To the memory of my beloved grandfather Ali Abidi

First and foremost, I would like to express my sincere gratitude to the very inspiring supervisor Dr. Petya KRASTEVA for her guidance, motivation and support. Thank you for giving me the opportunity to join SBB Lab for my master's internship first and to stay as a PhD student. Being an experimentalist as well, she provided continuous and valuable guidance to experiments and methodology. I felt trust and encouragement to explore new tracks to achieve our scientific goals. From the first steps, she gave me the right balance between freedom and guidance allowing me to work independently, she even gave me a more general lab responsibility that contributed to building my confidence and constructed a valuable experience. I would like next to thank my ex-colleague and friend Dr. Samira ZOUHIR for the enriching discussions and support. We started working hand in hand on the same project, which was a valuable and fruitful experience. Thank you for the day-to-day advice and for making the PhD a fun experience both inside and outside the lab. I would also like to thank my colleague and friend Dr. Marion DECOSSAS, from whom I learnt a lot both professionally and personally. Thank you for your great support and for many life lessons. I couldn't ask for a better person to share an office with. A big thanks to the friend and colleague Lucía TORREZ-SANCHEZ for supporting me and for the great discussions we had. My PhD experience would not have been the same without you. Thanks to the intern and ex-colleague Lucie PUYGRENIER, I was lucky to work with you. I would like to thank my colleague Dr. Axel SIROY for the valuable advice and discussions. Thanks to Dr. Stéphane ROCHE for being our host in our first lab in Gif-sur-Yvette Paris and for the constructive discussions about X-ray crystallography.

Big thanks to Dr. Rémi FRONZES and Dr. Esther MARZA for their help and support when we first moved our lab to the IECB and for their continuous guidance and advice. Many thanks to all group members working with Dr. Rémi FRONZES, Dr. Axel INNIS and Dr. Yaser HASHEM's for the informative discussions about the cryo-EM data collection and processing. Thanks also to Armel BEZAULT for his training in grid preparation and data collection.

These last years of my life were marked by this life-changing PhD journey, during which many people contributed to make it easier and enjoyable. To name a few of such people, I would like to give many thanks to Laure BATAILLE, Myriam MEDERIC and Thierry DAKHLI for the motivational and supportive discussions.

Thanks to the new friends I made in IECB Coralie ROBERT, Julien MARQUEVIELLE and Pierre NOTTELET for the scientific and non-scientific discussions and for the great moments that we shared together. I would also like to thank my friends Cyrine and Olfa. I want to thank you for the great moments, for the nights and days that we spent together. Thanks also to Mayen, Abir, Marwa and Houwaida for their support. I also want to thank my friends Eva, Mathilde and Yasmina, I was lucky that you were the first friends I made in France after coming from my native country Tunisia. From the time of the amphitheaters of Paris Saclay university, you were always there for me, and I hope to continue sharing each-others important life steps.

It is a real pleasure to thank my sister and best friend Imene and my brother-in-law and friend Hamza, to whom I owe a lot. Thank you for your unconditional love and support. Many thanks go to my brother Ahmed who taught me perseverance.

From the bottom of my heart, I thank my parents Neila and Hassan, to whom I owe everything. I could not have completed this dissertation without the support of my husband Ahmed, I am grateful for having you in my life. Thank you for your love, encouragement and patience. extracellulaires) [START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF], [2], [5]. Les EPS sont principalement composés d'acides nucléiques, de lipides, de protéines et de polysaccharides [START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF]. Parmi ces polysaccharides se trouve la cellulose, composant majeur de la matrice extracellulaire chez de multiples souches commensales ainsi que pathogènes [6], [START_REF] Zogaj | The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix[END_REF]. De par sa résistance mécanique, la cellulose confère aux bactéries une protection contre les environnements hostiles. Dans les biofilms bactériens, la sécrétion de cellulose par les bactéries à Gram-négatif nécessite la co-expression de gènes multiples dont les différentes sous-unités qui en résultent s'assemblent pour former un complexe de sécrétion traversant l'intégralité de la paroi bactérienne [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF], [START_REF] Krasteva | Insights into the structure and assembly of a bacterial cellulose secretion system[END_REF]. Ce complexe stable possède une partie périplasmique, une partie que réside dans la membrane interne et une partie cytoplasmique. La structure de ce mégacomplexe a été caractérisée en microscopie électronique grâce à la technique dite de particule isolée en coloration négative par le Dr. P. Krasteva et ses collaborateurs [START_REF] Krasteva | Insights into the structure and assembly of a bacterial cellulose secretion system[END_REF]. Malgré l'apport important de cette structure, la faible résolution du complexe n'a pas permis la distinction des différents composants protéiques. De plus, plusieurs travaux intéressés par l'étude de la sécrétion de la cellulose se sont focalisés sur les deux sous unités catalytiques, négligeant les sous unités cytoplasmiques régulatrices qui sont ainsi longtemps restées énigmatiques [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF]- [START_REF] Morgan | Observing cellulose biosynthesis and membrane translocation in crystallo[END_REF].

Table of figures

État de l'art

La sécrétion de cellulose chez les bactéries à Gram-négatif nécessite la co-expression de gènes multiples qui sont conservés et répandus chez de multiples bactéries commensales et pathogènes [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF]. Le système de gènes bactériens est typiquement organisé en un ou deux opérons appelés bcs (bacterial cellulose synthesis) [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF]. BcsQ a été localisée, l'adhésion intercellulaire y est initiée par la production de cellulose [START_REF] Quéré | BcsQ is an essential component of the Escherichia coli cellulose biosynthesis apparatus that localizes at the bacterial cell pole[END_REF].

BcsE

BcsE est une protéine cytoplasmique nécessaire à la sécrétion maximale de cellulose et qui possède un domaine de liaison au c-di-GMP -une molécule clé pour la signalisation intracellulaire [START_REF] Krasteva | Insights into the structure and assembly of a bacterial cellulose secretion system[END_REF], [START_REF] Quéré | BcsQ is an essential component of the Escherichia coli cellulose biosynthesis apparatus that localizes at the bacterial cell pole[END_REF]. La protéine BcsE se lie au c-di-GMP via le motif RxxD du domaine GIL (GGDEF I-site-Like), situé à son extrémité C terminale [START_REF] Fang | GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria[END_REF]. Ce motif similaire au site I des domaines GGDEF des diguanylate cyclases, les enzymes qui produisent le c-di-GMP à partir de GTP, est crucial à la fonction de BcsE. Des études de notre groupe et en collaboration ont aussi montré que la présence de BcsE dans le complexe est nécessaire pour la stabilité de la sous-unité catalytique BcsA et que les deux protéines interagissent directement à travers leurs modules de liaison au c-di-GMP (domaine GIL de BcsE et domaine PilZ de BcsA) [START_REF] Krasteva | Insights into the structure and assembly of a bacterial cellulose secretion system[END_REF]. Ces résultats nous ont alors montré l'importance d'étudier cette protéine en détail afin de déterminer son rôle et sa contribution dans l'assemblage et la formation du complexe Bcs.

Le second messager c-di-GMP contrôle la formation des biofilms

Dans la plupart des organismes bactériens, le changement entre l'état motile et l'état stationnaire sous forme de biofilm est exercé par le c-di-GMP, un second messager intracellulaire qui a des effets multiples permettant d'inhiber, directement ou indirectement, la motilité flagellaire et de stimuler la sécrétion des composants de la matrice extracellulaire tels que les exopolysaccharides [START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF], [START_REF] Petrova | A Novel Signaling Network Essential for Regulating Pseudomonas aeruginosa Biofilm Development[END_REF], [START_REF] Hengge | Principles of c-di-GMP signalling in bacteria[END_REF]. La présence intracellulaire du c-di-GMP est contrôlée par deux activités enzymatiques opposées : les diguanylate cyclases avec leur domaine GGDEF le synthétisent et des phosphodiestérases possédant un domaine EAL ou HD-GYP le dégradent [START_REF] Chan | Structural basis of activity and allosteric control of diguanylate cyclase[END_REF]- [START_REF] Schmidt | The Ubiquitous Protein Domain EAL Is a Cyclic Diguanylate-Specific Phosphodiesterase: Enzymatically Active and Inactive EAL Domains[END_REF].

Objectifs de la thèse

Conclusion et perspectives

Les dernières années représentent une révolution dans l'étude de la cellulose. Les études antérieures se sont intéressées aux sous-unités catalytiques des systèmes bactériens de biogenèse de la cellulose, pendant que les sous-unités régulatrices sont longtemps restées énigmatiques. Notre groupe de recherche a largement marqué cette révolution de par l'identification des rôles mécanistiques de sous-unité accessoires et essentielles, exprimées par les bactéries dans les biofilms bactériens. Ces travaux de recherche ont concerné différents

Introduction

Bacteria can adopt two main modes of growth in nature, the first being the free-swimming planktonic lifestyle, while the second is the formation of sessile biofilms [START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF], [2]. Biofilms are bacterial cells living in a community and embedded in a self-produced matrix, usually attached to a biotic or abiotic surface [START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF], [2]. The biofilm matrix provides the embedded bacteria with tolerance, fitness and protection against the external world, among other privileges [START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF], [3], [4].

Thereby from an evolutionary point of view, the bacteria have more survival reasons to optimize mechanisms for the secretion of a biofilm matrix. Biofilms can be seen as an empire

where bacterial cells live in a community surrounded by a fortress, the stronger the fortress walls, the more protected are the individual cells. Although the biofilm lifestyle is a common trait, the secreted biopolymers in the extracellular matrix, also known as the Extracellular Polymeric Substances (EPS), differ between the species by the type of matrix components, the underlying secretion machineries, as well as the external cues that orchestrate the mechanisms engaged in the EPS secretion [START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF], [2], [5]. The EPS typically includes polysaccharides, proteins, nucleic acids and lipids (Table 1) [2]. One thing is for sure, the key signaling molecule c-di-GMP (Bis-(3′-5′)-cyclic dimeric guanosine monophosphate), promotes the synthesis of many of the EPS components in biofilms [6], [START_REF] Römling | Cyclic di-GMP: the first 25 years of a universal bacterial second messenger[END_REF]- [START_REF] Krasteva | Versatile modes of cellular regulation via cyclic dinucleotides[END_REF]. Usually through a c-di-GMP-guided pathway, as a key example, many bacteria produce cellulose as a major component of their extracellular matrix. The polymer has a large number of advantages, such as excellent mechanical resistance and water retention and, together with curli, builds up the biofilms of important enterobacterial species such as Salmonella enterica Serovar Typhimurium (S.

Typhimurium) and Escherichia coli (E. coli), [6], [START_REF] Zogaj | The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix[END_REF].

In the present work, I decipher key components and mechanisms that drive cellulose secretion in the biofilm matrix. In this introduction part, I will provide the necessary background for better appreciation of our findings uncovering the mechanisms of bacterial cellulose secretion. I will provide an overview of cellulose as one of the major polysaccharides secreted by bacteria in the extracellular biofilm matrix. I will also discuss the conservation of cellulose secretion systems, as well as the components involved in this secretory mechanism with the second messenger c-di-GMP in the center of this sophisticated process. As unraveling all the Bcs (bacterial cellulose synthesis) systems is beyond the aim of this project, I rather spot an emphasis on cellulose secretion in Gram-negative bacteria and more precisely on the type II Bcs secretion system also known as the E. coli-like Bcs system.

The matrix of biofilms

Key steps for biofilm formation

The process of biofilm formation is cyclic and, although it can feature various degrees of complexity depending on external cues and the specific bacterial species, it can be summarized in three major steps (Figure 1). The overall process is typically under the control of intracellular c-di-GMP-dependent signaling events and comprises the attachment of planktonic cells to a surface, the multiplication and maturation into a multicellular three-dimensional structure and finally the dispersion of bacterial cells that can then colonize new surfaces [START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF], [START_REF] Petrova | A Novel Signaling Network Essential for Regulating Pseudomonas aeruginosa Biofilm Development[END_REF], [START_REF] Sauer | Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm[END_REF].

Biofilms are truly collaborative multicellular communities, where the extracellular matrix provides not only mechanical protection but also a medium for exchange of nutrients, horizontal gene transfer (including genes for antibiotics resistance) and immune system escape, when in a eukaryotic host.

Regarding biofilm dispersion, on one hand it allows the bacteria to disseminate, on the other, the bacterial cells that leave the biofilm become exposed and unprotected. The dispersion takes place when remaining in the biofilm displays more drawbacks than advantages, for example upon development of oxygen or waste product gradients that can lead to cell lysis [START_REF] Rumbaugh | Biofilm dispersion[END_REF]. To tackle such toxicity and survive, many signals can trigger the activation of phosphodiesterases (PDE) that degrade the c-di-GMP second messenger and thereby inhibit EPS secretion and increase the cells' motility [START_REF] Hengge | Principles of c-di-GMP signalling in bacteria[END_REF].

Figure 1. Key steps of the biofilm formation

Bacteria can reversibly attach to surfaces subsequently to the loss of motility and synthesis of extracellular adhesive substances providing strong anchors. Mature biofilms are then developed and are characterized by colony variance, complex architecture, and collaborative group behavior between functionally differentiated members. Finally, the dispersion leads to the release of highly motile planktonic cells, capable to colonize new surfaces [4], [START_REF] Rumbaugh | Biofilm dispersion[END_REF].

Cellulose, a major constituent of the bacterial biofilm maze, and more

The biofilm matrix is a hydrated mass that apart from intact cells contains a matrix of polysaccharides, proteins, nucleic acids and lipids [2]. The different secreted components play a plethora of functions that are relevant to the biofilm growth, some of which are summarized in Table 1. Although it has been generally difficult to analyze the extracellular matrix components, technological developments have allowed for the characterization of many of them. One of these components is cellulose, which is the second major component of S. Typhimurium and E. coli biofilms. Together with curli proteins that upon secretion assemble into amyloid fibers, cellulose was discovered to provide agar-grown biofilms with the ability to form sophisticated three-dimensional structures, such as the "rdar" (for red, dry, and rough) morphotype shown in figure 2 [START_REF] Zogaj | The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix[END_REF].

Table 1. Examples of EPS and their functions in bacterial biofilms

EPS component Function in biofilms

Polysaccharides

Figure 2. Rdar morphotypes of E. coli and Salmonella strains

Rdar morphotypes of Salmonella and E. coli strains grown on Congo red plates. Figure from [START_REF] Zogaj | The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix[END_REF] Looking at a larger picture, cellulose has well-known reputation as the major constituent of the plants cell walls, making it the most abundant biopolymer on the planet Earth [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF]. In addition to plants, however, cellulose is also produced by protists, fungi, animals, viruses, and of course bacteria, with Cyanobacteria being the first cellulose producers and the last common ancestor of cellulose biosynthesis genes [START_REF] Little | Revised Phylogeny of the Cellulose Synthase Gene Superfamily: Insights into Cell Wall Evolution[END_REF], [START_REF] Nobles | Cellulose in Cyanobacteria. Origin of Vascular Plant Cellulose Synthase[END_REF].

So, why do bacteria secrete cellulose? Simply put, for its exceptional water retention capacity, porosity, mechanical resistance and chemical simplicity, combined with its low antigenicity and ability to interact with additional components from the bacteria or their hosts [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF].

In the present project, I will spot the light on the enterobacterial cellulose secretion system, as a widespread nanomachine for bacterial biofilm formation.

Structure and forms of bacterial cellulose

Cellulose is an unbranched homopolysaccharide of D-glucose molecules connected via acetal linkages joining the C1 and C4 carbons of the glucopyranose rings in a ß-configuration [START_REF] Nishiyama | Crystal Structure and Hydrogen-Bonding System in Cellulose Iβ from Synchrotron X-ray and Neutron Fiber Diffraction[END_REF] (Figure 3A). The added glucose is rotated by approximatively 180° compared to its neighbor, as a result the repeating unit is the disaccharide cellobiose [START_REF] Gardner | The structure of native cellulose[END_REF] (Figure 3A-B). Along the linear polysaccharide, each glucose moiety forms two hydrogen bonds with each of its neighbors. The C3 hydroxyl of a given glucose unit engages in a hydrogen bond with the ring oxygen of the glucose neighbor attached at the C4 carbon, while the C6 donates a hydrogen bond to the C2 hydroxyl of the same neighboring moiety (Figure 3A). On the other hand, the ring oxygen and the C2 hydroxyl accept a hydrogen bond from the C3 and C6 hydroxyls of preceding glucose unit, respectively, i.e. the glucose unit attached to the C1 carbon [START_REF] Nishiyama | Crystal Structure and Hydrogen-Bonding System in Cellulose Iβ from Synchrotron X-ray and Neutron Fiber Diffraction[END_REF] (Figure 3A-B). These connections not only stabilize a coplanar conformation of the glucopyranose rings, moreover, the hydroxyl groups point away from the face of the pyranose rings resulting in a facility of engaging in different interactions with other cellulose polymers (Figure 3A). Even more, chemical modifications can occur on the building glucose units, such as the attachment phophoethanolamine (pEtN residues at the C6 position of the cellulose polymer produced by E. coli and Salmonella) [START_REF] Thongsomboon | Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose[END_REF] (Figure 3C). The chemical properties of the cellulose polymer all together bring the existence of different forms of bacterial cellulose. In bacteria, the cellulose is synthesized and extruded through the Bcs system and once at the cell surface, a subsequent assembly of the glucan chain can follow forming either a structured architecture also known as crystalline cellulose or more disordered structure known as amorphous cellulose.

Crystalline cellulose

Crystalline cellulose, has drawn the attention over the past decades due to its architecture reminding that of the plant cell walls. The different cellulose strands pack against each other forming hydrogen bonds between the equatorial hydroxyl groups and van der Waals forces between the glucopyranose rings (Figure 4A-B-C). Early studies using negative-stain, freezefracture electron microscopy revealed that in crystalline cellulose-secreting Gluconacetobacter xylinus, the cellulose synthase terminal complexes (TCs) form a line along the long axis of the cell surface which corresponds to the export sites of the elementary cellulose fibrils [31]- [START_REF] Benziman | Cellulose biogenesis: Polymerization and crystallization are coupled processes in Acetobacter xylinum[END_REF].

Once secreted in the extracellular environment, the elementary fibrils pack to form the cellulose ribbon, one per cell [31]- [START_REF] Benziman | Cellulose biogenesis: Polymerization and crystallization are coupled processes in Acetobacter xylinum[END_REF]. The cellulose biosynthesis involves a machinery encompassing the cell envelope, where a catalytic core composed of the BcsAB tandem is involved in the polymerization of the glucan chain using UDP (uridine diphosphate)-glucose as a substrate (Figure 6). In most systems of Gram-negative species, including crystalline cellulose secreting bacteria, the export of the nascent chain through the outer membrane (OM) involves an OM porin called BcsC. The structure of the latter subunit from pEtN-cellulose secreting E. coli has been solved recently and revealed that the protein adopts a ß-barrel fold at its C-terminus, however the pore diameter would be compatible with the extrusion of only a single-stranded cellulose chain [START_REF] Acheson | Architecture of the Cellulose Synthase Outer Membrane Channel and its association with the Periplasmic TPR Domain[END_REF]. Therefore, crystalline microfibril and ribbon formation must occur in the external environment, parallel to the longitudinal cell axis, however, it would be predetermined by the longitudinal TC microarray formation in the cell. Other factors, such as accessory subunits BcsHD thought to form individual cellulose chain conduits in the periplasm and a recently identified cytoskeletal element thought to stabilize the longitudinal TC array have also been shown to play key roles in cellulose crystallinity [START_REF] Sunagawa | Cellulose complementing factor (Ccp) is a new member of the cellulose synthase complex (terminal complex) in Acetobacter xylinum[END_REF]- [START_REF] Nicolas | Structure of the Bacterial Cellulose Ribbon and Its Assembly-Guiding Cytoskeleton by Electron Cryotomography[END_REF]. Nevertheless, the molecular mechanisms behind crystalline cellulose formation remain a missing piece in the puzzle and need to be better unraveled for the production of the bacterial cellulose for biotechnological applications.

Amorphous cellulose

Amorphous cellulose is disordered and non-structured cellulose that does not follow a pattern of regularly packed fibrils or layers (Figure 4B, bottom) and is generally believed to be easier to unravel when compared to the crystalline type. Interestingly, amorphous cellulose can also be commonly found in biofilms with high crystalline cellulose I content, marking the borders between large crystalline domains.

Types of bacterial cellulose secreting systems

The mechanisms governing the secretion of bacterial cellulose have sparked intense interest since the polymer's discovery in bacteria at the end of the 19th century [START_REF] Brown | XLIII.-On an acetic ferment which forms cellulose[END_REF]. Many studies have since aimed at the elucidation of cellulose biosynthesis mechanism and the subunits involved in the process. Consequently, many nomenclatures have been attributed to the different subunits, so in 2015 Römling and Galperin revisited the classification of bcs operons and proposed a novel unified nomenclature for bcs proteins and their encoding genes [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF]. The same study highlighted the diversity among the different coding operons among bacterial species and clades and divided the majority of bcs loci into three major types [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF] (Figure 5A-B) (see below for more details). The common point to all three systems is the catalytic subunit BcsA with its partner BcsB (Figures 5A-B-6). BcsA is the inner membrane protein composed of multiple transmembrane helices that stand above two cytosolic domains, a glycosyltransferase domain that catalyzes the polymerization of UDP-glucose into the β-1,4-linked glucan and a C-terminal c-di-GMP-sensing PilZ domain [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF], [START_REF] Morgan | Mechanism of activation of bacterial cellulose synthase by cyclic-di-GMP[END_REF], [START_REF] Lin | Identification of the uridine 5'diphosphoglucose (UDP-Glc) binding subunit of cellulose synthase in Acetobacter xylinum using the photoaffinity probe 5-azido-UDP-Glc[END_REF]. The majority of BcsB's amino acid sequence occupies a large periplasmic donut-shaped module, comprising two flavodoxin-like domains and two carbohydrate-binding domains, the latter of which are proposed guide the nascent polysaccharide to the outer membrane [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF], [START_REF] Morgan | Mechanism of activation of bacterial cellulose synthase by cyclic-di-GMP[END_REF], [START_REF] Abidi | Architecture and regulation of an enterobacterial cellulose secretion system[END_REF] (Figure 6). In addition, BcsB is anchored in the inner membrane by its C-terminal tail, composed of an amphipathic extracytosolic helix and a transmembrane anchor helix. Previous in vitro studies have demonstrated that, in both E.

coli and Rhodobacter sphaeroides (R. sphaeroides), this C-terminal tail is indispensable for cellulose polymerization, thereby, BcsB is characterized as the co-catalytic subunit, or copolymerase. In several cases, BcsA and BcsB genes are fused and likely post-translationally divided into two proteins [START_REF] Mcmanus | AcsA-AcsB: The core of the cellulose synthase complex from Gluconacetobacter hansenii ATCC23769[END_REF]. In addition to these core catalytic subunits, most bcs operons in Gram-negative bacteria encode a periplasmic hydrolase BcsZ and an outer membrane (OM) secretory subunit BcsC composed of an N-terminal tetratricopeptide repeat (TPR)-rich periplasmic domain and a C-terminal OM porin domain.

Most cellulose secretion systems feature multiple additional Bcs subunits, many of which are indispensable for cellulose secretion. In addition, certain subunits typically co-occur with others, which prompted the recent classification of cellulose secretion systems based on the accessory to the catalytic BcsAB tandem subunits [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF].

Type I cellulose secretion system

The type I cellulose secretion system is found in α, ß and γ subdivisions of proteobacteria. This type includes the core components genes bcsABZC, as well as the hallmark of this type of secretion system gene bcsD (Figure 5A-B). In G. xylinus, the encoded protein BcsD is proposed to localize to the periplasm and to contribute to the packing of the glucan chains into ordered cellulose ribbons also known as type I crystalline cellulose [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF], [START_REF] Saxena | Characterization of genes in the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization[END_REF]. Added to that, upstream of the bcsD gene is found bcsH (also known as "ccpAx" or "ORF2") whose protein product interacts with BcsD and is also suggested to play a role in crystalline fibril assembly [START_REF] Sunagawa | Cellulose complementing factor (Ccp) is a new member of the cellulose synthase complex (terminal complex) in Acetobacter xylinum[END_REF].

Nevertheless, whereas the BcsH subunit seems limited to the Komagataeibacter lineage, many types I bcs operons are found in non-crystalline cellulose-secreting bacteria, suggesting broader roles for the hallmark BcsD subunit. Additional Bcs components can also be found in these Type I systems, such as the BcsO, BcsP, and BcsQ subunits but their functions have not been characterized yet [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF].

Type II cellulose secretion system

The type II cellulose secretion systems are widespread among ß-and g-Proteobacteria and especially enterobacterial pathogens [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF]. In Salmonella and E. coli, the bcs locus is divided into two divergently arranged operons, bcsEFG and bcsRQABZC [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF] (Figure 5A-B). The discriminating components of this type are the cytosolic c-di-GMP-sensing subunit BcsE and the periplasmic membrane-anchored BcsG responsible for installing the pEtN modification in the secreted amorphous cellulose [START_REF] Thongsomboon | Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose[END_REF]. In addition, the BcsR, BcsQ and BcsF subunits, whose functions are discussed below in more details, are also encoded by the respective bcs operons.

Type III cellulose secretion system

The type III secretion systems are limited to some cyano-and α-Proteobacterial species [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF].

They lack the three subunits BcsD, BcsE and BcsG, whereas BcsC subunit is replaced by another outer membrane TPR-rich subunit, BcsK, which lacks the C-terminal porin module [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF] (Figure 5A-B).

Other types of cellulose secretion

There are additional types of cellulose secretion systems that don't fit into the standard picture of any of the types above or in some cases can feature a hybrid architecture. Examples include systems where the type I hallmark bcsD gene, can be encoded with the type II bcsE(F)G cluster.

In other examples, such as in the cellulose-producing cyanobacterium Thermosynechococcus vulcanus, both bcsB and bcsC genes are missing and the BcsA-mediated cellulose polymerization and extrusion are assisted by an efflux pump-like tandem composed of an innermembrane HlyD-like subunit and an outer membrane TolC-like exporter [START_REF] Maeda | Genetic identification of factors for extracellular cellulose accumulation in the thermophilic cyanobacterium Thermosynechococcus vulcanus: proposal of a novel tripartite secretion system[END_REF]. Further 'outsiders' involve Pseudomonas fluorescens SBW25, which is known to secrete acetylated cellulose and exhibits a hybrid wss operon, combining BcsQ-and BcsABZC-like components, together with an acetylation complex homologous to the alginate secretion system. In accordance with the omnipresence of cellulose secretion machineries among many diverse organisms, some Gram-positive bacteria are also found to secrete cellulose. Studies on Streptomyces coelicor have shown that this actinomycete secretes cellulose in order to adapt to osmotic responses or growth of the hyphal tips [START_REF] Xu | A Cellulose Synthase-Like Protein Involved in Hyphal Tip Growth and Morphological Differentiation in Streptomyces[END_REF], [START_REF] Liman | A laterally acquired galactose oxidase-like gene is required for aerial development during osmotic stress in Streptomyces coelicolor[END_REF] and uses a yet different cellulose secretion system, some components of which appear to have a fungal origin.

Covering all the so far described systems of cellulose secretion is beyond this work, however this part highlights both the diversity and the complexity of the cellulose secreting systems.

Moreover, with the ever-growing abundance of genomics data, the arborescence of Bcs secretion systems keeps growing and embraces now many Gram-positive bacteria, for whom many questions remain unanswered, especially concerning exopolysaccharide secretion through the thick peptidoglycan layer or the Gram-positive cell envelope devoid of an outer membrane [START_REF] Silhavy | The Bacterial Cell Envelope[END_REF].

Figure 5. Prevalent types of Bcs systems

The three major types of the Bcs systems and their corresponding operons organizations as classified in [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF], [START_REF] Abidi | Weaving of bacterial cellulose by the Bcs secretion systems[END_REF].

Mechanism for cellulose biosynthesis

Across the various organisms that secrete cellulose, a shared mechanism of cellulose polymerization is determined by an enzyme belonging to the glycosyl transferase family 2 [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF], [START_REF] Omadjela | BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis[END_REF], [START_REF] Römling | Molecular biology of cellulose production in bacteria[END_REF]. The latter catalyzes the reaction of cellulose synthesis by the addition of one glucose at a time to the nascent polysaccharide using a preactivated nucleotide sugar substrate, UDPglucose. UDP-glucose is made available by a dedicated enzyme, UDP-glucose pyrophosphorylase or UDPGP, which is essential for cellulose secretion [START_REF] Valla | Cloning of a gene involved in cellulose biosynthesis in Acetobacter xylinum: complementation of cellulose-negative mutants by the UDPG pyrophosphorylase structural gene[END_REF]. Upon incorporation of the glucose moiety from the substrate into the nascent polysaccharide, the UDP product is then released back in the cytosol for its recycling. The formerly attached glucose through an α-configuration to the UDP, is inverted to be consequently attached to the glycosidic chain in a ß-configuration [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF], [START_REF] Lairson | Glycosyltransferases: Structures, Functions, and Mechanisms[END_REF]. The catalytic reaction is basically a SN2-like substitution reaction, in which the nucleophile C4-hydroxyl group (acceptor), attacks the C1 carbon of the donor glucose (Figure 3A). This reaction is facilitated by both a deprotonation of the nucleophile and a stabilization of the substrate's pyrophosphate group via a divalent metal cation, usually Mg 2+ or Mn 2+ [10], [START_REF] Omadjela | BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis[END_REF], [START_REF] Brown | Radiometric and spectrophotometric in vitro assays of glycosyltransferases involved in plant cell wall carbohydrate biosynthesis[END_REF]. Once the glycosidic transfer is completed at the non-reducing end in the cytosol, the glucan chain exits the bacterial cell with the reducing end pointing first to the periplasm and subsequently the extracellular milieu [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF], [START_REF] Mcnamara | A Molecular Description of Cellulose Biosynthesis[END_REF].

Subunits for bacterial cellulose secretion

Polymerization and transport across the bacterial envelope

BcsAB tandem

The bcsA and bcsB genes are widespread in almost all cellulose synthase operons, and are usually found adjacently in a tandem [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF] (Figure 5A). The structures of the catalytic BcsAB have been thoroughly studied in R. sphaeroides, whose Bcs system belongs to the type III cellulose secretion systems and secreted an amorphous polymer [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF]. The BcsA subunit, or cellulose synthase protein, is roughly a 100 kDa-sized subunit formed by a non-continuous transmembrane region, a glycosyl transferase cytosolic domain and a C-terminal pilZ domain that binds the synthase-activating second messenger dinucleotide c-di-GMP (the mechanism underlying the c-di-GMP signaling is described in more details below) [START_REF] Krasteva | Insights into the structure and assembly of a bacterial cellulose secretion system[END_REF], [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF]. The transmembrane region is an α-helical transmembrane cellulose export domain (TMD), which in R. sphaeroides is formed by eight transmembrane helices (TM1-8), four of which are Nterminal and the other four are C-terminal to the central GT domain. In addition, a single TM helix of the partner BcsB protein completes the export domain and is indispensable for the catalytic mechanism [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF] (Figure 6). Whereas BcsA's conserved glycosyl transferase (GT) domain is inserted between TM4 and TM5 [START_REF] Cantarel | The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics[END_REF], its active site is capped by a so-called 'gating loop' inserted between TM7 and and TM8 of the export domain and requires c-di-GMP binding to the protein to allow for substrate entry. The c-di-GMP sensing is carried out by a C-terminal cytosolic PilZ domain adopting a ß-barrel fold [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF] (Figure 6). The GT domain interacts with the inner leaflet of the cytosolic membrane via three amphipathic interface helices (IF1-3). To date, all structures of the duo BcsAB co-crystallized with a translocating polysaccharide, which was apparently copurified with BcsAB as the polysaccharide was not added during the purification steps [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF]- [START_REF] Morgan | Observing cellulose biosynthesis and membrane translocation in crystallo[END_REF]. The structural studies revealed that BcsA's glycosyl transferase domain is able to accommodate 10 glucose units of the nascent polysaccharide, whose glucopyranose rings form van der Waals interactions with residues Met300, Phe301, Phe316 , Trp383 of IF2, Phe419, Phe426, Tyr433, Phe441,Val551, Val555, Trp558 [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF]. The equatorial hydroxyl groups of the glucose units form hydrogen bonds with Tyr80, Asn118, His276, Asn412, Arg423, Glu439, Tyr455, Ser476, Glu477 [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF]. While the van der Waals interactions of the translocation glucan chain encompass IF1, IF2, TM5, 5/6 loops, and TM8, the hydrogen bonds are made with TM3, TM4, GT, TM5, 5/6 loops, TM6, and IF3 [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF]. The structure of BcsA's GT domain harbors a conserved motif formed by three non-sequential aspartic acids, as well as a Glutamine-Glutamine/Arginine-a random residue-Arginine-Tryptophan motif (or D, D, D, Q(Q/R)xRW) [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF]. The latter signature is also characteristic in other ßglycosyltransferases [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF]- [START_REF] Morgan | Observing cellulose biosynthesis and membrane translocation in crystallo[END_REF]. In R. sphaeroides, the first two Aspartic Acids (Asp179, Asp246) of the "D,D,D,Q(Q/R)xRW" are involved in UDP coordination, while the third aspartic acid (Asp 343) likely constitutes the catalytic base. This third astaptate is part of a strictly conserved TED (Threonine-Glutamate-Aspartate) motif of the so-called 'finger helix', which is also involved in gating loop relaxation and polysaccharide translocation [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF]. It is conventional now that the polysaccharide extrusion through BcsA transmembrane channel is assisted by its membrane-anchored partner BcsB [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF]. A study demonstrated that cellulose synthesis in vitro requires both BcsA and BcsB, and in particular BcsB's C-terminal tail of an amphipatic and a transmembrane helices [START_REF] Omadjela | BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis[END_REF]. This interdependence is further supported by the fact that in some species, bcsB gene is fused with bcsA [START_REF] Saxena | Characterization of genes in the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization[END_REF]. The latter observation has also long supported the hypothesis of an equimolar assembly of BcsAB across the bacterial domain of life, however we will show here that such an assumption is over-simplistic and that at least in the E. coli-like Bcs biosynthetic platform, the BcsAB tandem can feature a markedly different stoichiometry allowing for additional functionalities within the secretion system. Crystal structure of the complex showing a 1:1 BcsAB assembly in cartoon representation and a co-purified cellulose polymer in sticks (from protein data bank entry 4p00) [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF].

BcsC, the ß-barrel porin

BcsC is a ~130 kDa outer membrane protein whose amino acid sequence starts by a signal peptide. The mature protein, afterwards the signal peptide cleavage, adopts ~19tetratricopeptide (TPR) repeats that most likely localize in the periplasm, guide the nascent polysaccharide and/or interact with the peptidoglycan, while the C-terminal ~400 residues adopt a ß-barrel conformation and function as an outer membrane porin for cellulose export (Figure 7A). Thus, BcsC is the second checkpoint export protein after the IM (Inner Membrane)

BcsA synthase. More generally, the scheme of the porin-like domain preceded by periplasmic TPR-rich modules is a recurrent theme across exopolysaccharide secretion systems. In the alginate biosynthetic system, the secretion of alginate requires the AlgK-AlgE duo, where AlgK provides the periplasmic TPR scaffold, and AlgE functions as the outer membrane porin. In addition, the PelE-PelB complex from the Pel system exhibits the same pattern with periplasmic TPR modules from (both PelE and PelB) and a porin domain (from PelB), as well as the PgaA protein from the PNAG (poly-N-acetylglucosamine) secretion machinery, which similarly to BcsC incorporates both TPR modules and a porin domain in the same polypeptide chain [START_REF] Low | Gram-negative synthase-dependent exopolysaccharide biosynthetic machines[END_REF].

The crystal structure of a truncated versions of BcsC have been reported recently. One of these structures encompasses the last TPR motif and the C-terminal ß-barrel from the E. coli homolog. The porin is formed by 16 ß-strands whose connecting loops are longer on the extracellular side and form a dome-shaped structure on the cell surface [START_REF] Acheson | Architecture of the Cellulose Synthase Outer Membrane Channel and its association with the Periplasmic TPR Domain[END_REF]. The lumen of the ~15 Å-wide channel is highly electronegative and is suggested to facilitate the insertion of the hydrated zwitterionic pEtN-cellulose [START_REF] Acheson | Architecture of the Cellulose Synthase Outer Membrane Channel and its association with the Periplasmic TPR Domain[END_REF] (Figure 7B). The channel permeability is controlled by the extracellular loop connecting 8 ß-barrel strands 15 and 16 (also called the 'gating loop')

and is stabilized by two conserved Tyrosines (Tyr1025 and Tyr1124) belonging to extracellular loop 6 and the gating loop, respectively [START_REF] Acheson | Architecture of the Cellulose Synthase Outer Membrane Channel and its association with the Periplasmic TPR Domain[END_REF]. Furthermore, both aromatic and hydrophilic residues line the lumen of the channel suggesting that facilitated cellulose diffusion relies on both aromatic stacking and hydrogen bonding with the polysaccharide [START_REF] Acheson | Architecture of the Cellulose Synthase Outer Membrane Channel and its association with the Periplasmic TPR Domain[END_REF]. Interestingly, the C-terminal 15 residues were found inserted as an elongated loop into the lumen of the porin domain, where it is stabilized by a number of interactions, most important of which is the stabilization of the conserved Y1157 residue midway across the channel [START_REF] Acheson | Architecture of the Cellulose Synthase Outer Membrane Channel and its association with the Periplasmic TPR Domain[END_REF]. Another recent study used SEC-SAXS (Size-exclusion chromatography-coupled, small angle X-ray scattering) on a construct covering most of the TPR repeats from another BcsC homolog and revealed an overall extended solenoid architecture typical to the TPR rich periplasmic scaffolds that can span most of the intermembrane width of the periplasm [START_REF] Nojima | Crystal structure of the flexible tandem repeat domain of bacterial cellulose synthesis subunit C[END_REF]. The same study also reported a crystal structure of five sequential TPR repeats from the N-terminus of the respective BcsC homolog. Interestingly, the five protomers in the asymmetric unit adopted three different conformations suggesting an overall flexible BcsC conformation that can adapt to variability in the intermembrane periplasmic distance [START_REF] Silhavy | The Bacterial Cell Envelope[END_REF], [START_REF] Nojima | Crystal structure of the flexible tandem repeat domain of bacterial cellulose synthesis subunit C[END_REF] (Figure 7A).

Accessory subunits

BcsG and the phosphoethanolamine modification

Together with BcsE and BcsF, BcsG is specific to the type II E. coli-like secretion systems. In E. coli it is associated with the bcsEFG operon, that is localized next to the bcsRQABZC [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF].

BcsG is roughly 60 kDa and its large C-terminal domain had been first predicted to lie in the periplasm and belong to the phosphatase alkaline family [START_REF] Galperin | Divergence and Convergence in Enzyme Evolution[END_REF]. The role of this subunit had remained elusive until recent studies showed that BcsG is responsible for adding phosphoethanolamine (pEtN) groups to the nascent polysaccharide, in both Salmonella and E.

coli [START_REF] Thongsomboon | Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose[END_REF], [START_REF] Hollenbeck | Phosphoethanolamine cellulose enhances curli-mediated adhesion of uropathogenic Escherichia coli to bladder epithelial cells[END_REF] (Figure 3C). The pEtN groups are likely added from the phostphatidylethanolamine (PE) lipids of the inner membrane. Solid-state nuclear magnetic resonance spectroscopy analysis revealed that a pEtN group attach to the C6 of the glucose building blocks and initial quantification analyses estimated that up to half of the residues can carry the pEtN modification in the secreted polysaccharide [START_REF] Thongsomboon | Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose[END_REF]. The pEtN transferase activity is indeed carried out by the periplasmic C-terminal domain whereas the N-terminal part for the protein in embedded in the inner membrane. In line with biofilm formation, by interacting with curli fibers, pEtN cellulose was also shown to play a key role in promoting the adhesion of uropathogenic E. coli cells to bladder cell in vitro [START_REF] Hollenbeck | Phosphoethanolamine cellulose enhances curli-mediated adhesion of uropathogenic Escherichia coli to bladder epithelial cells[END_REF]. The adhesion of cells to a surface is a crucial step of biofilm formation [2]. While curli are required for cell adhesion, pEtN cellulose strengthens the adhesion of curli to the host cell surface [START_REF] Hollenbeck | Phosphoethanolamine cellulose enhances curli-mediated adhesion of uropathogenic Escherichia coli to bladder epithelial cells[END_REF]. By consequence, pEtN cellulose together with curli was proposed to enhance Urinary Tract Infectious (UTI) E. coli pathogenesis [START_REF] Hollenbeck | Phosphoethanolamine cellulose enhances curli-mediated adhesion of uropathogenic Escherichia coli to bladder epithelial cells[END_REF]. Lately, high resolution BcsG structures of the C-terminal domain from Salmonella enterica and E. coli have been reported [START_REF] Sun | Structural and Functional Characterization of the BcsG Subunit of the Cellulose Synthase in Salmonella typhimurium[END_REF], [START_REF] Anderson | The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase[END_REF]. The crystallographic snapshots from both studies were in a full accordance, and both C-terminal domains adopt a typical phosphatase alkaline fold, with 7 stranded ß-sheets sandwiched between α-helices (Figure 8). Both structures exhibit almost identical folds (0.4Å R.M.S.D) and contain a Zn 2+ ion interacting with different moieties of amino acid Cys243, Ser278, Glu442, and His443, altogether form the active site (Figure 8) [START_REF] Sun | Structural and Functional Characterization of the BcsG Subunit of the Cellulose Synthase in Salmonella typhimurium[END_REF], [START_REF] Anderson | The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase[END_REF]. The function of BcsG is not fully understood yet, as a BcsG deletion in Salmonella typhimurium severely decreased not only the efficiency of cellulose secretion, but also affected BcsA insertion and/or stability in the membrane [START_REF] Sun | Structural and Functional Characterization of the BcsG Subunit of the Cellulose Synthase in Salmonella typhimurium[END_REF]. This observation stands in line with previous works where two-hybrid assay showed that BcsG interacts with its operon neighbors BcsF and BcsE, as well as with the synthase BcsA likely having structural and not only enzymatic roles in secretion system assembly and cellulose biogenesis [START_REF] Krasteva | Insights into the structure and assembly of a bacterial cellulose secretion system[END_REF], [START_REF] Thongsomboon | Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose[END_REF].

BcsZ

BcsZ is ubiquitous among Bcs systems and part of the conserved 3-4 component core, which also includes the catalytic BcsAB tandem and, in Gram-negative species, the OM pore BcsC [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF]. This peculiar subunit has been shown to exhibit an endo-β-1,4-glucanase activity, meaning that it can hydrolyze the nascent polysaccharide. This feature of having a polysaccharidedegrading enzyme as part of the cognate secretion system is not unique to cellulose secretion, as other EPS secreting systems such as the PNAG, Pel and alginate machineries encode similarly an EPS-digesting enzyme within the same gene clusters as the other core components [START_REF] Low | Gram-negative synthase-dependent exopolysaccharide biosynthetic machines[END_REF]. Similarly to the endocellulase BcsZ, PgaB and PelA exhibit both a hydrolase activity while AlgL displays a lyase function [START_REF] Low | Gram-negative synthase-dependent exopolysaccharide biosynthetic machines[END_REF]. Moreover, this feature is also preserved in higher kingdoms, as higher plants include an endoglucanase named Korrigan (KOR) required for the correct assembly of the elongating cell walls [START_REF] Nicol | A plasma membrane-bound putative endo-1,4-beta-D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis[END_REF].

Although this pattern is frequently observed in other systems, the biological relevance to have a glucanase around the nascent glucan chain has sparked many questions that remain to be answered. Hydrolases such as BcsZ in S. typhimurium has been shown to promote virulence, while in vitro experiments have showed that PslA and PelG disrupt the biofilm formed by P.

aeruginosa [START_REF] Ahmad | BcsZ inhibits biofilm phenotypes and promotes virulence by blocking cellulose production in Salmonella enterica serovar Typhimurium[END_REF], [START_REF] Baker | Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms[END_REF]. Furthermore, BcsZ could play a role in the packing of the cellulose microfibrils in G. xylinus. In fact, the deletion of bcsZ gene in G. xylinus resulted in irregular and twisted packing of the de novo synthesized fibrils suggesting that BcsZ could play a crucial role in cleaving the entanglements that can occur between secreted cellulose fibrils and thus BcsZ ensures the formation of a well-structured crystallized cellulose [START_REF] Nakai | Formation of Highly Twisted Ribbons in a Carboxymethylcellulase Gene-Disrupted Strain of a Cellulose-Producing Bacterium[END_REF]. The latter observation opens the door to another question regarding amorphous cellulose where wild-type levels of BcsZ are also required for optimal cellulose secretion.

BcsZ structures from both E. coli and G. xylinus adopt a (α/α)6-barrel fold forming a single domain, in which six pairs of antiparallel α -helices are found to form an inner and outer ring [START_REF] Yasutake | Structural characterization of the Acetobacter xylinum endo-β-1,4glucanase CMCax required for cellulose biosynthesis[END_REF], [START_REF] Mazur | Apo-and Cellopentaose-bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ[END_REF] (Figure 9A-B). Structural analysis on the E. coli homolog revealed that the connecting loops extend on one side of the barrel forming antiparallel ß-sheets, as a result the structure adopts a deep "sickle shaped" groove on one side where the two catalytic residues D243 and E55 are embedded deep down the groove [START_REF] Mazur | Apo-and Cellopentaose-bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ[END_REF]. Both BcsZ structures from E. coli and G. xylinus fit completely in the GH-8 family of enzymes and adopt an overall similar architecture, with a slight difference regarding the substrate polysaccharide [START_REF] Yasutake | Structural characterization of the Acetobacter xylinum endo-β-1,4glucanase CMCax required for cellulose biosynthesis[END_REF], [START_REF] Mazur | Apo-and Cellopentaose-bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ[END_REF]. BcsZ from G. xylinus needs cellopentaose or longer polysaccharide for its activity, as opposingly to its homolog BcsZ from E. coli, which requires at least an hexasaccharide [START_REF] Mazur | Apo-and Cellopentaose-bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ[END_REF], [START_REF] Kawano | Effects of endogenous endo-β-1,4-glucanase on cellulose biosynthesis in Acetobacter xylinum ATCC23769[END_REF]. Thus, a catalytically inactive E.coli BcsZ mutant solved in complex with cellopentaose is suggested to be in the post-hydrolysis state in which the protein contacts glucan chain at the non-reducing end as the reducing end has already left the catalytic center [START_REF] Mazur | Apo-and Cellopentaose-bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ[END_REF]. The common part between the BcsZ structures and other homologs from GH-8 family enzymes revealed that the interactions with the glucan chain are basically hydrogen bonds and stacking interactions with conserved aromatic residues.

Nevertheless, all studies on enterobacterial BcsZ were carried out before the discovery of the pEtN modification, so substrate specificity for the modified cellulose remains to be characterized.

BcsZ is not limited to Gram-negative bacteria, as a recent study identified the CcsZ (Clostridium cellulose synthase Z) protein and functional BcsZ homologs have been described even in the atypical efflux-type cellulose secretion systems of cyanobacteria [START_REF] Maeda | Genetic identification of factors for extracellular cellulose accumulation in the thermophilic cyanobacterium Thermosynechococcus vulcanus: proposal of a novel tripartite secretion system[END_REF], [START_REF] Scott | Identification of the Clostridial cellulose synthase and characterization of the cognate glycosyl hydrolase, CcsZ[END_REF]. It is true that there is no direct sequence homology between BcsZ and CcsZ and added to that CcsZ is a membrane tethered endo-β-1,4-glucanase that belongs to the GH-5 family instead of the GH-8 family of enzymes. Nevertheless, CcsZ has specificity for cellulosic materials and is believed to act in a similar manner in its cognate secretion systems [START_REF] Scott | Identification of the Clostridial cellulose synthase and characterization of the cognate glycosyl hydrolase, CcsZ[END_REF]. The discoveries around BcsZ together point toward a putative role in limited hydrolytic action on the cellulose chains in the periplasm or at the cell surface thus preventing aggregative microfibrils formation before the exopolysaccharide reaches the extracellular environment. Interestingly, whereas in most studies BcsZ is positioned in the periplasmic compartment, other studies have suggested fully extracellular role for the subunit [START_REF] Yasutake | Structural characterization of the Acetobacter xylinum endo-β-1,4glucanase CMCax required for cellulose biosynthesis[END_REF], [START_REF] Koo | Evidence that a beta-1,4endoglucanase secreted by Acetobacter xylinum plays an essential role for the formation of cellulose fiber[END_REF]. Indeed, more studies are necessary to better characterize the fine functional balance exerted by BcsZ homologs as they can be suitable targets to prevent biofilm formation or, alternatively, play a role in biotechnological applications to increase cellulose production.

BcsQ and BcsR

The type II cellulose secretion system of E. coli is characterized by two additional genes bcsR (formerly yhjR) and bcsQ (formerly yhjQ) leading the operon that contains the four core secretory componens BcsA, BcsB, BcsZ and BcsC (Figure 5A) [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF]. Whereas BcsR is a short ~7 kDa peptide without reliable structural models until our work, BcsQ belongs to the SIMIBI (Single-recognition particle, MinD and BioD) ATPases family conserved in both prokaryotes and Eukaryotes. Members of the latter family comprise MinD, responsible for the positioning the divisome in bacterial cells, FlhG/FlhF involved in bacterial flagellum assembly and SRP54-SR and Get3 NTPases responsible for membrane protein sorting [START_REF] Shan | ATPase and GTPase Tangos Drive Intracellular Protein Transport[END_REF], [START_REF] Bange | SIMIBI twins in protein targeting and localization[END_REF]. Regarding cellulose secretion, the closest homologs to BcsQ are WssA/WssJ proteins that are part of the acetylated cellulose secretion machinery in P. fluorescens SBW25 [START_REF] Quéré | BcsQ is an essential component of the Escherichia coli cellulose biosynthesis apparatus that localizes at the bacterial cell pole[END_REF], [START_REF] Spiers | Biofilm formation at the air-liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose[END_REF]. BcsR and BcsQ share predicted cytosolic localization and partake in interactions with several Bcs components [START_REF] Krasteva | Insights into the structure and assembly of a bacterial cellulose secretion system[END_REF].

Interestingly, BcsQ localizes at the cell pole, which corresponds to the site of cellulose secretion, as well as initial cell-cell-adhesion in E. coli [START_REF] Quéré | BcsQ is an essential component of the Escherichia coli cellulose biosynthesis apparatus that localizes at the bacterial cell pole[END_REF]. Remarkably, both BcsR and BcsQ are essential for cellulose secretion in E. coli but are absent in many Type I and Type III cellulose secretion systems suggesting different regulatory inputs for the enterobacterial Bcs machinery [START_REF] Krasteva | Insights into the structure and assembly of a bacterial cellulose secretion system[END_REF]. These secretion system-specific effects and the essentiality of the two subunits for pEtN cellulose biogenesis motivated us to decipher the roles of both BcsR and BcsQ and many mechanistic insights regarding the structures and functions of both components can be found in the Results chapter.

BcsE, the c-di-GMP sensor

Before the identification of BcsE as a c-di-GMP receptor in the Bcs system, the PilZ domaincontaining BcsA was the first described to require the interaction with c-di-GMP to perform its glycosyltransferase activity [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF], [START_REF] Ross | Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid[END_REF], [START_REF] Amikam | PilZ domain is part of the bacterial c-di-GMP binding protein[END_REF]. Later BcsE joined the group of c-di-GMP receptors upon the characterization of a conserved C-terminal domain containing a c-di-GMP-binging RxxD motif, which -when found on c-di-GMP-producing GGDEF domain-containing diguanylate cyclases -can serve as an autoinhibitory, or I-, site for allosteric regulation.

Conversely, BcsE was dubbed as a GGDEF I-site Like, or GIL-, domain protein by Fang and coworkers [START_REF] Fang | GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria[END_REF]. Although BcsE is not absolutely essential for cellulose secretion it plays a crucial role in maintaining the stability of the Bcs complex and contributes to optimal polysaccharide production [START_REF] Krasteva | Insights into the structure and assembly of a bacterial cellulose secretion system[END_REF], [START_REF] Fang | GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria[END_REF]. The bcsE gene leads the operon bcsEFG, which is hallmark for E. coli-like Type II cellulose secretion systems. The pEtN-cellulose modifications by BcsG on one hand, and the proximity of the bcsEFG genes to the bcsABZC cluster on the other, suggests that this second Bcs cluster not simply contributes to secretion and chemical modifications of the cellulose but rather plays key roles in the macroscopic architecture of the mature bacterial biofilms [START_REF] Krasteva | Insights into the structure and assembly of a bacterial cellulose secretion system[END_REF], [START_REF] Thongsomboon | Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose[END_REF].

BcsF

BcsF is a small subunit anchored to the inner membrane by a single transmembrane helix. The bcsF gene is not common to all proteobacteria, and where annotated is usually a downstream operon neighbor of the bcsE gene (Figure 5A) [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF]. It has been shown that BcsF, interacts with BcsA, BcsE and BcsG in vivo, however the mechnisms by which the interactions occur and the exact role of BcsF in cellulose secretion, needed to be decrypted. Here we examined further the role of this subunit and demonstrate that it is key to Bcs macrocomplex assembly integrity via membrane recruitment of the regulatory BcsRQE components (see Results section) [START_REF] Krasteva | Insights into the structure and assembly of a bacterial cellulose secretion system[END_REF], [START_REF] Thongsomboon | Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose[END_REF].

C-di-GMP, a versatile second messenger

Cyclic diguanylate or c-di-GMP is a monocyclic RNA dinucleotide second messenger that is almost ubiquitous in the prokaryotic world and has also been observed in eukaryotic social amoeba from the Dictyostelium clade [START_REF] Chen | The prokaryote messenger c-di-GMP triggers stalk cell differentiation in Dictyostelium[END_REF]. The discovery of c-di-GMP goes back to almost half a century now, when Benziman and coworkers first described that the in vitro cellulose synthase activity of G. xylinus was promoted by GTP and an unknown GTP-converting protein factor [START_REF] Aloni | Achievement of high rates of in vitro synthesis of 1,4-13-D-glucan: Activation by cooperative interaction of the Acetobacter xylinum enzyme system with GTP, polyethylene glycol, and a protein factor[END_REF]. The latter hypothesis was supported in a successive study, where digitonin solubilization showed that the GTP-converting protein associates with the membrane-bound enzyme and is promoted by Ca 2+ ions [START_REF] Aloni | Solubilization of the UDPglucose:1,4-beta-D-glucan 4-beta-D-glucosyltransferase (cellulose synthase) from Acetobacter xylinum. A comparison of regulatory properties with those of the membranebound form of the enzyme[END_REF]. Shortly after, the GTP-converting enzyme was isolated using agarose-conjugated GTP as an affinity matrix and its synthase-activating product identified as bis(3′ ,5′ )-cyclic diguanylic acid, or c-di-GMP [START_REF] Ross | Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid[END_REF]. Almost two decades later with the revolution of DNA (Deoxyribonucleic acid) sequencing methods and ever-growing data on genome assemblies, c-di-GMP has revealed itself as a universal second messenger in the center of a plethora of mechanisms in the bacterial world [START_REF] Krasteva | Sensing the messenger: The diverse ways that bacteria signal through c-di-GMP[END_REF], [START_REF] Low | Gram-negative synthase-dependent exopolysaccharide biosynthetic machines[END_REF], [START_REF] Jenal | Cyclic di-GMP: second messenger extraordinaire[END_REF]. Often through direct control, c-di-GMP orchestrates many biological processes including motility, virulence gene expression and secretion of extracellular polysaccharides [START_REF] Krasteva | Sensing the messenger: The diverse ways that bacteria signal through c-di-GMP[END_REF], [START_REF] Low | Gram-negative synthase-dependent exopolysaccharide biosynthetic machines[END_REF], [START_REF] Jenal | Cyclic di-GMP: second messenger extraordinaire[END_REF]. Many studies thoroughly reviewed the different roles of this small molecule and highlighted the layers of physiological responses controlled by c-di-GMP covering the transcriptional, translational and posttranslational levels [START_REF] Römling | Cyclic di-GMP: the first 25 years of a universal bacterial second messenger[END_REF], [START_REF] Krasteva | Sensing the messenger: The diverse ways that bacteria signal through c-di-GMP[END_REF], [START_REF] Low | Gram-negative synthase-dependent exopolysaccharide biosynthetic machines[END_REF], [START_REF] Jenal | Cyclic di-GMP: second messenger extraordinaire[END_REF]. Moreover, in order to adapt to the large spectrum of mechanisms, c-di-GMP is able to adopt variable stable conformations when bound to proteins which makes it difficult to predict its new targets [START_REF] Krasteva | Versatile modes of cellular regulation via cyclic dinucleotides[END_REF] (Figure 10). In this part of the present study, I will discuss targets and regulation of the near-ubiquitous second-messenger cdi-GMP. I also contribute to the understanding of c-di-GMP signaling in relation with cellulose secretion by the discovery of new dinucleotide-sensing mechanisms specific to the E. coli-like type II cellulose secretions in the Results chapter.

Figure 10. Examples highlighting the conformational adaptability of c-di-GMP

Based on the structural studies, c-di-GMP can adopt a variety of conformations when bound to proteins in order to adapt and confer specificity to the different targets.

Synthesis and degradation of c-di-GMP

C-di-GMP is synthesized from 2 molecules of GTP by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs) (Figure 11). Multiple-sequence analyses across bacterial genomes have led to the characterization of the domains that confer the activity of the c-di-GMP-metabolizing enzymes, either the diguanylate cyclase activity or the phosphodiesterase. DGCs contain GGDEF domains, while PDEs are characterized by EAL or HD-GYP domains, where the domain names come from conserved amino acid motifs [START_REF] Chan | Structural basis of activity and allosteric control of diguanylate cyclase[END_REF]- [START_REF] Schmidt | The Ubiquitous Protein Domain EAL Is a Cyclic Diguanylate-Specific Phosphodiesterase: Enzymatically Active and Inactive EAL Domains[END_REF]. Interestingly, many proteins feature both GGDEF and EAL domains, for that reason it is difficult to assign a specific function to such hybrid proteins from domain architecture and sequence conservation alone. The first structurally characterized protein featuring a GGDEF domain, PleD C. crescentus , not only allowed for the experimental confirmation of the diguanylate cyclase function, but also revealed an allosteric binding site, called I-site (from Inhibitory site) which is represented by an RxxD motif (i.e. an arginine and an aspartate spaced by two amino acids) and is involved in a negative feedback control [START_REF] Chan | Structural basis of activity and allosteric control of diguanylate cyclase[END_REF]. The degradation of c-di-GMP, on the other hand, can be achieved in two steps, with first phosphodiester cleavage leading to a linear pGpG product and a subsequent hydrolytic event producing 2 moieties of GMP. While HD-GYP domains typically break the c-di-GMP fully to GMP, some EAL domains can lead to the accumulation of pGpG (Figure 11). In Gram-negative bacteria, the HD-GYP proteins, are less common, whereas GGDEF and/or EAL domain-containing proteins are widespread among Gram-negative bacteria and a bit less in Gram-positive bacteria. Many bacteria feature multiple proteins containing c-di-GMP-metabolizing domains and oftentimes these modules can be 'degenerate', or catalytically inactive, yet important in c-di-GMP-mediated signal transduction.

For example, the E. coli K-12 genome encodes 29 proteins containing both GGDEF and/or EAL domains, and 4 of those exhibit degenerate domains [START_REF] Hengge | Systematic Nomenclature for GGDEF and EAL Domain-Containing Cyclic Di-GMP Turnover Proteins of Escherichia coli[END_REF]. Among the studies of everincreasing number of GGDEF/EAL proteins, different nomenclatures have been used, and a recent consensus nomenclature was proposed to name genes encoding catalytically active GGDEF and EAL domains as dgc and pde, respectively [START_REF] Hengge | Systematic Nomenclature for GGDEF and EAL Domain-Containing Cyclic Di-GMP Turnover Proteins of Escherichia coli[END_REF]. Surprisingly, c-di-GMPmetabolizing proteins are generally not redundant and despite the presence of multiple such actors in the cell, their function appears to be restricted to specific signaling pathways and physiological effects. For example, among the identified DGCs in cellulose-secreting E. coli K12 strains mostly DgcC is responsible for activating pEtN cellulose secretion, whereas in the E. coli 1094 strain studied in this work this role is taken by DgcQ enzyme [START_REF] Richter | Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose[END_REF], [START_REF] Da Re | A CsgD-Independent Pathway for Cellulose Production and Biofilm Formation in Escherichia coli[END_REF]. Bacterial two-hybrid assays have showed that cellulose-promoting DgcC interacts with the phosphodiesterase PdeK, and both enzymes interact with the co-catalytic partner BcsB [START_REF] Richter | Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose[END_REF]. To coordinate cellulose biogenesis, a model is thus proposed where interactions between the copolymerase BcsB, the c-di-GMP "source" DgcC and the c-di-GMP "sink" PdeK secure physical proximity to fine-tune cellulose biogenesis [START_REF] Richter | Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose[END_REF]. The idea of having a source and sink in physical proximity is consistent with a mathematical model from the same study where c-di-GMP molecules produced locally by the DgcC enzyme can dramatically increase the probability of c-di-GMP capture by the Bcs secretion machinery, and thus lead to cellulose secretion activation in vivo, where -as we show in the Results section -activating dinucleotide retention will be further secured by the multi-site c-di-GMP complexation by the cytosolic BcsERQ vestibule-forming complex and the PilZ module of the synthase BcsA [START_REF] Richter | Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose[END_REF], [START_REF] Abidi | Architecture and regulation of an enterobacterial cellulose secretion system[END_REF].

Finally, the co-localization of PdeK with the secretion machinery can be seen as a security valve that controls the flow of the intracellular c-di-GMP and consequently adapts the cellulose biogenesis to the cadence of the external stimuli and cellular metabolism.

Figure 11. Biochemistry of c-di-GMP

The three-dimensional structure of c-di-GMP is formed by two GMP moieties that are linked via 5'-3' macrocyclic ring. The synthesis of c-di-GMP is catalyzed by diguanylate cyclases containing GGDEF domains. c-di-GMP is degraded by proteins containing either EAL or HD-GYP domains. yielding pGpG or 2GMP molecules. The threedimensional structure of c-di-GMP is shown in the middle of the reaction in stick representation, carbon atoms are shown in gray, oxygen in red, nitrogen in blue and phosphorus in orange (from protein data bank entry 3hv8). Upon binding to effector molecules, c-di-GMP regulates diverse cellular processes generally leading to loss of motility, cell adhesion and persistence of biofilm forming communities [START_REF] Hengge | Principles of c-di-GMP signalling in bacteria[END_REF]- [START_REF] Schmidt | The Ubiquitous Protein Domain EAL Is a Cyclic Diguanylate-Specific Phosphodiesterase: Enzymatically Active and Inactive EAL Domains[END_REF].

Multileveled signaling of c-di-GMP in Bcs system

In order to orchestrate cellulose biogenesis in E. coli, c-di-GMP typically employs multilevel signal transduction mechanisms, which culminate with BcsA activation upon the binding of the c-di-GMP to the PilZ domain [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF] (more details are in the section below). In pEtN-cellulose secreting E. coli 1094 and Salmonella, the signaling cascade starts with expression of the stationary phase sigma factor RpoS, or σ S , in response to external stimuli [START_REF] Da Re | A CsgD-Independent Pathway for Cellulose Production and Biofilm Formation in Escherichia coli[END_REF] (Figure 12).

Under physiological conditions, the actual c-di-GMP levels in the cytosol are quite low, mainly due to efficient degradation of the dinucleotide by the phosphodiesterase PdeH [START_REF] Sarenko | More than Enzymes That Make or Break Cyclic Di-GMP-Local Signaling in the Interactome of GGDEF/EAL Domain Proteins of Escherichia coli[END_REF]. Stationary phase RpoS leads to the expression of a specific diguanylate cyclase DgcE, which counteract the effects of the phosphodiesterase PdeH and releases the diguanylate cyclase DgcM from inhibitory interactions with the phosphodiesterase PdeR. DgcM thus can engage in an interaction with the MlrA transcription activator and activate the transcription of CsgD [START_REF] Sarenko | More than Enzymes That Make or Break Cyclic Di-GMP-Local Signaling in the Interactome of GGDEF/EAL Domain Proteins of Escherichia coli[END_REF]- [START_REF] Pfiffer | Genetic dissection of Escherichia coli's master diguanylate cyclase DgcE: Role of the N-terminal MASE1 domain and direct signal input from a GTPase partner system[END_REF]. CsgD in in turn activates the expression of the rest of the csg genes involved in curli secretion and the diguanylate cyclase DgcC which ultimately produces the cellulose synthaseactivating c-di-GMP [START_REF] Römling | Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter[END_REF], [START_REF] Römling | AgfD, the checkpoint of multicellular and aggregative behaviour in Salmonella typhimurium regulates at least two independent pathways[END_REF].

Adding to the complexity of c-di-GMP signaling, studies on a collection of E. coli isolates showed that CsgD-/DgcC-dependent cellulose secretion is not the only mechanism for c-di-GMP control of the BcsA cellulose synthase [START_REF] Da Re | A CsgD-Independent Pathway for Cellulose Production and Biofilm Formation in Escherichia coli[END_REF]. For example, in the commensal E. coli 1094 strain used as a model organism in this work, cellulose secretion is specifically activated by a different diguanylate cyclase -the enzyme DgcQ -and appears to circumvent the CsgDdependent signaling cascade [START_REF] Da Re | A CsgD-Independent Pathway for Cellulose Production and Biofilm Formation in Escherichia coli[END_REF] (Figure 12). Both signaling pathways, CsgD-/DgcCdependent or independent, share in common the RpoS-dependent initiation, however it is unclear what the signaling mediators are for the downstream DgcQ activation [START_REF] Da Re | A CsgD-Independent Pathway for Cellulose Production and Biofilm Formation in Escherichia coli[END_REF].

Figure 12. Cellulose biosynthesis regulation in E. coli

The figure illustrates the two pathways regulating the pEtN-cellulose production in enterobacteria. On the left is the CsgD-/DgcC-dependent pathway, on the right is the CsgD-independent/DgcQ-dependent pathway. In both cases, the production of c-di-GMP is required for the cellulose biosynthesis. In particular, cellulose polymerization and extrusion requires the binding of the dinucleotide to the PilZ module of the synthase BcsA, which in turn is facilitated by the multi-site c-di-GMP complexation of the cytosolic regulatory complex BcsERQ (see Results section) [START_REF] Abidi | Architecture and regulation of an enterobacterial cellulose secretion system[END_REF], [START_REF]Structure and Multitasking of the c-di-GMP-Sensing Cellulose Secretion Regulator BcsE[END_REF]. Diguanylate cyclases (DGC) are shown in green, phosphodiesterases (PDE) are shown in pink [START_REF] Hengge | Principles of c-di-GMP signalling in bacteria[END_REF], [START_REF] Da Re | A CsgD-Independent Pathway for Cellulose Production and Biofilm Formation in Escherichia coli[END_REF].

C-di-GMP and the cellulose synthase BcsA

In most Bcs systems, cellulose secretion is initiated upon the activation of the synthase BcsA by the second messenger c-di-GMP. BcsA harbors the three modules, namely the transmembrane export domain, the family-2 glycosyltransferase responsible for the catalytic reaction, and the well-studied c-di-GMP-binding C-terminal PilZ domain [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF]. Different crystallographic snapshots of the BcsA R. sphaeroides with its co-catalytic partner BcsB, have been studied in great details. Comparisons between the different states reveal that cyclic dinucleotide binding induces several important conformational changes. First, in the c-di-GMP bound state, the PilZ ß-barrel rotates by approximatively 20° around a so-called 'hinge' helix, which is sandwiched at the interface between the β-barrel and the glycosyltransferase domain [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF], [START_REF] Morgan | Observing cellulose biosynthesis and membrane translocation in crystallo[END_REF].

An additional conformational change that characterizes the c-di-GMP-bound state involves Arg580 situated from the conserved R580xxxR motif that precedes the PilZ β-barrel and partakes in c-di-GMP complexation [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF]. In the c-di-GMP-free state the synthase is in an autoinhibitory state, where the so-called 'gating loop' caps the active site entry in a resting-state conformation stabilized by a salt bridge interaction between the Arg580 and an equally conserved Glu371 from the gating loop [START_REF] Morgan | Mechanism of activation of bacterial cellulose synthase by cyclic-di-GMP[END_REF]. Upon c-di-GMP binding, Arg580 rotates by approximatively 180° and is involved in a new interaction with the dinucleotide, which frees the gating loop and creates a large 22.5 x 12.4 Å opening to the active site, wide enough to allow for entry of the substrate UDP-glucose. Further increase in c-di-GMP concentrations do not increase the affinity of BcsA for UDP-glucose in vitro [START_REF] Omadjela | BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis[END_REF] consistent with c-di-GMP-based control of active site entry as evidenced by the crystal structures.

Nevertheless, control of active site accessibility is not the only function of the BcsA's gating loop. Some of the resolved c-di-GMP-bound crystal structures contain either a homolog of the substrate UDP-glucose, or the product UDP. In fact, in both latter states, the gating loop inserts deeply into the catalytic pocket, where many of the loop's residues and especially the conserved FxVTxK motif coordinate the uracil base and the pyrophosphate of the UDP moiety [START_REF] Morgan | Mechanism of activation of bacterial cellulose synthase by cyclic-di-GMP[END_REF] (Figure 13). Moreover, a crystallographic snapshot of a c-di-GMP-bound state in the pretranslocation conformation features a largely disordered gating loop with an overall trajectory reminding that of the UDP-bound but c-di-GMP-free resting state [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF], [START_REF] Morgan | Observing cellulose biosynthesis and membrane translocation in crystallo[END_REF]. Therefore, it is conceivable that both the release of c-di-GMP or of the product UDP can initiate gating loop relaxation. Furthermore, different studies have reported markedly different binding affinities between BcsA and c-di-GMP, more consistently in the micromolar range, which in a physiological context would suggest a more dynamic sampling rather than strong complexation [START_REF] Omadjela | BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis[END_REF], [START_REF] Weinhouse | c-di-GMP-binding protein, a new factor regulating cellulose synthesis in Acetobacter xylinum[END_REF], [START_REF] Ryjenkov | The PilZ Domain Is a Receptor for the Second Messenger c-di-GMP: THE PilZ DOMAIN PROTEIN YcgR CONTROLS MOTILITY IN ENTEROBACTERIA*[END_REF]. One may thus think that the c-di-GMP binding to the PilZ domain is highly dynamic, and that release of c-di-GMP in-between catalytic cycles can be even required to allow for efficient gating loop relaxation, product release and polysaccharide translocation, before c-di-GMP re-complexation causing the initiation of a new cycle. Substrate entry and insertion of the gating loop would allow the initiation of the catalysis, and upon the glycosyl transfer the gating loop would relax, retracting from the catalytic pocket to allow for the recycling of UDP to UDP-glucose in a mechanism depending simultaneously on the local concentrations of c-di-GMP, UDP-glucose, and UDP (Figure 13). As we show in this work, such dynamic process of activating dinucleotide recycling is likely greatly facilitated in the E.

coli-like Bcs system by the formation of a synthase-proximal c-di-GMP pool by multi-site complexation to the regulator BcsERQ vestibule surrounding the PilZ module of the synthase (see Results section).

Figure 13. Zoom-in of the active site of synthase BcsA

The different catalytic states are captured in the crystal structures of the R. sphaeroides BcsAB tandem from crystals grown or soaked with different ligands. Protein data bank accession numbers are indicated in each panel; presence of substrate homologs, products, c-di-GMP, translocation state of the polymer and gating loop conformation are indicated for each state [START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF]- [START_REF] Morgan | Observing cellulose biosynthesis and membrane translocation in crystallo[END_REF]. Green arrows indicate putative conformation transition pathways integrating all captured conformational states. Alternative pathways would depend on local variations of c-di-GMP, product and substrate molecules, as well as finger helix-mediated or spontaneous translocation. The cellulosic polymer, substrate homolog, UDP product, c-di-GMP and key residues from the gating loop, c-di-GMP coordinating PilZ-proximal linker, finger helix and conserved QRGRW motif are shown as sticks.

Cellulose synthase core within kingdoms

Plants are more famous for the processive synthesis of cellulose as a primary building material of their cell walls, thus making cellulose the most abundant biopolymer on the planet Earth.

However, the identification of plant cellulose synthases relied on gene homology to their bacterial bcsA homologs. Later, it became evident that the genes for cellulose synthesis in plants have likely evolved from cyanobacteria via lateral gene transfers during multiple ancient endosymbiotic events [START_REF] Little | Revised Phylogeny of the Cellulose Synthase Gene Superfamily: Insights into Cell Wall Evolution[END_REF], [START_REF] Nobles | Cellulose in Cyanobacteria. Origin of Vascular Plant Cellulose Synthase[END_REF]. Plants build cellulose through CesA variants, which belong to the glycosyl transferase 2 family like their bacterial counterparts BcsA [START_REF] Little | Revised Phylogeny of the Cellulose Synthase Gene Superfamily: Insights into Cell Wall Evolution[END_REF]. Globally, both cellulose synthases catalyze the glucose transfer from the UDP-glucose to the C4 hydroxyl of the nascent cellulose polymer through a process coupling the polymerization of the cellulose polymer and its extrusion through the plasma membrane (Figure 3A). Nevertheless, in plants the synthase complexes enrolled in polysaccharide secretion have been typically shown to form hexameric macroassemblies called 'rosettes' or CSCs (cellulose synthase complexes). Recent studies revealed that the rosettes likely accommodate six CesA trimers, or 18 CesA protomers, that play a major role in securing the packing of the secreted glucan chains into fundamental microfibrils composed of 18 glucan chains in a mechanism similar to the crystalline cellulose ribbon secretion in G. xylinus [START_REF] Nixon | Comparative Structural and Computational Analysis Supports Eighteen Cellulose Synthases in the Plant Cellulose Synthesis Complex[END_REF] (Figure 5A). A recent study using in situ cryo-electron tomography, revealed another similarity between cellulose synthesis in crystalline cellulosesecreting bacteria and plants by identifying a novel cytoplasmic structure in G. xylinus and G.

hansenii dubbed "the cortical belt", which spatially correlates with the extracellular cellulose ribbon and is proposed to tether the synthase terminal complexes in a manner similar to cortical microtubule tethering of plant CSCs [START_REF] Nicolas | Structure of the Bacterial Cellulose Ribbon and Its Assembly-Guiding Cytoskeleton by Electron Cryotomography[END_REF] (Figure 5B-C).

Applications of bacterial cellulose

Since its discovery, bacterial cellulose is increasingly stealing the spotlight from plant cellulose, especially the crystalline product secreted by bacteria from the Komagataeibacter lineage.

Although both plant and bacterial celluloses share similarities in their overall structure, the bacterial nanocellulose is much purer due to the lack of co-produced hemicelluloses and lignin.

Purity is only one of the many outstanding properties of bacterial cellulose, such as its excellent water retention capacity, high crystallinity, transparency, biodegradability and low antigenicity.

Moreover, nanocellulose exhibits tremendous thermal and chemical stability allowing efficient non-denaturing sterilization, as well as valuable mechanical properties, such as tensile strength and elasticity. The almost endless list of desirable properties from a materials science perspective, makes bacterial cellulose an iconic biopolymer in the center of many applications in diverse fields such as the food, cosmetics and medical industry (See table 2 for examples of applications). To date, the major limiting factor for use of bacterial cellulose at the industrial scale, is perceptibly its cost. To tackle this economic problem, we need to decode and optimize the molecular mechanisms governing cellulose biogenesis by the Bcs nanomachineries in nature.

Table 2. Examples of bacterial cellulose applications

Field Application

Food

Food thickening [START_REF] Paximada | Effect of bacterial cellulose addition on physical properties of WPI emulsions. Comparison with common thickeners[END_REF] Emulsification [START_REF] Shi | Utilization of bacterial cellulose in food[END_REF] Water-binding [START_REF] Shi | Utilization of bacterial cellulose in food[END_REF] Immobilization of probiotics [START_REF] Fijałkowski | Survival of probiotic lactic acid bacteria immobilized in different forms of bacterial cellulose in simulated gastric juices and bile salt solution[END_REF] Active food packaging [START_REF] Ludwicka | Bacterial Nanocellulose-A Biobased Polymer for Active and Intelligent Food Packaging Applications: Recent Advances and Developments[END_REF] Paper Higher quality papers [START_REF] Fillat | Bacterial cellulose for increasing barrier properties of paper products[END_REF] Flame retardation material [START_REF] Basta | Performance of improved bacterial cellulose application in the production of functional paper[END_REF] Electronics Flexible displays [START_REF] Barud | Optically transparent membrane based on bacterial cellulose/polycaprolactone[END_REF] Electronic paper displays [START_REF] Shah | Towards electronic paper displays made from microbial cellulose[END_REF] Organic light emitting diodes [START_REF] Pinto | Transparent composites prepared from bacterial cellulose and castor oil based polyurethane as substrates for flexible OLEDs[END_REF] Cosmetics Texturing agent [START_REF] Augustine | Biopolymers for Health, Food, and Cosmetic Applications[END_REF] Emulsion stabilizer [START_REF] Almeida | Bacterial Nanocellulose toward Green Cosmetics: Recent Progresses and Challenges[END_REF] Carrier of skin active substances [START_REF] Almeida | Bacterial Nanocellulose toward Green Cosmetics: Recent Progresses and Challenges[END_REF] Medicine Suture biomaterials [START_REF] Wu | Regenerated chitin fibers reinforced with bacterial cellulose nanocrystals as suture biomaterials[END_REF] Wound dressing [START_REF] Zheng | Latest Advances on Bacterial Cellulose-Based Antibacterial Materials as Wound Dressings[END_REF], [START_REF] Czaja | Microbial cellulose--the natural power to heal wounds[END_REF] Drug delivery [START_REF] De Lima Fontes | Effect of in situ modification of bacterial cellulose with carboxymethylcellulose on its nano/microstructure and methotrexate release properties[END_REF] Tissue engineering [START_REF] Lv | Bacterial Cellulose-Based Biomimetic Nanofibrous Scaffold with Muscle Cells for Hollow Organ Tissue Engineering[END_REF] Artificial cornea [START_REF] Wang | Preparation and in vitro characterization of BC/PVA hydrogel composite for its potential use as artificial cornea biomaterial[END_REF] Retinal pigment epithelium [START_REF] Gonçalves | Acetylated bacterial cellulose coated with urinary bladder matrix as a substrate for retinal pigment epithelium[END_REF] Blood vessels [START_REF] Scherner | In vivo application of tissue-engineered blood vessels of bacterial cellulose as small arterial substitutes: proof of concept?[END_REF] 50

3 Objectives of the thesis 51

This PhD aims to provide mechanistic insights into the cellulose secretion machinery involved in bacterial biofilm formation. We know now that bacterial cellulose can be produced by three major types of secretion systems sharing as a common feature the catalytic core BcsAB [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF].

This conserved assembly formed by the accessory BcsB co-polymerase and the glycosyl transferase, c-di-GMP-sensing PilZ and inner membrane export module of BcsA has been studied in great details [2][3][4]. Added to the catalytic subunits BcsAB, additional Bcs components as well as the chemical properties of the secreted exopolysaccharide delineate the separate types of cellulose secretion [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF], [START_REF] Mcnamara | A Molecular Description of Cellulose Biosynthesis[END_REF]. Several works spot the light on some of these additional subunits, such as the structural characterization of the OM membrane porin domain of BcsC and its role in the delivery of the polysaccharide to the extracellular compartment [START_REF] Acheson | Architecture of the Cellulose Synthase Outer Membrane Channel and its association with the Periplasmic TPR Domain[END_REF];

the structural characterization of the BcsG subunit and its role in the modification of the polysaccharide by the addition of pEtN groups [START_REF] Thongsomboon | Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose[END_REF], [START_REF] Sun | Structural and Functional Characterization of the BcsG Subunit of the Cellulose Synthase in Salmonella typhimurium[END_REF]; or the ever-increasing evidence of the crucial roles of c-di-GMP in the regulation of cellulose secretion and biofilm formation [START_REF] Krasteva | Sensing the messenger: The diverse ways that bacteria signal through c-di-GMP[END_REF], [START_REF] Low | Gram-negative synthase-dependent exopolysaccharide biosynthetic machines[END_REF], [START_REF] Jenal | Cyclic di-GMP: second messenger extraordinaire[END_REF]. What is missing in the big picture, are the structures and functions of the multiple additional Bcs subunits, some of which are indispensable for cellulose secretion, as well as the mechanism of assembly and signaling within the system in its integrity.

The E. coli-like cellulose secretion system is encoded by at least two operons, one of which encodes a second c-di-GMP sensing protein, BcsE, which is important for maximal cellulose secretion, yet little more was known about this regulator subunit [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF]. In addition, this operon encodes two more proteins whose disruption drastically affects cellulose production [START_REF] Krasteva | Insights into the structure and assembly of a bacterial cellulose secretion system[END_REF] the BcsG subunit, whose role as a pEtN transferases was only recently uncovered, as well as a short transmembrane peptide of unknown mechanism of function, BcsF. On the other side, the second operon starts with genes encoding the essential for cellulose secretion BcsR and BcsQ, followed by those for the core components BcsABZC.

A study by Krasteva and colleagues has demonstrated recently that in E. coli most of the inner membrane and cytosolic Bcs subunits assemble to form a stable, megadalton-sized secretory nanomachine with unique asymmetric architecture (BcsRQABEF or Bcs macrocomplex herein) [START_REF] Krasteva | Insights into the structure and assembly of a bacterial cellulose secretion system[END_REF]. Additionally, the latter study unveiled the functions of many of the accessory subunits, and in particular the in vivo contributions of each Bcs component to cellulose secretion, as well as protein partners that partake in binary protein-protein interactions [START_REF] Krasteva | Insights into the structure and assembly of a bacterial cellulose secretion system[END_REF]. Yet, the structural reconstruction of the Bcs macrocomplex, based on low resolution electron microscopy data on a sample embedded in negative-stain, did not allow for further mechanistic insights. In this context, the goal of the thesis was to study the structure of key components and multiprotein subcomplexes of the E. coli Bcs secretion system in higher resolution. To this aim, we resorted

Introduction to article 1

In many gram-negative bacteria, biofilm formation goes in hand with cellulose secretion. As discussed above, the latter usually requires the co-expression of a c-di-GMP-activatable inner membrane BcsAB tandem, an outer membrane porin BcsC and multiple accessory subunits which allow for the classification of Bcs systems [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF], [START_REF] Abidi | Weaving of bacterial cellulose by the Bcs secretion systems[END_REF]. The Type II E. coli-like cellulose secretion system is distinguished by two separate operons, namely bcsEFG and bcsRQABZC,

where bcsEFG operon is the hallmark of this type of secretion system [START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF]. Previous studies demonstrated that BcsR and BcsQ are essential for cellulose secretion, while BcsE is required for maximal cellulose production and has been shown to also bind cyclic-di-GMP [START_REF] Quéré | BcsQ is an essential component of the Escherichia coli cellulose biosynthesis apparatus that localizes at the bacterial cell pole[END_REF].

Interestingly, the latter cytosolic subunits, together with the additional partner BcsF were suggested to interact in different combinations as evidenced by cell-based bacterial two-hybrid complementation assays [START_REF] Krasteva | Insights into the structure and assembly of a bacterial cellulose secretion system[END_REF]. The aim of the study described in the article 1 was to further explore the interactions between the different cytosolic subunits, identify stable protein-protein interactions, design stable protein expression constructs, overexpress and purify the target proteins and multisubunit assemblies and study the structural and functional determinants of these subunits both in vitro and in cellulo.

To this end, we performed multiple recombinant (co-)expression/(co-)purification experiments on the BcsR, BcsQ and BcsE components, alone and in different combitions. We established point mutants and truncated variants that were instrumental for the stabilization of the protein complexes or subunits and their subsequent crystallization and structure determination. We reported the structure of the c-di-GMP-binding module of BcsE and showed that the previously postulated GIL domain is in fact a tandem of degenerate receiver (REC*) and diguanylate cyclase (GDDEF*) domains that are incompetent for phosphotransfer or c-di-GMP biogenesis, respectively. We further showed that BcsE senses both BcsQ and c-di-GMP through nonoverlapping interfaces on the degenerate GGDEF* module and that the remaining N-terminal domain is likely a degenerated P-loop ATPase module, which is responsible for membrane targeting of the BcsERQ regulatory complex via interactions with inner-membrane BcsF.

Furthermore, the expression and purification of BcsE and this N-terminal module, led to the identification of a new high-affinity binding partner, which was identified with the help of mass-spectrometry analysis on as the S10 or NusE protein, a key component of the conserved cellular transcription antitermination complex (TAC) [START_REF] Baniulyte | sous-complexes régulateurs, qui ont révélé une stoechiométrie inattendue de ces sous unités, une interaction à différents motifs du c-di-GMP et une régulation dépendante de l'ATP (adénosine triphosphate). Pour obtenir plus de détails mécanistiques du macrocomplexe dans son intégralité[END_REF]. We validated the latter discovery Nevertheless, the intricate mechanisms for secretion systems assembly and biofilm matrix secretion require further investigations, especially considering a rising biotechnological interest and many already enabled applications of these intriguing biological polymers.

Figure 15. Examples of bacterial exopolysaccharide secretion systems

Examples of three synthase dependent systems. Left, Pel system; middle PNAG (poly-N-acetylglucosamine) system; right, Alginate system. Shown are the subunits and their proposed topologies in Gram-negative bacteria [START_REF] Abidi | Weaving of bacterial cellulose by the Bcs secretion systems[END_REF]. The cellulose biosynthesis, however, is not limited to higher kingdoms, as bacteria also secrete cellulose as an extracellular component of their biofilm matrix. While the discovery of bacterial cellulose goes back to the 19th century, it is only a century later that a succession of structural works provided details of the widely conserved glucosyltransferase BcsA (Bacterial cellulose synthesis A), which is allosterically activated by the intracellular second messenger c-di-GMP (Bis-(3′-5′)-cyclic dimeric guanosine monophosphate). The catalytic subunit BcsA together with its co-synthetic partner BcsB, permits the polymerization and extrusion of the polysaccharide through the inner bacterial membrane. Nevertheless, various accessory subunits contribute to or are essential for synthase complex assembly and stability, as well as postsynthetic modifications and secretion of the nascent cellulose through the complex bacterial cell envelope. The E. coli (Escherichia coli) cellulose secretion machinery for instance, is characterized by nine subunits (BcsRQABZCEFG) that span from the cytosol to the extracellular space. A recent study provided mechanistic understanding of the cellulose secretion system assembly in E. coli and revealed that most of the Bcs subunits interact to form a secretory nanomachine.

However, its initial low-resolution reconstruction didn't allow to distinguish the regulatory subunits or their exact functional mechanisms, even though the interest in bacterial cellulose in the biotechnological and other fields continues to spark. To address these inadequacies, I studied the conserved E. coli-like Bcs system, structurally and biochemically.

I showed that the cytosolic subunits BcsR, BcsQ, and BcsE interact to form regulatory subcomplexes. I also provided multiple crystallographic snapshots of these accessory subcomplexes, which revealed unexpected subunit stoichiometry, multisite c-di-GMP recognition, and an unexpected ATP (adenosine triphosphate)-dependent regulation. To gain further mechanistic and structural details on the assembled macrocomplex, I resorted to single particle cryo-EM (cryo-Electron Microscopy). The structural study of the macrocomplex revealed a roughly 1 MDa (Megadalton)-sized asymmetric secretory assembly. I further show that the asymmetry of the Bcs macrocomplex is due to self-driven and membranecurvature-dependent BcsB homo-oligomerization through ß-sheet complementation in the periplasm. These findings allow to place key pieces of the puzzle of cellulose secretion system assembly and function into the bigger picture of bacterial signaling and biofilm formation.

Figure 1 .

 1 Figure 1. Key steps of the biofilm formation ........................................................................

Figure 2 .

 2 Figure 2. Rdar morphotypes of E.coli and Salmonella strains ...........................................

Figure 3 .

 3 Figure 3. Cellulose structure ..................................................................................................

Figure 4 .

 4 Figure 4. Bacterial cellulose structure ..................................................................................

Figure 5 .

 5 Figure 5. Prevalent types of Bcs systems ..............................................................................

Figure 6 .

 6 Figure 6. BcsAB cellulose synthase complex. .......................................................................

Figure 7 .

 7 Figure 7. Architecture of the outer membrane pore BcsC ..................................................

Figure 8 .

 8 Figure 8. Crystal structure of the BcsG periplasmic domain .............................................

Figure 9 .

 9 Figure 9. BcsZ homologs in E. coli and G. xylinus ...............................................................

Figure 10 .

 10 Figure 10. Examples highlighting the conformational adaptability of c-di-GMP ............

Figure 11 .

 11 Figure 11. Biochemistry of c-di-GMP. ..................................................................................

Figure 12 .

 12 Figure 12. Cellulose biosynthesis regulation in E. coli ........................................................

Figure 13 .

 13 Figure 13. Zoom-in of the active site of synthase BcsA .......................................................

Figure 14 .

 14 Figure 14. The similarity between cellulose synthesis in plants and bacteria ...................

Figure 15 .

 15 Figure 15. Examples of bacterial exopolysaccharide secretion systems ..........................

  Adherence to biotic or abiotic surfaces Formation of the three-dimensional biofilm architecture Retention of water to keep the microenvironment hydrated Mechanical stability of biofilms Proteins Formation and stabilization of the polysaccharide matrix network Link the bacterial surface to the other EPS Electron donor or acceptor to ensure a redox activity in the biofilm matrix DNA Intercellular connection Bacterial cells adhesion Facilitate the exchange of genetic information in the biofilm matrix Enzymes Create pores to improve nutrients access Disrupt the biofilm matrix for disseminationAdapted from[2]

Figure 3 .

 3 Figure 3. Cellulose structure A. The structure of cellulose is shown in stick representation, carbon atoms are shown in gray, oxygen in red. The individual glucose units are connected via glycosidic ß-bonds between C1 and C4. Cellulose is elongated at its nonreducing end of the acceptor glucose and each glucose unit is rotated 180° compared to its neighbor. B. The cellobiose repeating unit is shown in Fischer projection. C. Structure of phosphoethanolamine.

Figure 4 .

 4 Figure 4. Bacterial cellulose structure A. Structure of the crystalline cellulose formed through extensive inter and intramolecular hydrogen bonds and Van der Waals forces. Image by Luca Laghi, reproduced under license CC BY-SA 3.0 (https://creativecommons. org/licenses/by-sa/3.0/legalcode). B. Cryo-electron micrographs of secreted bacterial cellulose. Figures from [37] Above micrograph: a biofilm-embedded Gluconacetobacter hansenii cell surrounded by crystalline cellulose ribbons. Below micrograph: amorphous pEtN-cellulose, marked by an asterisk, secreted by the commensal E. coli 1094 strain. C. Crystalline cellulose secreted by Gluconacetobacter xylinus-containing medium. The cellulose pellicle formed at the water-air interface is shown by a green arrow.

3 Figure 1 .

 31 Figure 1. Bacterial cellulose secretion. (A) Prevalent types of bacterial cellulose secretion systems and associated metabolic processes. UDPGP: UDP-glucose pyrophosphorylase, also called UTP-glucose-1-phosphate uridylyl transferase or GalU; UDP: uridine diphosphate; ATP: adenosine triphosphate; PP: pentose-phosphate pathway; DHAP: dihydroxyacetone phosphate; TCA cycle: tricarboxylic acid cycle; c-di-GMP: cyclic diguanosine monophosphate; PDE: phosphodiesterase; DGC: diguanylate cyclase. (B) Inter-and intrastrand hydrogen bonding in crystalline cellulose I. Image by Luca Laghi, reproduced under license CC BY-SA 3.0 (https://creativecommon s.org/licenses/by-sa/3.0/legalcode). (C) Cryo-electron micrographs of secreted bacterial and plant cellulose. Data: courtesy of William J. Nicolas, partially reported in (Nicolas et al. 2021) and reproduced under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/legalcode). Left: a bio lm-embedded Gluconacetobacter hansenii cell surrounded by crystalline cellulose ribbons (marked by white arrowheads); middle: amorphous phosphoethanolamine (pEtN)-cellulose (marked by an asterisk), secreted by the commensal Escherichia coli 1094 strain; right: plant cellulose micro brils observed as electron-dense laments in onion cell wall in situ. Scale bars: 100 nm.

Figure 6 .

 6 Figure 6. BcsAB cellulose synthase complex.

Figure 7 .

 7 Figure 7. Architecture of the outer membrane pore BcsC A-B. Crystal structure of E. coli BcsC 707-1157 (from protein data bank entry 6tzk) encompassing TPR19, a connecting linker region and the outer membrane porin domain [34]. The protein is shown in cartoon, the extracellular loops and luminal C-terminal tail are also shown as transparent surface. The lumen constriction proximal to the extracellular surface is seen in B, the gating π-stacking residues Y 1025 and Y 1124 from extracellular loops EL6 and EL8 are shown as sticks.

Figure 8 .

 8 Figure 8. Crystal structure of the BcsG periplasmic domain The overall fold of the catalytic module is shown in cartoon representation. The Zn ++ coordination in the active with the key residues involved in cation coordination and/or essential for the pEtN modification are shown as sticks (Figure generated in PyMoL, Schrodinger LLC).

Figure 9 .

 9 Figure 9. BcsZ homologs in E. coli and G. xylinus A. Crystal structure of a cellopentaose-bound catalytically inactive mutant of E. coli BcsZ [65]. The oligosaccharide and catalytic dyad are shown as sticks. B. Crystal structure of the G. xylinus BcsZ homolog [64].

Figure 14 .

 14 Figure 14. The similarity between cellulose synthesis in plants and bacteria A. Proposed structure of a CesA rosette complexes based on [90], where multiple CesA protomers will secure crystalline microfibril bundling upon exit of the nascent cellulose strains from the cell reproduced under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/legalcode) B. Cryo-electron tomography visualization of the cellulose ribbon and the underlying cortical belt proposed to organize the linear assembly of Bcs terminal complexes for crystalline cellulose secretion in the Komagataeibacter lineage. Data from [37] CB, cortical belt; IM, inner membrane; PG, peptidoglycan; OM, outer membrane; CR, crystalline cellulose ribbon. C. Schematic representation of the G. xylinus / G. hansenii type I cellulose secretion system.

  to cryo-EM to study the structure of the assembled Bcs macrocomplex, studies performed primarily by my colleague Samira Zouhir. Personally, I focused on studies started by a shortterm intern in the group, Meryem Caleechurn, aimed at deciphering the structure and function of the BcsRQ and BcsERQ regulatory complexes by X-ray crystallography. My work involved significant protein engineering efforts, screening and optimization of expression construct, purification protocols and crystallization conditions, and, following successful crystallization and structure solving, the design and execution of multidisciplinary functional studies in validating structure-derived hypotheses on the subunits' function. On the other hand, I also provided key evidence on the self-polymerization mechanisms driving Bcs macrocomplex assembly in the inner membrane by solving the cryo-EM structure of isolated BcsB multimers, consistent with the assymetric Bcs macrocomplex assembly and non-canonical BcsA:BcsB stoichiometry.

4. 2 2 4. 2 . 1 Introduction to article 2

 22212 ArticleThe article 2 is a continuation to article 1 in further deciphering the structure and assembly of the assembled Bcs macrocomplex. Moreover, we solved multiple crystallographic snapshots of the regulatory Bcs subcomplexes. Combining structural and functional investigations, we uncover the mechanism of asymmetric Bcs secretion system assembly and periplasmic crown polymerization and reveal unexpected subunit stoichiometry, multisite c-di-GMP recognition, and ATP-dependent regulation.The work was a close collaboration with Dr. Samira Zouhir, who focused on the cryo-EM studies of the assembled Bcs macrocomplex and the final stages of structural model refinements, whereas I concentrated on the stabilization and crystallization of the BcsRQ and BcsE*RQ multicomponent complexes, the characterization of newly discovered c-di-GMP binding motifs in the BcsE regulator, as well as the validation by cryo-EM of the mechanism of periplasmic crown polymerization via intersubunit structural complementation between neighboring BcsB subunits. Together with Dr. Petya Krasteva, we also conducted all functional assays of cellulose secretion, ligand complexation, ATP-ase activity, and biochemical detection of Bcs subunit expression. This work also benefited from contributions and useful discussions with former colleagues Stéphane Roche and Meryem Caleechurn.Carbohydrates are arguably the most important energy carrier and the most abundant biological components in all kingdoms of life. Evolutionary speaking, many bacteria dedicate a part of their metabolism to produce a plethora of carbohydrates, depending on both the disposable building blocks and the surrounding environment. Gram-negative and Gram-positive bacteria use their cell factories to secrete various classes of biopolymers and polysaccharides are oftentimes the main class of secreted extracellular substances[START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF],[2],[6],[START_REF] Zogaj | The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix[END_REF]. The polysaccharides either remain linked to the cell surface a capsular coat or are freely secreted into the extracellular milieu to contribute to the scaffolding of the biofilm matrix. Along the previous chapters, I spot the light on the cellulose as a main biofilm polysaccharide secreted by many bacteria, due to its unique characteristics that contribute to maintain the cell-cell and cellsurface adhesion and establish a highly protective and biocompatible environment for the multicellular communities. Through the present studies, I focused on the multicomponent cellulose secretion machinery in Gram-negative bacteria, particularly the E. coli-like Type II cellulose secretion system, which comprises a total of nine subunits (BcsRQABZCEFG) that span from the cytosol to the extracellular space. Along the results chapter, we marked a milestone in the mechanistic understandings for cellulose secretion system assembly and nucleotide-dependent regulation. Before our discoveries, several studies had shown that processive glucose polymerization is performed by the glycosyl transferase domain of BcsA, whose active site is made accessible by binding of dimeric c-di-GMP to an adjacent PilZ domain. The transport through the IM is coupled to the polymerization and is energized by the high-energy phosphoanhydride bonds of the preactivated synthase substrate, UDP-glucose. The nascent polysaccharide chain is subsequently extruded, one molecule at a time, through the IM transport domain of BcsA assisted by the C-terminal tail-anchor of BcsB. The latter partner adopts a donut-shaped periplasmic architecture and is proposed to guide the polysaccharide on its way to the outer membrane secretory component BcsC. Additional studies revealed that the cellulose secreted by E. coli-like systems is covalently decorated by pEtN residues in a post synthetic way by the periplasmic subunit BcsG. Another component is the presumably periplasmic endonuclease BcsZ, which is suggested to be required for degradation of cellulose in case it remains accumulated in the periplasm, for cleavage and restructuring of nascent glucan chains to allow microfibril formation to take place outside the cell, or for release of the polysaccharide from the cell surface. On the intracellular side of the bacterial membrane, the regulatory subunits BcsR, BcsQ and BcsE remained enigmatic. When we started our investigations, we had little information available about these components. We knew that E. coli-like cellulose secretion in vivo is absolutely dependent on the presence of the two small cytosolic proteins BcsR and BcsQ, and is boosted the third cytosolic protein BcsE, as well as by the short membrane-embedded polypeptide BcsF. A study by Krasteva and colleagues revealed that most of the IM and cytosolic Bcs components (BcsRQABEF) form a megadaltonsized secretory macrocomplex with a multimeric, layered, and asymmetric architecture.However, the structures, localization, and functional roles of the individual subunits remained largely unresolved. Through our two related studies (article 1 and article 2), we provided structural and functional insights into the individual subunits, together with the integral complex assembly. Based on crystallographic and functional data, we showed that BcsE actually features a tripartite architecture. In it, an N-terminal catalytically incompetent ATPaselike domain aids BcsE dimerization, participates in BcsF-mediated membrane recruitment of the essential BcsR and BcsQ subunits and interacts with conserved TAC (transcription antitermination complex) components suggesting additional regulatory roles at the gene expression level. Moreover, we showed that the previously hypothesized GIL domain providing a second c-di-GMP sensor to the system is in fact a degenerate receiver-GGDEF domain tande, (BcsE REC*-GGDEF* ), where the divergent diguanylate cyclase module binds both c-di-GMP and BcsQ through mutually independent interfaces. Contrasting degrees of sequence conservation between the N-terminal module and the REC-GGDEF tandem, as well as the identification of organisms where the corresponding BcsE parts are encoded by separate genes, point toward multidomain BcsE evolution and function integration via separate gene fusion events. The cdi-GMP-bound BcsE REC*-GGDEF* -BcsRQ R156E complex brought up more surprises, as the BcsE variant in the multicomponent complex adopts a strikingly different conformation from the structure of c-di-GMP-bound BcsE REC*-GGDEF* . While the canonical I-site RXXD motif on the catalytically incompetent diguanylate cyclase module coordinates a c-di-GMP moiety, the degenerate receiver domain and a so-called 'interstitial helix', linking it to the GGDEF module, undergo a 144° rotation and 45Å displacement to contribute a distinct conserved RXXD motif (R 306 ATD) and coordinate a second intercalated dinucleotide molecule via virtually identical arginine/aspartate-dependent interactions. We further revealed the importance of the REC domain I-site bound to the complexed c-di-GMP, using solution-based isothermal titration calorimetry experiments, where truncated (BcsE GGDEF* ) or point-mutated BcsE (BcsE REC*- GGDEF* •A 306 ATA) exhibited drastically altered thermodynamic profiles of the ligand binding reactions. Finally, the crystal structure of the BcsE GGDEF* -BcsRQ complex showed an additional, crystallographic c-di-GMP-binding interface involving multiple π-stacking and polar interactions with conserved residues from BcsE (Arg 503 His 504 ), BcsR (Arg 51 Trp 52 ) and BcsQ (Arg 219 ). Even though the biological relevance of this third dinucleotide binding site remains unverified, it is possible that it contributes additional weak interactions within the assembled cellulose secretion machinery, where the BcsERQ complex forms a swaddle-like vestibule around the BcsA PilZ domain and through multi-site c-di-GMP complexation can provide a synthase-proximal pool of circulating dinucleotide for enzyme activation. Regarding the assembled cellulose secretion machinery, we provided nearly atomic resolution data showing that BcsB polymerizes via a ß-sheet complementation mechanism among neighboring subunits and propose that polymerization would be simultaneously limited by the polymerization-induced membrane curvature due to BcsB's superhelicity and C-terminal anchors, the intrinsic membrane surface tension and the protein-protein interactions with the rest of the secretion system components. The integral assembled Bcs secretion macrocomplex thus contains a periplasmic crown of up to six BcsB copies, a single BcsA synthase, a likely dimer of BcsF inner membrane peptides and the BcsR2Q2E2 cytosolic regulatory complex, which supports the synthase through BcsRQ-BcsA PilZ interactions on one side and is anchored to the inner membrane through BcsE NTD -BcsF interactions on the other. Placing the cellulose machinery in a wider picture, the Bcs system belongs to the larger family of synthase-dependent exopolysaccharide secretion systems. Members of the latter include the alginate, the PNAG (poly-N-acetylglucosamine) and the Pel exopolysaccharide biosynthesis systems, many of which are found in both Gram-positive and Gram-negative bacteria. The common trait among the synthase-dependent family is the processive coupling of the polymerization with the export of the glycan chain. Opposingly to the Bcs system, the glycosyl-transferase and inner membrane translocation can be performed by separate components, as for example the PelF and PelG subunits of the Pel systems. In line with the Bcs system, in which the glycosyl transfer and the extrusion through the IM are carried out by the single subunit BcsA, alginate and PNAG systems employ Alg8 and PgaC respectively (Figure15). Another common feature imposes itself among the members of the synthase-dependent exopolysaccharide family is the c-di-GMP dependent activation engaging one or more dinucleotide-sensing protein modules such as Alg44 PilZ , PelD GGDEF domains and a composite binding site at the PgaC-PgaD interface for the alginate, Pel and PNAG systems, respectively. Additionally, AlgK, PelE, PelB and PgaA, on one hand, and AlgL, PelA, PgaB on the other, substitute the TPR-rich BcsC modules and the hydrolase BcsZ, respectively. Within the last years, numerous studies uncovered the structures of different modules of the latter synthase-dependent exopolysaccharides systems, whereas genomic studies allowed to establish comparisons-based parallels in a wide range of bacteria.

Figure 1 . 6 Final conclusion and perspectives 118

 16118 Figure 1. Bacterial cellulose secretion. (A) Prevalent types of bacterial cellulose secretion systems and associated metabolic processes. UDPGP: UDP-glucose pyrophosphorylase, also called UTP-glucose-1-phosphate uridylyl transferase or GalU; UDP: uridine diphosphate; ATP: adenosine triphosphate; PP: pentose-phosphate pathway; DHAP: dihydroxyacetone phosphate; TCA cycle: tricarboxylic acid cycle; c-di-GMP: cyclic diguanosine monophosphate; PDE: phosphodiesterase; DGC: diguanylate cyclase. (B) Inter-and intrastrand hydrogen bonding in crystalline cellulose I. Image by Luca Laghi, reproduced under license CC BY-SA 3.0 (https://creativecommon s.org/licenses/by-sa/3.0/legalcode). (C) Cryo-electron micrographs of secreted bacterial and plant cellulose. Data: courtesy of William J. Nicolas, partially reported in (Nicolas et al. 2021) and reproduced under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/legalcode). Left: a bio lm-embedded Gluconacetobacter hansenii cell surrounded by crystalline cellulose ribbons (marked by white arrowheads); middle: amorphous phosphoethanolamine (pEtN)-cellulose (marked by an asterisk), secreted by the commensal Escherichia coli 1094 strain; right: plant cellulose micro brils observed as electron-dense laments in onion cell wall in situ. Scale bars: 100 nm.

Table of contents 1 SUMMARY OF THE THESIS IN FRENCH ............................................................. 10 1

  .1 CONTEXTE ET MOTIVATIONS ......................................................................................1.2 ÉTAT DE L'ART .......................................................................................................... 1.2.1 Les sous unités catalytiques .................................................................................. 1.2.1.1 BcsA et BcsB ................................................................................................ 1.2.2 Les sous unités régulatrices .................................................................................. 1.2.2.1 BcsR et BcsQ ................................................................................................ 1.2.2.2 BcsE ..............................................................................................................

1.2.3 Le second messager c-di-GMP contrôle la formation des biofilms ...................... 1.2.4 Objectifs de la thèse .............................................................................................. 1.3 RESULTATS ET DISCUSSION ........................................................................................ 1.3.1 Étude de la structure cristallographique de BcsRQ ............................................. 1.3.2 Étude de la structure cristallographique de BcsRQ avec deux domaine Cterminaux de BcsE ............................................................................................................ 1.3.3 Étude de la structure du complexe de sécrétion par cryo-EM .............................. 1.4 CONCLUSION ET PERSPECTIVES .................................................................................. 2 INTRODUCTION ......................................................................................................... 17 2.1 THE MATRIX OF BIOFILMS .......................................................................................... 2.1.1 Key steps for biofilm formation............................................................................. 2.1.2 Cellulose, a major constituent of the bacterial biofilm maze, and more .............. 2.2 STRUCTURE AND FORMS OF BACTERIAL CELLULOSE .................................................. 2.2.1 Crystalline cellulose .............................................................................................. 2.2.2 Amorphous cellulose ............................................................................................. 2.3 TYPES OF BACTERIAL CELLULOSE SECRETING SYSTEMS ............................................. 2.3.1 Type I cellulose secretion system .......................................................................... 2.3.2 Type II cellulose secretion system ......................................................................... 2.3.3 Type III cellulose secretion system ....................................................................... 2.3.4 Other types of cellulose secretion ......................................................................... 2.4 MECHANISM FOR CELLULOSE BIOSYNTHESIS ............................................................. 2.5 SUBUNITS FOR BACTERIAL CELLULOSE SECRETION ....................................................

1.1 Contexte et motivations

  Les biofilms bactériens sont des communautés collaboratives, protégés par une matrice extracellulaire et formés par la plupart des espèces bactériennes[START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF],[2]. C'est une forme de vie préférée par les espèces bactériennes qui est impliquée dans la tolérance aux antibiotiques,

	l'acquisition de résistances par des processus de transfert horizontal de gènes et le
	développement de maladies chroniques [2]-[4]. Les biofilms représentent un problème de santé
	majeur dans les hôpitaux et dans l'industrie agroalimentaire. Les bactéries vivant en
	communautés dans les biofilms sont protégées par une matrice extracellulaire qui est constituée
	majoritairement d'eau et d'EPS (Extracellular Polymeric Substances, soit polymères

2.2 Les sous unités régulatrices

  BcsA et BcsBLe gène bcsA et le gène bcsB sont répandus dans presque tous les opérons de Bcs et se trouvent généralement en tandem[START_REF] Römling | Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions[END_REF]. Les structures des sous unités-catalytiques BcsAB ont été étudiées en détail chez R. sphaeroides[START_REF] Morgan | Crystallographic snapshot of cellulose synthesis and membrane translocation[END_REF]-[START_REF] Morgan | Observing cellulose biosynthesis and membrane translocation in crystallo[END_REF]. BcsA est une protéine d'environ 100 kDa qui correspond à la cellulose synthase du système Bcs et qui est formée par une région

	1.2.1 Les sous unités catalytiques
	1.2.1.1

Les protéines codées par les gènes bcs comprennent des protéines catalytiques comme BcsA et BcsB, qui mènent la polymérisation et l'export de la cellulose à travers la membrane interne ; des protéines pour la modification enzymatique dans le périplasme et l'export à l'extérieur de la cellule comme BcsG, BcsZ et BcsC ; et plusieurs protéines régulatrices dont le mécanisme exact reste inconnu comme BcsR, BcsQ, BcsE et BcsF [8]-[12]. transmembranaire N-terminale, sous laquelle se trouve un domaine cytoplasmique de glycosyl transférase suivie d'un domaine pilZ qui se lie au c-di-GMP (di-guanosine monophosphate cyclique) [10], [13]. La région transmembranaire est un domaine d'export de la cellulose formé de huit hélices transmembranaires (TM1-8) [10]-[13]. Quatre de ces hélices TM sont Nterminales et les quatre autres sont C-terminales. BcsB quant à elle est considérée comme le partenaire catalytique de BcsA. En effet, des études ont montré que l'hélice transmembranaire de BcsB liée à BcsA, est essentielle à la polymérisation et au passage de la cellulose à travers la membrane interne des bactéries. BcsB est formée par un module périplasmique contenant deux domaines de liaison aux glucides et deux autres domaines adoptants le repliement jelly roll et insérée dans la membrane interne via une hélice transmembranaire qui vient compléter les huit hélices TM1-8 de BcsA [10]-[13]. 1.

1.2.2.1 BcsR et BcsQ

BcsR et BcsQ sont des sous-unités cytoplasmiques essentielles à la sécrétion de cellulose in vivo. Il a notamment été montré par nos collaborateurs qu'au niveau des pôles de la bactérie où

3 Résultats et discussion 1.3.1 Étude de la structure cristallographique de BcsRQ L

  'un des premiers travaux que j'ai pu entamer dans le contexte de mes recherches était l'étude de l'interaction de BcsR et de BcsQ. Une fois le protocole de coexpression/copurification mis au point, j'ai procédé à l'optimisation des conditions de cristallisation des deux protéines. Dans ce cadre j'ai pu obtenir des cristaux qui diffractent et qui nous ont permis de résoudre la structure du complexe à haute résolution. Ces résultats sont importants non seulement parce que la structure du complexe BcsRQ a été résolue pour la première fois, mais aussi parce qu'ils représentent un outil indispensable pour l'étude des interactions possibles entre BcsRQ et leur partennaire BcsE. La structure de BcsRQ a montré une dimérisation du complexec'est-à-dire un dimère de deux hétérodimères BcsRQ avec une surface assez importante d'interaction.

Dans ce contexte, j'ai combiné études structurales et fonctionnelles afin d'étudier la sécrétion de la cellulose et son rôle dans la persistance des biofilms bactériens. Pour ceci, j'ai entrepris d'étudier trois sous-unités clés pour l'assemblage et la fonction du système de sécrétion de cellulose chez la bactérie modèle E. coli. Dans un premier temps l'objectif était de déterminer les structures à haute résolution des protéines cytoplasmiques BcsR, BcsQ et BcsE, vu les rôles importants qu'elles pourraient jouer. D'une autre part, afin de déterminer la structure du complexe de Bcs avec une meilleure résolution, j'ai utilisé la cryo-EM (cryo-microscopie électronique) par la technique dite de « particules isolées ».

1.

Comme la protéine BcsQ est prédite à fonctionner comme une ATPase (Adénosine TriPhosphatase), notre objectif était de collecter différents états conformationnels de la protéine qui pourraient exister. Pour cela une analyse de trois types de cristaux a été effectuée. La protéine BcsRQ sans nucléotides et la protéine purifiée et cristalisée en présence d'ADP (Adénosine DiPhosphate) et/ou d'AppCp (analogue non hydrolysable de l'ATP). A partir de la collecte de jeux complets de données de diffraction sur les différents cristaux, nous avons constaté qu'il n'existait qu'un seul état conformationnel, celui avec la protéine en complexe avec l'ATP et cela malgré l'ajout d'EDTA (Éthylènediaminetétraacétique).

1.

3.2 Étude de la structure cristallographique de BcsRQ avec deux domaine C- terminaux de BcsE Quand

	problème, j'ai généré des constructions de BcsE en gardant soit la partie C-terminale soit la
	partie N-terminale en se basant sur les prédictions de structures secondaires. Les résultats
	obtenus révèlent deux constructions qui sont copurifiés avec BcsRQ, à savoir BcsE 349-523 et
	BcsE 217-523 . La seconde étape était alors l'étude de la structure et du mécanisme d'intéraction
	de BcsRQE, pour ceci j'ai opté pour la technique de cristallographie aux rayons X et j'ai pu
	obtenir des cristaux qui diffractent d'abord de BcsRQE 349-523 et de BcsRQ R156E E 217-523 . Les
	structures cristallographiques ont montré que BcsE se lie effectivement à la partie C-terminale
	de BcsQ. En plus de révéler les déterminants structuraux d'interaction avec BcsQ, les deux
	structures de BcsRQE ont fourni des informations inattendues quant à l'interaction avec le
	second messager c-di-GMP. Dans la structure BcsRQE 349-523 le c-di-GMP se trouve lié en tant
	que dimère intercalé à chaque domaine GGDEF. Cependant, dans le complexe BcsRQ R156E E 217-
	523 , BcsE subit un changement conformationnel par rapport à la structure de BcsE 217-523 seul
	(résolue par Samira Zouhir), contribuant ainsi à la découverte d'un motif d'interaction avec le
	c-di-GMP jamais identifié jusque-là.
	dans un premier temps était de
	mettre au point un protocole pour la coexpression/copurification de BcsRQE. Ceci nous a
	permis de confirmer nos attentes que BcsE interagit avec BcsRQ en se liant à la partie C-
	terminale de BcsQ. D'autant plus, nos résultats confirment que BcsR et BcsQ sont localisées
	dans le cytoplasme car elles sont récupérées dans la fraction soluble. Toutefois quand BcsR,
	BcsQ et BcsE interagissent, leur extraction nécessite un protocole d'extraction semblable à un
	protocole d'extraction d'une protéine membranaire contrairement à BcsRQ seuls. En effet, pour
	extraire et purifier le complexe BcsRQE, nous sommes amenés à utiliser des détergents. Ceci
	rendait non seulement la procédure de cristallisation difficile voire impossible, mais en plus le
	complexe protéique BcsRQE n'était pas produit en quantité suffisante. Pour surmonter le

j'ai commencé le travail sur les sous unités cytoplasmiques, l'interaction entre BcsE et BcsRQ n'avait pas été démontré. Comme pour BcsRQ, le but

1.3.3 Étude de la structure du complexe de sécrétion par cryo-EM

	Dans l'optique d'étudier l'architecture et l'assemblage du système de sécrétion dans son
	intégralité, j'ai utilisé la technique d'analyse de particules isolées par cryo-EM. Ceci a permis
	non seulement de confirmer l'architecture non-canonique atypique de ce système de sécrétion
	mais aussi de déterminer la structure de la protéine BcsB. Nous avons montré que cette dernière
	fait partie du coeur catalytique et cette structure nous a également permis de comprendre
	l'architecture asymétrique particulière du système de sécrétion, dû à une polymérisation de sa
	partie périplasmique. Bien que la structure d'une protéine homologue eût déjà été résolue, nos
	travaux ont montré pour la première fois la propriété d'auto-assemblage du macrocomplexe de
	cette caractéristique particulière de ce système de sécrétion de cellulose.

niveaux, à savoir l'expression, l'assemblage et la régulation du système de sécrétion de la cellulose. De plus, ces travaux sont un élément clé pour mieux comprendre le comportement des bactéries dans les biofilms, ainsi que les liens mécanistiques aboutissant à l'acquisition du phénomène de résistance aux antibiotiques. Le système bactérien de sécrétion de la cellulose fait partie d'un ensemble de systèmes conservés chez les Eucaryotes et les Procaryotes. Les différents résultats de recherches cités ci-dessus peuvent ainsi contribuer à long terme au développement de thérapies de nouvelle génération, sans risques associés de forte pression évolutive et de développement de résistances.Nos travaux présentent également un intérêt majeur dans un tout autre domaine. En effet, la cellulose possède un fort intérêt biotechnologique, en tant que nano-matériel inoffensif, biocompatible et à coût réduit.

References

Maison du doctorat de l'Université Paris-Saclay 2 ème étage aile ouest, Ecole normale supérieure Paris-Saclay 4 avenue des Sciences, 91190 Gif sur Yvette, France

Acknowledgments

Abbreviations

by in cellulo two-hybrid functional complementation assays and protein co-purification experiments and revealed that BcsE competes with a second TAC component, NusB, for S10 complexation. Although the exact role of these interactions with the TAC machinery remain to be further investigated, the in silico detection of putative intrinsic terminators in the bcsR and bcsQ loci leading the second Bcs operon, led us to speculate that BcsE might not only contribute to c-di-GMP complexation and recruitment of the essential BcsRQ complex to the inner membrane but also be potentially targeted to the site of BcsRQ expression via the TAC machinery for efficient co-translational BcsERQ complex assembly.