinia chrysanthemi 3937[END_REF]) and methylesterase

PemA [START_REF] Laurent | Characterization and overexpression of the pem gene encoding pectin methylesterase of Erwinia chrysanthemi strain 3937[END_REF] de-esterify pectin and release PGA which facilitates further degradation.

Endo-Pels (PelA, PelB, PelC, PelD, PelE, PelI, PelL and PelZ) cleave glycosidic linkages between two neighboring galacturonic acids and produce a planar product with an unsaturated bond between C4 and C5 at the nonreducing end (Fig. 3) [START_REF] Robert-Baudouy | Pectic enzymes of Erwinia chrysanthemi, regulation and role in pathogenesis[END_REF][START_REF] Abbott | Structural biology of pectin degradation by Enterobacteriaceae[END_REF]).

An endo-polygalacturonase PehN [START_REF] Hugouvieux-Cotte-Pattat | PehN, a polygalacturonase homologue with a low hydrolase activity, is coregulated with the other Erwinia chrysanthemi polygalacturonases[END_REF][START_REF] Abbott | Structural biology of pectin degradation by Enterobacteriaceae[END_REF], a rhamnogalactruonate lyase RhiE [START_REF] Laatu | Rhamnogalacturonate lyase RhiE is secreted by the out system in Erwinia chrysanthemi[END_REF] and a cellulase CelZ [START_REF] Zhou | Enhancement of expression and apparent secretion of Erwinia chrysanthemi endoglucanase (encoded by celZ) in Escherichia coli B[END_REF]) may be also involved in plant cell wall degradation.

Fig. 3: Pectin catabolism in D. dadantii [START_REF] Blot | The oligogalacturonate-specific porin KdgM of Erwinia chrysanthemi belongs to a new porin family[END_REF]. Extracellular pectinases (Pae-pectin acetylesterase, Pem-pectin methylesterase, Pel-pectate lyase, and Peh-polygalacturonase) degrade the pectin and the polygalacturonate (PGA) into oligogalacturonides (GAn) which then enter the cell. GAn are degraded by the periplasmic pectinases and pass through the inner membrane via one of the two transport systems, TogMNAB or TogT. GAn are then catabolized in the cytoplasm, producing pyruvate and 3-phosphoglyceraldehyde which integrate the general cellular metabolism.

Among these enzymes, the endo-Pels are considered, along with the endo-polygalacturonases, as a key factor in plant tissue maceration during soft rot infection [START_REF] Pickersgill | Crystal structure of polygalacturonase from Erwinia carotovora ssp. carotovora[END_REF].

Depolymerized pectic fragments are passively transported into the periplasmic space through the anion-specific oligosaccharide porins of KdgM and KdgN (Fig. 3) (Rodionov et al., 2004;[START_REF] Condemine | Differential regulation of two oligogalacturonate outer membrane channels, KdgN and KdgM, of Dickeya dadantii (Erwinia chrysanthemi)[END_REF]. Once transported into the periplasm, the pectic oligomers are further degraded by periplasmic pectinases into short oligomers, mainly dimers and trimers.

I.3.3.2 Periplasmic pectin degradation

Within the periplasm of D. dadantii, there are three major classes of pectinases:

Pectin esterases: the pectin methylesterase PemB anchored to the periplasmic face of the outer membrane [START_REF] Shevchik | Characterization of pectin methylesterase B, an outer membrane lipoprotein of Erwinia chrysanthemi 3937[END_REF] and the pectin acetylesterase PaeX located in the periplasm [START_REF] Shevchik | PaeX, a second pectin acetylesterase of Erwinia chrysanthemi 3937[END_REF]).

An exo-pectate lyase: PelX degrades the oligogalacturonates by attacking the reducing end and yieds unsaturated digalacturonates (Shevchik et al., 1999a).

Three exo-polygalacturonases: PehV, PehW and PehX, which complement the activity of the PelX, degrade the oligogalacturates by attacking their nonreducing end and release digalacturonates [START_REF] Nasser | Analysis of three clustered polygalacturonase genes in Erwinia chrysanthemi 3937 revealed an anti-repressor function for the PecS regulator[END_REF]Shevchik et al., 1999a).

I.3.3.3 Intracellular transport and cytoplasmic degradation

The products of extracellular and periplasmic pectin degradation are small (di-and tri-)

oligogalacturonides. These carbohydrates are subsequently transported into the cytoplasm where they are ultimately degraded by the cytoplasmic pectate lyase PelW and the oligogalacturonate lyase (Ogl) into galacturonate (GA) and 5-keto-4-deoxyuronate (DKI). These products finally degrade into pyruvate and 3-phosphoglyceraldehyde that enter the citric acid cycle and produce energy. Four distinct transporters are involved into the transport of small oligogalacturonides and monomers across the inner membrane: ExuT, KdgT, TogT and TogMNAB (Fig. 3) (Hugouvieux- [START_REF] Hugouvieux-Cotte-Pattat | Two transporters, TogT and TogMNAB, are responsible for oligogalacturonide uptake in Erwinia chrysanthemi 3937[END_REF]Abbott and Boraston, 2007). In fact, among these four independent systems, TogMNAB is the most prominent transporter during pectinolysis. In the cytoplasm, two enzymes are involved in degradation of pectic fragments: an exo-pectate lyase PelW, which degrades the short oligogalacturonates into unsatured digalacturonates; an oligogalacturonate lyase Ogl, which degrades unsaturated and saturated digalacturonates and generates galacturonate and DKI (Shevchik et al., 1999b).

Cotte

II Protein translocation and secretion pathways

The main pathogenicity factor of D. dadantii is due to the secretion of various enzymes, such as pectinases and cellulases into the extracellular environment. These secreted enzymes degrade the plant cell wall components and cause soft-rot disease. However, the bacterial cell enveloppe imposes a barrier for these proteins that are synthesized in the cytosol but function outside the cell. Various specialized transport mechanisms have evolved in Gram-negative bacteria to allow proteins to travel from the cytoplasm over the cell envelope to the exterior. is covalently attached to the outer membrane via LPPs [START_REF] Tommassen | Assembly of outer-membrane proteins in bacteria and mitochondria[END_REF].

II.1 The cell envelope of Gram-negative bacteria

The cell envelope of Gram-negative bacteria is composed of an inner membrane (IM) or cytoplasmic membrane (CM) and an outer membrane (OM), which are separated by the peptidoglycan-containing periplasm (Fig. 4). The IM is a symmetrical bilayer composed of phospholipids. Because of its hydrophobic nature, it is impermeable for hydrophilic compounds and also protons are unable to migrate freely over this membrane. IM proteins can be peripherally associated with the membrane by means of electrostatic and/or hydrophobic interactions or be present as integral membrane proteins spanning the phospholipids bilayer via hydrophobic α-helical segments. The OM faces the periplasm on one side and the extracellular medium on the other. This membrane is an asymmetrical bilayer composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet (Fig. 4), which renders the OM relatively resistant to detergents. The OM is semi-permeable due to the presence of channels formed by proteins, generically called "porins" (Fig. 4). These channels allow the passive diffusion of small hydrophilic molecules into the periplasm. Porins and other integral outer membrane proteins (OMPs) are characterized by the presence of amphipathic anti-parallel βstrands, which form a barrel-like structure with a hydrophobic surface facing the lipids, whereas the interior of the barrel is hydrophilic. The OM also contains lipoproteins, which are anchored to the membrane via their lipid moiety. Between the two membranes is located a hydrophilic periplasmic compartment containing a peptidoglycan layer (Fig. 4). This is a rigid structure consisting of linear polymeric sugar chains that are covalently linked via short oligopeptides. The peptidoglycan layer is important for the cell shape and rigidity. Apart from the peptidoglycan layer, the periplasm also harbors chaperones, degradative enzymes, proteins involved in nutrient acquisition, and proteins involved in peptidoglycan synthesis.

II.2 Protein translocation across and insertion into the inner membrane

Two distinct systems are involved in the transport of proteins across the IM into the periplasm, namely the general secretion (Sec) system and the twin-arginine translocation (Tat) system (Fig. 5). In bacteria, most exported proteins are moved across IM by the Sec pathway [START_REF] De Keyzer | The bacterial translocase: a dynamic protein channel complex[END_REF]. This system transports proteins into the periplasm in an unfolded conformation, but it can also integrate proteins into the IM. By contrast, the Tat system exports folded proteins across the IM [START_REF] Berks | Protein targeting by the bacterial twin-arginine translocation (Tat) pathway[END_REF].

II.2.1 Sec system

The Sec system consists of an IM peripherally associated ATPase SecA that acts as a molecular motor, and a translocon that constitutes the protein-conducting channel. The translocon is formed by a heterotrimeric complex, SecYEG (de [START_REF] De Keyzer | The bacterial translocase: a dynamic protein channel complex[END_REF]. The translocation processes are assisted by the YidC and SecDF (YajC) complex that transiently interacts with the translocon (de [START_REF] De Keyzer | The bacterial translocase: a dynamic protein channel complex[END_REF][START_REF] Du Plessis | The Sec translocase[END_REF]. Substrates of the Sec systems are synthesized at ribosomes in the cytosol. To reach their destination, these proteins need to be recognized and targeted to the Sec translocase that translocates them across or inserts them into the cytoplasmic membrane.

II.2.1.1 Recognition and targeting

Substrates of Sec systems are synthesized as precursor proteins with an N-terminal signal sequence, which targets them to the translocase. A typical Sec signal sequence contains a positively charged N-terminal region (n-region), a nonpolar hydrophobic core (h-region) and a more polar C-terminal region (c-region) containing the signal peptidase cleavage site (Fig. 6). Generally, integral membrane proteins do not have a signal sequence, and instead their hydrophobic transmembrane domains (TMDs) function as an internal signal for targeting and insertion. Two different pathways are employed for recognition and targeting of proteins to the translocation site: one employs the chaperone SecB for post-translational targeting (Fig. 5b), and the other utilizes the signal recognition particle (SRP) for co-translational targeting (Fig. 5a) [START_REF] Müller | Protein traffic in bacteria: multiple routes from the ribosome to and across the membrane[END_REF]. In bacteria, the post-translational pathway is used by proteins secreted across the membrane, while co-translational pathway is mainly utilized by IM proteins [START_REF] De Gier | The E. coli SRP: preferences of a targeting factor[END_REF].

SecB is a molecular chaperone that binds to newly synthesized precursor polypeptides (preproteins) and stabilizes them in an unfolded and non-aggregated state after they exit from the ribosome translation tunnel [START_REF] Breukink | In vitro studies on the folding characteristics of the Escherichia coli precursor protein prePhoE. Evidence that SecB prevents the precursor from aggregating by forming a functional complex[END_REF][START_REF] Bechtluft | Direct observation of chaperone-induced changes in a protein folding pathway[END_REF]. Although SecB does not directly bind to the signal sequence of preproteins (Randall and Hardy;2002), the presence of this sequence is indispensable for export [START_REF] Zhou | The structural view of bacterial translocation-specific chaperone SecB: implications for function[END_REF]. The peptide-binding study defined a general SecB-binding motif that consists of approximately nine amino acid residues enriched in aromatic and basic residues, whereas acidic residues are strongly disfavored [START_REF] Knoblauch | Substrate specificity of the SecB chaperone[END_REF]. SecB is a homotetramer that is organized as a dimer of dimers (Fig. 7A) [START_REF] Zhou | The structural view of bacterial translocation-specific chaperone SecB: implications for function[END_REF][START_REF] Natale | Sec-and Tat-mediated protein secretion across the bacterial cytoplasmic membrane-distinct translocases and mechanisms[END_REF]. Biochemical data revealed two types of preprotein binding sites in SecB tetramer: one that interacts with polypeptides with extended β-sheet stretches and another interacts with hydrophobic polypeptide regions [START_REF] Randall | The interaction between the chaperone SecB and its ligands: evidence for multiple subsites for binding[END_REF][START_REF] Crane | Sites of interaction of a precursor polypeptide on the export chaperone SecB mapped by site-directed spin labeling[END_REF]. Consistent with these biochemical data, the putative peptide-binding groove in crystal structure of SecB tetramer seems to contain two subsites that are involved in recognition of distinct features in the preprotein. One subsite is a deep cleft lined with mostly conserved aromatic residues that may recognize the hydrophobic and aromatic regions of the polypeptides, while the other subsite forms a shallow open groove with a hydrophobic surface that might be involved in the binding of β-pleated sheets (Fig. 7B) [START_REF] Natale | Sec-and Tat-mediated protein secretion across the bacterial cytoplasmic membrane-distinct translocases and mechanisms[END_REF][START_REF] Zhou | The structural view of bacterial translocation-specific chaperone SecB: implications for function[END_REF].

Subsequently, SecB directs the bound preprotein into the translocation pathway via its specific interaction with membrane-bound SecA [START_REF] Randall | Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA[END_REF].

A.

B.

Fig. 7:

The structure of SecB homo-tetramer (A) and the peptide-binding groove within the SecB molecule (B).

The homo-tetramer (A) is organized as a dimer of dimers. Each subunit is colored differently; one dimer consists of the green and red subunits while the other consists of the blue and yellow subunits. The unfolded preprotein would bind to a channel consisting of two subsites (1 and 2) (B) [START_REF] Natale | Sec-and Tat-mediated protein secretion across the bacterial cytoplasmic membrane-distinct translocases and mechanisms[END_REF][START_REF] Zhou | The structural view of bacterial translocation-specific chaperone SecB: implications for function[END_REF].

Preproteins with very hydrophobic signal peptides and most IMPs are targeted to Sec system by SRP in a co-translational manner. In fact, unlike in eukaryotes, translational arrest was not been observed in Gram-negative bacteria. However, it was suggested that the shorter traffic distances and the faster translocation rates in bacteria overcome the need for translation arrest [START_REF] Driessen | Protein translocation across the bacterial cytoplasmic membrane[END_REF]. The E. coli SRP is composed of a complex of a 4.5S RNA and a 48 kDa GTPase P48 or Ffh (for fifty-four homolog) that interacts specifically with the signal sequence or hydrophobic TMSs of nascent proteins [START_REF] Driessen | Protein translocation across the bacterial cytoplasmic membrane[END_REF]. SRP also binds to the ribosomal large subunit near its exit gate [START_REF] Schaffitzel | Structure of the E. coli signal recognition particle bound to a translating ribosome[END_REF] and brings the ribosome-nascent chain (RNC) complex to the membrane through its interaction with FtsY (SRP receptor). FtsY binds to membrane via the anionic phospholipids but also binds directly to the SecYEG channel [START_REF] Angelini | Membrane binding of the bacterial signal recognition particle receptor involves two distinct binding sites[END_REF][START_REF] Kuhn | The bacterial SRP receptor, SecA and the ribosome use overlapping binding sites on the SecY translocon[END_REF]. Both Ffh and FtsY are GTP-binding proteins and are capable of GTP hydrolysis, which is activated by the interaction of these two proteins [START_REF] Egea | Substrate twinning activates the signal recognition particle and its receptor[END_REF]. Upon GTP hydrolysis by both SRP and its receptor, the RNC-SRP-FtsY complex dissociates, and the released RNC complex is transferred to the translocase (see, for review, du [START_REF] Du Plessis | The Sec translocase[END_REF].

II.2.1.2 Mechansim and structural insights in preprotein translocation

The SRP and SecB-mediated targeting routes converge at the translocase. SecA couples with the stepwise translocation of preprotein across the SecYEG channel in the bacterial IM.

(1) SecA, an ATPase dependent motor protein

SecA is a soluble protein that localizes to both the cytosol and the IM. It functions as an

ATPase dependent motor protein. It binds to the membrane via low-and high-affinity interaction with anionic phospholipids and the SecYEG complex, respectively [START_REF] Du Plessis | The Sec translocase[END_REF]. It interacts with nearly all other components involved in protein translocation, and its ATP hydrolysis activity is suppressed in its resting state, whereas it is stimulated by its binding to the membrane or interaction with acidic phospholipids and more strikingly in the presence of SecYEG and preproteins [START_REF] Kusters | SecA, a remarkable nanomachine[END_REF]. It is believed that SecA undergoes large conformational changes to drive protein translocation. The crystal structure of SecA from various organisms have been solved [START_REF] Hunt | Nucleotide control of interdomain interactions in the conformational reaction cycle of SecA[END_REF][START_REF] Sharma | Crystal structure of Mycobacterium tuberculosis SecA, a preprotein translocating ATPase[END_REF][START_REF] Osborne | A large conformational change of the translocation ATPase SecA[END_REF][START_REF] Vassylyev | Crystal structure of the translocation ATPase SecA from Thermus Thermophilus reveals a parallel, head-to-head dimer[END_REF][START_REF] Papanikolau | Structure of dimeric SecA, the Escherichia coli preprotein translocase motor[END_REF]. Most of these structures display SecA packed as a dimer (Fig. 8) with an antiparallel orientation except for the T. thermophilus SecA that was crystallized as a parallel dimer [START_REF] Vassylyev | Crystal structure of the translocation ATPase SecA from Thermus Thermophilus reveals a parallel, head-to-head dimer[END_REF]. The SecA protomer can be subdivided into several structural subdomains (reviewed by Kusters andDriessen, 2011 and[START_REF] Du Plessis | The Sec translocase[END_REF], i.e., nucleotide-binding folds 1 and 2 (NBF1 and NBF2), the preprotein cross-linking domain (PPXD), the α-helical scaffold domain (HSD), the α-helical wing domain (HWD) and the C-terminal linker (CTL). The actual motor function of SecA is performed by the "DEAD motor" core that consists of NBF1 and NBF2. The interface between these two domains forms the nucleotide binding site. ATP can be bound to the interface and hydrolyzed to induce the conformational changes necessary for preprotein translocation. The PPXD domain can be crosslinked to the leader and mature domain of preprotein. The HSD domain contacts all other domains of SecA and therefore likely plays an important role in the catalytic cycle of SecA. The HWD domain seems to be flexible and loosely linked with the rest of the molecule. The CTL domain has been shown to be involved in lipid binding, in Zn 2+ and in SecB binding. Recently, it was shown that the amino terminus of SecA was also involved in the SecB binding [START_REF] Randall | Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA[END_REF]. (2) SecYEG complex, the protein-conducting channel

The pore of the translocase is formed by a complex of the SecY, SecE and SecG proteins. The heterotrimeric organization of the translocation pore is conserved throughout the three kingdoms of life [START_REF] De Keyzer | The bacterial translocase: a dynamic protein channel complex[END_REF]. Among the three components, SecY is the central component of translocon, in which a polypeptide-conducting channel is formed. SecY has 10 transmembrane segments (TM1-TM10), six cytosolic regions (C1-C6) and five periplasmic regions (P1-P5). It interacts independently with SecE and SecG to form a heterotrimeric SecYEG complex. The interaction of SecE and SecY protects SecY from degradation by the membrane-bound protease FtsH [START_REF] Kihara | FtsH is required for proteolytic elimination of uncomplexed forms of SecY, an essential protein translocase[END_REF] that is involved in the degradation of unassembled membrane protein complexes. SecE is a 14 kDa integral membrane protein. In E. coli, SecE consists of three transmembrane segments (TMSs) while the homologue Sec61γ has only one TMS [START_REF] Kusters | SecA, a remarkable nanomachine[END_REF]. Cysteine-scanning mutagenesis studies identified contacts between SecE TMS3

and SecY TMS 2, 7 and 10 [START_REF] Kaufmann | Cysteinedirected cross-linking demonstrates that helix 3 of SecE is close to helix 2 of SecY and helix 3 of a neighboring SecE[END_REF][START_REF] Veenendaal | Mapping the sites of interaction between SecY and SecE by cysteine scanning mutagenesis[END_REF][START_REF] Veenendaal | The core of the bacterial translocase harbors a tilted transmembrane segment 3 of SecE[END_REF]. Despite its small size, SecE is essential for viability and translocation, while SecG, a small protein of ~12kDa containing two TMS, is not essential for viability or translocation. It was hypothesized that SecG facilitates the binding and insertion of SecA into the translocon by undergoing transient topological inversions during protein translocation [START_REF] Nagamori | Membrane topology inversion of SecG detected by labeling with a membrane-impermeable sulfhydryl reagent that causes a close association of SecG with SecA[END_REF]. However, another study showed that a topologically fixed SecG was fully functional in protein translocation [START_REF] Van Der Sluis | Topologically fixed SecG is fully functional[END_REF]. Cysteine-scanning mutagenesis showed that SecG is in close proximity to C3 and TMS4 of SecY (van der [START_REF] Van Der Sluis | Topologically fixed SecG is fully functional[END_REF]. To date, no evidence has been found for a physical interaction between SecE and SecG. A number of studies demonstrated the oliogomeric states of SecYEG, such as dimers, trimers, or tetramers [START_REF] Manting | SecYEG assembles into a tetramer to form the active protein translocation channel[END_REF][START_REF] Tziatzios | The bacterial protein-translocation complex: SecYEG dimers associate with one or two SecA molecules[END_REF][START_REF] Mitra | Structure of the E. coli protein-conducting channel bound to a translating ribosome[END_REF]. Interestingly, a structural analysis suggested that monomeric SecA-SecYEG is sufficient for translocation [START_REF] Zimmer | Structure of a complex of the ATPase SecA and the protein-translocation channel[END_REF]. There is evidence that an interacting partner (ribosome or SecA) as well as simple integration into the lipid bilayer induces dimerization/oligomerization of SecYEG (Matra et al., 2005;[START_REF] Scheuring | The oligomeric distribution of SecYEG is altered by SecA and translocation ligands[END_REF]. Currently, two models are proposed for the orientation of the dimeric translocon: the 'front-to-front' and 'back-to-back' orientation (Fig. 9) [START_REF] Du Plessis | The Sec translocase[END_REF]. In the 'front-to-front' orientation, the lateral gates of the two SecYEG complexes are facing each other (Fig. 9A), while in the 'back-to-back' orentation, the two SecYEG complexes are contacted via the TMS of SecE that embraces the SecY subunits as a clamp. However, the functional relevance of these two orientations remains puzzle (du Plessis et

A. B.
al., 2011). The SecYEG complex can also associate transiently with another heterotrimeric membrane protein complex YidCSecDF (YajC) which stimulates preprotein translocation [START_REF] Nouwen | SecDFyajC forms a heterotetrameric complex with YidC[END_REF]. Although YajC associates with SecDF, it is not essential for functionality, but cells lacking SecD and SecF are cold sensitive, which prevents growth, and are severely defective in protein translocation [START_REF] Driessen | Protein translocation across the bacterial cytoplasmic membrane[END_REF]. However, the SecDF is not needed for translocation per se. The role of SecDF has remained unclear, although it is proposed to function in later stages of translocation and in membrane protein biogenesis [START_REF] Driessen | Protein translocation across the bacterial cytoplasmic membrane[END_REF][START_REF] Du Plessis | The Sec translocase[END_REF]. Recently, the crystal structure of T. thermophilus SecDF revealed a pseudo-symmetrical, 12-helix transmembrane domain belonging to the resistance nodulation and cell division (RND) superfamily and two major periplasmic domains, P1 and P4 (Fig. 10) [START_REF] Tsukazaki | Structure and function of a membrane component SecDF that enhances protein export[END_REF]. They proposed that SecDF functions as a membrane integrated chaperone, powered by proton motive force (PMF) to achieve ATP-independent protein translocation. This function depends on the ability of the periplasmic P1 domain to interact with a substrate and to undergo a structural transition.

YidC is functionally and structurally homologous to Oxa1 in mitochondria and Alb3 in chloroplasts [START_REF] Samuelson | YidC mediates membrane protein insertion in bacteria[END_REF][START_REF] Kuhn | The Alb3/Oxa1/YidC protein family: membranelocalized chaperones facilitating membrane protein insertion?[END_REF]. It facilitates the insertion of a subset of membrane proteins via the translocon [START_REF] Kol | Subunit a of the F(1)F(0) ATP synthase requires YidC and SecYEG form membrane insertion[END_REF][START_REF] Du Plessis | The Sec translocase[END_REF]. YidC is also able to act as an insertase on its own [START_REF] Serek | Escherichia coli YidC is a membrane insertase for Sec-independent proteins[END_REF][START_REF] Dalbey | Assembly of bacterial inner membrane proteins[END_REF].

(3) Mechanism of protein translocation It is believed that multiple rounds of ATP binding and hydrolysis lead to the stepwise translocation of the preprotein (Fig. 11) [START_REF] Facey | Biogenesis of bacterial inner-membrane proteins[END_REF]. The preprotein is targeted by SecB or SRP to the tranlocon. The translocation starts with the binding of ATP to SecA. A hairpin loop of the signal sequence with the early mature protein region is inserted into the translocation pore. This step can also be stimulated by the PMF, which likely affects the orientation of the signal sequence within the channel or the conformation of the SecY protein and facilitate opening of the lateral gate region [START_REF] Facey | Biogenesis of bacterial inner-membrane proteins[END_REF]. ATP hydrolysis results in a dissociation of SecA from the preprotein and weakens the SecA-SecYEG binding affinity.

SecA is then likely released from the translocon but may rebind to the partially translocated preprotein trapped in the SecYEG pore and thereby causing a translocation of about 5 kDa of preprotein through the pore [START_REF] Facey | Biogenesis of bacterial inner-membrane proteins[END_REF]. Repeated cycles of ATP binding and hydrolysis, as well as polypeptide binding and release, result in the stepwise translocation of the polypeptide across the membrane (Fig. 11).

Fig. 11:

The model for the different steps of Sec-mediated protein translocation [START_REF] Facey | Biogenesis of bacterial inner-membrane proteins[END_REF].

These steps are coupled to ATP hydrolysis cycles and steps 3-5 are repeated until the polypeptide is fully translocated.

Once the preprotein has been transported into periplasm, the N-terminal signal sequences of precursors are cleaved by the type II signal peptidase for lipoproteins and the type I for the others [START_REF] Dev | Rapid assay and purification of a unique signal peptidase that processes the prolipoprotein from Escherichia coli B[END_REF][START_REF] Tuteja | Type I signal peptidase: an overview[END_REF]

II.2.2 Protein insertion in the inner membrane

Most IMPs insert into the membrane via the Sec system [START_REF] Du Plessis | The Sec translocase[END_REF][START_REF] Dalbey | Assembly of bacterial inner membrane proteins[END_REF]. They are synthesized without a cleavable signal sequence but with hydrophobic TMS required for targeting and insertion into IM. Indeed, SRP recognizes and binds to a hydrophobic segment of a membrane protein as it emerges from the ribosome at the membrane face and target the complex to its receptor FtsY located at the membrane. After GTP hydrolysis, the IMPs are transferred to the SecYEG translocation channel, and SRP and FtsY dissociate from each other.

ATP Pi

ADP ADP ATP ATP

Pi ATP

ADP

Both translocase SecYEG and insertase YidC are required to insert newly synthesized proteins into membrane [START_REF] Dalbey | Assembly of bacterial inner membrane proteins[END_REF]. SecYEG typically mediates the insertion of proteins into the membrane and the lateral partitioning of the TMSs into the lipid phase (Fig. 12a). The insertase YidC, suggested to play a direct role during the insertion of Sec-independent substrates, is responsible for inserting small proteins into the membrane (Fig. 12c) [START_REF] Winterfeld | Substrate-induced conformational change of the Escherichia coli membrane insertase YidC[END_REF][START_REF] Dalbey | Assembly of bacterial inner membrane proteins[END_REF]. YidC can also function as an integrase and cooperate with the Sec translocase to mediate membrane protein insertion (Fig. 12b). In some cases, YidC inserts the Nterminal domain of protein, SecYEG then promotes the insertion of the C-terminal region of the protein (Fig. 12d). The topology of membrane proteins is established during its insertion step and obeys the positive-inside rule [START_REF] Von Heijne | Membrane-protein topology[END_REF][START_REF] Dalbey | Assembly of bacterial inner membrane proteins[END_REF].

II.2.3 Tat system

The Tat system is found in the cytoplasmic membranes of many eubacteria, some Archaea, and the chloroplasts and mitochondria of plants [START_REF] Müller | Twin-arginine-specific protein export in Escherichia coli[END_REF]. In bacteria, the Tat system mediates export of periplasmic proteins in folded conformation [START_REF] Müller | Twin-arginine-specific protein export in Escherichia coli[END_REF]. Tat translocases consist of two or three membrane integrated subunits, i.e., TatA and TatC or TatA, TatB and TatC that together form a receptor and a protein conducting machinery for Tat substrates [START_REF] Natale | Sec-and Tat-mediated protein secretion across the bacterial cytoplasmic membrane-distinct translocases and mechanisms[END_REF]. The energy for translocation is provided by the proton motive force (PMF) [START_REF] Müller | Twin-arginine-specific protein export in Escherichia coli[END_REF].

II.2.3.1 Tat substrates

Generally, Tat substrates bear N-terminal signal sequences that resemble the overall organization of Sec signal sequences. They also contain a positively charged n-region, a hydrophobic h-region and a short polar c-region with the peptidase cleavage site (Fig. 6).

However, there are some differences between Sec and Tat signals [START_REF] Natale | Sec-and Tat-mediated protein secretion across the bacterial cytoplasmic membrane-distinct translocases and mechanisms[END_REF]: i) Tat signal sequence is characterized by the consensus motif Z-R-R-x-Φ-Φ at the interface of the N-and H-regions and a replacements of one or both arginine residues which are highly conserved markedly decrease the transport efficiency;

ii) bacterial Tat signal sequences are usually longer than their Sec counterparts;

iii) h-region of Sec signal sequences are more hydrophobic than those of Tat signal peptides due to more glycine and threonine residues; iv) c-region of Tat signals often contains basic residues while Sec signals are almost never charged in c-region, which are support to function as a Sec-avoidance motif. [START_REF] Tullman-Ercek | Export pathway selectivity of Escherichia coli twin arginine translocation signal peptides[END_REF] showed that Tat specificity depends both on the overall charge of the c-region and the N-terminal section of the mature protein. Some Tat substrates of E. coli do not have a Tat signal sequence of their own. They are transported in multimeric complexes together with protein containing a Tat signal sequence by a so-called "hitchhiker mechanism" [START_REF] Rodrigue | Co-translocation of a periplasmic enzyme complex by a hitchhiker mechanism through the bacterial tat pathway[END_REF].

The Tat system exports proteins that are folded prior to transport and somehow senses and rejects unfolded substrates, suggesting that the Tat machinery might have an innate 'quality control' activity that confers the ability to recognize the folded state of a substrate protein [START_REF] Robinson | Transport and proofreading of proteins by the twin-arginine translocation (Tat) system in bacteria[END_REF]. This is particularly important for the Tat substrates required cofactor insertion. In E. coli, about 6% of all secreted protein are predicted to be Tat-dependent and the majority are cofactor-containing redox proteins including hydrogenases, formate dehydrogenases, trimethylamine N-oxide (TMAO) reductase, dimethyl sulfhoxide (DMSO) reductase and nitrate reductases [START_REF] Palmer | Export of complex cofactor-containing proteins by the bacterial Tat pathway[END_REF][START_REF] Lee | The bacterial twin-arginine translocation pathway[END_REF], all of which function in anaerobic respiration.

It was suggested that the signal sequence plays a certain role in cofactor assembly of a Tat substrate, either by interaction with an accessory protein or by specific protein-protein interaction with unfolded portion of the mature protein sequence [START_REF] Berks | Protein targeting by the bacterial twin-arginine translocation (Tat) pathway[END_REF].

In addition to cofactor-containing proteins, in E. coli, there are also Tat substrates which lack cofactors, such as Sulf I and the periplasmic amidases AmiA and AmiC [START_REF] Berks | Protein targeting by the bacterial twin-arginine translocation (Tat) pathway[END_REF], indicating that co-factor assembly is not the only reason for Tat dependent transport.

The Tat system also allows integration of certain IMPs. Targeting to the IM requires both the hydrophobic C-terminal transmembrane helices (C-tail) and the N-terminal Tat signal peptide [START_REF] Hatzixanthis | A subset of bacterial inner membrane proteins integrated by the twin-arginine translocase[END_REF]. In addition, Tat system was also shown to be involved in the targeting of pre-folded or fast-folding lipoproteins [START_REF] Shruthi | Twin arginine translocase pathway and fastfolding lipoprotein biosynthesis in E. coli: interesting implications and applications[END_REF].

II.2.3.1 Tat translocase

Fig. 13: The predicted topology of the E. coli Tat components. Predicted helical regions are shown as boxes. [START_REF] Lee | The bacterial twin-arginine translocation pathway[END_REF] The Tat system of E. coli consists of three individual IMPs: TatA, TatB and TatC (Fig. 13).

The orthologues in the thylakoidal membrane of chloroplasts are termed Tha4, Hcf106 and cpTatC, respectively [START_REF] Lee | The bacterial twin-arginine translocation pathway[END_REF]. TatC is the largest and most highly conserved component consisting of 258 amino acids. It contains six predicted transmembrane helices with both N-and C-teminus located on the cytoplasmic side of the membrane (Fig. 13) [START_REF] Lee | The bacterial twin-arginine translocation pathway[END_REF]. It is generally accepted that the TatC protein alone, or in a complex with TatB, forms the initial recognition site for a Tat signal sequence [START_REF] Alami | Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli[END_REF]. However, the precise signal sequence-binding site of TatC has not yet been identified, it seems that the entire N-terminal half of TatC is involved in recognition [START_REF] Holzapfel | The entire Nterminal half of TatC is involved in twin-arginine precursor binding[END_REF], with the first cytosolic loop of TatC being of particular importance [START_REF] Kreutzenbeck | Escherichia coli twin arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities[END_REF][START_REF] Strauch | Escherichia coli tatC mutations that suppress defective twinarginine transporter signal peptides[END_REF]. Both TatA and TatB have a similar topology, with an N-terminal transmembrane α-helix that is linked by a hinge region to an extended amphipathic helix followed by an unstructured C-terminal tail (Fig. 13) [START_REF] Lee | The bacterial twin-arginine translocation pathway[END_REF].

In Gram-negative bacteria, two separate complexes are found at a steady state: a TatABC complex and homo-oligomeric TatA complex [START_REF] Lee | The bacterial twin-arginine translocation pathway[END_REF][START_REF] Robinson | Transport and proofreading of proteins by the twin-arginine translocation (Tat) system in bacteria[END_REF]. Within the TatABC complex, TatB and TatC form a functional unit with a ratio 1:1 [START_REF] Oates | The Escherichia coli twin-arginine translocation apparatus incorporates a distinct form of TatABC complex, spectrum of modular TatA complexes and minor TatAB complex[END_REF][START_REF] Orriss | TatBC, TatB and TatC form structurally autonomous units within the twin arginine protein transport system of Escherichia coli[END_REF]. The TatBC plays important roles in substrate binding but the role of TatA in the complex is unclear. However, the complex appears to be less stable in the absence of TatA [START_REF] Dabney-Smith | Oligomers of Tha4 organize at the thylakoid Tat translocase during protein transport[END_REF][START_REF] Behrendt | The TatBC complex formation suppresses a modular TatB-multimerization in Escherichia coli[END_REF]. Further analysis revealed that the major binding site for folded substrates is formed by TatB [START_REF] Maurer | TatB Functions as an Oligomeric Binding Site for Folded Tat Precursor Proteins[END_REF]. The TatA complexes have very unusual properties: they are remarkably heterogeneous and range in size from 50 kDa to well over 500 kDa. TatA forms different size homo-oligomeric complexes in the membrane, which suggests that TatA forms a size-fitting protein-conducting channel [START_REF] Mcdevitt | Subunit composition and in vivo substrate-binding characteristics of Escherichia coli Tat protein complexes expressed at native levels[END_REF][START_REF] Greene | Cysteine scanning mutagenesis and disulfide mapping studies of the TatA component of the bacterial twin arginine translocase[END_REF]. The functionally essential regions of TatA are located within the cytoplasmic region and the hinge region [START_REF] Hicks | The Escherichia coli twin-arginine translocase: conserved residues of TatA and TatB family components involved in protein transport[END_REF]2005). Since the TatABC and TatA complexes show little or no propensity to associate after isolation, it is generally accepted that, at the steady state, the Tat system comprises two distinct complexes, one containing TatABC and the other containing only TatA [START_REF] Robinson | Transport and proofreading of proteins by the twin-arginine translocation (Tat) system in bacteria[END_REF].

II.2.3.2 Translation mechanism

The bacterial and the thylakoidal Tat systems transport fully folded proteins across the IM into the periplasm of bacteria and across the thylakoid membrane into the luminal space of the chloroplast, respectively. Tat transport is somehow triggered by PMF. The translocation process can be divided into two separate stages: firstly, targeting and binding of the substrates to the TatBC receptor complex, and secondly, PMF dependent recruitment of the TatA complex to form the fully assembled and active Tat translocase. Several models have been proposed to explain the transport mechanism. Most of the models supposed that TatA complex form a hydrophilic channel through which the passenger domain of the Tat substrate is transported across the membrane [START_REF] Sargent | Purified components of the Escherichia coli Tat protein transport system form a double-layered ring structure[END_REF][START_REF] Alami | Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli[END_REF]. However, [START_REF] Dabney-Smith | Oligomers of Tha4 organize at the thylakoid Tat translocase during protein transport[END_REF] suggest that the TatA-recruitment does not depend on the substrates size, which is not consistent with the hypothesis of preformed TatA pores. Thus, another non-pore model is proposed (Fig. 14) [START_REF] Natale | Sec-and Tat-mediated protein secretion across the bacterial cytoplasmic membrane-distinct translocases and mechanisms[END_REF]. Prior to the contact with the Tat translocase, Tat substrates seem target to the membrane via an unassisted insertion of the signal sequence into the lipid bilayer. The region of the twin-arginine motif in Tat substrate signal sequences binds to the TatBC complex (Fig. 14A). Upon substrate binding and in the presence of PMF, TatA is recruited to the TatBCsubstrate ternary complex (Fig. 14B). Multiple hydrophobic TMDs of TatA assemble near the bound Tat substrate. This may destabilize the membrane bilayer. The interaction between TatA and Tat substrates may induce the conformational change in TatBC (Fig. 14C), which pulls the Tat substrate across membrane weakened by TatA (Fig. 14D). In the moment of releasing of the Tat substrate, a transient hole may be formed which could account for the large proton flux accompanying Tat substrate (Fig. 14E). After transport, the membrane has to be sealed immediately, TatA disassembles from TatBC and Tat substrate is released from the membrane by a cleavage of type I or type II signal sequences peptidases (Fig. 14F).

II.3 Protein folding and maturation in the periplasm

Once transported over the IM into the periplasm by the Sec or by the Tat system, the proteins residing in or transiting through the periplasm are folded into their native-conformation in periplasm. In the periplasm of E. coli, three groups of factors might facilitate their folding [START_REF] Mogensen | Interactions between folding factors and bacterial outer membrane proteins[END_REF] (Fig. 15), i.e., proteases, molecular chaperones and folding catalysts. Indeed, this categorization is not strict, since many of the proteins display several activities (Fig. 15). For example, DegP is a protease that degrades unfolded or misfolded proteins in the periplasm but also has chaperone activity, and peptidyl-prolyl isomerases (PPlases) SurA and FkpA act also as chaperones. The molecular chaperones are proteins that assist the noncovalent folding or unfolding of target proteins. In E. coli, several chaperones, i.e., DegP, Skp and SurA assist in the maturation of OMPs (Sklar et al., 2007b). Two types of folding catalysts are found in the periplasm, namely protein disulphide isomerases and cis-trans PPlases. The former catalyze the formation and rearrangement of disulfide bonds, which promotes the oxidative folding of secreted proteins, while the latter catalyse the cis-trans isomerization of peptide bonds. The disulphide isomerases DsbA and DsbC function together with the IMPs DsbB and DsbD. DsbA and DsbB catalyze the introduction of disulfide bonds into folding proteins, whereas DsbC and DsbD provide a proofreading system for the restoration of misfolded proteins with non-native disulfide bonds [START_REF] Nakamoto | Catalysis of disulfide bond formation and isomerization in the Esherichia coli periplasm[END_REF][START_REF] Mogensen | Interactions between folding factors and bacterial outer membrane proteins[END_REF][START_REF] Inaba | Disulfide bond formation system in Escherichia coli[END_REF]. DsbC of D. dadantii is also involved in catalyzing the formation of disulfide bonds in the secretion of pectate lyases [START_REF] Shevchik | Differential effect of dsbA and dsbC mutations on extracellular enzyme secretion in Erwinia chrysanthemi[END_REF] Fig. 15: The periplasmic and outer membrane folding factors [START_REF] Mogensen | Interactions between folding factors and bacterial outer membrane proteins[END_REF]. In the periplasm of E. coli, three groups of proteins might assist in protein folding: protease, molecular chaperones and folding catalysts. In the OM of E. coli, both proteinaceous and lipidic components may assist in OMP folding.

II.4 Lipoproteins

Bacterial lipoproteins are anchored to the IM or OM via an N-terminal lipid moiety N-acyldiacyl-glyceryl cysteine. Lipidation and folding of these proteins occur after their translocation over the IM via the Sec system or in some case by the Tat system [START_REF] Gralnick | Extracellular respiration of dimethyl sulfoxide by Shewanella oneidensis strain MR-1[END_REF]. Prior to cleavage of the signal sequence, a diacylglycerol group is transfered by enzymes Lgt from phosphatidylglycerol to the sulfhydryl group of the cysteine located at the N-terminal of lipoproteins [START_REF] Sankaran | Lipid modification of bacterial prolipoprotein. Transfer of diacylglyceryl moiety from phosphatidylglycerol[END_REF]. After cleavage of the signal sequence by type II signal peptidase, the free α-amino group of the cysteine is acylated by the enzyme Lnt [START_REF] Sankaran | Lipid modification of bacterial prolipoprotein. Transfer of diacylglyceryl moiety from phosphatidylglycerol[END_REF], yielding the mature lipoprotein.

In E. coli, lipoproteins are sorted to the IM or OM depending on the lipoprotein-sorting signal located at position 2 of mature lipoprotein. An aspartate at this position retains the lipoprotein in the IM, whereas the absence of this IM retention signal results in transport to OM by Lol system (Tokuda andMatsuyama, 2004, Lewenza et al., 2006). However, in P. aeruginosa, +3 and +4 positions were identified as sorting residues. A lysine and serine residue at +3 and +4, respectively, retained the protein in the IM, whereas a leucine and isoleucine resulted in translocation to the OM [START_REF] Narita | Amino acids at positions 3 and 4 determine the membrane specificity of Pseudomonas aeruginosa lipoproteins[END_REF][START_REF] Tanaka | Characterization of the Pseudomonas aeruginosa Lol system as a lipoprotein sorting mechanism[END_REF]. open the hydrophobic LolA cavity to accommodate the lipoprotein. When the LolA-lipoprotein complex interacts with the OM receptor LolB, the lipoprotein is transferred to LolB and inserted into the OM. Further transport to the outer leaflet of the OM, if applicable, occurs through unknown mechanisms.

The Lol system comprises an IM ATP-binding cassette (ABC) transporter LolCDE complex, a periplasmic carrier protein LolA and an OM receptor protein LolB. The model (Fig. 16) [START_REF] Bos | Functioning of outer membrane protein assembly factor Omp85 requires a single POTRA domain[END_REF] states that lipoproteins destined for the OM interact with the LolCE, and the binding results in an increase in the affinity of LolD for ATP. Upon hydrolysis of ATP, the lipoprotein is released from LolCE to the periplasmic chaperone LolA [START_REF] Ito | A novel ligand bound ABC transporter, LolCDE, provides insights into the molecular mechanisms underlying membrane detachment of bacterial lipoproteins[END_REF]. LolA chaperones OM lipoproteins across the periplasm and delivers them to LolB, which causes lipoprotein releasing from LolA and integration of the lipoprotein into the inner leaflet of the OM [START_REF] Bos | Functioning of outer membrane protein assembly factor Omp85 requires a single POTRA domain[END_REF].

In some bacteria, cell surface-exposed lipoproteins also are present. However, whether the transport over the OM occurs through an extension of the Lol system or by an unrelated transport system is unclear. For example, in K. oxytoca, cell surface-exposed lipoprotein pullulanase contains an aspartate at position +2, indicating that it is not transported by Lol system. Indeed, it requires the T2SS named Pul for transport [START_REF] Pugsley | The complete general secretory pathway in gram-negative bacteria[END_REF]. In N. meningitidis, the cell surfaceexposed lipoproteins LbpB and TbpB could be transported form the IM to OM by the Lol system. However, no component of the Lol system is involved in the translocation of the surface-exposed lipoprotein from the periplasmic side of the OM to the cell surface. In addition, the sequenced genomes do not reveal the presence of a type II secretion apparatus [START_REF] Van Ulsen | Protein secretion and secreted proteins in pathogenic Neisseriaceae[END_REF]. They may be transported to the cell surface via an extension of the Lol system.

II.5 Integral outer membrane proteins

There are two major types of proteins in the OM, lipoproteins and integral OMPs. These two types of proteins require different systems for folding and insertion into the OM. Integral βbarrel OMPs need the SurA and Skp-DegP pathways to transport nascent OMPs across the periplasmic space to the OM (Sklar et al., 2007b). It is well kown that integral OMPs require Bam complex for folding and insertion into the OM [START_REF] Werner | YaeT (Omp85) affects the assembly of lipid-dependent and lipidindependent outer membrane proteins of Escherichia coli[END_REF][START_REF] Wu | Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli[END_REF][START_REF] Doerrler | Loss of outer membrane proteins without inhibition of lipid export in an Escherichia coli YaeT mutant[END_REF]Raetz, 2005, Malinverni et al., 2006;Sklar et al., 2007a). One known exception is secretin PulD. It has been shown that Bam complex is not required for insertion of PulD into the OM [START_REF] Collin | YaeT-independent multimerization and outer membrane association of secretin PulD[END_REF][START_REF] Guilvout | In vitro multimerization and membrane insertion of bacterial outer membrane secretin PulD[END_REF]. Recently, [START_REF] Collin | Sorting of an integral outer membrane protein via the lipoprotein-specific Lol pathway and a dedicated lipoprotein pilotin[END_REF] showed that PulD is sorted via the lipoprotein-specific Lol pathway.

Bam complex is composed of five proteins: BamA (formerly YaeT in E. coli and Omp85 in N. meningitidis) and four accessory lipoproteins, BamB (YfgL), BamC (NlpB), BamD (YfiO) and BamE (SmpA) [START_REF] Wu | Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli[END_REF][START_REF] Vuong | Analysis of YfgL and YaeT interactions through bioinformatics, mutagenesis and biochemistry[END_REF][START_REF] Knowles | Membrane protein architects: the role of the BAM complex in outer membrane protein assembly[END_REF]. BamA is found in all Gram-negative bacteria and folds into two distinct domains: an N-terminal soluble domain assembled from five POTRA (polypeptide translocation associated) domains and a C-terminal trans-membrane domain assumed to fold as a β-barrel [START_REF] Misra | First glimpse of the crystal structure of YaeT's POTRA domains[END_REF]. It was proposed that the first two POTRA domains are involved in interaction with OMPs. The POTRA-OMP interactions may be mediated through β-augmentation, in which a β-strand of POTRA is augmented by the addition of a β-strand from the substrates [START_REF] Kim | Structure and function of an essential component of the outer membrane protein assembly machine[END_REF][START_REF] Knowles | Fold and function of polypeptide transport-associated domains responsible for delivering unfolded proteins to membranes[END_REF]. The C-terminal phenylalanine of substrates seems to be important for the recognition of BamA [START_REF] Robert | Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal moti[END_REF]. Indeed, the importance of having five POTRA domains in all BamA remains unclear. Several studies suggested that POTRA domains also act as a scaffold for the binding of accessory factors and the number of POTRA domains correlate with the correct substrate folding [START_REF] Kim | Structure and function of an essential component of the outer membrane protein assembly machine[END_REF][START_REF] Bos | Functioning of outer membrane protein assembly factor Omp85 requires a single POTRA domain[END_REF][START_REF] Knowles | Fold and function of polypeptide transport-associated domains responsible for delivering unfolded proteins to membranes[END_REF].

The accessory lipoproteins BamBCDE are less conserved since not all of them are present in every Bam complex [START_REF] Gatsos | Protein secretion and outer membrane assembly in Alphaproteobacteria[END_REF][START_REF] Volokhina | The beta-barrel outer membrane protein assembly complex of Neisseria meningitidis[END_REF][START_REF] Anwari | A modular BAM complex in the outer membrane of the alpha-proteobacterium[END_REF]. Among them, BamD seems to be the only essential lipoprotein (Sklar et al., 2007a;[START_REF] Fardini | Investigation of the role of the BAM complex and SurA chaperone in outer-membrane protein biogenesis and type III secretion system expression in Salmonella[END_REF][START_REF] Volokhina | The beta-barrel outer membrane protein assembly complex of Neisseria meningitidis[END_REF]. Recently, the 3D structure of BamB, BamD and BamE became available [START_REF] Heuck | Augmenting β-augmentation: structural basis of how BamB binds BamA and may support folding of outer membrane proteins[END_REF][START_REF] Sandoval | Crystal structure of BamD: an essential component of the β-Barrel assembly machinery of gram-negative bacteria[END_REF][START_REF] Kim | Crystal structure of Escherichia coli BamB, a lipoprotein component of the β-barrel assembly machinery complex[END_REF]. It was suggested that the Cterminal domain of BamD provides a scaffold for interaction with Bam components, while the N-terminal domain is involved in interaction with the substrates [START_REF] Sandoval | Crystal structure of BamD: an essential component of the β-Barrel assembly machinery of gram-negative bacteria[END_REF]. BamB, a nonessential component of the Bam complex, could simultaneously bind to BamA and prefolded β-barrel proteins, thereby enhancing the folding and membrane insertion capacity of the Bam complex [START_REF] Heuck | Augmenting β-augmentation: structural basis of how BamB binds BamA and may support folding of outer membrane proteins[END_REF]. The functional significance of the BamC and BamE remain to be elucidated.

Fig. 17: Models proposed for outer membrane proteins (OMPs) biogenesis in E. coli [START_REF] Knowles | Membrane protein architects: the role of the BAM complex in outer membrane protein assembly[END_REF].

OMPs destined for the β-barrel assembly machinery (Bam) complex are transported across the IM by the Sec system. Following export, the nascent OMPs are recruited by two proposed chaperone pathways, the SurA and the Skp-DegP pathways, and are transported through the periplasm to the OM. Five models are proposed: 1) the pore-folding model; 2) the complex pore-folding model; 3) the barrel-folding model; 4) the chaperonefolding model; 5) the accessory -folding model.

Several models were proposed to explain how BamA facilitates assembly and insertion of the β-barrel OMPs (Fig. 17) [START_REF] Knowles | Membrane protein architects: the role of the BAM complex in outer membrane protein assembly[END_REF]: i) pore-folding model, the Bam complex functions as a single monomeric complex and incorporates the nascent OMP into the β-barrel pore, the POTRA and/or accessory components thread the OMP into the pore; ii) the complex pore-folding model, the central pore is formed by a multimeric Bam complex, the release of folded OMP into the membrane occurs owing to the opening of the oligomeric assembly and the periplasmic components help folding and deposit the protein directly into the membrane; iii) the barrel-folding model, the BamA provides a template for OMP barrel folding in the vicinity of the membrane; iv) the chaperone folding model, the periplasmic chaperones, in particular DegP, fold the nascent OMPs and protect them from degradation, and the Bam complex only function in the insertion into the membrane; v) the accessory folding model, the Bam complex only functions to fold the nascent OMP, and the folded OMP is then released to DegP in a quality-control mechanism to remove misfolded OMPs. The folded OMPs are then inserted into the OM probably facilitated either by DegP or by an additional periplasmic factor.

II.6 Protein secretion systems in Gram-negative bacteria

Gram-negative bacteria have evolved several secretion systems to deliver extracellular proteins across the bacterial cell envelope into the external medium or directly into the target cell.

The secreted proteins fulfil various functions, e.g., acquisition of nutrients, motility and intercellular communication. Moreover, pathogenic bacteria use these systems to secrete harmful toxins, adhesins, degradative enzymes and other secreted effectors to help the colonization of host organism. There are six secretion systems, from type I to type VI (T1SS to T6SS), which have been identified in Gram-negative bacteria to date [START_REF] Desvaux | Secretion and subcellular localizations of bacterial proteins: a semantic awareness issue[END_REF][START_REF] Bleves | Protein secretion systems in Pseudomonas aeruginosa: A wealth of pathogenic weapons[END_REF][START_REF] Filloux | Protein secretion system in Pseudomonas aeruginosa: an essay on diversity, evolution and function[END_REF]. These secretion systems can be classified into two categories: i) the Sec or Tat independent pathways, including T1SS, T3SS; T4SS and T6SS [START_REF] Holland | Type I protein secretion in bacteria, the ABC-transporter dependent pathway[END_REF][START_REF] Gerlach | Protein secretion systems and adhesins: the molecular armory of Gram-negative pathogens[END_REF][START_REF] Durand | Structural biology of bacterial secretion systems in Gram-negative pathogens-potential for new drug targets[END_REF]. They transport secreted proteins from the bacterial cytoplasm out of the cells in one-step; ii) the Sec or Tat dependent pathways, including T2SS and T5SS. The secretion by these systems is a two-step process. In the two later cases, the substrates are synthesized with a N-terminal signal peptide, which are recognized by the Sec or Tat translocons and are translocated across the IM [START_REF] Voulhoux | Involvement of the twinarginine translocation system in protein secretion via the type II pathway[END_REF][START_REF] Chevalier | Membrane targeting of a bacterial virulence factor harbouring an extended signal peptide[END_REF].

Subsequently, the mature proteins (without the N-terminal peptide) are targeted to one of these two secretion systems that mediate their transport through the OM. The main structural and functional features of T1SS, T3SS, T4SS, T5SS and T6SS will be summarized briefly in the following sections. Since D. dadantii T2SS is the subject of this thesis, this system will be addressed in more details in a special section.

II.6.1 Type I secretion system (T1SS)

Fig. 18: Model of the T1SS [START_REF] Delepelaire | Type I secretion in gram-negative bacteria[END_REF]. The T1SS consists of an IM protein, ABC transporter, an OM protein, OMP and membrane fusion protein, MFP. The secretion signal (colored in yellow) is located at the Cterminus of unfolded secreted protein and is not cleaved during secretion.

The type I secretion system (T1SS) belongs to a large family of ATP-binding cassette (ABC) transporters involved in export (ABC exporter or effluxer)/import (ABC importer) of various substrates across bacterial cell membrane. The T1SS is widespread in Gram-negative bacteria and transports unfolded proteins of various sizes, including pore-forming hemolysins (HlyA), adenylate cyclases, lipases, proteases, surface layers and hemophores (HasA), from the cytoplasm to the extracellular medium in one step [START_REF] Delepelaire | Type I secretion in gram-negative bacteria[END_REF]. This system is a heterotrimeric complex consisting of an ABC transporter, a channel forming outer membrane protein (OMP) and a membrane fusion protein (MFP) connecting the above two proteins (Fig. 18). It has been shown that T1SS-secreted proteins have a C-terminal secretion signal and this signal interacts with the ABC protein [START_REF] Blight | Heterologous protein secretion and the versatile Escherichia coli haemolysin translocator[END_REF]. However, several studies pointed out that additional domains may be involved in the interaction with the secretion complex. Firstly, efficient secretion of heterologous proteins fused to E. coli HlyA or D. dadantii PrtB requires C-terminal fragments much larger than the minimal secretion signals [START_REF] Létoffé | Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B[END_REF][START_REF] Palacios | Subset of hybrid eukaryotic proteins is exported by the type I secretion system of Erwinia chrysanthemi[END_REF]. Secondly, hemophore (residues, 1-174) without its Cterminal signal retains its capacity to interact with HasD (ABC protein) and induce the assembly of the tripartite complex [START_REF] Cescau | Probing the in vivo dynamics of type I protein secretion complex association through sensitivity to detergents[END_REF]. Recently, [START_REF] Masi | Multiple signals direct the assembly and function of a type I secretion system[END_REF] identified multiple regions distributed along HasA (residues, 1-174) as additional HasD binding sites. II.6.1.1 ABC protein, the motor of the system ABC protein binds and hydrolyses ATP, and uses the energy to drive the transport of secreted proteins across membranes. It is suggested that ABC protein functions as homodimers [START_REF] Fetsch | Vanadate-catalyzed phtocleavage of the signture motif of an ATPbinding cassette (ABC) transporter[END_REF]Zaitseva et al., 2005ab) and is also responsible for substrate recognition and specificity. ABC exporter comprises four domains: two transmembrane domains (TMDs) and two cytosolic nucleotide-binding domains (NBDs). The NBD is formed by various conserved motifs such as the Walker A and B motifs, the C-loop or ABC signature motif, the Q-and Proloop, and the D-and H-loops (Higgins and Linton, 2004), while TMD displays very little sequence homology. The homodimerization through TMDs form the substrate translocation pathway, while the NBDs control the conformation of the TMDs through ATP-induced dimerization and hydrolysis-induced separation [START_REF] Zaitseva | A structural analysis of asymmetry required for catalytic activity of an ABC-ATPase domain dimer[END_REF][START_REF] Procko | The mechanism of ABC transporters: general lessons from structural and functional studies of an antigenic peptide transporter[END_REF][START_REF] Gyimesi | ATP hydrolysis at one of the two sites in ABC transporters initiates transport related conformational transitions[END_REF] (Fig. 19). The export process is driven by ATP binding and/or hydrolysis at the NBD. Without nucleotide, the NBDs are apart or open. Unpon binding of ATP, both NBDs close to form a tight dimer with two ATPase active sites at the interface [START_REF] Hollenstein | Structure of an ABC transporter in complex with its binding protein[END_REF].

When the NBDs are open, the substrate-binding cavity formed by the TMDs faces inwards the cytosolic NBDs. When the NBDs bound to ATP and closed, the substrate-binding cavity faces outwards (Fig. 19) [START_REF] Procko | The mechanism of ABC transporters: general lessons from structural and functional studies of an antigenic peptide transporter[END_REF]. Both ABC exporter and importer probably use the same basic mechanism. [START_REF] Procko | The mechanism of ABC transporters: general lessons from structural and functional studies of an antigenic peptide transporter[END_REF]. Binding of ATP triggers the formation of dimer and moves the coupling helices closer together and flips the TMDs to an outward-facing conformaiton. Hydrolysis of ATP revert the TMDs to adopt an inward-facing conformation.

II.6.1.2 Membrane fusion protein (MFP)

MFP or adaptor proteins span across the periplasm bridging the two membrane components (ABC transporter/OMP). The crystal structure of several proteins of MFP family have been solved and revealed that they consist of a membrane-proximal (MP) domain, a β-barrel domain, a lipoyl domain, and an α-hairpin domain [START_REF] Akama | Crystal structure of the membrane fusion protein, MexA, of the multidrug transporter in Pseudomonas aeruginosa[END_REF][START_REF] Mikolosko | Conformational flexibility in the multidrug efflux system protein AcrA[END_REF][START_REF] Symmons | The assembled structure of a complete tripartite bacterial multidrug efflux pump[END_REF][START_REF] Yum | Crystal structure of the periplasmic component of a tripartite macrolide-specific efflux pump[END_REF]. The α-hairpin domain was suggested to be involved in interaction with OMP TolC, while the other domains are related to interaction with IMP [START_REF] Lobedanz | A periplasmic coiled-coil interface underlying TolC recruitment and the assembly of bacterial drug efflux pumps[END_REF][START_REF] Symmons | The assembled structure of a complete tripartite bacterial multidrug efflux pump[END_REF]. Furthermore, [START_REF] Xu | The tip region of the MacA alphahairpin is important for the binding to TolC to the Escherichia coli MacAB-TolC pump[END_REF] revealed that the conserved residues located at the tip region of the α-hairpin of MacA (MPF in efflux pump MacAB-TolC) play an essential role in the binding of TolC, which support the idea of tip-to-tip binding between MacA and TolC [START_REF] Yum | Crystal structure of the periplasmic component of a tripartite macrolide-specific efflux pump[END_REF][START_REF] Xu | Crystal structure of the periplasmic region of MacB, a noncanonic ABC transporter[END_REF][START_REF] Xu | The tip region of the MacA alphahairpin is important for the binding to TolC to the Escherichia coli MacAB-TolC pump[END_REF].

II.6.1.3 Outer membrane protein (OMP)/TolC

The OMP forms the exit pore for both the ABC transport pathway and the drug efflux pumps.

The 3D structures of three OMPs, i.e., TolC, OprM and VceC from E. coli, P. aeruginosa and V. cholerae, respectively, have been solved [START_REF] Koronakis | Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export[END_REF][START_REF] Akama | Crystal structure of the membrane fusion protein, MexA, of the multidrug transporter in Pseudomonas aeruginosa[END_REF][START_REF] Federici | The crystal structure of the outer membrane protein VceC from the bacterial pathogen vibrio cholerae at 18 Å resolution[END_REF]. Despite very little sequence similarity, they are structurally conserved. Take TolC as an example, it adopts a trimeric structure organized into two barrels (Fig. 20). A 12-stranded, 40 Å-long β-barrel inserts into the OM to form an open pore of 30 Å in diameter. The unusual αhelical barrel, 100 Å in length, protrudes into the periplasm. The tip of the TolC periplasmic entrance interacts with the apex of AcrB [START_REF] Xu | The tip region of the MacA alphahairpin is important for the binding to TolC to the Escherichia coli MacAB-TolC pump[END_REF]. TolC is open to the outside medium but is closed by densely packed α-helical coiled coils, inner H7/H8, and outer H3/H4 at its periplasmic entrance. Opening of the periplasmic entrance is therefore the key to the function of TolC.

An allosteric mechanism has been proposed for TolC opening [START_REF] Koronakis | Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export[END_REF]. This mechanism envisages that the inner coiled-coils α-helices (H7 and H8) undergo an iris-like movement to realign with the outer coiled-coils (H3 and H4), thereby enlarging the pore in a "twist-to-open" transition [START_REF] Koronakis | Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export[END_REF][START_REF] Andersen | Transition to the open state of the TolC periplasmic tunnel entrance[END_REF]. Recently, the crystal structure of two open states of TolC, TolC RS (370 pS, represents the conductance of membrane) and TolC YFRS (1,000 pS) have been solved, which presents sequential snapshots of the transition to advanced opening [START_REF] Pei | Structures of sequential open states in a symmetrical opening transition of the TolC exit duct[END_REF]. They supposed that disruption of the interprotomer links is the key to movements at the periplasmic tip [START_REF] Pei | Structures of sequential open states in a symmetrical opening transition of the TolC exit duct[END_REF]. In the moderated conductance state of TolC RS (370 pS compared with the 80 pS of TolC WT ), disruption of the bonds of Arg 367 -Asp 153 and Arg 367 -Thr 152 causes inner helix H7 at the periplasmic tip to undergo a significant twist and expansion around H8. The magnitude of twist and expansion is similar in the advanced open-state TolC YFRS (1,000 pS), with interprotomer distances of 18.9Å (TolC RS ) and 21.3 Å (TolC YFRS ). [START_REF] Izoré | Biogenesis, regulation and targeting of the type III secretion system[END_REF]. The system is composed of three major parts: the basal body, the needle and the translocon. The basal body is a multi-ring structure that anchors the system to the two bacterial membranes. Upon its formation, the needle protein potentially travels through the ring (step 1) and self-polymerizes on the outside of the bacterium. Subsequently, translocon proteins (step 2) are secreted, and finally, toxins/effectors are injected into the eukaryotic cytoplasm (step 3).

The type III secretion system (T3SS) is used by various Gram-negative bacteria, especially those involved in animal and plant pathogenesis, e.g., Yersinia, Shigella, Salmonella, Pseudomonas, enteropathogenic and enterohemorrhagic Escherichia coli (EPEC, EHEC) and Erwinia, to inject effector proteins directly into the eukaryotic host cell cytoplasm [START_REF] Moraes | Piecing together the type III injectisome of bacterial pathogens[END_REF]Worrall et al., 2010). The T3S machinery, also known as the injectisome or needle complex, is genetically, structurally and functionally related to bacterial flagella [START_REF] Blocker | Type III secretion systems and bacterial flagella: insights into their function from structural similarities[END_REF][START_REF] Erhardt | Bacterial nanomachines: the flagellum and type III injectisome[END_REF]. One of the differences between the two systems is that the T3SS apparatus contains an OM multimeric protein complex, formed by the protein called secretin.

The T3SSs are composed of at least 20 different proteins. However, the core of injectisome proteins, whose sequence are conserved in all known examples, do not exceed nine proteins [START_REF] Cornelis | The type III secretion injectisome[END_REF]. These proteins assemble into three major parts (Fig. 21), including a basal body of the system, a hollow needle-like structure and a 'translocon' [START_REF] Izoré | Biogenesis, regulation and targeting of the type III secretion system[END_REF][START_REF] Matteï | Membrane targeting and pore formation by the type III secretion system translocon[END_REF]. The assembly of the needle complex occurs in an orderly manner, in which the base substructure is assembled followed by the assembly of the inner rod and needle substructure [START_REF] Diepold | Deciphering the assembly of the Yersinia type III secretion injectisome[END_REF].

II.6.2.1 The basal body of the T3SS

The basal structure of the injectisome includes a pair of rings in the inner and outer membranes, joined together by a rod. The IM ring (IMR) is proposed to be the first ring to be assembled within the base [START_REF] Kimbrough | Assembly of the type III secretion needle complex of Salmonella typhimurium[END_REF]. However, [START_REF] Diepold | Deciphering the assembly of the Yersinia type III secretion injectisome[END_REF], by studying the Yersinia Ysc system, showed that the formation of the OM ring (OMR) precede that of the IMR. The IMR consists of two proteins (YscJ/MxiJ/PrgK and YscD/MxiG/PrgH in Yersinia, Shigella, and Salmonella spp, respectively) that oligomerize to form two concentric rings of different diameters (Fig. 22). The OMR, associated with the OM and the peptidoglycan layer, is a 12-14-mer of the YscC that belongs to subfamily of secretins [START_REF] Tamano | Supramolecular structure of the Shigella type III secretion machinery: the needle part is changeable in length and essential for delivery of effectors[END_REF][START_REF] Blocker | Structure and composition of the Shigella flexneri 'needle complex', a part of its type III secreton[END_REF]Burghout et al., 2004b). The proper location and insertion of secretins into the OM require the assistance of a protein of the YscW family, called pilotin (Burghout et al., 2004a). In recent reconstructions of Salmonella and Shigella needle complexes, the secretin protrudes into the periplasmic space, forming a "neck" region that is potentially formed by its Nterminus and directly contacts the IMR (Fig. 22) [START_REF] Hodgkinson | Threedimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout[END_REF][START_REF] Schraidt | Topology and organization of the Salmonella typhimurium type III secretion needle complex components[END_REF]Schraid and Marlovits, 2011). This observation was confirmed by experiments in which the crystal structure of the soluble N-terminal domain of EscC (the E. coli secretin) was docked into the S. typhimurium T3SS electron density map [START_REF] Spreter | A conserved structural motif mediates formation of the periplasmic rings in the type III secretion system[END_REF]. The reconstructuion of the S. flexneri needle complex indicates a 12-fold symmetry for the OM secretin-containing region and 24-fold symmetry for the IM region (Fig. 22A) [START_REF] Hodgkinson | Threedimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout[END_REF]. However, a recent high-resolution reconstruction of the S. typhimurium needle complex revealed a 15-fold symmetry for the secretin and a 24-fold symmetry for the IM ring, which yields a three-fold symmetry for the overall complex (Fig. 22B) (Schraid and Marlovits, 2011). Subsequent to assembly of the basal body, this structure must be rendered competent for secretion by 'export apparatus' which involves five membrane embedded proteins and several cytoplasmic membrane associated proteins (Fig. 21) [START_REF] Izoré | Biogenesis, regulation and targeting of the type III secretion system[END_REF]. Although all of the components of the 'export apparatus' are essential for T3SS, it is unknown whether these membrane proteins exert a common function or even if they exist as a single complex within the bacterial envelope. One of the components of the 'export apparatus', YscU in Yersinia was found to work as a substrate specificity switch [START_REF] Sorg | YscU recognizes translocators as export substrates of the Yersinia injectisome[END_REF]. This protein and its homologues possess N-terminal transmembrane domain and a long cytoplasmic C-terminal domain that undergoes auto-cleavage. YscU cleavage is required to acquire the conformation allowing recognition of translocators. The ATPase is implicated in chaperone/substrate recognition and subsequent dissociation and unfolding of substrates. This process utilizes ATP hydrolysis to energize effector secretion but also depends on PMF [START_REF] Worrall | Structural overview of the bacterial injectisome[END_REF][START_REF] Izoré | Biogenesis, regulation and targeting of the type III secretion system[END_REF].

II.6.2.2 The needle formation and regulation

Once the basal body is in place, the T3SS needle is synthesized. The T3SS needle is formed by a single small protein of the YscF/PrgI/MxiH family that is synthesized in the cytoplasm and polymerized only after secretion to the surface. It was found that the monomeric forms of needle proteins are not stable. Thus, within the bacterial cytoplasm, the needle-forming protein must be stabilized by their cognate chaperones to prevent its self-assembly or degradation. For example, in P. aeruginosa, the needle protein PscF is maintained in its monomeric form within the bacterial cytoplasm by a bimolecular chaperone, PscE-PscG [START_REF] Plé | Cochaperone interactions in export of the type III needle component PscF of Pseudomonas aeruginosa[END_REF]. Once the needle proteins are secreted to the surface, the needle is built by several hundred subunits that polymerize into an approximately 60 nm in length and 8 nm in diameter (with internal diameter 2-2.5 nm) structure. The length of the needle is highly regulated. It was proposed that YscP protein, which could act as a molecular ruler, determines the length of the Yersinia T3SS needle [START_REF] Journet | The needle length of bacterial injectisomes is determined by a molecular ruler[END_REF][START_REF] Wagner | The helical content of the YscP molecular ruler determines the length of the Yersinia injectisome[END_REF]. [START_REF] Wagner | The helical content of the YscP molecular ruler determines the length of the Yersinia injectisome[END_REF] further showed that the needle length correlates with the helical content of the ruler part of YscP. The polymerization of protomer into T3SS needles is spontaneous but requires a conformational change in the Cterminal helix of the protein to a β-sheet [START_REF] Poyraz | Protein refolding is required for assembly of the type three secretion needle[END_REF].

After the needle complex is fully assembled, the secretion machine switches substrate specificity, becoming competent for the secretion of translocon components and effector proteins. The translocon is a proteinaceous pore-forming complex that inserts directly into the host cellular membrane. It is composed of one hydrophilic and two hydrophobic translocator proteins.

II.6.2.3 The translocon

Two hydrophobic translocators (PopB/PopD and YopB/YopD in Pseudomonas and Yersinia, respectively) form the pore itself within the host cell membrane, while the hydrophilic protein (PcrV and LcrV, respectively) form a tip connecting the distal end of the needle to the membrane spanning translocon [START_REF] Goure | Protective anti-V antibodies inhibit Pseudomonas and Yersinia translocon assembly within host membranes[END_REF][START_REF] Mueller | The type III secretion system tip complex and translocon[END_REF]. These components are dispensable for secretion but are essential for the injection of type III effectors into the target cytoplasm.

Therefore, the translocon components are considered to be the first substrates secreted by the T3SS needle upon cell contact. Before secretion, all three translocon components remain associated with their respective chaperones and are stored in the cytoplasm (Fig. 23) [START_REF] Matteï | Membrane targeting and pore formation by the type III secretion system translocon[END_REF]. In the bacterial cytoplasm, the two hydrophobic translocators are associated with a common chaperone that shares a considerable sequence identity even in distant species. Upon cell contact, the chaperones are released and the unfolded translocators presumably travel through the interior of needle. Once having reached outmost extremity of the conduit, they are refolded and oligomerized to form the translocation pore (Fig. 23). Indeed, several studies have analyzed the structure of the pore by different methods [START_REF] Ide | Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli[END_REF][START_REF] Dacheux | Pore-forming activity of type III systemsecreted proteins leads to oncosis of Pseudomonas aeruginosa-infected macrophages[END_REF][START_REF] Schoehn | Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas[END_REF]. The pore size was estimated between 3.0 and 5.0 nm. An equimolar mix of the two translocators PopB and PopD of the P. aeruginosa T3SS in vitro led to the formation of ring structures, suggesting that PopB and PopD oligomerize to form ring-like complexes [START_REF] Faudry | Synergistic pore formation by type III toxin translocators of Pseudomonas aeruginosa[END_REF].

II.6.2.4

The assembly of the system and protein secretion The assembly of the T3SS apparatus appears to be a conserved, tightly regulated and inducible process. Recently, it was shown that the assembly of the injectisome starts with the formation of the stable secretin ring in the OM, and proceeds inwards through discrete attachment steps of YscD and YscJ at the IM. After the formation of the membrane rings, the components of the cytosolic ATPase-C ring complex assemble at the cytosolic side of the injectisome. Afterwards, the needle and translocon can be assembled (Fig. 23 and 24) [START_REF] Diepold | Deciphering the assembly of the Yersinia type III secretion injectisome[END_REF][START_REF] Matteï | Membrane targeting and pore formation by the type III secretion system translocon[END_REF]. However, [START_REF] Wagner | Organization and coordinated assembly of the type III secretion export apparatus[END_REF] showed that the assembly process is initiated by the deployment of a subset of the conserved IMP components of the export apparatus. Upon assembly completion, the T3SS switches into a state that can be termed as 'standby' mode. The switch from a standby mode to active injection may be triggered by the contact with the host cell membrane [START_REF] Enninga | Imaging the assembly, structure and activity of type III secretion systems[END_REF]. The needle could transmit a signal back to the bacterium by altering its helical state or changing in the packing of the needle [START_REF] Kenjale | The needle component of the type III secretion of Shigella regulates the activity of the secretion apparatus[END_REF][START_REF] Deane | Molecular model of a type III secretion system needle: impliactions for host-cell sensing[END_REF]. The cytoplasmic face of the T3SS reconginzes the effectors and initates the transport. The signal leading to substrate recognition has been proposed to be encoded either in the N-terminal peptide or in the 5'-signal in its mRNA [START_REF] Blaylock | Yersinia enterocolitica type III secretion of YopR requires a structure in its mRNA[END_REF][START_REF] Arnold | Targeting effectors: the molecular recognition of Type III secreted proteins[END_REF]. Chaperone dependent processes might also serve as additional signal or signal enhancer.

II.6.3 Type IV secretion system (T4SS) comprising full-length VirB7 (in green) and the VirB9 CTD (in yellow) and VirB10 CTD (in red). The structure of the core complex determined using cryo-EM and single particle analysis is rendered as a cut-out volume (in grey). The core complex is composed of the full-length VirB7, VirB9 and VirB10 proteins.

The T4SSs are widely distributed among Gram-negative and-positive bacteria and fulfil a wide variety of fuction, such as mediating the conjugative transfer of plasmids and other mobile DNA elements to bacterial recipient cells, or delivering protein or DNA substrates to eukaryotic target cell. Another type of T4SSs mediates DNA uptake (transformation) or release, thereby promoting further genetic exchanges [START_REF] Wallden | Type IV secretion systems: versatility and diversity in function[END_REF][START_REF] Waksman | Molecular architecture of bacterial type IV secretion systems[END_REF].

Although different T4SSs have different functions, they share the same central components and probably assemble and function in a similar manner. In Gram-negative bacteria, many T4SSs are similar to the A. tumefaciens VirB-VirD T4SS (Fig. 25), which is one of the best-studied T4SSs.

This system comprises 12 proteins named VirB1 to VirB11 and VirD4 [START_REF] Christie | Biogenesis, architecture and function of bacterial type IV secretion systems[END_REF].

These proteins can be grouped according to their functions and/or cellular locations: an extracellular pilus composed of a major (VirB2) and a minor (VirB5) subunit; three ATPases located at the IM, i.e., VirB4, VirB11, and VirD4; an IM channel formed by VirB6, VirB8, and the NTDs of VirB9 and VirB10; an OM complex composed of VirB7, the CTDs of VirB9 and VirB10 [START_REF] Chandran | Structure of the outer membrane complex of a type IV secretion system[END_REF][START_REF] Waksman | Molecular architecture of bacterial type IV secretion systems[END_REF].

II.6.3.1 Structure of the core complex

The structure of T4SS core complex from the conjugative pKM101 plasmid has been determined [START_REF] Fronzes | Structure of a type IV secretion system core complex[END_REF]. This structure showed that VirB7, VirB9 and VirB10 homologues form a tightly associated 'core complex' that is inserted in both the inner and outer membranes. The core complex is a cylindrical structure of 185 Å in diameter and 185 Å in length containing two layer termed O (outer) and I (inner) linked by thin stretches of density (Fig. 25b and Fig. 26) [START_REF] Fronzes | Structure of a type IV secretion system core complex[END_REF].

II.6.3.2 Structure of the outer-membrane complex

Crystal structure of the T4SS OM complex containing the entire O layer has been solved recently [START_REF] Chandran | Structure of the outer membrane complex of a type IV secretion system[END_REF]. In this structure, the O layer has two parts (Fig. 27), i.e., the main body and the cap. The cap is made of a hydrophobic ring of two-helix bundles contributed by 14 CTDs of TraF/VirB10 defining a 32 Å channel. The main body is made of the rest of CTDs of TraF/VirB10, TraO/VirB9 and TraN/VirB7. It has a 172 Å diameter. When the complex is viewed from the extracellular milieu, the VirB10 CTD forms an inner ring surrounded by the VirB9-VirB7 complex. The VirB7 forms spokes that radially cross the entire assembly. Because VirB10 homologues span the entire space between two membranes and are inserted in both, they are suggested to play regulatory roles, probably sensing conformational changes due to substrate binding in the cytosol and relaying them to regulate OM channel opening and closing [START_REF] Wallden | Type IV secretion systems: versatility and diversity in function[END_REF][START_REF] Waksman | Molecular architecture of bacterial type IV secretion systems[END_REF]. Indeed, [START_REF] Banta | An Agrobacterium VirB10 mutation conferring a type IV secretion system gating defect[END_REF] recently showed that a VirB10 mutation disrupts a gating mechanism in the core chamber, which confirmed the role of VirB10 in regulating passage of secretion substrates across the OM.

II.6.3.3 Structure of the inner-membrane complex

The NTDs of VirB9 and VirB10 constitute the I layer of the T4SS core complex which is

A. B.
inserted in the IM via the VirB10 N-terminal transmembrane segment. It was suggested that two membrane proteins VirB6 and VirB8 are also involved in formation of the IM pore. VirB6 is probably a central component of the IM channel. It is a polytopic IM protein with a periplasmic N terminus, five TMS and a cytoplasmic CTD [START_REF] Jakubowski | Agrobacterium tumefaciens VirB6 domains direct the ordered export of a DNA substrate through a type IV secretion system[END_REF]. VirB8 is a bitopic IM protein consisting of an N-terminal TMS and a large periplasmic CTD [START_REF] Terradot | Structure of two core subunits of the bacterial type IV secretion system, VirB8 from Brucella suis and ComB10 from Helicobacterpylori[END_REF].

Both VirB6 and VirB8 were shown to directly contact the substrate during secretion in A.

tumefaciens [START_REF] Cascales | Definition of a bacterial type IV secretion pathway for a DNA substrate[END_REF].

II.6.3.4 The cytoplasmic ATPase

Three ATPases, VirB4, VirB11 and VirD4, energize the system for apparatus assembly and substrate translocation through the channel. VirB4 is one of the less characterized with no crystal structure solved to date. Various oligomerization states of this protein have been reported [START_REF] Rabel | The VirB4 family of proposed traffic nucleoside triphosphatases: common motifs in plasmid RP4 TrbE are essential for conjugation and phage adsorption[END_REF][START_REF] Arechaga | ATPase activity and oligomeric state of TrwK, the VirB4 homologue of the plasmid R388 type IV secretion system[END_REF][START_REF] Durand | Biochemical dissection of the ATPase TraB, the VirB4 homologue of the Escherichia coli pKM101 conjugation machinery[END_REF]. However, ATPase activity has only been reported for hexameric forms [START_REF] Arechaga | ATPase activity and oligomeric state of TrwK, the VirB4 homologue of the plasmid R388 type IV secretion system[END_REF][START_REF] Durand | Biochemical dissection of the ATPase TraB, the VirB4 homologue of the Escherichia coli pKM101 conjugation machinery[END_REF], indicating that VirB4 functions as a hexamer, similarly to VirB11 and VirD4. VirB11 belongs to a family of ATPases termed 'traffic ATPase', which are associated with Gram-negative bacterial typeII, type III, type IV and typeVI secretion systems [START_REF] Fronzes | Structure of a type IV secretion system core complex[END_REF]. VirB11 homolgues are hexameric peripheral IM proteins [START_REF] Krause | Sequence-related protein export NTPase encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of helicobacter pylori share similar hexameric ring structures[END_REF]. The crystal structure of H. pylori VirB11 homologue (HP0525) revealed that each monomer consists of two domains formed by the N-and C-terminal halves of the protein (Fig. 28B) [START_REF] Yeo | Crystal structure of the hexameric traffic ATPase of the helicobacter pylori type IV secretion system[END_REF][START_REF] Savvides | VirB11 ATPase are dynamic hexameric assemblies: new insights into bacterial type IV secretion[END_REF]. The nucleotide-binding site is at the interface between the two domains.

A.

B.

In the hexamer, the NTD and CTD form two separate rings defining a chamber of ~50 Å in diameter (Fig. 28B).

The ATPase VirD4 functions as coupling protein (CP) in DNA-transferring systems, coupling DNA substrate processing and translocation, and as substrate receptor in effector translocation systems where it recruits substrates to the T4SS for secretion [START_REF] Wallden | Type IV secretion systems: versatility and diversity in function[END_REF]. CPs are anchored in the bacterial IM by their N-terminal membrane helix. The crystal structure of a soluble fragment of T4SS CP, namely TrwB, revealed a globular hexameric assembly composed of two distinct domains with a ~20 Å-wide channel in the middle [START_REF] Gomis-Rüth | The bacterial conjugation protein TrwB resembles ring helicases and F1-ATPase[END_REF] (Fig. 28A). It was suggested that TrwB might operate as a motor, pumping DNA through its central channel using the energy derived from ATP hydrolysis [START_REF] Tato | The ATPase activity of the DNA transporter TrwB is modulated by protein TrwA: implications for a common assembly mechanism of DNA translocating motors[END_REF]. Substrates recognition occurs through specific interactions between the CPs and signal sequences harbored by their substrates [START_REF] Waksman | Molecular architecture of bacterial type IV secretion systems[END_REF]. For the DNA substrate, signal sequences were carried by relaxase components of DNA transfer intermediates. For effector proteins, secretion signals have been initially positioned near the C termini and presumed to consist of clusters of hydrophobic or positive charged residues [START_REF] Schulein | A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells[END_REF]. Howerever, recent findings suggested that the substrate recognition is mediated by a combination of C-terminal signal, additional intrinsic motifs, and other cellular factors, e.g., chaperones and accessory proteins (Alvarez-Martinez and [START_REF] Alvarez-Martinez | Biological diversity of prokaryotic type IV secretion system[END_REF].

II.6.3.5 The pilus

VirB2 and VirB5 form pilus structure extending from the extracellular surface. The pilus might be used as a conduit for substrates or for adhesion [START_REF] Waksman | Molecular architecture of bacterial type IV secretion systems[END_REF]. The VirB2 of A. tumefaciens forms the major pilus component and is cyclized via an intramolecular covalent head-to-tail peptide bond [START_REF] Eisenbrandt | Conjugative pili of IncP plasmids and the Ti plasmid Tpilus are composed of cyclic subunits[END_REF]. No structural information for VirB2-like proteins is available. However, a crystal structure is available for the minor pilus component VirB5 homologue in pKM101 TraC [START_REF] Yeo | Structural and functional characterization of the VirB5 protein from the type IV secretion system encoded by the conjugative plasmid pKM101[END_REF]. The structure reveals a three-helix bundle flanked by a smaller globular part (Fig. 25b). VirB5 was shown to localize at the tip of the T pilus [START_REF] Aly | The VirB5 protein localizes to the T-pilus tips in Agrobacterium tumefaciens[END_REF] and to play a role in adhesion and host recognition [START_REF] Yeo | Structural and functional characterization of the VirB5 protein from the type IV secretion system encoded by the conjugative plasmid pKM101[END_REF].

II.6.4 Type V secretion system (T5SS)

The T5SS comprises the most wide-spread and simplest secretion system in Gram-negative bacteria [START_REF] Hodak | Current challenges in autotransport and two-partner protein secretion pathways[END_REF]. This system allows secretion of proteins across the OM via a transmembrane pore formed by a β-barrel structure without any need of energy [START_REF] Yen | Protein-translocating outer membrane porins of Gram-negative bacteria[END_REF][START_REF] Desvaux | Type V protein secretion: simplicity gone awry?[END_REF]. The T5SSs are subdivided into two branches, i.e., autotransporter (Type Va) and two-partner secretion pathway (Type Vb) [START_REF] Jacob-Dubuisson | Protein secretion through autotransporter and twopartner pathways[END_REF][START_REF] Cotter | Trimeric autotransporters: a distinct subfamily of autotransporter proteins[END_REF]. Proteins secreted via these systems have similarities in their primary structures composed of three domains (Fig. 29) [START_REF] Hodak | Current challenges in autotransport and two-partner protein secretion pathways[END_REF]: i) the Nterminal signal sequence for IM translocation; ii) the functional passenger domain; and iii) the Cterminal translocation unit.

II.6.4.1 Autotransporter protein secretion (AT)

The ATs typically consist of a 20-400-kDa passenger domain that possesses the effector functions and a 10-30-kDa β-domain facilitating translocation across the OM [START_REF] Dautin | Protein secretion in gram-negative bacteria via the autotransporter pathway[END_REF]. The ATs can be classified into two groups, i.e., classical AT and trimeric AT [START_REF] Cotter | Trimeric autotransporters: a distinct subfamily of autotransporter proteins[END_REF]. The differences between classical AT and trimeric AT are that [START_REF] Cotter | Trimeric autotransporters: a distinct subfamily of autotransporter proteins[END_REF][START_REF] Van Ulsen | Protein folding in bacterial adhesion: secretion and folding of classical monomeic autotransporters[END_REF][START_REF] Lyskowski | Structure and biology of trimeric autotransporter adhesins[END_REF]:

i) the C-terminal translocator domain of the classical AT is monomeric whereas the trimeric AT is trimeric (Fig. 30);

ii) the structure of the passengers is rather different, the passenger domain of classical ATs generally form a right-handed β helical structure, while different folds have been observed in various trimeric ATs passenger domains (Fig. 31).

iii) the passenger domain is released from the translocator domain into the extracellular milieu by a proteolytic cleavage in most classical ATs,, whereas in trimeric ATs, all passenger domain studied so far remain covalently attached to their β barrel membrane anchor.

Fig. 30: Crystal structures of C-terminal fragments of the classical autotransporter NalP [START_REF] Oomen | Structure of the translocator domain of a bacterial autotransporter[END_REF] and the trimeric autotransporter Hia [START_REF] Meng | Structure of the outer membrane translocator domain of the Haemophilus influenzae Hia trimeric autotransporter[END_REF]. In the NalP structure, β strands are colored yellow and α helix is colored red. In the Hia structure, individual subunits are colored blue, yellow, and orange. [START_REF] Emsley | Structure of Bordetella pertussis virulence factor P.69 pertactin[END_REF]. (B) A remarkable pertactin-like 24-turn β helical structure of Hemoglobin protease Hbp passenger domain [START_REF] Otto | Crystal structure of hemoglobin protease, a heme binding autotransporter protein form pathogenic Escherichia coli[END_REF]. (C) The trimeric crystal structure of the Y. enterocolitica collagen binding (YadA) passenger domain [START_REF] Nummelin | The Yersinia adhesin YadA collagen-binding domain structure is a novel left-handed parallel β-roll[END_REF]) and (D) H.

influenzae Hia adhesion binding domain (HiaBD1) [START_REF] Yeo | Structural basis for host recognition by the Haemophilus influenzae Hia autotransporter[END_REF].

Despite these aforementioned differences, the mechanism of ATs is generally assumed to be

HiaBD1

Neck the same for both classical ATs and trimeric ATs. All AT proteins are synthesized as pre-proproteins and exported across the IM via a Sec-dependent process, initiated by its N-terminal signal sequence, and finally released in the periplasm in a pro-protein form. Once in the periplasm, it was originally supposed that ATs mediate their own transport across OM. However, recent research suggests that they are chaperoned by certain periplasmic proteins, such as SurA and DegP to the Bam (Omp85) complex located in the OM which may assist in translocation [START_REF] Van Ulsen | Protein folding in bacterial adhesion: secretion and folding of classical monomeic autotransporters[END_REF][START_REF] Adler | Autotransporters and their role in the virulence of Burkholderia pseudomallei and Burkholderia mallei[END_REF]. Indeed, several ATs have been shown to interact directly with the periplasmic chaperones including SurA and DegP [START_REF] Ieva | Interaction of an autotransporter passenger domain with BamA during its translocation across the bacterial outer membrane[END_REF]Ruiz-Perez et al., 2009) and Bam complex [START_REF] Sauri | The Bam (Omp85) complex is involved in secretion of the autotransporter haemoglobin protease[END_REF][START_REF] Lehr | C-terminal amino acid residues of the trimeric autotransporter adhesin YadA of Yersinia enterocolitica are decisive for its recognition and assembly by BamA[END_REF]. BamA forms a 16-stranded β barrel with a pore size of ~2.5 nm which is significantly larger than the AT β barrel (~1.2 nm). This would allow for translocation of partially folded ATs. However, the mechanism by which the passenger domain is translocated across the OM remains to be elucidated. Three models have been proposed to explain the autotransportation mechanism (Fig. 32) [START_REF] Hodak | Current challenges in autotransport and two-partner protein secretion pathways[END_REF][START_REF] Dautin | Protein secretion in gram-negative bacteria via the autotransporter pathway[END_REF][START_REF] Lyskowski | Structure and biology of trimeric autotransporter adhesins[END_REF], i.e., i) haripin model, in which translocation might be initiated by the formation of a hairpin at the C-terminus of the passenger domain that is maintained while proximal segments of the polypeptide are threaded through the pore in a linear fashion; ii) Omp85/YaeT (Bam complex) assisted secretion model, in which the transport of the passenger domain across the OM and the integration of the β domain into the OM are catalyzed by the Bam complex; iii) multimeric model, in which the passenger domain folds at least partially in the periplasm and is then transported across the OM through a large channel created by the oligomerization of the β domain.

II.6.4.2 The two-partner system (TPS)

Although the TPS resembles the AT in several aspects, for example, exoproteins contain all the information for translocation through cell envelope, translocation of exoproteins across the OM via a transmembrane pore formed by a β-barrel, there are fundamental differences in the mechanism of secretion. Unlike AT, the passenger domain and the pore forming β-domain of TPS are encoded by two separate genes, refered as member of TpsA and TpsB families, respectively (Fig. 29A) [START_REF] Hodak | Current challenges in autotransport and two-partner protein secretion pathways[END_REF]. Most of the TpsA proteins share two common features, i.e., an N-terminal conserved region containing ~300 residues, termed the "TPS domain" and stretches of repeated sequences that have been predicted to form β-helical structures [START_REF] Junker | Pertactin βhelix folding mechanism suggests common themes for the secretion and folding of autotransporter proteins[END_REF][START_REF] Kajava | The turn of the screw: variations of the abundant beta-solenoid motif in passenger domains of Type V secretory proteins[END_REF]. The crystal structure of two TpsA proteins, fiamentous hemagglutinin adhesin FHA and H. influenzae adhesin HMW1, revealed that the TPS domain folds into a β-helix (Fig. 33A) [START_REF] Clantin | The crystal structure of filamentous hemagglutinin secretion domain and its implications for the two-partner secretion pathway[END_REF][START_REF] Yeo | The structure of the Haemophilus influenzae HMW1 pro-piece reveals a structural domain essential for bacterial twopartner secretion[END_REF]. TpsB transporters specifically recognize their cognate TpsA partners in the periplasm and mediate their translocation across the OM through a hydrophilic channel. TpsB belongs to the Omp85 (BamA)/TpsB superfamily involved in assembly of proteins into, or translocation across the OM [START_REF] Delattre | Substrate recognition by the POTRA domains of TpsB transporter FhaC[END_REF]. All the members of this superfamily share a similar organization, with one to seven POTRA domains followed by a C-terminal TM β barrel. The FHA/FhaC pair of B. pertussis represents a paradigm model TPS system. The crystal structure of FhaC revealed a 16 β-barrel that is occluded by an N-terminal α-helix and a hairpin-like loop, along with a periplasmic module composed of two POTRA domains (Fig. 33B) [START_REF] Clantin | Structure of the membrane protein FhaC: a membrane of the Omp85-TpsB transporter superfamily[END_REF].

According to the FhaC structure and functional experiments, [START_REF] Clantin | Structure of the membrane protein FhaC: a membrane of the Omp85-TpsB transporter superfamily[END_REF] proposed a model for FHA transport across the OM (Fig. 34). FHA is exported across the IM in a Secdependent manner and transits through the periplasm in an extended conformation. It was shown that DegP chaperones the extended FHA polypeptide in the periplasm [START_REF] Baud | Role of DegP for twopartner secretion in Bordetella[END_REF]. The N-terminal TPS domain of FHA initially interacts in an extended conformation with the FhaC POTRA1 domain in the periplasm. This interaction would then bring the region corresponding to the first repeats of the central β-helical domain of FhaC in proximity to the tip of loop L6. The recently identified T6SS is a widely distributed protein export machine. It is capable of cell-contact-dependent targeting of effector proteins between Gram-negative bacterial cells [START_REF] Pukatzki | The type VI secretion system: translocation of effectors and effector-domains[END_REF][START_REF] Schwarz | What is type VI secretion doing in all those bugs?[END_REF]. This system not only functions in pathogenesis, but also was found to plays a role in biofilm formation and killing of other bacterial cells [START_REF] Enos-Berlage | Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus[END_REF][START_REF] Jani | Type VI secretion: not just for pathogenesis anymore[END_REF][START_REF] Zheng | Genetic analysis of anti-amoebae and anti-bacterial activities of the type VI secretion system in Vibrio cholerae[END_REF]. T6SS gene clusters contain from 15 to more than 20 genes and secrete substrates lacking N-terminal hydrophobic signal sequences [START_REF] Bingle | Type VI secretion: a beginner's guide[END_REF][START_REF] Bönemann | Tubules and donuts: a type VI secretion story[END_REF]. The proteins encoded by T6SS gene clusters are assembled into a multi-component apparatus (Fig. 35) [START_REF] Cascales | The type VI secretion toolkit[END_REF]Pukatzki et al., 2009;[START_REF] Bönemann | Tubules and donuts: a type VI secretion story[END_REF]. Bioinformatic analysis has identified a core of 13 genes that may constitute the minimal member needed to produce a functional apparatus [START_REF] Boyer | Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources?[END_REF].

Conformational changes in

T6SS core components include two IM components, IcmF (intracellular multiplication protein F) and DotU/IcmH orthologs (Zheng et al., 2007), an AAA+ (ATPases associated with various cellular activites) family ATPase called ClpV [START_REF] Bönemann | Tubules and donuts: a type VI secretion story[END_REF]. ClpV associates with several other conserved T6SS proteins and its ATPase activity has been reported to be essential for T6SS function. In most T6SSs, hemolysin co-regulated protein (Hcp) and valine-glycine repeat protein G (VgrG) are both exported by T6SS and required for the function of the T6SS machine [START_REF] Leiman | Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin[END_REF][START_REF] Hachani | Type VI secretion system in Pseudomonas aeruginosa: secretion and multimerization of VgrG proteins[END_REF]. However, how T6SS machines assembles and the mechanism by which effectors are delivered via the secretory apparatus have remained elusive. Current models of the T6SS derive from the observation that several of its components share structural homology to bacteriophage T4 proteins [START_REF] Pukatzki | Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin[END_REF][START_REF] Leiman | Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin[END_REF][START_REF] Bönemann | Tubules and donuts: a type VI secretion story[END_REF], it has been proposed that target cell recognition and effector delivery occur in a process analogous to bacteriophage entry (Fig. 36) [START_REF] Kanamaru | Structural similarity of tailed phages and pathogenic bacterial secretion systems[END_REF][START_REF] Bönemann | Tubules and donuts: a type VI secretion story[END_REF]. Indeed, the structure of Hcp and VgrG provide evidence for an evolutionary relationship between T6SSs and the cell-puncturing machinery of bacteriophage.

The crystal structure of Hcp revealed the formation of hexameric rings [START_REF] Mougous | A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus[END_REF], which readily polymerize in solution to form nanotubes with external and internal diameters of 85 Å and 40 Å, respectively and are up to 100 nm long [START_REF] Mougous | A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus[END_REF][START_REF] Jobichen | Structural basis for the secretion of EvpC: a key type VI secretion system protein form Edwardsiella tarda[END_REF]. This structure shows an evolutionary relationship to the T4 tail tube protein gp19 [START_REF] Leiman | Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin[END_REF]. VgrG proteins show structural similarities to the spike complex of the T4 bacteriophage, which is composed of the (gp27) 3 -(gp5) 3 complex. This complex is used by phage to puncture the bacterial cell surface, which allows to inject DNA into the cytoplasm [START_REF] Pukatzki | Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin[END_REF][START_REF] Leiman | Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin[END_REF]. VgrG may form a trimeric 'needle' at the distal end of a tail tubelike structure formed by the polymerisation of hexameric rings of Hcp [START_REF] Pukatzki | The type VI secretion system: translocation of effectors and effector-domains[END_REF].

However, because the triple β-helix of VgrG is too narrow to function as a protein channel, it might dissociate from the Hcp tubule after it has punctured the target cell membrane [START_REF] Bönemann | Tubules and donuts: a type VI secretion story[END_REF]. In addition, some VgrGs carrying an additional C-terminal extension may act as genuine effectors. These VgrGs have been named 'evolved VgrGs' [START_REF] Pukatzki | The type VI secretion system: translocation of effectors and effector-domains[END_REF].

Recently, another T6SS component HsiF [START_REF] Lossi | Structure-function analysis of HsiF, a gp25-like component of the type VI secretion system in Pseudomonas aeruginosa[END_REF] shows similarity to a T4 phage protein gp25, which was proposed to have lysozyme activity. However, lysozyme activity for HsiF proteins was not detectable. Several other components of T6SS are also characterized. The IM proteins IcmF interacts with the IM protein DotU/IcmH and the lipoprotein SciN that is tethered to the OM and is exposed to the periplasm [START_REF] Sexton | Legionella pneumophila DotU and IcmF are required for stability of the Dot/Icm complex[END_REF][START_REF] Aschtgen | SciN is an outer membrane lipoprotein required for type VI secretion in enteroaggregative. Escherichia coli[END_REF]. This IcmF-DotU/IcmH-SciN complex might form the core of a transmembrane-spanning structure [START_REF] Bönemann | Tubules and donuts: a type VI secretion story[END_REF]. The cytoplasmic domain of IcmF has a conserved Walker A motif implicated in nucleotide binding and hydrolysis. Thus, IcmF may be involved in energizing of T6SS (Ma et al., 2009b).

Based on the overall similarities in architecture between VipA and VipB tubules and the viral tail sheath structure, it was proposed that contraction of VipA/VipB tubules could energize the injection of putative Hcp tubules with the VgrG tip on top into target cells (Fig. 36). ClpV might be required to export VipA and VipB subunits into the periplasm, followed by VipA/VipB tubule reformation and engulfment of the Hcp tube (Fig. 36B). Alternatively, it might need to remove cytosolic VipA/VipB tubules that do not harbor an engulfed Hcp tube (Fig. 36C).

III Type II secretion system (T2SS)

The T2SS was first discovered in Klebsiella oxytoca. This system, called the Pul system, is required for secretion of the starch-hydrolyzing lipoprotein, pullulanase PulA [START_REF] D'enfert | Cloning and expression in Escherichia coli of the Klebsiella pneumoniae genes for production, surface localization and secrtion of the lipoprotein pullulanase[END_REF]. This system have then been demonstrated in numerous other Gram-negative bacteria, including human pathogens such as Vibrio cholerae (Eps) and Pseudomonas aeruginosa (Xcp), fish pathogens such as Aeromonas hydrophila (Exe) and plant pathogens such as Erwinia chrysanthemi, Erwinia carotovora (Out) and Xantomonas campestris (Xps) [START_REF] Cianciotto | Type II secretion: a protein secretion system for all seasons[END_REF]. In these bacteria, the T2SSs secrete important virulence factors directly involved in the pathogenesis. Sequencing of bacterial genomes has identified T2S-like genes in numerous other species [START_REF] Cianciotto | Type II secretion: a protein secretion system for all seasons[END_REF]. This system is highly conserved in various Gram-negative bacteria and notably among human and plant pathogens where this machinery is often a major virulence factor [START_REF] Cianciotto | Type II secretion: a protein secretion system for all seasons[END_REF].

III.1 Genetic organization of the T2SS

The number of genes identified as being essential for theT2SS, also referred to as Gsp (general secretory pathway), varies from 12 to 16 dependent on species [START_REF] Filloux | The underlying mechanisms of type II protein secretion[END_REF]. The homologous genes and gene products have been designated in most species by letters A to O and S. However, in Pseudomonas, the letters P to Z and A have been used instead. gsp genes are often organized into one or two operons including the most conserved genes gspC to M and O coding for essential core components (Fig. 37). Some other gsp genes, coding for accessory components presented in only certain T2SSs, such as gspAB, gspN and gspS, are often located separately from the core gsp genes (Fig. 37). Generally, the T2SSs exhibit a similar genetic organization.

However, in some species, the genetic organization varies. The gspC and gspD genes of P. aeruginosa form an operon that is divergent from the operon containing the gspE-M genes (de Groot., 2001) (Fig. 37). In X. campestris, gspCD genes were found to be localized next to gspM at the end of the gsp operon (Fig. 37). Mutations in core gsp genes prevent secretion and result in the accumulation of the exoproteins in the periplasm. Thus, the products of all core gsp genes are essential for formation of the function T2S machinery. [START_REF] Filloux | The underlying mechanisms of type II protein secretion[END_REF].

III.2 Structural organization and function of the T2SS

Secretion by the T2SS is a two-step process (Fig. 38). The proteins to be secreted (exoproteins) are firstly synthesized with an N-terminal cleavable signal sequence. These precursors are targeted and transported through the IM via the Sec (in unfolded state) or Tat (in folded state) translocon [START_REF] Johnson | Type II secretion: from structure to function[END_REF][START_REF] Filloux | Protein secretion system in Pseudomonas aeruginosa: an essay on diversity, evolution and function[END_REF]. The signal peptide is then cleaved by an appropriate leader peptidase and the mature proteins are released into the periplasm. Once in the periplasm, the exoproteins are folded before being recognized by the secretion machine and translocated across the OM by the T2SS.

The T2S machinery also named secreton, is a multiprotein complex that spans both the IM and OM of bacteria [START_REF] Johnson | Type II secretion: from structure to function[END_REF]. It is composed of 12 core components. The OM secretin GspD forms a secretion pore. A cytoplasmic ATPase GspE, along with the IM proteins GspF, L, M form an IM platform. The major (GspG) and minor (GspH, I, J, K) form a pilus-like fiber. The IM protein GspC may play as a linker by connecting the two sub-secreton complexes, the IM platform GspE-F-L-M and the OM secretin complex. The pre-pseudopilin peptidase/methylase GspO processes the pseudopilins prior to their integration into the apparatus [START_REF] Cianciotto | Type II secretion: a protein secretion system for all seasons[END_REF][START_REF] Johnson | Type II secretion: from structure to function[END_REF]. Beyond these core components, there are some accessory components presented in only some species, including the pilotin GspS, GspN, GspA and GspB.

III.3 Description of the different parts of theT2S secreton III.3.1 The secretin GspD: the channel of the machinery

The OM protein GspD belongs to the secretin family. Secretins form large homomultimeric protein complexes in the OM of Gram-negative bacteria that are crucial for translocation of proteins and assembled fibers across the OM. The secretins are involved in the T2SS, T3SS, the type IV pili system (T4PS) and the filamentous bacteriophage assembly (Korotkov et al., 2011a).

III.3.1.1 The T2SS secretin, GspD

Sequence comparisons and further functional and structural studies showed that the secretin GspD consists of two main regions: a conserved C-terminal domain and a variable N-terminal domain (Fig. 39A) [START_REF] Genin | A superfamily of proteins involved in different secretion pathways in gram-negative bacteria: modular structure and specificity of the N-terminal domain[END_REF][START_REF] Nouwen | Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy[END_REF]Korotkov et al., 2011a). In addition, some secretins contain a small S-domain (30-70 amino acids) near the secretin C-terminus (Fig. 39A) to which a secrtin-specific pilotin binds [START_REF] Nouwen | Secretin PulD: association with pilot PulS, structure and ion-conducting channel formation[END_REF][START_REF] Guilvout | Multimerization-defective variants of dodecameric secretin PulD[END_REF]. The conserved C-terminal domain contains several putative transmembrane β-strands and forms a β-barrel in a multimeric state that makes the actual channel in the OM. Indeed, the C-domain of the GspD homologue XcpQ from P. aeruginosa was predicted to contain 13 putative transmembrane β-strands (equivalent to 50% of the XcpQ C domain) [START_REF] Bitter | Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa[END_REF]. However, circular dichroism (CD) indicated that only 25% of the PulD C domain could have β-strand configuration, which suggests that a large part of C domain is not organized in a classical OM β-barrel structure and, therefore, is probably not embedded in the membrane [START_REF] Chami | Structural insights into the secretin PulD and its trypsin-resistant core[END_REF].

The N-terminal domain of GspD extends into the periplasm and could be implicated in interaction with other T2SS components, and/or with secreted proteins. The structural study (Korotkov et al., 2009a) showed that the N-terminal domain of the secretin GspD from enterotoxigenic Escherichina coli (ETEC) consists of four subdomains (N0 to N3). The crystal structure of the first three N-terminal domains N0-N1-N2 showed that these three periplasmic domains are arranged into two lobes (Fig. 39B): a compact N-terminal lobe containing the N0 and N1 subdomains, and a second lobe containing the N2 subdomain (Korotkov et al., 2009a). In the structure, the N0 subdomain interacts with the N1 subdomain via an antiparallel pair of β strands involving β3 of N0 and β6 of N1. Their interface residues are hydrophobic throughout the T2SS GspD family, which suggests that the compact N0-N1 lobe of peri-GspD is most likely a common feature in T2SSs. The N0 subdomain is composed of two α helices flanked on one side by a mixed three-stranded β sheet and on the other by a two stranded antiparallel sheet (Fig. 39B) (Korotkov et al., 2009a). This fold is similar to the signaling domain of TonB-dependent receptors [START_REF] Brillet | A beta strand lock exchange for signal transduction in TonB-dependent transducers on the basis of a common structural motif[END_REF][START_REF] Ferguson | Signal transduction pathway of TonB-dependent transporters[END_REF]. TonB-dependent receptors are bacterial OM proteins that bind and transport ferric chelates, called siderophores, vitamin B 12 , nickel complexes and carbohydrates [START_REF] Noinaj | TonB-dependent transporters: regulation, structure and function[END_REF]. The transport requires energy in the form of PMF and an IM complex TonB-ExbB-ExbD, to transduce this energy to the OM. This process involves the interaction between TonB box of the OM receptor and TonB of the IM complex. Superimposition of the TonB-dependent OM receptor FpvA signaling domain onto the N0 domain of ETEC-GspD showed that an extra β strand could be bound by the N0 domain in a fashion similar to the interaction between the TonB box and the signaling domain (Fig. 40) (Korotkov et al. 2009a). In the structure of ETEC-GspD, the β2 of N0 is accessible and not blocked by the tight interactions between the N0 and N1 subdomains. Thus, the authors suggested that β2 of N0 might interact with a β strand from another protein during the functioning of the T2SS.

The N1 and N2 subdomains consist of two or three α helices and a three-stranded β-sheets (Fig. 38B). This structure is similar to the eukaryotic type I KH (hnRNP K homology, named for the human heterogeneous nuclear ribonucleoprotein K) domain that is typically involved in binding RNA and DNA [START_REF] Valverde | Structure and function of KH domains[END_REF]. This fold is also present in ring-forming proteins of the T3SS [START_REF] Spreter | A conserved structural motif mediates formation of the periplasmic rings in the type III secretion system[END_REF]. The N3 subdomain was suggested to have a structure similar to that of the N1 and N2 subdomains (Korotkov et al., 2009a). The crystal structure of two Nterminal domains (N0 and N1) of the secretin EscC from the EPEC T3SS reveals that they adopt folds similar to those observed in the N0 and N1 domains of the EPEC GspD T2SS secretin (Fig. 39C) [START_REF] Spreter | A conserved structural motif mediates formation of the periplasmic rings in the type III secretion system[END_REF]. However, the mutual orientation of these domains in the T2SS and T3SS secretins is unexpectedly different. It is unknown whether these structural differences simply indicate the different organization in these two secretins or represent different conformations of a flexible unit adopted by both secretins during secretion or assembly (Korotkov et al., 2011a). domains of ETEC T2SS secretin GspD (B) (Korotkov et al., 2009a) and comparison of N0 and N1 domains of ETEC T2SS secretin GspD and EPEC T3SS secretin EscC (C) [START_REF] Reichow | Structure of the cholera toxin secretion channel in its closed state[END_REF]. SP, signal peptide. aeruginosa (Korotkov et al., 2009a). The N0 subdomain (cyan) superimposed onto the signaling domain (red) of the FpvA. TonB box residues of FpvA are shown in yellow.

III.3.1.2 Channel formed by the secretin GspD homomultimeric complex

Eelectron microscopy (EM) studies revealed that 12-14 identical secretins form toroidal complexes with a central cavity that ranges from 50 to 90 Å in diameter dependent on the species [START_REF] Bitter | Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa[END_REF][START_REF] Nouwen | Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy[END_REF]Burghout et al., 2004). As a channel of this size in the OM would cause leakage of periplasmic proteins, so the opening and closing of the secretion channel should be tightly regulated. EM analysis of the T2SS secretin PulD from K. oxytoca [START_REF] Nouwen | Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy[END_REF] and cryo-EM of the secretin pIV from the filamentous bacteriophage assembly system [START_REF] Opalka | Structure of the filamentous phage pIV multimer by cryo-electron microscopy[END_REF] revealed a central channel plug. The N terminus of secretins seems to fold back into the cavity formed by the C terminus and plugs the channel [START_REF] Nouwen | Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy[END_REF]. However, the following cryo-EM studies of PulD indicated that the Cdomain probably forms the plug that occludes the channel [START_REF] Chami | Structural insights into the secretin PulD and its trypsin-resistant core[END_REF]. 3D reconstruction of PulD shows a cylindrical dodecameric arrangement of secretin subunits with two rings. An open ring ('saucer' appearance) is present in the outer leaflet of the OM, with weak connections to a second ring ('cup' appearance) in the periplasm and the inner leaflet of the OM (Fig. 41). The periplasmic entrance of the cylinder is wide open, but a continuous density closes off the periplasmic part of the structure from the OM region (Fig. 41). [START_REF] Chami | Structural insights into the secretin PulD and its trypsin-resistant core[END_REF] suggested that docking of secreted proteins might displace the centrally located plug, thus creating a continuous channel through which the proteins can be secreted. Indeed, the secreted protein PelB of D. dadantii binds directly to GspD during secretion (Shevchik et al., 1997).

Moreover, recently, both surface plasmon resonance (SPR) and EM studies have shown that the periplasmic domain of ETEC GspD and V. cholerae GspD interact with the B-pentamer of heatlabile enterotoxin (LT) and the B-pentamer of cholera toxin (CT), respectively [START_REF] Reichow | Structure of the cholera toxin secretion channel in its closed state[END_REF][START_REF] Reichow | The binding of cholera toxin to the periplasmic vestibule of the type II secretion channel[END_REF]. However, the interactions between secreted proteins and secretin are not sufficient to cause the pore opening since the addition of pullulanase to the mixture liposome/PulD did not change the channel conductivity [START_REF] Nouwen | Secretin PulD: association with pilot PulS, structure and ion-conducting channel formation[END_REF]. In addition, various studies have shown interaction between secretin and other proteins, such as GspG [START_REF] Lee | Associations of the major pseudopilin XpsG with XpsN (GspC) and secretin XpsD of Xanthomonas campestris pv. campestris type II secretion apparatus revealed by cross-linking analysis[END_REF], GspK-GspI-GspJ complex [START_REF] Reichow | Structure of the cholera toxin secretion channel in its closed state[END_REF], GspC [START_REF] Korotkov | Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae[END_REF][START_REF] Login | A 20-residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi[END_REF] and GspB [START_REF] Condemine | Overproduction of the secretin OutD suppresses the secretion defect of an Erwinia chrysanthemi OutB mutant[END_REF][START_REF] Ast | Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer[END_REF][START_REF] Strozen | Involvement of the GspAB complex in assembly of the type II secretion system secretin of Aeromonas and Vibrio species[END_REF]. These interactions may be also directly or undirectly involved in the pore opening control. To date, how the pore opening remains puzzled. Based on the crystal structure, Korotkov et al. (2009a) modeled cylindrical arrangements of periplasmic GspD domains. Although they did not obtain a plausible model using the entire peri-GspD structure, both the N0-N1 and N2 lobe produced models that showed that they form an assembly with cyclic C12 symmetry (Fig. 42). In the N0-N1 ring, β4 from one N0 subdomain approaches β1 of a neighboring N0 subdomain resulting in the formation of an extended β sheet (Fig. 42C), while helix α1 of one N1 subdomain approaches helix α5 of a neighboring N1 subdomain in a manner similar to the corresponding helices in the rings of N2 subdomain. The inner diameter of N0 ring (~70 Å) is sufficient to allow entry of molecules like the CT and LT AB5 enterotoxins (~ 64 Å), while the N1 ring (~ 46 Å) seems to be too narrow for passage of the folded B pentamer (Fig. 42A). The authors suggested that the pentamer might be transiently unfolded to interact with strand β2 of the N0 subdomain, thereby permitting a passage of the pentamer through the N1 and N2 rings. Alternatively, the secretin undergoes conformational changes during translocation of proteins. This might widen the N1 ring (Korotkov et al., 2009).

Recently, a number of EM studies on secretins from various export systems have been published [START_REF] Hodgkinson | Threedimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout[END_REF][START_REF] Reichow | Structure of the cholera toxin secretion channel in its closed state[END_REF][START_REF] Jain | Structural characterization of outer membrane components of the type IV pili system in pathogenic Neisseria[END_REF][START_REF] Burkhardt | Structure and function of PilQ, a secretin of the DNA transporter from the thermophilic bacterium Thermus thermophilus HB27[END_REF]. It is now recognized that features common to all secretins include a cylindrical arrangement of 12-15 subunits, a large periplasmic vestibule with a wide opening at one end and a periplasmic gate at the other (Korotkov et al., 2011a). The very recent cryo-EM study on the T2SS secretin EspD from V. cholerae (VcGspD) showed an assembly of ~155 Å in diameter and ~200 Å in length, with a largely unobstructed periplasmic vestibule (Fig. 43) [START_REF] Reichow | Structure of the cholera toxin secretion channel in its closed state[END_REF]. The opening to the periplasmic vestibule is ~75 Å in diameter, and the channel was narrowed to ~55 Å in internal diameter by a constriction (Fig. 43). The periplasmic vestibule from the extracellular chamber of the channel was sealed off by the periplasmic gate. Following the periplasmic gate, the extracellular domain appears as a chamber of ~100 Å in diameter. [START_REF] Reichow | Structure of the cholera toxin secretion channel in its closed state[END_REF] have placed the dodecameric ring models composed of N0-N1, of N2 and of N3 in the density map of VcGspD. The N0-N1 ring fits into the widest area at the bottom of the periplasmic part of the secretin, and the N2 and N3 rings can be positioned directly above the N0-N1 ring in the map. In addition, the N3 ring fits to the constriction site of the cryo-EM map (Fig. 44A). They also placed the structure of the cholera toxin heterohexamer AB 5 into the VcGspD secretin density (Fig. 44B). The AB 5 structure (~65 Å) fits well within the ~75 Å-wide vestibule created by the N0-N2 subdomains, without any steric hindrance. However, the AB 5 cannot fit through the constriction formed by N3 (~55 Å in diameter) without a major conformational change in the secretin (Fig. 44B). A recent study showed that CT B5 binds in the lower part of the periplasmic vestibule of the secretin VcGspD, just below the channel constriction [START_REF] Reichow | The binding of cholera toxin to the periplasmic vestibule of the type II secretion channel[END_REF]. It was proposed that during the process of secretion, the secreted proteins interact with the secretin constriction. This interaction could provide the trigger that causes large conformational changes in the T2SS secretin, which results in opening of the periplasmic gate [START_REF] Reichow | The binding of cholera toxin to the periplasmic vestibule of the type II secretion channel[END_REF].

III.3.1.3 The secretin multimerization and insertion into the OM

The general OM assembly factor Omp85/YaeT (BamA) may be one of the factors that facilitate secretin insertion and multimerization [START_REF] Voulhoux | Role of a highly conserved bacterial protein in outer membrane protein assembly[END_REF]. Indeed, the absence of Omp85 prevents N. meningitidies secretin PilQ multimerization and decreases the amount of PilQ detected on the surface by immunofluorescence [START_REF] Voulhoux | Role of a highly conserved bacterial protein in outer membrane protein assembly[END_REF]. However, a study of K. oxytoca secretin showed that PulD multimerization and membrane insertion are independent of BamA (YaeT) [START_REF] Collin | YaeT-independent multimerization and outer membrane association of secretin PulD[END_REF].

In certain T2SSs, lipoprotein pilot GspS is necessary for the correct targeting and insertion of the secretin into the OM [START_REF] Shevchik | Functional characterization of the Erwinia chrysanthemi OutS protein, an element of a type II secretion system[END_REF][START_REF] Guilvout | Bacterial outer membrane secretin PulD assembles and inserts into the inner membrane in the absence of its pilotin[END_REF]. In D. dadantii, pilotin OutS has been shown to bind to the C-terminal 62 residue of the secretin OutD [START_REF] Shevchik | Functional characterization of the Erwinia chrysanthemi OutS protein, an element of a type II secretion system[END_REF]. Recently, [START_REF] Nickerson | Outer membrane targeting of secretin PulD relies on disordered domain recognition by a dedicated chaperone[END_REF] identified the 28 C-terminal residues of the S domain as a minimal binding site that is sufficient for OM targeting of PulD in vivo. The region upstream of this binding site is not required for targeting or multimerization, but is needed for secretin function in T2SS. In the absence of pilotin or S domain of secretin, the secretin misslocates to the IM [START_REF] Guilvout | Bacterial outer membrane secretin PulD assembles and inserts into the inner membrane in the absence of its pilotin[END_REF][START_REF] Guilvout | Multimerization-defective variants of dodecameric secretin PulD[END_REF]. In vitro studies showed that secretin PulD does not require PulS for multimerization or membrane insertion and PulD multimerization occurs very quickly without PulS [START_REF] Guilvout | In vitro multimerization and membrane insertion of bacterial outer membrane secretin PulD[END_REF]. The PulD multimers would be too large to diffuse through the peptidoglycan [START_REF] Demchick | The permeability of the wall fabric of Escherichia coli and Bacillus subtilis[END_REF]. Thus, PulS binds to S domain of PulD to protect PulD monomers from degradation and to target them to OM via the conserved OM lipoprotein-specific Lol pathway [START_REF] Collin | Sorting of an integral outer membrane protein via the lipoprotein-specific Lol pathway and a dedicated lipoprotein pilotin[END_REF]. Some secretins are lipoproteins per se, and they do not depend on separate pilotins for correct OM targeting. For example, GspD HxcQ , the lipoprotein secretin from the second T2SS of P. aeruginosa, was showed to be self-piloted to the OM via its N-terminal lipid anchor [START_REF] Viarre | HxcQ liposecretin is self-piloted to the outer membrane by its N-terminal lipid anchor[END_REF].

In some other bacteria T2SSs, secretin assembly requires other auxiliary factors, for example, the GspA and GspB complex has been shown to be required for correct localization and multimerization of the secretin GspD multimer in the OM of A. hydrophila [START_REF] Ast | Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer[END_REF][START_REF] Strozen | Involvement of the GspAB complex in assembly of the type II secretion system secretin of Aeromonas and Vibrio species[END_REF].

III.3.2 The pseudopilins and prepilin peptidase

Five proteins of T2SS, i.e., GspG,-H,-I,-J and -K, share sequence homology with pilin subunits of type IV pili and are called pseudopilins. These pseudopilins assemble a pilus-like structure, called pseudopilus, spanning the periplasmic compartment and/or acting as a piston to push the secreted protein through the OM pore [START_REF] Hazes | Towards a systems biology approach to study type II/IV secretion systems[END_REF][START_REF] Reichow | The binding of cholera toxin to the periplasmic vestibule of the type II secretion channel[END_REF].

Both the T2S pseudopilins and the type IV pilins are synthesized as precursors, which are processed by a specific prepilin petidase [START_REF] Filloux | The underlying mechanisms of type II protein secretion[END_REF][START_REF] Johnson | Type II secretion: from structure to function[END_REF]. The conserved G1, F+1 and E+5 residues are shown in uppercase. The position of the leader sequence cleavage site is indicated with an arrow. The leader peptide and the hydrophobic region are shown [START_REF] Filloux | The underlying mechanisms of type II protein secretion[END_REF].

III.3.2.1 GspO, a specialised prepilin peptidase/methylase

The precursors of pseudopilins contain a conserved N-terminal leader peptide composed of six or seven residues followed by a hydrophobic domain of about 20 residues (Fig. 45) [START_REF] Filloux | The underlying mechanisms of type II protein secretion[END_REF]. This leader peptide is recognized and processed by the prepilin peptidase/methylase PilD/GspO and the liberated N-terminus is further methylated. This modification is also catalyzed by the prepilin peptidase, which is thus a bifunctional enzyme [START_REF] Filloux | The underlying mechanisms of type II protein secretion[END_REF][START_REF] Johnson | Type II secretion: from structure to function[END_REF]. The topology study of prepilin peptidase OutO from D. dadantii demonstrated that GspO is polytopic IM protein with eight transmembrane segments [START_REF] Reeves | beta-Lactamase topology probe analysis of the OutO NMePhe peptidase and six other Out protein components of the Erwinia carotovora general secretion pathway apparatus[END_REF]. The first cytoplasmic loop of this protein is large and contains a tetracysteine consensus motif, C-X-X-C _X21_C-X-X-C. However, this motif does not seem to be required for peptidase activity (LaPointe et al., 2000). The sole residue required for peptidase activity is aspartate located in the second and third cytoplasmic loop. Thus, GspO is a bifunctional aspartic acid protease that cleaves the leader peptides of pseudopilin subunits and N-methylates the newly generated Nterminus [START_REF] Johnson | Type II secretion: from structure to function[END_REF]. The processing site is located immediately before the hydrophobic region after a highly conserved glycine residue (G-1) (Fig. 45). Then, the mature pseudopilin subunits are helically packed via interactions between their hydrophobic N-terminal domains and are assembled into pseudopilus. A conserved glutamate residue within the hydrophobic domain (E+5) could play a key role in the assembly process of pilin and pseudopilin subunits [START_REF] Aas | Substitutions in the N-terminal alpha helical spine of Neisseria gonorrhoeae pilin affect type IV pilus assembly, dynamics and associated functions[END_REF][START_REF] Campos | Detailed structural and assembly model of the type II secretion pilus from sparse data[END_REF].

(GspG) (GspH) (GspI) (GspJ) (GspK)

III.3.2.2 The pseudopilus: the piston of the machinery

Except for GspK, the pseudopilins have an N-terminus similar to type IV pilins (G(F/M)XXXE) followed by 15-20 hydrophobic amino acids (Fig. 45). GspK is considered to be atypical because it lacks the phenylalaine (+1) and glutamate (+5) residues (Fig. 45) [START_REF] Filloux | The underlying mechanisms of type II protein secretion[END_REF] and has a higher molecular weight (>30kDa) than classical pilins and pseudopilins (15-30 kDa) [START_REF] Bleves | The secretion apparatus of Pseudomonas aeruginosa: identification of a fifth pseudopilin, XcpX (GspK family)[END_REF]. In addition, in most T2SS GspK has a pair of cysteine residues that form a disulfide bridge [START_REF] Pugsley | Disulfide bond formation in secreton component PulK provides a possible explanation for the role of DsbA in pullulanase secretion[END_REF][START_REF] Korotkov | Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system[END_REF], whereas other pseudopilins are cysteine-free. The relative in vivo stoichiometry between pseudopilins has been estimated in P. aeruginosa to be 16 (GspG):1(GspH):1(GspI):4(GspJ). Thus, GspG is considered as the major pseudopilin and the others GspH-GspK are the minor pseudopilins [START_REF] Durand | Structural biology of bacterial secretion systems in Gram-negative pathogens-potential for new drug targets[END_REF].

(1) The major pseudopilin GspG GspG is cotranslocationally inserted into the IM using the SRP/Sec pathway and does not require any components of the T2SS IM platform to be targeted into the IM [START_REF] Francetic | Signal recognition particle-dependent inner membrane targeting of the PulG pseudopilin component of a type II secretion system[END_REF][START_REF] Arts | Interaction domains in the Pseudomonas aeruginosa type II secretory apparatus component XcpS (GspF)[END_REF]. Given the strong conservation of their N-terminal sequences, it is likely that other (pseudo)pilins also use the SRP/Sec pathway [START_REF] Francetic | Signal recognition particle-dependent inner membrane targeting of the PulG pseudopilin component of a type II secretion system[END_REF]. Following insertion into the IM, GspG was suggested to be processed by the prepilin peptidase GspO. It has been speculated that this process causes a conformational change of GspG, which enables GspG to interact with other components of the T2SS [START_REF] Gray | In vivo cross-linking of EpsG to EpsL suggests a role for EpsL as an ATPase-pseudopilin coupling protein in the Type II secretion system of Vibrio cholerae[END_REF]. The IM components suggested to act as a platform for the assembly of the subunits into a (pseudo)pilus-like structure, using the energy provided by the ATPase GspE. Indeed, upon overexpression, both GspG homologs from the K. oxytoca (PulG) and P. aeruginosa (XcpT) could be assembled in a multifibrillar structure (pseudopilus), crossing the bacterial cell envelop and exposed on the cell surface [START_REF] Sauvonnet | Pilus formation and protein secretion by the same machinery in Escherichia coli[END_REF][START_REF] Durand | Type II protein secretion in Pseudomonas aeruginosa: the pseudopilus is a multifibrillar and adhesive structure[END_REF][START_REF] Durand | XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus[END_REF]. Although these pseudopili obtained by overproduction of pseudopilins cannot be considered as physiologically relevant structures, they reveal that GspG pseudopilins have a propensity to pack into helical complexes reminiscent of type IV pilus. None of the other four pseudopilins have been shown to form a pseudopilus, which suggests that the assembly of such a structure is a unique property of GspG [START_REF] Durand | XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus[END_REF]. The structures of the soluble domain of PulG and XcpT, GspG homologs from K. oxytoca and P. aeruginosa have been solved, respectively [START_REF] Köhler | Structure and assembly of the pseudopilin PulG[END_REF][START_REF] Alphonse | Structure of the Pseudomonas aeruginosa XcpT pseudopilin, a major component of the type II secretion system[END_REF]. Both structures globally present the same features as the type IV pilins. All type IV pilins share a common fold with an extended N-terminal hydrophobic α-helix, a four-stranded anti-parallel β-sheet and a variable domain called the αβ-loop containing a disulfide bridge (Fig. 46). However, GspG lacks a highly variable αβ-loop region and do not have disulfide bridge stabilizing the protein fold.

Recently, Korotkov et al. (2009b) showed that the major pseudopilin GspG binds to calcium ion to obtain sufficient stability necessary for its function. However, the major pseudopilin HxcT of the second T2SS of P. aeruginosa is unable to assemble a hyper-pseudopilus, which suggests different pseudopilus architecture [START_REF] Durand | The assembly mode of the pseudopilus: a hallmark to distinguish a novel secretion system subtype[END_REF]. The formation of an extracellular pseudopilus blocks protein secretion, which suggests that the overproduced pseudopilus cross the OM via the secretin channel and thus disturb the release of secreted protein through the same channel at the same time [START_REF] Durand | Type II protein secretion in Pseudomonas aeruginosa: the pseudopilus is a multifibrillar and adhesive structure[END_REF]. Consequently, it is supposed that in physiological conditions, the pseudopilus formed by the pseudopilins should be retained in the periplasm. The length of T2SS pseudopilus might be controlled by the incorporation of minor pseudopilin subunits. Indeed, it was demonstrated that GspK constrols the pilus elongation since the pilus length was found to vary conversely to the concentration of GspK subunits [START_REF] Durand | XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus[END_REF]. GspK was also found to interact with GspG and induce disassembly of the pseudopilus. Results obtained with K. oxytoca and P. aeruginosa pseudopilins

A. B. C. D.
suggested that GspI plays a major role at the initiation step of GspG assembly. Alternatively, it may anchor the T2SS pilus to the cell envelop [START_REF] Sauvonnet | Pilus formation and protein secretion by the same machinery in Escherichia coli[END_REF][START_REF] Vignon | Type IV-like pili formed by the type II secreton: specificity, composition, bundling, polar localization and surface presentation of peptides[END_REF][START_REF] Durand | XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus[END_REF]. GspI may be loacted at the basis of the pseudopilus, in connection with IM components of the machinery. However, more recent studies suggest that GspI together with GspJ and GspK could constitute the tip of the pseudopilus [START_REF] Korotkov | Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system[END_REF].

(2) The minor pseudopilins GspHIJK

The crystal structure of the N-terminally truncated variant of EpsH, a GspH homolog from V.

cholerae, comprises an N-terminal α-helix and C-terminal β-sheet consistent with the type IVa pilin fold (Fig. 47) (Yanez et al., 2008b). However, structural comparisons revealed major differences between the minor pseudopilin EpsH and the major pseudopilin GspG from K.

oxytoca. EpsH contains a large β-sheet in the variable domain, where GspG contains an α-helix.

Importantly, EpsH contains at its surface a hydrophobic crevice between its variable and conserved β-sheets, wherein a majority of the conserved residues within the EpsH family are clustered. In addition, the authors proposed a tentative model of a T2SS pseudopilus with EpsH at its tip, the conserved crevice faces away from the helix axis and may be involved in the interaction with one or more other T2SS proteins.

The crystal structure of a complex formed by the V. vulnificus GspI and GspJ (EpsI and EpsJ)

represents the first atomic resolution structure of a complex of two different pseudopilin components from the T2SS (Yanez et al., 2008a). It gives the evidence that the two minor pseudopilins EpsI and EpsJ interact directly with each other. In the crystal structure, EpsI comprises of one N-terminal α-helix followed by four anti-parallel β-strands arranged with one β-sheet (Fig. 48A). EpsJ consists of one N-terminal α-helix followed by ten β-strands, of which nine occur within two β-sheets, plus a short β2' strand which is not part of a β-sheet (Fig. 48A).

The interface between EpsI and EpsJ is highly conserved with a large number of hydrophobic residues involved in heterodimer formation. In addition, the arrangement of EpsI and EpsJ in the heterodimer would correspond to a right-handed helice, characteristic for proteins assembled into a pseudopilus. The recently solved nanobody-aided structure of the EpsI:EpsJ complex confirmed this viewpoint [START_REF] Lam | Nanobody-aided structure determination of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus[END_REF]. The main progress in the understanding of the assembly of pseudopilins into the pseudopilus is achieved by the crystal structure of the GspK-GspI-GspJ complex from enterotoxigenic E. coli (ETEC) [START_REF] Korotkov | Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system[END_REF]. GspK consists of two domains, a pilin domain and a unique α-domain (Fig. 47). The former domain has the characteristic components of the pilin fold: an N-terminal α-helix, a short 'variable' segment and a four-stranded C-terminal antiparallel β-sheet. GspK, GspI and GspJ form a stable quasihelical heterotrimer. The N-terminal α-helices of the three subunits interact with each other in the center of the complex (Fig. 48B), in a fashion that is very similar to the models of the type IV pilus fibers [START_REF] Köhler | Structure and assembly of the pseudopilin PulG[END_REF][START_REF] Craig | Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions[END_REF]. GspK is able to form a stable complex with the pseudopilins GspI and GspJ, which suggests that GspK may participate in several interactions with other pseudopilins during pseudopilus assembly and/or function since it has been already demonstrated to interact with the major pseudopilin GspG [START_REF] Durand | XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus[END_REF].

The GspK-GspI-GspJ heterotrimer seems to be at the tip of the pseudopilus, with GspK and

GspJ positioned in such a way that no additional pseudopilin subunit can be added 'upward'.

III.3.2.3 Assembly mode of the pseudopilus

It seems that two distinct complexes contribute to the pseudopilus structure: the pseudopilus core formed by the homomultimerization of the major pseudopilin through interaction between hydrophobic domains and a tip heteromeric complex formed by at least three of the minor pseudopilins. EM and modeling image analysis of the PulG-containing pseudopilus suggested that pseudopilus is most likely assembles into a left-handed helical pilus with the long Nterminal α-helix of each subunit packing into the core of the pilus structure [START_REF] Köhler | Structure and assembly of the pseudopilin PulG[END_REF].

However, [START_REF] Campos | Detailed structural and assembly model of the type II secretion pilus from sparse data[END_REF][START_REF] Campos | Modeling pilus structures from sparse data[END_REF] recently proposed that the overall organization of T2SS pilus is right-handed, consistent with protomer organization in gonococcal T4P structure [START_REF] Craig | Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions[END_REF]. Based on the high-resolution structure of the PulG monomer completed by modeling and the scanning transmission electron microscopy (STEM) analysis of single PulG filaments, [START_REF] Campos | Detailed structural and assembly model of the type II secretion pilus from sparse data[END_REF] proposed a one-start helix T2S pilus assembly model validated by cysteine cross-linking and charge inversion experiments (Fig. 49). In this model, each protomer (P) interacts directly with three upper (P +1 , P +3 , P +4 ) and three lower (P -1 , P -3 , P -4 ) protomers in the filament, through hydrophobic (P -1 and P +1 ) and electrostatic (all six) interaction.

In the first step, the protomer P is tethered to the nascent filament via electrostatic interactions with protomer P +1 (Asp 48 -Arg 87 , Glu 44 -Arg 88 , and Glu 5 -Phe 1 ). The hydrophobic patch on protomer P +3 and P +4 exposed to aqueous environment (Fig. 49A, B andD) could contribute to these contacts or be masked by an assembly factor (PulF/L/M). Upon insertion, Lys 35 and Lys 28 of protomer P could interact with Glu 5 of P +3 , which stabilizes the P +3 in its final state (Fig. 48B,C, and E ) and promotes the most frequently modeled TMS arrangement. The minor pseudopilins in the GspJ-I-K tip also are arranged in a right-handed helix [START_REF] Forest | The type II secretion arrowhead: The structure of GspI-GspJ-GspK[END_REF]. Recently, [START_REF] Douzi | The XcpV/GspI pseudopilin has a central role in the assembly of a quaternary complex within the T2SS pseudopilus[END_REF] revealed an additional interaction between GspH and GspJ, which suggested the existence of a quaternary complex (GspH-GspI-GspJ-GspK) at the tip of the pseudopilus. During the pseudopilus biogenesis, GspH may be located at the base of the minor pseudopolins complex and could form the hinge between the tip complex and the core pseudopilus formed by the GspG. Indeed, several studies favor a linker position for GspH:

1) Yanez et al. (2008b) suggested to place GspH at the tip of the GspG pseudopilus with its specific conserved crevice by docking experiments;

2) GspH was shown to interact with pseudopilins GspG and GspJ [START_REF] Kuo | Roles of the minor pseudopilins, XpsH, XpsI and XpsJ, in the formation of XpsG-containing pseudopilus in Xanthomonas campestris pv. campestris[END_REF][START_REF] Douzi | The XcpV/GspI pseudopilin has a central role in the assembly of a quaternary complex within the T2SS pseudopilus[END_REF]. GspI is the central component and initiator of pseudopilus formation by interacting with both GspJ and GspK with two different interaction sites. GspH then enters the ternary complex GspJ-GspI-GspK via its interaction with GspJ. The tip quaternary complex is then able to accommodate the major pseudopilin GspG via a hydrophobic interaction with GspH.

The elongation of the pseudopilus might be arrested by contact of the tip complex with the secretin. Indeed, it was shown by SPR that the trimeric pseudopilus tip complex GspK-GspI-GspJ interacts with the periplasmic domain of secretin [START_REF] Reichow | Structure of the cholera toxin secretion channel in its closed state[END_REF]. In addition, it was shown that both minor pseudopilin GspJ [START_REF] Douet | Systematic analysis, by the yeast two-hybrid, of protein interaction between components of the type II secretory machinery of Erwinia chrysanthemi[END_REF] and major pseudopilin GspG [START_REF] Gray | In vivo cross-linking of EpsG to EpsL suggests a role for EpsL as an ATPase-pseudopilin coupling protein in the Type II secretion system of Vibrio cholerae[END_REF] interact with GspL. This indicates that energy transduced by GspL may be required to extract the N-terminal hydrophobic α-helix of both the major and minor pseudopilins from the IM while the pseudopilus is formed in the periplasmic compartment.

III.3.3 GspE-F-L-M: the inner membrane complex

GspE, GspF, GspL and GspM constitute a complex in the IM that is presumably used as a platform for assembling and anchoring the pseudopilus to the IM [START_REF] Py | An inner membrane platform in the type II secretion machinery of Gram-negative bacteria[END_REF]. Moreover, the IM platform is assumed to either initiate the signal transduction for gating the pore or to generate and transmit the energy that is necessary for protein secretion via conformational changes.

III.3.3.1 The motor GspE

GspE belongs to the large superfamily of 'typeII/IV secretion NTPases', or 'secretion superfamily ATPases'. This superfamily involves the ATPases, which function in multiple macromolecular transport system, such as T2SS and T4SS, type IV pilus (T4P) assembly, DNA uptake, and archeal flagellae assembly systems [START_REF] Planet | Phylogeny of genes for secretion NTPases: identification of the widespread tadA subfamily and development of a diagnostic key for gene classification[END_REF][START_REF] Peabody | Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella[END_REF][START_REF] Craig | Type IV pilus structure and bacterial pathogenicity[END_REF]. The ATPases belonging to the T2SS and T4P share the following features: i) a

Walker A box with the P-loop GX4GK(S/T) that is used to bind NTP; ii) an atypical Walker B box which is thought to be the determinant for nucleoside recognition; iii) the Aspartate box located between the Walker A box and Walker B box, may be involved in the formation and stabilization of the nucleoside-binding fold by interacting with Mg 2+ ; iv) a conserved Histidine box for which no function has yet been assigned; v) a tetracysteine motif, which is reminiscent of a zinc-binding domain, appears to be essential for function [START_REF] Possot | The conserved tetracysteine motif in the general secretory pathway component PulE is required for efficient pullulanase secretion[END_REF].

GspE are multi-domain proteins (Fig. 50A), which contain domains N1 to C2. Domain N0 is only present in a few homologs including XpsE and XcpR in X. campestris and P. aeruginosa, respectively. The crystal structure of an N-terminally truncated version of GspE∆90 and N1 subdomain of GspE (N1-EpsE) in complex with the cytoplasmic domian of GspL (cyto-EpsL) from V. cholerae have been solved, respectively [START_REF] Robien | Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae[END_REF][START_REF] Abendroth | The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae[END_REF].

The former structure revealed an organization in two major domains for the monomer, i.e., the N2 domain and the complete C-terminal domain with its three subdomains C1, CM and C2 (Fig. 50B) [START_REF] Robien | Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae[END_REF], while the latter showed that GspE and GspL form a hetero-tetramer, in which GspL is the central dimer and GspE binds on the periphery (Fig. 50C) [START_REF] Abendroth | The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae[END_REF]. The N2 and C domains are separated by a linker of 15 amino-acid residues. The C1 side view (middle), and the cytoplasmic face (right) of the hexameric ring model of EpsE [START_REF] Robien | Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae[END_REF].

domain contains all four conserved boxes that characterize traffic ATPases from the T2SS and T4P assembly machinery, i.e., the Walker A, Walker B, Asp and His boxes. The small C M subdomain is a hairpin-like meandering loop with a conserved tetracysteine motif that binds a metal cation near the sharply bent proximal end of the loop. The C2 subdomain is spatially interposed between the C1 and C M subdomains [START_REF] Robien | Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae[END_REF]. Another crystal structure of the N-terminal domain of GspE from X. campestris was reported in two conformational forms, i.e., open form and closed form (Fig. 50D) [START_REF] Chen | Structure and function of the XpsE N-terminal domain, an essential component of the Xanthomonas campestris type II secretion system[END_REF]. GspE family proteins have been shown to form hexameric rings (Fig. 50E and 51) [START_REF] Robien | Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae[END_REF][START_REF] Shiue | XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL[END_REF].

Moreover, [START_REF] Patrick | Oligomerization of EpsE coordinates residues from multiple subunits to facilitate ATPase activity[END_REF] modeled GspE as a hexameric ring with C2 symmetry. ATP binding triggers the oligomerization of GspE as well as its association with N-terminal domain of GspL [START_REF] Shiue | XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL[END_REF] (Fig. 51). Indeed, a double-mutant GspE mutated at K331 and R504, which is defective in ATP binding, no longer oligomerized in the presence of Mg-AMPPNP (non-hydrolysable analog of ATP), nor did it associate with GspL. Unlike the GspE (K331M, R504A) double-mutant, single mutation in GspE at K331 in Walker A motif causes reduction in its ATPase activity by 17-fold without significantly influencing its interaction with GspL, which indicates that GspE-GspL association precedes ATP hydrolysis (Fig. 51). Acidic phospholipids, specifically cardiolipin, together with cytoplasmic domain of GspL, synergistically stimulated both the oligomerization and intrinsic ATPase activity [START_REF] Camberg | Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids[END_REF]. Further analysis

showed that residues at the C-terminus of GspL cytoplasmic domain adjacent to the predicted transmembrane helix interfered with the cardiolipin-stimulated ATPase activity of GspE. This suggests that the membrane proximal region of the cytoplasmic domain of GspL may participate in fine-tuning the interaction of GspE with phospholipids and thereby regulate its oligomerization and ATPase activity [START_REF] Camberg | Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids[END_REF]. et al., 2011).

In brief, GspE is active as an oligomer, likely a hexamer. The binding, hydrolysis and release of nucleotide cause GspE to undergo dynamic structural changes, thus converting chemical energy to mechanical work, ultimately resulting in extracellular secretion. GspE associates with the T2SS through interaction with the N-terminal domains of GspL and GspF [START_REF] Py | An inner membrane platform in the type II secretion machinery of Gram-negative bacteria[END_REF]Arts et al., 2007a).

III.3.3.2 GspF, L and M: the platform stabilizers

(1) GspF

GspF is a polytopic integral IMP. It consists of a small periplasmic loop and two larger cytoplasmic domains connected by three transmembrane segments. The N-terminal domain is located in the cytoplasm whereas the C-terminus is located in the periplasm ( [START_REF] Thomas | The general secretion pathway of Erwinia carotovora subsp. carotovora: analysis of the membrane topology of OutC and OutF[END_REF]Arts et al., 2007a). Using a yeast two-hybrid system, [START_REF] Py | An inner membrane platform in the type II secretion machinery of Gram-negative bacteria[END_REF] revealed that the cytoplasmic N-terminal domain of OutF interacts with OutE and the cytoplasmic domain of OutL. Furthermore, OutL was required for the formation of OutE-OutF complex whereas the OutE-OutL complex formation is independent of OutF. This leads to the suggestion that GspE and GspL might first interact together and then associate with the GspF. XcpS, a GspF homolog of P. aeruginosa, was found unstable in the absence of other Xcp components, but could be stabilized by co-expression of the XcpR (GspE) and XcpY (GspL) components of the machinery (Arts et al., 2007a), which indicates an interaction between these three proteins. The cytoplasmic loop located at the C-terminal part of XcpS was shown to be involved in the stabilization by XcpR and XcpY. These results suggest that the two cytoplasmic domains of GspF could participate in the interaction with GspE and GspL and contribute to the stability of the IM complex.

Recently solved crystal structure of the N-terminal cytoplasmic domain of EpsF (cyto-EpsF) (GspF of V. cholerae) showed that it adopts an all-helical fold with six helices forming two layers (Fig. 52) (Abendroth et al., 2009a). Two cyto-EpsF domains form a tight dimer and contain two symmetry-equivalent calcium sites (Fig. 52). In vivo cross-linking experiments further support for dimer formation of cyto-EpsF (Abendroth et al., 2009a). Sequence analysis of the GspF family suggests that the second cytoplasmic domain of members of this family adopts the same conformation as the first cytoplasmic domain. (2) GspL and GspM

GspL is a bitopic IM protein. It consists of a large cytoplasmic domain, a single transmebrane helix and a small periplasmic domain (Fig. 53). The crystal structure of the cytoplasmic domain of the GspL V. cholerae, cyto-EpsL, unexpectedly revealed structural homology with the actinlike ATPase superfamily (Fig. 54) (Abendroth et al., 2004a). Cyto-EpsL consists of three β-rich domains with domains I and III corresponding to the conserved domains 1A and 2A of the actinlike ATPases. However, cyto-EpsL is incapable of forming actin-like filaments because it misses the non-conserved domains 1B and 2B of the actin-like proteins (Fig. 54). Moreover, cyto-EpsL has an additional domain II, which has a SHS2 (strand-helix-strand-strand) topology. This topology is only observed for domain 1C of the cell division protein FtsA, where it likely participates in intermolecular interactions that result in recruitment of other proteins essential for cell division [START_REF] Rico | Role of two essential domains of Escherichia coli FtsA in localizsation and progression of the division ring[END_REF][START_REF] Johnson | Type II secretion: from structure to function[END_REF]. Structural homology and sequence conservation suggested that domain II of cyto-EpsL is also involved in protein-protein interactions. Indeed, the crystal structure of N1 subdomain of EpsE in complex with cyto-EpsL shows that domains II and III of cyto-EpsL are involved in the interaction with the N1 subdomain of EpsE and the majority of the protein-protein interface involves domain II of cyto-EpL (Fig. 50C) [START_REF] Abendroth | The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae[END_REF]. Contrary to the actin-like ATPase, no nucleotidebinding site was found in cyto-EpsL by sequence analysis of structural homologues of the superfamily or in the crystal structure (Abendroth et al., 2004a). Instead, EpsL together with acidic phospholipids synergistically stimulated both the oligomerization and intrinsic ATPase activity of EpsE [START_REF] Shiue | XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL[END_REF][START_REF] Camberg | Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids[END_REF]. Many studies have shed light on the GspL dimer formation. The yeast two-hybrid studies on the periplasmic domain of E. chrysanthemi GspL revealed that this domain interacts with itself [START_REF] Py | Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL[END_REF]. With a similar approach, [START_REF] Douet | Systematic analysis, by the yeast two-hybrid, of protein interaction between components of the type II secretory machinery of Erwinia chrysanthemi[END_REF] suggested that both the cytoplasmic and the periplasmic domains of E. chrysanthemi OutL are involved in the formation of dimer. The crystal structure of cyto-EpsL revealed a homodimer interface (Fig. 55A) (Abendroth et al., 2004a). In agreement with of this, the X-ray structure of N1-EpsE and cyto-EpsL complex showed that the cyto-EpsL is dimeric and forms a heterotetramer with the N1-EpsE [START_REF] Abendroth | The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae[END_REF]. Recently, the structure of the periplasmic domain of GspL from V.

parahaemolyticus (Vp peri-EpsL) was solved (Abendroth et al., 2009b). Each Vp peri-EpsL chain folds as a compact globular unit and consists of two α-helices and four β-strands forming an anti-parallel β-sheet (Fig. 55B). This is an unusual variant of the ferredoxin-like fold, observed for the first time in peri-EpsM (Abendroth et al., 2004b) (Described below). The Vp peri-EpsL was found to form a dimer in solution and in crystals. The dimer interface is made up by residues from the N-terminal helices c α1 and c α1' and the N-terminal strands c βA and c βA' (Fig. 55B) (Abendroth et al., 2009b). The interface residues of the Vp peri-EpsL dimer are well conserved in the GspL family, suggesting conservation of the dimer too. Therefore, the dimer interface might spread out over the cytoplasmic, the periplasmic and membrane domains and all contributions might be required simultaneously for a stable dimerization. GspM is a small IMP with a short cytoplasmic segment, one transmembrane helix and a periplasmic C-domain (Fig. 53). The crystal structure of the periplasmic domain of GspM from V. cholerae (EpsM, residue 44-165) has been solved (Abendroth et al., 2004b). It adopts a novel variation of the ferredoxin-like fold. Monomeric EpsM consists of two compact contiguous αββsubdomains forming a sandwich of two α-helices and a four-stranded β-sheet (Fig. 56A), while in the "classic" ferredoxin fold, the arrangement of secondary structure elements along the polypeptide chain is βαβ-βαβ (Fig. 56B).

Both full-length EpsM [START_REF] Sandkvist | Direct interaction of the EpsL and EpsM proteins of the general secretion apparatus in Vibrio cholerae[END_REF] and the soluble periplasmic domain are capable of forming dimer (Abendroth et al., 2004b). This suggests that neither the transmembrane domain nor the short cytoplasmic N-terminus is required for homodimerization. However, it remains to be confirmed that the full-length protein forms the same dimer interface as the periplasmic domain. Most of dimer interface of the crystal dimer formed by residues from helix

A. B.
α2. A deep cleft with a polar rim and a hydrophobic bottom, made by conserved residues, are located between the two monomers. Additional electron density was found in the clef between the subunits (Abendroth et al., 2004b), suggesting that this region might serve as a binding site for an unknown ligand or protein partner. Further experimental analysis mapped the region involved in EpsM dimerization to its C-terminal region between amino acids 100 and 135, thus confirming the X-ray crystal structure model [START_REF] Johnson | Mapping critical interactive sites within the periplasmic domain of the Vibrio cholerae type II secretion protein EpsM[END_REF]. As aforementioned, the overall folds of the preplasmic regions of GspM and GspL are similar and consist of a permutation of ferredoxin-like fold. However, their roles in secretion process are not elucidated. Several investigations demonstrated that GspL and GspM play an important role in the stabilization of the IM platform. GspL and GspM of V. cholerae were found to form a stable binary complex and protected each other from proteolytic degradation when produced in E.

coli, indicating that these two proteins interact with each other in vivo (Sandvist et al., 1999).

Similar results were obtained in P. aeruginosa and K. oxytoca [START_REF] Michel | Mutual stabilization of the XcpZ and XcpY components of the secretory apparatus in Pseudomonas aeruginosa[END_REF][START_REF] Possot | Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE[END_REF]. Studies in yeast-two hybrid system showed that the periplasmic domain of OutM and

OutL from E. chrysanthemi interact with each other [START_REF] Py | An inner membrane platform in the type II secretion machinery of Gram-negative bacteria[END_REF][START_REF] Douet | Systematic analysis, by the yeast two-hybrid, of protein interaction between components of the type II secretory machinery of Erwinia chrysanthemi[END_REF]. Based on the results on GspE, L, M of T2SS, Abendroth et al. (2009b) proposed architecture of the T2SS IM complex (Fig. 57). The cyto-GspL and N1-GspE form heterotetramer. Each heterotetramer is linked to two adjacent N2-C1-C2 domains of GspE in the hexamer. The TM helices of GspL in each heterotetramer cross the IM and the periplasmic domains of GspL form dimers.

GspL was also shown to interact with minor pseudopilin GspJ [START_REF] Douet | Systematic analysis, by the yeast two-hybrid, of protein interaction between components of the type II secretory machinery of Erwinia chrysanthemi[END_REF]. Recently, in vivo cross-linking study showed an interaction between GspL and the major pseudopilin GspG [START_REF] Gray | In vivo cross-linking of EpsG to EpsL suggests a role for EpsL as an ATPase-pseudopilin coupling protein in the Type II secretion system of Vibrio cholerae[END_REF]. Previous studies have shown that GspL interacts with GspE. Together with these results, [START_REF] Gray | In vivo cross-linking of EpsG to EpsL suggests a role for EpsL as an ATPase-pseudopilin coupling protein in the Type II secretion system of Vibrio cholerae[END_REF] proposed that GspL may function as a scaffold to link GspG and GspE and thereby transduces the energy generated by ATP hydrolysis to support secretion.

III.3.4 Connection between IM and OM sub-complexes: GspC

Most of the proteins composing the T2SS are associated with or integrated in the IM, except for GspD and GspS, which are located in the OM. This suggests that the certain components of the T2SS ensure a permanent or transient junction between the two cellular membrane to allow for a structural integrity of the secretion machinery. It has been suggested that the IM protein GspC may connect the two sub-complexes, the IM platform consisting of GspE-F-L-M and the OM secretin pore formed by GspD by interacting with both the secretin GspD and the two IM components GspL and GspM.

GspC is a bitopic IM protein that appears to be one of the least conserved component of all the core components of the T2SS [START_REF] Gérard-Vincent | Identification of XcpP domains that confer functionality and specificity to the Pseudomonas aeruginosa type II secretion apparatus[END_REF]. It consists of a short cytoplasmic Nterminal cytoplasmic sequence, a single membrane-spanning helix (TMS), followed by a large hydrophilic region which protrudes into the periplasm (Fig. 58). The periplasmic region contains a so-called homology region (HR) and a PDZ domain near their C termini in the majority of GspC. In some GspC family members, the PDZ domain is absent or replaced by a coiled-coil (CC) domain [START_REF] Gérard-Vincent | Identification of XcpP domains that confer functionality and specificity to the Pseudomonas aeruginosa type II secretion apparatus[END_REF][START_REF] Peabody | Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella[END_REF]. The PDZ domains are named after the three eukaryotic proteins in which they were first found: PSD-95 (a 95 kDa protein involved in signaling at the post-synaptic density), DLG (the Drosophila melanogaster Discs large protein) and ZO-1 (the zonula occludens 1 protein involved in maintenance of epithelial polarity) [START_REF] Hung | PDZ domains: structural modules for protein complex assembly[END_REF]. These domains have also been referred to as DHR (Discs large homology repeat) domains or GLGF repeats (after the highly conserved four-residue GLGF sequence within the domain). They are small globular modules of about 80-100 amino acid residues consisting of six β-strands (βA-βF) and two αhelices (αA and αB) (Fig. 59) [START_REF] Harris | Mechanism and role of PDZ domains in signaling complex assembly[END_REF]. The PDZ domains function as proteinprotein interaction modules and typically recognize the short (about five residues long) extreme C-termini of target proteins (Jelen et al., 2003). Peptide ligands bind in an extended groove between strand βB and helix αB thus serving as an additional antiparallel β-stranded within the PDZ domain (Fig. 59A). This mechanism is referred to as β-strand addition [START_REF] Remaut | Protein-protein interaction through beta-strand addition[END_REF]. The highly conserved GLGF motif is located within the loop connecting strands βA and βB. It is involved in hydrogen bond formation with the carboxyl group of the Cterminal residues of the interacting peptide [START_REF] Harris | Mechanism and role of PDZ domains in signaling complex assembly[END_REF]. Certain PDZ domains associate with partner proteins via internal protein sequence that structurally mimics the Cterminus [START_REF] Brenman | Cloning and characterization of postsynaptic density 93, a nitric oxide synthase interacting protein[END_REF]. For instance, the PDZ domain of neuronal nitric oxide synthase (nNOS) specifically interacts with PDZ domains of PSD-95 and syntrophin [START_REF] Brenman | Cloning and characterization of postsynaptic density 93, a nitric oxide synthase interacting protein[END_REF]. A stable β-hairpin (called the β-finger) of the nNOS PDZ domain appeared to be responsible for binding to PDZ domains in a fashion similar to that of peptides despite the absence of a carboxy-terminal carboxylate moiety (Fig. 59B) [START_REF] Hillier | Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex[END_REF]. The PDZ domain of GspC appears to be involved in interaction with certain exoproteins and therefore determines the secretion specificity, as shown by swapping the PDZ domains of OutC from E. chrysanthemi and E. carotovora which switches the specificity of the secretion system [START_REF] Bouley | The PDZ domain of OutC and the N-terminal region of OutD determine the secretion specificity of the type II out pathway of Erwinia chrysanthemi[END_REF]. The crystal structures of two variants of the PDZ domain of EpsC, a short (residues 219-305; called s-PDZ-C) and a long (residue 201-305; called also l-PDZ-C), have been solved [START_REF] Korotkov | Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae[END_REF]. The structure of the s-PDZ-C of EpsC has the typical PDZ domain fold with six characteristic β-strands (βA-βF) flanked by helices αA and αB (Fig. 60A).

While the structure of the l-PDZ-C of EpsC has a unique feature that is not present in any other PDZ domain of known structure. The chain of l-PDZ-C starts with the long helix α1 followed by a kink and the shorter helix α2 (Fig. 60B). This N-terminal extension together with helix αA forms a well-ordered helical sub-domain in the structure of l-PDZ-C. Both variant structures show that the PDZ domain of EpsC adopts a more open form than in previously reported structures. In addition, no members of the GspC family contain a GLGF motif preceding strand βA. These indicate that the PDZ domain of this bacterial protein binds proteins in a manner which is altogether different from that seen in any other PDZ domain so far [START_REF] Korotkov | Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae[END_REF]. The fact that two variant crystal structures show a significant movement of αB, may reflect the occurrence of conformational changes in EpsC upon protein binding and release when the T2SS performs its intricate function (Fig. 60C) [START_REF] Korotkov | Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae[END_REF]. The coiled-coil domains are composed of two or more α helices arranged in parallel or antiparallel manner and usually implicated in protein-protein interactions [START_REF] Burkhard | Coiled coils: a highly versatile protein folding motif[END_REF].

It has been suggested that both the coiled-coil and PDZ domains are involved in homomultimerization of GspC components in a non-specific maner [START_REF] Gérard-Vincent | Identification of XcpP domains that confer functionality and specificity to the Pseudomonas aeruginosa type II secretion apparatus[END_REF].

Certain GspC, such as XphA, which is much shorter and possesses neither a PDZ nor coiled-coil domain, is still a functional protein [START_REF] Michel | XphA/XqhA, a novel GspCD subunit for type II secretion in Pseudomonas aeruginosa[END_REF]. This is consistent with the previous results that lack of the C-terminal coiled-coil domain in XcpP (GspC of P. aeruginosa) does not prevent the protein from assembling into a functional T2SS [START_REF] Gérard-Vincent | Identification of XcpP domains that confer functionality and specificity to the Pseudomonas aeruginosa type II secretion apparatus[END_REF] and deleting the PDZ domain of OutC of E. chrysanthemi results in a partially functional protein [START_REF] Bouley | The PDZ domain of OutC and the N-terminal region of OutD determine the secretion specificity of the type II out pathway of Erwinia chrysanthemi[END_REF].

Studies of GspC of P. aeruginosa and X. campestris showed that substitution of the N-terminal region, either with the first TMS of TetA or with a signal peptide, completely abolished protein function [START_REF] Bleves | Structure-function analysis of XcpP, a component involved in general secretory pathway-dependent protein secretion in Pseudomonas aeruginosa[END_REF][START_REF] Lee | Functional dissection of the XpsN (GspC) protein of the Xanthomonas campestris pv. campestris type II secretion machinery[END_REF]. This indicates that the TMS of GspC not only functions anchor, but also implicates in the functionality of the protein. This was further confirmed by [START_REF] Login | The single transmembrane segment drives self-assembly of OutC and the formation of a functional type II secretion system in Erwinia chrysanthemi[END_REF]. They showed that the TMS of OutC is more than just a membrane anchor. It mediates an efficient self-association necessary for protein function. Sitedirected mutagenesis of the TMS revealed that cooperative interactions between three polar residues, Gln 29 , Arg 15 , and Arg 36 , located at the same helical face provide adequate stability for OutC self-assembly [START_REF] Login | The single transmembrane segment drives self-assembly of OutC and the formation of a functional type II secretion system in Erwinia chrysanthemi[END_REF]. The disruption of OutC TMS self-association resulted in a loss of protein activity in vivo. However, studies with the PulC (GspC of K. oxytoca)

suggested that the TMS of this protein could be replaced with a lipid anchor, without a loss of functionality [START_REF] Possot | Membrane association and multimerization of secreton component pulC[END_REF]. Thus, the exact function and necessity of the TMS may differ between bacterial backgrounds.

Little is known about structural properties of the HR domain. Recently, in collaboration with Prof. Pickersgill from London University, the 3D structure of HR domain of OutC was solved. It revealed a structure that similar to PilP of T4P [START_REF] Golovanov | The solution structure of a domain from the Neisseria meningitidis lipoprotein PilP reveals a new betasandwich fold[END_REF]. The protein adopts a beta-sandwich type fold with two beta sheets, each composed by 3 antiparallel strands, stacked on each other (Gu et al., 2012b). A short 3 10 helix is formed at the N-terminus, which is also found in the only other β-sandwich -type fold protein PilP [START_REF] Golovanov | The solution structure of a domain from the Neisseria meningitidis lipoprotein PilP reveals a new betasandwich fold[END_REF]. The exact function of this domain is still unclear. The HR domain of GspC of V. cholerae was shown to be engaged in interactions with the periplasmic domain of the secretin EpsD [START_REF] Korotkov | Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae[END_REF].

This result is in agreement with previous studies suggesting that GspC could interact with the OM secretin [START_REF] Bleves | Structure-function analysis of XcpP, a component involved in general secretory pathway-dependent protein secretion in Pseudomonas aeruginosa[END_REF][START_REF] Lee | Association of the cytoplasmic membrane protein XpsN with the outer membrane protein XpsD in the type II protein secretion apparatus of Xanthomonas campestris pv. campestris[END_REF][START_REF] Possot | Membrane association and multimerization of secreton component pulC[END_REF]. Recently, we showed that a short peptide of the HR domain of OutC (OutCsip) interacts in vitro with two distinct sites of OutD: N0 and N2-N3' subdomains [START_REF] Login | A 20-residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi[END_REF].

In conclusion, Studies on the GspC from various bacterial species have revealed that this protein is most likely performing multiple functions in the T2SS, Namely: i) it was shown that the the periplasmic region of GspC proximal to the TMS interacts with the GspL-M hetero-multimer and thus GspC may be part of a larger IM subcomplex consisting of GspC-F-L-M [START_REF] Possot | Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE[END_REF][START_REF] Robert | Identification of XcpZ domains required for assembly of the secreton of Pseudomonas aeruginosa[END_REF][START_REF] Robert | Role of XcpP in the functionality of the Pseudomonas aeruginosa secreton[END_REF][START_REF] Tsai | A reversibly dissociable ternary complex formed by XpsL, XpsM and XpsN of the Xanthomonas campestris pv. campestris type II secretion apparatus[END_REF][START_REF] Lee | Functional dissection of the XpsN (GspC) protein of the Xanthomonas campestris pv. campestris type II secretion machinery[END_REF];

ii) several studies point out interactions between GspC and the OM secretin GspD [START_REF] Lindeberg | Complementation of deletion mutants in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species-specific secretion of proteins via the type II pathway[END_REF][START_REF] Possot | Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE[END_REF]Rober et al., 2005;[START_REF] Korotkov | Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae[END_REF], furthermore, our recent studies showed that a short peptide of the HR domain of OutC (OutCsip) interacts in vitro with two distinct sites of OutD: N0 and N2-N3' subdomains [START_REF] Login | A 20-residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi[END_REF];

iii) deletion and domain exchange studies of GspC and GspD proteins from Erwinia indicated that both the PDZ domain of GspC and the N-terminal domain of GspD are involved in the secretion specificity of the T2SS [START_REF] Bouley | The PDZ domain of OutC and the N-terminal region of OutD determine the secretion specificity of the type II out pathway of Erwinia chrysanthemi[END_REF]; iv) cross-linking analysis revealed an association of the major pseudopilin GspG with the GspC in functional strains of X. campestris pv.campestris [START_REF] Lee | Associations of the major pseudopilin XpsG with XpsN (GspC) and secretin XpsD of Xanthomonas campestris pv. campestris type II secretion apparatus revealed by cross-linking analysis[END_REF].

Thus, GspC is thought to link three subcomplexes, namely the secretin GspD in the outer membrane, the pseudopilins and the IM platform and allows to form an integral T2S apparatus.

III.3.5 Auxiliary proteins GspN, GspA and GspB

GspN is a bitopic IM protein. It has been identified in many bacteria, e.g., in K. oxytoca, V.

cholerae, E. carotovora, but not in D. dadantii. Non-polar PulN mutant of the T2SS of K.

oxytoca reconstructed in E. coli remained functional [START_REF] Possot | Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE[END_REF].

GspA and GspB proteins are not present in all the species carrying a T2SS, indicating that the role of GspAB is not indispensable to assembly of the T2SS. For example, a recent study showed that GspAB present in both Aeromonas and Vibrio and is essential for formation of a functional T2SS in Aeromonas but is dispensable in Vibrio [START_REF] Strozen | Involvement of the GspAB complex in assembly of the type II secretion system secretin of Aeromonas and Vibrio species[END_REF]. GspA is a membrane protein with a conserved ATP binding site which suggested to be a traffic ATPase [START_REF] Schoenhofen | Purification and characterization of the Nterminal domain of ExeA: a novel ATPase involved in the type II secretion pathway of Aeromonas hydrophila[END_REF], whereas GspB has a sequence similar to that of TonB which associates with the IM and is required to transduce IM energy to OM TonB-dependent receptors. This suggested the involvement of GspB in an energy-transducing process. It was demonstrated that GspA and GspB form a complex within the cytoplasmic membrane and such complex from A. hydrophila (ExeA/ExeB) may help the translocation of the ExeD secretin from the IM to the OM [START_REF] Ast | Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer[END_REF][START_REF] Strozen | Involvement of the GspAB complex in assembly of the type II secretion system secretin of Aeromonas and Vibrio species[END_REF]. Indeed, in the abesence of ExeAB, ExeD remains in the IM as a monomer. Interestingly, the periplasmic domain of ExeA was found to contain a putative peptidoglycan-binding motif and the interaction with peptidoglycan may help achieve such a function [START_REF] Howard | Interactions between peptidoglycan and the ExeAB complex during assembly of the type II secretin of Aeromonas hydrophila[END_REF]. Moreover, this inteaction caused the periplasmic ExeA domain to form a large multimers, possibly a ring-like structure (presumable also containing ExeB) on petidoglycan [START_REF] Li | ExeA binds to peptidoglycan and forms a multimer for assembly of the type II secretion apparatus in Aeromonas hydrophila[END_REF]. Such ring could act as a scaffold to direct the assembly of the secretin through ExeA-D and/or ExeB-D interaction. Indeed, GspB homolog OutB was found to be required for the secretion in E. chrysanthemi and interact with secretin OutD. The two proteins are also characterized by a mutual stabilization [START_REF] Condemine | Overproduction of the secretin OutD suppresses the secretion defect of an Erwinia chrysanthemi OutB mutant[END_REF].

However, contrary to the essential role of GspAB in assembly of the secretin and secretion of T2SS substrates in Aeromonas species, GspB is not required for pullulanase secretion, at least when the T2SS of K. oxytoca were reconstructed in E. coli [START_REF] Possot | Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE[END_REF]. In vibrio, mutation of gspA (which also apparently inactivated gspB) did not prevent secretion of T2SS substrates. However, a significant reduction in the amount of assembled secretin multimer were detected in the absence of GspAB [START_REF] Strozen | Involvement of the GspAB complex in assembly of the type II secretion system secretin of Aeromonas and Vibrio species[END_REF]. The precise function of GspAB play in the T2SS secretion remains to be elucidated.

III.4 Cell location of the T2SS components

Fluorescence microscopy of living V. cholerae cells revealed that green fluorescent protein (GFP) chimeras of GspM (EpsM) and GspL (EpsL) localized predominantly to one of the poles of the V. cholerae cell [START_REF] Scott | Directed polar secretion of protease from single cells of Vibrio cholerae via the type II secretion pathway[END_REF]. Moreover, EpsM was found to have all the information needed to be localized at the pole of the cell because when expressed in E. coli, EpsM can localize to the pole in the absence of other T2SS components. Interestingly, EpsL was associated with the cell pole only upon co-expression with EpsM. These results indicate that not all the proteins of T2SS contain the information required for polar targeting. And the assembly of the machinery is an ordered event with a cascade protein-protein interaction in which the GspM protein may determine the membrane location of the machinery, recruiting the GspL component, which in turn brings GspE into association with the membrane. Coherent with the polar location of GspM and GspL, this study also revealed a polar secretion of the V. cholerae toxins [START_REF] Scott | Directed polar secretion of protease from single cells of Vibrio cholerae via the type II secretion pathway[END_REF]. However, [START_REF] Buddelmeijer | Green fluorescent chimeras indicate nonpolar localization of pullulanase secreton components PulL and PulM[END_REF] showed that both GFP-PulL and GFP-PulM

III.5 Exoprotein recognition

Secrection by the T2SS is a two-step process. Exoproteins bearing singal peptide are firstly recognised either by the Sec or the Tat system and translocated across the IM. Then they are folded in the periplasm and become the substrates for the T2SS. The T2SS could distinguish its substrates from other periplasmic proteins and translocted them through OM. However, little is known how the T2S apparatus recognizes T2S substrates and no obvious amino acid sequence motif has been identified in T2S substrates as a notable secretion signal. Indeed, proteins secreted by the same bacterium often do not share any obvious sequence homology. This process may be based on the recognition of a structural secretion motif by some of the Gsp components.

III.5.1 Species-specific recognition of exoproteins

T2SS is often well conserved between various Gram-negative bacteria. However, T2SSdependent exoproteins from one specie can not be generally secreted via the T2SS of antother specie. For example, the K. oxytoca Pul system cannot secrete P. aeruginosa or E. chrysanthemi proteins (de [START_REF] De Groot | Conservation of xcp genes, involved in the two-step protein secretion process, in different Pseudomonas species and other gram-negative bacteria[END_REF]He et al., 1991a), whereas, P. aeruginosa can not secrete pullulanase [START_REF] De Groot | Conservation of xcp genes, involved in the two-step protein secretion process, in different Pseudomonas species and other gram-negative bacteria[END_REF]. Heterologous secretion has been observed only in certain closely related organisms. For example, the bacteria P. alcaligenes and P. aeruginosa could secrete exoproteins of each other [START_REF] De Groot | Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition[END_REF]. E. chrysanthemi and E. carotovra use the T2SS (Out system) whose individual components share a high degree of sequence conservation, to secrete similar Pels. However, the E. chrysanthemi T2SS fails to secrete Pel1 from E. carotovora even though Pel1 possesses 71% indentity to E. chrysanthemi PelC [START_REF] Lindeberg | External loops at the C terminus of Erwinia chrysanthemi pectate lyase C are required for species-specific secretion through the out type II pathway[END_REF]. Two pectate lyases, PelI of E. chrysanthemi and Pel-3 of E. carotovora, share 67% identity. When PelI and Pel-3 were expressed from plasmids in E. carotovora, they were secreted with a similar efficiency. However, when expressed in E. chrysanthemi, PelI was secreted but Pel-3 was not. This indicates that the Out systems of E.carotovora and E.

chrysanthemi differ on their capacity to distinguish between the secretion substrates [START_REF] Bouley | The PDZ domain of OutC and the N-terminal region of OutD determine the secretion specificity of the type II out pathway of Erwinia chrysanthemi[END_REF]. Heat-labile enterotoxin (LT) from ETEC and cholera toxin (CT) from V. cholerae are very similar in structure, they share 83% sequence identity in the mature B subunit. Despite the high degree of homology between LTB and CTB, it was shown that mutations affect their secretion differently in distinct T2SS, indicating that each substrate has evolved closely with its T2SS to enable its secretion [START_REF] Mudrak | Specificity of the type II secretion systems of enterotoxigenic Escherichia coli and Vivrio cholerae for heat-labile enterotoxin and cholera toxin[END_REF]. In conclusion, these results suggested a different level of T2SS species-specifity. Exoprotein recognition process may depend on the presence of a secretion motif within the secreted protein. However, comparisons of the sequences of the diverse proteins which are secreted by T2SS did not reveal an obvious common linear motif in various T2S substrates. The fact that Gsp-dependent exoproteins are translocated across the OM in a folded conformation led to suggest that a three-dimensional "structural motif" may exist in T2SS substrates.

III.5.2 Folding and secretion motif of exoproteins

Several studies suggested that T2SS exoproteins are translocated across the OM in a folded conformation.The studies with V. cholerae and A. hydrophila have shown that the B pentamer of the cholera toxin is formed in the periplasm before secretion while aerolysin is secreted as a dimer [START_REF] Hirst | Conformation of protein secreted across bacterial outer membranes: a study of enterotoxin translocation from Vibrio cholerae[END_REF]. The studies with E. chrysanthemi cellulase and pectate lyases and K. oxytoca pullulanase have shown that the disulfide bond formation is a prerequisite for T2SS-dependent secretion [START_REF] Pugsley | Translocation of a folded protein across the outer membrane in Escherichia coli[END_REF][START_REF] Bortoli-German | Periplasmic disulphide bond formation is essential for cellulase secretion by the plant pathogen Erwinia chrysanthemi[END_REF][START_REF] Shevchik | Differential effect of dsbA and dsbC mutations on extracellular enzyme secretion in Erwinia chrysanthemi[END_REF].

The lipase of P. aeruginosa was not secreted in a mutant strain devoid of the oxidoreductase DsbA, indicating the importance of the disulfide bond formation to the secretion of the enzyme [START_REF] Urban | DsbA and DsbC affect extracellular enzyme formation in Psudomonas aeruginosa[END_REF]. Therefore, the secretion motif may be formed and presented to the secretory apparatus on the folding of the exoprotein. This motif probably involve distal regions brought into close proximity by the folding of the protein.

Many studies have attempted to identify the structural motif presented as secretion signal in T2SS substrates. Two non-adjacent regions in the K. oxytoca pullulanase, A (close to the signal peptide) and B (in the C-terminal region), together direct the secretion of PulA-BlaM and PulA-CelZ hybrid proteins [START_REF] Sauvonnet | Identification of two regions of Klebsiella oxytoca pullulanase that together are capable of promoting beta-lactamase secretion by the general secretory pathway[END_REF]. Furthermore, [START_REF] Francetic | Towards the identification of type II secretion signals in a nonacylated variant of pullulanase from Klebsiella oxytoca[END_REF] showed that A and B fused to the PelB signal peptide (sp) can also promote secretion of BlaM and CelZ but not that of nuclease NucB or several other reporter proteins. PelBsp-PulA, a nonacylated variant of PulA made by replacing the lipoprotein signal peptide (sp) with the signal peptide of pectate lyase PelB from E. chrysanthemi, was efficiently secreted into the medium.

However, the deletion of most of region A or all of region B, either individually or together, had only a minor effect on PelBsp-PulA secretion. These led to identify an additional PulA region (region C) that could contain part of the PulA secretion signal or be important for its correct presentation [START_REF] Francetic | Towards the identification of type II secretion signals in a nonacylated variant of pullulanase from Klebsiella oxytoca[END_REF]. Indeed, the deletion of region C abolished PelBsp-PulA secretion without dramatically affecting its stability, and PelBsp-PulA-NucB chimeras were secreted only if the PulA-NucB fusion point was located downstream from region C [START_REF] Francetic | Towards the identification of type II secretion signals in a nonacylated variant of pullulanase from Klebsiella oxytoca[END_REF]. Studies of cellulase Cel5 from E. chrysanthemi suggested that, prior to secretion, a folded form of the cellulose-binding domain (CBD) interacts physically with the catalytic domain, resulting in a transient secretion-specific conformation [START_REF] Chapon | Alteration of a single tryptophan residue of the cellulose-binding domain blocks secretion of the Erwinia chrysanthemi Cel5 cellulase (ex-EGZ) via the type II system[END_REF].

Furthermore, [START_REF] Chapon | Type II protein secretion in gram-negative pathogenic bacteria: the study of the structure/secretion relationships of the cellulase Cel5 (formerly EGZ) from Erwinia chrysanthemi[END_REF] suggested that the catalytic domain ought to fold into a wildtype conformation before Cel5 is secreted. Since a mutation of R57H, which is located at the heart of the catalytic domain of Cel5, exhibits distinct but close conformation from the wild-type, is not secreted, indicating the strong constraints that secretion exerts upon folding. In polygalacturonase PehA of E. carotovora , two regions, one close to the C-terminus between residues 342 and 369 and another between residues 84 and 135 in the large central loop were required together to promote secretion, indicating that these two regions include a targeting motif [START_REF] Palomäki | A putative threedimensional targeting motif of polygalacturonase (PehA), a protein secreted through the type II (GSP) pathway in Erwinia carotovora[END_REF]. These two motifs are structurally dissimilar, which suggests that there are two distinct recognition regions in the T2SS. In PelC of E. chrysanthemi, a single C-terminal region composed of loops was considered to be involved in a targeting motif whereas two helical regions, which were also reported to be required for secretion, were suggested to be necessary for the proper positioning of the loop region [START_REF] Lindeberg | External loops at the C terminus of Erwinia chrysanthemi pectate lyase C are required for species-specific secretion through the out type II pathway[END_REF].

As reported above, the secretion motif may be composed of different parts of the polypetide chain which are brought together during protein folding. However, the nature of the conformational signal has not yet been elucidated even though the 3D structure of several T2Sdependent exoproteins have been solved. There is evidence that these proteins are rich in β-sheet content (Sandkvist, 2001b). For example, studies on P. aeruginosa showed that exotoxin A 60-120 region appeared to be rich in anti-parallel β-sheet [START_REF] Lu | A specific targeting domain in mature exotoxin A is required for its extracellular secretion from Pseudomonas aeruginosa[END_REF]. Several 3D structures of the proteins, i.e., PelC, PelE, Cel5, PelA, Pel9A and PelI secreted by E. chrysanthemi T2SS

Out, showed that they are all rich in β-sheet [START_REF] Yoder | Unusual structural features in the parallel beta-helix in pectate lyases[END_REF][START_REF] Lietzke | The three-dimensional structure of pectate lyase E, a plant virulence factor from Erwinia chrysanthemi[END_REF][START_REF] Chapon | Type II protein secretion in gram-negative pathogenic bacteria: the study of the structure/secretion relationships of the cellulase Cel5 (formerly EGZ) from Erwinia chrysanthemi[END_REF][START_REF] Thomas | Structure of pectate lyase A: comparison to other isoforms[END_REF][START_REF] Jenkins | The crystal structure of pectate lyase Pel9A from Erwinia chrysanthemi[END_REF][START_REF] Creze | The crystal structure of pectate lyase peli from soft rot pathogen Erwinia chrysanthemi in complex with its substrate[END_REF]. However, the putative secretion motif of PelC seems to be composed of loops [START_REF] Lindeberg | External loops at the C terminus of Erwinia chrysanthemi pectate lyase C are required for species-specific secretion through the out type II pathway[END_REF]. In addition, one of putative PehA targeting motif, the A region, is composed of two large loop structure [START_REF] Palomäki | A putative threedimensional targeting motif of polygalacturonase (PehA), a protein secreted through the type II (GSP) pathway in Erwinia carotovora[END_REF]. Thus, nature of the secretion motif in the proteins secreted by the T2SS remains to be identified.

III.5.3 Secretion specificity and GspC/GspD

Several studies have shown that two components of the secreton GspC/GspD are not individually exchangeable, even between closely related species such as P. aeruginosa and P.

alcaligenes XcpP/XcpQ (de [START_REF] De Groot | Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition[END_REF], or E. chrysanthemi and E. carotovora

OutC/OutD [START_REF] Lindeberg | Complementation of deletion mutants in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species-specific secretion of proteins via the type II pathway[END_REF]. These indicated that GspC and GspD could be involved in the specific recognition of the exoproteins. Indeed, the pectate lyase PelB of E. chrysanthemi but not PelC of E. carotovora was demonstrated to interact with the N-terminal domain of OutD (Shevchik et al., 1997). Recently, surface plasmon resonance (SPR) studies showed that the periplasmic domain of ETEC GspD interacts with the B-pentamer of heat-labile enterotoxin [START_REF] Reichow | Structure of the cholera toxin secretion channel in its closed state[END_REF] while EM showed that the B-pentamer of cholera toxin bound within the V. cholerae GspD periplasmic vestibule [START_REF] Reichow | The binding of cholera toxin to the periplasmic vestibule of the type II secretion channel[END_REF]. The N-terminus is the most variable region of the secretin, consistent with the notion of specificity for recognition of the exoproteins. [START_REF] Bouley | The PDZ domain of OutC and the N-terminal region of OutD determine the secretion specificity of the type II out pathway of Erwinia chrysanthemi[END_REF]

III.6 Comparison between the T2SS and the type IV pili

The type IV pili (T4P) are cell surface filament that traverses the OM of Gram-negative bacteria and contribute to prokaryotic cell survival and pathogenesis. These polar filaments are composed of thousands of copies of pilin and are 5-8 nm in diameter and up to several micrometers long [START_REF] Chiang | Disparate subcellular localization patterns of Pseudomonas aeruginosa Type IV pilus ATPase involved in twitching motility[END_REF]Nudleman et al., 2006). T4P serve remarkably diverse functions, including bacterial motility, adhesion, microcolony and biofilm formation, DNA uptake during natural transformation, phage transduction (reviewed in [START_REF] Craig | Type IV pilus structure and bacterial pathogenicity[END_REF][START_REF] Pelicic | Type IV pili: e pluribus unum?[END_REF]. It was also shown that the contribution of T4P to virulence was related to its direct involvement in the secretion of toxins [START_REF] Hager | Type IV pili-mediated secretion modulates Francisella virulence[END_REF][START_REF] Han | Type IV fimbrial biogenesis is required for protease secretion and natural transformation in Dichelobacter nodosus[END_REF]. Based on length and sequence of the pilin subunit, T4P can be further classified into two subclasses, termed T4aP and T4bP [START_REF] Craig | Type IV pili: paradoxes in form and function[END_REF]. Pilins of T4aP have short leader peptides (less than 10 aa) and a characteristic length of about 150-160 aa. T4aP are involved in adherence, twitching motility and a form of flagellar-independent surface motility involved in dissemination from the point of attachment. Pilins of T4bP have long leader peptides (about 15-30 aa) and are either long (180-200 aa) or surprisingly short (only about 40-50 aa). T4bP are found predominantly in enteric pathogens, function in adhesion and bacterial aggregation [START_REF] Ayers | Architecture of the type II secretion and type IV pilus machineries[END_REF]. Systematic genetic studies showed that the proteins associated with T4bP are more diverse than their T4aP counterparts [START_REF] Pelicic | Type IV pili: e pluribus unum?[END_REF]. Even so, a conserved core of proteins, common to both T4aP and T4bP is identified (Figs. 61B and61C), namely: i) one or several pilin proteins; ii) a specific peptidase that processes the prepilins and prepilin-like proteins; iii) an ATPase that powers T4P assembly; iv) an OM secretin responsible for mediating the passage of the pilus fiber across the OM; v) IM complex that is critical for the assembly of functional machinery. The T2SS and T4P, especially T4aP, share a number of protein homologs that are predicted, or have been demonstrated, to localize and function in a similar manner (Fig. 61). Most strikingly, T2SS requires the presence of proteins that are highly homologous to type 4 pilins [START_REF] Bose | Type 4 pilus biogenesis and type II-mediated protein secretion by Vibrio cholerae occur independently of the TonB-facilitated proton motive force[END_REF]. In both systems, the prepilins or prepilin-like proteins are processed by a homologous type 4 prepilin peptidase. In addition, both systems have an OM protein, known as the secretin, that has been demonstrated to multimerize and form an OM channel through which proteins can pass during the extracellular translocation step. Both systems also have at least one ATPase, together with conserved polytopic IM protein, named GspE/PilB and GspF/PilC, in T2SS and T4P respectively. These proteins are thought to assure extrusion of growing (pseudo)pilus from the IM [START_REF] Py | An inner membrane platform in the type II secretion machinery of Gram-negative bacteria[END_REF][START_REF] Morand | Type IV pilus retraction in pathogenic Neisseria is regulated by the PilC proteins[END_REF]. They have an IM complex composed of several conserved proteins that are critical for the assembly of functional machinery [START_REF] Sampaleanu | Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex[END_REF]. However, some secreton components that are needed for T2SS assembly appear to be absent from the T4P system (Table . 1). Additionally, certain components are only present in T4P systems (Table . 1). These observations probably reflect the ancient separation during divergent evolution of the T2S and T4P systems. Nevertheless, the comparison between T2S and T4P systems, especially with current research findings, will help us to better understand the assembly and function mechanism of the two systems. white (Korotkov et al., 2011a).

Fig. 63: Electron microscopy structures of secretins. (A) Cryo-EM reconstruction of T2SS secretin (i) [START_REF] Reichow | Structure of the cholera toxin secretion channel in its closed state[END_REF] and T3SS secretin (ii, iii) [START_REF] Hodgkinson | Threedimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout[END_REF]. (B) EM structures of PilQ secretins from N. meningitidis (i) [START_REF] Collins | Structure of the Neisseria meningitidis outer membrane PilQ secretin complex at 12 Å resolution[END_REF], N. gonorrhoeae (ii) [START_REF] Jain | Structural characterization of outer membrane components of the type IV pili system in pathogenic Neisseria[END_REF] and T. thermophilus (iii) [START_REF] Burkhardt | Structure and function of PilQ, a secretin of the DNA transporter from the thermophilic bacterium Thermus thermophilus HB27[END_REF].

Although some differences in symmetry and nature of the constriction or plug were found, they share a similar basic overall structure (Fig. 63) [START_REF] Ayers | Architecture of the type II secretion and type IV pilus machineries[END_REF]Korotkov et al., 2011a). For example, the crystal structures of the N-terminal domains (N0-N1-N2) of the T2SS secretin GspD from ETEC and the periplasmic domains (N0-N1) of the T3SS secretin EscC from EPEC revealed that the two N-terminal domains N0-N1 from two systems adopt a similar fold, although the mutual orientation of these domains with respect to each other is unexpectedly different (Fig. 39C) (Korotkov et al., 2009a;[START_REF] Spreter | A conserved structural motif mediates formation of the periplasmic rings in the type III secretion system[END_REF]. Recent cryo-EM structure of the secretin GspD from the V. cholera T2SS revealed a multimeric channel of 12-fold symmetry with a periplasmic gate in its closed state (Fig. 43) (page 60) [START_REF] Reichow | Structure of the cholera toxin secretion channel in its closed state[END_REF]. Similar results were initially observed through transmission EM studies of the T4aP secretin PilQ [START_REF] Collins | Analysis of the PilQ secretin from Neisseria meningitidis by transmission electron microscopy reveals a dodecameric quaternary structure[END_REF][START_REF] Collins | Three-dimensional structure of the Neisseria meningitidis secretin PilQ determined from negative-stain transmission electron microscopy[END_REF]. However, subsequent cryo-EM and 2D crystal analyses suggested that the T4aP secretin adopts fourfold symmetry that is closed at both ends but lacks the periplasmic gate found in reconstructions of the secretins from other secretion systems (Fig. 63B, i) [START_REF] Collins | Structure of the Neisseria meningitidis outer membrane PilQ secretin complex at 12 Å resolution[END_REF]. Very recent transmission EM analysis of the T4P secretin PilQ from N.

gonorrhoeae showed a double ring structure with a 14-fold symmetry for the central ring, which consists of PilQ (Fig. 63B, ii) [START_REF] Jain | Structural characterization of outer membrane components of the type IV pili system in pathogenic Neisseria[END_REF]. Another recent study revealed a PilQ complex with a width of 15 nm and a length of 34 nm consisting of an extraordinary stable "cone" and "cup" structure and five ring structures with a large central channel (Fig. 63B, iii), which differs from all T2SS, T3SS and T4P secretin complexes known to date [START_REF] Burkhardt | Structure and function of PilQ, a secretin of the DNA transporter from the thermophilic bacterium Thermus thermophilus HB27[END_REF]. The authors suggested that the 500 residue long N-terminal aa form a complex structure comprising of five staged ring systems spanning the periplasmic space [START_REF] Burkhardt | Structure and function of PilQ, a secretin of the DNA transporter from the thermophilic bacterium Thermus thermophilus HB27[END_REF]. This model is coherent with those of the secretins from the T2SS and T3SS. The ring models generated by using the crystal structures of the periplasmic domains of the secretins from the T2SS (N0-N1-N2) and T3SS (N0-N1), suggested that the N-terminus of secretins could mediate the formation of periplasmic rings (Korotkov et al., 2009a;[START_REF] Spreter | A conserved structural motif mediates formation of the periplasmic rings in the type III secretion system[END_REF].

Top view of secretins from various export systems showed ring-like structures ranging from 12-20 nm in diameter, with large central channels ranging from 5-10 nm in diameter [START_REF] Burkhardt | Structure and function of PilQ, a secretin of the DNA transporter from the thermophilic bacterium Thermus thermophilus HB27[END_REF][START_REF] Reichow | Structure of the cholera toxin secretion channel in its closed state[END_REF][START_REF] Collins | Structure of the Neisseria meningitidis outer membrane PilQ secretin complex at 12 Å resolution[END_REF]. To maintain the integrity of the OM, secretins need to be gated. It was suggested that the interactions between the secretin and other components of the T2S/T4P machinery are involved in the gating of the secretin. Indeed, the IM lipoprotein PilP of the Neisseria T4aP and the IM protein GspC of the T2SS have been shown to interact with their respective secretins [START_REF] Balasingham | Interactions between the lipoprotein PilP and the secretin PilQ in Neisseria meningitidis[END_REF][START_REF] Login | A 20-residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi[END_REF]. This group of proteins that is also involved in the IM complexes, was recently suggested to be designated as 'secretin dynamic-associated' (SDA) proteins [START_REF] Ayers | Architecture of the type II secretion and type IV pilus machineries[END_REF]. In the T4bP, a putative SDA protein BfpU has also been shown to interact with the N-terminal domain of the cognate secretin [START_REF] Daniel | Multiple interactions between the transmembrane division proteins of Bacillus subtilis and the role of FtsL instability in divisome assembly[END_REF]. A recent study on the bacteriophage secretin pIV identified two gate regions within the C-terminal secretin domain, called GATE1 and GATE2. Amino acid substitutions in these regions resulted in a leaky channel phenotype [START_REF] Spagnuolo | Identification of the gate regions in the primary structure of the secretin pIV[END_REF]. Several mutations resulted in a similar phenotype have also been identified in the N3 domain that corresponds to the periplasmic constriction site in VcGspD structure. This was consistent with the notion that this domain may be involved in triggering the opening of the periplasmic gate of the secretin complexes [START_REF] Reichow | Structure of the cholera toxin secretion channel in its closed state[END_REF][START_REF] Reichow | The binding of cholera toxin to the periplasmic vestibule of the type II secretion channel[END_REF]. meningitidis T4PS pilotin PilW (orange) is composed of six TPR motifs arranged as a superhelix [START_REF] Trindade | Structure of a widely conserved type IV pilus biogenesis factor that affects the stability of secretin multimers[END_REF].

In many cases, the assembly of secretins is assisted by a specific small OM lipoprotein, pilotin.

In T4P, the pilotin PilF from P. aeruginosa is required for the OM targeting and multimerization of T4aP secretin PilQ (Watson et al., 1996b). Homologous pilotins PilW from N. meningitidis and Tgl from M. xanthus appear to be required for multimerization of the cognate secretins, but not for their targeting to the OM [START_REF] Carbonnelle | Type IV pilus biogenesis in Neisseria meningitidis: PilW is involved in a step occurring after pilus assembly, essential for fibre stability and function[END_REF]Nudleman et al., 2006). Recent structure data showed that unlike the T3SS pilotin MxiM from S. flexneri, which adopts a conical shape β-barrel structure interrupted by an α-helix (Fig. 64a) [START_REF] Okon | Structural characterization of the type-III pilot-secretin complex from Shigella flexneri[END_REF], T4aP pilotins are composed of six tetratricopeptide repeats (TPRs) arranged as a super-helix (Fig. 64b) [START_REF] Koo | PilF is an outer membrane lipoprotein required for multimerization and localization of the Pseudomonas aeruginosa Type IV pilus secretin[END_REF][START_REF] Trindade | Structure of a widely conserved type IV pilus biogenesis factor that affects the stability of secretin multimers[END_REF]. The concave surfaces on the structures were suggested as potential binding sites for the T4aP secretin whereas the hydrophobic cleft at the top of the βbarrel of MxiM serves as binding site for either lipids or the C-terminal domain of of MixD.

However, unlike T2SS and T3SS secretins, T4P secretins lack the C-terminal pilotin binding domain, S-domain (Fig. 62). It is still unknown which part of T4aP secretin interacts with a pilotin.

III.6.2 The (pseudo)pilins

The pseudopilus and pilus are essential components of both T2SS and T4P, respectively. One major (pseudo)pilin and several minor (pseudo)pilins are involved in the assembly of (pseudo)pilus. However, in both systems, the (pseudo)pilus itself is composed mainly by major (pseudo)pilin subunits while the precise roles of the minor (pseudo)pilins remain to be investigated. For example, the major T4aP pilin PilA (in P. aeruginosa) or PilE (in Neisseria spp.)

homopolymerizes to form the pilus fiber, whereas the minor pilins PilE, PilV, PilW, PilX, FimT-U (in P. aeruginosa) or PilH, PilI, PilJ, PilK, PilX (in Neisseria spp.) contribute to pilus assembly in ways that are still poorly understood [START_REF] Hazes | Towards a systems biology approach to study type II/IV secretion systems[END_REF]. Recent study in P.

aeruginosa suggested that the minor pilins PilV, PilW and PilE may be required for the formation of one or more subcomplexes that allow FimU and PilX to promote pilus assembly and stimulate the opening of the secretin pore [START_REF] Giltner | Pseudomonas aeruginosa minor pilins are incorporated into type IV pili[END_REF]. This indicates that at least certain minor pilins could play a role in the initiation but not termination, of pilus assembly.

FimT, unlike the other minor pilins, does not have significant role in pilus assembly. [START_REF] Giltner | Pseudomonas aeruginosa minor pilins are incorporated into type IV pili[END_REF] also demonstrated that the minor pilins are incorporated not simply at the tips of new pili, but also throughout growing pili, which suggests that the minor pilins and major pilin together form the pilus fiber. In the T2SS, the pseudopilus core is formed by the homomultimerization of the major pseudopilin GspG through interactions between hydrophobic domains. A tip heteromeric complex is formed by at least three of the minor pseudopilins. The minor pseudopilin GspI may be invovled in initiation of peseudopilus formation while GspK may control pilus elongation (see Chapter III.3.2.2).

The pilins are synthesized as precursors, prepilins, which typically have a conserved hydrophotic N-terminal sequence in an extended α-helix containing a leader sequence with putative prepilin peptidase GspO/PilD cleavage and methylation sites (Fig. 45, page 62).

Processing and methylation of pilins by peptidase require three highly conserved residues: Gly (-1), Phe (+1) and Glu (+5) [START_REF] Strom | structure-function and biogenesis of the type IV pili[END_REF]. The prepilins are co-translationally targeted by the signal recognition particle to the Sec machinery and remain in the IM as bitopic proteins due to their hydrophobic N-terminal α-helix [START_REF] Pelicic | Type IV pili: e pluribus unum?[END_REF]. After processed by peptidase, pilins could be extracted from the IM and added to the base of the growing pilus. The mechanism of this process remains poorly understood. An IM complex coupled to a cytoplasmic ATPase is needed for this process. In the T2SS, cytoplasmic ATPase GspE may probably drive both extension and retraction. In the T4aP, cytoplasmic ATPase PilB may energize pilus extension while two additional ATPase, PilT and PilU play a role in pilus retraction, since inactivation of either gene results in a hyperpiliated phenotype [START_REF] Hazes | Towards a systems biology approach to study type II/IV secretion systems[END_REF]. The architecture of T2SS and T4P pilus is that subunits assemble through interactions between the conserved N-terminal α-helix of the pilins, which form a hydrophobic core in the filament.

Meanwhile the less conserved C-terminal globular domains provide specific subunit-subunit interfaces and define the exposed surface of the pilus to provide functional variability [START_REF] Craig | Type IV pilus structure and bacterial pathogenicity[END_REF]. Several high resolution structures of T4P and T2SS pilin subunits have been determined by X-ray crystallography and NMR, which provides a molecular basis for overall (pseudo)pilus assembly and functionality (see chapter III.3.2.3). The T4P pilins share a common fold with an extended N-terminal α1-helix, a globular domain, an αβ-loop and D-region (Fig. 65a). The α1 mediates pilus assembly by forming a hydrophobic helical bundle in the filament core. The protruding half of α1, α1-N is primarily hydrophobic, whereas the buried half, α1-C is amphipathic. The hydrophobic face of α1-C interacts with the globular head domain while the hydrophilic face is exposed on the surface of the protein. Due to the presence of two helixdisrupting residues: a glycine or proline at position 22 and a proline at position 42, which introduce subtle kinks in the α-helix, the α1 of the full-length GC and PAK pilin structures have an S-shaped curve (Figs. 65a and65b). Indeed, the proline at position 22 is highly conserved in major pseudopilins from the T2SS and also introduce kink in the α-helix. [START_REF] Campos | Detailed structural and assembly model of the type II secretion pilus from sparse data[END_REF] showed that PulG P22A mutant was significantly less piliated than the wild type, which suggested that the kink induced by Pro is an important structural element facilitating the α-helix bundle packing.

The superposition of three independent structures of pilins revealed the flexibility in α1. When the α1-C portions of three structures are superimposed, the positions of N-termini diverge by 10 Å (Fig. 65e) [START_REF] Craig | Type IV pilus structure and bacterial pathogenicity[END_REF]). The α1 structures of the T4b pilins may be less curved than in T4a pilins because of lacking residues proline or glycine at positions 22 and 42.

The dominant features of the globular heads domain are the N-terminal α1-helix, α1-C, and a four-(T4P) or three-(major pseudopilin GspG, minor pseudopilins GspH, GspI and GspJ) stranded anti-parallel β-sheet (Alphone et al., 2010) (Figs. 65 and 47). The αβ-loop and D-region flank the β-sheet of the T4P pilins (Fig. 65a). The αβ-loop connects the N-terminal α-helix to the β-sheet. In the T4P, the D-region is bound by the two conserved C-terminal cysteines, which forms a disulfide bridge that is essential for pilus assembly [START_REF] Kirn | Delineation of pilin domains required for bacterial association into microcolonies and intestinal colonization by Vibrio cholerae[END_REF]. Protein interaction studies in both the T2S and the T4P systems have led to the identification of IM complexes, GspL/M/C and PilM/N/O/P, respectively, which are critical for the assembly of the respective systems [START_REF] Py | An inner membrane platform in the type II secretion machinery of Gram-negative bacteria[END_REF][START_REF] Ayers | PilM/N/O/P proteins form an inner membrane complex that affects the stability of the Pseudomonas aeruginosa type IV pilus secretin[END_REF]. Since these components do not share any sequence similarity, it was thought that they could play specific functions within each of these systems [START_REF] Filloux | The underlying mechanisms of type II protein secretion[END_REF][START_REF] Hazes | Towards a systems biology approach to study type II/IV secretion systems[END_REF]. However, the recent structural studies revealed a structural similarity between these components, suggesting that they could play similar roles in (pseudo)pilus assembly [START_REF] Sampaleanu | Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex[END_REF][START_REF] Karuppiah | Structure of the PilM-PilN, inner membrane Type IV pilus biogenesis complex from Thermus thermophilus[END_REF].

III.6.3 The inner membrane complex

It was suggested that the T4P PilM/N/O/P complex is homologous to GspL/M/C in the T2SS (Fig. 66). Additionally, a detailed analysis of PilM/N/O and their ortholog, GspL/M, in the V. cholerae T2SS revealed significant similarities in their secondary structures and threedimensional folds (Fig. 66) [START_REF] Sampaleanu | Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex[END_REF]. PilM is a cytoplasmic protein with an actin-like fold. The crystal structure of PilM could be divided into two subdomains, 1A/C and 2A/B (Fig. 67A) (Karupphiah and Derrick, 2011), according to a previous designation for FtsA (Van de Ent et al., 2000). The cytoplasmic domain of the T2SS protein GspL aligns well with the PilM structure (Fig. 67C; rmsd backbone atoms 2.8 Å). And the periplasmic portion of GspL exhibits the similar fold as PilN. The PilN peptide binds in a narrow channel between the 1A and 1C subdomains while ATP is bound in a deep cleft on the PilM surface, with the triphosphate moiety buried more deeply than the ribose and adenine rings. According to these results, [START_REF] Karuppiah | Structure of the PilM-PilN, inner membrane Type IV pilus biogenesis complex from Thermus thermophilus[END_REF] proposed a model for the interactions of PilM and PilN and PilO (Fig. 68). PilM would first bind ATP, and then the PilM-ATP binary complex would associate with the N-terminus of PilN. The structure of the PilO periplasmic domain consists of two Nterminal coiled coils (CC domain) and a C-terminal ferredoxin-like domain that is similar to that adopted by the T2SS protein GspM (Fig. 69) [START_REF] Sampaleanu | Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex[END_REF]Abendroth et al., 2004).

Thus, these recent structural studies revealed the similar structural organization of soluble domains of PilM/N/O and GspL/M, respectively. More, these results suggested that the bitopic GspL could be equivalent to PilM (cytoplasmic) fused to PilN (periplasmic).

The in vivo mutual stability data offered the first indication that PilN/O may interact (Ayers et al., 2009). [START_REF] Sampaleanu | Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex[END_REF] provided evidence for a direct interaction between them in vitro. The authors showed that PilO may assist in the proper folding of PilN and that the periplasmic domain of PilN and PilO interact to form a stable heterodimer.

PilP is predicted to be a lipoprotein with its lipid attachment site at Cys16, which anchors it to the IM [START_REF] Balasingham | Interactions between the lipoprotein PilP and the secretin PilQ in Neisseria meningitidis[END_REF]. PilP is thought to be a part of the IM complex of T4P and has been initially assigned as pilotin since it stabilizes the secretin PilQ complex [START_REF] Drake | PilP, a pilus biogenesis lipoprotein in Neisseria gonorrhoeae, affects expression of PilQ as a high-molecular-mass multimer[END_REF].

However, subsequent studies suggested that it was inappropriate to define PilP as pilotin since PilP was not required for PilQ stability [START_REF] Balasingham | Interactions between the lipoprotein PilP and the secretin PilQ in Neisseria meningitidis[END_REF]. Moreover, the reduced amounts of PilQ (monomer and complex) in the pilP deletion mutant were merely due to the fact that pilP and pilQ genes are cotranscribed. Thus, PilP may fulfil some other roles. Indeed, it is important for pilus biogenesis. In addition, the structure of PilP is very different from that of the pilotin MxiM from the T3SS [START_REF] Golovanov | The solution structure of a domain from the Neisseria meningitidis lipoprotein PilP reveals a new betasandwich fold[END_REF] and the pilotin OutS from the T2SS (Gu et al., 2012a, accepted). The structure of PilP from Neisseriae revealed a novel β-sandwich structure, made up of a single 3 10 -helix and seven β-strands [START_REF] Golovanov | The solution structure of a domain from the Neisseria meningitidis lipoprotein PilP reveals a new betasandwich fold[END_REF]. Although the lipoprotein PilP was not clearly needed to stabilize the secretin PilQ complex, the N-and Cterminal regions of PilP have been shown to interact with the central portion of the PilQ monomer [START_REF] Balasingham | Interactions between the lipoprotein PilP and the secretin PilQ in Neisseria meningitidis[END_REF]. Recently, [START_REF] Ayers | PilM/N/O/P proteins form an inner membrane complex that affects the stability of the Pseudomonas aeruginosa type IV pilus secretin[END_REF] showed that loss of any one of PilM/N/O/P has a negative impact on the stability of the other gene products, which indicated the interactions between them. These observations suggest that PilP on one hand, interacts with IM proteins PilN or PilO, and on the other hand, interacts with the OM secretin PilQ, and thus connects the IM and OM subcomplexes, similar to the putative function of GspC. Furthermore, our recent structure analysis of the HR domain of GspC revealed a fold similar to PilP, indicating that these two proteins could play similar functions within the T2SS and T4P, respectively (see results section, Gu et al., 2012b).

Objective of this thesis

The type II secretion system (T2SS) is widely employed by pathogenic Gram-negative bacteria to secrete toxins and lytic enzymes facilitating host invasion. The phytopathogen Dickeya dadanti (ex. Erwinia chrysanthemi) uses this system, named Out, to secrete several cellwall degrading enzymes and to cause soft-rot disease of many plants. The T2SS consists of 12-15 proteins that build together a multiprotein complex spanning the entire cell envelope.

Currently, how the individual components assemble to form functional machinery and how exoproteins are transported across the OM are still unclear. The OM pore formed by the secretin GspD is thought to allow translocation of folded exoproteins. Gating of this pore should be tightly regulated. Notably, the IM component GspC, interacts with the secretin GspD and could be involved in the gating of the OM secretin pore. It seems therefore that GspC and GspD interaction plays a crucial role in structural and functional integrity of the secretion machinery by connecting the IM components of the T2SS to the OM secretin pore. Functional and structural characterization of these two components and molecular analysis of their interactions are necessary to better understand the assembly and function of the T2SS. Certain previous studies investigated the putative interactions between GspC and GspD. However, which regions and residues are involved in these interactions and how these two proteins interact within a functional T2SS are still unclear. Therefore, molecular and functional analysis of these proteinprotein interactions is the main objective of this thesis. This study will allow to better understand the assembly and function of this nano-machine.

Previous results of our laboratory (the thesis of Dr. F. Login) have shown that the N-terminal domain of OutD (aa 28-285) interacts with the non-PDZ periplasmic region of OutC (corresponding mainly to HR) by an in vitro GST pull-down assay and by NMR spectroscopy.

The first task of this thesis is to identify the minimal regions involved in OutC-OutD interaction using in vitro copurification. The results of this study have been published in the article: "A 20residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi" [START_REF] Login | A 20-residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi[END_REF] in Molecular Microbiology and will be presented in Chapter I.

To better understand molecular mechanisms that govern OutC-OutD interactions, very recently, the structural analysis of OutC and of OutC-OutD interactions was performed by NMR in collaboration with Prof. Pickersgill from Queen Mary University of London. This pioneer structural analysis (3D structures of neither HR nor GspC-GspD complex were not yet available for other T2SS) opens a new possibility to exploit the T2SS and provides us with an excellent guideline for the functional analysis of the system. In Chapter II, we therefore used alanine and cysteine substitutions and disulfide cross-linking to examine the predictions on the structure of HR domain of OutC and the OutC HR -OutD N0 complex in vivo. This work constitutes a part of the article titled "Solution structure of homology region (HR) domain of the type II secretion system" (Gu et al., 2012b) in Journal of Biological and Chemistry.

To probe the putative HR/OutD interfaces in the functional secretion system and to assess the biological relevance of the HR/N0 interface indicated by recent crystal structure of GspC HR -GspD N0-N1 complex (Korotkov et al., 2011b), we performed in vivo cysteine-scanning mutagenesis and disulfide cross-linking analysis to search for residues that are directly involved in OutC-OutD interaction and are important for the protein function. The results will be presented in Chapter III and in the form of the article: "Cysteine scanning mutagenesis and disulfide mapping analysis of the arrangement of GspC and GspD protomers within the T2SS"

(Wang et al., accepted).

Targeting and proper assembly of the secretin into the OM often require accessory proteins.

The pilotin GspS and the IM protein GspB play such a role towards a cognate secretin. To unravel the molecular mechanism of GspS action and its interaction with the secretin, we undertook a structural and functional analysis of the pilotin OutS. This work will be presented in Chapter IV, as a part of the manuscript: "Structural and functional insights into the pilotsecretin complex of the type II secretion system" (Gu et al., 2012a). Several studies suggested the interaction between the secretin OutD and IM protein OutB. However, there was no direct evidence for this interaction. In Chapter V, we examined the interaction between OutD and OutB.

Lastly, we tried to construct the protein derivatives of OutC and OutD suitable for NMR, crystallography and X-ray analysis. The results will be presented in Chapter VI.

Materials and methods

I Bacterial strains and growth conditions

I.1 Bacterial strains E. coli strains NM522 (△ (lac-proAB) △ (mcrB-hsdSM) 5 supE thi, (F' proAB lacI q , lacZ△M15) and BL21 (DE3) (Fˉ dcmˉ ompT rBˉ mBˉ Ionˉ λ (DE3)) were used for general cloning and protein production purposes, respectively. D. dadantii strains constructed and used are listed in Table . 2.

I.2 Growth conditions and conservation

The bacteria were usually grown in rich medium LB (10 g•l -1 tryptone, 5 g•l -1 Yeast extract and 10 g•l -1 NaCl, pH 7) or minimal medium M63 (13.6 g•l -1 KH 2 PO 4 , 2 g•l -1 (NH 4 ) 2 SO 4 , 0.2 g•l - 1 MgSO 4 , 0.5 mg•l -1 FeSO 4 supplied with 2 g•l -1 glycerol, pH 7.2). The solid media were realized by adding agar at 15 g•l -1 . The cultures were grown under aeration with shaking at 150 rpm or 120 rpm at 30 °C for E. coli and 28 °C for D. dadantii. If necessary, arabinose and IPTG were added at 0.03% and 1 mM, respectively. Antibiotics were used at the following final concentrations: ampicillin, 150 μg•ml -1 , kanamycin, 100 μg•ml -1 , chloramphenicol, 50 μg•ml -1 .

For conservation, the bacteria cells were grown in rich medium Luria-Bertani (LB) overnight.

For D. dadantii, glycerol was added to a final concentration of 40% and the cells were conserved at -80°C. For E. coli, glycerol was added to a final concentration of 20% and the cells were conserved at -20°C.

II Genetic and molecular biology methods

II.1 Plasmids construction and DNA manipulations

Plasmids and primers used in the study are listed in appropriate chapters. DNA cloning and manipulation were performed using standard methods. Site-directed mutagenesis was performed using the QuickChange kit (Stratagene). The nucleotide sequences of mutant and amplified genes were checked (Cogenics). Plasmids were introduced by the CaCl 2 procedure into E. coli [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF] or by electroporation into D. dadantii [START_REF] Enderle | Electroporation of freshly plated Escherichia coli and Pseudomonas aeruginosa cells[END_REF].

II.2 Preparation of the stock of φEC2 bacteriophage

The φEC2 phage lysate was prepared from the donor bacteria in exponential growth phase (OD 600 of approximately 1). This culture was infected by φEC2 phage stock (0.2 ml bacteria + 0.2 ml bacteriophage). After 20 min of adsorption at room temperature (without agitation), 5 ml of soft agar (0.4% in LB medium), which was melt and maintained in liquid state, was added into the bacteria phage mixture. The mixture was then poured on plates LCG (solid medium GL + glucose + CaCl2). After overnight incubation at 30°C, phage lysates were harvested by scraping the top layer after adding 5 ml of LB. The retained cells were lysed by adding chloroform. The mixture was vortexed until homogeneous, agar and cell debris were removed by centrifugation (10 min, 8000 g) and the phage stocks were stored at 4°C.

II.3 Transduction

Transduction is a mechanism for transfer of bacterial DNA from one cell to another by using a virus (bacteriophage) as a vector.

0.2 ml of phage stock was added to 0.2 ml of overnight grown culture. After incubation for 20 min at room temperature without agitation, the mixture was supplemented with 0.8 ml of LB and grown at 30°C for 2-3 h for the expression of transduced genes of resistance to antibiotics usually used as markers for transduction. The cells were then spread on the plate containing an appropriate antibiotic and grown at 30°C for 48 h.

II.4 Construction of D. dadantii mutant strains

The D. dadantii mutant strains carrying single cysteine substitution on chromosome were constructed by exchange-eviction mutagenesis [START_REF] Ried | An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in Gram-negative bacteria by marker exchange-eviction mutagenesis[END_REF]. The nptI-sacB-sacR cartridge that confers kanamycin (Kan) resistance and sucrose sensitivity was inserted into a PstI site of outC, cloned on a plasmid and then introduced into the chromosome by gene exchange recombination. The resulting mutant strain A2365 was Km R , sucrose-sensitive and secretiondeficient. The plasmids pTdB-OC (Amp R ) carrying outC gene coding for a cysteine substitution of interest were transformed into this strain. The cells were first grown on the GL plates with Amp. Then, the cells were grown overnight in LB without antibiotics and next in LB supplemented with sucrose to a final concentration 10%. The outC allele containing single cysteine substitution was exchanged for the chromosomal allele containing the nptI-sacB-sacR cartridge by selection for sucrose tolerance. The culture was subsequently diluted and plated onto GL without antibiotics. Next, the sensetivity of colonies to Amp and Kan was checked on replica plates. The resulting D. dadantii mutant strains should lose the plasmid (Amp S ) and nptI-sacB-sacR chromosomial cartridge (Kan S ). The desired point mutations were then checked by PCR using chromosomic DNA of corresponding D. dadantii mutant strain as template, and the primers OutC (GCCAGAGCATGAATATCGTG) and ROutC (GAACTCCTGAATATCGGTTC). The D. dadantii mutant strains constructed are listed in Table . 2 and the secretion ability of the mutant strains was analyzed by complementation tests.

III Biochemical methods

III.1 Complementation tests

To assess the secretion ability of the mutant D. dadantii strains, secretion of pectinases and cellulase was compared with that of the wild type strain. To test the functionality of the mutant OutC and OutD proteins, the D. dadantii ∆outC and ∆outD mutant strains respectively (Table . 2)

were transformed with pTdB-OC or pTdB-OD plasmids carrying a corresponding outC or outD mutant gene. Secretion efficiency was estimated by both plate assays and immunoblotting assays.

The plate assays were performed on the solid medium containing M63 + glycerol + carboxymethyl cellulose (CMC) + MgSO 4 , for cellulase activity and on solid medium containing M63 + glycerol + PGA, for pectate lyase activity. To estimate the cellulase secretion, after growth for 24 h at 30°C, plates were covered with a saturated solution of Congo red (0.2%) that binds to CMC. After washing with 1M NaCl, the colonies secreting cellulase were surrounded by a translucent yellow halo on a red background. To estimate the pectate lyase secretion, after growth for 24 h at 30°C, plates were covered with a saturated solution of copper acetate (10%).

The colonies secreting pectate lyases are surrounded by a translucent halo on a blue background.

The size of halo represents the secretion ability.

For immunoblotting assays, D. dadantii cells were grown at 28°C in LB supplemented with galacturonate for variable time periods (12-16 hours) until the early stationary phase. Cells were pelleted by centrifugation at 10,000 g for 2 minutes, the supernatant was collected and cells were resuspended in the same volume of LB. The culture supernatants and cell suspensions were boiled with Laemmli sample buffer. Then, the proteins were separated by SDS-PAGE and revealed with antibodies generated against diverse pectate lyases secreted by Out system, namely PelB, PelD and PelI. Ratio of pectinases in the culture supernatant reflects efficiency of the secretion and hence functionality of the corresponding mutant OutC and OutD proteins.

III.2 GST copurification and pull-down assays

The principle of copurification and pull-down is shown in Fig. 71. GST fused to OutC derivative of interest was used as a bait protein and an OutD derivative fused to His was used as a prey protein. The GST-fused bait protein can be then captured by Glutathione Sepharose resin.

If the two proteins interact with each other, the prey protein can also be co-purified.

III.2.1 GST copurification assay

For copurification assays, the bait and prey proteins were coexpressed in the same cell. The couples of GST-OutC and OutD His derivatives were coexpressed in E. coli BL21 (DE3) carrying one of the pGX-oC X-Y -oD X-Y plasmids (oC X-Y and oD X-Y for any OutC and OutD derivatives).

Bacteria were grown in 100 ml LB. At an OD600 of ~0.5, IPTG was added to 1 mM. After 3 h of additional growth, the cells were pelleted by centrifugation for 8 min at 5000 g and frozen at -80°C. To perform copurification, the cells were resuspended in 2.5 ml of buffer TSE (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM PMSF). After sonication, cell extracts were supplemented with Triton X-100 to 1% (v/v) and the final volume was adjusted to 3 ml.

Then, cell extracts were incubated for 20 min at 12 rpm to solubilize the proteins. Unbroken cells were eliminated at 6000 g for 10 min and 1.5 ml of such cell lysate were added to 0.3 ml of Glutathione Sepharose which had been washed with water and equilibrated with TSE buffer supplemented with 1% (v/v) Triton X-100. After 1 h of incubation with mixing at 12 rpm and at 15°C, the resin was spun for 2 min at 1000 g, and then washed three times with 1 ml of TSE buffer supplemented with Triton X-100 to 1% (v/v). At this stage, the bound proteins could be eluted with Laemmli sample buffer, separated by SDS-PAGE and probed by immunoblotting with either OutD antibodies, or Ni-NTA conjugated with peroxidase, or Tetra-His-antibodies (Quiagen). Alternatively, the GST-fused protein could be released from GST by PreScission protease. To do this, the washed resin was supplemented with TSE buffer containing Triton X-100 1% (v/v) and PreScission protease (~ 40 u/ml). After an overnight incubation with mixing at 8 rpm and at 15°C, the supernatant containing the proteins of interest released from GST were collected and used for further analysis.

III.2.2 GST pull-down assay

In contrast to copurification assay, in GST pull-down assay, the bait and prey proteins were expressed separately and the matrix consisting of GST-OutC derivative bound on Glutathione

Sepharose has been prepared and checked in advance. The beads with bound GST-OutC derivatives or with GST alone used a negative control have been systematically checked by SDS-PAGE prior to the binding assay and then diluted if necessary with non-charged resin to reach a similar amount of various fusion proteins. Then, an equal amount of prey protein of interest (OutD His derivative) was loaded onto the resin with immobilized GST fusions, incubated for 1 h and then washed three times with TSE. The bound proteins were eluted with Laemmli sample buffer, separated by SDS-PAGE and probed by immunoblotting with either OutD antibodies, or Ni-NTA conjugated with peroxidase, or Tetra-His-antibodies (Quiagen).

III.3 Limited proteolysis

Limited proteolysis was carried out either with purified OutC and OutD derivatives or with copurified OutC-OutD complexes by using trypsin. 3 μl of trypsin at 0.1 mg/ml were added to 150 μl samples and incubated at 25 °C for various time periods (2, 5, 15, 30 and 60 min).

Proteolysis was stopped by adding 3 μl of 100 mM PMSF. The samples were then analyzed by The principle of cysteine disulfide cross-linking is shown in Fig. 72. If the two introduced cysteine residues are in close proximity, the disulfide bond can be formed either spontaneously in an appropriate oxidative environment (e.g., the periplasm) or upon application of an oxidizing agent. The maximal distance found in proteins between α-carbons linked by disulfide bond is approximately 7 Å, with an average distance of 5-6 Å (Lynche and Koshland, 1991). This method allows therefore to estimate the proximity between the selected protein sites in the natural background (e.g., within the functional T2SS) and to identify sites involved in proteinprotein interactions.

III.4.1 In vivo disulfide cross-linking analysis

We firstly performed oxidative disulfide cross-linking by coexpressing OutC and OutD mutant proteins carrying cysteine substitutions in D. dadantii. However, due to a high background noise detectable by western blot, it was often difficult to exploit these results. Therefore, more frequently, we employed such an analysis in E. coli by coexpressing cysteine variants of OutC and OutD from the same plasmid. To enable a proper targeting and assembly of OutD into the OM, the pilotin OutS was also coexpressed with OutC and OutD variants from pACT3-oS plasmid. Indeed, we considered that coexpression of OutC and OutD in E. coli can mimic some OutC-OutD interaction within the functional T2SS. To assess the extent of disulfide cross-linking, spontaneous formation of disulfide bonds in steady-state cultures was examined. We considered that oxidizing environment of the periplasm is adequate to generate disulfide bonds between proximal residues during the bacterial growth. Indeed, induction of disulfide bonding by the addition of an external oxidant copper phenanthroline (CuP) increased the extent of cross-linking but significantly dropped its specificity.

More exactly, plasmids pTdB-oCoD that coexpress outC and outD coding for paired combinations of cysteine substitutions in OutC and OutD were transformed into E. coli NM522 cells bearing the pACT-oS plasmid. To test the effect of the secreted protein on disulfide crosslinking, pTPLB-oCoD plasmids coexpressing the pectate lyase PelB together with the paired combinations of cysteine substitutions in OutC and OutD were used instead of pTdB-oCoD that is lacking pelB. To assess the effect of the T2SS IM components OutL, OutM and OutE, the pK-ELM plasmid or the empty pBADIK vector were combined with pTdB-oCoD (or pTPLB-oCoD)

and pACT-oS plasmids in the same E. coli NM522 cells. Bacteria were grown aerobically at 120 rpm for 14-15 h at 30°C in LB supplemented if necessary with 1 mM IPTG, 0.3 mg ml -1 arabinose and appropriate antibiotics. Cells from 1 ml culture were pelleted at 10,000 g for l min and washed with TBS buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl). Then, to block free thiol groups and prevent further disulfide bond formation, the cells were incubated in the same volume of 20 mM 2-iodoacetamide (IA) in TBS for an additional 30 min at 25°C. The cells were then pelleted, resuspended in 200 μl Laemmli sample buffer without 2-mercaptoethanol and lysed in boiling water for 10 min. The samples were then additionally incubated for 15 min at 37°C with benzonase (Sigma-Aldrich), separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-OutC and anti-OutD antibodies.

III.4.2 Development of optimal conditions for in vitro disulfide cross-linking

The over-expression vectors pGX-oC 60-272 -oD 28-285 carrying various combinations of cysteine substitution in these OutC and OutD derivatives were constructed. The expression procedure was the same as in copurification assay described above. The cells were harvested by centrifugation and resuspended in 1.5 ml of TSE buffer supplemented with 1 mM PMSF. To search for optimal oxidation conditions favoring formation of the OutC-OutD complex, we firstly used different concentrations of the oxidative agent, copper phenanthroline (CuP). Once cells have been broken, the cell extracts were adjusted to 3 ml with TSE and then separated into 3 aliquots (A, B, C). In A and B, CuP was added at 0.25 mM and 1.25 mM, respectively, while C was used as a control.

After incubation for 30 min at 12 rpm at 15°C, unbroken cells were eliminated by centrifugation and the supernatants were loaded to 0.3 ml of Glutathione Sepharose which has been preliminarily equilibrated with TSE. The following procedures were the same as in an usual copurification. We found that the amounts of cross-linked products were increased in the presence of CuP but similarly at 0.25 mM and 1.25 mM. We subsequently probed the effect of 1.5 mM CuP as oxidizing agent on various steps of the purification process, this agent was either added: a) in the cell extract just after the cells were broken; or b) in the cell supernatants after elimination of unbroken cells by centrifugation; or c) in Glutathione Sepharose with bound GST fusion proteins; or d) in proteins released from the GST fusion by PreScission protease. The other procedures were the same as in an usual copurification. All the samples were analyzed by SDS-PAGE in non-reducing condition (without β-mercaptoethanol).

III.5 Cell fractionation

Subcellular fractionation was carried out on cells grown to exponential or early stationary phase (OD 600 1.0 to 1.4). Release of periplasmic proteins was performed by osmotic shock, and cell membrane fractionation was performed by sucrose gradient centrifugation, as described previously [START_REF] Shevchik | Characterization of pectin methylesterase B, an outer membrane lipoprotein of Erwinia chrysanthemi 3937[END_REF]. After breaking the cells in a French press and removing the debris at 5000 g for 10 min, crude membrane fractions were isolated by centrifugation (200 000 g for 2 h). The pellet and the supernatant correspond to the membrane fraction and the soluble fraction respectively. The membranes were then solubilized in 20 mM HEPES-NaOH pH 7.2 containing 65% (W/V) sucrose, loaded at the bottom of a centrifugation tube and covered by a step-gradient in the same buffer containing continuous layers of sucrose (60 to 35%). The vesicles corresponding to the inner and outer membrane were separated by centrifugation at 200 000 g for 48 h. Fractions were collected from the bottom of the gradient and the presence of OutD was revealed by immunodetection after SDS-PAGE. The position of the OM porins was determined by staining of the gel and the position of the IM fractions was determined by assaying of NADH oxidase activity (Osborn et al., 1972) and immunodetection of the IMP TolA.

III.6 SDS-PAGE and immunoblotting

III.6.1 SDS-PAGE

SDS-PAGE was usually performed according to Laemmli [START_REF] Laemmli | Cleavage of structural proteins during assembly of the head of bacteriophage T4[END_REF]. Protein samples were electrophoresed on 10%, 12% or 15% SDS-PAGE gels, depending on protein size. For analysis of disulfide cross-linking complexes, the samples were treated as indicated above (page 108); importantly, β-mercaptoethanol was absent in this case. After electrophoresis, proteins were either stained with Coomassie Brilliant Blue G-250 (CBB) or were analyzed by immunoblotting. For CBB staining, the gels were soaked in stain solution (0.5% CBB, 10% v/v acetic acid and 40% v/v methanol) for half an hour, followed by destaining in 10% acetic acid until the background was destained completely. Two types of SDS-PAGE were used in this thesis, namely Tris-Glycine-SDS-PAGE [START_REF] Laemmli | Cleavage of structural proteins during assembly of the head of bacteriophage T4[END_REF] and Tris-Tricine-SDS-PAGE (Schägger, 2006). Tris-Glycine-SDS-PAGE was preferentially used for separation of proteins larger than 30 kDa, and Tris-Tricine-SDS-PAGE for proteins smaller than 20 kDa.

III.6.2 Immunoblotting (Western blot)

After electrophoretic separation by SDS-PAGE, proteins were transferred onto a PVDF membrane in a semi-dry apparatus. The membrane was then saturated in TBS containing 4%

gelatine for 1 h at 30 °C and washed three times with TBS-T (TBS, 0.1% Tween 20). Then the membrane was incubated with the primary antibodies of interest diluted in TBS-T (1:6000 diluted anti-OutC, 1:3000 diluted anti-OutL, 1:4000 diluted anti-OutD, 1:6000 diluted anti-PelB, 1:10000 diluted anti-PelI, 1:4000 diluted anti-PelD and 1:6000 diluted anti-BlaM) for 1 h at room temperature. Non bound primary antibodies were removed by washing of the membrane with TBS-T (7 min, for 3 times). Secondary antibodies, anti-rabbit IgG, labeled with peroxidase and diluted 1/60000 in TBS-T, were then added. After incubation for 1 h at room temperature, the membrane was washed with TBS-T (7 min, for 3 times). Detection of peroxidase activity was performed using the ECL kit (GE Healthcare). The membrane was covered with substrate containing luminol. A light emitted as a result of oxidation of the substrate by peroxidase was captured by exposure of a film.

Results

Chapter I: Identification of the interaction regions between OutC and OutD

I.1 Preface

The outer membrane pore-forming secretin GspD and the inner membrane protein GspC are two key components of the type II secretion complex, involved in secretion specificity. GspC and GspD could interact to provide the structural and functional integrity of the secretion machinery across the two membranes of the Gram-negative bacteria [START_REF] Lindeberg | Complementation of deletion mutants in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species-specific secretion of proteins via the type II pathway[END_REF]Shevchik et al., 1997). However, there was no direct evidence which regions and sites of GspC and GspD are involved in the interaction. In this study, we examined the interaction between the secretin OutD and an inner membrane component OutC using both in vitro and in vivo approaches. We showed that a 20-residue peptide of OutC named OutCsip interacts with two distinct sites of OutD, one located in N0 domain and another overlapping N2-N3' domains and suggested an alternating mode of these interactions during the secretion process.

This study was mainly accomplished in our laboratory and complementary NMR experiments have been performed in the Lab. of Pr. R.W. Pickersgill (London University). The experimental results of Mr. Login (Ph.D thesis) showed that the N-terminal domain of OutD (residues 28-285) interacts with the non-PDZ periplasmic region of OutC (mainly corresponding to HR). In addition, he found that the N-terminal domain of OutD interacts with another region of OutC, the TMS of OutC or the region proximal to the TMS.

In this study, I participated in or completely performed the following parts of the work:

(1) The N-domain of OutD consists of four subdomains (N0 to N3). Thus, to identify the minimal regions of OutC and OutD involved in the interactions, I constructed certain truncated derivatives of OutC and OutD and corresponding coexpression vectors (an exhaustive list of the plasmids used in this study is present in the supplementary section, Table S1). Then, I used these derivatives in GST copurification assays to map the interaction regions within OutC and OutD (the section "A short region of OutC interacts with the two distinct sites within the N-domain of OutD" in the enclosed paper).

(2) I assessed the binding of OutCsip to two distinct sites of OutD and performed competition binding assays with multiple protein derivatives (the section "The two binding sites of the N-domain of OutD recognize different regions of OutCsip" in the enclosed paper).

(3) I assessed the functional relevance of the OutCsip region in a complementation test with E.

chrysanthemi ∆outC strain (the section "A single amino acid substitution in the OutCsip region blocks secretion in E. chrysanthemi" in the enclosed paper)

(4) I studied the effect of the V143S mutation on the OutC-OutD interaction in pull-down assays (the section "The substitution V143S prevents interaction of OutCsip with OutD 116- 285 " in the enclosed paper)

The details of these results are present as a part of the following article:

I.2 Article 1:

A 20-residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi.

Chapter II: Structure-functional analysis of the HR domain of

OutC

II.1 Preface

OutC (GspC) is one of the twelve core components of the T2SS. It is thought to be involved in both recognition of substrate and interaction with the outer-membrane secretin, OutD (GspD).

OutC is a bitopic inner membrane protein. It consists of a short N-terminal cytoplasmic sequence, a single trans-membrane segment followed by two periplasmic regions: a so-called homology region (HR) and a C-terminal PDZ domain [START_REF] Login | The single transmembrane segment drives self-assembly of OutC and the formation of a functional type II secretion system in Erwinia chrysanthemi[END_REF]. The PDZ domain is involved in secretion specificity but is not an essential module since it is absent or replaced by a coiled-coil domain in certain GspCs [START_REF] Bouley | The PDZ domain of OutC and the N-terminal region of OutD determine the secretion specificity of the type II out pathway of Erwinia chrysanthemi[END_REF]Peabody et al., 200). The crystal structure of the PDZ domain of GspC from V. cholerae and D. dadantii has been solved and suggested an unusual mechanism of substrate recognition [START_REF] Korotkov | Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae[END_REF]Fries et al., non-published). Neither the exact function of HR nor its structure are unknown; however it was shown to interact in vitro with the N-terminal region of the secretin GspD (see Chapters I and III). This chapter reports the solution structure of the HR domain of OutC and explores its interaction with the secretin. This structure was solved in collaboration with the team of Pr. R.W.Pickersgill from Queen Mary University of London. The HR domain adopts a β-sandwichlike fold consisting of two β-sheets each composed of three anti-parallel β-strands. This structure is strikingly similar to the periplasmic region of PilP, an inner membrane lipoprotein from the type IV pilus system highlighting the common evolutionary origin of these two systems. We elucidated an interaction between the HR domain of OutC and N0 domain of OutD (β1 HR -α2/β3 N0 ) in vitro and demonstrated the importance of the interactions involving β1 of the HR domain in vivo. The biological relevance of these structural predictions was assessed by site directed mutagenesis followed by functional assays in D. dadantii and in vivo disulfide-bonding analysis.

In this study, I performed following experimental work:

(1) I assessed the biological relevance of highly conserved residues, which constitutes a hydrophobic core of the HR domain, by in vivo site directed mutagenesis and cysteine mediated cross-linking analysis (the section "Several conserved residues form the hydrophobic core of HR" in the enclosed paper)

(2) I explored the functional importance of HR residues involved in the HR-N0 interface presumed by NMR experiments. To perform this analysis, I used alanine and cysteine site directed mutagenesis followed by functional assays in D. dadantii (the section "Assessment of HR residues involved in HR-N0 interaction in vivo" in the enclosed paper)

(3) I probed by in vivo disulfide cross-linking analysis the biological relevance of HR-N0 and N0-N1 interfaces predicted by structural studies (the section "In vivo assessment of HR-N0 and N0-N1 interactions" in the enclosed paper).

The details of these results are present as a part of the following article.

II.2 Article 2:

Solution structure of homology region (HR) domain of the type II secretion system.

Chapter III: Mapping of the critical interaction sites between

OutC and OutD by in vivo disulfide-bonding analysis

III.1 Preface

The results presented in Chapter I (Article 1) revealed that a short segment of the HR domain of OutC, consisting of strands β6 HR and β7 HR , interacts in vitro with two distinct sites of OutD, one located in the N0 domain and another overlapping the N2-N3' domains. Recent structural studies addressed this question by crystallographic and NMR analysis and presumed another mode of interaction (Chapter II (Article 2) and Korotkov et al., 2011). The solution and the crystal HR/N0 interfaces both involve strand β1 HR of HR but two different sites in N0, strands β3 N0 and β1 N0 , respectively. To assess the functional relevance of these presumed interaction sites, we exploited these recent structural data and used cysteine mutagenesis and in vivo disulfide-bonding analysis to map the interactions between the HR domain of OutC and the periplasmic region of OutD in their native environment, within the T2SS of D. dadantii. The results suggest the presence of at least three distinct sites of interactions between the periplasmic domains of OutC and OutD, namely, 1) β1 HR -β1 N0 , 2) β7 HR -β2 N0 and 3) β7 HR -β10 N2 . The secretion substrate diminishes certain interactions and provokes an important rearrangement of the HR structure. The inner membrane components OutE/L/M differently affect various interaction sites, reinforcing ones but diminishing the others, suggesting a possible switching mechanism of their interaction in the course of secretion. Disulfide mapping shows that organization of GspD and GspC protomers is incompatible with rotational symmetry and suggests a radial symmetry of their organization within the T2SS.

I participated in all the aspects of this study. Moreover, I performed the most part of experimental work presented in this paper.

The details of these results are present in the following article.

III.2 Article 3:

Cysteine scanning mutagenesis and disulfide mapping analysis of the arrangement of GspC and GspD protomers within the T2SS

III.3 Un-published results and discussion

Disulfide cross-linking analysis of the HR domain of OutC revealed an unexpected behavior of the cysteine substitutions of residues located at the second β-sheet. The corresponding cysteine variants of OutC generated self-bonding patterns incoherent with the structure of this domain. Namely, cysteine substitutions of the buried L145 (β6) and L154 (β7) provoked an efficient self-bonding whereas the contiguous, but solvent exposed, V144 and V153 did not (Wang et al., submitted). Such a behavior could reflect an "inverted" orientation of the corresponding β-strands (β6 and β7) and probably the entire second β-sheet consisting of three anti-parallel β-strands (β5 to β7). Alternatively, it could not be completely excluded that substitution of the hydrophobic residues (Leu and Val) by nucleophilic Cys severely affected hydrophobic core of the HR domain and provoked such an aberrant behavior. Several evidences suggest a biological relevance of this phenomenon. First, such "inverted" cross-linking patterns were reinforced within the functional T2SS of D. dadantii. Second, the secreted protein PelB and the inner membrane components OutE/L/M generated a rather opposite effect on such an "inversion", suggesting that it could be functionally reversible. Third, cysteine substitutions of the hydrophobic residues located on the first β-sheet, V100 (strand β1) and I113 (strand β2) did not provoke an aberrant cross-linking indicating that substitutions of these hydrophobic residues are well tolerated. Nevertheless, to better understand this phenomenon, we performed some III. N1B, compare lanes 2 with 3). These results are inconsistent with the HR structure where the side chains of V144 and V153 are solvent exposed and are not close to that of I113 (Fig. 1 in Wang et al., submitted). However, these data confirm the notion that, within the functional T2SS, the β6 and β7 strands of the HR domain could take an "inverted" orientation. Such intramolecular disulfide bonds were therefore formed between the "correctly" arranged buried side chain of I113C (β2) and those of V144C or V153C located at the "inverted" β-strands β6 and β7, respectively. In order to test a competition between "correct" and "inverted" side chains, involved in an intramolecular bonding with either V144C or V153C. We could provide a following explanation of these apparently puzzling results. In the triple I113C/V144C/V153C mutant, the solvent exposed side chains of V144C and V153C are close enough since located on neighboring β-strands (β6 and β7, respectively) and thus, efficiently form an intramolecular disulfide bond (V144C-V153C), which is compatible with the protein function. Consequently, the sulfhydryl of I113C becomes free and thus, could be partially engaged in an intermolecular bonding with the same group from the neighboring OutC protomer. Therefore, functionality of this triple mutant is improved in comparison with the corresponding double mutants. On the contrary, in the triple I113C/V143C/V153C mutant, I113C is fully engaged in a more proficient intramolecular bonding either with the buried V143C or with the solvent exposed V153C, which completely arrests the protein function. Moreover, these data show that multiple cysteine substitutions per se are well compatible with the functionality of OutC and thus, do not disturb folding of the HR domain.

Chapter IV: Assessment of the secretin-pilotin interaction in vivo

IV.1 Preface

Secretins form oligomeric ring-like structures in the OM of Gram-negative bacteria, which facilitate the translocation of proteins and other macromolecular complexes across the OM.

Besides the T2SS, the secretins are also present in the type III secretion and type IV pili systems and are involved in extrusion of the filamentous bacteriophages. Given the complexity and size of secretin assemblies, with molecular weights in the range between 0.5 and 1.0 MDa, it is not surprising that auxiliary proteins have been identified that are important for secretin targeting, oligomerization, insertion and proper assembly in the OM. A specialized class of small lipoproteins named pilotins plays these functions towards the cognate secretins. For example, in certain T2SSs, the pilotin GspS is necessary for the correct targeting and insertion of the secretin GspD into the OM. Although the pilotins from different export pathways are thought to perform roughly similar functions in the biogenesis of their cognate secretins, they appear to be unrelated in sequence and structure. Notably, the crystal structure of the pilotin MxiM from the T3SS of Shigella flexneri adopts a conical shape β-barrel structure interrupted by an α-helix [START_REF] Okon | Structural characterization of the type-III pilot-secretin complex from Shigella flexneri[END_REF], while the pilotins from the T4P of Pseudomonas aeruginosa and Neisseria meningitidis are composed of six tetratricopeptide repeats arranged as a superhelix [START_REF] Koo | PilF is an outer membrane lipoprotein required for multimerization and localization of the Pseudomonas aeruginosa Type IV pilus secretin[END_REF][START_REF] Trindade | Structure of a widely conserved type IV pilus biogenesis factor that affects the stability of secretin multimers[END_REF]. This indicates that the pilotins from various export pathways could have somewhat different modes of function. No pilotin structure from the T2SS has been available until July 2011. To unravel the molecular mechanisms of OutS (GspS) action and its interaction with the secretin, we undertook a structure-functional analysis of the pilotin OutS from the T2SS of D. dadantii. Once again, this study was performed in collaboration with the team of Pr. R.W.Pickrsgill from Queen Mary University of London. The crystal structure of OutS that comprises an arrangement of four α-helices is profoundly different from the pilotins from the T3SS and T4P. We showed that OutS binds tightly to 18 residues close to the C-terminus of the secretin OutD causing this unstructured region to become helical on forming the complex. To assess the pilotin/secretin interaction in vivo, we constructed truncated and/or mutated variants of these proteins and performed a series of in vivo experiments. They confirm an essential role of the ultimate C-terminal sequence of OutD. Furthermore, we showed that the region upstream of this binding site is not required for targeting of the secretin but is essential for its function.

In this study, I constructed several OutD derivatives and examined their functionality and targeting to the OM by flotation sucrose gradient centrifugation (the section "Assessment of the pilotin/secretin interaction in vivo" in this paper).

The details of these results are present as a part of the following article:

IV.2 Article 4:

Structural and functional insights into the pilotin-secretin complex of the type II secretion system.

Chapter V: Interaction between OutB and OutD V.1 Introduction

The T2SS is composed of at least 12 core components which could be subdivided into three groups, i.e., 1) the OM pore forming secretin GspD, 2) the IM membrane components GspC-L-M-F and the ATPase GspE, 3) the pseudopilins GspG, H, I, J, K and prepilin peptidase GspO.

Besides this conserved core of 12 proteins, in certain bacteria, some additional Gsp proteins are required for assembly of a functional T2S machinery. For example, an OM lipoprotein GspS named pilotin, is required for the correct targeting and insertion of the secretin into the OM in Erwinia, Klebsiella and some other bacteria; an IM protein with putative ATPase activity GspA, has been found in E. coli [START_REF] Francetic | The cryptic general secretory pathway (gsp) operon of Escherichia coli K-12 encodes functional proteins[END_REF][START_REF] Francetic | Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion[END_REF], Aeromonas species (ExeA) and Vibrio species (EpsA) [START_REF] Heidelberg | DNA sequence of both chromosomesof the cholera pathogen Vibrio cholerae[END_REF]; an IM protein GspB has been described in Klebsiella species (PulB) (d'Enfert and Pugsley, 1989), Erwinia species (OutB) [START_REF] Condemine | Some of the out genes involved in the secretion of pectate lyases in Erwinia chrysanthemi are regulated by kdgR[END_REF][START_REF] Lindeberg | Complementation of deletion mutants in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species-specific secretion of proteins via the type II pathway[END_REF], Aeromonas species (ExeB) [START_REF] Jahagirdar | Isolation and characterization of a second exe operon required for extracellular protein secretion in Aeromonas hydrophila[END_REF] and Vibrio species (EpsB) [START_REF] Heidelberg | DNA sequence of both chromosomesof the cholera pathogen Vibrio cholerae[END_REF]. In Klebsiella and Erwinia species, gspB gene is clustered with gspS, and an equivalent of gspA gene was not found in their chromosome. In Aeromonads and Vibrios, gspB forms an operon with gspA. Indeed, in A. hydrophila, GspA and GspB are found to form an IM complex that is required for an efficient secretion of aerolysin [START_REF] Schoenhofen | An ExeAB complex in the type II secretion pathway of Aeromonas hydrophila: effect of ATP-binding cassette mutations on complex formation and function[END_REF]. Further studies demonstrated that ExeAB complex is required for a correct localization and multimerization of the ExeD secretin in the OM [START_REF] Ast | Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer[END_REF][START_REF] Strozen | Involvement of the GspAB complex in assembly of the type II secretion system secretin of Aeromonas and Vibrio species[END_REF]. In the absence of ExeAB, ExeD remains in the IM as a monomer. ExeA contains a putative peptidoglycan-binding motif, the interaction between ExeA and peptidoglycan may cause the periplasmic ExeAB domains to form a large multimers, possibly a ring-like structure on peptidoglycan. Such ring could be necessary for transport and assembly of the secretin multimer in the OM [START_REF] Li | ExeA binds to peptidoglycan and forms a multimer for assembly of the type II secretion apparatus in Aeromonas hydrophila[END_REF]. In contrast to Aeromonads, where gspAB are absolutely required for the function of T2SS, in Vibrios, inactivation of gspA resulted in only minor reduction in secretion of T2SS substrates. However, the amount of secretin multimer was more significantly decreased in these mutants, indicating that although GspAB of Vibrios facilitate the assembly of the secretin, other yet unidentified factors may provide redundancy for secretin assembly [START_REF] Strozen | Involvement of the GspAB complex in assembly of the type II secretion system secretin of Aeromonas and Vibrio species[END_REF]. In many bacteria carrying a functional T2SS, no obvious GspAB homologues have been found and in some bacteria, such as Erwinia and Klebsiella, GspB but no GspA is present. In K. oxytoca, pulB mutation has no apparent effect on secretion (D'Enfert and Pugsley, 1989). In E. chrysanthemi, the absence of OutB results in the reduction of secretion by 30% [START_REF] Condemine | Some of the out genes involved in the secretion of pectate lyases in Erwinia chrysanthemi are regulated by kdgR[END_REF]. An overproduction of OutD was shown to suppress the phenotype of an outB mutant [START_REF] Condemine | Overproduction of the secretin OutD suppresses the secretion defect of an Erwinia chrysanthemi OutB mutant[END_REF]. Similar result was also found in A. hydrophila [START_REF] Ast | Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer[END_REF]. Overexpression of the secretin ExeD is sufficient to suppress the secretion defect of ExeAB mutant. In E. coli, cloning of the gspAB operon into the same plasmid with gspC-O caused a marked increase in GspG levels, which suggests that GspA and/or GspB might play a regulatory role [START_REF] Francetic | Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion[END_REF]. However, considering a significant sequence similarity between GspA/B of Aeromonas, Vibrio and E. coli, it seems unlikely that these proteins could play so different functions in these bacteria, i.e., peptidoglycan binding and secretin assembly in the first but regulation in the latter. Therefore, to date, the precise role of GspB in T2SS remains unclear.

OutB is an IMP with a deduced molecular mass of about 23.5 kDa, while with an apparent molecular mass of 29 kDa. This aberrant mobility could result from its amino acid composition (9% proline) [START_REF] Condemine | Overproduction of the secretin OutD suppresses the secretion defect of an Erwinia chrysanthemi OutB mutant[END_REF]. Topological analysis revealed that OutB have a bitopic topology with the N-terminus anchored in the IM and a large C-terminal periplasmic domain. The similarity between ExeB and energy-transducing protein TonB led [START_REF] Schoenhofen | An ExeAB complex in the type II secretion pathway of Aeromonas hydrophila: effect of ATP-binding cassette mutations on complex formation and function[END_REF] to suggest that ExeB could function with the putative ATPase ExeA to transduce metabolic energy to open the secretion pore. Interestingly, V. vulnificus encodes a 718 amino acids (aa) protein that contains a GspA domain within the N-terminal 530 aa of the protein and a GspB domain within the C-terminal 188 aa, which indicates that GspA and GspB really function together [START_REF] Strozen | Involvement of the GspAB complex in assembly of the type II secretion system secretin of Aeromonas and Vibrio species[END_REF]. The periplasmic domain is well conserved between ExeB and OutB, suggesting that both proteins could play similar functions within respective T2SS, although GspA is apparently lacking in E. chrysanthemi. outB and pulB are clustered with outS and pulS, respectively, which indicates that these two proteins may collaborate to pilot the cognate secretin to the OM. Indeed, OutS binds tightly to 18 residues close to the C-terminus of the secretin to target and insert the secretin into the OM (Gu et al., 2012a, accepted). In the absence of pilotin or S domain, the secretin miss-locates to the IM [START_REF] Guilvout | Bacterial outer membrane secretin PulD assembles and inserts into the inner membrane in the absence of its pilotin[END_REF]Gu et al., 2012a, accepted). Several experiments have suggested an interaction between OutB and OutD [START_REF] Condemine | Overproduction of the secretin OutD suppresses the secretion defect of an Erwinia chrysanthemi OutB mutant[END_REF]. In E. chrysanthemi, the presence of OutB stabilizes OutD. OutD expressed in E. coli can be partially protected from proteolytic degradation by the coexpression of OutB. OutB can be cross-linked with OutD by formaldehyde. However, there is no direct evidence on the interaction between GspB and GspD, and, the exact role of GspB in T2SS remains unclear.

In this chapter, we examined the interaction between the secretin OutD and OutB using in vitro co-purification. To elucidate the role of OutB, we studied the effect of OutB on the interactions between the HR domain of OutC and the two distinct sites of OutD, one located in N0 domain and another overlapping N2-N3' domains.

V.2 Results

V.2.1 Effect of OutB on the interactions of OutC with two distinct sites of

OutD

It seems reasonable to expect that certain protein/protein interactions between the T2SS components occur during the assembly of the system, while others take place in the functional machinery. These interactions may be either permanent or transient, and could induce conformational changes of proteins in the course of the secretion process. The fact that OutB and OutD stabilized each other indicates an interaction between OutB and OutD in the functional secretion machinery [START_REF] Condemine | Overproduction of the secretin OutD suppresses the secretion defect of an Erwinia chrysanthemi OutB mutant[END_REF]. To test whether the interaction between OutB and OutD affects the interaction patterns between OutD and OutC, we constructed the plasmid pACT3 expressing either outS or outS with outB under the control of Ptac. Then, pT7-6 plasmids coexpressing various combinations of cysteine variants of OutC and OutD (Table . V. 1) either coexpressed or not with PelB were introduced into E. coli NM522, which carry a pACT3 plasmid either with outS or with outS and outB. Therefore, we could test the effect of the secreted protein (PelB) and OutB on the extent of disulfide cross linking between various cysteine variants of OutC and OutD. Thus, we could estimate whether PelB and/or OutB interfere with the corresponding OutC-OutD interactions. Overnight cultures were harvested and treated as described in Materials and methods. The expression level of proteins was verified by immunoblotting and the cross-linked patterns with various gene combinations were assessed by non-reducing SDS-PAGE gel.

(1) Effect of OutB on the interaction of OutCsip and N0 of OutD OutCsip region consists of two β strands (β6 and β7) located on HR domain. An efficient disulfide cross-linking between V153C (β7 strand) of HR and T53C (β2 strand) of N0 domain of OutD suggests that the β7 of HR is proximal to the β2 of N0 during the secretion process.

However, no obvious cross-linked complex was detected between V144C (β6 strand) of HR and T53C (β2 strand) of N0, which indicates that these two positions are not juxtaposed in vivo. We showed that the interaction between these sites of OutC and OutD depend on the presence of other Out components (OutE/L/M) and the secreted protein (chapter III). We further tested whether the presence of OutB affects the extent of crosslinks between various cysteine variants Therefore, these experiments suggest that OutB differently affects the affinity of the two OutC-OutD interacting sites, slightly increasing the interaction between V153C (β7 of HR) and T53C (β2 of N0) but decreasing that between V144C (β6 of HR) and V271C (β12 of N3). This may result from the direct interaction of OutB with OutC or with OutD. Therefore, OutB could probably compete for the same interaction site(s) within OutC and OutD. Previous study suggested an interaction between OutB and OutD [START_REF] Condemine | Overproduction of the secretin OutD suppresses the secretion defect of an Erwinia chrysanthemi OutB mutant[END_REF]. To provide a direct evidence for an interaction between OutB and OutD, we performed an in vitro copurification.

V.2.2 Mapping of the interaction site between OutB and OutD

OutB protects OutD from proteolytic degradation when the two proteins are coexpressed in E.

coli [START_REF] Condemine | Overproduction of the secretin OutD suppresses the secretion defect of an Erwinia chrysanthemi OutB mutant[END_REF]. This effect does not require the N-terminal portion of OutB. In addition, family sequence alignment shows that the N-terminal region of GspB is dissimilar while the C-terminal portion is much more conserved (Fig. Identical residues are in red, strongly similar and weakly similar residues are in green and blue respectively.

The OutB region used in copurification assay is indicated by a green bar.

| OutB GKPGDDAAQGNEQEPTSKPEQTVRTTKK PulB ---G-----AIEESPQAE---------- ExeB ---------------------------- EpsB ----------------------------

V.3 Discussion and conclusions

In bacteria that contain both gspA and gspB, e.g., Aeromonads and Vibrios, gspA and gspB are clustered into the same operon, which indicates that GspAB acts in partnership. Indeed, in A.

hydrophila, ExeA and ExeB were shown to form a large heteromultimeric complex in the IM, which is required for a correct localization and multimerization of the ExeD secretin in the OM [START_REF] Ast | Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer[END_REF][START_REF] Strozen | Involvement of the GspAB complex in assembly of the type II secretion system secretin of Aeromonas and Vibrio species[END_REF]. Moreover, in V. vulnificus, gspA and gspB genes are fused into the single gene. In the absence of GspAB complex, the secretion of aerolysin and assembly of the secretin multimer in A. hydrophila were abrogated [START_REF] Ast | Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer[END_REF]. However, deletion of gspA and gspB in Vibrio species had a minimal effect on the secretion while it decreased assembly of the secretin multimer. Further cross-complementation experiments confirmed that GspAB perform the same role in Vibrios as does in Aeromonads, although they are differently required for assembly of the functional secretion machinery. It was suggested that the C-terminal peptidoglycan-binding motif of GspA could be directly involved in the assembly of secretin in the OM. The interaction between GspA and peptidoglycan may result in the reorganization of the peptidoglycan meshwork to allow monomeric GspD subunits or the secretin multimer itself to pass through to reach the OM, or may possibly provide a scaffold for assembly of the secretin [START_REF] Li | ExeA binds to peptidoglycan and forms a multimer for assembly of the type II secretion apparatus in Aeromonas hydrophila[END_REF][START_REF] Strozen | Involvement of the GspAB complex in assembly of the type II secretion system secretin of Aeromonas and Vibrio species[END_REF]. The role of GspB in assembly of the secretin is not clear. GspB may fulfil the part of the pilotin role that GspS plays in K. oxytoca and E. chrysanthemi, since no obvious GspA homologue is present and gspB is clustered with gspS in these bacteria. These indicate that two proteins may collaborate to pilot the secretins to the OM. The GspB is not required for the T2SS secretion by the Pul system of K. oxytoca reconstructed in E. coli.

However, it significantly improves secretion of E. chrysanthemi Out system. Thus, the role of GspB remains a puzzle.

Our in vitro and in vivo results have shown that a short peptide of OutC (OutCsip,(139)(140)(141)(142)(143)(144)(145)(146)(147)(148)(149)(150)(151)(152)(153)(154)(155)(156)(157)(158) interacts with two distinct sites of OutD, one located in N0 subdomain and another overlapping N2-N3' subdomain (chapter I). Further cysteine scanning and disulfide cross-linking analysis confirmed these interactions in vivo and showed that the secreted protein and the other T2SS components (OutE/L/M) differently affect these interactions (Chapter III). These data suggest that various OutC-OutD interactions could correspond to various functional states of the secretion machinery. We proposed that during secretion process, certain T2SS components and/or substrates could affect differently the interactions between OutC and various sites of To examine whether HR of OutC and OutB bind to different sites of N0 domain of OutD and to test if the interactions between them are cooperative or competitive, the binding affinities of these three proteins should be assessed by using copurification competition assays. In this type of experiments, the binding of various OutD derivatives on GST-HR or GST-OutB matrixes could be affected (either increased or diminished by the presence of the third partner, either OutB or HR of OutC, respectively). It should also be noted that the effect of OutB on the β1 HR -β1 N0 interaction site was not examined in this work. This interface has been suggested by a recent structural study (Korotokov et al., 2011) and was recently assessed by cysteine bonding analysis (chapter III). It is therefore necessary to test the effect of OutB on this OutC-OutD interaction site. We showed that OutB diminished the interaction between HR and N2-N3' domains of OutD.

Although no data indicates the interaction between OutC and OutB, we cannot exclude the possibility that OutB and N2-N3' of OutD compete for binding to HR of OutC. To examine this, a direct interaction between OutB and OutC should be assessed by using in vitro copurification.

It should be also interesting to construct protein derivatives suitable for NMR or X-ray crystallography to determine the interface between the C-terminal of OutB and N0 domain of OutD.

Chapter VI: Construction of OutC and OutD protein derivatives suitable for structural analysis

VI.1 Introduction

The previous studies and this work demonstrated that the periplasmic regions of OutC and

OutD interact together. To understand the molecular mechanisms of these interactions, we undertook structural studies of these proteins, notably, in collaboration with the team of Prof. R.

Pickersgill from Queen Mary University. Previously, in collaboration with this team, the crystal structures of the pectate lyase PelL, pectin methylesterase PemA and the PDZ domain of OutC have been solved (Jenkins et al., 2001;2004). Unfortunately, a similar crystal structure of the PDZ domain of OutC homologue, EpsC from V. cholerae had been published just before finishing our work [START_REF] Korotkov | Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae[END_REF], which compromised the publication of our PDZ structure. When I started this thesis work, neither the atomic resolution structures of the periplasmic region of GspD and HR domain of OutC alone nor in complex were available.

Since such data are essential to understand molecular organization and assembly of GspC and GspD within the T2SS, we also undertook to prepare OutC and OutD derivatives suitable for the X-ray crystallography and Nuclear Magnetic Resonance (NMR) analysis. Since during this thesis work, the crystal structures of certain periplasmic domain of GspD and GspC have been solved in parallel by another research team, it became necessary to readapt our strategy of the structural analysis of OutC and OutD. Therefore, some experimental approaches used to generate protein derivatives suitable for further structural analysis have been initiated and developed but stopped because they became obsolete.

X-ray crystallography and NMR are widely used techniques to determine structures of proteins. X-ray crystallography makes use of the diffraction pattern of X-rays which are shot through an object. The pattern is determined by the electron density within the crystal. The diffraction is the result of an interaction with the high energy X-rays and the electrons in the atom. X-ray crystallography requires the growth of protein crystals up to 1 mm in size from a highly purified protein source. Since crystal growth is an experimental technique and there exists no rules about the optimal conditions for a protein solution to result in a good protein crystal, preparing protein crystals suitable for structural analysis is currently the bottleneck in structure determination by this method. A pure (>97%) and homogeneous protein sample is crucial to successful crystallization attempt.

NMR is a technique to study biological macromolecules in solution and does not require the protein crystal. It is based on the fact that nuclei of atoms have magnetic properties that can be utilized to yield chemical information. In order to analyze the nuclear magnetic resonance data, it is important to find out the correspondence between chemical shifts and atoms. To avoid problems with overlapping peaks generated by the hydrogen atoms using homonuclear magnetic resonance, NMR heteronuclear (hydrogen/nitrogen or hydrogen/nitrogen/carbon) is most commonly used. The HSQC (heteronuclear single quantum correlation) is a heteronuclear NMR with isotopic labeling to N 15 and HMQC (heteronuclear multiple quantum correlation) is NMR heteronuclear labeling with 15 N and C 13 isotope.

VI.2 Results

Since OutC and OutD are membrane proteins, they need the addition of detergents to be soluble in aqueous solution. In this case, membrane proteins form mixed micelles with detergents. Stoichiometry and properties of membrane proteins in such mixed micelles depend on the protein and detergent nature. The presence of detergents often interferes with regular arrangements of the protein complexes in the crystal resulting in diffuse diffraction pattern. An alternative approach consists in using of only soluble regions of membrane proteins. Several soluble derivatives of OutC and OutD have been tested in previous study for crystallization trails, but failed to yield crystals (Login, Ph.D. thesis). A possible reason is that the presence of many flexible regions impairs crystallization. To bypass this problem, we have applied two strategies, i.e., limited proteolysis of large protein derivatives to find more stable domains and formation of covalently linked complexes, and at the final step, combination of these two approaches.

VI.2.1. Identification of stable OutC and OutD derivatives by limited proteolysis

Copurification assays showed that the periplasmic domain of OutD (OutD 28-285 ) is efficiently copurified with the periplasmic domain of OutC (OutC 60-272 ) . This suggests formation of a complex between these two proteins. However, affinity of such a complex is too low, since the two proteins were then eluted as two separate peaks in gel filtration (Login et al., 2010, supplementary data). The interaction interface could be buried and therefore partially protected from proteolysis. To examine this hypothesis and to generate the derivatives of OutC and OutD suitable for a structural analysis, we employed limited proteolysis. The secondary structure of HR is deduced from the recently solved 3D structure of OutC (Gu et al., 2012b, reviewed) while that of PDZ is deduced from the 3D structure of OutC homologue, EpsC of V. cholerae [START_REF] Korotkov | Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae[END_REF]. The secondary structure of peri-OutD is deduced from the 3D structure of peri-GspD (Korotkov et al., 2009a).

SRGVNEEVPGYNAKIVSIRPDRVVLQYQGRYEVLGLYSQEDSGSDGVPGAQVNEQLQQRA C2-1 SRGVNEEVPGYNAKIVSIRPDR-------------------------------------- C2-2 ----------------------VVLQYQGRYEVLGLYSQEDSGSDGVPGAQVNEQLQQRA C2-3 ------------------------------------------------------------ 190 200 210 220 230 240 | | | | | | α2 βA βB αA βC βD αB OutC 60-272 STTMSDYVSFSPIMNDNKLQGYRLNPGPKSDSFYRVGLQDNDMAVALNGLDLRDAEQAKK C2-1 ------------------------------------------------------------ C2-2 STTMSDYVSFSPIMNDNKLQGYR------------------------------------- C2-3 -----------------------------------VGLQDNDMAVALNGLDLRDAEQAKK 250 260 270 | | | βE βF OutC 60-272 AMERMADVHNFTLTVERDGQRQDIYMEFGGDE C2-1 -------------------------------- C2-2 -------------------------------- C2-
YGFSVVPMDNGVLKVIRSKDAKSSSIPLANNEQPGIGDELVTRVVPLNNVAARDLAPLLR D1-1 YGFSVVPMDNGVLKVIRSKDAKSSSIPLANNEQPGIGDELVTRVVPLNNVAARDLAPLLR D1-2 ----------------------SSSIPLANNEQPGIGDELVTRVVPLNNVAARDLAPLLR D2 YGFSVVPMDNGVLKVIRSKDAKSSSIPLANNEQPGIGDELVTRVVPLNNVAAR------- D3-1 ------------------------------------------------------------ D3-2 ------------------------------------------------------------ 150 160 170 180 190 200 | | | | | | β7 β8 α5 β9 α6 OutD 28-285 QLNDNAGAGTVVHYEPSNVLLMTGRAAVIKRLVDIVNTVDKTGDREMVTVPLTYASAEDV D1-1 QLNDNAGAGTVVHYEPSNVLLMTGRAAVIKRLVDIVNTVDKTGDREMVTVPLTYASAEDV D1-2 QLNDNAGAGTVVHYEPSNVLLMTGRAAVIKRLVDIVNTVDKTGDREMVTVPLTYASAEDV D2 ------------------------------------------------------------ D3-1 -------------------------------LVDIVNTVDKTGDREMVTVPLTYASAEDV D3-2 ------------------------------------------------------------ 210 220 230 240 250 260 | | | | | | β10 β11 α7 OutD 28-285 AKLVNDLNKSDEKNALPSTMLANVVADGRTNSVVVSGEENARQRAVEMIRQLDRKQVVQG D1-1 AKLVNDLNKSDEKN---------------------------------------------- D1-2 AKLVNDLNKSDEKNALPSTMLANVVADGRTNSVVVSGEENARQRAVEMIRQLDRKQVVQG D2 ------------------------------------------------------------ D3-1 AKLVNDLNKSDEKN---------------------------------------------- D3-2 -------------------------------------------------------QVVQG 270 280 | | β12 OutD 28-285 GTKVIYLKYAKALDLIEVDKLAAALEHHHHHH D1-1 -------------------------------- D1-2 GTKVIYLKYAKALDLIEVDKLAAALEHHHHHH D2 -------------------------------- D3-1 -------------------------------- D3-2 GTKVIYLKYAKALDLIEVDKLAAALEHHHHHH N0 domain N1 domain N1 domain N2 domain N3 domain

VI.2.2 Construction of OutC-OutD derivatives suitable for disulfide crosslinking

In another approach, we introduced the site-specific cysteine substitutions into the presumed interaction sites of OutC and OutD to obtain an OutC-OutD complex stabilized by an intermolecular disulfide bond. We suggested that OutC and OutD derivatives bound together via an intermolecular disulfide bond could be more stable and thus suitable for crystallography or NMR studies. Positions for such cysteine substitutions were suggested by our previous functional studies and in vivo disulfide cross-linking analysis (Chapter III).

(

1) Construction of appropriate over-expression vectors

Functional analysis showed that V143S substitution in OutC blocks secretion in D. dadantii.

Moreover, our in vitro copurification showed that V143S mutation prevents the interaction of OutCsip peptide with the N1-N3' region of OutD [START_REF] Login | A 20-residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi[END_REF]. This suggests that V143 of OutC could be directly involved in interaction with OutD. By a search for extragenic suppressor mutations, we found two point mutations in OutD, i.e., A174V and G190D, which partially restore secretion in D. dadantii outCV143S mutant (not published). The allele-specific suppression could indicate a direct interaction of these residues. In addition, sequence alignment showed that G190 is highly conserved residue. Thus, cysteine residues were firstly introduced by site-directed mutagenesis in place of V143 of OutC and G190 of OutD in the expression vector pGX-oC 60-272 -oD 28-285 .

( We subsequently examined whether application of the oxidative agent on the other steps of copurification process is more efficient and generates a higher amount of disulfide complexes. In parallel, our collaborators identified a stable folded region corresponding to residues 77-172 of OutC, which gave more clear NMR signals and was used for further structural analysis. We therefore stopped further construction and analysis of the OutC and OutD derivatives by limited proteolysis. Recently, the 3D structure of HR domain of OutC (aa 77-172) and the structure of HR domain of OutC in complex with N0 subdomain of OutD (aa 28-112) have been solved by NMR (Chapter II). We largely exploited these structural data together with the structure of Nterminal domain of GspD in ETEC (Korotkov et al., 2009a) to perform cysteine scanning mutagenesis and in vivo disulfide bonding analysis in order to map the OutC-OutD interaction sites.

In another approach, we tried to generate stable derivatives of OutC and OutD cross-linked together by an intermolecular disulfide bond. Positions suitable for such disulfide cross-linking were indicated by in vivo disulfide bonding analysis that has been performed in parallel (Chapter III). However, such a comprehensive in vivo analysis took a long time and we have started the in vitro cross-linking approach before the completed results of the corresponding in vivo analysis was available. Indeed, many technical approaches have been used with OutCV143C and OutDG190C variants in order to purify these derivatives bound by an intermolecular disulfide.

However, subsequent in vivo assays (chapter III) showed that the corresponding disulfide bonding is poorly relevant in vivo. Therefore, biological significance of such a complex would be rather low. Nevertheless, multiple practical approaches employed in the course of this study could be useful in the future to generate and purify cross-linked complexes of some other protein derivatives.

Using of another couple of cysteine variants of OutC and OutD, V153C and T53C, respectively, was not successful either. In this case, homodimer of the corresponding OutD derivative was the main cross-linking product while OutC-OutD heterodimer was faintly detectable, which compromised its further purification. A probable explanation is that an appropriate conformation and/or mutual arrangement of OutC and OutD are necessary for V153C and T53C becoming proximal enough to form a disulfide bond. Such conditions could occur within the functional T2SS but are not fulfilled when the isolated periplasmic regions of OutC and OutD are assembled in vitro. In any case, in the used in vitro conditions, T53C is mainly involved in the OutD-OutD interface and is not accessible for an interaction with V153 of OutC. These results even if negative are rather well compatible with the in vivo disulfidebonding analysis (Chapter III). Indeed, the latter suggested that the OutC-OutD interactions are not formed through static interfaces, since the same sites of OutC and OutD are often involved in self-interactions with the equivalent sites of neighboring protomers (i.e., homodimerization of OutDT53C) and in an interaction with another protein partner (i.e., OutCV153C-OutDT53C complex). The in vivo disulfide-bonding analysis also showed that extent of OutC-OutD crosslinking was rather low in comparison with that of self-bonding of the corresponding OutC and OutD variants. This suggests that self-interactions of OutC and OutD protomers are more prevalent and could constitute initial or "basal" interactions within the secretion machine while the OutC-OutD contacts are transient and loose. Thus, the interactions representative of the "stand by" state of the system would generate more efficient cross-linking than the transient but functionally important contacts.

Recently, the interfaces between HR of GspC/OutC and N0 of GspD/OutD have been revealed

both by NMR and X-ray crystallography (Chapter II and Gu et al., 2012b;Korotkov et al., 2011a). In these studies, isolated domains of these two proteins have been used. In the latter study, the structure of the complex has been stabilized by using specific nanobodies. Our recent in vivo disulfide bonding analysis suggests that the corresponding interface is functionally relevant. It should be interesting to generate corresponding derivatives of OutC and OutD carrying appropriate cysteine substitutions and probe the efficiency of their disulfide crosslinking in vitro and try to purify such a complex. This would allow to test a general feasibility of this approach.

General conclusions and perspectives

The enterobacterium Dickeya dadantii (ex. Erwinia chrysanthemi) causes soft-rot disease of various plants. The tissue maceration results from the degradation of the pectin of plant cell walls by pectinolytic enzymes. These pectinases are secreted by the type II secretion system, named

Out. The Out system consists of 14 proteins, OutB to OutM and OutS. This thesis focuses on the functional and structural characterization of the two core components of the system OutC and OutD, and molecular analysis of the interaction between them.

The first axis of this thesis is to understand structure-function relationships between OutC and OutD and characterize their interaction sites.

In the first part (Chapter I), we used truncation analysis of OutC and OutD to map the minimal regions involved in OutC-OutD interaction. This work employed pull-down assays and mutagenesis analysis, and followed the previous study realized in the laboratory (Mr. Login, Ph.D. thesis). Together with the previous results, we showed that a 20-residue peptide named This suggests that N0/HR interface presumed by the NMR experiments is not biologically relevant. Alternatively, an optimal arrangement of residues or the distance adequate for disulfide formation was not achieved. Single cysteine substitution of G99 on β1 of HR, and I64 and S65 on β3 of N0 provoked homodimerization of the mutant OutC and OutD, respectively, which suggests that in the functional T2SS, neighboring HR domains of OutC and neighboring N0 domains of OutD could interact via the β1 strand and β3 strand, respectively. We further assessed by disulfide bonding analysis the mutual arrangement of N0 and N1 domains of OutD within the functional T2SS. We showed that the crystal interface suggested by the recent structural study (Korotkov et al., 2009) consisting of β3 of N0 and β6 of N1 is functionally relevant.

Recent crystal structure of GspC HR -GspD N0-N1 complex (Korotkov et al., 2011b) revealed that the corresponding interface includes an antiparallel arrangement of strand β1 of GspC HR and strand β1 of GspD N0 . To assess its biological relevance in the functional secretion system and to probe the other putative HR/OutD interfaces suggested by the previous in vitro study [START_REF] Login | A 20-residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi[END_REF], we performed in vivo cysteine scanning and disulfide cross-linking analysis (Chapter III). At least three distinct GspC-GspD interactions were detected, namely 1) β1 HR -β1 N0 , 2) β7 HR -β2 N0 , 3) β7 HR -β10 N2 . None of these interactions is formed through static interfaces since the same sites are also involved in self-interactions with equivalent neighboring domains. Disulfide self-bonding of critical interaction sites abolishes the secretion implying a transient nature of these interactions. The secretion substrate diminishes certain interactions and provokes an important rearrangement of the HR GspC structure. The inner membrane components OutE/L/M differently affect various interaction sites, reinforcing ones but diminishing the others, suggesting a possible switching mechanism of their interaction in the course of secretion.

Disulfide mapping shows that organization of GspD and GspC protomers is incompatible with rotational symmetry and suggests a radial symmetry of their organization within the T2SS.

The second axis of this thesis is to study the mechanism of the targeting and assembly of the secretin OutD in the outer membrane. Two auxiliary proteins, i.e., OutB and OutS are involved in this process. Firstly, we undertook structure-function analysis of OutS and OutS-OutD complex (Chapter IV). This study revealed that the OutS structure is very different from that of the T3SS (MxiM) or T4P (PilW). The OutS structure comprises four α-helices which is the remarkable insertion of one α-helical hairpin into a second open α-helical hairpin with bent final helix. Both in vitro and in vivo results showed that the pilotin OutS binds tightly to 18 residues close to the C-terminus of the secretin causing this unstructured region to become helical on binding to the pilotin. Furthermore, our in vivo results showed that the pilotin OutS can also interact with the region upstream of this binding site, which is not required for targeting, but is required for the secretin function in the T2SS. These results are coherent with the data from the pilotin PulS of K. oxytoca that were recently published [START_REF] Nickerson | Outer membrane targeting of secretin PulD relies on disordered domain recognition by a dedicated chaperone[END_REF][START_REF] Nickerson | Outer membrane targeting of secretin PulD relies on disordered domain recognition by a dedicated chaperone[END_REF].

The role of the second auxiliary protein OutB in the targeting and assembly of the secretin remains less evident. Our study (Chapter V), notably in vitro copurification assay showed a direct interaction between the periplasmic domain of OutB and the N0 domain of OutD.

Moreover, the presence of OutB differently alters the affinity of the HR domain of OutC towards two sites of OutD, N0 and N2-N3. These data presume a complex interplay between various domains of OutD, OutC and OutB.

Thus, our results suggest the existence of two distinct interfaces between OutD N0 /OutC HR , one involving the strand β1 of N0 and the strand β1 of HR and another including the strand β2 of N0 and the strands β6 and β7 of HR. We showed that the latter site of HR is also implicated in the interaction with the N2-N3' domains of OutD. In addition, the two mentioned sites of HR as well as several sites within the periplasmic domains of OutD are also involved in the self-interactions with an equivalent neighboring domain (in Fig. 73 panels A and B, these self-interactions are indicated by the dots of same color). These data together with the other recent research findings allow us to propose an improved model of the type II secretion system (Fig. 73C). OutC could be organized in dodecamer with C6 symmetry and neighboring protomers can interact via β1 HR -β1 HR interface (indicated by pink dots, Fig. 73A). The OutD dodecamer could also be organized with a C6 symmetry (via β2 N0 -β2 N0 , β10 N2 -β10 N2 and β11 N2 -β11 N2 ) (indicated by orange dots, Fig. 73B). The dodecamer of OutC surrounds the periplasmic portion of the OutD dodecamer (Fig. 73B) through twelve OutC/OutD heterodimeric interactions via the first β sheet of HR and β1 of OutD in standby state. OutC and OutD could be important for secreted proteins recognition and capture them within the large periplasmic vestibule of OutD. The presence of the secreted proteins could provoke an "inverted" orientation of the second β sheet of HR and reinforce homodimerization of OutC through this "inverted" interface (indicated by orange dots, Fig. 73A).

On the other hand, the secreted proteins could diminish the self-interactions of neighboring N0 domains of OutD via β2 N0 -β2 N0 but increase those through β1 N0 -β1 N0 (indicated by orange and mangeta dots, respectively, Fig. 73B). A decreasing of β1 HR -β1 HR self-contact HR and N0 (via β7 HR -β2 N0 ) would be replaced by the interaction of HR with the other interaction site located in N2-N3' (β7 HR -β10 N2 ) (Fig. 73C, right panel). This interaction could trigger a conformation change in the secretin that causes opening of the periplasmic gate. Upon opening of the secretin pore, the extending pseudopilus may push the exoprotein through the opened gate and serve as a plug to prevent channel leakage. How the GspD periplasmic gate closes, and the pseudopilus retracts and/or disassembles to reset the system remain to be elucidated.

Understanding of this mechanism requires elucidating of the functions and interactions of other components of the secretion system.

To give a perspective of this work, the following studies should be performed. These studies will allow to a better understand of the assembly and mechanism of the T2SS.

i)

The exact function of OutB should be elucidated. Our results (Chapter V) showed that the periplasmic domain of OutB (OutB 112-220 ) interacts with N0 domain of OutD.

Cysteine cross-linking analysis indicates that the two distinct sites of N0, i.e., located at strands β1 (F34C) and β2 (T53C), respectively are involved in interaction with strands β1 (G99C) and β7 (153C) of HR, respectively (Chapter III). We have showed that the presence of OutB increases the formation of cross-linked complexes between OutC V153C (strand β7 of HR) and OutD T53C (strand β2 of N0), indicating a certain cooperation between OutB/N0 and the latter HR/N0 interaction. The effect of OutB on another HR/N0 interacting site including of β1 of HR and β1 of N0 should be examined and the affinity of HR of OutC and OutB to N0 of OutD should be compared using copurification (pull-down) assay. We also showed that OutB decreases the interaction between V144C (β6) of HR and V271C (β12) of N3.

Although no data indicates the interaction between OutC and OutB, we cannot exclude the possibility that OutB and N3 of OutD compete for binding to the same site of HR domain of OutC. To probe this idea, a direct interaction between OutB and

OutC should be examined. The structure determination of the periplasmic domain of OutB (either by NMR or X-ray crystallography) is in progress in collaboration with the team of Prof. Pickersgill from Queen Mary University of London. This work will allow to confirm the interaction between the periplasmic domain of OutB and N0 domain of OutD and reveal the molecular mechanism of this interaction.

ii)

The precise interaction sites between secreted protein and OutC and OutD, respectively, should be determined. Our results showed that the presence of secreted protein differently alters homodimerization via various sites of the periplasmic regions of OutC and OutD and affects the interaction between OutC and OutD. To explain the latter phenomenon, we firstly suggested that the interaction between a secreted protein and the PDZ domain of OutC changes the conformation of HR that weakens the affinity of OutC to OutD. However, a secreted protein CelZ, whose secretion is PDZ independent, gives the same effect on the interactions between OutC and OutD as PelB. Thus, the secreted protein may probably interact with some other region(s) of OutC or/and OutD. To map the precise interaction regions, multiple complementary approaches could be employed: a) NMR experiments to study in vitro interactions between various domains of OutC and OutD and the secreted protein.

Most of these protein derivatives suitable for such type of study have been constructed in the course of this thesis; b) experiments employing bacterial twohybrid assay to study equivalent interactions between various OutC, OutD domains and secreted proteins in vivo; c) another approach using site directed in vivo photocross linking. Indeed, the two latter approaches are currently exploited in the laboratory.

iii)

The interaction between OutD and the IM components OutL and OutM should be examined. Our results showed that the presence of OutE-L-M differently alters the cross-linking patterns of various OutC-OutD interacting sites. These effects may be due to the direct interaction between OutC and OutL-M, Indeed, we showed a strong effect of OutE-L-M on OutC homodimerization, indicating an interaction between them. However, we could not exclude that these effect may also due to the direct interactions between OutD and OutL-M. Indeed, the thesis of Mr. Login showed that the periplasmic domain of OutM interacts with the periplasmic domain of OutD (Login, Ph.D. thesis).

iv)

The precise stoichiometry of Out proteins should be determined. The actual representation of the T2SS suggests that the secretin GspD could form a dodecameric ring in the OM. The secretion ATPase GspE is suggested to be a hexamer and associates with the T2SS through forming a 1:1 complex with the cytoplasmic domain of GspL, which indicates a hexameric arrangement of GspL. The relative in vivo stoichiometry between pseudopilins has been estimated to be 16 (GspG):1(GspH):1(GspI):4(GspJ). The stoichiometry of the other T2SS components remains unknown. Although we proposed that OutC and OutD could form a 1:1 complex in the functional T2SS, we cannot exclude the other possibility, e.g., 1:2 or 2:1 ratio between OutC and OutD. To determine the stoichiometry of OutC and OutD, we could use immunodetection to estimate the relative amount of these proteins within the functional T2SS. The purified OutC and OutD proteins could be used as control to determine the concentration of these proteins in D. dadantii cells.

Fluorescence fluctuation spectroscopy could be also used to quantify the stoichiometry of protein complexes in living cells via analysis of brightness. The brightness provides a spectroscopic marker for observing protein interactions and their stoichiometry directly inside cells.

Fig. 1 :

 1 Disease symptoms caused by D. dadantii. Fig. 2: The plant cell wall and the structure of pectin. Fig. 3: Pectin catabolism in D. dadantii. Fig. 4: Schematic representation of the Gram-negative bacterial cell envelope. Fig. 5: Schematic overview of the E. coli Sec-and Tat translocases. Fig. 6: Schematic comparison of typical N-terminal signal sequences from substrates of the Tat and Sec systems. Fig. 7: The structure of SecB homo-tetramer and the peptide-binding groove within the SecB molecule. Fig. 8: Crystal structure of SecA dimer with an antiparallel orientation of the M. tuberculosis. Fig. 9: Models proposed for the dimer of the SecYEG. Fig. 10: Structures of T. thermophilus SecDF. Fig. 11: The model for the different steps of Sec-mediated protein translocation. Fig. 12: The machineries involved in inner membrane proteins insertion. Fig. 13: The predicted topology of the E. coli Tat components. Fig. 14: Model for the membrane passage of Tat substrates. Fig. 15: The periplasmic and outer membrane folding factors. Fig. 16: Model for lipoprotein transport through the bacterial cell envelope. Fig. 17: Models proposed for outer membrane proteins (OMPs) biogenesis in E. coli. Fig. 18: Model of the T1SS. Fig. 19: Mechanism of a typical ABC transporter. Fig. 20: Structural overview of the TolC periplasmic entrance. Fig. 21: Schematic diagram of the T3SS of Yersinia spp. Fig. 22: Three-dimension reconstruction of the S. flexneri and S. typhimurium needle complex (NC). Fig. 23: Schematic diagram illustrating needle and translocon formation, as well as toxin secretion steps in the T3SS of P. aeruginosa. Fig. 24: Model of assembly of the Yersinia injectisome. Fig. 25: Secretion pathway and structure of the T4SS. Fig. 26: Cryo-EM structure of the TraN/VirB7, TraO/VirB9, and TraF/ VirB10C-ST core complex. Fig. 27: The T4S system outer-membrane complex. Fig. 28: Crystal structure of T4SS ATPases VirD4 and VirB11. Fig. 29: Proteins composing the two-partner system (TPS) and autotransporter (AT). Fig. 30: Crystal structures of C-terminal fragments of the classical autotransporter NalP and the trimeric autotransporter Hia. Fig. 31: The crystal structure of different autotransporter (AT) passenger domain. Fig. 32: Models for the autotransport mechanism. Fig. 33: Crystal structures of TPS domain of filamentous hemagglutinin FHA and FhaC. Fig. 34: Proposed model of FHA transport across the outer membrane. Fig. 35: Structure and function of the T6SS. Fig. 36: Potential similarities in the injection mechanisms of tailed bacteriophages and T6SS. Fig. 37: Genetic organization of the T2SS. Fig. 38: Model of structural organization of the T2SS. Fig. 39: Schematic representation of D. dadantii T2SS secretin OutD and crystal structure of N0-N1-N2 domains of ETEC T2SS secretin GspD. Fig. 40: Comparison of Peri-GspD N0 subdomain with TonB-dependent OM receptor FpvA from P. aeruginosa. Fig. 41: 3D reconstruction of the K. oxytoca secretin PulD. Fig. 42: Modeled cylindrical arrangements of periplasmic GspD domains. Fig. 43: Cryo-EM reconstruction of V. cholerae T2SS secretin GspD. Fig. 44: Fitting models. Fig. 45: Sequence alignment of the N-terminal domains of the PilA pilin subunit of type IV pili and pseudopilins of the Xcp-type II secretory pathway from P. aeruginosa.

Fig. 46 :

 46 Fig. 46: Comparison of major pseudopilins with type IV pilins. Fig. 47: The crystal structures of pseudopilins. Fig. 48: The crystal structure of pseudopilin complexes. Fig. 49: The pseudopilus assembly model. Fig. 50: GspE domain organization and crystal structures. Fig. 51: Model depicting sequence of events in T2SS initiated by ATP binding to GspE. Fig. 52: The dimer of N-terminal cytoplasmic domain of EpsF (cyto1-EpsF56-171) from V. cholerae. Fig. 53: Schematic representation of GspL and GspM from D. dadantii. Fig. 54: Crystal structure of cyto-EpsL and the closest structural homologues ParM and FtsA of cyto-EpsL. Fig. 55: The crystallographic dimer of GspL. Fig. 56: The structure of the periplasmic domain of EpsM from V. cholerae and Comparison of the EpsM and ferredoxin folds. Fig. 57: A possible architecture of the T2SS IM GspE: GspL subcomplex. Fig. 58: Schematic representation of the OutC and XcpP domains. Fig. 59: Structure of the PDZ domain bound to peptide and internal peptide motif. Fig. 60: The structure of the PDZ domain of EpsC from V. cholerae. Fig. 61: T2S and T4P system architectures. Fig. 62: Domain composition of secretins in different systems. Fig. 63: Electron microscopy structures of secretins. Fig. 64: Structures of pilotins. Fig. 65: Crystal structures of the type IV pilins. Fig. 66: Comparison of the T4P and T2S IM sub-complexes. Fig. 67: Structure of T. thermophiles PilM and comparison with other bacterial actin-like proteins. Fig. 68: Model for the interaction of PilM with PilN and PilO. Fig. 69: Crystal structure of P. aeruginosa PilO∆68 and comparison of the ferredoxin-like folds in monomer of PilO and EpsM. Table. 1: Nomenclature used for Gsp and Xcp T2SS, Pil T4aP in Gram-negative bacteria P. aeruginosa. Materials and methods Fig. 71: Principle of copurification and pull-down. Fig. 72: Principle of cysteine disulfide cross-linking. Table. 2:D. dadantii strains used in this thesis. Results-Chapter V Fig. V. 1: The effect of OutB on the interactions between HR of OutC and two distinct sites of OutD, N0 and N2-N3'. Fig. V. 2: Family sequence alignment of GspB proteins. Fig. V. 3: Schematic diagrams of OutB, OutC and OutD and their truncated derivatives used in the study. Fig. V. 4: Mapping the interaction region(s) between OutB and OutD. Table.V. 1: Plasmids employed in this chapter. Table.V. 2: Primers used in this chapter.Results-Chapter VIFig. VI. 1: The schematic diagrams of OutC and OutD and their truncated derivatives used in this chapter. Fig. VI. 2: Generation of OutC and OutD fragments by limited proteolysis with trypsin. Fig. VI. 3: Sequence alignment of OutC or OutD and the fragments released by limited proteolysis with trypsin whose sequences were determined by N-terminal (Edman) analysis. Fig. VI. 4: Analysis of the spontaneous disulfide bond formation in vitro. Fig. VI. 5: Development of the optimal condition for disulfide cross-linking in vitro. Fig. VI. 6: Analysis of the solubility of the cross-linked OutC-OutD complexes. Fig. VI. 7: In vitro cysteine bonding analysis between OutC 60-272 V153C and OutD 28-285

  Fig. 46: Comparison of major pseudopilins with type IV pilins. Fig. 47: The crystal structures of pseudopilins. Fig. 48: The crystal structure of pseudopilin complexes. Fig. 49: The pseudopilus assembly model. Fig. 50: GspE domain organization and crystal structures. Fig. 51: Model depicting sequence of events in T2SS initiated by ATP binding to GspE. Fig. 52: The dimer of N-terminal cytoplasmic domain of EpsF (cyto1-EpsF56-171) from V. cholerae. Fig. 53: Schematic representation of GspL and GspM from D. dadantii. Fig. 54: Crystal structure of cyto-EpsL and the closest structural homologues ParM and FtsA of cyto-EpsL. Fig. 55: The crystallographic dimer of GspL. Fig. 56: The structure of the periplasmic domain of EpsM from V. cholerae and Comparison of the EpsM and ferredoxin folds. Fig. 57: A possible architecture of the T2SS IM GspE: GspL subcomplex. Fig. 58: Schematic representation of the OutC and XcpP domains. Fig. 59: Structure of the PDZ domain bound to peptide and internal peptide motif. Fig. 60: The structure of the PDZ domain of EpsC from V. cholerae. Fig. 61: T2S and T4P system architectures. Fig. 62: Domain composition of secretins in different systems. Fig. 63: Electron microscopy structures of secretins. Fig. 64: Structures of pilotins. Fig. 65: Crystal structures of the type IV pilins. Fig. 66: Comparison of the T4P and T2S IM sub-complexes. Fig. 67: Structure of T. thermophiles PilM and comparison with other bacterial actin-like proteins. Fig. 68: Model for the interaction of PilM with PilN and PilO. Fig. 69: Crystal structure of P. aeruginosa PilO∆68 and comparison of the ferredoxin-like folds in monomer of PilO and EpsM. Table. 1: Nomenclature used for Gsp and Xcp T2SS, Pil T4aP in Gram-negative bacteria P. aeruginosa. Materials and methods Fig. 71: Principle of copurification and pull-down. Fig. 72: Principle of cysteine disulfide cross-linking. Table. 2:D. dadantii strains used in this thesis. Results-Chapter V Fig. V. 1: The effect of OutB on the interactions between HR of OutC and two distinct sites of OutD, N0 and N2-N3'. Fig. V. 2: Family sequence alignment of GspB proteins. Fig. V. 3: Schematic diagrams of OutB, OutC and OutD and their truncated derivatives used in the study. Fig. V. 4: Mapping the interaction region(s) between OutB and OutD. Table.V. 1: Plasmids employed in this chapter. Table.V. 2: Primers used in this chapter.Results-Chapter VIFig. VI. 1: The schematic diagrams of OutC and OutD and their truncated derivatives used in this chapter. Fig. VI. 2: Generation of OutC and OutD fragments by limited proteolysis with trypsin. Fig. VI. 3: Sequence alignment of OutC or OutD and the fragments released by limited proteolysis with trypsin whose sequences were determined by N-terminal (Edman) analysis. Fig. VI. 4: Analysis of the spontaneous disulfide bond formation in vitro. Fig. VI. 5: Development of the optimal condition for disulfide cross-linking in vitro. Fig. VI. 6: Analysis of the solubility of the cross-linked OutC-OutD complexes. Fig. VI. 7: In vitro cysteine bonding analysis between OutC 60-272 V153C and OutD 28-285 T53C

  pull down et une analyse structurale d'OutC et OutD et de leurs interactions par RMN. Nos résultats indiquent la présence d'au moins trois sites d'interaction entre les régions périplasmiques d'OutC et d'OutD et suggèrent que ces interactions s'établissent par un mécanisme d'addition des brins β. Nous avons démontré qu'un site situé sur le domaine HR d'OutC pouvait interagir avec deux sites distincts d'OutD suggérant un mode d'interaction alternatif. La présence d'exoprotéines et/ou des composants de membrane interne du système OutE-L-M, modifie différemment l'affinité de ces trois sites d'interaction entre OutC et OutD. Nous proposons que ces interactions alternatives entre divers sites d'OutC et OutD pourraient refléter une succession d'étapes fonctionnelles lors du processus de sécrétion.

Fig. 1 :

 1 Fig. 1: Disease symptoms caused by D. dadantii: (A) in potato (B) in carnation (C) in chicory and (D) in chrysanthemum.

Fig. 2

 2 Fig. 2 The plant cell wall and the structure of pectin. (A) Schematic representation of the plant cell wall. (B) Schematic diagram of the structure of pectin. HGA: homogalacturonan; RG-I: rhamnogalacturonan I; RG-II:

Fig. 4 :

 4 Fig. 4: Schematic representation of the Gram-negative bacterial cell envelope. The inner membrane (IM) is a symmetrical bilayer composed of phospholipids (PL). A typical structure of IM proteins (IMPs) is α-helix. The outer membrane is an asymmetrical bilayer, of which the inner leaflet is composed of PL and the outer leaflet of lipoplysaccharide (LPS). A typical structure of OM proteins (OMPs) is β-barrel. Lipoproteins (LPPs) are attached to both the inner and outer membranes. The peptidoglycan layer (PG) is located in the periplasm and

Fig. 5 :

 5 Fig. 5: Schematic overview of the E. coli Sec-and Tat translocases (Natale et al., 2008). (a) Co-translational and (b) post-translational translocation of unfolded proteins by the Sec-translocase, and (c) Translocation of folded proteins by the Tat translocase.

Fig. 6 :

 6 Fig. 6: Schematic comparison of typical N-terminal signal sequences from substrates of the Tat and Sec system (Natale et al., 2008): n-region, a postive charged amino-terminal; h-region, an uncharged hydrophobic core; cregion, a polar carboxyl-terminal region that contains a type I signal petidase cleavage site. Z and x stand for any polar residue and Φ for hydrophobic residue.

Fig. 8 :

 8 Fig. 8: Crystal structure of SecA dimer with an antiparallel orientation of M. tuberculosis. A central pore at the centre of the dimer is indicated by a circle. The different domains of SecA are represented by different colors: dark blue and light blue represent the nucleotide binding folds 1 and 2 (NBF1 and NBF2), respectively. The preprotein cross-linking domain (PPXD) is shown in red, while α -helical scaffold domain (HSD) is shown in green, the α-helical wing domain (HWD) is shown in yellow, and the C-terminal linker domain (CTL) was not resolved in this structure (du Plessis et al., 2011).

Fig. 9 :

 9 Fig. 9: Models proposed for the dimer of the SecYEG. (A) The front-to-front model with the lateral gates formed by TM2 (green) and TM7 (yellow) of SecY opposing each other. SecE (red) braces the two translocons on each side. (B) The back-to-back arrangement for two SecYEG translocons. In this instance, the two translocons are aligned with the large TM of SecE. Both translocons can act independently of each other for membrane protein insertion and protein translocation. Indicated are the locations of the central pores as well as the TM2/TM7 lateral gate (du Plessis et al., 2011).

Fig. 10 :

 10 Fig. 10: Structures of T. thermophilus SecDF (Tsukazaki et al., 2011). (A) The crystal structure of full-length SecDF viewed from the membrane side and (B) the periplasmic side.

Fig. 12 :

 12 Fig. 12: The machineries involved in inner membrane proteins insertion (Dalbey et al., 2011). (a) SecYEG mediates the insertion of proteins into the membrane. (b) YidC and SecYEG function together to insert and integrate membrane proteins. (c) YidC acts as an independent insertase. (d) YidC and SecYEG work sequentially to insert membrane proteins.

Fig. 14 :

 14 Fig. 14: Model for the membrane passage of Tat substrates (Natale et al., 2008). A) Binding of Tat substrates with Tat(B)C complex. B) Recruitment of TatA. C) Conformational change of Tat(B)C and transport of the substrate through the membrane which is weakened by TatA. D) Flux of proton. E) Seal of the membrane hole. F) Disassembly of TatA form TatBC and release of substrate by a cleavage peptidase.

Fig. 16 :

 16 Fig. 16: Model for lipoprotein transport through the bacterial cell envelope (Bos et al., 2007). After their transport via the Sec system and subsequent lipidation at the N-terminal cysteine (C), lipoproteins with an aspartate (D) at the +2 (C-D) retain in the IM. Lipoproteins with another amino acid at the +2 position (X) are destined for the OM. Energy from ATP hydrolysis by LolD is transferred to LolC and LolE and utilized to

Fig. 19 :

 19 Fig. 19: Mechanism of a typical ABC transporter[START_REF] Procko | The mechanism of ABC transporters: general lessons from structural and functional studies of an antigenic peptide transporter[END_REF]. Binding of ATP triggers the formation

Fig. 20 :Fig. 21 :

 2021 Fig. 20: Structural overview of the TolC periplasmic entrance (Pei et al., 2011). (A) Side view of the resting closed state of trimeric TolC. The individual protomers are colored blue, magenta, and green. The outer membrane embedded β barrel is open to the extracellular medium, but the coiled coils taper to close the periplasmic entrance of the α-helical barrel. H3, H4, H7 and H8 are the periplasmic α-helices that rearrange during the openning of TolC. Dashed lines indicate cross-sections through TolC, at the levels of the following: 1, the narrowest constriction of the pore formed by a ring of Asp 374 residues; 2, the constraining bond network; and 3, the periplasmic tip. (B) Cross-sections indicated by dashed lines in A, viewed through the pore toward the equatorial domain and OM β barrel. The gray backgroud outlines the surface representation.

Fig. 22 :

 22 Fig.22: Three-dimension reconstruction of the S. flexneri (A) (Hodgkinson et al., 2009) and S. typhimurium (B) (Schraid and Marlovits, 2011) needle complex (NC). (A. a) Surface representation. (A. b) Slices through the 3D reconstructions at the linker (1), IMR shoulder (2), shoulder-connector transition (3), connector (4), connector-OMR3 transition (5), OMR2 (6), OMR2-OMR1 transition (7) and OMR1 (8). (A. c) Longitudinal cutaway map. The hollow arrow on the left side of connector indicates the point at which the structure is cut to give the view in d. (A. d) The top view surface (view from outside the cell) and (A. e) The bottom view surface (view from the cytoplasm). (B) Applying symmetries to substructures of different components of the NC, that is, a 24-fold for the IR1 and a 15-fold for the neck and OR, led to higher resolutions. (B. a) Surface representation and (B. c) top and bottom view of S. typhimurium NC. (B. d) Tilted view (45°) of IR1 after symmetrization (C24) revealed structural details to subnanometer resolution.

Fig. 23 :

 23 Fig. 23: Schematic diagram illustrating needle and translocon formation, as well as toxin secretion steps in the T3SS of P. aeruginosa (Matteï et al., 2011). (A) Once the base rings (green) are in place, the needle protein PscF is released from its chaperones (PcsG and PscE) and polymerizes to form the T3SS needle. (B) The hydropilic translocator PcrV (V antigen) is released from its cytoplasmic partner (PscG) and forms the cap of the PscF needle. (C) Two hydrophobic translocator proteins PopB and PopD release PcrH. (D) Upon formation of the Pop translocon on the eukaryotic membrane, toxins produced in the bacterial cytoplasm release their cognate chaperones and are injected through the translocon pore and into the target cytoplasm.

Fig. 24 :

 24 Fig. 24: Model of assembly of the Yersinia injectisome (Diepold et al., 2010). Formation of the injectisome is initiated by formation of the secretin ring in the OM. Next, YscD attaches to YscC, which allows the subsequent completion of the membrane and supramembrane (MS) ring by attachment of YscJ. After the formation of the membrane ring structures, the ATPase-C ring complex, consisting of YscN, K, L, and Q assembles at the cytoplasmic side of the injectisome. Afterwards, the needle consisting of YscF and LcrV can be assembled.

Fig. 25 :

 25 Fig. 25: Secretion pathway and structure of the T4SS (Waksman and Fronzes, 2010). (a) The T4SS composition and assembly. The components are represented according to their proposed localization. Most (but not all) of the interactions that have been confirmed biochemically are indicated by the physical proximity of the schematic representations of each protein. (b) T4SS component structures. The atomic structures are shown in ribbon representation. They include full-length VirB5 (in orange), the VirB8 periplasmic domain (in light green), full-length VirB11 (in violet), the VirD4 soluble domain (in magenta) and the T4S OM complex

Fig. 26 :

 26 Fig. 26: Cryo-EM structure of the TraN/VirB7, TraO/VirB9, and TraF/ VirB10C-ST core complex (Fronzes et al., 2009). (A) Side view. (B) Cut-away side view. Electron density is color-coded from red to blue to indicate regions of strong to weaker density, respectively. (C) Top view (view from outside the cell). (D) Bottom view (view from the cytoplasm).Each layer forms a double-walled ring-like structure that defines hollow chambers inside the complex. The I layer is anchored in the IM and resembles a cup with a cytoplasmic opening of 55 Å in diameter. It consists of the N-terminal domains (NTDs) of the VirB9 and VirB10 homologues. The O layer consists of a main body and a narrower cap defining a 10 Å-diameter

Fig. 27 :

 27 Fig. 27:The T4S system outer-membrane complex[START_REF] Chandran | Structure of the outer membrane complex of a type IV secretion system[END_REF]. Ribbon diagram (A) and spacefilling cut-away (B) of the tetradecameric complex. TraF CT /VirB10 CT , TraO CT /VirB9 CT and TraN/VirB7 subunits are colored green, cyan and magenta, respectively. In (B), dimensions and labelling of the various parts of the complex are provided.

Fig. 28 :

 28 Fig. 28: Crystal structure of the T4SS ATPases VirD4 and VirB11. (A) The hexameric (i) and monomeric (ii) crystal structures of E. coli VirD4 homologue TrwB (Gomis-Rüth et al., 2001). (B) the H. pylori VirB11 homologue Hp0525[START_REF] Savvides | VirB11 ATPase are dynamic hexameric assemblies: new insights into bacterial type IV secretion[END_REF]. CTD, C-terminal domain; NTD, N-terminal domain.

Fig. 29 :

 29 Fig. 29: Proteins composing the two-partner system (TPS) (A) and autotransporter (AT) (B) (Hodak and Jacob-Dubuisson, 2007). (A) TpsA proteins are the secreted components containing the conserved N-terminal, ~250-residue-long TPS domain, followed by a long region forming essentially β-strands. The TpsB transporter is composed of a C-terminal β-barrel and an N-terminal periplasmic region containing a POTRA domain. (B) The AT proteins contian an N-terminal passenger domain rich in β-strands, similarly to TpsA proteins, and a Cterminal β-barrel. A linker region forming an α-helix separates the two domains.

Fig. 31 :

 31 Fig. 31: The crystal structure of different autotransporter (AT) passenger domains. (A) Pertactin passenger domain reveals 16-turn-β helix fold[START_REF] Emsley | Structure of Bordetella pertussis virulence factor P.69 pertactin[END_REF]. (B) A remarkable pertactin-like 24-turn β helical

Fig. 32 :

 32 Fig. 32: Models for the autotransport mechanism (Dautin and Bernstein, 2007). (a) The hairpin model, (b) the Omp85 model and (c) the multimer model. The passenger domain (blue), the β barrel (dark green) and Omp85 (pink) are colored differently.

Fig. 33 :

 33 Fig. 33: Crystal structures of TPS domain of fiamentous hemagglutinin FHA (A) and FhaC (B). (A) Ribbon representation of the overall structure of Fha30. The helix β-sheets PB1, PB2 and PB3, the β-hairpin β7/β8, and the β-sheet β16/β25 are shown (Clantin et al., 2004). (B) Ribbon representation of FhaC viewed from the membrane plane. Putative position of OM boundary is indicated with horizontal lines, with the extracellular side (E) at the top and the periplasm (P) at the bottom. The α helix H1 is colored red, POTRA 1 light blue, POTRA 2 purple.[START_REF] Clantin | Structure of the membrane protein FhaC: a membrane of the Omp85-TpsB transporter superfamily[END_REF].

Fig. 34 :

 34 Fig. 34: Proposed model of FHA transport across the outer membrane (Clantin et al., 2007). (A) The TPS domain of FHA in an extended conformation interacts with POTRA1 of FhaC. (B) The channel opens after conformational changes of loop L6, and translocation initiates, with FHA adopting transiently an extended β hairpin structure. (C) FHA progressively folds and elongates into its β-helical fold. (D) After the C terminus of FHA has reached the surface, the TPS domain dissociates from POTRA 1 and is translocated. The folding of the TPS domain caps the N terminus of the FHA β helix.

  Fig. 35: Structure and function of the T6SS (Bönemann et al., 2010). VipA/VipB tubules are suggested to act as tail sheath proteins by engulfing Hcp tubes. A pilus-like structure formed by Hcp and VgrG, punctrues the lipid bilayer of target cells. The role of T6SSs in bacterial virulence is either based on the exposure of evolved VgrG-linked effector domains (left part) or the delivery of effector proteins to cytosol of target cells (right part).

Fig. 36 :

 36 Fig. 36: Potential similarities in the injection mechanisms of tailed bacteriophage and T6SS (Bönemann et al., 2010). (A) Contraction of the T4 tail sheath structure upon contact with host cells provides the energy to push the tail needle through the OM of target cells. Contraction of VipA/VipB tubules is suggested to have a similar function leading to the export of Hcp and VgrG. (B and C) Putative roles of the ClpV in causing contraction of periplasmic VipA/VipBs.

Fig. 37 :

 37 Fig. 37: Genetic organization of the T2SS. The homologue genes coding for T2SS components are represented by the same color[START_REF] Filloux | The underlying mechanisms of type II protein secretion[END_REF].

Fig. 38 :

 38 Fig. 38: Model of structural organization of the T2SS. 1. Sec or Tat systems promote export of exoproteins through the inner membrane (IM). 2. T2SS transports exoproteins across the outer membrane (OM).

Fig. 39 :

 39 Fig. 39: Schematic representation of D. dadantii T2SS secretin OutD (A) and crystal structure of N0-N1-N2

Fig. 40 :

 40 Fig. 40: Comparison of peri-GspD N0 subdomain with TonB-dependent OM receptor FpvA from P.

Fig. 41 :

 41 Fig. 41: 3D reconstruction of the K. oxytoca secretin PulD (Chami et al., 2005). (A) The intact complex (white mesh) and the digested fragment (blue). The C domain is represented by blue; the N and S domains are represented by gray. The top perspective view of the native PulD complex (B) and the bottom perspective view of trypsin-resistant PulD complex (D) are shown. The corresponding axial back-projection is shown in (C) and (E), respectively.

Fig. 42 :

 42 Fig. 42: Modeled cylindrical arrangements of periplasmic GspD domains (Korotkov et al., 2009a). (A) The models of dodecameric N0-N1 ring and N2 ring. The structure of heat-labile enterotoxin (LT B5) is shown for size comparison. (B) Bottom view of the C12 ring of the N0-N1 subdomains with the LT B5 toxin structure. (C) Stereo figure of the N0:N1 interface viewed perpendicular to the C12 symmetry axis in the ETEC GspD model shown in (A). (D) Stereo figure of the subunit interface viewed parallel to the C12 symmetry axis in the ETEC GspD ring of N2 domains shown in (B).

Fig. 43 :

 43 Fig.43: Cryo-EM reconstruction of V. cholerae T2SS secretin GspD[START_REF] Reichow | Structure of the cholera toxin secretion channel in its closed state[END_REF]. In side view, three domains are identified from bottom to top as the periplasmic domain, the outer membrane domain and the extracellular cap. In slice view, the channel contains an extracellular gate, an extracellular chamber, a periplasmic gate and a perplasmic vestibule with a constriction.

Fig. 44 :

 44 Fig. 44: Fitting models (Reichow et al., 2010). (A) Fitting of 12-fold symmetric ring models of the VcGspD N-terminal periplasmic domains (N0-N3) into the VcGspD density map. (B) Fitting of the cholera toxin AB 5 heterohexamer (A subunit, yellow; B subunits, gold) into the VcGspD periplasmic vestibule.

Fig. 45 :

 45 Fig. 45: Sequence alignment of the N-terminal domains of the PilA pilin subunit of type IV pili and pseudopilins of the Xcp-type II secretory pathway from P. aeruginosa. XcpT,-U, -V, -W and -X are more generally named GspG, -H, -I, -J and -K, respectively. The residues in the pseudopilins that are identical to those in the PilA sequence are shown in bold. Here R and K, and also I and L, are treated as 'identical' residues.

Fig. 46 :

 46 Fig. 46: Comparison of major pseudopilins with type IV pilins. (A) The soluble domain of the pseudopilin PulG from K. oxytoca and the soluble domain of the pseudopilin XcpT from P. aeruginosa (B), (C) the soluble domain of the pilin PilA from P. aeruginosa strain PAK and (D) the soluble domain of the pilin PilA from P. aeruginosa strain K122-4. The N-terminal α-helices are colored in blue, β-sheet of the α/β roll in green and the αβ-loop in red. In yellow are indicated the 'D' domain of the pilin structures (Alphonse et al., 2010).

Fig. 47 :

 47 Fig. 47:The crystal structures of pseudopilins[START_REF] Korotkov | Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system[END_REF]. The N-terminal a-helices are represented in blue, the conserved β-sheet is in green, the variable region is in purple and the α-domain of GspK is in light blue. Above, ribbon diagrams of the individual structures of GspI, GspJ and GspK from ETEC, the structures of the major pseudopilin GspG from K. oxytoca and the minor pseudopilin GspH from V. cholerae. All structures are shown in similar orientations based on superposition of the shared C-terminal βsheet (green).

Fig. 48 :

 48 Fig. 48: The crystal structure of pseudopilin complexes. (A) The crystal structure of the V. vulnificus EpsI: EpsJ complex (Yanez et al., 2008a). a) A ribbon diagram of one VvEpsI 2 : EpsJ 2 heterotetramer. VvEpsI, blue; VvEpsI', cyan; VvEpsJ, brown; and VvEpsJ', red. One chloride ion, located between VvEpsJ and VvEpsJ', is shown in green. b) A 'top' view of one VvEpsI 2 :EpsJ 2 heterotetramer with buried accessible surfaces of the intersubunit contacts indicated. c) A close-up view of one VvEpsI: EpsJ heterodimer, in the same orientation as the heterotetramer shown in (a), with VvEpsI colored dark blue and VvEpsJ colored brown. d) The VvEpsI:EpsJ heterodimer shown in (c) rotated by 180°. (B) The crystal structure of the ETEC GspK-GspI-GspJ complex[START_REF] Korotkov | Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system[END_REF]. a) The modeled N-terminal a-helical extensions of GspK (blue), GspI (violet) and GspJ (green). The observed heterotrimer crystal structure is shown in surface representation and modeled α-helices are shown as cartoons. The insert shows that residues GspJ-Glu5 and GspI-Phe1, and also GspI-Glu5 and GspK-Met1, are in close proximity when the a-helices are extended. (b) The complex of GspK-GspI-GspJ by downward addition of GspG (orange) or GspH (yelleow) (c).

Fig. 49 :

 49 Fig. 49: The pseudopilus assembly model (Campos et al., 2010). (A) Electrostatic envelop of the pilus and PulG monomer, inserted in the membrane (gray rectangle). Arrows indicate the PulG tethering via Asp 48 -Arg 87 and Glu 44 -Arg 88 salt bridges, the hydrophobic patch (outlined in green), and Glu 5 . (B) Ribbon view of the pilus and the incoming protomer. (C) The incoming protomer (P) incorporation is associated with the membrane extraction of the P +1 , driving by the PulE ATPase, adding 10.4Å to the pilus. (D and E) Zoom views of the rectangle in C showing two alternative interaction of Glu 5 of P +3 with Phe 1 of P +4 (D) of Lys 35 and Lys 28 of P, stabilizing the pilus in its optimal state (E).

Fig. 50 :

 50 Fig. 50: GspE domain organization and crystal structures. (A) Schematic representation of domain structure of GspE with the subdomains N0 (red), N1 (gray), N2 (cyan), C1 (dark blue), C M (yellow) and C2 (green). The N0 subdomain is restricted to a subset of GspE family member. (B) Crystal structure of N-terminally truncated version of GspE△90 monomer from V. cholerae[START_REF] Robien | Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae[END_REF]. The position of a bound molecule of AMPPNP (non-hydrolysable analog of ATP) is shown. (C) Crystal structure of the N1 subdomain of GspE (N1-EpsE) in complex with the cytoplasmic domain of GspL (cyto-EpsL) from V. cholerae[START_REF] Abendroth | The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae[END_REF]. (D) Superposition of the two structures of GspE N0-N1 from X. campestris with domain N1 colored light green and domain N0 in its closed and open conformations colored in dark green and orange, respectively[START_REF] Chen | Structure and function of the XpsE N-terminal domain, an essential component of the Xanthomonas campestris type II secretion system[END_REF]. (E) Hexameric ring model of EpsE. View from the proposed membrane-facing side (left),

Fig. 51 :

 51 Fig.51: Model depicting sequence of events in T2SS initiated by ATP binding to GspE[START_REF] Shiue | XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL[END_REF].Steps blocked by mutant GspE are indicated by mutated residue(s) in boxes. Filled circles depict ATP.[START_REF] Shiue | Mutation of a key residue in the type II secretion system ATPase uncouples ATP hydrolysis from protein translocation[END_REF] identified R286 as a key residue in GspE. This residue plays a pivotal role in coupling ATP hydrolysis to protein translocation. Mutation of R286 to Ala caused the basal GspE ATPase activity elevated. However, the mutant GspE (R286A) is non-functional in protein secretion via T2SS[START_REF] Shiue | Mutation of a key residue in the type II secretion system ATPase uncouples ATP hydrolysis from protein translocation[END_REF]. Detailed analyses indicated that GspE (R286A) lost the ability of the coordinating N-and C-domains of GspE. The authors proposed that R286 serves as a sensor for different states of the nucleotide. Once mutated, the 'sensor' function of R286 is broken. Thus, the structural alteration resulted from the ATP binding or ATP binding-induced oligomerization could not propagate to the N-domain of GspE as judged by lack of conformation change at N-domain required for its association with the N-domain of GspL. Thus, GspE lost the ability to associate with the cytoplasmic membrane via GspL. Consequently, ATP hydrolysis by GspE was uncoupled from protein secretion. Recently,[START_REF] Patrick | Oligomerization of EpsE coordinates residues from multiple subunits to facilitate ATPase activity[END_REF] showed that several other conserved arginines are essential for ATP hydrolysis. Replacements of these arginines abrogate the T2S process due to reduction of the ATPase activity but do not have any measurable effect on ATP binding or oligmerization of GspE. Similarly, some additional mutations within the intersubunit interface disrupt the function of GspE, indicating that a precise arrangement of residues from different subunits around the active site is essential for the action of GspE (Patrick

Fig. 52 :

 52 Fig. 52: The dimer of N-terminal cytoplasmic domain of EpsF (cyto1-EpsF 56-171 ) from V. cholerae (Abendroth et al., 2009a). Each cyto-EpsF 56-171 subunit consists of a six-helix bundle. The dimer is shown in three different orientations, green spheres indicate calcium ions in the dimer interface.

IMFig. 53 :Fig. 54 :

 5354 Fig. 53: Schematic representation of GspL and GspM from D. dadantii.

Fig. 55 :

 55 Fig. 55: The crystallographic dimer of GspL. (A) The dimer of cyto-EpsL. The dimer interface, transmembrane helices (green dots) and the putative EpsE-binding region are indicated (Abendroth et al., 2004a). (B) The Vp peri-EpsL dimer in the crystal. The subunits A' and A are positioned left and right, respectively. The anti-parallel four-stranded β-sheet per subunit is extended to form an eight-stranded antiparallel β-sheet in the dimer(Abendroth et al., 2009b).

Fig. 56 :

 56 Fig. 56: The structure of the periplasmic domain of EpsM from V. cholerae (A) and comparison of the EpsM and ferredoxin folds (B) (Abendroth et al., 2004b). (A) The structure of the dimer (a). Each EpsM consists of a sandwich of a four-stranded antiparallel β-sheet and two helices. Helices α2 and α2* mediate most of the intermolecular contacts, the putative ligand-binding groove is located between β3 and β3*. (b) The monomer consists of two αββ-subdomains, which have a very similar structure. (c) The superposition of the two subdomains in the same orientation as of the green subdomain in (b). (B) Comparison of the EpsM and ferredoxin folds. Structurally corresponding secondary structure elements have the same color and the secondary structure elements after virtual transformation are in lighter colors.

Fig. 57 :

 57 Fig.57: A possible architecture of the T2SS IM GspE: GspL subcomplex (Abendroth et al., 2009b). The view is from the cytoplasm outwards with the cytoplasm in the center and the periplasm at the periphery. The hexamer of full-length of GspE (purple) and the three dimers of cyto-GspL (shades of green per dimer) shown in the center are in the cytoplasm. Predicted TM helices of GspL are shown as hatched rods, located in the IM.On the outside are depicted three peri-GspL dimers in the periplasm (in darker shades of green). A tentative position of the peri-GspM dimer is depicted in light and darker yellow.

Fig. 58 :

 58 Fig. 58: Schematic representation of the OutC and XcpP domains. The positions are according to Login et al. (2010) and Gérard-Vincent et al. (2002).

Fig. 59 :

 59 Fig. 59: Structure of the PDZ domain bound to peptide and internal peptide motif. (A) Ribbon representation of the PDZ domain of PSD95 (blue) with KQTSV peptide forming antiparallel β-sheet with βB strand (red arrow). (B) Complex of the syntrophin PDZ domain (shown as blue and green solvent-accessible surface representation) and nNOS PDZ domain (shown as red ribbon representation with β-finger indicated) (Jelen et al., 2003).

Fig. 60 :

 60 Fig. 60: The structure of the PDZ domain of EpsC from V. cholerae (Korotkov et al., 2006). (A) The structure of the short version of the PDZ domain of EpsC (s-PDZ-C), spanning residues 219 to 305. (B) The structure of the long version of the PDZ domain of Eps-C (l-PDZ-C), ranging from residue 201 to 305. (C) The superposition of the two PDZ-C structures.

(

  GspL and GspM of K. oxytoca) localized circumferentially in the E. coli cell envelope, with occasional brighter foci when other secreton factor were present. Furthermore, overproduction of GFP-PulM or GFP-PulL results in their polar localization. These results suggest that polar accumulation may be due to overproduction of the proteins and do not represent the normal location of GspM and GspL. Coherent with the non-polar localzation of the K. oxytoca T2SS components,[START_REF] Buddelmeijer | Type II secretion system secretin PulD localizes in clusters in the Escherichia coli outer membrane[END_REF] further showed that chromosome-encoded PulD-mCherry (PulD fused to a monomeric red fluorescent protein) formed fluorescent foci on the periphery of the cell in the presence of high levels of its cognate chaperone, the pilotin PulS.Recently,[START_REF] Lybarger | Docking and assembly of the type II secretion complex of Vibrio cholerae[END_REF] showed that fluorescently tagged, chromosomally expressed GspC and GspM of V. cholerae form discrete foci along the lengths of the cells(Lybarer et al., 2009). Furthermore, they showed that those fluorescent foci in both chromosomal GFP-GspCand GFP-GspM-expressing strains of V. cholerae disperse upon deletion of gspD, suggesting that GspD is crucial for proper localization of the IM proteins GspC and GspM and perhaps their assembly into the T2S complex(Lybarer et al., 2009).

Fig. 61 :

 61 Fig.61: T2S and T4P system architectures[START_REF] Ayers | Architecture of the type II secretion and type IV pilus machineries[END_REF]. They have a similar organization, consisting of inner and outer membrane subcomplexes. Proteins predicted to have similar structures are represented with similar colors. OM subcomplexes consist of a dodecameric secretin with its associated pilotin. The SDA (secretin dynamic-associated protein) mediates secretin function and also links the OM components with those in the IM. IM subcomplexes typically consist of the platform protein, one or more bIMPs (bitopic inner membrane protein), the ATPase(s) and, in the T2S/T4aP system, a cytoplasmic protein with an actin-like fold.

Fig. 62 :

 62 Fig. 62: Domain composition of secretins in different systems. The N-terminal signal sequences recognized and cleaved by a signal peptidase are represented by gray and prolipoprotein signal peptidases are represented by yellow. The homologous domains are represented by the same colors, N0: purple, N1-N2-N3: light green, Secretin domain: blue, pilotin-interaction domain (S domain): dark blue and unknown topology domains:

Fig. 64 :

 64 Fig. 64: Structures of pilotins. (a) The structure of the S. flexneri T3SS pilotin MxiM (green) in complex with the C-terminal fragment of the secretin MxiD (dark blue) (Okon et al., 2008). (b) The structure of the N.

Fig. 65 :

 65 Fig. 65: Crystal structures of the type IV pilins. (a) Full-length PilE structure of N. gonorrhoeae. (b) Fulllength PilA structure of P. aeruginosa K. (c) Soluble domain structure of P. aeruginosa K122-4 PilA. (d) Soluble domain structure of major pilin TcpA of V. cholera T4bP. (e) Superposition of the three independent full-length PilA structures shows flexibility in α1-N (Craig et al., 2004).

Fig. 66 :

 66 Fig. 66: Comparison of the T4P and T2S IM sub-complexes (Sampaleanu et al., 2009). (a) and (b) represent the models while (b) and (d) represent the schematic domain organization of the IM sub-complexes found in T4P (a and b) and T2S (c and d). Similar proteins are rendered in the same color. The ferredoxin-like fold is shown as a pair of diamonds. Domains for which structures are available are surrounded by thick outlines while regions predicted to be unstructured are shown with broken lines.

Fig. 67 :

 67 Fig. 67: Structure of T. thermophiles PilM and comparison with other bacterial actin-like proteins. (A). Structure of PilM (magenta) overlaid with the PilN peptide (blue) and ATP. (B) Superposition of of PilM and ligands with FtsA (cyan) and EpsL (green) (C) (Karupphiah and Derrick, 2011).

Fig. 68 :

 68 Fig. 68: Model for the interaction of PilM with PilN and PilO (Karupphiah and Derrick, 2011).

Fig. 69 :

 69 Fig. 69: Crystal structure of P. aeruginosa PilO ∆68 (A) and comparison of the ferredoxin-like folds (B) in monomer of PilO (a) and EpsM (b) (Sampaleanu et al., 2009). (A) The PilO periplasmic domain consists of two N-terminal coiled coils (CC domain) and a C-terminal ferredoxin-like domain (Core domain). (B) The ferredoxin-like domain is similar to that adopted by the T2SS protein EpsM.

Fig. 71 :

 71 Fig. 71: Principle of copurification and pull-down.

Fig. 72 :

 72 Fig. 72: Principle of cysteine disulfide cross-linking.

  additional experiments. The two β-sheets of the HR domain of OutC are connected together through several hydrophobic interactions, including the side chains of well-conserved Leu93, Leu97, Ile113 (the first β-sheet) and V143, Leu154 and Leu156 and semi-conserved Leu/Ile145, Ile/Leu/Val135 and Ile/Val138 (the second β-sheet) (Gu et al., 2011a). Consistent with the HR structure, double I113C/V143C substitution provoked an efficient intramolecular disulfide bond within OutC and fully arrested secretion, indicating that a covalent cross-linking of the two β-sheets of HR abolishes the protein function (Gu et al., 2011a). It should be noted that all the tested single cysteine variants of OutC (except for G99C) retained functional (Fig. 4A, lanes 2 to 9 and Wang et al., submitted). Unexpectedly, when we combined I113C (buried) with either V144C or V153C (both are solvent exposed), the corresponding double substitutions, I113C/V144C and I113C/V153C, were completely defective in secretion (Fig. III. N1A, lanes 8 and 9). On nonreducing gel, these two mutants migrated slightly faster than the wild-type OutC, indicating an efficient intramolecular disulfide bonding within HR (Fig. III. N1B, lane 3 and not shown). A certain amount of homodimer, which is usually formed by the single I113C mutant, was fully disappeared in these double mutants, indicating that the sulfhydryl group of I113C was fully involved in a more proficient intramolecular disulfide bond with either V144C or V153C (Fig.

  we constructed the two triple OutC mutants, I113C/V143C/V153C and I113C/V144C/V153C. The former protein was fully non-functional and formed at least one intramolecular disulfide bond (Fig. III. N1A, lane 10 and Fig. III. N1B, lane 4). Notably, the homodimer usually formed by I113C was fully absent, indicating that its sulfhydryl was completely involved in the intramolecular bonding with either V143C or V153C. On the contrary, the triple I113C/V144C/V153C mutant became functional. It is remarkable since each of the corresponding double mutants (I113C/V144C and I113C/V153C) was completely nonfunctional (Fig. III. N1A, compare lanes 8 and 9 with 11). Notably, similar to single I113C variant, a certain amount of homodimer formed by I113C was detected with this triple mutant (Fig. III. N1B, compare lanes 2 and 5), indicating that sulfhydryl of I113C became available and was not

Fig

  Fig. III: N1 Functional (A) and disulfide bond analysis of OutC variants carrying multiple cysteine substitutions within the T2SS of D. dadantii. (A) The secretion activity of OutC variants (indicated on top of gels) was estimated in a complementation assays with D. dadantii ∆outC strain A3556. Bacteria carrying a plasmid with OutC variants were grown to steady-state, then, culture supernatant (s) and cells (c) were separated and analyzed by immunoblotting with PelD and PelI-antibodies. The amount of secreted proteins (PelD, PelE and PelI) in the culture supernatant reflects secretion efficiency. (B) Disulfide-bonding analysis of the same OutC variants. Cells from the same cultures as in A were treated with iodoacetamide to block remaining free thiol groups and the extent of disulfide-bonding was assessed by non-reducing SDS-PAGE followed by immunoblotting with OutD-antibodies. A certain amount of homodimer characteristic for the thiol of I113C was only detected with OutCI113C and OutCI113C/V144C/V153C (As shown on Fig. 3B of the Article 3, homodimers formed by various cysteine variants of OutC have different and thus, characteristic, apparent molecular mass.) Double I113C/V153C and triple I113C/V143C/V153C and I113C/V144C/V153C variants (indicated by a dot) migrated slightly faster than the wild type OutC monomer, indicating formation of an intramolecular disulfide bond. Positions of monomers (1-m) and dimers (2-m) are indicated by arrowheads.

Primer

  Nucleotide sequence (5'→3') OutB-BH-5' CTGGGATCCCCCGCCAAATTGGTAACAG OutB-XB-3' CGGAATTCCGGCTCTAGAGCATGATGTGCAGTTGCTG of OutC and OutD. We found that the presence of OutB slightly increased the amount of OutC-OutD complex between V153C (β7) of HR and T53C (β2) of N0 subdomain of OutD (Fig. V. 1. IA, lanes 5 and 7). However, OutB had no obvious effect on cross-linking between V144C (β6) of HR and T53C (β2) of N0 subdomain of OutD (Fig. V. 1. IA, lanes 1 and 3). (2) Effect of OutB on the interaction of OutCsip and N2-N3' of OutD An in vivo disulfide cross-linking analysis revealed that the second interacting site consisting of OutCsip and N2-N3' domain, such as V144C (β6 of HR) of OutC interacts with V271C (β12 of N3) of OutD while V153C (β7 of HR) of OutC interacts with V232C (β10 of N2) of OutD. This indicates that these domains (HR of OutC and N2 and N3' of OutD) are arranged in a manner such that β6 of OutC is more proximal to β12 of N3 of OutD whereas β7 of OutC is closer to β10 of N2. We therefore tested whether OutB affects these crosslinking patterns. We observed that the presence of OutB led to a substantial decrease in the formation of complex between V144C (β6 of HR) of OutC and V271C (β12 of N3) of OutD (Fig. V. 1. IIIA, lanes 1 and 3). However, the presence of OutB only slightly affected the formation of the complex between V153C (β7 of HR) of OutC and V271C (β12 of N3) of OutD (Fig. V. 1. IIIA, lanes 5 and 7). OutB had no obvious effect on the crosslinking ability between V144C (β6) and V153C (β7) of OutC and V232C (β10 of N2) of OutD (Fig. V. 1. IIA, lanes 1 and 3, 5 and 7).

Fig. V. 1 :

 1 Fig. V. 1: The effect of OutB on the interactions between the HR of OutC and two distinct sites of OutD, N0 (I) and N2-N3' (II and III). E. coli NM522 cells expressing the indicated variants of OutC and OutD either alone or with PelB, or with OutB, or with PelB and OutB (indicated on top of the panels) were grown overnight and treated as described in Materials and methods to preserve spontaneously formed disulfide bonds. (A) The samples were separated by SDS-PAGE under non-reducing conditions (without β-mercaptoethanol) and probed with OutC-antibodies. (B) The samples were separated by SDS-PAGE under reducing condition (with β-mercaptoethanol) and either probed with OutB-antibodies or with OutC-antibodies to estimate the amounts of OutB and OutC variants. The deduced compositions of the immunoreactive species (OutC dimer and OutC-OutD complex) are indicated by arrows.

  V. 2). These indicate that C-terminal region of OutB which extends into the periplasm could play a similar function within GspBs, for example, they could be involved in the interaction with the periplasmic region of OutD. To test this hypothesis, the C-terminal region of OutB (OutB 112-220 ) was fused to GST and coexpressed with a His-tagged N-terminal region of OutD (OutD 28-285 ) from the same plasmid pGEX-6P-3. Then the two proteins were co-purified from the same whole cell extract by glutathione-affinity chromatography. In these experiments, GST-tagged OutB was bound to Glutathione Sepharose and used as a bait to catch other proteins that interact with OutB (e.g., OutD). Our previous results showed that OutD 28-285 bound efficiently to GST-OutC 128-272 , but not to GST-OutC 161-272 . We therefore used these two GST fusions as a positive and a negative control, respectively (Fig. V. 3 and Fig. V. 4A, lanes 1 and 3). In copurification assay, OutD 28-285 bound efficiently to GST-OutC 128-272 and to GST-OutB 112-220 (Fig. V. 4A, lanes 1 and 2), but not to GST-OutC 161-272 (Fig. V. 4A, lane 3). This indicates that the C-terminal region of OutB (OutB 112- 220 ) interacts with the N-terminal region of OutD (OutD 28-285 ). To map the interaction site(s) within OutD, we employed two shorter OutD derivatives, OutD 28-112 and OutD 116-285 , one consisting of the N0 domain and the other comprising the N1, N2 and the beginning of N3 domains (Fig. V. 3C). In copurification assay, we found that only OutD 28-112 (N0) bound to GST-OutB 112-220 (Fig. V. 4B, lane 5). This suggests that the C-domain of OutB (OutB 112-220 ) interacts with the N0 domain of OutD (OutD 28-112 ). Therefore, N0 domain of OutD interacts with two sites within HR domain of OutC (Chapter III) and the C-terminal domain of OutB. The competition experiments above (Fig. V. 1. IA) suggested that at least some of these interactions are compatible and even cooperative.

Fig. V. 2 :

 2 Fig. V. 2: Family sequence alignment of GspB proteins. OutB from Erwinia chrysanthemi, PulB from Klebsiella oxytoca, ExeB from Aeromonas hydrophila and EpsB from Vibrio cholera are aligned by ClustalW.

  --------------QEATPDTKP---AKLVTGWQTAKPGELPYIAF PulB PVNAGPEIQAEP----------------EIAIDEDN------------------LPPLRY ExeB PNDSAPAANGSQGLAERIMNALNSTPLMEETAPQAQSESQAMPISALPLELKQRVPPLAY EpsB LRVQAIMRDQSS----------------EPATPSTPTS-AAVSLTQHSDRYQGQLPALNF .

Fig. V. 3 :

 3 Fig. V. 3: Schematic diagrams of OutB (A), OutC (B) and OutD (C) and their truncated derivatives used in this chapter.

Fig. V. 4 :

 4 Fig. V. 4: Mapping the interaction region(s) between OutB and OutD. (A) C-terminal domain of OutB interacts with N-terminal domain of OutD. (B) C-terminal domain of OutB interacts with N0 subdomain of OutD. Whole-cell extracts of E. coli BL21 (DE3) coexpressing GST-OutB 112-220 or GST-OutC derivatives with OutD derivatives (indicated on top) were used in copurification assays on Glutathione Sepharose. Bound proteins were eluted with Laemmli sample buffer, separated by SDS-PAGE and then stained with Coomassie G250. GST-OutB 112-220 or GST-OutC derivatives are indicated by asterisks, OutD derivatives are indicated by arrows.

  OutD, thus provoke a switch from one functional state to another. In Chapter III, we have examined the effects of secreted proteins and IM components OutE-L-M on the interactions between OutC and various sites of OutD. In this study, we showed that OutB interacts directly with OutD and more precisely with N0 domain (Fig. V. 4). Such interaction may induce the conformational change of OutD and therefore affect the interactions between OutC and OutD. Alternatively, OutB could probably compete with OutC for the same interaction site(s) with OutD. Disulfide cross-linking analysis showed that the presence of OutB slightly increases the formation of OutC-OutD complex between V153C (β7) of OutC and T53C (β2) of N0 domain of OutD (Fig. V. 1A, lanes 5 and 7) whereas it decreases that between V144C (β6) of OutC and V271C (β12) of N3 domain of OutD. Disulfide cross-linking requires a close proximity and proper orientation of the two cysteine residues to allow formation of a disulfide bond between them. The presence of OutB increased the formation of OutC-D complex suggests a conformational change either in OutC or OutD or both that improve these interactions. Such a conformational change may occur via their direct interaction with OutB. Since no data suggests an interaction between OutC and OutB, we proposed that the conformational change occurs in OutD via an interaction between the periplasmic domain of OutB and the periplasmic domain of OutD. Indeed, copurification assays revealed an interaction between the periplasmic region of OutB (OutB 112-220 ) and N0 domain of OutD (OutD 28-112 ). Together with in vivo disulfide cross-linking results (Fig. V. 1 .I), we showed that N0 domain of OutD interacts with both the HR domain of OutC and the periplasmic region of OutB. Moreover, our results indicated that these interactions are compatible and even cooperative since in the presence of OutB, the cross-linking between OutC (β7) and N0 domain (β2) becomes ever more pronounced, indicating a possible cooperative effect. Thus, different faces of N0 may be involved in interactions with HR and OutB. The presence of OutB decreases the formation of the complex between OutC (β6) and N2-N3' (β12) of OutD (Fig. V. 1. III). The effect of OutB on this OutC-OutD interaction site seems to be indirect since no interaction were detected between OutB (OutB 112-220 ) and N1-N2-N3' of OutD (OutD 116-285 ) (Fig. V. 4B). Alternatively, OutB could probably compete with N2-N3' of OutD for binding to HR of OutC.

  Fig.VI. 1: The schematic diagrams of OutC (A) and OutD (B) and their truncated derivatives used in this chapter.

Fig. VI. 2 :

 2 Fig. VI. 2: Generation of OutC and OutD fragments by limited proteolysis with trypsin. OutC 60-272 and OutD 28- 285 were either coexpressed or OutC 60-272 was expressed alone (as indicated on top) and used in copurification assays on Glutathione Sepharose. Proteins were eluted with PreScision protease and then incubated for various periods time (indicated at the top of panels) with 2 μg/ml of trypsin. Samples were analysed by Tricine SDS-PAGE. The proteins were transferred to a PDVF membrane and then stained with Coomassie G250. The bands of interest (indicated by red star for OutC fragments and blue star for OutD fragments) were cut off and subjected to the N-terminal (Edman) analysis.

  Fig. VI. 3: Sequence alignment of OutC (A) or OutD (B) and the fragments released by limited proteolysis with trypsin (Fig. VI. 2) whose sequences were determined by N-terminal (Edman) analysis. The C-termini of the fragments were deduced from their molecular weight by using trypsin cleavage sites. The sequence of fragment C1 was not obtained while several bands, i.e., C2, D1 and D3 correspond to more than one peptide.

  ---------------------------------------------------------D2 AEFSASFKGTDIQEFINTVSKNLNKTVIIDPTVRGTISVRSYDMMNEGQYYQFFLSVLDV D3-1 ------------------------------------------------------------D3-2---------------------------------------------------------

)

  Disulfide cross-linking in vitro GST-OutC 60-272 and OutD 28-285 carrying V143C and G190C substitutions in OutC and OutD, respectively, were coexpressed in E. coli BL21 (DE3) and whole cell extracts were used to copurify the OutC-OutD cross-linked species. We firstly tested the formation of spontaneously formed corss-linked products. A certain amount of OutD homodimer was detected whereas only little OutC homodimer and OutC-OutD heterodimer was found (Fig. VI. 4). To improve the formation of disulfide bound complexes, we applied an external oxidant, copper phenanthroline (CuP) which increases oxidation potential of the medium. After sonication, cell extracts were incubated with different concentrations of CuP and then the proteins of interest were copurified

Fig. VI. 4 :

 4 Fig. VI. 4: Analysis of the spontaneous disulfide bond formation in vitro. V143C and G190C substitutions were introduced in GST-OutC 60-272 and OutD 28-285 , respectively (indicated on top of panels). Proteins were coexpressed and whole cell extracts were used in copurification assays on Glutathione Sepharose and then eluted with PreScision protease. The eluted proteins were separated by SDS-PAGE under non-reducing conditions and then stained with Coomassie G250. D-D: cross-linked OutD 28-285 homodimer, C-D: cross-linked OutC-OutD heterodimer, C-C: OutC 60-272 homodimer. The nature of these complexes has been revealed by immunoblotting with OutC-and OutD-antibodies (Fig. VI. 6B).

Therefore, 1

 1 .5 mM CuP was applied of various steps of the copurification process (Materials and methods and Fig. VI. 5B). The results clearly show that using of the oxidative agent at the final step of the copurification assay was much more efficient (Fig. VI. 5B, lane 4). Homodimer of OutC was the major cross-linked product while homodimers of OutD and heterodimers OutC-OutD were less abundant. Nevertheless, the amounts of all these complexes seem to be sufficient to envisage their further purification by ion-exchange and/or hydrophobic-interaction chromatography. In an attempt to perform this, we assessed the solubility of these protein complexes by their precipitation/solubilization with ammonium sulfate (Fig. VI. 6A). The results show that contrary to OutD-OutD dimer, OutC-OutD and OutC-OutC complexes

Fig. VI. 5 :

 5 Fig. VI. 5: Development of optimal conditions for disulfide cross-linking in vitro. (A) Effect of copper phenanthroline (CuP) concentration on the formation of OutC-D complexes. Indicated concentrations of CuP were added to cell extracts, incubated for 20 min and then the proteins were purified on Glutathione Sepharose and eluted with PreScision protease (panel I). The proteins remain bound to the resin were then eluted with Laemmli sample buffer without β-mercaptoethanol (panel II). (B) Effect of CuP on formation of OutC-D complexes on different steps of purification process. CuP (1.5 mM) was added either to the whole cell extract (a), or soluble cell fraction (b), or resin with bound GST fusion proteins (c), or proteins eluted from the resin with PreScision protease (d).

Fig. VI. 6 :

 6 Fig. VI. 6: Solubility assays of the cross-linked OutC-OutD complexes. (A) OutC 60-272 V143C and OutD 28- 285 G190C proteins were copurified on Glutathione Sepharose and treated with CuP as above (Fig. VI. 5B, lane 4). Then the proteins were precipitated with ammonium sulfate (90%) and resolubilized in the elution buffer to appreciate their suitability for further purification steps. S: the soluble fraction, C: the insoluble fraction. (B) Nature of cross-linked complexes was assessed by immunoblotting with ani-OutC (lane 1), anti-OutD (lane 2)and anti-GST (lane 3) antibodies.

Fig. VI. 7 :

 7 Fig. VI. 7: In vitro cysteine bonding analysis between OutC 60- 272 V153C and OutD 28-285 T53C . The eluted proteins were either treated with CuP (+) or not (-), separated by SDS-PAGE under non-reducing conditions and then stained with Coomassie G250. D-D: cross-linked OutD 28-285 homodimer, C-D: crosslinked OutC-OutD heterodimer, C-C: OutC 60-272 homodimer.

  OutCsip (secretin interacting peptide, residues 139-158) interacts in vitro with two distinct sites of the N-terminal (periplasmic) region of OutD: one located in the N0 subdomain and another overlapping the N2-N3' subdomains. We found that two distinct regions of OutD interact either with the same site or with two partially overlapping sites located within OutCsip segment.Moreover, the binding of one interacting region (N0 of OutD) to OutCsip increases the binding of the other interacting region (N2-N3' of OutD). Conversely, the binding of N2-N3' to OutCsip reduces the binding of N0 to OutCsip. Mutagenesis analysis shows that OutCsip is critical for the function of the T2SS. A single substitution, V143S, located within OutCsip prevents its interaction with N2-N3' and fully inactivates the T2SS. The thesis of Mr. Login showed that the N0 subdomain of OutD interacts also with a second binding site within OutC located in the region proximal to the transmembrane segment. We thus suggest that successive interactions between these distinct regions of OutC and OutD may have functional importance in switching the secretion machine.To better understand molecular mechanisms that govern OutC-OutD interactions, we performed structural analysis of OutC and of OutC-OutD interactions by NMR in collaboration with the team of Prof. Pickersgill from Queen Mary University of London (Chapter II). The solution structure showed that the HR domain of OutC adopts a β-sandwich-like fold constituted by two β-sheets, each composed of three antiparallel β-strands. The two β-sheets are connected together through several conserved hydrophobic interactions, notably, I113-V143 couple. This has been further confirmed by in vivo cysteine bonding analysis. The solution structure of the complex formed by the HR domain of OutC and N0 domain of OutD suggested that the HR/N0 interface involves a pair of antiparallel β strands, β1 of HR and β3 of N0. However, in vivo cysteine bonding analysis detected only low amounts of the corresponding OutC-OutD complex.

Fig. 73 :

 73 Fig. 73: Proposed models. (A) A model of a dodecameric ring with C6 symmetry formed by HR domains of OutC. The HR domain comprises two β-sheets which are represented by dark blue indicated 1 consisting of β1, β2 and β3, and light blue indicated 2 consisting of β5, β6 and β7, respectively. The two distinct sites involving in homodimerization are represented by orange and magenta dots. The homodimerization of HR the first β-

Table .

 .

	VI. 1: Plasmids employed in this chapter.
	General conclusions and perspectives
	Fig. 73: Proposed models.

  have shown that both the PDZ domain of OutC and the N-terminal domain of OutD are required for specific recognition of secreted proteins. And the proteins secreted by E. chrysanthemi Out system could be divided into three main groups: i) most of the pectate lyases, for example, PelB, C, D, E and PelL, whose secretion is dependent on the presence of the PDZ domain of OutC and the N-terminal domain of Thus, Bouley et al. (2001) proposed that diverse exoproteins possess a variable number of targeting signals which are recognized by different regions of OutC and OutD.

		OutD;
	ii)	Cel5, that is secreted in a PDZ-independent manner but require the presence of the N-
		terminal domain of OutD for secretion;
	iii)	PemA and PelI, whose secretion is PDZ-independent and is mediated as much by OutD
		of E. chrysanthemi as by OutD of E. carotovora.

Table . V

 . . 1: Plasmids employed in this chapter.

	Plasmid	Relevant characteristic(s)	Reference
	pT7-6	The Φ10 promoter of T7, Amp R	Tabor Richardson, 1985	and
	pACT3	The tac promoter, Cm R	Dykxhoorn et al., 1996
	pACT3-oS	pACT3 carrying outS under Ptac	Chapter III
	pACT3-oSoB	pACT3 carrying outS and outB under Ptac	This chapter
	Vectors coexpressing OutC and OutD cysteine variants under the control of PpelC
	pTdB-oC V144C -oD T53C	outC (V144C) and outD (T53C)	Chapter III
	pTdB-oC V144C -oD V232C	outC (V144C) and outD (V232C)	Chapter III
	pTdB-oC V144C -oD VI271C	outC (V144C) and outD (V271C)	Chapter III
	pTdB-oC V153C -oD T53C	outC (V153C) and outD (T53C)	Chapter III
	pTdB-oC V153C -oD V232C	outC (V153C) and outD (V232C)	Chapter III
	pTdB-oC V153C -oD V271C	outC (V153C) and outD (V271C)	Chapter III
	Vectors coexpressing PelB with OutC and OutD cysteine variants under the control of PpelC
	pTPLB-oC V144C -oD T53C	pelB followed by outC (V144C) and outD (T53C)	Chapter III
	pTPLB-oC V144C -oD V232C	pelB followed by outC (V144C) and outD (V232C)	Chapter III
	pTPLB-oC V144C -oD V271C	pelB followed by outC (V144C) and outD (V271C)	Chapter III
	pTPLB-oC V153C -oD T53C	pelB followed by outC (V153C) and outD (T53C)	Chapter III
	pTPLB-oC V153C -oD V232C	pelB followed by outC (V153C) and outD (V232C)	Chapter III
	pTPLB-oC V153C -oD V271C	pelB followed by outC (V153C) and outD (V271C)	Chapter III

Table . V

 . . 2: Primers used in this chapter. Mutated or introduced bases are in bold.

Table .

 . VI. 1: Plasmids employed in this chapter.

	Plasmid	Relevant characteristic(s)	Reference
	Vectors co-expressing GST-OutC and OutDHis derivatives
	pGX-oC 60-272 -oD 28-285	pGEX-6P-3 carrying GST-outC (aa 60 to 272) followed by outD-6His (aa 28 to 285)	Chapter I
	pGX-oC 60-272 -oD 28-285 (G190C)	pGEX-6P-3 carrying GST-outC (aa 60 to 272) followed by outD-6His (aa 28 to 285 with G190C)	This chapter
	pGX-oC 60-272(V143C) -oD 28-285

  The vector expressing either GST-OutC 60-272 or GST-OutD 28-285 or coexpressing GST-OutC 60- 272 together with OutD 28-285 (Table.VI. 1 and Fig. VI. 1) were transformed into E. coli BL21 (DE3) cells. The proteins were purified or copurified by affinity chromatography on Glutathione Sepharose (Materials and methods). Then we performed a limited proteolysis with trypsin of either purified OutC 60-272 or OutD 28-285 or a complex of these two protein derivatives (Materials and methods). Limited proteolysis assays revealed that OutD 28-285 alone appeared far more susceptible to proteolysis than OutD 28-285 in complex with OutC 60-272 . These results suggested that OutC 60-272 can stabilize the structure of OutD 28-285 and prevent its degradation (data not shown). This implies that these two proteins interact with each other in vitro. Therefore, we used this phenomenon to identify the protein regions protected from proteolysis. Several relatively

	stable derivatives of OutC and OutD were obtained by limited proteolysis (Fig. VI. 2). The N-
	terminal (Edman) analysis was performed to identify the sequence of these derivatives. Certain
	of these derivatives have been characterized (Fig. VI. 3) and sent to our collaborators in London
	Univeristy for structural analysis.

Cette thèse est accessible à l'adresse : http://theses.insa-lyon.fr/publication/2012ISAL0010/these.pdf © [X. Wang], [2012], INSA de Lyon, tous droits réservés

Acknowledgements

PVDF Polyvinylidene fluoride RG-I Rhamnogalacturonan-I RG-II Rhamnogalacturonan-II RND Nodulation and cell division RNC

Table . 1: Nomenclature used for Gsp and Xcp T2SSs, Pil T4aP in Gram-negative bacteria P. aeruginosa [START_REF] Hazes | Towards a systems biology approach to study type II/IV secretion systems[END_REF]Ayer et al., 2010).

III.6.1 The secretin

The secretin family is conserved in different secretion and export systems (Fig. 62) and is known to form oligomeric ring-like structures in the OM of Gram-negative bacteria. It consists of a variable N-terminal domain that resides in the periplasm and a conserved C-terminal domain that inserts in the OM where it forms a pore conduit for pili or secreted proteins (see the Chapter III.3.1.2, the secretins of T2SS). The micromolecular complexes formed by several secretins from T2SS, T3SS, T4P and filamentous phage extrusion systems have been studied by EM analysis [START_REF] Reichow | Structure of the cholera toxin secretion channel in its closed state[END_REF][START_REF] Hodgkinson | Threedimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout[END_REF]Jian et al., 2011;[START_REF] Opalka | Structure of the filamentous phage pIV multimer by cryo-electron microscopy[END_REF].

Recently, atomic resolution structures of periplasmic domain of secretins from T2SS and T3SS

have been solved (see Chapter III.3.1.1, page 55) (Korotkov et al., 2009a;[START_REF] Spreter | A conserved structural motif mediates formation of the periplasmic rings in the type III secretion system[END_REF]