Je Remercie Également

Sylvie Nessler

Nathalie Campo

Martin Picard

Por Tudo Joana

Lina ! Pela Cumplicidade Imediata

Maarten Van Dijl

Emma Dave

Edda Klipp

Andreas Otto

David Humphreys

Neesha Dedi

Gilles Malherbe

Dörte Becher

Marco Fonseca

Thiago Feder

Keywords: Bacillus subtilis, Protein secretion, Membrane protein complex, Protease, Lantibiotic resistance, Quality control Sécretion protéique, complexe protéique membranaire, protéase, résistance aux lantibiotiques, contrôle qualité

Bacillus subtilis is a model organism for Gram-positive bacteria and a workhorse used for largescale production of industrial enzymes. Secretory and membrane proteins are mainly translocated through the SecA-YEG (Sec) pathway. Although this highly conserved pathway has been extensively studied, there is a lack of knowledge about the protein interactions and complexes involved in this mechanism in B. subtilis. Therefore, to better understand these membrane protein complexes, we SPA-tagged a set of proteins involved in the Sec pathway for tandem affinity purification. Through the combination of this technique with mass spectrometry and Blue Native PAGE, we studied the protein interactions within the (holo)translocon as well as with the accessory proteins. Our results showed that, under their SPA-tagged forms, SecDF and SpoIIIJ, could not be retrieved together after immune-capture, as opposed to what was shown in E. coli. This result indicated that SecDF and SpoIIIJ, unexpectedly, do not form a stable complex in B. subtilis. The other experiments using SPA-tagged proteins (SecG, BdbB, and BdbC) did not provide evidence for the existence of stable complexes involving proteins known to be part of the translocon complex, except for one protein, the so-called signal peptide peptidase, SppA.Using the SPA-tag technique, we have identified a membrane protein complex, involving SppA and another membrane protein, YteJ. While YteJ function is unknown, SppA is a known membrane bound serine protease, described as responsible for recycling of the signal peptides of the translocated proteins left in the membrane and also involved in the resistance to nisin, an antibacterial peptide Of the lantibiotic family. Interestingly, sppA and yteJ genes are organized in operon, for which we confirmed the regulation by two sigma factors, σ A and σ W , using transcriptional fusions. The fluorescent microscopy experiments revealed a diffused membrane localization for both proteins, and therefore their co-localization was difficult to prove by this technique. Using deletion mutant strains, we showed that both SppA and YteJ are involved in the lantibiotic resistance: the sppA -strain was more sensitive while the yteJ -strain showed a more sensitive phenotype than wild type, but less pronounced than the sppA -strain. The sppA-dependent resistance was confirmed by the ectopic overexpression of SppA that restored the lantibiotic resistance. However, the overexpression of SppA also induced the formation of elongated cells. Interestingly, this phenotype can be suppressed by the simultaneous overexpression of YteJ, indicating an YteJ-dependent negative control of SppA activity. Furthermore, using the elongated cell phenotype, we tested the overexpression of SppA along with truncated versions of YteJ. The results pinpointed to the inhibitory role of the C-terminal domain of YteJ. Finally, we performed in vitro biochemical studies with purified His-tagged SppA and YteJ which showed that B subtilis SppA and YteJ form a membrane complex when expressed together in E. coli. That the SppA protease activity of the purified complex His-SppA-YteJ was almost null, and that, purified from the membrane, the full length SppA protease activity was lower than that of a soluble truncated version of SppA. This full-length SppA activity was strongly reduced in the presence of YteJ, supporting the hypothesis of an inhibition by YteJ. Furthermore, we showed that the ability or inability of SppA to digest fully folded proteins probably depends on the structure of the proteins, suggesting a possible role in protein quality control. In conclusion, our in vivo and in vitro studies showed that YteJ, via one of its domain, acts as a negative regulator of the protease activity of SppA in this complex. Although we have documented a role for this complex in the lantibiotic resistance, a possible role of SppA in protein quality control might not be excluded I Titre : Caractérisation du complexe membranaire impliquant la signal peptide peptidase SppA et YteJ chez Bacillus subtilis.

Mots clés :

INTRODUCTION :

Les complexes membranaires impliqués dans la sécrétion des protéines chez B. subtilis ont été identifiés mais ont été peu étudiés biochimiquement contrairement à leurs homologues chez E. coli. Objectifs de l'étude :Dans le cadre de l'ITN « ProteinFactory », dont le but était d'améliorer les capacités de production/sécrétion des protéines chez les bactéries, nous avons tenté d'identifier les complexes membranaires impliqués dans la sécrétion de protéines chez B. subtilis en nous focalisant sur les protéines de la voie « Sec » (Figure 1). En effet, si les connaissances fondamentales ont été obtenues chez E. coli, peu de données existent sur les interactions physiques entre ces protéines chez B. subtilis, ou encore leur dynamique en fonction de la protéine sécrétée ou de la phase de croissance, par exemple. Grâce à l'emploi de deux techniques biochimiques : le BN-PAGE et l'étiquetage de protéines ciblées pour réaliser de la purification par affinité en tandem (SPA-tag), nous avons tenté de répondre à ces questions.

Le modèle d'étude : Bacillus subtilis est une bactérie modèle pour l'étude des bactéries à Gram positif ; elle est capable de produire des endospores afin de résister aux conditions difficiles et est aussi un modèle pour l'industrie par sa capacité à sécréter un grand nombre de molécules, et notamment des protéines (Figure 2). III trouvent dans la même portion de gel (Figure 4). Nous en avons conclu que SppA et YteJ co-migrent dans ce type de gel ce qui suggère que les deux protéines forme potentiellement un complexe protéique membranaire. YteJ, protéine inconnue, est une protéine de la famille RDD, famille de fonction inconnue, potentiellement un transporteur.

Par SPA-tag :

La séquence d'une étiquette permettant une double chromatographie séquentielle par affinité a été fusionnée en 5' de la séquence du gène de la protéine d'intérêt par intégration chromosomique du plasmide pMUTIN-SPA-LIC. Des souches sppA-SPA et yteJ-SPA furent ainsi obtenues mais seule la souche yteJ-SPA a montré une expression de YteJ-SPA détectable par Western-blot. La purification de YteJ-SPA à partir des membranes, solubilisées en présence de DDM, a été réalisée et les protéines copurifiées ont été analysées par spectrométrie de masse. La protéine la plus abondante ayant été copurifiée avec YteJ-SPA était SppA. Nous avons pu estimer d'après ces données de spectrométrie de masse que le ratio entre SppA et YteJ dans le complexe était de 1:1 ou 2:1. IV 2.2 La sécrétion des protéines : Afin de tester le rôle de SppA et de YteJ, potentiellement via son interaction avec SppA, dans la sécrétion des protéines, nous avons utilisé la protéine modèle AmyM dont le gène est présent sur le plasmide pCS73. Aucune des souches comportant les délétions de sppA et/ou yteJ, n'ont montré de différence dans la quantité finale de AmyM sécrétée dans le milieu (Figure 6). Des expériences de « pulsechase assays » avec ces mêmes souches mutantes réalisées par des collaborateurs (Pr JM Van Dijl, Groningen) ont également conclu à l'absence de différence dans la cinétique de sécrétion de AmyQ.

Résistance à la subtilin

Après avoir testé la nisine, peptide de la famille des lantibiotiques, nous avons testé un autre peptide de cette famille, la subtiline, que nous avons produite à l'aide d'une souche sécrétrice de B. subtilis (ATCC6633). Les résultats obtenus avec la nisine et la subtiline ont été identiques, quoique plus reproductibles avec la subtiline (Figure 7). La présence de subtiline (ou nisine) dans le milieu de culture LB sur les cellules de la souche sauvage provoque un retard de croissance mesurable par densité optique et ce retard est accentué chez la souche sppA à partir de 25% de subtiline. La souche yteJ montre un phénotype intermédiaire à partir de 30%. La résistance aux lantibiotiques liée à SppA décrite dans la littérature est donc confirmée, et nos expériences montrent que YteJ semble avoir un rôle dans cette résistance. V Afin de vérifier que l'absence de SppAet celle de YetJ -était bien la raison directe de la sensibilité accrue à la subtiline chez les souches délétées de ces gènes, nous avons utilisé ces souches et les avons complémentées en réintroduisant, en ectopique au locus amyE, une copie du gène délété, sppA ou yetJ, ou encore de l'operon sppA-yteJ, sous un promoteur inductible à l'IPTG. Ces souches furent testées en présence de 30% de subtiline, concentration retenue car provoquant une différence observable de résistance à la subtiline entre les souches. Dans ces conditions, des concentrations croissantes d'IPTG ont été ajouté dans le milieu LB, afin de tester la dose-réponse de l'expression de ces gènes pour la résistance à la subtiline (Figure 8).

A B

VI

A partir de 25 µM d'IPTG, les courbes de croissance de la souche Phs-sppA se rapproche de la souche sauvage et de la souche Phs-yteJ, qui, elle, se comporte comme la souche sauvage. Ceci prouve que la résistance à la subtiline dépend bien directement de SppA. En revanche, à partir de 100 µM IPTG, nous avons systématiquement observé que les cellules de la souche Phs-sppA lysaient rapidement après l'entrée en phase stationnaire. Il semblerait donc que la surexpression de SppA, seule, provoque un effet délétère sur les cellules. Cet effet n'est en revanche pas observé lorsque les deux protéines SppA et YteJ sont surexprimées en même temps comme c'est le cas de la souche Phs-sppA-yteJ (Figure 9). Nous pouvons noter que, alors que sans IPTG cette souche est plus sensible à la subtiline, une meilleure résistance à la subtiline pour cette souche par rapport à la souche sauvage est observable dès l'ajout de 10 µM d'IPTG dans le milieu de culture.

Ces expériences nous ont permis de confirmer le rôle direct de SppA dans la résistance à la subtiline, possiblement via son activité protéasique/peptidasique. Cependant, une surexpression de SppA devient délétère au-delà d'un seuil, ce qui se traduit par une lyse prématurée des cellules. En revanche, la surexpression simultanée des deux protéines ne provoque aucun problème aux cellules de cette souche et lui permet même d'être plus résistante que la souche sauvage.

Régulation de l'activité protéase de SppA

La surexpression de SppA dans la souche Phs-sppA nous avait montré que, pour des concentrations de 100 µM d'IPTG, des effets délétères sur les cellules pouvaient être observés à travers une biomasse plus faible en phase stationnaire et une chute de la densité optique certainement due à une lyse prématurée des cellules. Cet effet était totalement annihilé par la surexpression concomitante de YteJ. Nous avons voulu observé au microscope l'effet sur la morphologie cellulaire.

Effet de la surexpression de SppA sur la morphologie cellulaire de cellules de B. subtilis

Les effets sur la morphologie cellulaire furent observés sur des cellules prises en phase exponentielle de croissance, en présence de 100 µM d'IPTG, au microscope à contraste de phase ; les membranes révélées avec une coloration au FM 4-64 et l'ADN chromosomique coloré au DAPI (Figure 10).

Figure 9 : Croissance des cellules de la souche sauvage (BSB1) et de la souche délétée de l'opéron sppA-yetJ complémentés par cet opéron en ectopique sous le contrôle d'un promoteur Phyperspank (Phs) inductible à l'IPTG (notées Phs-sppA-yteJ). L'effet de la subtiline (30%) est testé en fonction de concentrations croissantes d'IPTG (0 à 100 µM).

VII

Pour la souche Phs-sppA, les cellules sont apparues en filament indiquant un problème de séparation des deux cellules filles après division. La coloration au FM 4-64 a montré les septums, visibles, mais plus éloignés les uns des autres que les septums observés pour la souche sauvage BSB1. De même la coloration au DAPI a révélé que les chromosomes semblaient moins compacts que ceux de la souche sauvage. Pour les souches Phs-yteJ et Phs-sppA-yteJ n'ont pas montré de différences morphologiques par rapport à la souche sauvage. Ceci a confirmé que la surexpression de SppA, qui permet de retrouver une résistance à la subtiline, entraine des problèmes morphologiques aux cellules et affecte le bon déroulement du processus de division cellulaire. Nous avons vérifié que c'était bien l'activité protéase de SppA qui en était la raison, en surexprimant une version inactive de SppA après l'introduction d'une mutation K119A dans le site catalytique (Figure 11). Avec SppA K199A les cellules sont apparues normales, ce qui nous a permis de conclure que c'était bien l'activité protéase de SppA qui provoquait les défauts morphologiques. VIII L'effet de l'activité SppA surexprimée sur la morphologie cellulaire est annulé par la surexpression simultanée de YteJ avec SppA dans la souche Phs-sppA-yteJ (Figure 10). Nous avons donc émis l'hypothèse que YteJ jouait un rôle régulateur de l'activité de SppA. Après expression chez E. coli, excepté pour SppA ∆1-52 qui a été purifiée à partir de la fraction cytosolique, les autres protéines, étant membranaires, ont été purifiées à partir des membranes de E. coli, isolées par ultracentrifugation et solubilisées avec 1% DDM. Les échantillons furent chargés sur colonne de Ni-NTA agarose (Qiagen). Après lavage, les protéines furent éluées avec 200 mM imidazol et concentré sur Amicon Ultra-15 Centrifugal Filters. L'analyse par SDS-PAGE du résultat des purifications de ces quatre protéines est montré en Figure 13

Expression et purification de

IX

Nous avons pu conclure que SppA apparait en 2 à 3 bandes (identifiées comme SppA par spectrométrie de masse), une seule bande pour la forme tronquée SppA ∆1-52 , et que la purification de His-SppA entraine YteJ (identifiée par spectrométrie de masse) lorsqu'elle est co-exprimée avec YteJ. Ceci prouve que l'interaction entre SppA et YteJ peut également exister chez E. coli.

Activité protéase de SppA

Afin de tester l'activité protéase de SppA ∆1-52 , nous avons utilisé des protéines disponibles au laboratoire. L'activité protéase de SppA ∆1-52 , comme annoncé dans la littérature, est apparue efficace envers certaines protéines (HNS, Rok), partielle envers CcpA et faible voire nulle sur le lysozyme et la BSA (Figure 14). SppA ∆1-52 a montré une activité protéase dégradant seulement l'extension (linker) d'une GFP, mais pas sur la GFP elle-même. Nous pourrions conclure que certaines structures ou séquences d'acides aminés permettent ou non l'activité de SppA. L'activité de SppA et de SppA/YteJ a alors été testée avec cette GFP ext en tant que substrat et comparée à celle de SppA ∆1-52 (Figure 15). A concentration de protéine identique, l'activité de SppA ∆1-52 (sans domaine transmembranaire) est beaucoup plus forte que celle de SppA (forme avec domaine transmembranaire). Cette différence peut refléter un effet du domaine transmembranaire qui pourrait participer lui-même à la régulation de l'activité de SppA. En revanche, l'activité de SppA dans le complexe SppA/YteJ est apparue comme nulle durant 90 minutes. Cependant, SppA dans ce complexe est active puisque qu'une dégradation de GFP ext fut observée après 16 heures de réaction. Nous avons conclu que, dans le complexe SppA/YteJ, YteJ agit en tant que régulateur/inhibiteur de l'activité protéase de SppA.

Le domaine C-terminal de SppA des différentes formes purifiées

Le domaine C-terminal de SppA est absent de SppA active mais présent dans un mutant catalytique (K199A) ce qui suggère que ce domaine participe à la régulation de l'activité de SppA. De façon intéressante, nous avons remarqué que la purification de SppA conduit à l'obtention de plusieurs bandes sur gel de SDS-PAGE séparées de quelques kDa. Par spectrométrie de masse, nous avons pu identifier que toutes ces bandes sont des formes de SppA (Figure 16). Par ailleurs, cette analyse a aussi révélé la présence du domaine C-terminal de SppA, à travers la détection de deux peptides y appartenant, uniquement dans SppA purifiée en complexe avec YteJ. SppA dans le complexe SppA/YteJ possède donc encore son domaine C-terminal, comme pour le mutant catalytique SppA K199A . Ce résultat est en accord avec le résultat précédent qui montrait que SppA dans le complexe SppA/YteJ est inactive.

Le domaine C-terminal de YteJ

Afin d'étudier plus précisément comment YteJ régule l'activité de SppA, nous avons construit des souches qui surexpriment SppA et différentes versions tronquées du domaine C-terminal de YteJ. Des zones structurales de YteJ ont été ciblées : dernière hélice alpha prédite (12 derniers acides aminés) ou

REFERENCES:

1. Tjalsma, H., et al., Proteomics of protein secretion by Bacillus subtilis: separating the "secrets" of the secretome. Microbiol Mol Biol Rev, 2004. 68(2): p. 207-33. 2. Nam, S.E., A.C. Kim, and M. Paetzel, Crystal structure of Bacillus subtilis signal peptide peptidase A. Journal of Molecular Biology, 2012. 419(5): p. 347-358. Self-Processing, and Regulation. Biochemistry, 2013. 52: p. 8811-8822. 4. Kingston, A.W., X. Liao, and J.D. Helmann, Contributions of the σ(W) , σ(M) and σ(X) regulons to the lantibiotic resistome of Bacillus subtilis. Molecular microbiology, 2013. 90(3): p. 502-18. 5. Saito, A., et al., Post-liberation cleavage of signal peptides is catalyzed by the site-2 protease (S2P) in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 2011. 108(33): p. 13740-5. 6. Shao, L., et al.,

2.2.5

The deletion of sppA and yteJ affects the resistance to lantibiotics .............................

2.2.6

The deletions of sppA and yteJ do not affect the resistance to vancomycin, erythromycin or protect against lysozyme ... and their products for the benefit of mankind in different areas and, at the same time, contributes to a resource-efficient and sustainable economy [START_REF] Gavanji | Application of recombinant DNA technology-A Review[END_REF][START_REF] Chu | Industrial choises for protein production by large scale cell culture[END_REF]. The biotech industry is hugely important for the EU, as it is a sector that employs around 7.5 million people and it has an annual turnover over 100 € billion (European Commission 2017). Within this industry, the production of recombinant enzymes and biopharmaceutical proteins is of major importance. The global market for such recombinant proteins has grown significantly in the past decade, worth 180 € millions in 2017 and is forecast to exceed 225 € million by 2021. The constant growth of this market leads to a continuous need for optimization of the production platforms that can deliver protein products in greater yields, with higher quality and at lower costs. Therefore there is an overwhelming need for the development of next-generation super-secreting cell factories ( [START_REF] Dijl | Bacillus subtilis: from soil bacterium to super-secreting cell factory[END_REF][START_REF] Browning | Escherichia Coli 'TatExpress' strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway[END_REF][START_REF] Matthews | Reexamining opportunities for therapeutic protein production in eukaryotic microorganisms[END_REF].

LIST OF APPENDIX TABLES

LIST OF APPENDIX FIGURES

This thesis was carried out within the frame of the EU-funded innovative training network (ITN), Protein Factory (2015Factory ( -2018)), of the Horizon 2020 Marie Skłodowska-Curie actions program. This project brings together 6 academic Institutes, which provide the expertise in synthetic biology and protein secretion, and 5 non-academic partners which include some of the world's premier biotechnology companies (Novozymes, DSM, Abera Bioscience, UCB-Celltech and FGen) with the main objective of creating super-secreting strains, bypassing major production bottlenecks such as secretion stress. A variety of different host expression systems for the production of recombinant proteins has been described over the years, and each has its own advantages and disadvantages. The host organisms should be precisely selected for each specific product, considering the product development and running cost, yield, and easiness of downstream processing, time to market, necessity of correct glycosylation, and available infrastructure [START_REF] Chu | Industrial choises for protein production by large scale cell culture[END_REF]. The two most popular industrial workhorses for the production of biopharmaceuticals and industrial enzymes are Escherichia coli and Bacillus subtilis, respectively [START_REF] Tu | Finite element modeling of kirschner pin and bone thermal contact during drilling[END_REF]. These bacteria are the two model organisms used in Protein Factory. Both are fast growing bacteria able to replicate in cheap media to high cell density, their genomes are far better studied than other microorganisms which makes available many genetic tools, both are amenable to genetic manipulation and can express and produce proteins with high yields. Due to E. coli very high level of expression of recombinant proteins, that riches up to 30% of total cellular protein, this bacterium is preferably used for the production of biopharmaceuticals, such as the human growth hormone (e.g. Humatrope) or insulin (e.g. Lispro) [START_REF] Ramadan | Production of Biopharmaceuticals in E. Coli : Current Scenario and Future Perspectives[END_REF]. E. coli is a Gram-negative bacterium, characteristically having a periplasm and an outer membrane bilayer composed of lipopolysaccharides. As such, the secreted proteins are exported from the cytoplasm to the periplasmic space from which they can be recovered by the rupture of the outer membrane, which gives advantages in downstream processing [START_REF] Blount | The unexhausted potential of E. Coli[END_REF]. However, the presence of this outer membrane can be a limitation as it works as a permeability barrier and obstructs the secreted proteins of being released to the medium. The overexpression of the recombinant proteins in this host can origin misfolded proteins that accumulate in the cytoplasm as inclusion bodies [START_REF] Villaverde | Protein aggregation in recombinant bacteria: biological role of inclusion bodies[END_REF]. To recover these proteins the inclusion bodies need to be solubilized and the proteins refolded into bioactive molecules, a process that bring major costs to the production process with a final poor recovery of the recombinant proteins [START_REF] Sørensen | Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli[END_REF]. At an industrial scale and depending on the proteins to be obtained, in the case of secreted proteins, using B. subtilis as a host organism might be a better option. As a non-pathogenic Gram-positive bacteria, B. subtilis have a cytoplasmic membrane surrounded by a permeable cell envelope that allows the release of secretory proteins directly into the culture medium. Therefore, B.

subtilis was used as a model organism for the development of this thesis project and, will be described in detail in the next section.

Bacillus subtilis, a Gram-positive bacterium

Bacillus subtilis is a Gram-positive rod-shaped bacterium that belongs to the Firmicute phylum (Bacillaceae family). The main classes of this phylum are the Clostridia, Bacilli (Bacillus, Listeria, Enterococcus, Staphylococcus and Streptococcus) and Mollicute [START_REF] Wolf | Phylogeny of firmicutes with special reference to mycoplasma (mollicutes) as inferred from phosphoglycerate kinase amino acid sequence data[END_REF]). B. subtilis was discovered in 1835 by Christian Gottfried Ehrenberg and originally named "Vibrio subtilis", however in 1872 Ferdinand Cohn renamed it "Bacillus subtilis". B. subtilis is considered as an obligate aerobe, but can also grow anaerobically by respiration on nitrate or by fermentation on glucose [START_REF] Nakano | Characterization of anaerobic fermentative growth of Bacillus Subtilis: identification of fermentation end products and genes required for growth[END_REF][START_REF] Hoffmann | The anaerobic life of Bacillus subtilis : cloning of the genes encoding the respiratory nitrate reductase system[END_REF]. The B. subtilis genome exhibits a low % GC content and, although Gram-positive and Gram-negative bacteria diverged about one billion years ago, it shares similarities with the E. coli genome sequence, as about one-quarter of the entire B. subtilis

Box1: Project context

This thesis is part of the EU-funded innovative training network (ITN), part of the Horizon 2020 Marie Skłodowska-Curie actions program, entitled Protein Factory. A project that intends to overcome the major bottlenecks of recombinant protein production, such as secretion stress, through the creation of engineered super-secreting strains, capable of secreting an unprecedented range of target molecules. Bacillus subtilis was the model organisms used in this project.

genome have clear orthologous counterparts in E. coli [START_REF] Kunst | The complete genome sequence of the gram-positive Bacterium Bacillus subtilis[END_REF]. Besides the core of the translation and transcription machineries, other operons involved in major integrated functions, such as ATP synthesis (atp operon) and electron transfer machinery (cta and qox operons), are well conserved between the two organisms [START_REF] Kunst | The complete genome sequence of the gram-positive Bacterium Bacillus subtilis[END_REF]. In contrast, these two bacteria are structurally different, B. subtilis, as a Gram-positive bacterium, possesses a single cytoplasmic membrane and a thick cell wall matrix of peptidoglycans and teichoic acids, thus lacking a true periplasmic space [START_REF] Matias | Cryo-Electron microscopy reveals native polymeric cell wall structure in bacillus subtilis 168 and the existence of a periplasmic space[END_REF][START_REF] Weidenmaier | Teichoic acids and related cell-wall glycopolymers in gram-positive physiology and host interactions[END_REF], while E. coli has a characteristic Gram-negative envelope composed by three principal layers, the inner (IM) and outer (OM) membrane flanking their cell wall within the periplasmic space [START_REF] Salton | Studies of the bacterial cell wall: IV. The composition of the cell walls of some gram-positive and gram-negative bacteria[END_REF][START_REF] Weidenmaier | Teichoic acids and related cell-wall glycopolymers in gram-positive physiology and host interactions[END_REF].

B. subtilis natural niche is the rhizosphere soil, it grows associated to the plant roots, a healthy and mutually beneficial association. By one side, B. subtilis benefits from the components excreted by plant roots (such as amino acids, organic acids, sugars, phenolic acids and other secondary metabolites), in turn it contributes to the plant defence against pathogenic bacteria [START_REF] Gaskins | Rhizosphere bacteria and their use to increase plant productivity: a review[END_REF][START_REF] Haney | Associations with Rhizosphere bacteria can confer an adaptive advantage to plants[END_REF]. II. endospore, a small robust, enduring and metabolically inactive cells are produce in response to nutritional limitations. Spores can survive years in the absence of nutrients, and can resist to a wide variety of extreme conditions such as heat, cold, drying and radiations [START_REF] Burbulys | Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay[END_REF][START_REF] Gohlke | The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter[END_REF].

During the sporulation process, cells secrete extracellular killing factors that induce the lysis of nonsporulating cells that have not developed immunity to these toxins.

This lysing process releases nutrients into the medium that the surviving sporulating cells can feed on, and thus this behaviour is referred to cannibalism (González-Pastor 2011; Gonzalez-Pastor 2003); III. competent cells, a type of cells able to scavenge DNA from environment to use as a nutrient source or, when possible, integrated into the chromosome by recombination to gain new functions [START_REF] Anagnostopoulos | Requirements for transformation in Bacillus subtilis[END_REF];

Connected to the cell fate is their social behaviour, bacteria do not grow as dispersed individual organisms, they communicate and cooperate with each other, originating bacterial communities. This social behaviour gives to the cell the ability to perceive environmental changes and react accordingly [START_REF] López | Extracellular signals that define distinct and coexisting cell fates in Bacillus subtilis[END_REF]. The biofilms are an example of how bacterial cells can form multicellular structures able to adhere to surface encased in a self-produced extracellular matrix, as it is visible on Figure 1 [START_REF] Vlamakis | Sticking Together: building a biofilm the Bacillus subtilis way[END_REF][START_REF] Kearns | A master regulator for biofilm formation by Bacillus Subtilis[END_REF][START_REF] Aleti | Surfactin variants mediate species-specific biofilm formation and root colonization in Bacillus[END_REF].

Undomesticated B. subtilis strains form robust, multicellular biofilms that exhibit complex architectural features [START_REF] Lemon | Biofilm development with an emphasis on Bacillus subtilis[END_REF]. Swarming is another social behaviour through which a synchronised group of flagellum-driven bacterial cells moves on a surface [START_REF] Partridge | Swarming: flexible roaming plans[END_REF][START_REF] Kearns | Swarming motility in undomesticated Bacillus subtilis[END_REF][START_REF] Ke | Water surface tension modulates the swarming mechanics of Bacillus subtilis[END_REF]. Bacteria communicate between each other and respond to fluctuations in external cues as a global bacterial population by a complex sensing system called quorum sensing. This mechanism allows the cells to produce signal molecules to activate distinct pathways and trigger changes in gene expression in response to changes in cell density, leading bacteria to adapt to new scenarios [START_REF] Bibliography Abisado | Bacterial Quorum Sensing and Microbial Community Interactions[END_REF][START_REF] Camilli | Bacterial small-molecule signalling pathways[END_REF][START_REF] Ke | Water surface tension modulates the swarming mechanics of Bacillus subtilis[END_REF].

B. subtilis: a Gram-positive model

In 1958, John Spizizen showed for the first time that the strain B. subtilis 168, a nonpathogenic and auxotrophic for tryptophan strain, is transformable by exogenous chromosomal DNA [START_REF] Spizizen | Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate[END_REF]. This discovery opened a door that allowed the scientific community to study and deeply understand the genetics and molecular biology of B. subtilis, adopting this organism as a genetic tool, which is one of the most studied microorganisms along with E. coli and Salmonella typhimurium. The 4215 kb entire genome of B. subtilis was sequenced in 1997 and completely re-sequenced and annotated in 2009, with 4406 predicted ORFs (Open Reading Frame) [START_REF] Kunst | The complete genome sequence of the gram-positive Bacterium Bacillus subtilis[END_REF][START_REF] Barbe | From a consortium sequence to a unified Sequence: The Bacillus subtilis 168 reference genome a decade later[END_REF]. Studies showed that only around 260 genes were found to be essential for growth, corresponding to proteins involved in DNA replication, protein synthesis (i.e. ribosomes, synthesis, translation, secretion, protein quality control, lipid biosynthesis, cell metabolism and cell division) [START_REF] Kobayashi | Essential Bacillus subtilis genes[END_REF][START_REF] Commichau | Essential genes in Bacillus subtilis: a re-evaluation after ten years[END_REF][START_REF] Koo | Construction and analysis of two genome-scale deletion libraries for Bacillus subtilis[END_REF]. The acquirement of such information, as the genome sequence, has allowed more comprehensive and global experimental approaches such as transcriptomic, proteomics and interactomics [START_REF] Mäder | Transcriptome and proteome analysis of Bacillus subtilis gene expression modulated by amino acid availability transcriptome and proteome analysis of Bacillus subtilis Gene expression modulated by amino acid availability[END_REF][START_REF] Otto | Systems-wide temporal proteomic profiling in glucose-starved Bacillus subtilis[END_REF][START_REF] Marchadier | An expanded protein-protein interaction network in Bacillus subtilis reveals a group of hubs: exploration by an integrative approach[END_REF][START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF]. Along with the creation of dedicated and reliable B. subtilis databases. These databases are either part of global efforts such as BsubCyc or specialized to specific scientific problems such as transcriptional regulation in B. subtilis (DBTBS) or proteome databases (KEGG, UniProt and PDB) [START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF]Kanehisa et al., 2016;Burley et al., 2017). SubtiWiki, a highly detailed database, has been developed to gather together all information of B. subtilis, from specific databases of genes, messenger RNAs, protein expression, protein-protein interactions to regulatory and metabolic pathways [START_REF] Michna | subtiwikia database for the model organism bacillus subtilis that links pathway, interaction and expression information[END_REF][START_REF] Zhu | SubtiWiki in 2018: from genes and proteins to functional network annotation of the model organism Bacillus subtilis[END_REF].

B. subtilis: a bacterium of industrial interest

In addition to its GRAS (Generally Recognized as Safe) status, B. subtilis and close relatives are a source of products with industrial importance for the production of numerous proteins such as amylases (used in the bread industry), proteases, cellulases (used in the detergent industry) or antibiotics such as bacitracin and polymyxin (van [START_REF] Dijl | Bacillus subtilis: from soil bacterium to super-secreting cell factory[END_REF][START_REF] Westers | Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism[END_REF]H. Cao et al. 2017). This capability is mainly due to two intrinsic characteristics of this group of bacteria:

I.

In their natural habitat, the soil, they have the ability to produce a wide range of hydrolytic enzymes that allows them to breakdown soil-based macromolecules into vital nutrients (Pohl and Harwood 2010b).

II.

They are also able to export and secrete proteins directly into the extracellular medium at concentrations as high as grams per litre, due in part to the single membrane system of Grampositive bacteria, in contrast to the double membrane system of Gram-negative bacteria.

The fact that B. subtilis is easy to grow at high cell densities and its natural ability to release secretory proteins into the culture medium is of great industrial and commercial interest. It reduces downstream processing costs, makes purification more straightforward and reduces the likelihood of contamination with cytoplasmic proteins (van [START_REF] Dijl | Bacillus subtilis: from soil bacterium to super-secreting cell factory[END_REF]H. Cao et al. 2017) .

Box2: B. subtilis

Commonly found in the soil, B.subtilis, is a rod shaped ubiquitous bacterium that secretes numerous enzymes to the culture medium, enabling the bacterium to survive in a continuously changing environment. It is considered as a GRAS organism, it can be easily cultivated up to high cell densities and handled in the laboratory. Along with its high genetic tractability and amenability to adopt different life-styles, B. subtilis has become the Grampositive model organism, fundamental for research studies, and a cell factory used for largescale production of industrial enzymes.

Protein translocation across cytoplasmic membrane in bacteria

Membrane and exported proteins are crucial players for maintenance and survival of bacterial organisms and their correct compartmentalization is a key step for molecular function (Figure 2).

After being synthesized by ribosomes in the bacteria cytoplasm, these proteins need to be inserted in the cytoplasmic membrane, anchored to the membrane or released into the extracellular medium [START_REF] Green | Bacterial secretion systemsan overview[END_REF][START_REF] Schneewind | Protein secretion and surface display in grampositive bacteria[END_REF][START_REF] Costa | Secretion systems in gram-negative insights[END_REF]. Exported proteins have important functions as, among others, hydrolysis of extracellular polymers (proteins, nucleic acids, polysaccharides), synthesis of the cell wall and intercellular matrix, such as biofilm, modification of the host cell, and sensing of the environmental conditions [START_REF] Ivankov | How many signal peptides are there in bacteria?[END_REF]). In the present chapter, I will describe all the export routes that membrane and secretory proteins can undertake in bacteria, with greater emphasis on the Gram-positive B. subtilis.

The general protein secretion pathway: the Sec system

In bacteria, the Sec system is highly conserved and it represents the major route for protein transportation through the hydrophobic membrane barrier. The Sec system allows the translocation of proteins in an unfolded conformation, without loss of small molecules such as ions and valuable metabolites. This system was originally identified in E. coli back in the early 90's. By the use of liposomes, a preprotein substrate (proOmpA) mixed with Sec purified proteins, respectively SecB, SecA and SecY/E proteins, these early studies showed that these components form a binding cascade from SecB to SecA to SecY/E and that they may be enough for the basic reactions involved in the targeting and the translocation of a cargo protein [START_REF] Brundage | The purified E. coli integral membrane protein SecYE is sufficient for reconstitution of seca-dependent precursor protein translocation[END_REF][START_REF] Hartl | The binding cascade of SecB to SecA to SecY/E mediates preprotin targeting to the E. coli plasma membrane[END_REF]). The Sec translocase, in its minimal form, consists of a protein-conducting channel formed by the heterotrimeric membrane protein complex SecYEG, and the essential ATPase SecA that acts as a molecular motor mediating protein transport in this pathway [START_REF] Brundage | The purified E. coli integral membrane protein SecYE is sufficient for reconstitution of seca-dependent precursor protein translocation[END_REF][START_REF] Akimaru | Reconstitution of a Protein Translocation System Containing Purified SecY, SecE, and SecA from Escherichia coli[END_REF][START_REF] Beckwith | The Sec-Dependent Pathway[END_REF].

In general, the main components of the Sec system are highly conserved, and homologous of the SecYEG complex (Figure 3) have been found in the cytoplasmic membrane of archaea, the chloroplast thylakoid membrane and the eukaryotic endoplasmic reticulum [START_REF] Pohlschröder | Protein translocation in the three domains of life: variations on a theme[END_REF].

However, recent studies on the Gram-positive Sec system revealed that a large number of Grampositive bacteria possess accessory Sec components that are not found in other organisms. A second SecA paralog, termed SecA2, and a second SecY paralog, termed SecY2, are present and although being involved in protein translocation, in most cases, they are not essential [START_REF] Prabudiansyah | The Canonical and accessory sec system of gram-positive bacteria[END_REF]. This accessory pathway is found in different Gram positive bacterial species as Mycobacterium, Listeria, Clostridium difficile, Streptococcus, Staphylococcus and Bacillus. Bacillus subtilis is devoid of accessory SecA2 protein, however this is present in some Bacillus species such as Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus smithi, and Bacillus methanolicus.

SecA2 promotes the export of a small subset of proteins and plays an important role in virulence [START_REF] Kurtz | The SecA2 secretion factor of mycobacterium tuberculosis promotes growth in macrophages and inhibits the host immune response[END_REF][START_REF] Sullivan | The Mycobacterium Tuberculosis SecA2 system subverts phagosome maturation to promote growth in macrophages[END_REF]. SecY2 is predicted to form an accessory membrane channel that is responsible for the export of specific proteins, which cannot be exported by the canonical SecYEG [START_REF] Prabudiansyah | The Canonical and accessory sec system of gram-positive bacteria[END_REF]. These accessory SecA2/Y2 pathway appear to mediate the transport of the surface protein GspB, the superoxide dismutase SodA and the catalase peroxidase KatG [START_REF] Bensing | An accessory Sec Locus of Streptococcus gordonii is required for export of the surface protein GspB and for normal levels of binding to human platelets[END_REF][START_REF] Kurtz | The SecA2 secretion factor of mycobacterium tuberculosis promotes growth in macrophages and inhibits the host immune response[END_REF]. [START_REF] Collinson | The structure of the bacterial protein translocation complex SecYEG[END_REF]H Tjalsma et al. 2000). These factors are responsible for maintaining the substrate in an unfolded secretory state and for targeting the pre-protein to the translocon formed by the SecYEG complex.

II.

Translocation: The secreted or membrane proteins are pushed into the SecYEG channel. This movement is driven by the translocon motor SecA, an ATPase that interacts with the substrate proteins and binds to the SecYEG complex [START_REF] Bauer | A 'Push and Slide' mechanism allows sequence-insensitive translocation of secretory proteins by the SecA ATPase[END_REF]. The repeated cycles of binding, hydrolysis of ATP, conformational changes of the dimeric SecA leads to the translocation of the secretory protein through the SecYEG complex [START_REF] Robson | Energy transduction in protein transport and the ATP hydrolytic cycle of SecA[END_REF].

III.

Release and maturation: Subsequently, the signal peptide is cleaved resulting in the release of the protein from the membrane. Finally the protein undergoes a maturation process carried out by other accessory proteins, such as the foldase PrsA, or the Bdb thiol-disulfide oxido-reductases [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF][START_REF] Wely | Translocation of proteins across the cell envelope of gram-positive bacteria[END_REF][START_REF] Driessen | Protein translocation across the bacterial cytoplasmic membrane[END_REF].

Substrate recognition

A crucial early event in the Sec secretion pathway is the identification of substrates that are destined for secretion by cytoplasmic components. The first evidence of a substrate recognition system for protein translocation dates back to the 70's, when Milstein and his colleagues (1972) provided insights into how secreted immunoglobulins were made in precursor form with an amino-terminal hydrophobic sequence that was cleaved to the mature form of the protein, raising the signal sequence hypothesis [START_REF] Milstein | A possible precursor of immunoglobulin light chains[END_REF]. Few years later, Blobel and Dobberstein provided substantial and persuasive evidences into how secreted proteins are recognized. They showed, through in vitro studies, that the product synthesized from the mRNA for the light chain of lgG was larger by 4,000 g/mol than the actual secreted light chain [START_REF] Blobel | Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membranebound ribosomes of murine myeloma[END_REF].

The non-mature preproteins have a hydrophobic N-terminal extension, the signal sequence or signal peptide (SP), that can be recognised by the targeting system (as SRP, SecB and CsaA) responsible for directing the preproteins to the cytoplasmic membrane [START_REF] Ivankov | How many signal peptides are there in bacteria?[END_REF]. The signal sequences are typically between 15 up to 30 amino acids long and they are characterized by three different domains:

i. N-domain: a 5-to 8-residue-long positively charged amino terminal that interacts with the translocation machinery and the negatively charged phospholipids of the membrane;

ii.

H-domain: a 8-to 12-residues-long hydrophobic core region decisive for the signal peptide function. The presence of these hydrophobic residues promotes an α-helical conformation and consequently a formation of a hairpin-like structure that might result in the insertion of the signal peptide into the membrane;

iii. C-domain: a 5-to 7-residue long sequence containing a cleavage site for specific signal peptidases (SPase) responsible for removing the signal peptide from the exported protein, during or shortly after its translocation.

Although, the signal peptides have this structure in common there is a high variability of the amino acids sequence Figure 5). In general, the signal peptides of Gram-positive bacteria are typically longer and more hydrophobic than the SP of Gram-negative bacteria [START_REF] Zanen | Signal peptide hydrophobicity is critical for early stages in protein export by Bacillus subtilis[END_REF][START_REF] Harwood | Bacillus protein secretion: an unfolding story[END_REF]. Responsible for the targeting of membrane proteins and secretory proteins to the correct secretion machinery. The SP can be classified according to the SPase cleavage sites and the pathway by which the preproteins are predicted to be exported. The Sec-dependent SPs, which are the most abundant type of SPs, contain a characteristic AXA motif that allows the targeting to the Sec translocation machinery. The signal peptides present in B. subtilis can be classified into five distinct classes: twinarginine (SRRxFLK) signal peptides, secretory (Sec-type) signal peptides, lipoprotein signal peptides, pseudopilin-like signal peptides, and bacteriocin and pheromone signal peptides (H [START_REF] Tjalsma | Proteomics of protein secretion by Bacillus subtilis: separating the 'secrets' of the secretome[END_REF]). The Tat-dependent SPs have a twin arginine motif (RR/KR) specific that directs them to the Tat secretion machinery [START_REF] Natale | Secand Tat-Mediated protein secretion across the bacterial cytoplasmic membrane distinct translocases and mechanisms[END_REF]. The difference between the SP of the secretory proteins and lipoproteins is the presence of a conserved lipobox. This sequence contains a Nterminal invariable cysteine residue that have to be diacylglycerol-modified before their attachment to the outer surface of the membrane [START_REF] Harwood | Bacillus protein secretion: an unfolding story[END_REF]. In B. subtilis four proteins have been identified with pseudopilins SPs (ComGC, ComGD, ComGE, and ComGG) which are cleaved by the SPase ComC [START_REF] Chung | All seven ComG Open reading frames are required for DNA binding during transformation of competent Bacillus subtilis[END_REF]. Finally, the fifth class of SPs refers to the ribosomally synthesized pheromones and lantibiotics, which contain only a N-and C-domains and completely devoid of a hydrophobic H-domain (Fig. 4). These types of proteins are exported and cleaved by ABC transporters [START_REF] Tjalsma | Signal peptide-dependent protein transport in Bacillus subtilis: A Genome-Based Survey of the Secretome[END_REF].

Since the SPs are an essential prerequisite for a successful targeting and secretion of secreted proteins, it is a good starting point for the optimization of secretion of heterologous proteins. In general, the signal peptides of Gram-positive bacteria are typically longer and more hydrophobic than the SP of Gram-negative bacteria [START_REF] Zanen | Signal peptide hydrophobicity is critical for early stages in protein export by Bacillus subtilis[END_REF][START_REF] Harwood | Bacillus protein secretion: an unfolding story[END_REF]. Zanen et al, have showed that the hydrophobicity is critical for early signal peptide recognition. B. subtilis is not able to secrete preproteins with signal peptides of low hydrophobicity but secretory proteins with signal peptides with high hydrophobicity would be more efficiently translocated in this bacterium [START_REF] Zanen | Signal peptide hydrophobicity is critical for early stages in protein export by Bacillus subtilis[END_REF].

As an attempt to optimize the secretion of heterologous proteins, [START_REF] Brockmeier | Systematic screening of all signal peptides from bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in gram-positive bacteria[END_REF] created a library of all natural Sec type SPs from B. subtilis fused to reporter proteins that were then tested with a screening approach, making use of two lipolytic enzymes-cutinase from Fusarium solani pisi and a cytoplasmatic esterase of metagenomic origin. They observed that the best SP of one target protein may not be the best or even work for other secreted proteins. As an example, the fusion of the SP of AmyE to the cutinase did not inevitably result in an improvement of the secretion. As a conclusion, the authors showed that the secretion efficiency for a certain target protein in B. subtilis is determined by complex modifications and interactions between the signal peptide and the mature protein [START_REF] Brockmeier | Systematic screening of all signal peptides from bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in gram-positive bacteria[END_REF].

Intracellular chaperoning and targeting to the Sec translocase

The targeting of the secretory preproteins to the Sec pathway in bacteria is an important step for a correct protein exportation. During this stage, preproteins interact with cytosolic chaperones to undertake the correct route, to be maintained in the correct unfolded state and to ensure a driving force that provides directionality to the secretion, because polypeptides can slide by Brownian motion in either direction inside the channel [START_REF] Rapoport | A gene encoding a holin-like protein involved in spore morphogenesis and spore germination in Bacillus subtilis[END_REF]. The protein targeting to the channel can occur by two distinct routes, by a co-translational or post-translational route (Figure 6).

Co-translational translocation route

In the co-translational path, the new emerging protein chain is simultaneously synthesized, pushed directly from the ribosome exit tunnel through the translocon and exported or laterally inserted into the lipid bilayer. This co-translational route is performed by the signal recognition particle (SRP), which is a highly conserved ribonucleoprotein complex, in all three kingdoms of life, and which is responsible for recognizing the hydrophobic signal sequence of the ribosome-nascent chain (RNC) as it emerges from the ribosomes and targeting it to the cytosolic membrane [START_REF] Koch | Signal Recognition Particledependent protein targeting, universal to all kingdoms of life[END_REF]. In general, SRP is composed of a small cytoplasmic RNA (scRNA) and signal sequence binding subunit [START_REF] Ménétret | Architecture of the ribosome-channel complex derived from native membranes[END_REF][START_REF] Collinson | Channel Crossing: How are proteins shipped across the bacterial plasma membrane?[END_REF]. First, this protein complex binds to a membrane receptor, forming a heterodimeric SRP-receptor complex, which helps the targeting of the emerging nascent pre-protein from the ribosome to the membrane after the binding to the signal peptide [START_REF] Saraogi | Co-translational protein targeting to the bacterial membrane[END_REF]. E. coli SRP comprises a scRNA domain and a single essential protein, Ffh. After the recognition of the hydrophobic signal sequence of the emerging protein, the SRP interacts with the SRP membrane receptor FtsY, which leads to the transfer of the emerging protein to the Sec translocase for translocation or insertion into the membrane [START_REF] Angelini | FtsY, the bacterial signalrecognition particle receptor, interacts functionally and physically with the SecYEG translocon[END_REF][START_REF] Leake | Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging[END_REF]. Ffh and FtsY belongs to the SRP-GTPase family, depending on GTP-binding, and both are essential for SRP-dependent protein secretion and cell viability (S. [START_REF] Chen | A molecular modeling study of the interaction between SRP-Receptor complex and peptide translocon[END_REF][START_REF] Neher | SRP RNA controls a conformational switch regulating the SRP-SRP receptor interaction[END_REF]. B. subtilis has a co-translational route too. As the E.

coli SRP, B. subtilis SRP consists of a scRNA and a GTPase Ffh, however it additionally contains two molecules of the Histone-like protein Hbsu [START_REF] Nakamura | Bacillus subtilis histone-like protein , HBsu , is an integral component of a SRP-like particle that can bind the Alu domain of small cytoplasmic RNA[END_REF]. The SRP can be divided into two functional domains: the S domain recognizes the N-terminal signal sequences as soon as the preprotein emerges from the ribosomal tunnel exit and the Alu domain imposes an elongation arrest by blocking the elongation factor entry site, retarding translation [START_REF] Kempf | Structure of the complete bacterial SRP Alu domain[END_REF][START_REF] Rodnina | Protein elongation, co-translational folding and targeting[END_REF]. Therefore, by stopping the elongation, SRP prevents membrane proteins from being prematurely released from the ribosome before the ribosome-nascent chain complex (RNC) has correctly engaged with the translocation channel [START_REF] Kempf | Structure of the complete bacterial SRP Alu domain[END_REF]. In B. subtilis SRP, the HbsU protein binds to the SRP Alu-Like domain, being essential to this process. In a study using β-lactamase as a reporter protein, the repression of hbs gene and depletion of Hbsu leads to slow growth and defect in the translocation with a high accumulation of the preprotein form of the β-lactamase and an induced scRNA upregulation [START_REF] Yamazaki | Depletion of Bacillus subtilis Histonelike protein, HBsu, causes defective protein translocation and induces upregulation of small cytoplasmic rna[END_REF].

Post-translational translocation route

The post-translational translocation route involves the binding of specialized energytransducing factors, such as ATPases. Although in eukaryotes the translocation occurs across the endoplasmic reticulum (ER) membrane and is realised by a larger complex formed by trimeric Sec61 complex associated with additional components, the post-translocational mechanism works as in bacteria [START_REF] Osborne | Protein translocation by the Sec61/Secy channel[END_REF]. It needs a chaperone that acts as a molecular clamp to grab the emerging polypeptide, directing to the pore and preventing its backsliding. This mechanism occurs by cycles of hydrolyses of ATP, that consequently increase the affinity for unfolded polypeptide [START_REF] Collinson | Channel Crossing: How are proteins shipped across the bacterial plasma membrane?[END_REF]. The SecB protein is the secretory chaperone in E. coli. This protein binds to the mature regions of a subset of preproteins maintaining them in a translocation-competent state (unfolded and extended state) and to the carboxy terminus of SecA, which suggests that SecB promotes protein translocation of the associated preproteins by its specific binding to SecA. Therefore, the SP interacts post-translationally with SecA, the protein motor of the pore SecYEG, that subsequently undergoes different conformational states and cycles of ATP and ADP binding states promoting the translocation through the SecYEG pore by a "push and slide" movement [START_REF] Bauer | A 'Push and Slide' mechanism allows sequence-insensitive translocation of secretory proteins by the SecA ATPase[END_REF]. Then the SecD, SecF and YajC, associated membrane proteins, prevents the slide back of the translocated protein [START_REF] Corey | Unlocking the bacterial SecY translocon article unlocking the bacterial secy translocon structure[END_REF].

SecB protein is absent from B. subtilis and all other Gram-positive bacteria, and many studies have focused on identifying a functional homologue of this protein in this group of microorganisms.

To date these studies have failed to identify a convincing homologue. It might be important to note that SecB is not essential for E. coli viability and it is only needed for the secretion of a specific subset of proteins, many of which are outer membrane proteins (van [START_REF] Wely | Translocation of proteins across the cell envelope of gram-positive bacteria[END_REF] subtilis [START_REF] Müller | Interaction of Bacillus subtilis CsaA with SecA and precursor proteins[END_REF]. Furthermore, it was shown that the intracellular and general chaperones GroES and GroEL or DnaK, DnaJ and GrpE can work on pre-proteins and maintain them in a translocation-competent form. By inactivating the gene encoding HrcA, a negative regulator of the groE and dnaK operons, the authors bserved that the synthesis of all these chaperones. This increases the yields of the single-chain antibody (SCA) fragment and a reduced inclusion body formation (S. C. [START_REF] Wu | Enhanced secretory production of a singlechain antibody fragment from Bacillus Subtilis by coproduction of molecular chaperones[END_REF].

SecA and the Sec translocase

In B. subtilis, the membrane bound SecYEG complex forms the translocon (De [START_REF] Keyzer | The Bacterial Translocase: A dynamic protein channel complex[END_REF]. As the name implies, this complex includes the SecY, SecE, and SecG proteins. SecY is the largest component of the translocase and is essential for translocation and viability. The secY gene is located within a ribosomal operon, the spc operon. SecY is predicted to span the cytoplasmic membrane 10 times and is found in randomly dispersed foci that dynamically assemble and disassemble in the membrane [START_REF] Dajkovic | Dynamic organization of SecA and SecY secretion complexes in the B. subtilis membrane[END_REF]. SecE is a small integral membrane protein that is essential for translocation and viability [START_REF] Jeong | Isolation and characterization of the SecE homologue gene of Bacillus subtilis[END_REF]. In E.

2016).

SecA is an ATPase located on the cis side of the membrane which is responsible for both the preprotein targeting and the coupling of the energy required for translocation. It is the motor component responsible for pushing the preprotein through the SecYEG pore, as illustrated on Figure 7 [START_REF] Bauer | A 'Push and Slide' mechanism allows sequence-insensitive translocation of secretory proteins by the SecA ATPase[END_REF]. Therefore, SecA is considered as part of the core of the translocation channel as the motor protein of the SecYEG translocon interacts with both proteins involved in the translocation, the secretory preprotein and the membrane proteins that compose the translocon [START_REF] Bauer | A 'Push and Slide' mechanism allows sequence-insensitive translocation of secretory proteins by the SecA ATPase[END_REF][START_REF] Tanaka | Crystal structures of SecYEG in lipidic cubic phase elucidate a precise resting and a peptide-bound state[END_REF]. [START_REF] Mulder | Construction of an artificial SecYEG operon allowing high level secretion of α-amylase[END_REF], constructed an artificial SecYEG operon under the control of an inducible promoter. They demonstrated that the yield of α-amylase could be improved by increasing the production of the SecYEG proteins [START_REF] Mulder | Construction of an artificial SecYEG operon allowing high level secretion of α-amylase[END_REF]. To investigate the bottleneck of the Sec pathway in B. subtilis [START_REF] Chen | Combinatorial Sec pathway analysis for improved heterologous protein secretion in Bacillus subtilis: identification of bottlenecks by systematic gene overexpression[END_REF] developed a combinatorial approach to evaluate the effect of a systematic overexpression of 23 genes or operons involved or related to this route for the production of AmyL and AmyS. Contrary to [START_REF] Mulder | Construction of an artificial SecYEG operon allowing high level secretion of α-amylase[END_REF] results, this new approach showed that the overexpression of the translocase genes did not significantly increase the production of heterologous proteins, but the overexpression of post-translocation molecular chaperone (PrsA) leads to an optimization of AmyL and AmyS secretion (J. [START_REF] Chen | Combinatorial Sec pathway analysis for improved heterologous protein secretion in Bacillus subtilis: identification of bottlenecks by systematic gene overexpression[END_REF]. Furthermore, in 2016, Dajkovic and colleagues revealed by internal reflection microscopy (TIRFM) that SecA and SecY form foci throughout the cytoplasmic membrane. These foci were in constant flux with freely diffusing SecA and SecY molecules, concluding, this way that the secretion system is remarkably dynamic and is in constant reorganization [START_REF] Dajkovic | Dynamic organization of SecA and SecY secretion complexes in the B. subtilis membrane[END_REF].

Protein release and maturation

The final step of the Sec translocation consists in the cleavage of the signal peptide (SP) by

Signal peptidases (SPases), resulting in the release of the protein from the membrane to the media or lipid modified by the diacylglyceryl-transferase and cleaved by the lipoprotein-specific signal peptidase. SPases are membrane proteins responsible for the cleavage of the signal peptide and they are divided in Type I, Type II and TypeIV (Figure 8).

In bacteria, SPaseI cleaves the majority of SP of the secretory pro-proteins, mainly proteins released to the medium or cell wall proteins. Lep is the only type I signal peptidase that E. coli has, while B. subtilis has five: SipS, SipT, SipU, SipV, and SipW. SipS and SipT are the main signal peptidases and their deletion leads to cell death [START_REF] Tjalsma | The plasmid-encoded Signal Peptidase SipP can functionally replace the major signal peptidases SipS and SipT of Bacillus subtilis[END_REF].

SPasesI are general signal peptidases, catalysing the release of exported proteins through the Sec pathway and, also, by the Tat pathway, which facilitates the export of fully folded proteins [START_REF] Lüke | Proteolytic processing of Escherichia coli twin-arginine signal peptides by LepB[END_REF]). These peptidases, that have a large C-terminal domain characterized by two transmembrane helixes and an active site extended to the extra-cytoplasmic membrane surface, are unusual serine protease, as the active site exhibits a Ser-Lys dyad configuration instead of the canonical Ser-His-Asp triad architecture [START_REF] Dalbey | Membrane proteases in the bacterial protein secretion and quality control pathway[END_REF].

The SPaseII, the lipoprotein signal peptidase, has a crucial role in recognizing and processing the lipid-modified bacterial membrane proteins, which have to be diacylglycerol-modified at an Nterminal cysteine residue prior to their attachment to the outer surface of the membrane [START_REF] Pohl | Heterologous Protein Secretion by Bacillus Species: from the cradle to the grave[END_REF]. In B. subtilis, Lgt is the diacylglyceryl-transferase responsible for this lipid modification, prior to the SP cleavage by the SPaseII Lsp, contrary to E. coli this protein is not essential for cell viability [START_REF] Tjalsma | The potential active site of the lipoprotein-specific (Type II) signal peptidase of Bacillus subtilis[END_REF]. This peptidase is an aspartic acid protease, whose catalytic residues are fixed just beneath the extra-cytoplasmic membrane surface.

The SPaseIV, the prepilin signal peptidase, are responsible for the processing of the precursors of type IV pilins and related pseudopilins involved in the secretion of proteins across the outer membranes of Gram-negative bacteria (type II secretion) or in DNA uptake by Gram-positive bacteria.

In contrast to the other types of peptidases, this type has a specific recognition sequence, Gly-Phe-Thr-Leu-Ile-Glu, where cleavage occurs between the Gly and Phe residues, and is also responsible for Nmethylation of the phenylalanine at position +1 relative to the cleavage site. Similar to SPaseII, these peptidases are intramembrane-cleaving aspartic acid proteases, characterize by eight transmembrane helixes with the active site positioned on the cytoplasmic side close to the membrane, within short cytoplasmic loops [START_REF] Strom | Identification of active-site cysteines in the conserved domain of PilD, the bifunctional Type IV Pilin leader peptidase/n-methyltransferase of Pseudomonas aeruginosa[END_REF]. Beyond the cleavage of the signal peptide and subsequent release of the mature protein, there can be other limitation step for the optimization of the secretion, which implies an optimization of the rate of the signal peptides processing by the signal peptidases. Therefore, Tjalsm et al (2000) described different signal peptidases present in B. subtillis which have different affinities for different substrates, suggesting that the overexpression of the right combination of signal peptides will lead to an increase of the protein of interest [START_REF] Tjalsma | Bacillus subtilis contains four closely related type i signal peptidases with overlapping substrate specificities[END_REF]H Tjalsma et al. 2000). The rate of signal peptide processing by signal peptidases has also been considered as a possible limiting factor for protein production. [START_REF] Vitikainen | Quantitation of the capacity of the secretion apparatus and requirement for PrsA in growth and secretion of alpha-amylase in Bacillus subtilis[END_REF] showed that the proportion of mature AmyQ in SipToverproducing cells was higher than in the wild type [START_REF] Vitikainen | Quantitation of the capacity of the secretion apparatus and requirement for PrsA in growth and secretion of alpha-amylase in Bacillus subtilis[END_REF].

After this cleavage reaction the released SP need to be recycled, otherwise these peptides could have an inhibitory action to protein translocation. SppA and RasP have been described as possible signal peptide peptidases responsible for this degradation in B. subtilis [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF][START_REF] Neef | Intramembrane protease RasP boosts protein production in Bacillus[END_REF].

Gram-positive bacteria are known by their characteristic cell envelope architecture that facilitates the release of secretory proteins directly into the culture medium. Therefore, it is expected that this architecture would be compatible with the protein export process. However, a closer look at the cell wall reveals that it offers a very challenging environment for proteins to fold after emerging from the translocase. Unfolded proteins that emerge from the translocase must be able to fold into their native configuration, a crucial step not only for their functionality but also for their stability. The folding process should be quick without forming intra-or inter-molecular interactions that could block the translocation machinery and/or block cell wall synthesis, either of which would compromise cell viability. B. subtilis contains several post-translocational and quality control proteases in the membrane and cell wall, as well as extracellular proteases that rapidly target heterologous proteins or misfolded native proteins with exposed protease recognition sites. In B. subtilis, key roles are played by PrsA, the folding catalyst, various thiol-disulphide oxidoreductases (BdbA, BdbB, BdbC and BdbD), cleaning proteases such as the membrane-associated HtrA and HtrB proteases and extracellular proteases as WprA.

PrsA is a peptidyl-prolyl cis-trans isomerase (PPIase), an essential membrane anchored lipoprotein, bound to the outer surface of the cytoplasmic membrane, that assists proteins with cisprolyl residues to fold [START_REF] Kontinen | A gene (PrsA) of Bacillus subtilis involved in a novel, late stage of protein export[END_REF][START_REF] Kontinen | The PrsA lipoprotein is essential for protein secretion in Bacillus subtilis and sets a limit for high-level secretion[END_REF]. Its deletion results in altered cellular morphology and cell death given that this protein is required for the folding of one or more proteins involved in cell wall synthesis [START_REF] Kontinen | The PrsA lipoprotein is essential for protein secretion in Bacillus subtilis and sets a limit for high-level secretion[END_REF]. Moreover, several studies have revealed that there is a linear relationship between the amount of PrsA and the yield of certain secretory proteins (notably amylases), making PrsA a popular target when engineering host strains dedicated to the production of native, as well as heterologous secretory protein [START_REF] Vitikainen | Secretion of heterologous proteins in Bacillus subtilis can be improved by engineering cell components affecting post-translocational protein folding and degradation[END_REF].

Due to the oxidized environment into which secretory proteins are translocated, wich favours disulfide bond formation, B. subtilis expresses four thiol-disulphide oxidoreductase/isomerase enzymes (BdbA, BdbB, BdbC and BdbD) involved in the disulphide bond formation [START_REF] Sarvas | Post-translocational folding of secretory proteins in gram-positive bacteria[END_REF]). However, secretory proteins from B. subtilis and other Gram-positive bacteria generally lack disulphide bonds. Nevertheless, these enzymes have an important role in the production of heterologous proteins that require such bonds.

HtrA and HtrB are serine proteases, belonging to the quality control protease family, combating the extracellular folding stress, and preventing potentially fatal obstruction of the secretory translocase and cell wall synthesis [START_REF] Darmon | A novel class of heat and secretion stress-responsive genes is controlled by the autoregulated CssRS two-component system of Bacillus subtilis[END_REF][START_REF] Antelmann | The extracellular proteome of Bacillus Subtilis under Secretion Stress Conditions[END_REF]. It was recently suggested that, in addition to their already described roles as quality control proteases, HtrA and HtrB might also function to release lipoproteins from their membrane anchorage, to degrade membrane proteins and also to facilitate the folding of native secretory proteins (Krishnappa et al. 2013).

WprA is one of the eight extracellular proteases encoded in B. subtilis (AprE, Bpr, Epr, Mpr, NprB, NprE, Vpr), which is also localized on the cell wall [START_REF] Krishnappa | Degradation of extracytoplasmic catalysts for protein folding in Bacillus subtilis[END_REF]. It is a serine protease involved in the degradation of peptides for recycling and nutrient provision and the proteolytic processing of other proteins [START_REF] Babe | Purification and biochemical analysis of Wpra, a 52-Kda serine protease secreted by B. subtilis as an active complex with its 23 kDa propeptide[END_REF]. Although it was believed that, correctly folded Sec-secreted proteins would be highly resistant to the extra-cytoplasmic proteases, [START_REF] Krishnappa | Degradation of extracytoplasmic catalysts for protein folding in Bacillus subtilis[END_REF] have shown that many of these proteins are in fact degraded. They showed that the deletion of WprA leads to an increase in the amount of PrsA secreted into the medium, demonstrating that WprA has a role in the degradation of PrsA [START_REF] Krishnappa | Degradation of extracytoplasmic catalysts for protein folding in Bacillus subtilis[END_REF].

Twin-Arginine Transport (Tat) Pathway

In contrast to other transport systems, the twin-arginine translocase (Tat) is an unusual protein-targeting export pathway, found in all the kingdoms of life, that uniquely facilitates the translocation of fully folded and cofactor-attached proteins across the cytoplasmic membrane [START_REF] Sargent | The Twin-Arginine transport system : moving folded proteins across membranes[END_REF][START_REF] Berks | The Twin-Arginine protein translocation pathway[END_REF]. The active translocation is driven only by the transmembrane proton motive force (PMF) [START_REF] Anné | Protein secretion in gram-positive bacteria: from multiple pathways to biotechnology[END_REF][START_REF] Patel | Protein transport by the bacterial Tat pathway[END_REF]. This system was identified for the first time in the thylakoids of plant chloroplasts in 1997, it was shown that a metallo-enzyme was translocated across the thylakoid membrane in a folded conformation through a ∆pH-dependent pathway [START_REF] Settles | Old and New pathways of protein export in chloroplasts and bacteria[END_REF]. This pathway has been evolutionarily conserved in plant chloroplasts where it mediates protein import across the thylakoid membrane and is essential for the formation of a functional photosynthetic apparatus [START_REF] Alcock | Assembling the Tat Protein Translocase[END_REF]. Later in 1998, Weiner and his colleagues proved the existence of this pathway in prokaryotes. They identified, in E. coli, a Sec-independent pathway which is required for the correct localization of periplasmic redox proteins and for the targeting of DMSO reductase to the membrane [START_REF] Weiner | A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins[END_REF].

Consequently, Tat pathway plays a key role in a range of different cellular processes, because it mediate the transport, in its majority, of proteins that bind cofactors in the cytoplasm and, thus, fold

Box3: Sec Pathway

The Sec pathway is highly conserved in bacteria and represents the main route for protein transportation. It mediates the translocation of secretory and membrane proteins in an unfolded conformation. This secretion process can be divided into three steps: targeting, translocation and release. The secreted proteins are synthesised as precursors (pre-proprotein) with a N-terminal signal peptide (SP), which is recognized by cytoplasmic factors (Signal Recognition Particle-SRP) or chaperones (CsaA) involved in targeting of the preprotein to the translocon SecYEG. The translocation of these proteins across the channel is driven by the translocon motor SecA, an ATPase that interacts with the substrate proteins and binds to the SecYEG complex. The repeated cycles of binding, hydrolysis of ATP, conformational changes of a dimeric SecA leads to the translocation of the secretory protein through the SecYEG complex. Subsequently, the signal peptide is cleaved resulting in the protein release from the membrane. Finally, the protein undergoes a maturation process carried out by accessory proteins, such as the foldase PrsA, or the Bdb thiol-disulfide oxidoreductases [START_REF] Driessen | Protein translocation across the bacterial cytoplasmic membrane[END_REF][START_REF] Kang | Molecular engineering of secretory machinery components for high-level secretion of proteins in Bacillus species[END_REF][START_REF] Tjalsma | Proteomics of protein secretion by Bacillus subtilis: separating the 'secrets' of the secretome[END_REF]).

before transport. Such co-factored proteins have essential roles in photosynthetic and respiratory energy metabolism, iron and phosphate nutrition, resistance to heavy metals and antimicrobial peptides, cell separation, symbiotic nitrogen fixation and membrane biosynthesis (Ben C. [START_REF] Berks | The Tat protein translocation pathway and its role in microbial physiology[END_REF][START_REF] Buck | The importance of the twin-arginine translocation pathway for bacterial virulence[END_REF].

Similar to the Sec pathway, Tat substrates are targeted to the Tat apparatus by an N-terminal signal peptide. Although Tat and Sec signal peptides share a similar overall architecture (Figure 9), Tat signal peptides specifically exhibit a larger hydrophobic region, a general low hydrophobicity and bear a conserved S/TRR-x-FLK (with x representing a polar aminoacid) eponymous motif that contains the characteristic pair of adjacent arginine residues (twin arginines) [START_REF] Alcock | Assembling the Tat Protein Translocase[END_REF][START_REF] Goosens | A Tat Ménage à Trois -the role of Bacillus subtilis TatAc in twin-arginine protein translocation[END_REF]. A fact that gives to this pathway its name. The twin arginine residues are critical for the Tat translocation in chloroplasts, but may not be an absolute prerequisite in bacteria, as the rate of translocation is affected by mutating a single arginine, whereas mutation of both completely abolishes the transport. In general, two types of integral membrane proteins compose the Tat translocon [START_REF] Monteferrante | Mapping the twin-arginine protein translocation network of Bacillus subtilis[END_REF][START_REF] Goosens | A Tat Ménage à Trois -the role of Bacillus subtilis TatAc in twin-arginine protein translocation[END_REF][START_REF] Alcock | Assembling the Tat Protein Translocase[END_REF]):

 TatC: a highly hydrophobic and relatively large membrane protein with six transmembrane helices, whose N-and C-termini faces the cytoplasm;

 TatA-like proteins (TatA, TatB or TatE): smaller proteins with a single hydrophobic transmembrane domain (TMD) at the N-terminus immediately followed by an amphipathic helix (APH) and a C-terminal tail densely charged. The TatA-like components resulted from gene duplication and they have evolved towards different functions, namely substrate binding (TatB)

and pore formation (TatA). E. coli possesses a third TatA paralog, TatE, which is functionally equivalent to TatA but is present at a much lower concentration in the cell and is not essential for Tat transport.

The Tat transport requires (i) a docking complex composed by TatB and TatC, which together form a multivalent receptor complex that binds Tat substrates [START_REF] Fröbel | Transmembrane insertion of twin-arginine signal peptides is driven by TatC and regulated by TatB[END_REF]; and (ii) a pore complex, which results from the assembly of multiple protomers of TatA [START_REF] Gohlke | The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter[END_REF].

Translocation is initiated when a substrate protein with the correct signal peptide is recognized and bound to the docking complex located in the membrane (Figure 10). As soon as it binds, the TatC component of the docking complex inserts the substrate proteins into the membrane [START_REF] Blümmel | Structural features of the tatc membrane protein that determine docking and insertion of a twin-arginine signal peptide[END_REF]. This binding event triggers the proton motor force (PMF) and, consequently, the recruitment of pore-forming TatA protomers from a pool in the membrane to form the active TatABC-containing translocation site [START_REF] Sargent | The Twin-Arginine transport system : moving folded proteins across membranes[END_REF][START_REF] Alcock | Assembling the Tat Protein Translocase[END_REF][START_REF] Wojnowska | Precursor-receptor interactions in the twin arginine protein transport pathway probed with a new receptor complex preparation[END_REF]. Upon completion of transport, the substrate is released from the translocation site and the signal peptide is cleaved by signal peptidase. TatA with a green fluorescent protein (GFP). When substrates are scarce, TatA is localized in the membrane surrounding the cells, while it is found in large oligomers when the substrate is present in high concentrations [START_REF] Leake | Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging[END_REF][START_REF] Rose | Substrate-dependent assembly of the tat translocase as observed in live Escherichia coli cells[END_REF].

The Tat machinery transports folded proteins of widely varying size across the membrane, these substrates may vary from 20 Å up to 70 Å of diameter comparing to an unfolded polypeptide chain with 12 A° diameter transported by the Sec pathway [START_REF] Gohlke | The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter[END_REF][START_REF] Berks | The Twin-Arginine protein translocation pathway[END_REF]. The variation of the TatA channel diameter according to large or small substrates, ensuring a tight seal substrate transportation across the membrane without an uncontrolled ion leakage, it is a challenge faced by the Tat machinery. The membrane-weakening and pulling mechanism hypothesis explains how the Tat system manage this process. The model suggests that the N-terminal of the TatA oligomers, that form the Tat translocon site, are capable of destabilize the membrane by weakening or even rupturing the membrane upon substrate-binding, allowing the TatC component to pull the substrate through the destabilized membrane [START_REF] Frobel | Transmembrane insertion of twin-arginine signal peptides is driven by TatC and regulated by TatB[END_REF].

In In the case of Gram-positive bacteria as the actinomycetes (including the Streptomycetes, Mycobacterium and Corynebacterium), the Tat system is similar to the Gram-negative bacteria and is composed by TatA, TatB and TatC components [START_REF] Anné | Protein secretion biotechnology in gram-positive bacteria with special emphasis on Streptomyces lividans[END_REF]. In contrast, Staphylococci and Bacillus have a smaller core Tat pathway, mainly composed by a single pair of TatA and TatC and devoid of TatB. B. subtilis have genes that encode for two TatC (TatCd and TatCy) and three TatA (TatAd, TatAy and TatAc). These Tat components can assemble into two distinct Tat translocases that operate in parallel, working independently of each other and translocating specific substrates. Both systems appear to be localized at the cell poles and division sites [START_REF] Monteferrante | Mapping the twin-arginine protein translocation network of Bacillus subtilis[END_REF]. The first of the two systems, TatAy-TatCy (TatAyCy), was expressed constitutively over many tested conditions and exports several substrates, including YwbN, an iron-dependent DyP-peroxidase, the Rieske iron-sulfur protein QcrA and the alkaline phosphatase YkuE. In contrast, the second system, TatAd-TatCd (TatAdCd), whose the only known substrate is the phosphodiesterase (PhoD), is only expressed in conditions of phosphate limitation [START_REF] Goosens | A Tat Ménage à Trois -the role of Bacillus subtilis TatAc in twin-arginine protein translocation[END_REF][START_REF] Patel | Protein transport by the bacterial Tat pathway[END_REF][START_REF] Jongbloed | Bifunctional TatA subunits in minimal Tat protein translocases[END_REF].

In bacteria the Tat and Sec pathways work in parallel. Even if their systems have specific components, they share associated proteins, allowing a crosstalk between the cellular systems [START_REF] Goosens | The Tat system of gram-positive bacteria[END_REF]. This have been documented in E. coli, the production of heterologous membrane bound Rieske iron-sulfur protein, depends on the secretion of both pathways, as a portion of this protein was shown to use a Sec-YidC-dependent membrane insertion mechanism while the remaining portion was translocated in a Tat-dependent manner [START_REF] Keller | Co-Operation between different targeting pathways during integration of a membrane protein[END_REF]. In B.

subtilis, the TatAy-TatCy membrane complex interacts with the cell wall and quality control protease WprA. Through yeast two-hybrid experiments, it was shown that WprA resulted in a decrease of the amount of the TatAy-TatCy -translocated EfeB and YkuE proteins, suggesting a functional WprA-TatAy-TatCy-EfeB/ YkuE interaction [START_REF] Monteferrante | Mapping the twin-arginine protein translocation network of Bacillus subtilis[END_REF].

Box4: Tat pathway

The twin-arginine translocase (Tat) is a unusual protein-targeting export pathway, found in all the kingdoms of life, that uniquely facilitates the translocation of fully folded and cofactor-attached proteins across the cytoplasmic membrane [START_REF] Sargent | The Twin-Arginine transport system : moving folded proteins across membranes[END_REF][START_REF] Berks | The Twin-Arginine protein translocation pathway[END_REF].

An event, that remarkably has no requirement for ATP as energy source, the active translocation is driven only by the transmembrane proton motive force (PMF) [START_REF] Anné | Protein secretion in gram-positive bacteria: from multiple pathways to biotechnology[END_REF][START_REF] Patel | Protein transport by the bacterial Tat pathway[END_REF]. This pathway have this name due to its signal peptides distinguish themselves by bearing a conserved S/TRR-x-FLK (with x representing a polar aminoacid) eponymous motif that contains the characteristic pair of adjacent arginine residues (twin arginines) [START_REF] Alcock | Assembling the Tat Protein Translocase[END_REF][START_REF] Goosens | A Tat Ménage à Trois -the role of Bacillus subtilis TatAc in twin-arginine protein translocation[END_REF]. The variation of the Tat machinery pore is remarkable, since it is able to transport folded proteins of widely varying size across the membrane, from 20 Å up to 70 Å of diameter compared to unfolded polypeptide chains with 12 A° diameter transported by the Sec pathway [START_REF] Gohlke | The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter[END_REF][START_REF] Berks | The Twin-Arginine protein translocation pathway[END_REF].

Flagella Export Apparatus (FEA)

Bacteria are motile microorganisms. To survive they need to swim or swarm depending on their environment. The bacterial motor that allows this movement is called the flagellum, a rotating semi-rigid helical filament. The flagellum was observed for the first time in 1681, when Antony van Leeuwenhoek, the father of protozoology and bacteriology, observed his own faeces [START_REF] Dobell | Antony van Leeuwenhoek and his 'Little Animals[END_REF]. In one of his letters describing this observations, he clearly described a flagellated microscopic parasite named two centuries later as Giardia intestinalis, and wrote that this "little animal" has "sundry little paws" capable of strongly stirring the medium, a clear observation of flagella [START_REF] Dobell | Antony van Leeuwenhoek and his 'Little Animals[END_REF].

The bacterial flagellum is a highly efficient molecular nanomachine with approximately 120-140 nm of diameter and about 10 to 20 µm long, composed by three main structures : the basal body, the hook and the filament, which are made of about 25 different proteins [START_REF] Erhardt | Bacterial Nanomachines: The Flagellum and Type III Injectisome[END_REF]. The basal body, a membrane-anchored structure that works as a structural anchor, a reversible rotary motor and flagellar protein export system [START_REF] Minamino | Molecular motors of the bacterial flagella[END_REF]. The hook is a curved, thicker and flexible cylinder that joins the basal body to the filament, contributing to the synchronized rotation of the several flagella on the cell [START_REF] Depamphilis | Fine structure and isolation of the hook-basal body complex of flagella from Escherichia coli and Bacillus subtilis[END_REF]. The filament is a helical propeller composed by thousands of copies of the protein monomer flagelin (F. [START_REF] Wang | A structural model of flagellar filament switching across multiple bacterial species[END_REF]. All the components of these protein complexes are exported by the flagellum protein export apparatus, using ATP and PMF as the energy source [START_REF] Forster | Protein transport across the cell wall of monoderm gram-positive bacteria[END_REF][START_REF] Diepold | Type III Secretion Systems: The bacterial flagellum and the injectisome[END_REF]. The flagellum is a great example of how membrane protein super-complexes are of importance for cell integrity.

Due to cell envelope differences, Gram-positive and Gram-negative flagellum are structurally different. As the basal body is a transmembrane structure that attaches the filament and, the hook to the cell, it is the structure that has more differences between the two types of bacteria. While the As described above, the flagellar membrane complex, named basal body, works as a rotary motor and flagellar protein export system, the two key elements for the existence and well-functioning of the flagellum. The B. subtilis flagellar motor comprises an inner membrane FliF ring that sits on top of a cytoplasmic gear-like rotor complex composed by FliG, a C-ring protein complex FliM and FliY responsible for controlling the flagellar direction, and the stator transmembrane complex composed by MotA and MotB proteins [START_REF] Depamphilis | Fine structure and isolation of the hook-basal body complex of flagella from Escherichia coli and Bacillus subtilis[END_REF][START_REF] Minamino | Molecular motors of the bacterial flagella[END_REF]. The stator complex allows the flow of protons across the membrane. This flux leads to a conformational change of MotA, which promotes the electrostatic interaction with the C-terminus of FliG, generating the torque for flagellar rotation [START_REF] Blair | Flagellar movement driven by proton translocation[END_REF][START_REF] Berg | The rotary motor of bacterial flagella[END_REF]. Through FliF, the torque movement is transferred to the rod complex, a conduction duct that drives the rotation from the basal body to the hook and filament [START_REF] Gabel | The speed of the flagellar rotary motor of Escherichia coli varies linearly with protonmotive force[END_REF][START_REF] Mukherjee | The structure and regulation of flagella in Bacillus subtilis[END_REF]. B. subtilis encodes five homologs of rod proteins -FlgB, FlgC, FlhO, FlhP and FliE. The four proteins, FlgB, FlgC, FlhO and FlhP, were co-purified with the B. subtilis hook-basal body, results that support their role in the rodshape structure [START_REF] Depamphilis | Fine structure and isolation of the hook-basal body complex of flagella from Escherichia coli and Bacillus subtilis[END_REF][START_REF] Kubori | Purification and characterization of the flagellar hookbasal body complex of Bacillus subtilis[END_REF]). The study of mutants depleted for FlhO and FlhP showed that these two proteins are part of the upper part of the rod structure, close to the hook assembly, since their deletion leads to the secretion of the hook subunits, to the media, abolishing the hook formation [START_REF] Courtney | Molecular characterization of the flagellar hook in Bacillus subtilis[END_REF]. In contrast, the deletion of fliE does not abolish the hook formation, but reduces rod and hook proteins secretion. Therefore, FliE is not only a structural component of the rod, but a regulatory component necessary for full and correct secretion of the flagellar components [START_REF] Burrage | The assembly order of flagellar rod subunits in Bacillus subtilis[END_REF].

The flagellar protein secretion system translocates the flagellar components, across the membrane, in an unfolded conformation, to the channel of the growing flagellum (a structure with 20 Å of diameter) to maintain the proteins unfolded while they move to the flagellum tip up to 10-20 µm long [START_REF] Evans | A chain mechanism for flagellum growth[END_REF]. This translocation process requires cellular energy, using ATP and PMF as the energy source. Despite their differences, the flagellar secretion apparatus and the type III secretion system (T3SS) of pathogenic bacteria, the injectisome, are remarkably conserved. These two systems are structurally similar and their secretion mechanism are analogous, and most part of the membrane and ATPase complex components share sequence homology [START_REF] Carpenter | Bacillus subtilis flagellar proteins Flip , FliQ , FliR and FlhB are related to Shigella flexneri virulence factors[END_REF][START_REF] Diepold | Type III Secretion Systems: The bacterial flagellum and the injectisome[END_REF][START_REF] Deng | Assembly, structure, function and regulation of Type III secretion systems[END_REF].

The secretion machinery comprises a membrane gate complex, inserted into the FliF M-ring and composed by FlhA, FlhB, FliP, FliQ, and FliR, five higly conserved transmembrane proteins, and a cytoplasmic ATPase complex formed by FliH, FliI, and FliJ [START_REF] Erhardt | Bacterial Nanomachines: The Flagellum and Type III Injectisome[END_REF].

The transmembrane protein FliO, part of this apparatus too, is responsible for an efficient assembly of the export gate complex. FliP and FliR form the core structure of the gate complex, a starting point for the assembly of the remaining components (Figure 14) [START_REF] Fukumura | Assembly and stoichiometry of the core structure of the bacterial flagellar Type III export gate complex[END_REF]. Recently, by performing purification experiments, it was shown that salmonella FliP forms a homohexamer dependent on the FliO complex help, their interactions are required for a FliP correct assembly [START_REF] Fukumura | Assembly and stoichiometry of the core structure of the bacterial flagellar Type III export gate complex[END_REF].

Figure 14. Model for the assembly process of the Gram-negative flagellar type III export apparatus. The export apparatus is composed of a transmembrane export gate complex made of FlhA, FlhB, FliP, FliQ, and FliR and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ. The FliP dimers form a homohexamer with the help of the FliO complex, followed by the assembly of FliQ, FliR, and FlhB and finally of FlhA during MS ring formation in the cytoplasmic membrane. Then, the FliM/FliN complex binds to FliG to form the C ring on the cytoplasmic face of the MS ring. Finally, the FliH/FliI/FliJ ATPase ring complex is formed at the flagellar base through interactions of FliH with FlhA and FliN (FliY in B. subtilis), allowing export of substrates though the central cavity of the FliP6 ring complex [START_REF] Fukumura | Assembly and stoichiometry of the core structure of the bacterial flagellar Type III export gate complex[END_REF].

FlhA and FlhB are responsible for coordinating the flagellar protein export, through their Cterminal cytoplasmic domains, and form a docking complex platform for the substrates, ATPase complex (FliH, FliI and FliJ) and chaperones associated to this system [START_REF] Fukumura | Assembly and stoichiometry of the core structure of the bacterial flagellar Type III export gate complex[END_REF]). The Salmonella and E. coli FlhA forms a homononamer. In E. coli, it was shown that this protein plays also a key role in the energy transduction mechanism, acting as an ion channel to conduct H+ and Na+. FlhA works along with the cytoplasmic ATPase complex or as a genetic backup that compensates for the loss or inactivation of the ATPase complex [START_REF] Minamino | The bacterial flagellar Type III Export gate complex is a dual fuel engine that can use both H + and Na + for flagellar protein export[END_REF]. During the flagellum growth, the exported substrates remain unfolded while they move through the flagellum channel, from the bottom to the end, it is other example of the great importance of protein-protein interactions. [START_REF] Evans | A chain mechanism for flagellum growth[END_REF] demonstrated that this mechanism uses the entropic force of each unfolded substrate and that these substrates gather together thus creating a multi-subunit chain through the flagellum channel by the isolation of the growing filaments [START_REF] Evans | A chain mechanism for flagellum growth[END_REF].

Through these experiments, they also revealed that, each subunit is linked head-to-tail to the other via an interaction between the juxtaposed amino N-terminal and carboxyl C-terminal helices, as it is possible to see on Figure 15 [START_REF] Evans | A chain mechanism for flagellum growth[END_REF]. The first subunit on the filament bottom is docked to the export machinery and its C-terminal is free to be captured by a free unfolded subunit, through sequential rounds of linkage. Hence, a multi-subunit chain is created until the flagellum tip, where it is predicted that the N-terminal of each unfolded subunit crystallizes [START_REF] Evans | A chain mechanism for flagellum growth[END_REF]. Furthermore, [START_REF] Evans | A chain mechanism for flagellum growth[END_REF] confirmed, by thermodynamic analysis, that subunit link regions in the chain resist to the pulling force generated, an energy that is automatically adjusted over the increasing length of the growing flagellum [START_REF] Evans | A chain mechanism for flagellum growth[END_REF].

Type IV Secretion Systems

The type IV Secretion System (T4SS) is a long and thin multi-subunit, membrane-spanning translocation system that is present on the surface of many bacterial species. T4SSs have evolved from a self-transmissible, single-stranded DNA conjugation system with VirB4-like AAA + ATPase to systems with an enormous diversity in their overall structure and the types of substrates secreted.

Therefore, this pathway serves to translocate a highly diverse family of substrates, since it has a functional astounding collective capacity of (Figure 16 A-E):

 recognizing and translocating single-stranded (ss) DNA substrates across the membrane to bacterial or eukaryotic target cell, whereby direct cell contact is required (conjugation machines);

 delivering effector proteins (effector translocator systems) to the cytosol of eukaryotic target cells in a contact-dependent mechanism;

 exporting/importing molecules from/to the extracellular milieu;

 delivering killing toxins to bacterial neighbours;

 contributing to biofilm development.

Consequently, T4SSs are involved in a variety of functions including pilus formation, toxin and other protein secretion, gene transfer, and biofilm formation [START_REF] Grohmann | Micro Review Type IV secretion in gram-negative and gram-positive bacteria[END_REF]. This type of secretion system can be found in Gram-positive as well in Gram-negative bacteria and in some archaea. Furthermore, many pathogenic bacteria use this secretion system as virulence factor and several intracellular symbionts use it for supporting their colonization and propagation in the eukaryotic host (Figure 16 E) [START_REF] Cascales | The versatile bacterial Type IV secretion systems[END_REF].

Box5:Flagella Export Apparatus (FEA)

Bacteria are motile microorganisms, to survive they need to swim or swarm depending on their environment. The bacterial motor that allows this movement it is called the flagellum, a rotating semi-rigid helical filament. The bacterial flagellum is a highly efficient molecular nanomachine with approximately 120-140 nm of diameter and about 10 to 20 µm long, composed by three main structures : the basal body, the hook and the filament, which are made of about 25 different proteins [START_REF] Erhardt | Bacterial Nanomachines: The Flagellum and Type III Injectisome[END_REF]. All the components of these protein complexes are exported, in an unfolded conformation, by the flagellum protein export apparatus, using ATP and PMF as the energy source.  The ConQ coupling protein is responsible for the docking of the conjugative DNA to the T4SS translocation channel and belongs to the FtsK clade of the FtsK-HerA superfamily of ATPases [START_REF] Leonetti | Critical components of the conjugation machinery of the integrative and conjugative element ICEBs1 of Bacillus subtilis[END_REF]. ConQ, as the ATPases of its superfamily, forms doughnut-shaped hexamer with a central channel large enough to hold single or double-stranded DNA (Figure 17), through its two N-terminal transmembrane helices and a cytoplasmic C-terminal ATPase domain [START_REF] Iyer | Comparative genomics of the FtsK-HerA superfamily of pumping ATPases: implications for the origins of chromosome segregation, cell division and viral capsid packaging[END_REF]). This protein is essential to the mobilization and conjugation by ICEBs1 (C. A. [START_REF] Lee | The Bacillus subtilis conjugative transposon ICEBs1 mobilizes plasmids lacking dedicated mobilization functions[END_REF].

 The Gram-positive cell wall is a thick and dense layer. The presence of a cell wall hydrolase is a key element that allows the correct assembly of the translocation channel, and, allowing the

Holins

The term "holin" or hole formation protein originally refers to phage-encoded pore forming membrane proteins that control access of phage-encoded endolysins to the peptidoglycan layer. Recent studies showed that these proteins and their homologues may also be important for a variety of other functions in Gram-positive phage-free bacteria such as [START_REF] Saier | Holins in bacteria, eukaryotes, and archaea: multifunctional xenologues with potential biotechnological and biomedical applications[END_REF]: The detailed pore formation mechanism, its regulation and the pore dynamics remain unknown. Studies have shown that until a signal triggers their oligomerization, the small-pore-forming pin-holins accumulate in the cytoplasmic membrane until it dissipates the PMF. As a consequence this leads to a membrane permeabilization and large-pore formation that allows the release of endolysins, which are produced fully folded in the cytoplasm (Figure 18) [START_REF] Young | Bacteriophage Holins: deadly diversity[END_REF][START_REF] Saier | Holins in bacteria, eukaryotes, and archaea: multifunctional xenologues with potential biotechnological and biomedical applications[END_REF].


When the endolysins reach the cell wall they cleave various bonds in the peptidoglycan polymer, depending on the type of endolysin. The genes that encode all the elements involved on this holin-

Box6: Type IV Secretion Systems (T4SSs)

The type IV Secretion Systems (T4SSs) are long and thin multi-subunit, membranespanning translocation systems that are present on the surface of many bacterial species. This

A guarantee of a correct protein translocation in B. subtilis

As described in the previous chapter, membrane and secreted proteins carry out a multiplicity of essential functions that directly affect the cellular protein homeostasis, so that the maintenance of this proteome is of extreme importance for the cell viability. These proteins, from their synthesis until reaching their final destiny, pass through different structural and folding states. To avoid cell damage coming from defective and misfolded proteins, which can be caused by different cellular stresses and leads to an increase of the cytotoxicity level, the cells use specific machineries to overcome these problems, called quality control mechanism. One part of the quality control strategies is protein degradation carried out by protease complexes, with the main goal of removing deleterious species and recycling of amino acids.

Proteases enzymes, that preform protein catabolism by hydrolysis of peptide bonds, are part of these defense mechanism to eliminate the damaged and misfolded proteins, regulating this way the fate, activity, localization and protein-protein interactions of many different proteins [START_REF] Lo | Proteases : multifunctional enzymes in life and disease[END_REF]. Some bacterial proteases act as chaperones too, and have a key role in cellular protein quality control (e.g. B. subtilis WprA feeding protease). Initially, these enzymes were differentiated into two classes, based on their interaction with the substrate, the endopeptidases, which target internal peptide bonds, and exopeptidases (aminopepti-dases and carboxypeptidases), the action of which is directed to the NH2 and COOH terminal domains. Later studies allowed a more detailed classification. Based on their mechanism of catalysis, proteases can be divided into six distinct classes, aspartic, glutamic, and metalloproteases, cysteine, serine, and threonine proteases [START_REF] Lo | Proteases : multifunctional enzymes in life and disease[END_REF]. Some bacterial proteases do not work as individual catalytic devices to hydrolyze the substrate, they have the ability to interact with each other forming protease complexes. Some of these complexes are crucial, as the case of the AAA + proteases (ATPase associated with cellular activities), to the proper functioning and maintenance of the bacterial cell, as well as to its response to the stress, since these multimeric machines are able to recognize and degrade misfolded, damaged or aggregated proteins [START_REF] Dougan | AAA + proteins and substrate recognition , it all depends on their partner in crime[END_REF].

A bacterial cytoplasmic quality control system: ClpXP protease

Cytoplasmic protease complexes, as ClpXP, are responsible for the degradation of defective or denatured membrane or secretory proteins that, due to stress conditions, cannot be translocated into or out of the membrane and are arrested in the cytoplasm. ClpXP consists of two subunits, the ClpX subunit, which has ATPase activity and form a hexameric ring, and the ClpP, which is a doubleheptameric serine protease (Figure 20). The interaction between these proteins was observed for the first time in the early 1990s, Gottesma et al. named and identified E. coli ClpX as the protein that, in the presence of ClpP, induces the ATP-dependent degradation of λ0 protein. They showed that the clpX and clpP genes form an operon and are co-transcribed in a single heat-induced mRNA [START_REF] Gottesman | ClpX, an alternative subunit for the ATP-Dependent Clp protease of Escherichia coli. sequence and in vivo activities[END_REF]. As for E. coli ClpP, it has been demonstrated that B. subtilis clpP is also induced by heat shock stress. Additionally, it was shown that B. subtilis clpP is induced by salt and ethanol stress, as well as after a puromycin treatment. Indeed after such a stress exposure the amount of clpP specific mRNA increased; however the highest increase was shown after heat shock stress [START_REF] Gerth | Stress induction of the Bacillus subtilis ClpP gene encoding a homologue of the proteolytic component of the Clp protease and the involvement of ClpP and ClpX in stress tolerance[END_REF]. Therefore, these experiments suggested that B. subtilis ClpP is involved in the cell response to puromycin treatment or heat shock stress, through the degradation of abnormal proteins resulting from the stress. It was also demonstrated that the deletion of clpP or clpX in B. subitilis grown in stress conditions, induced the production of elongated cells (Figure 19) and an impaired growth, indicating that B. subtilis ClpXP have substrates involved in cell morphology or cell division and emphasized the importance of ClpXP as proteases or chaperones in stress tolerance [START_REF] Gerth | Stress induction of the Bacillus subtilis ClpP gene encoding a homologue of the proteolytic component of the Clp protease and the involvement of ClpP and ClpX in stress tolerance[END_REF]. The interaction between this two proteins is essential, as the ClpX protein is responsible for recognition and binding of the substrates, and it allows the degradation of larger protein substrates as for example disulphide-bonded substrates which require the translocation of three polypeptide chains.

The crystal structure of the ClpX structure revealed a small pore almost completely closed, that will need to expand and adapt to the different substrate sizes [START_REF] Baker | ClpXP, an ATP-powered unfolding and proteindegradation machine[END_REF][START_REF] Gersch | AAA+ chaperones and acyldepsipeptides activate the ClpP Protease via conformational control[END_REF].

So, how does the ClpX narrow pore expand according to the substrate size?

Each ClpX protomer has two domains, the large and the small domains, that assemble, through the binding of the small domain of one protomer to the large domain of the next one, in an asymmetric hexamer that can bind a maximum of four molecules of ATP and/or ADP [START_REF] Glynn | Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine[END_REF]). Due to its asymmetry and domain-domain orientation the ClpX hexamer can be divided into subunits of two main classes, the "loadable" or L subunits and the "unloadable" or U subunits [START_REF] Glynn | Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine[END_REF][START_REF] Baker | ClpXP, an ATP-powered unfolding and proteindegradation machine[END_REF]. In the L subunit, the large and small AAA+ domains of each protomer connect in a nucleotide-binding conformation, corresponding to four subunits of the hexamer. Interestingly, on the two U subunits can occur a rotation of 80° between the bound domains of the protomers (large and small AAA+ of each protomer), originating movements as large as 30 Å that destroy the nucleotide-binding site [START_REF] Glynn | Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine[END_REF][START_REF] Baker | ClpXP, an ATP-powered unfolding and proteindegradation machine[END_REF]. Consequently, the two U subunits, create two flexible sides of the hexamer that allow the adaptation of the pore to different substrate sizes, extending the pore to accommodate larger substrates and then refold and shortening it to contact with smaller substrates [START_REF] Glynn | Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine[END_REF][START_REF] Baker | ClpXP, an ATP-powered unfolding and proteindegradation machine[END_REF]. This movement can be compared to the unhinging of the jaws of a snake, which allows it to swallow large prey, as it is demonstrated on Figure 22. This mechanism starts by the recognition of substrates by ClpX, through the binding to short unstructured peptide sequences called degradation tags or degrons, which are typically located at the N-terminal or C-terminal end of a target protein, or to adaptor proteins, that modulate and enhance the substrate recognition by the respective ATPase partner [START_REF] Dougan | AAA + proteins and substrate recognition , it all depends on their partner in crime[END_REF][START_REF] Kirstein | Adaptor protein controlled oligomerization activates the AAA + protein ClpC[END_REF]. The adaptor proteins bind to specific substrates and form a delivery complex with the ATPase partner, by binding to its N-terminal domain [START_REF] Kirstein | Adaptor protein controlled oligomerization activates the AAA + protein ClpC[END_REF][START_REF] Molière | Chaperone-Protease systems in regulation and protein quality control in Bacillus subtilis[END_REF][START_REF] Elsholz | Functional diversity of AAA + protease complexes in Bacillus subtilis[END_REF]. The ClpX adaptor proteins of B. subtilis are YjbH and CmpA [START_REF] Kirstein | Adapting the machine: adaptor proteins for Hsp100/Clp and AAA+ Proteases[END_REF][START_REF] Elsholz | Functional diversity of AAA + protease complexes in Bacillus subtilis[END_REF].

YjbH is a cytosolic protein involved in sensing various stresses and is an adaptor protein for the ClpXP catalyzed degradation of Spx factor, being, this way, involved in the resistance against nitrosating agent. The name of transcription factor Spx was originated from the identification of genetic mutations on its encoding gene (yjbD), that caused a suppression clpP or clpX [START_REF] Nakano | loss-of-function mutations in YjbD Result in ClpX-and ClpP-independent competence development of Bacillus Subtilis[END_REF]. High concentrations of Spx were observed in the clpP mutant, higher than in the wild-type strain, indicating that ClpP is necessary to maintain the Spx concentration low [START_REF] Nakano | loss-of-function mutations in YjbD Result in ClpX-and ClpP-independent competence development of Bacillus Subtilis[END_REF].

YjbH exposes the Spx degradation tag, the ClpX recognition site, mediating its degradation. This recognition site, part of the Spx 28-amino-acid C-terminal segment, is buried by its own structure buries, a structure that is disrupted by YjbH interaction, thereby promoting Spx degradation by ClpXP [START_REF] Garg | The YjbH protein of Bacillus Subtilis enhances ClpXP-catalyzed proteolysis of Spx[END_REF][START_REF] Chan | adaptor bypass mutations of Bacillus subtilis spx suggest a mechanism for YjbH-enhanced proteolysis of the regulator Spx by ClpXP[END_REF]. This way, under normal conditions, the YjbH-ClpXP interaction suppress Spx activity through its degradation, while under stress conditions, e.g. heat or oxidative stress, leads to the YjbH aggregation that promotes the stabilization and accumulation of Spx [START_REF] Chan | adaptor bypass mutations of Bacillus subtilis spx suggest a mechanism for YjbH-enhanced proteolysis of the regulator Spx by ClpXP[END_REF][START_REF] Elsholz | Functional diversity of AAA + protease complexes in Bacillus subtilis[END_REF].

On the other hand, CmpA adaptor protein, together with ClpXP, are involved in the qualitycontrol pathway that maintain the integrity of the spore envelope, by removing defective sporulating cells [START_REF] Tan | A qualitycontrol mechanism removes unfit cells from a population of sporulating bacteria[END_REF][START_REF] Elsholz | Functional diversity of AAA + protease complexes in Bacillus subtilis[END_REF]. Under correct sporulation conditions, the cells produce spores with a proper envelope, ClpXP degrades CmpA and the sporulation continues.

However, in those cells harbouring defects or misassembled spore envelopes, CmpA stabilizes and delivers the coat morphogenetic protein SpoIVA for degradation by ClpXP, that, consequently, destabilizes the forespore and leads to cell lysis, thereby eliminating the defective cells and ensuring the correct spores assembly within the population [START_REF] Tan | A qualitycontrol mechanism removes unfit cells from a population of sporulating bacteria[END_REF][START_REF] Elsholz | Functional diversity of AAA + protease complexes in Bacillus subtilis[END_REF].

This structural and activity coordination between the six-fold symmetric ClpX ATPase and seven-fold symmetric ClpP peptidase allows the unfolding of bound substrates in an ATP consuming reaction and the transportation of the unfolded polypeptide into the ClpP proteolytic chamber, that are consequently degraded to small peptides that exit the chamber by diffusion, as represented at Figure 20 [START_REF] Glynn | Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine[END_REF][START_REF] Baker | ClpXP, an ATP-powered unfolding and proteindegradation machine[END_REF][START_REF] Elsholz | Functional diversity of AAA + protease complexes in Bacillus subtilis[END_REF].

Membrane-bound proteases, an important quality control step for membrane and secretory proteins.

The bacterial cell envelope, membrane and cell wall, the external layer of bacterial cells, is very important for the integrity and viability of bacteria, as it provides physical protection, determines the cell shape and is the principal stress-bearing element [START_REF] Scheffers | Bacterial cell wall synthesis: new insights from localization studies[END_REF][START_REF] Thomas | The bacterial cell envelope[END_REF]. These are sophisticated, dynamic and complex structures that, which in addition to protecting, allows selective passage of nutrients from the outside, translocate secretory proteins and waste products from the inside. Gram-negative bacteria cell envelope, such for E. coli, is composed of two membranes, the cytoplasmic membrane and an outer membrane, and between these structures a membrane-enclosed periplasm, a dynamic and metabolically highly active environment. The outer membrane of Gram-negative bacteria contains lipopolysaccharides in its outer leaflet and phospholipids in the inner leaflet.

In contrast, Gram-positive bacteria have a cytoplasmic membrane and a cell wall. The cell wall is a complex structure composed of surface proteins, teichoic acids and a thick layer of peptidoglycan (PG). Peptidoglycan, also called murein, is a heteropolymer composed of long glycan chains, made up of alternating β-1,4-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) subunits, which are cross-linked by peptide bridges to form a strong but flexible structure (ref). Peptidoglycan is present in almost all bacteria, in B. subtilis these glycan strands are considered to be long (between 50 and 250 disaccharide units) when compared to, for instance, Staphylococcus aureus strands (between 3 and 10 disaccharide units), except in Mycoplasma and a few other species that lack detectable cell walls [START_REF] Vollmer | Peptidoglycan structure and architecture[END_REF]. To recoup the absence of periplasm, Gram-positive bacteria are able to lipo-modify homologues of periplasmic proteins and attach them to the outer leaflet of the cytoplasmic membrane, as well as retain some secretory proteins in the cell wall by ionic or covalent attachment to the peptidoglycan [START_REF] Sarvas | Post-translocational folding of secretory proteins in gram-positive bacteria[END_REF][START_REF] Schneewind | Sec-secretion and sortase-mediated anchoring of proteins in gram-positive bacteria[END_REF]. This way, the Gram-positive cell wall can be described as a thicker but dynamic and flexible structure rich in cell surface proteins, including quality control proteases, extracytoplasmic chaperones, autolysins, surface layer proteins, and substrate binding proteins (Pohl and Harwood 2010a;[START_REF] Schneewind | Sec-secretion and sortase-mediated anchoring of proteins in gram-positive bacteria[END_REF]. In addition to peptidoglycan, an important class of cell surface glycopolymers in Gram-positive bacteria are the phosphate rich teichoic acids (TAs). These molecules play a role in a large variety of functions, such as in maintaining the physicochemical properties of the cell surface, cation homeostasis, resistance to antimicrobial peptides and lytic enzymes, acting as phage receptors, in cell division, biofilm formation and host adhesion.

There are two types of TAs, distinguished by the way they are covalently linked to the surface, the lipo-teichoic acids (LTAs), which are anchored to the cytoplasmic membrane, extending from the cell into the peptidoglycan layer, and the wall teichoic acids (WTAs), which are covalently attached to the peptidoglycan layers and extend beyond them. although the last one is present in minor amounts [START_REF] Freymond | Poly(Glucosyl-N-Acetylgalactosamine 1-Phosphate), a wall teichoic acid of Bacillus subtilis 168: its biosynthetic pathway and mode of attachment to peptidoglycan[END_REF]). These anionic polymers and the lipo-teichoic acids create a high density of negative charge in the cell wall, an environment into which secretory proteins emerge in an unfolded state from the translocase [START_REF] Hyyrylainen | D-Alanine substitution of teichoic acids as a modulator of protein folding and stability at the cytoplasmic membrane/cell wall interface of Bacillus Subtilis[END_REF].

As described before in this introduction the Gram-positive architecture is compatible to protein export, a closer look at the cell envelope reveals that it offers a very challenging environment for proteins. From the correct insertion of membrane proteins and their maintenance to the unfolded proteins that emerge from the translocase, which should fold into their native configuration quickly without forming intra or intermolecular interactions that could block the translocation machinery and/or block cell wall synthesis sites, either or which would compromise cell viability. This reinforce the importance of having membrane quality control systems. As is the case of HtrA, HtrB and WprA for the Sec pathway, as they are serine proteases, belonging to the quality control protease family, combating the extracellular folding stress, and preventing potentially fatal obstruction of the secretory translocase. Another serine protease of the Sec pathway is the Signal peptide peptidase A (SppA), that will be described in the next subchapter.

Signal peptide peptidase A (SppA)

As detailed in the second chapter of this introduction the rapid removal and recycling of the cleaved signal peptide is crucial for maintaining a proper protein translocation, an action engaged by a membrane protease named signal peptide peptidase. The E. coli signal peptide peptidase, encoded by the gene sppA, was purified and characterized in 1987 by Ichihara and his colleagues [START_REF] Ichihara | Molecular cloning and sequencing of the SppA gene and characterization of the encoded protease IV, a Signal Peptide Peptidase, of Escherichia coli[END_REF][START_REF] Suzuki | Characterization of the SppA gene coding for Protease IV, a Signal Peptide Peptidase of Escherichia coli[END_REF]. They showed that this protein forms a tetramer, in which each protomer is estimated to have 67 kDa, and it is able to digest, in the presence of the cell envelop, the accumulated signal peptides and non-specifically other proteins [START_REF] Ichihara | Molecular cloning and sequencing of the SppA gene and characterization of the encoded protease IV, a Signal Peptide Peptidase, of Escherichia coli[END_REF].

Through sequence comparison between E. coli and B. subtilis genomes, a homologue of the E.

coli sppA gene was found in B. subtilis, the yteI. YteI was renamed signal peptide peptidase A (SppA) of B. subtilis as E. coli SppA (SppAEC), since these two proteins have 49% of sequence similarity [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF]. In contrast, B. subtilis SppA (SppABS) is a smaller membrane protease with about 36 kDa that assembles as an octamer, which protomer have a globular and extension part [START_REF] Nam | Crystal structure of Bacillus subtilis signal peptide peptidase A[END_REF].

As characteristic of serine proteases, the SppA catalytic domain consists in a dyad of a serine and a lysine [START_REF] Nam | Crystal structure of Bacillus subtilis signal peptide peptidase A[END_REF]. In order to improve the structure resolution, SppAEC and SppABS, were purified without the N-terminal transmembrane region and crystalized. The SppAEC protomer structure showed that the active site residues are in opposite domains, the lysine general base (K209) was identified in the N-terminal domain while the serine nucleophile (S409) is in the C-terminal domain, which means that they just interact with the chains are side-positioned [START_REF] Kim | Crystal structure of a bacterial Signal Peptide Peptidase[END_REF]P. Wang et al. 2008). Differently, the crystal structure of SppABS revealed that a serine (S147) residue, located at the globular region of the protomer, and a lysine (K199), located at the extension region, are quite distant from each other, as shown on Fig. 23. Demonstrating that SppABS on its own is not able to catalyse a substrate, as the active-site catalytic dyad is promoted by the interaction between the S147 of one promoter and the K199 of the adjacent protomer [START_REF] Nam | Crystal structure of Bacillus subtilis signal peptide peptidase A[END_REF]. In 2013, Nam and colleagues, revealed an important step of SppABS activity regulation through its crystal structure. They solved the crystal structure of the SppABS ∆2-54 deleted of the first 54 amino acids, corresponding to the N-terminal transmembrane domain, and of an active-site mutant (K199A) of SppABS ∆2-54 , which is an inactive form of SppA. The structure of the SppABS ∆2-54 inactive form revealed the presence of the C-terminal region bound to the catalytic groove, meaning that SppABS ∆2-54 self-processes its own C-terminal [START_REF] Nam | Structure of Signal Peptide Peptidase A with C -Termini bound in the active sites: insights into specificity, self-processing, and regulation[END_REF]. They also demonstrate that this C-termini peptide possibly have a role in the regulation of proteolysis, as it is able to compete with a synthetic peptide, based on the bound peptide, for the SppABS active sites [START_REF] Nam | Structure of Signal Peptide Peptidase A with C -Termini bound in the active sites: insights into specificity, self-processing, and regulation[END_REF]. In agreement with the information obtained for SppAEC, in vitro studies demonstrated that SppABS not only digests the signal peptides as it is able to digest fully folded proteins. An active form of purified SppABS was able to digest overnight a folded E. coli lipoprotein (BamD) involved in outer membrane protein assembly [START_REF] Nam | Crystal structure of Bacillus subtilis signal peptide peptidase A[END_REF]. Furthermore the SppABS function and role in secretion was studied by [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF] where was shown that the deletion of SppA induced an increase of pre-AmyQ accumulated in the cytoplasm and, consequently, a slight decrease in the secreted mature AmyQ, a normally highly secreted protein. Suggesting, this way, that SppA has a role on the processing of the pre-proteins under hyper-secreting conditions. B. subtilis have extracytoplasmic function σ factors (ECF) σ M , σ W and σ X that induce a stress response against lantibiotics, contributing for the resistance to this antibiotic peptides. SppABS is expressed under the control of a σ A factor (a constitutive expression through the different growth phases) and also under the control of σ M , which can indicate possible connection to stress response and lantibiotic resistance [START_REF] Huang | Identification of target promoters for the Bacillus subtilis SigmaX factor using a consensus-directed search[END_REF][START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF]). The possible involvement of SppABS in cleaving and consequently protecting the cell against peptide antibiotics that insert themselves into the cytoplasmic membrane was investigated by Kingston and colleagues (Kingston, Liao, and Helmann 2013a). Through diffusion disks assays, they demonstrated that the deletion of SppA decreases the strain sensitivity to nisin, lantibiotic that binds to lipid II and inhibiting cell wall synthesis and forming pores in the cytoplasmic membrane, which suggests a possible role on antibiotic cell protection (Kingston, Liao, and Helmann 2013a).

Scope of the Thesis

Around 5-10% of the bacterial proteins produced are transported from the cytoplasm to the extracellular compartment to perform several important functions, such as the provision of nutrients, cell-to-cell communication, and release of toxins and detoxification of the environment. In B. subtilis, the major route for the export of these proteins involves mainly the highly conserved Sec pathway [START_REF] Harwood | Bacillus protein secretion: an unfolding story[END_REF][START_REF] Tjalsma | Proteomics of protein secretion by Bacillus subtilis: separating the 'secrets' of the secretome[END_REF]).

The Sec pathway, described in the introduction section 1.2.1 of the introduction, mediates the translocation across the hydrophobic membrane barrier of secretory and membrane proteins in an unfolded conformation. Briefly, this secretion process can be divided into three steps: targeting, translocation and release. First the secreted proteins are synthesised as precursors (pre-pro-protein) with a N-terminal signal peptide (SP), which is recognized by cytoplasmic factors (Signal Recognition Particle-SRP) or chaperones (CsaA) involved in targeting the pre-protein to the translocon SecYEG.

The translocation of these proteins across the channel is driven by the translocon motor SecA, an ATPase that interacts with the substrate proteins and binds to the SecYEG complex. The repeated cycles of binding, hydrolysis of ATP, conformational changes of a dimeric SecA leads to the translocation of the secretory protein through the SecYEG complex. Subsequently, the signal peptide is cleaved resulting in the release of the protein from the membrane. Finally, the protein undergoes a maturation process carried out by other accessory proteins, such as the foldase PrsA, or the Bdb thioldisulfide oxido-reductases [START_REF] Driessen | Protein translocation across the bacterial cytoplasmic membrane[END_REF][START_REF] Kang | Molecular engineering of secretory machinery components for high-level secretion of proteins in Bacillus species[END_REF][START_REF] Tjalsma | Proteomics of protein secretion by Bacillus subtilis: separating the 'secrets' of the secretome[END_REF].

Although these mechanisms involved in the Sec pathway has been extensively studied in B.

subtilis, the knowledge about the protein complexes is limited. For example, it was shown, in E. coli, that the holotranslocon is a large complex where the YidC and SecYEG are linked by a subcomplex consisting of YajC and SecDF. SecDF regulates the interaction and activity of SecA with SecYEG.

Physical interactions, organization, activity and bioenergetics of these two sub-complexes SecYEG-YidC and SecDF-YajC were studied in vivo and in vitro [START_REF] Duong | Biogenesis of the gram-negative bacterial envelope[END_REF][START_REF] Schulze | Membrane protein insertion and proton-motiveforce-dependent secretion through the bacterial holo-translocon SecYEG-SecDF-YajC-YidC[END_REF].

Although paralogous proteins have been identified in B. subtilis, such detailed data on these complexes are not available. This raises questions about the organisation of the holotranslocon in B.

subtilis.. In the first chapter of results, we will show the efforts made to detect physical interactions involving the B. subtilis (holo)translocon proteins as well as the possible interactions between the accessory proteins involved in the maturation of secreted proteins. The goal was also to discoverunknown protein partners interacting with these complexes. In this chapter we showed a possible protein complex between a known protease, called SppA, and an unknown protein YteJ.

The chapter 2 of the results is dedicated to the study of the membrane complex SppA/YteJ. The signal peptide peptidase A (SppA), a component of the Sec machinery of B. subtilis, was named as such because sequence comparison with that of E. colirevealed that these two proteins share 49% of sequence similarity [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF]. SppA is a membrane bound serine protease that forms a homo-octameric dome-shaped complex described to be involved in the recycling of the remaining signal peptides (SPs), cleaved off the translocated proteins, in the membrane. SppA is also involved in the resistance to nisin, an antibacterial peptide of the lantibiotic family [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF]M. Cao et al. 2002;[START_REF] Nam | Structure of Signal Peptide Peptidase A with C -Termini bound in the active sites: insights into specificity, self-processing, and regulation[END_REF]. Its function and role in protein secretion were studied by [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF], who showed that the deletion of SppA induced a mild decrease in the secretion of mature secretory proteins, suggesting that SppA has a role on the processing of the pre-proteins under hypersecreting conditions [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF]. However, [START_REF] Saito | Post-Liberation cleavage of signal peptides is catalyzed by the site-2 protease (S2P) in Bacteria[END_REF], showed that the disruption of E. coli sppA gene did not affect in vivo cleavage of the SPs and that the B. subtilis RasP, a S2P protease, is involved in proteolysis of SPs of some secretory proteins and. Recently in 2017, Neef et al. gave a proof that the overexpression of RasP in B. subtilis improved the secretion of an heterologous and highly secreted AmyAc amylase by tenfold, proving that the overexpression of a bacterium membrane protease can boost the protein production [START_REF] Neef | Intramembrane protease RasP boosts protein production in Bacillus[END_REF]. These results changed the view on the SppA function, rising new questions about this topic. In B. subtilis sppA gene is in an operon with another gene called yteJ. From its sequence, the protein YteJ, of unknown function, is predicted to be an integral membrane protein belonging to the RDD family, a family of proteins of unknown functions. To better understand the influence of YteJ on SppA function and to try to clarify SppA role, we divided this study in two main partsthe in vivo and in vitro study. In vivo, we tried to uncloak the main role of the SppA/YteJ complex in the cell, starting to study the effect of the deletion of these two genes, their regulation by two sigma factors (σ A and σ W ), their cellular localization by fluorescent microscopy, the involvement of both SppA and YteJ in the lantibiotic resistance.

RESULTS

Identification of membrane protein complexes involved in the B. subtilis Sec pathway

SPA-tag constructions

In order to study the interactions within the B. subtilis (holo)translocon, as well as the possible interactions between the accessory proteins or unknown protein partners interacting with these protein complexes, we constructed strains harbouring the 3'-translational SPA sequence fusions in the genetic background of the BSB1 strain. This strain is tryptophan-prototrophic (trp+) derivative of the 168 trpC2 strain as described in [START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF].

A set of proteins of the Sec secretion pathway (see Table 1 below) was chosen to be SPAtagged for tandem affinity purification. For that purpose, the 3' ends of the corresponding genes were fused to the SPA-tag sequence present in the plasmid pMutin-SPA-LIC. The constructed plasmids were used to transform the BSB1 strain by a Campbell-like integration. Moreover, the pMutin-SPA-LIC is an integrative plasmid that contains an IPTG inducible / LacI repressible Pspac promoter that, once integrated in the chromosome, is just upstream of the downsteam genes to ensure their expression.

The construction of these plasmids had some setbacks: it had been only possible to obtain nine strains out of ten, as PrsA-SPA strain was not obtained. PrsA is an essential protein for extracellular folding of some secreted proteins after their translocation. The addition of the SPA-tag might have impaired its activity, therefore leading to an unviable strain. The final strains harbouring the 3'-translational SPA fusions are listed in Table S2. Growth curves (Figure 26) and Western-blot (Figure 27) analyses were performed to check the possible growth defect due to the constructs and the production of the corresponding tagged proteins respectively. Strains were grown in Luria Bertani (LB) medium and the OD was recorded. The growth of none of the strains was affected by the SPA-tag fused sequence to any of the proteins chosen (Figure 26). To check the expression of the SPA-tagged proteins in these strains by Western-blot, samples of cells were taken in exponential, transition and stationary phases. Although the nine strains harboured the 3'-translational SPA sequence fusions to the selected genes, only six proteins (SecDF, SecG, SpoIIIJ, YetJ, BdbB and BdbC) were detected by Western-blot using anti-FLAG anti-bodies (Figure 27).

The SipS-SPA, SipT-SPA and SppA-SPA were not detected (Figure 27 (B)-C and (C)-D, F).

However, for these three proteins, the C-terminal domain (fused to the SPA-tag in our constructs) is predicted to be located on the extracellular side of the cytoplasmic membrane, while for all the other protein fusions detected successfully the C-terminal domain is predicted to be inside the cytoplasm.

Therefore, it might be possible that the SPA-tag sequence was cleaved by extracellular proteases when the three proteins are translocated and inserted into the membrane. To study these three proteins we decided to construct strains harbouring a N-terminal SPA-tag fusions instead of the C-terminal fusion.

A SPA-tag sequence was fused to the 5' end of the corresponding full-length genes and cloned into another plasmid, pSG-SPANter, which allows the integration of the fused gene at the amyE locus by homologous recombination. Unfortunately, although several attempts were done, these constructions could not be obtained.

Purifications of the SPA-tagged proteins

The tandem affinity purification approach is based on two sequential affinity purifications as the SPA-tag is composed of two affinity tags (FLAG and CaM peptide). This double affinity purification allows the elimination of more contaminants compared to a single affinity purification.

This technique is also use in native conditions to purify protein complexes, i.e. without using crosslinkers that sometimes induce "false" interactions. The C-terminal SPA-tagged proteins (SecDF, SecG, SpoIIIJ, YteJ, BdbB and BdbC) that were proved to be well expressed (see western-blot Figure 27) were purified using this procedure. For the identification of the co-purified proteins by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), the purified samples were loaded on SDS-PAGE for a short migration. Large bands (0.5 cm), which comprises all the co-purified proteins, were cut out of the gel and were sent for analysis by LC:MS/MS.

According to [START_REF] Schulze | Membrane protein insertion and proton-motiveforce-dependent secretion through the bacterial holo-translocon SecYEG-SecDF-YajC-YidC[END_REF], SecDF, an ATPase, and YidC, an membrane protein translocase, form a subcomplex that interacts with the SecYEG in E. coli, and all together constitute the holo-translocon [START_REF] Schulze | Membrane protein insertion and proton-motiveforce-dependent secretion through the bacterial holo-translocon SecYEG-SecDF-YajC-YidC[END_REF]. Therefore, we expected that SecDF and SpoIIIJ, respective B. subtilis homologous of E. coli SecDF and YidC, form a sub-complex in B. subtilis as well. As such, we expected to identify and detect, by LC-MS/MS, these proteins together in the same sample after the SPA-purification of SecDF-SPA or SpoIIIJ-SPA. However, SecDF was not found in the co-purified proteins of the SpoIIIJ-SPA purified sample, and conversely, SpoIIIJ was not detected either in the SecDF-SPA purified sample (data not shown). These results showed that, as opposed to E.coli [START_REF] Schulze | Membrane protein insertion and proton-motiveforce-dependent secretion through the bacterial holo-translocon SecYEG-SecDF-YajC-YidC[END_REF], SecDF and SpoIIIJ do not co-purify in B. subtilis. Perhaps, in B. subtilis this two proteins form a weak interaction that could be disrupted by the presence of a SPA-tag. Although counter-intuitive, the difference could come from the detergent (n-Dodecyl β-D-maltoside-DDM) concentration used to solubilize the membranes, since in E. coli 2% DDM was used [START_REF] Schulze | Membrane protein insertion and proton-motiveforce-dependent secretion through the bacterial holo-translocon SecYEG-SecDF-YajC-YidC[END_REF] and our experiments were performed with 1% DDM. In B. subtilis, these complexes might be more susceptible to the DDM concentration and even 1% is too high so that the weak interactions might be abolished. In order to test this hypothesis, we used four different concentrations 0.5%, 0.75%, 1% and 2% of DDM to solubilize the B. subtilis membranes from the SecDF-SPA and SpoIIIJ-SPA strains. To maximise the chances to get these two proteins co-purified in at least one condition,

we also used only one affinity chromatography, i.e. the calmoduline resin. After purification in these different conditions of DDM concentrations, to check the quantity levels of the captured proteins, SpoIIJ-SPA or SecDF-SPA, we analysed each purified sample by Western-blot (Figure 28 (A)).

These results showed that the purification of both proteins were more or less the same whatever the different concentrations of DDM (Figure 28 (A)). The LC-MS/MS results corresponding to the analysis of these samples, that have identified numerous proteins (mainly contaminants), are focused only on the presence or absence of the other possible partners (SpoIIIJ for SecDF-SPA and SecDF for SpoIIIJ-SPA) and are showed as two tables in Figure 28 (B). These results showed that the SpoIIIJ was not detected in the SecDF-SPA purified samples. However, low amounts of SecDF were detected in all the SpoIIIJ-SPA samples. Altogether, these results showed that SecDF and SpoIIIJ do not form a stable complex in B. subtilis in the conditions tested. The small amounts of SecDF found in the SpoIIIJ-SPA purified samples might be an indication of a weak interaction between the two proteins or a simple contamination as only one affinity column was used. In either cases we could conclude that these two proteins form a stable complex, at least in our tested conditions. The other experiments using SPA-tagged proteins (SecG, BdbB, and BdbC) did not provide evidence for the existence of any stable complexes involving proteins known to be part of the translocon complex.

The only exception came with the finding of SppA, the so-called signal peptide peptidase, that co-purified with YteJ-SPA. This result was expected as preliminary results obtained in our lab from Blue native PAGE (BN-PAGE) experiments had indicated that these two proteins might form a complex. Two tandem affinity purifications of the YteJ-SPA protein and LC-MS/MS analysis showed that the relative quantities of the two proteins could be inferred from the LC-MS/MS data. The SppA/YteJ ratios found were 2:1 and 1:1. As both genes are in operon, it can be expected that the two proteins are translated in a 1:1 ratio, which would be in accordance with our experimental results.

Optimisation of the BN-PAGE technique

The BN-PAGE (for Blue Native PAGE) can be used to isolate protein complexes from the membrane fraction, to determine the molecular weight of native protein complex, to obtain information about their oligomeric states and to identify physiological protein-protein interactions [START_REF] Wittig | Blue Native PAGE[END_REF]. We therefore used the BN-PAGE as a complementary technique to identify the components of membrane protein complexes involved in the Sec pathway. As this electrophoretic technique is performed in native conditions, theoritically, the different protein We used the SecDF-SPA strain to analyse if the BN-PAGE can used for our study as SecDF-SPA is well expressed and can be easily detected by Western-Blot. Again different concentrations of DDM were used to solubilize the SecDF-SPA cell membranes. To obtain better results we have loaded the maximum quantity of protein possible (in this case 17.5µg, all the samples were loaded with same amount) and to avoid contamination between samples, we have loaded the samples with four empty wells between. In BN-PAGE, proteins often appear as a smear and not as single bands as in SDS-PAGE gels (Figure 29). Nonetheless, at low DDM concentrations the smears corresponding to SecDF-SPA appeared higher in the gel, indicating that SecDF-SPA is part of a higher molecular weight complex. In contrast, at higher DDM concentrations this complex is dissociated as SecDF-SPA appeared lower in the gel (Figure 29). For the analysis of BN-PAGE by LC-MS/MS each gel lane was divided in ten small pieces, in-gel trypsin digested to identify proteins present in each part of the lane (Figure 30). The results we obtained from the LC-MS/MS analysis of these samples indicated that 1% DDM is the best concentration to solubilize the membrane samples of B. subtilis, as it was the condition with a higher number of proteins detected. We needed a membrane protein complex that can be used as a control in our experiment, and, according to preliminary data obtained at our laboratory SppA and YteJ could co-migrate together in BN-PAGE. In our experiment, in the presence of 1% DDM, the LC-MS/MS results showed that the presence together of SppA and YteJ in the gel fraction number 4 (Figure 30).

Interestingly, YteJ was identified in the piece of gel in which the highest number of SppA peptides were detected. At lower or higher DDM concentrations, YteJ was not detected.

Unfortunately, peptides belonging to SpoIIIJ were identified in almost all pieces of gel, with an equal number of spectra in each fraction, indicating that SpoIIIJ might dissociate easily from a possible membrane complex. Moreover, for an unknown reason, SecDF was not detected in any piece of gel, in any DDM conditions (Figure 30). Altogether, these results reinforced the hypothesis that SppA and YteJ form a stable complex, while, if SpoIIIJ and SecDF form a membrane protein complex, it is unstable.

In conclusion, using SPA purification combined with BN-PAGE analysis, we were unsuccessful to reveal any expected complexes of the Sec pathway. We focused on using native condition to capture native complex; however, it might be necessary to use cross-linking agents to study these complexes in B. subtilis.

As we have identified one complex with both techniques, the SppA/YteJ complex, the next chapter will be entirely dedicated to the study of this membrane protein complex, that was supposed to be related to the Sec pathway through the signal peptide peptidase function of SppA.

The SppA/YteJ membrane complex

The function of B. subtilis SppA has been studied, as described at the introduction (Signal peptide peptidase A (SppA) section). However, yteJ which in an operon with sppA encodes for a protein of unknown function and unknown structure. As a first step towards the understanding of this unknown protein, we analysed (i) its genomic context using bioinformatics tools, such as BLAST [START_REF] Singh | BLAST-based structural annotation of protein residues using protein data bank[END_REF] and microbial genomic context viewer [START_REF] Overmars | MGcV: The microbial genomic context viewer for comparative genome analysis[END_REF], and (ii) its

The sppA yteJ operon is regulated by sigma factors σ A and σ W

Bacterial sigma factors interact with the RNA polymerase core and enable its binding to specific promoter sequences, inducing the initiation of transcription [START_REF] Britton | Genome-Wide analysis of the stationary-phase sigma factor ( sigma-h ) regulon of Bacillus subtilis[END_REF]. Sigma-A (σ A ) is the major sigma factor required for expression of many essential housekeeping genes and the majority of the RNAs necessary during bacterial growth [START_REF] Britton | Genome-Wide analysis of the stationary-phase sigma factor ( sigma-h ) regulon of Bacillus subtilis[END_REF].

The extracytoplasmic function (ECF) subfamily of sigma factors is a distinct group that allows bacteria to adequately respond to stressful environmental conditions and that are involved in the control of cell envelope-related functions, such as secretion, synthesis of exopolysaccharides or macromolecules [START_REF] Missiakas | MicroReview the extracytoplasmic function sigma factors : role and regulation[END_REF]. B. subtilis has seven members of this sigma factor family, identified through bacterial genome sequencing. The sigma-W factor (σ W ), a member of this subfamily, is normally induced in response to cell envelope stress caused by antibiotics, alkaline and salt shocks [START_REF] Zweers | Definition of the W regulon of Bacillus subtilis in the absence of stress[END_REF]).

In 1998, Huang and colleagues showed that the B. subtilis sppA and yteJ genes are members of the σ W regulon [START_REF] Huang | Identification of target promoters for the Bacillus subtilis SigmaX factor using a consensus-directed search[END_REF]. Based on the data from [START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF], the expression of the B. subtilis sppA-yteJ operon seems to be dependent on σ W but also possibly on σ A , the house keeping sigma factor. To test the relative importance of the σ A and σ W -dependent promoters in the regulation of sppA yteJ expression, we constructed transcriptional fusions with these promoters These constructs were inserted ectopically at the amyE locus together with a spectinomycin resistance cassette. The constructs were confirmed by PCR and sequencing. The final strains harbouring either the σ AW -dependent promoters or the single σ W -dependent promoter were named GHBs48 (BSB1 amyE::Paw-sfgfp) and GHBs 54 (BSB1 amyE::Pw-sfgfp), respectively. For the sake of clarity, these strains were labelled Pσ AW and Pσ W , respectively. By following the fluorescence intensity of the sfGFP, directly related to its expression, during growth we estimated the relative quantitative contribution of each promoter in the sppA yteJ expression. As described above, the σ W factor is involved in cell-membrane and cell-wall stress, therefore we used two antimicrobial peptides that perturb the cell membrane integrity, nisin and subtilin, to induce its activation. Nisin and subtilin, two members of the class I of the lantibiotic family, insert themselves at the cell membrane by binding to the lipid II, a peptidoglycan precursor. They arrest the Lipid II in the cell membrane, abrogating the peptidoglycan formation [START_REF] Cotter | Bacteriocinsa viable alternative to antibiotics?[END_REF][START_REF] Spieß | Autoinduction specificities of the lantibiotics subtilin and nisin[END_REF]. Consequently these peptides induce the formation of pores in the cell membrane and affect the cell wall formation, which leads to cell weakening and lysis [START_REF] Cotter | Bacteriocinsa viable alternative to antibiotics?[END_REF]. Since the σ W -dependent promoter is fused to the sfgfp in both constructions, inducing its expression will be revealed by the sfGFP production level, and therefore the contribution of the σ A can be inferred. We tested a range of different concentrations of these peptides (from 0 to 6 µg of nisin, and from 0 to 30% v/v of subtilin), and the results are shown in Figure 33 and Figure 34. The strains were grown in LB medium, supplemented with 100 µg.mL -1 spectinomycin, at 37°C with vigorous shaking in flasks. Overnight cultures were diluted 20-fold in 100 µL per well of pre-warmed fresh CH defined medium (pre-culture) into 96-well microtiter plates.

The CH pre-cultures were grown until an OD600nm of 0.3-0.4 and then diluted 400-fold into a prewarmed fresh medium. The antimicrobial peptides were added when the cell cultures reached early exponential phase, around 0. At the top of Figure 33, the graphs labelled 0 µg corresponds to the controls, i.e. the Pσ AW and Pσ W strains growing in LB without nisin. Both strains showed identical growth rates, while the intensity of measured sfGFP fluorescence remained low. With 1 µg of nisin (second line panel of Figure 33), although the three characteristic phases of the bacterial growth (exponential, transition and stationary phase) can be observed, when compared with the controls 0 µg, the growths of both strains are affected. Just after induction, a decrease in the cell growth was observed followed by a rapid growth recovery. However, these cells do not reach the same optical density as the controls and as in stationary phase, the OD of the nisin-treated cultures showed a slight decrease due to cell lysis. When treated with 2 µg of nisin the strain growths were affected, as a drop in the OD600nm was measured just after induction. Probably a part of the cells died, by cell lysis, and it took around 200 min to the remaining living cells to produce enough biomass to be measurable by OD. The corresponding ratio of sfGFP fluorescence intensity/OD600nm showed, 60 min (represented by a dashed line) after the induction, a clear peak, meaning that the remaining living cells were producing higher amount of sfGFP. This was interpreted as a boost of activity of the sigma-dependent promoters. For higher concentrations of nisin, from 3 µg up to 6 µg, a longer lag phase was observed during which the OD600nm was not measurable. Regardless of nisin concentrations, the levels of the fluorescence/OD ratio increased during the first 60 min. The highest levels were recorded with 3 µg.ml -1 of nisin, while the addition of 4 or 6 µg.ml -1 of nisin led to lower levels. For 2 and 3 µg.mL -1 of nisin, a peak of fluorescence/OD ratio was observed -i.e. same duration for the increase and decrease of the signal, while for higher concentrations (4 and 6 µg), a longer lag phase and even a plateau than what we observed for lower nisin concentrations. Whatever the nisin concentration applied to the cells, the fluorescence/OD600nm ratios followed the same patterns and levels in the two strains, P W and P AW . Therefore, whether the sfgfp gene was under the control of a single promoter sequence, i.e. in the P W strain, or the two adjacent promoters sequences found natively in the chromosome, i.e. in the P AW strain, the results were identical. The exception was a slight increase of this ratio that was observed for the highest concentrations of nisin (6 µg.ml -1 ), which indicated a very marginal contribution of  A to the overall control of the operon expression. We concluded that the main promoter responsible for the genetic regulation of the sppA yteJ operon was the  W -dependent promoter. It should be noted that the fluorescence/OD ratio decreased to reach back the minimal levels after a period of time which increased proportionally to the nisin concentration. Moreover, whatever the nisin concentration considered, when the signal ratio was back to the minimum level, the cell growth concomitantly resumed. As  W is activated in response to nisin, one might suggest that the nisin concentrations decreased below the activation threshold of  W , and that these concentration levels were compatible with cell division. Similar results were obtained when cells were treated with subtilin (Figure 34), an antimicrobial peptide of the same family. At low concentration of subtilin (5%), we did not observe any difference between the control and the treated strains (first and second lines of the Figure 34), neither for the growth nor for the sfGFP production. When we used 10% of subtilin, the sfGFP expression was induced, displaying a small peak in the fluorescence intensity. When increasing the treatment to 15% of subtilin we reproduce the results observed with 2 µg of nisin. After 60 min (represented by the dashed line) of subtilin treatment, a clear peak on the sfGFP fluorescence intensity ratio was observed. As observed for nisin, treating the strains with high concentrations of subtilin, 20% and 30%, leads to an increase in the lag phase and a  W -dependent response 60 min after induction, with higher and wider peaks of fluorescence intensity. In the presence of 30% of subtilin, cells did not grow. but a high fluorescence peak with a larger plateau, than the lower concentrations of subtilin, was observed. In conclusion, we observed that regardless of the nisin or subtilin concentrations, the major contributor for the regulation of the expression of the sppA yteJ operon was σ W .

Deletion of the sppA and yteJ genes

To better understand the role of the B. subtilis SppA/YteJ complex, we deleted these two genes either separetly or together to study the effect of their deletion(s). The sppA, yteJ and both genes were deleted from their native locus and replaced by a chloramphenicol resistance cassette (cat). The expression of this cassette remained under the control of the sppA yteJ native promoter, as described at the section 4.3.2 of the Material and Methods chapter. These constructs were confirmed by PCR and sequencing. The final strains were named: GHBs 023 (BSB1 ∆yteJ::cat), GHBs 025 (BSB1 ∆sppA::cat) and GHBs 028 (BSB1 ∆sppA yteJ::cat). For the sake of clarity, strains GHBs 023, 025 and 028 will be named ∆yteJ, ∆sppA and ∆sppA yteJ, respectively. The growth of the strains were recorded by measuring, at regular intervals, the optical density at 600 nm (OD600nm). The Figure 35 shows the growth curves plotted for each strain. The wild type strain (BSB1) and the deletion mutants presented the same growth rate and cell density at all growth stages. These results allowed us to conclude that the deletion of the sppA and/or yteJ genes did not affect the growth of the mutant strains in LB medium. This is in agreement with the result reported in [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF] for the deletion of sppA. We checked by microscopy whether cell morphology was affected. Cells were grown exactly as previously described and samples were collected during the exponential (exp), transition (tran) and stationary (stat) phases of the growth cycle and analysed by microscopy (Figure 36). Cells were stained with the membrane dye FM4-64, allowing the observation of the cell membranes and septum.

The cell morphology of the ∆sppA yteJ strain was not affected, as shown in with the ∆sppA yteJ mutant cells (right side of the panel), both appear to be normal B. subtilis rodshaped growing cells with 4-10 μm long [START_REF] Yu | Monitoring bacterial growth using tunable resistive pulse sensing with a pore-based technique[END_REF], with a well-defined division septum and cell poles. Identical cell morphology was observed in transition and stationary phases. Cell morphology of each single deletion mutant strains were also checked by microscopy and as the double deletion mutant no phenotypic differences were observed comparatively to the BSB1 strain. With these results

we concluded that the deletion of sppA, yteJ or sppA yteJ do not affect cell morphology. [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF] showed that the deletion of sppA affected the maturation of a highly secreted protein, AmyQ. AmyQ, a secreted protein through the Sec pathway, is maintained in an unfolded pre-protein form in the cytoplasm and reaches its folded mature form after being translocated through the cell membrane and released to the extracellular medium. [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF] observed that the sppA deletion caused a delay in the processing of the AmyQ mature form which led to an accumulation of the pre-AmyQ, which resulted in a mild decrease in the secretion of AmyQ. These results suggested that the SppA activity was important in the efficient processing of the pre-secreted proteins, supposedly by degrading the signal peptides from secreted proteins [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF].

Role of the SppA/YteJ complex in protein secretion

While the role of SppA in protein secretion was documented, we wondered what was the role of YteJ,

and also what was the role of the SppA/YteJ complex in this cellular process. To address these issues, we checked the secretion levels of a highly secreted protein, the maltogenic amylase (AmyM). Dr.

Van Rij, from DSM (Delft, The Netherlands), provided us with a multi copy plasmid containing the gene encoding AmyM (pCS73) and the corresponding empty plasmid (pCS74), all containing a neomycin (neo) resistance cassette. Each one of these plasmids was transformed into the B. subtilis sppA, yteJ or sppA yteJ deletion strains and the BSB1 wild type strain, giving rise to eight new strains (listed in Table S5 in the appendix section) from GHBs029 to GHBs040. AmyM protein has a molecular weight of 75.2 kDa and can be followed by SDS-PAGE.

To analyse the extracellular fraction, the cells were harvested in exponential phase of growth and centrifuged. The obtained cell-free supernatant was supplemented with TCA to precipitate the extracellular secreted proteins that were then analysed by SDS-PAGE. Before running the gel, the concentration of the protein samples were measured by the Bradford method and equally loaded. The results are shown in Figure 37. The two first lanes correspond to the control samples, the BSB1 wildtype strain and BSB1 with pCS74, respectively. These two strains do not contain the amyM gene. The sample BSB1 containing pCS74 empty plasmid was used as a control to check if the presence of a multicopy plasmid affects the normal protein content. Compared to the BSB1 sample without plasmid, first lane, the presence of the empty plasmid do not affect the protein content, as both lanes showed the same protein pattern. The remaining four lanes on this gel correspond to the extracellular protein samples of the strains expressing AmyM, thus containing the pCS73 plasmid, including the respective control BSB1 and deletion mutant strains. All these samples showed a band around 75 kDa that corresponds to the molecular weight of AmyM.

All three deleted strains showed the same intensity for the AmyM band on the gel. This indicated that the deletion of sppA, yteJ or the operon sppA yteJ had no impact on the overall amount of the hyper-secreted AmyM protein.

The delay in the processing of the AmyQ mature form observed by [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF] was a mild effect and the results were obtained through pulse-chase protein labelling. These facts can explain why it is not possible to see a difference on the final amount of the mature form in our experiments. To further study the premature and mature form of the secreted protein and analyse the differences between the amount of secreted protein in the wild-type and mutant strains, we needed to use specific antibodies for this protein. The production of AmyM antibodies, from the protein purification up to rabbit immunization, was part of a secondment project developed in collaboration with one of our industrial partners of the ProteinFactory ITN, UCB-Celltech company (Slough, UK), including an internship of six weeks on UCB premises. The details of this work are described in the Appendix 6.2. With the serum of antibodies against AmyM we analysed the extracellular fraction, obtained as previously explained, by Western Blot (WB). To do the WB we ran the samples in a 10%SDS-PAGE gel exactly as described for the previous experiment. In the first two lanes, corresponding to the controls, we cannot (as expected) observe a band corresponding to AmyM, since these strains do not produce AmyM. The remaining samples, expressing AmyM, showed a band around 75KDa corresponding to the molecular weight of AmyM and some lower bands corresponding to nonspecific antibodies reaction or possible AmyM degradation. However, once more, this technique did not show significant or measurable differences in the amount of secreted AmyM, between the strains. We also tried to use a second reporter protein, the highly secreted endo-1,4-beta-xylanase (XynA). The xynA gene was also provided by Dr. Van Rij, on a multi copy plasmid, and we proceeded to the same experiment as just described above for AmyM. Unfortunately, it was not possible to detect XynA on SDS-PAGE , maybe due to low expression and concentration of this secreted protein in the medium.

Figure 38. Analysis of AmyM extracellular amount by Western Blot. This WB was performed with antibodies against AmyM. pCS74 empty plasmid; pCS73 plasmid encoding amyM gene. The BSB1 wild type strain, BSB1 with pCS74 empty plasmid and with the pCS73 AmyM plasmid were used as controls. The black arrow points for the correct band of AmyM.

Effect of the sppA, yteJ or sppA yteJ overexpression in the secretion

Since we could not observe a difference in secretion caused by the deletion of the sppA and yteJ genes, we wondered what would be the effect of their overexpression. Therefore, we constructed overexpressing strains by re-inserting, in the WT BSB1 background, each gene or the full operon in the amyE ectopic locus under the control of the hyperspank promoter (Phs), an IPTG inducible promoter (as described in the material and methods section 4.3.3). These constructs were confirmed by PCR and sequencing. The final strains are GHBs 059 (BSB1 amyE::Phs yteJ /spec), GHBs 066 (BSB1 amyE::Phs sppA /spec) and GHBs 071 (BSB1 amyE::Phs sppA yteJ /spec). For the sake of clarity, the strains GHBs 059, 066 and 071 will be named BSB1 PhsyteJ, BSB1 PhssppA and BSB1 PhssppA yteJ, respectively. To address the effect of the overexpression of SppA, YteJ and of the SppA/YteJ complex in protein secretion we transformed these strains with either the multi copy plasmids containing the gene encoding AmyM (pCS73) or an empty plasmid (pCS74) as a control, as we proceeded before with the deletion mutants. This way, we could followed by SDS-PAGE the levels of AmyM secreted into the extracellular medium. It gave rise to nine new strains (listed in Table S5 in the appendix section) from GHBs073 up to GHBs081.

The overexpressing strains, listed above, were grown in LB at 37°C, supplemented with the correct antibiotics when needed, and the cells were harvested in exponential phase and centrifuged.

The extracellular cell-free medium was TCA precipitated and analysed by 10% SDS-PAGE gels.

Before loading the samples onto the gel, the concentrations in total protein were measured by the Bradford method and, then, loaded with the same concentration of 0. The second lane of the SDS-PAGE shown on Figure 39 corresponds to the extracellular medium sample of the BSB1 control strain, without the pCS73 (AmyM) plasmid. As this strain do not contain the amyM gene, AmyM is not produced, so the band around 75 kDa corresponding to AmyM is absent. In contrast, BSB1 with the pCS73 plasmid, on the third lane, shows a band with the expected size for AmyM, confirming that AmyM is produced. However, in the fourth lane, we can observe that when sppA is overexpressed the amount of AmyM in the extracellular medium is significantly reduced, as the 75 kDa band, corresponding to AmyM, present in this sample is fainter than the band shown on the third lane control. Interestingly, comparing to the control sample, when yteJ or sppA yteJ are overexpressed the amount of AmyM present in the extracellular medium increases. The last two lanes of the gel (Figure 39), corresponding to the yteJ or sppA yteJ overexpressing samples respectively, show a AmyM band more intense than the AmyM band present in the control of the second lane. With these results, we can conclude that when the amount of SppA increases in the cell the amount of secreted AmyM decreases. As SppA is a membrane protease, it might degrade directly the AmyM produced or it could be an indirect effect. When YteJ is overexpressed, AmyM levels seem to be higher than those observed with BSB1 strain, as if AmyM was less degraded. Furthermore, when

SppA is co-overexpressed with YteJ, the levels of AmyM are higher than BSB1. If SppA is able to degrade AmyM directlynot yet provenone can hypothesize that the SppA protease activity might be inhibited or regulated by YteJ.

The deletion of sppA yteJ affects the long term survival

The ability to replicate in a specific environment is named bacterial fitness. The fitness of a bacteria can be tested by analysing the capacity of survival of two strains, one antibiotic sensitive strain and one antibiotic resistant strain, under the same conditions for a predefined time. Furthermore, at the end of the predefined time, the number of bacteria is counted and the one which has produced a higher number is by definition "Fitter".

In order to characterise the ∆yteJ, ∆sppA and ∆sppA yteJ deletion mutant strains we studied their capacity of survival by performing some fitness tests. First the strains were grown in liquid rich medium (LB), at 37°C for 48h. At different time points (after 18h, 24h and 48h), samples of the cultures were plated out onto LB agar plates. The number of colonies were counted after the plates have been incubated for 18h at 37°C. As shown in Figure 40, after 18h of growth, the number of colonies counted is just slightly affected by the deletion of sppA, yteJ and the full operon, as compared with the number of WT colonies. The lowest number of colonies was counted for the ∆sppA strain,

showing around 200 CFU/ml, which is 1.5-times less than the wild type. After 24h of growth in the same medium, the capacity of survival of all strains was affected, because the number of colonies was lower than the numbers after 18h of growth, even for the WT control strain. However, the capacity of survival of the deletion mutants was even more affected, as in the absence of these genes the number of surviving colonies is lower than the WT. The ∆sppA strain was the more affected, showing the lowest value of colonies counted, around 150 CFU/ml lower than the wild type control.

After of 48h of growth, as shown on Figure 40, although all the strains showed a lower number of surviving colonies, the mutant strains have a lower capacity of survival. The ∆sppA yteJ and ∆sppA strains are both the less fit, as both have the lowest number of colonies counted. The yteJ strain was the least affected strain compared to the other mutant strains. The results of these tests showed that in LB medium the survival of the deletion strains is affected comparing to the wild type strain. We concluded that the deletion of sppA was the most deleterious one for survival, indicating that SppA function provides an advantage for long term survival. The same conclusion applied to the deletion of sppA yteJ operon. However, as the deletion of yteJ was the less deleterious, we concluded that YteJ function was not important for survival. The survival ability of the ∆sppA yteJ mutant was as much affected as for the ∆sppA mutant strain. As our main objective was to understand the function of the SppA/YteJ complex we performed further fitness tests with only BSB1 and ∆sppA yteJ strains. We wondered whether the composition of the media may affect the long-term survival of the strains. New fitness tests in LB rich media (Figure 41A) and in M9 minimal medium (M9 supplemented with glucose) were realised (Figure 41B). After 24h and 48h, samples were collected and plated on LB agar plates, and colonies were counted after 18h of growth at 37°C. Figure 41A shows the long-term survival of BSB1 and the ∆sppA yteJ strains. The deletion of the sppA yteJ operon clearly affected the long-term survival of the cells. Figure 41B shows the longterm survival of the same strains in M9 minimal medium. After 24h in the M9 medium, the number of CFU/ml for BSB1 was the same as when cells were grown in LB; however the number of CFU/ml for ∆sppA yteJ was already half of that observed in LB. After 48h, the CFU/ml number for BSB1 was half of that observed in LB indicating that the M9 medium is not as good as LB for long-term survival, even for the wild-type cells. After 48h in the M9 medium, the ∆sppA yteJ cells showed the lowest number of surviving cells. We can conclude that the medium composition and richness affect the longterm survival of B. subtilis cells and that this effect is more pronounced for the ∆sppA yteJ strain. [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF] showed that the deletion of B.subtilis sppA affected protein secretion.

Taking in account this fact, and what we observed in our results, we wanted to test if the BSB1 strain secreted a protein whose function or quantity was altered in the ∆sppA yteJ strain. We performed complementation assays for which the WT and ∆sppA yteJ strain were grown together in LB medium, at 37°C. Using the same procedure as described above, separated cultures of BSB1 and ∆sppA yteJ strains, as well as a mix of 50% BSB1 and 50% ∆sppA yteJ cells were grown in the same conditions.

After 24 and 48h of growth in the LB medium, cells were diluted and plated on LB agar medium.

However the mixed culture was plated on a second plate of LB agar supplemented with For both 24 and 48 hours of growth ( Figure 42) we observed that the ability to survive of the ∆sppA yteJ strain is lower than that of the WT strain. For the complementation experiment with the inoculation using an equal number of BSB1 and ∆sppA yteJ cells, after 24h, the total number of cells was approximately twice that of the ∆sppA yteJ cells. This indicated that during the first 24h, cells of both strains grew equally well.

However after 48h, the ∆sppA yteJ cells represented only 15% of the total cells. This experiment indicated that the defect of long-term survival of the ∆sppA yteJ cells was not due to the lack of a specific secreted protein or another secreted component in the medium, as BSB1 cells did not complement it.

The general conclusion is that the function of SppA is important for the long-term survival of B. subtilis cells and, that, even if the protease active sites of SppA are located outside the cell, SppA activity cannot help the survival of other cells around, but this activity seems important for the cell itself.

The deletion of sppA and yteJ affects the resistance to lantibiotics

In 2013, Kingston and his colleagues demonstrated that the deletion of B. subtilis SppA leads to a strain sensitivity to nisin, an antimicrobial peptide (Kingston, Liao, and Helmann 2013a). To investigate the possible involvement of the SppA/YteJ complex in the resistance to antimicrobial peptides we tested the resistance capacity of deletion ∆sppA and ∆yteJ deletion mutant strains to nisin and subtilin. Nisin and subtilin are two antimicrobial peptides of the same lantibiotic family [START_REF] Spieß | Autoinduction specificities of the lantibiotics subtilin and nisin[END_REF]. This experience was performed in 96-well plates, where the strains grew over 16 hours in the presence of nisin or subtilin, optical density at 600nm (OD600nm) was measured at regular intervals.

The BSB1 strain was used as a control strain. Overnight cultures of BSB1 and ∆sppA, ∆yteJ and ∆sppA yteJ strains were grown at 37 ᵒC, in LB and LB supplemented with Cm 5 µg/ml respectively, In Figure 43, BSB1, ∆sppA and ∆yteJ strains showed identical growth rates when grown in LB without nisin. With 3µg/ml of nisin (graph middle left), although the three characteristic phases of the bacterial growth (exponential, transition and stationary phase), the growth of the strains was affected. The strains took around 100 extra minutes to resume growth showing a longer lag phase than in the control conditions. We defined the time necessary to resume growth as the time needed for the strain to reach an OD600 of 0.1. In the presence of 3µg/ml of nisin, the deletion of yteJ led to a slightly increased resistance to nisin, as it took around 20 minutes less than the BSB1 strain to reach an OD600 of 0.1. On the contrary the deletion of sppA turn the cells more susceptible to nisin, as it took around 20 minutes more than the BSB1 strain to reach an OD600 of 0.1. When the concentration of nisin was increased to 4 µg/ml in the medium, the same results were observed but with an exacerbate difference between the strains. When treated with 6µg/ml of nisin the BSB1 strain showed a lag phase of 250 minutes longer than in the control condition without nisin. In this condition, the ∆yteJ strain showed the same lag phase and growth rate as the BSB1 strain, while the ∆sppA strain took slightly longer to resume growth. The ∆sppA strain is therefore more susceptible to nisin than the two other strains. In the presence of 8µg/ml of nisin, the strains did not grow.

To simplify we did not represent the ∆sppA yteJ results, as this mutant strain behaved exactly as the yteJ mutant strain. With these results we concluded that the deletion of sppA affects de resistance to nisin. However the deletion of yteJ or the full operon, yteJ sppA, showed non reproducible results with either an intermediate phenotype or a nisin resistance higher than that of the BSB1 strain.

Although the trend for the nisin resistance of the BSB1 strain compared to the deletion strains were reproducible, the effect of a specific nisin concentration could vary from one experiment to another. The stability of nisin might explain these differences. Therefore we decided to test another lantibiotic of the same class with a highly similar ring structure, subtilin. [START_REF] Spieß | Autoinduction specificities of the lantibiotics subtilin and nisin[END_REF]. The experiments were performed as explained before for nisin. As shown in Figure 44, the results obtained with subtilin were similar to that observed with nisin. With 20% of subtilin (graph top right, Figure 44), although we observed the three characteristic phases of the bacterial growth (exponential, transition and stationary phase), when compared with the control conditions 0% (graph top left), the growth of the strains was altered. The cells took slightly longer to resume growth, did not reach the same optical density in stationary phase as in the control conditions. The growth of the two deletion strains showed a small delay compared to the BSB1 strain. When treated with 25% of subtilin, the BSB1 and ∆yteJ mutant strains had identical growth rates, and similar 'lag phase' of around 200 min. The ∆sppA strain showed an even longer lag phase, around 100 extra minutes to resume growth, indicating a higher susceptibilty to subtilin.

Increasing the subtilin to 30% led to a striking difference between the growths of the three strains.

Comparing BSB1 with ∆yteJ, we can see that the deletion of yteJ led to a decrease in the resistance to subtilin, as it took around 200 minutes more than the BSB1 strain to reach an OD600 of 0.1. The deletion of sppA turn the strain even more susceptible, as it took 300 minutes more than the BSB1 strain to reach an OD600 of 0.1. The deletion of yteJ showed an intermediate phenotype, between the BSB1 and ∆sppA strains. When the concentration of subtilin was increased to 34%, the BSB1 strain was able to resume growth after 300 min while it took 600 min for the ∆sppA strain and while the ∆yteJ strain showed almost no growth. Altogether these results led us to conclude that the deletion of either yteJ or sppA turned the cells more susceptible to subtilin. In the presence of 38% subtilin, the strains did not grow. As stated before for the nisin results, with subtilin we did not show the ∆sppA yteJ results, as this strain had the same behaviour as the yteJ strain. With these results, comparable to the nisin results, we can conclude that the deletion of sppA affects de resistance to subtilin. However, depending on the concentration of subtilin, the deletion of yteJ or of the full operon showed again non reproducible results (as for nisin) with either an intermediate phenotype or a subtilin resistance higher than that of the BSB1 strain.

Figure 45 illustrates the effect of increasing subtilin concentrations on the growth rate. It revealed that the growth rates were strongly affected when the concentration of subtilin was higher than 30%. As the results obtained with subtilin were more reproducible than the experiments done with nisin, and as a 30% subtilin concentration showed significant differences in the lag phase duration of the three strains without affecting too much the growth rates (and so allowing to discriminate strains based on their subtilin resistance capacity), we selected this concentration to setup the lantibiotic resistance test used later in this work.

The deletions of sppA and yteJ do not affect the resistance to vancomycin, erythromycin or protect against lysozyme

Our results showed that the SppA/YteJ complex was involved, mainly through SppA, in the resistance to lantibiotics, polycyclic antimicrobial peptides that are capable of inserting themselves into the cellular membrane. To check whether the SppA/YteJ complex had a role in the resistance to other classes of antibiotics we repeated the previous experiments with two other classes of antibiotics.

First, we chose to test an antibiotic of peptidic nature, ancomycin, a glycopeptide antibiotic which acts by inhibiting the cell wall synthesis of Gram-positive bacteria. The experiment was performed as described in the previous section 2.2.5, testing a range (from 0 to 1 µg/ml) of vancomycin concentrations [START_REF] Adimpong | Antimicrobial susceptibility of Bacillus strains isolated from primary starters for african traditional bread production and characterization of the bacitracin operon and bacitracin biosynthesis[END_REF].

In Figure 46, the first graph (top left) of the panel corresponds to the controls, i.e. the BSB1 and ∆sppA and ∆yteJ mutant strains grown in LB without vancomycin. The three strains showed identical growth rates. When treated with 0.1 µg of vancomycin (graph top right), the growth was not altered, meaning that this concentration it is not high enough to induce a response. When 0.2 µg/ml of vancomycin was used (graph middle left), the wild-type strain showed a long lag phase; the ∆sppA and ∆yteJ mutant strains did not grow. We observed similar results for higher concentrations of vancomycin (0.5 and 1 µg/ml). Comparing these results with the results obtained for nisin and subtilin, we can conclude that the deletion of sppA or yteJ does not affects the resistance to vancomycin. To fully conclude on vancomycin, a range of concentrations between 0.1 to 0.2 µg/ml should be tested. To test a different class of antibiotic not related to cell wall or membrane synthesis or integrity, we chose erythromycin. Erythromycin is part of the macrolide family of antibiotics, which decreases the bacterial protein production. The experiment was performed as described earlier, testing a range (from 0-1 µg/ml) of different concentrations of erythromycin, taking into account the minimal inhibitory concentration (MIC) of this antibiotic against B. subtilis, 4 µg/ml [START_REF] Adimpong | Antimicrobial susceptibility of Bacillus strains isolated from primary starters for african traditional bread production and characterization of the bacitracin operon and bacitracin biosynthesis[END_REF].

The results obtained with erythromycin (Figure 47) are similar to that observed with vancomycin. In the absence of erythromycin, the BSB1, ∆sppA and ∆yteJ strains showed identical growth rates.

However, when we started to treat with 0.1 µg of erythromycin (Figure 47, graph top right) strains did not grow, meaning that at this concentration the antibiotic have a bactericidal effect. All the concentrations used of erythromycin showed the same results. Furthermore, for all concentrations of erythromycin used so far, the WT strain started to grow after 600 min, reaching an OD600 of 0.1.

Comparing these results with the results obtained with the lantibiotics, we concluded that the deletion of sppA or yteJ do not affects de resistance to erythromycin. SppA is a serine protease which is, according to [START_REF] Nam | Crystal structure of Bacillus subtilis signal peptide peptidase A[END_REF], capable of digesting fully folded proteins. Therefore, we decided to test if the SppA/YteJ complex could be involved in a mechanism that protects the cell against the activity of lysozyme, glycoside hydrolase that is used as an antimicrobial enzyme due to its capacity to digest peptidoglycan, the major component of the Gram-positive bacterial cell wall. The experiment was performed as previously described, testing different concentrations of lysozyme, from 0 to 250 µg/ml. The lysozyme results are shown in Figure 48. Whatever the concentrations of lysozyme added in the medium, no marked differences were observed between the BSB1 and the ∆sppA and ∆yteJ strains. (At 150 µg/ml lysozyme, the ∆yteJ strain took slightly longer to reach OD600 0.1 compared to the BSB1 and ∆sppA strain. As the results obtained for vancomycin and erythromycin, it seems that SppA/YteJ complex does not protect the cells against the lysozyme action. As a general conclusion about the role of SppA, we have confirmed that sppA is important in the lantibiotic resistance mechanism; however the protecting effect of SppA against lantibiotics can not be extended to any other antibiotic molecules that we have tested. The vancomycin peptide structure (a tricyclic glycopeptide) might prevent SppA to degrade it; and SppA protease activity seems to be unable to degrade the fully folded lysozyme. At this stage of the study, the role of YteJ in the complex is more difficult to understand as, depending on the concentrations of lantibiotic used, the positive or negative effect of the deletion of yteJ could be observed.

Overexpression of sppA results in the recovery of the BSB1 phenotype

in the presence of subtilin.

To check if the higher susceptibility to lantibiotics was specifically due to the deletion of sppA and yteJ we constructed strains overexpressing these two genes, either individually or as an operon.

The overexpressing strains were constructed by re-inserting, into the mutant genetic background, each gene or the full operon at the amyE locus under the control of the hyperspank promoter, an IPTG inducible promoter. The final strains were named as GHBs 061 (BSB1 ∆yteJ::cat amyE::Phs yteJ /spec), GHBs 067 (BSB1 ∆sppA::cat amyE:: Phs sppA /spec) and GHBs 072 (BSB1 ∆sppA yteJ::cat amyE::Phs sppA yteJ /spec). For the sake of clarity, strains GHBs 061, 067 and 072 will be named PhsyteJ, PhssppA and PhssppA yteJ, respectively.

Cells were grown in 96 well plates, over 16 hours in the presence of 30% subtilin and different concentrations of IPTG, and the optical density at 600 nm (OD600nm) was measured at regular intervals.

The BSB1 strain was used as the control strain. Overnight cultures of the BSB1 and PhsyteJ, PhssppA

and Phs sppA yteJ deletion mutant strains were grown at 37ᵒC with aeration and vigorous shaking in flasks. BSB1 in LB and the overexpressing mutants in LB supplemented with 5 µg/ml Cm and 100 µg/ml Spec. The overnight cultures were diluted 20-fold into 100 µl (in the 96 well plate) of fresh pre-warmed LB medium and grown until the beginning of the exponential phase. Then the cells were re-diluted 400-fold into a new plate in the following conditions: LB as control, LB supplemented with 30% subtilin and LB supplemented with 30% subtilin and different concentrations of IPTG.

The Figure 49 shows the results for the overexpression of sppA and yteJ. In LB (0% subtilin 0 µM IPTG), the BSB1, PhsyteJ and PhssppA strains showed identical growth rates. In the presence of 30% subtilin and 0 µM IPTG, the PhssppA (no SppA is produced) showed a longer lag phase, around 100 extra minutes, compared to BSB1 and PhsyteJ. When 10 µM of IPTG was added, inducing the overexpression of sppA and yteJ, we could observe that the lag phase of strain PhssppA decreased.

Increasing the concentrations of IPTG to 25 and 50 µM induced an even shorter lag phase for PhssppA strain. The PhssppA resistance to 30 % subtilin was almost recovered at 50µM ITPG. With 50µM of IPTG, the PhsyteJ showed a very slight increase in the resistance to 30% subtilin, as compared to the BSB1 strain. However, when induced with 100 µM, the three strains first grew similarly but then the high overexpression of sppA rapidly led to cell lysis in the stationary phase.

With these results, we can conclude that the deletion of sppA affects the resistance to subtilin directly through the enzymatic activity of SppA, as the complementation with sppA overexpression allowed the recovery of the subtilin resistance. However, a new phenomenon appeared with the early cell lysis observed when 100 µM IPTG were used to induce sppA. We also tested the overexpression of the operon, PhssppA yteJ ( Figure 50). From 10 up to 100 µM IPTG, the subtilin resistance recovery of the PhssppA yteJ strain was complete with the lowest concentration of IPTG (10 µM). However, the most remarkable result was the fact that in the presence of IPTG the overexpression of the sppA yteJ operon showed a shorter lag phase than the BSB1 strain. The PhssppA yteJ cells seemed to recover quicker from subtilin action than the WT strain. These results reinforced the idea that YteJ may have a role in the regulation of SppA, as when they were overexpressed together, the cells were more efficient to recover from subtilin action. Moreover, the results brought another hint in the relation between the two proteins. As the premature cell lysis, that was clearly observed when SppA was overexpressed alone, was not observed when YteJ was co-expressed with SppA, we hypothesized that YteJ might negatively regulate the activity of SppA, preventing its deleterious action.

The overexpression of sppA results in a change of the cell morphology.

The overexpression of sppA led to premature cell lysis. As the effect was drastic, we checked by microscopy if the overexpression of sppA, yteJ or sppA yteJ would affect the morphology of the cells. The cells were grown overnight at 37 ᵒC with aeration and vigorous shaking in flasks, BSB1 was inoculated into fresh LB media and the overexpression mutants were inoculated into LB medium supplemented with 100 μg/ml Spec and 5 µg/ml Cm. Overnight cultures were diluted 400-fold into fresh pre-warmed LB media without any supplements. Biomass (OD600) was measured at regular intervals and samples were collected during the early stationary (stat) phase of the growth cycle and observed by microscopy. The early stationary phase was chosen as, when induced with 100 µM IPTG, at this growth stage it is possible to observed a growth defect for PhssppA cells (see Figure 49). Cells were stained with the membrane dye FM4-64, allowing the observation of the cell membranes and septum, and with DAPI, allowing the observation of chromosomal segregation. The cells of the BSB1 strain (Figure 51-first line on the panel) appeared to be normal B. subtilis rod-shaped growing cells, 4-10 μm long [START_REF] Yu | Monitoring bacterial growth using tunable resistive pulse sensing with a pore-based technique[END_REF], well-defined division septa, cell poles and well segregated chromosomes.

The overexpression of sppA affected cell morphology (second line Figure 51). The PhssppA cells were longer than the BSB1 cells, with septal formation problems and an abnormal chromosomal segregation, as the cells did not divide equally and the chromosomal DNA was not well condensed in the middle of the cell. On other hand, the PhsyteJ and PhssppA yteJ cells appeared as normal B. subtilis rod-shaped cells with a well-defined division septa, cell poles and well segregated chromosomes. We concluded that the presence of a high amount of SppA leads to a morphological and division defect.

However, when overexpressed together with YteJ, it seems that YteJ somehow counteracted the morphological defect caused by the overexpression of SppA.

The observed phenotype, "elongated/early lysing cells" of the PhssppA strain, could be the result of an membrane overloading by the overexpressed membrane protein SppA itself or by its protease activity. To discriminate between these two hypotheses, we constructed an inactive form of SppA and inserted it at the amyE locus under the control of the hyperspank promoter (IPTG inducible). The active site of SppA is composed of a catalytic dyad, between a serine (S147) of one SppA protomer and a lysine (K199) of the adjacent protomer. To obtain an inactive form of SppA, we mutated the codon for the Lys199 changing it to a codon for alanine (K199A), as described in [START_REF] Nam | Crystal structure of Bacillus subtilis signal peptide peptidase A[END_REF]. The final strain was named GHBs 088 (BSB1 amyE::PhssppA K199A /spec), and to simplify Phs sppA K199A .

Figure 52 shows the cell morphology expected for BSB1 without (Figure 52A) and with (Figure 52B) IPTG: normal B.subtilis rod-shaped cells, while Phs sppA showed damaged long cells.

Interestingly, Phs sppA K199A (Figure 52B) cells did not show a cell growth defect but the normal cell morphology observed for the BSB1 strain. These results allowed us to conclude that the cell morphology defect is not a consequence of overexpressing a membrane protein but that the morphology defect was due to the protease activity of SppA.

SppA and YteJ cell localization.

To better characterize the SppA/YteJ complex, we tried to study the cell localization of these two proteins. We constructed two strains expressing a translation fusion of each gene, sppA and yteJ, with sfGFP, by insertion at the amyE locus under the control of the hyperspank promoter. These constructs are described in the material and methods section 4.3.4. These constructs were confirmed by PCR and sequencing. The final strains are GHBs 064 (BSB1 amyE::PhsyteJ-sfgfp /spec), GHBs 069 (BSB1 ∆sppA::cat amyE::Phs sfgfp-sppA /spec). For the sake of clarity, strains GHBs 064 and 069 will be named YteJ-sfGFP and sfGFP-SppA, respectively.

In Figure 53, for both strains YteJ-sfGFP and sfGFP-SppA, the sfGFP signal was found mostly at the cell septum. However, both proteins showed a cytoplasmic signal too, perhaps due to problems of translocation of the fused proteins. sfGFP-SppA was observed in foci around the cell membrane or poles. These results indicated that this protein complex was possibly located at the cell septum, but our observations did not allow us to conclude with a high level of confidence.

SppA and YteJ in vitro purification.

To analyse the role of YteJ in the complex and the SppA protease activity in vitro, we produced and purified B. subtilis SppA, YteJ and SppA/YteJ (from an artificial operon) from E. coli.

For that, from the pJ411 plasmid, we constructed plasmids harbouring a copy of each gene or the full operon fused to a His-tag sequence, under the control of the IPTG-inducible pT7 promoter, and a kanamycin resistance cassette. These plasmids were used to transform E. coli. The final genes sequences on these plasmids were confirmed by PCR and sequencing. The his-tagged proteins were purified as described on the section 4.9.6 of material and method. To simplify, we will refer to the histagged purified proteins as SppA, YteJ and SppA/YteJ.

Purifying active membrane proteins is a challenging task, as it means extracting proteins from the membrane maintaining its native environment to allow the proteins to preserve its fully folded conformation. This process can destabilize the protein structure impairing its activity. A strategy to avoid this problem and study a membrane protein activity is to purify the protein without its transmembrane domains, transforming the membrane protein in a soluble protein that can be recovered from the cell cytoplasm. This strategy was used by [START_REF] Nam | Crystal structure of Bacillus subtilis signal peptide peptidase A[END_REF], which allowed to obtain the crystallographic structure of SppA and discover that this protein is able to digest fully folded proteins [START_REF] Nam | Crystal structure of Bacillus subtilis signal peptide peptidase A[END_REF][START_REF] Nam | Structure of Signal Peptide Peptidase A with C -Termini bound in the active sites: insights into specificity, self-processing, and regulation[END_REF]. As such, we constructed and purified, as described by [START_REF] Nam | Crystal structure of Bacillus subtilis signal peptide peptidase A[END_REF] SppA truncated of its transmembrane domain, the first 52 amino acids from SppA N-terminus. This protein was overexpressed and purified from E. coli cytoplasm and the protein was named SppA ∆1-52 . In this study, we used SppA ∆1-52 as a control for SppA activity. The purified SppA ∆1-52 (lane 2, Figure 55A) showed an expected band at around 28 kDa. In the case of the full length SppA (lane 3), we observed at least three bands, one at 36 kDa which was expected as the theoretical molecular weigth (MW) is 36. YteJ-His (lowest band ≈20 kDa), indicating that these two proteins formed a membrane protein complex even when produced in E.coli.. Comparing with the full length SppA sample (lane 3), the SppA in the SppA/YteJ complex (lane 4)i.e. pulled down with the YteJ-His, was mainly observed as a single band. This single band corresponded to the highest band of SppA full length (≈36 kDa). In addition, all the full length proteins, purified from the E. coli membrane solubilized with DDM showed additional faint bands that corresponded to contaminants.

SppA has been described to form an octomer [START_REF] Nam | Crystal structure of Bacillus subtilis signal peptide peptidase A[END_REF], a structure obtained by crystallography of SppA ∆1-52 . We wondered whether this octomeric structure could also be that of the full length membrane SppA. The same question applied toYteJ. To get rid of contaminants we performed a second setp of purification: a gel filtration using a Superdex 200 HR 10/300 GL columnthat allows the separation of proteins from 10 up to 600 kDa. The curves and SDS-PAGE gels

showing the gel filtration results can be found Figure S 2. These results showed that SppA possibly forms an octomer around 300 kDa, as described in the literature, and YteJ may form an octomer around 160 kDa.

When loaded on the gel filtration column, the complex SppA/YteJ dissociated as the two proteins were eluted separately from the column, as two separated octomers. This result indicated that, in the conditions used for the gel filtration experiment, the individual complex of each protein, SppA and YteJ, can maintain their structure complex. However, the interaction between the two YteJ and SppA octomers appeared to be weaker than the intra-protein interactions. One possible explanation for this weakness is that the complex SppA/YteJ could be made of two subunits, each subunit is an octomer of each protein, and that the dissociation between the subunits can occur. Analysis of the purified samples on 12.5% SDS-PAGE gels, coomassie blue stained. For both gels, A and B, the first lane corresponds to protein markers, which the bands correspondent to 40, 30, 25 and 20 kDa are highlighted.

SppA can digest fully folded proteins or not

In 2012, Nam and its colleagues showed that the active soluble SppA was able to digest fully folded proteins, based on one example, the E.coli lipo-protein BamD [START_REF] Nam | Crystal structure of Bacillus subtilis signal peptide peptidase A[END_REF].

To test the SppA ∆1-52 activity we mixed it with different proteins, available in our laboratory, and incubated overnight at 37°C in the same buffer conditions as described by [START_REF] Nam | Crystal structure of Bacillus subtilis signal peptide peptidase A[END_REF] Interestingly, SppA ∆1-52 was not able to digest the bovine serum albumin protein (BSA) and lysozyme (Figure 56A lanes 2, 3, 6 and 7, respectively), as the band intensities remained the same for the BSA or lysozyme with or without the presence of SppA ∆1-52 . However, in the case of GFP we can see a shift on the size of the core band when treated with SppA ∆1-52 . This GFP, hereafter called GFP -ext , was modified by the addition of a around 3 kDa C-terminal tail (24 amino acids, usually used as a linker for GFP fusion). The shift on the band size corresponded to the digestion of this 3 kDa tail, the core of the GFP remaining intact. We then tested different proteins, four are shown on Figure 56B. SppA ∆1-52 could not digest the E. coli DNA single strand binding protein (SSB) (lane 3). When treated with SppA ∆1-52 , the bands corresponding to the following proteins disappeared: B. subtilis nucleoid associated protein (Rok) and E. coli DNA-binding protein (Ec-HNS). These two proteins were completely digested (Figure 56B, lane 5 and 7), therefore we aslo proved that SppA can digest fully folded proteins. However, the B. subtilis catabolite control protein A (CcpA), when mixed overnight with SppA, disappeared as a whole protein leaving a lower MW band still present. This is an example of a partial digestion of a protein by SppA ∆1-52 (lane 9). These results allowed us to conclude that, as described in the literature, SppA ∆1-52 can digest fully folded proteins. However, as the digestion was sometimes not possible for some substrates, occured sometimes only partially for others, it is highly probable that the protease activity of SppA ∆1-52 .depends on the structure of the substrate.

YteJ inhibits SppA activity

Based on the previous results we decided to use the GFP containing C-terminal unstructured tail as substrate to study the protease activity of SppA as well as SppA in the SppA/YteJ complex.

Their activity were tested overnight at 37°C (Figure 57A). SppA ∆1-52 and SppA were able to digest the substrate, as a MW downshift of the GFP ext was observed. However, when in complex with YteJ and mixed with the GFP ext substrate, SppA did not completely digest the substrate, as two bands corresponding to both, GFP and GFP ext , were visible on the gel. Altogether these results revealed the negative effect of the presence of YteJ on the activity of SppA.

As it seemed that the activity of SppA was slower when SppA was in complex with YteJ, we performed more precise kinetic experiments following the protease activity of the different forms of SppA by taking samples at intervals of one hour at 37°C. As the Bradford assay to determine the concentration of SppA was not very accurate with membrane proteins, we chose to estimate the quantities of SppA for our "different" SppAs on SDS-PAGE. The band intensities were used to adjust the quantity of SppAs mixed with the GFP ext substrate. In Figure 57B and C, after 15 min SppA ∆1-52 was able to digest almost 100% of the substrate, while SppA was only able to digest 50% of the substrate after 60 min. The protease activity of the full length membrane SppA appeared therefore lower than that of the soluble SppA. The presence of the transmembrane domain of SppA might be involved in the regulation of SppA activity. For one hour, in complex with YteJ, SppA was not able to digest the substrate. This result indicated that YteJ may act as a negative regulator of SppA activity.

An active SppA is only purified without its C-terminal, that the enzyme self-processes as described in [START_REF] Nam | Structure of Signal Peptide Peptidase A with C -Termini bound in the active sites: insights into specificity, self-processing, and regulation[END_REF]. The authors also showed that an inactive SppA, SppA K199A , was purified with its unprocessed C-terminal domain.

On our SDS-PAGE analysis, SppA appeared as three bands (lane 2, Figure 58A), while the inactive SppA K199A appeared as a single band, whose MW was higher than that of the active SppA (lane 3). When SppA is expressed together with YteJ, we observed a band for SppA which was higher than active form of SppA but slightly lower than the SppA K199A inactive form. The hypothesis was that:

1) when active SppA is expressed and purified alone, it can autocatalitycally remove its own C-terminal domain and appeared as multiple bands on gel (different stages of self-processing of the Cterminus)

2) SppA in the SppA/YteJ complex probably still contains its C-terminus and appears as a higher MW band comparable to that of the inactive SppA K119A .

To confirm this hypothesis we performed a MS/MS analysis of the major bands for SppA and SppA+YteJ shown on Figure 58A. The results showed that (i) the 3 bands of lane 2 were SppA, although the peptides from the C-terminal could not be detected (ii) for SppA purified in complex with

The C-terminal domain of YteJ is involved in the regulation of SppA activity

Following the previous results, we wanted to understand how YteJ is able to inhibit the SppA activity in vivo. To try to understand which domains of YteJ blocks SppA activity we used the morphological defective phenotype, caused by the overexpression of SppA, as a tool. With that objective we have constructed new mutant strains overexpressing sppA yteJ, with different truncated versions of YteJ and analyzed their cell morphology and compared with Phs sppA strain (Figure 59 A).

Therefore, when overexpressing these constructs if YteJ is still able to inhibit SppA we should expect normal cell like BSB1 wild type strains, by other hand if we truncate the domain that blocks SppA we should expect to see long filamentous cells with division defects. Based in its secondary protein structure, the C-terminal of YteJ was deleted with three different lengths. In the first strain, YteJ was truncated from its short C-terminal α-helix corresponding to the last twelve amino acids (Figure 59 B). Secondly, we have truncated YteJ from thirty five amino-acids from its C-terminal region (Figure 59C). At the last construct we have deleted the full C-terminal domain and substituted by the SPA tag (Figure 59 D). These constructs were re-inserted at the amyE ectopic locus, into the BSB1 wild type background, under the control of the hyperspank promoter, an IPTG inducible promoter. These constructions included their natural RBS and a spectinomycin (Spec) resistance cassette. These constructs were confirmed by PCR and sequencing. The final strains are GHBs 085 (BSB1 amyE::Phyper-spank sppA yteJ ∆129-164 spec), GHBs 087 (BSB1 amyE::Phyper-spank sppA yteJ ∆152-164 spec), GHBs 088 (BSB1 amyE::Phyper-spank sppA yteJ ∆92-164 SPA spec). In order to simplify the understanding, from here, the strains GHBs 085, 087 and 088 will be named Phs(sppA-yteJ ∆129-164 ),

Phs(sppA-yteJ ∆152-164 ) and Phs(sppA-yteJ ∆92-164 SPA), respectively. Overnight cultures were diluted 400-fold into fresh pre-warmed LB media without any supplements. Biomass (OD600) was measured at regular intervals and samples were collected during the early stationary (stat) phase of the growth cycle and observed by microscopy, as described at the previous section. Cells were stained with the membrane dye FM4-64, allowing the observation of the cell membranes and septum, and with DAPI, allowing the observation of chromosomal segregation.

The cells of the BSB1 strain (Figure 60-first line on the panel) appeared to be normal B. subtilis rodshaped cells, well-defined division septa, cell poles and well segregated chromosomes. The overexpression of sppA affected the cell morphology creating elongated cells, as the observed in our previous results (second line Figure 60). Surprisingly, when we overexpressed sppA-yteJ ∆152-164 (fourth line Figure 60), YteJ with the short truncation at its C-terminal, we could observe an effect in the cell morphology. Even though the cells were not so long as PhssppA cells, the cell were longer than the BSB1 cells, with an evident septa mal formation and an abnormal chromosomal segregation.

Furthermore, the overexpressed sppA-yteJ∆129-164 (fifth line

SppA digests subtilin

Our previous results showed that SppA ∆1-52 can digest fully folded proteins, depending on the structure of the substrate. In this chapter of results we have also demonstrated that SppA have an important role in the lantibiotic resistance mechanism. Assuming that the lantibiotics are peptides, we were wondering if in this resistance mechanism SppA is directly digesting the lantibiotics. As such we used the previous purified and active SppA ∆1-52 to test our hypothesis. SppA ∆1-52 was incubated overnight in a subtilin-containing reaction buffer (20 mM Tris-HCl pH7.5, 200mM NaCl) at 37°C, as well as the controls, SppA ∆1-52 alone in the reaction buffer, subtilin solution and a positive control with proteinase K. Proteinase K is a broad range serine protease, the same family that SppA (MORIHARA and TSUZUK 1975). Then these compounds were tested with the wild type BSB1 and ΔsppA strain. The experiment was done as described at section. At Figure 61, the first graph top left of the panel corresponds to the controls, i.e. the BSB1 and ∆sppA mutant strain grown in LB without subtilin. The two strains showed identical growth rates. When treated with 30% subtilin (graph top right), the growth of the strains was affected, with a visible difference in the resistance to subtilin between the BSB1 and the ∆sppA. ∆sppA took longer to recover its growth and reach an OD600 of 0.1. The subtilin was stable and did not degrade overnight at 37°C, as it induced the same type of growth delay (graph middle left), than the fresh subtilin used. When the subtilin was treated overnight with the SppA ∆1-52 , we can observe that the growth is similar to the control without subtilin (graph top left), without differences between the strains. The same was obtained for with the subtilin treated with proteinase K.

With these results we can conclude that the subtilin was degraded by both SppA ∆1-52 and proteinase K.

Therefore, we can hypothesize that, in vivo, the direct degradation of subtilin by SppA is part of the mechanism of action against lantibiotics activity.

DISCUSSION AND PERSPECTIVES 3 DISCUSSION AND PERSPECTIVES

The ability to transport newly synthesized proteins across and into the bacterial cytoplasmic membrane is an essential mechanism to maintain cell integrity. This transportation mechanism involves universally conserved protein-conducting machineries that can transport a wide variety of potential substrates. For example, the Sec translocon mediates the translocation of secretory and membrane proteins in an unfolded conformation. The SecYEG, the core channel of the bacterial Sec translocon, is the minimal membrane-embedded unit required for protein transport [START_REF] Collinson | The structure of the bacterial protein translocation complex SecYEG[END_REF].

However, the full translocation process from the recognition of the pre-protein, its translocation and final maturation involve a dynamic between the protein-channel and associated proteins, which interact and work together (de [START_REF] Keyzer | The Bacterial Translocase: A dynamic protein channel complex[END_REF]. Although the mechanisms involved in the Sec pathway has been extensively studied, the knowledge about B. subtilis protein complexes is limited.

The possible protein-protein interactions within the B. subtilis Sec pathway were studied and will be discussed in section 3.1. As SppA was initially identified as an accessory protein involved in the Sec pathway, the section 3.2 will focus on its interaction with YteJ, its role and activity in the cell.

Protein interactions of the B. subtilis Sec pathway

In E. coli, it has been established that the Sec holo-translocon is a supramolecular complex composed of the membrane protein complexes, SecYEG (Sec translocon), SecD/SecF, YajC and YidC [START_REF] Schulze | Membrane protein insertion and proton-motiveforce-dependent secretion through the bacterial holo-translocon SecYEG-SecDF-YajC-YidC[END_REF][START_REF] Corey | Unlocking the bacterial SecY translocon article unlocking the bacterial secy translocon structure[END_REF]Botte et al. 2016). Based on the multiple interactions found in E. coli, we intended to study these interactions in B. subtilis.

Our experiments of sequential peptide affinity purifications with the SPA-tagged proteins SecDF and SpoIIIJ in B. subtilis showed that the B. subtilis SecDF, homologous to a fusion of the E.

coli SecD and SecF, and B. subtilis SpoIIIJ, homologous to E. coli YidC, did not co-purify. According to these results, it seems that SecDF and SpoIIIJ do not form a stable complex in B. subtilis, unlike what was shown in E. coli. However, the absence of co-purification of B. subtilis SecDF and SpoIIIJ does not completely rule out the possibility that these two proteins interact. Their interaction can be weaker than that in E. coli, and might have been abolished by the use of a too high concentration of detergent, the n-dodecyl β-D-maltoside (DDM). In our procedure, we classically used a DDM concentration of 1% which might affect protein interactions, although 2% DDM concentration was used in E. coli to solubilize the membranes. We therefore tested a range of different DDM concentrations, from 0.5% to 2%, and also reduced the purification steps to only one affinity column to avoid losing some protein partners. Analysed by LC-MS/MS, the results of these experiments showed that the SpoIIIJ did not co-purified with the SecDF-SPA protein. However, in the reciprocal experiment, after affinity purification of SpoIIIJ-SPA, low amounts of SecDF were detected regardless of the DDM concentrations. This suggested that B. subtilis SecDF and SpoIIIJ may eventually interact.

If so, this interaction might be rather weak as the addition of a second affinity column had previously abolished it. Moreover, as this result was only obtained when the SPA tag was added to SpoIIIJ, it also suggests that the SPA tag fused to SecDF might have precluded the interaction. However, in conclusion, if B. subtilis SecDF and SpoIIIJ interact, these two proteins do not form a stable complex like in E. coli.

Unfortunately, in our attempt to study the translocon SecYEG, B. subtilis strains expressing SecY-SPA and SecE-SPA could not be obtained. Therefore we analysed only B. subtilis SecG-SPA by tandem affinity purification. It turned out that SecG-SPA did not co-purify with the expected SecY or SecE, nor SecDF, unlike what had been observed in E. coli. Again, we cannot rule out the possibility that the SPA tag fused to SecG have induced a dissociation of the other proteins, SecY and SecE, or weakened their interactions. However, it is highly unlikely as the translocon, SecYEG, is essential to the cell and as any defect in the formation of this complex should harm the cell. Our result rather reflects the fact that the translocon, SecYEG, or the holotranslocon, SecYEG-SecDF-SpoIIIJ, might not be a very stable protein complex in B. subtilis.

Combined together, these results suggest that B. subtilis do not possess a stable holotranslocon as the E. coli SecYEG-SecDF-YajC-YidC supracomplex. In E. coli the existence of this holo-translocon as a supracomplex was shown by different methods including BN-PAGE and the direct interactions within the proteins forming the holotranslocon were further refined by crosslinking experiments [START_REF] Schulze | Membrane protein insertion and proton-motiveforce-dependent secretion through the bacterial holo-translocon SecYEG-SecDF-YajC-YidC[END_REF][START_REF] Corey | Unlocking the bacterial SecY translocon article unlocking the bacterial secy translocon structure[END_REF]. To study the "holotranslocon" in B. subtilis, cross-linkers should be used and added to the cells before purification of any of the translocon components.

After translocation, the secretory proteins are released from the membrane and undergo a maturation process carried out by other accessory proteins to the Sec machinery, as for example the thiol-disulfide oxido-reductases (Bdb proteins), which during protein folding, are responsible for rearranging incorrect disulfide bonds [START_REF] Tjalsma | Proteomics of protein secretion by Bacillus subtilis: separating the 'secrets' of the secretome[END_REF][START_REF] Driessen | Protein translocation across the bacterial cytoplasmic membrane[END_REF]. Structural studies showed that the E. coli thiol-disulfide oxido-reductases DsbC and DsbD form a complex.

DsbD binds to the central cleft of the V-shaped DsbC dimer causing a shift into a close conformation and it activates DsbC (Fabianek et al. 2000;Haebel et al. 2002). We also investigated this connection between the B. subtilis BdbB and BdbD, two thiol-disulfide oxido-reductases homologous to E. coli DsbC and DsbD. We did not find evidences that these two proteins form a complex either, as our results showed that, when SPA-tagged, BdbB or BdbD did not co-purify with each other.

SppA/YteJ membrane protein complex

As shown in Chapter II of the results, we have found evidences of a stable complex between the B. subtilis SppA, the signal peptide peptidase, and the unknown membrane protein YteJ.

The first evidence for the existence of this complex was obtained by BN-PAGE experiments, which showed that these two proteins co-migrated together, suggesting that they might interact. The tandem affinity purification of YteJ-SPA showed that SppA was co-purified with YteJ-SPA, indicating again that these two proteins interact and form a stable complex. For the in vitro study, when the his6-sppA yteJ operon was expressed in E. coli, the purification of the his-tagged SppA clearly showed the presence of YteJ, reinforcing the idea that a membrane protein complex made of B.

subtilis SppA and YteJ can be formed in E. coli.

The existence of this complex in the cytoplasmic membrane of B. subtilis prompted us to study whether YteJ could play a role in this complex. I will discuss our results in the following order:

1. the role of SppA in protein secretion ?

2. the role of SppA (and YteJ) in the resistance to nisin, subtilin and LP9

3.

the protease activity of SppA is regulated by YteJ 4. SppA/YteJ, a complex involved in cell division ?

5.

YteJ, another function?

Role of SppA and YteJ in protein secretion:

In 1999, Bolhuis et al. observed that the deletion of sppA induced a slight decrease in the secretion of mature secretory proteins, and more importantly a delay in the maturation process of the hypersecreted protein AmyQ. As SppA was believed to be involved in the degradation of the signal peptides of the secreted proteins, this result was interpreted as a consequence of the presence of remnant signal peptides in the membrane, that would cause a slow-down of the processing of the preproteins under hyper-secreting conditions. Here, in this study, we have shown that the deletion of sppA and yteJ do not affect the secretion of a highly secreted protein, AmyM. However, [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF] used pulse chase experiments to show the maturation delay of the hypersecreted AmyQ.

Therefore, our deletion strains were also tested by pulse chase experiments (see The results obtained with the SppA, or YteJ overexpression strains, overexpressing, showed a negative effect on the AmyM secretion level when SppA is overexpressed, while the overexpression of YteJ, or SppA and YteJ together, did not lead to any difference. This indicated that, SppA, able to degrade secreted proteins, could be detrimental when overexpressed for the production of secreted proteins. Our result is in complete disagreement with previous results in Bacillus licheniformis for which the overexpression of SppA proved to be beneficial for the secretion levels of nattokinase and AmyL [START_REF] Cai | A novel strategy to improve protein secretion via overexpression of the sppa signal peptide peptidase in Bacillus Licheniformis[END_REF]. In this study, sppA was under the control of the constitutive promoter P43, a promoter which is thought to be active during the lag and exponential phase of growth but not during the stationary phase. In our study, we used the promoter Phs induced by the addition of IPTG, therefore SppA was expressed, regardless of the growth phase. This might explain the difference of results between the two studies, as in both cases, the extracellular secreted protein contents were assessed in late stationary phase. In conclusion, the role of SppA in protein secretion appeared to be quite limited as worst negative effects of 20 % or best positive effects of 60% in the protein secretion levels were reported [START_REF] Bolhuis | Signal peptide peptidase-and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins[END_REF]) [START_REF] Cai | A novel strategy to improve protein secretion via overexpression of the sppa signal peptide peptidase in Bacillus Licheniformis[END_REF], as opposed to the positive effects of 250-1000 % obtained after the overexpression of RasP [START_REF] Neef | Intramembrane protease RasP boosts protein production in Bacillus[END_REF].

Role in the resistance to nisin, subtilin and LP9

The role of SppA was more obvious in the resistance to nisin, subtilin and LP9 as the deletion of sppA induced a higher sensitivity of the cells to these antibiotic peptides. These results were in agreement with previous observations that showed that sppA, a member of the σ W regulon, was involved in the lantibiotic resistance (Kingston, Liao, and Helmann 2013b).

Our experiments with the sppA complementation strain confirmed the direct role of SppA in this resistance as the complemented cells showed a lantiobic or LP9 resistance identical to the wildtype cells. We also tested the direct digestion of subtilin by SppA by incubating the purified SppA in a subtilin-containing LB medium for 12 hours at 37°C, and we observed that the ΔsppA cells grew as well as the wild-type cells in this SppA-treated medium (Figure 61).

The role of YteJ appeared more complicated to understand with the antibiotic peptide experiments. The observed phenotypes of the ΔyteJ cells showed either a slightly higher sensitivity to the antibiotic peptides than the wild-type cells, or a greater resistance. It seemed to be dependent on the concentrations of the peptide used. If the resistance to the lantibiotic peptides or LP9 is only due to the protease activity of SppA, the ΔyteJ phenotype might be explained by the SppA regulatory role of YteJ. We showed that YteJ negatively regulates the activity of SppA; however, YteJ has to release its inhibitory action on SppA, presumably by responding to a signal, induced by the antibiotic peptides. In the absence of YteJ, the activity of SppA might not be timely regulated, hence the lower antibiotic resistance. This hypothesis could not account for the higher antibiotic resistance observed in ΔyteJ cells in some cases.

Regulation of the sppA yteJ operon

In parallel with the antibiotic resistance study, we also studied the transcription regulation of the operon sppA yteJ. In [START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF], a second promoter, σ A -dependent, was detected upstream of the σ W -dependent promoter. Our experiments using sGFP fusions with one or potentially two promoters showed that the major promoter was the σ W -dependent promoter. As such, we confirmed that sppA and yteJ are part of the σ W regulon, a regulon that is expressed when cells are subjected to an envelope or cell wall stress. Interestingly, the operon structure of these two genes, sppA and yteJ, is well conserved across the bacilli class, which includes Bacillus subtilis but also Listeria monocytogenes.

3.2.4 SppA/YteJ, a complex involved in quality control of cell division?

As shown in the chapter II of the results, we observed that high levels of expression of SppA create a severe cell growth defect, giving rise to a filamentous phenotype (see Figure 51). This phenotype could be due to the overexpression of a membrane protein. The overexpression of membrane proteins and proteases normally result in the accumulation of cytoplasmic aggregates containing the overexpressed proteins, accumulation of chaperones (e.g. DnaK), soluble proteases (e.g.

ClpXP) or precursors of secreted and membrane proteins (Wagner et al., 2007). To discard this hypothesis, we constructed a strain that overexpressed an inactive form of SppA, the SppA K199A . The analysis of the morphology of the cells showed a normal cell morphology (see Figure 52). These results allowed us to conclude that the activity of SppA is responsible for the cell growth defect. In J. [START_REF] Wu | Coordination of cell division and chromosome segregation by a nucleoid occlusion protein in bacillus subtilis[END_REF]. It was shown too that the B. subtilis minJ deletion mutant, comparing with the wild type strain, grew as long filaments of cells with sporadic septa (Figure 62), as we observed in our mutants [START_REF] Patrick | MinJ (YvjD) is a topological determinant of cell division in Bacillus subtilis[END_REF]. However, in both cases the segregation of the chromosomal DNA was not affected, as it is possible to observe a well divided nucleoids on Figure 62, the contrary of our results, that showed long filamentous cells with a unique long nucleoid (Figure 51). Other types of proteins could be the targets of an overexpressed, active and unregulated SppA, as for example the penicillin binding proteins (Pbp). Mutants of some Pbps show similar phenotype of elongated cells as well (Figure 63). In conclusion, the clear cell morphology phenotype defect observed in a strain overexpressing an active SppA points out that proteins involved in the division process or cell wall synthesis are affected. However, no specific substrate for SppA can be inferred from this observed phenotype, as too many proteins can be involved. 123

3.2.5

The protease activity of SppA and its regulation by YteJ 3.2.5.1 The protease activity itself: [START_REF] Nam | Crystal structure of Bacillus subtilis signal peptide peptidase A[END_REF] performed in vitro studies with a soluble form, truncated B. subtilis SppA Δ2- 52 , deleted from its transmembrane domain (amino acids from position 2 to 52). These authors showed that SppA ∆2-54 could digest signal peptides as well as fully folded proteins. Their conclusion was based on their assay of a successful overnight digestion of a fully folded E. coli lipoprotein (BamD) [START_REF] Nam | Crystal structure of Bacillus subtilis signal peptide peptidase A[END_REF]. In our study, we have shown that B. subtilis SppA ∆2-54 is not able to digest all fully folded proteins, as we tested SppA activity on seven different proteins available in the lab as shown in Figure 53. Our study of the activity of SppA ∆2-54 with more proteins indicates a new feature about the enzymatic properties of SppA. As some proteins showed a SppA proteolytically-resistant domain, our hypothesis is that some protein structures or specific sequences of amino-acids might have the ability to resist to the proteolytic activity of SppA. To be demonstrated this hypothesis would require a specific experimental setting involving, for example, a synthetic peptide that includes a variety of structures or amino acid sequences.

The complex SppA/YteJ and its activity:

In vitro, a soluble SppA, truncated from its N-terminal transmembrane domain, SppA ∆2-54 , has been crystallized as an octomer [START_REF] Nam | Crystal structure of Bacillus subtilis signal peptide peptidase A[END_REF]. In our hands, the full-length SppA, expressed and purified from the E. coli membrane fraction, is also an octomer. Its partner, B. subtilis YteJ contains two to three transmembrane spanning domains, depending on the prediction structural programs. YteJ was purified as an apparent molecular weight corresponding to an octomer as well.

The results from the two SPA purification experiments showed that the ratio was more probably a 1:1 between SppA and YteJ, which would be in accordance with the fact that the two genes are in operon.

It would imply that the SppA/YteJ complex would be made of two octomers of each protein. Although the two proteins were purified as a complex from the sequential affinity purification, in which each protein was fused to a different tag, the subsequent gel filtration experiment showed a complete dissociation of the two proteins during the chromatography, suggesting a relative weakly interaction between the two octomers. This interaction between multimers of different proteins is reminiscent of the membrane protein complex formed by the protease FtsH and HflK and HflC [START_REF] Ito | Cellular functions , mechanism of action , and regulation of FtsH protease[END_REF]. FtsH is a hexamer with most of its protease domain facing the cytoplasm. HflK and HflC, two membrane proteins that associate first as heterodimers, interact with E. coli FtsH, to form a huge complex of 1 MDa, the FtsH holoenzyme. It was shown that HflKC inhibits the protease activity of FtsH for one of its substrate, SecY [START_REF] Kihara | A protease complex in the Escherichia coli plasma membrane: HflKC (HflA) forms a complex with FtsH (HflB), regulating its proteolytic activity against SecY[END_REF].

We also showed that, in vitro, in the SppA/YteJ complex, the SppA protease activity was strongly diminished when compared to that of SppA alone. In vivo, the overexpression of SppA created morphological defect to the cells, whereas the cell morphology was unchanged compared to the wild-type cells, when YteJ was co-expressed with SppA. Our conclusion is that YteJ is a regulator of the protease activity of SppA. In vivo, we further showed that the last 25 amino acids of the Cterminal domain of YteJ were involved in this inhibition mechanism, and that removing the last 45 amino acids of YteJ led to a completely unregulated SppA. [START_REF] Nam | Structure of Signal Peptide Peptidase A with C -Termini bound in the active sites: insights into specificity, self-processing, and regulation[END_REF] had proposed that an active SppA processes its own C-terminus, and therefore that SppA C-terminus would act as a negative regulator domain, presumably preventing SppA to become active too early during translocation. Our findings provide another level of regulation for SppA through its interaction with YteJ. Our MS data showed that the C-terminal domain of SppA was detectable by mass spectrometry only when SppA was purified as a complex with YteJ. The two peptides belonging to the C-terminal domain of SppA that were detected are highlighted in the Figure 58 are 309 SEIDFLNMR 317 and 318 EILSQSGSPR 327 .

Therefore one could conclude that SppA in the SppA/YteJ complex is maintained in an inactive form by the presence of YteJ, and SppA in the SppA/YteJ complex is unable to self-process its C-terminus as an active SppA would do. However, as the SppA last seven amino acids, i.e. the peptide 328 MMYLYAK 335 , have not been found in our MS analysis, we cannot be certain that SppA, even in the complex SppA/YteJ, has not self-processed at least part of its C-terminus. Noticeably, when SppA was purified, it invariably showed multiple bands on SDS-PAGE, at least 3 visible bands, separated by an apparent molecular weight difference of 3 to 4 kDa, the highest molecular weight band corresponding exactly to the band of SppA in the SppA/YteJ complex (See Figure 58). This suggested that SppA, purified alone from the E. coli membrane fraction, was able to self-process its C-terminus partially, and that this process was slow. On the opposite, [START_REF] Nam | Structure of Signal Peptide Peptidase A with C -Termini bound in the active sites: insights into specificity, self-processing, and regulation[END_REF] noticed that, for the N-terminal transmembrane domain truncated SppA, SppA ∆2-54 , the self-processing of the C-terminus of SppA was probably very fast and complete in E. coli even before the enzyme was purified. It suggests that, in our work, the N-terminal transmembrane domain of the full length SppA may also slow down the self-processing of the C-terminus. Accordingly, the protease activity of the full-length SppA was slower than that of the soluble form, SppA ∆1-52 . E. coli FtsH has been shown to self-process the last seven amino acids of its own C-terminal domain in vivo, that this self-processing was growth phase-dependent, and that the deletion of hflKC slowed down the C-terminus self-processing of FtsH [START_REF] Akiyama | Self-Processing of FtsH and Its Implication for the Cleavage Specificity of This Protease[END_REF]). However, for E. coli FtsH, both full length and processed proteins are active. In the case of B. subtilis SppA,YteJ inhibits SppA by preventing its C-terminus self-processing. By interacting with SppA, YteJ is able to maintain SppA in its inactive form presumably until a physiological harm caused by antibiotic peptides requires the activation of SppA. In this view, SppA activity, that could be detrimental to the cell if not regulatedsee next paragraph -, would act only when necessary.

Finally, this raises the question of the nature of the signal that triggers the activation of SppA and how this happens. For example, the PDZ domain of E. coli DegS acts as an inhibitory domain of DegS until it binds hydrophobic peptides of the C-terminal of misfolded proteins leading to the release of the inhibition [START_REF] Walsh | OMP peptide signals initiate the envelope-stress response by activating degs protease via relief of inhibition mediated by its PDZ domain[END_REF]. This model of activation could be applied to YteJ and SppA, the C-terminal domain of YteJ acting as the inhibitory/activating domain for SppA.

YteJ, another function?

YteJ, by analogy to the PDZ domain for DegS, could be viewed as a sensor of a signal induced when the cell is subjected to a cell wall stress due to antibiotic peptides. The overall YteJ amino-acid sequence analysis shows that the protein belongs to the RDD superfamily, which is characterized by the presence of three highly conserved residues, arginine-aspartate-aspartate. This Therefore one can hypothesize that YteJ might act as a cation antiporter in B. subtilis as in H.

domain
andaensis. In case of salt stress and/or leakage of cations induced by lantibiotic peptides, thought to create holes in the membrane, YteJ would become more active inducing a conformation change in its own structure that, in turn, would induce the release of the YteJ C-terminus inhibitory effect on SppA.

The complex SppA/YteJ would be a two-protein system dedicated to fight the lantibiotic effects. In vivo experimental evidences that B. subtilis YteJ acts as a cation antiporter are needed. Within the B.

subtilis genome, YteJ and its closest related proteins (YxaI, YckC and YczC sharing over 30% identity) share a comparable predicted structure made of three transmembrane domains and these proteins belong to the RDD family; however, none of their genes seem to be in operon with another gene. Therefore, SppA and YteJ might be a unique example of a two-protein system involving a possible cation transporter regulating the activity of a protease. In vitro detailed enzyme kinetic studies combined with structural studies involving mutations in SppA as well as in the C-terminus of YteJ could reveal how this activating mechanism takes place.

Model for the interaction of SppA and YteJ

We propose the following model in which SppA and YteJ formed a membrane protein complex in which the two proteins are arranged as two homoactomers bound to each other (Figure 65). Based on our experimental data, SppA in the SppA/YteJ complex would be inactive because YteJ might interfere with the active site of SppA. In this conformation, SppA could not self-process its own C-terminal domain, a domain that has been shown to block its active site. Upon a signal, whose nature remains to be determined, YteJ, probably undergoing a conformational change, would release its inhibitory effect on SppA that in turn would become active. As lantibiotic peptides are able to create holes in the cytoplasmic membrane and that YteJ amino acid sequence shows homology with a cation antiporter of a distant relative of B. subtilis, the nature of the signal that could be sensed by YteJ could be related to ion homeostasis.

Main results

All of these in vitro and in vivo observations suggest that SppA and YteJ form a strong membrane protein complex. This study established that YteJ plays a key regulatory role in B.

subtilis SppA activity, highlighting a possible new mechanism of a protein regulating a protease. The main conclusions of this work are listed below.

 BN-PAGE experiments showed us that SppA and YteJ co-migrate together in a native gradient SDS-PAGE gel, the first evidence of the protein-protein interactions of these two proteins.

 SppA was co-purified with YteJ-SPA.

 In B.subtilis, the deletion of sppA affects the resistance to antimicrobial peptides, while deleting yteJ reveals an intermediate phenotype. The overexpression of SppA restores the resistance to subtilin.

 The high levels of expression of SppA create a cell growth defect and the presence of YteJ counteracts this effect.

 SppA activity is responsible for the cell morphology defect, as when we overexpressed an inactive version of SppA the cells have a normal phenotype.

 In vitro experiments showed that SppA is able to digest fully folded proteins, depending on their structure.

 YteJ inhibit SppA activity, possibly through its C-terminal region.

MATERIAL AND METHODS

In this chapter, the molecular biology, microbiology, protein purification and microscopy methods used throughout this study are described.

Techniques for DNA manipulation

Oligonucleotide

Oligonucleotides or primers were designed using the Geneious software (Gateway® cloning).

All oligonucleotides, upon arrival, were solubilized in sterilised deionized water and stored at -20°C as 100 pmol/µl stock solution. A detailed list of all primers used in this study is given in Table S1 Appendix.

Polymerase chain reaction (PCR)

The amplification of DNA fragments was done in vitro in by Polymerase Chain Reaction (PCR). Different DNA polymerases were used according to specific requirements: the High-Fidelity DNA polymerase Q5 TM DNA polymerase (NEB) was used for gene amplification; the Invitrogen TM Platinum TM SuperFi TM polymerase was used to amplify long fragments for building genetic constructs requiring a very accurate replication; and for colony-screening purposes the Thermo Scientific TM DreamTaq polymerase was used. All the enzymes used on this study were handled according to the supplier recommendations.

Purification of PCR products

PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN), according to the manufacturer's instructions.

Electrophoresis of DNA

The DNA was analysed by electrophoresis of DNA. This technique was performed according to standard methods using 1% agarose gels and Midori Green Advance (NIPPON Genetics) as fluorescent dye for detecting DNA.

Purification of DNA from agarose gel

For cloning purposes, after DNA electrophoresis, the fragments of interest were purified from the agarose gel slices using the QIAquick Gel Extraction Kit (QIAGEN) according to the manufacturer's instructions.

Purification of plasmid DNA

Plasmid DNA was extracted and purified from E. coli or B. subtilis using the QIAprep Spin

Miniprep Kit (QIAGEN), according to manufacturer's instructions. For the isolation of plasmid DNA from B. subtilis, lysozyme from chicken egg white (Sigma-Aldrich) was included in the resuspension buffer of the kit, at a final concentration of 2 mg/ml and the cell suspensions incubated at 37 °C for 15 min. All plasmid DNA was eluted with sterile deionized H2O.

Chromosomal DNA extraction

The genomic DNA of B. subtilis was extracted using the GenElute Bacterial Genomic DNA (SigmaAldrich) kit, according to supplier's instructions.

Digestion of DNA with restriction enzymes

The DNA strands were digested by restriction endonucleases (NEB). DNA was digested from 30 min up to 16 hours and inactivated for 20 min, in the conditions recommended by the manufacturer and using the buffers provided with the enzymes. The digestion products were either analysed by DNA electrophoresis, purified from the remaining components of the restriction digestion reaction, as explained in section 1.3, or directly added to the ligation reaction mix.

Ligation of DNA fragments

Digested DNA fragments were ligated by the T4 DNA ligase (INVITROGEN) in the conditions recommended by the manufacturer. The ligation reaction volume was typically 20µL and contained the vector and insert DNA in an approximate molar ratio of 1:3. The ligation mixtures were incubated at room temperature (RT), from 1h up to an overnight, and then used to transform E. coli.

Gibson assembly technology

This method, developed by Dr. Daniel Gibson and his colleagues, allows assembly of multiple DNA fragments with overlapping regions in a single-tube isothermal-reaction [START_REF] Gibson | Enzymatic assembly of DNA molecules up to several hundred kilobases[END_REF].

Typically, the adjacent fragments were designed to contain a homology region of 20 to 40 base pairs at their extremities. The amplified fragments were added to a assembly master mix (NEBuilder R HiFi DNA Assembly Master Mix), which possesses three different enzymatic activities -exonuclease, DNA polymerase, and DNA ligase activities that work in a single buffer. The exonuclease component creates single-stranded 3´overhangs that facilitate the annealing of fragments that share complementarity at one end (overlap region). The DNA polymerase incorporates nucleotides to fill in any gaps within each annealed fragment. The DNA ligase seals the nicks and covalently bounds the DNA adjacent segments. In more detail, 10µl of assembly master mix were mixed with 0.02-0.5pmols of DNA fragments, when up to three fragments are being assembled into a vector, and 0.2-1.0pmoles

of DNA fragments when from four to six fragments are being assembled, as recommended by the supplier's. The reaction mixture was incubated at 50•C for 15 min, when two or three fragments were being assembled, or 60 min, when more fragments were assembled. The total volume of the resulting product was then transformed into competent E. coli MC1061 strains.

Ligation-Independent Cloning (LIC)

Ligation-independent cloning (LIC) is a method that allows the ligation of a PCR fragment to a plasmid without using ligase or an restriction enzyme [START_REF] Botella | PBaSysBioII: an integrative plasmid generating Gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis[END_REF]. First the The pMUTIN-SPALIC vector was linearized by digestion with the restriction enzyme AscI and treated with T4 DNA polymerase. T4 polymerase has a exonuclease activity 3'→5' that creates the cohesive extremities in the presence of dATP for the vector, creating a linearized vector with ssDNA extremities.

Oligonucleotides used to amplify the insert (400bp of the end of the gene of interest) were constructed in order to contain specific extensions, LIC-P1 and LIC-P2 (Fig. 57-A) respectively, which are homologous with the pMUTIN-SPALIC extremities. The insert weas treated with T4 polymerase too, creating the ssDNA extremities. The two products were then annealed at room temperature with each other to form a new pMUTIN-SPA plasmid with the PCR fragment inserted upstream of the SPA sequence. The obtained plasmid is sufficiently stable for direct transformation into competent E. coli MC1061 strains. The extracted product plasmid was then transformed into B. subtilis and inserted into the chromosome by single cross-over (Fig. 57-B). pMUTIN-SPA has a Pspac inducible promotor to assure that after its integration into the chromosome the downstream genes are expressed. In this study, we used the LIC methodology to generate C-terminal SPA tag (sequential peptide affinity) translational fusions with genes of interest, through a pMUTIN-SPALIC integrative plasmid containing a LIC site generated synthetically. The transformation of DNA into E. coli MC1061, BL21 or ER2566 chimiocompetent strains were performed by heat shock. 20-50µl of the frozen competent stocked cells were mixed with the LIC sample, ligation mix or 1-5µl of 10pg to 100 ng/µl of plasmid DNA and incubated on ice during 20 minutes. The mixture was heat shocked for 45s at 42˚C and then put on ice again for 2 min. 500 µL of fresh LB were added to the cells and incubated at 37˚C for 45 min. Finally, the cells were harvested by centrifugation (1 min at 12000 xg), resuspended in 100 µL of fresh LB and plated on selective agar medium.

Transformation of Bacillus subtilis using natural competence

Transformation of B. subtilis cells was performed using a quick procedure protocol which is based on the natural competence of vegetative cells. A fresh plate was prepared by spreading the stock culture on plate, supplemented with the appropriate selection markers, and incubated overnight at 37˚C. After overnight incubation, a single colony was used to inoculate 2 ml of MC complemented medium (see appendix,section 6.1) and the culture was grown for 5h, at 37°C in a shaking incubator (200 rpm). Then 400µl of culture were mixed with 10µl of plasmid DNA or 1µl of chromosomal DNA and grew for additional 2 hours at 37°C with agitation (200rpm). The culture was plated on LB plates supplemented with the appropriate antibiotic and incubated overnight at 37°C.

Bacterial strains and growth conditions

E. coli and B. subtilis strains used in this work are listed in Table S2 and Table S3 respectively. B. subtilis strains used were derived from the BSB1 reference strain, which is a tryptophan-prototrophic (trp+) derivative of B. subtilis 168-trpC2 strain [START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF].

Nutrient Luria-

Strain construction

SPA tagged strains construction

The utilization of a SPA tag allows the purification of proteins in their native conditions. In order to study the possible protein-protein interactions in between the proteins involved in the Sec machinery, we have constructed translational fusions of a SPA-tag DNA sequence to the to the 3' end of the gene of interest. The cloning of a selected group of proteins involved in the Sec machinery (secDF, secG, spoIIIJ, bdbB, bdbC, sipT, sipS, sppA, yteJ and prsA), into the BSB1 background, were carried out according the LIC system described in section 4.1.11. The fragments contained ≥400bp of the end of the gene of interest, excluding the stop codon, and were amplified from chromosomal DNA using the appropriate primers listed in Table 2. The primers had a designed LIC tail sequence homologous to the LIC sequence present on the pMUTIN-SPA LIC plasmid. The vector and the insert were processed as described on the section 4.1.11 and Figure 66. After the annealing, of the vector and insert, at room temperature, the mixture was transformed into E.coli MC1061 and the transformants were selected in 100 µg.ml-1 ampicillin. The final product plasmids were extracted from E. coli and used to transform BSB1 strain. B. subtilis transformants were selected on LB agar plates supplemented with 0.6 µg.ml -1 Erythromycin. The 400 bp homology with the targeted gene allows the plasmid to insert into the chromosome by a single crossover in B.subtilis. To select the positive clones, the colonies that grew in the presence of 0.6 µg.ml -1 Erythromycin were re-streaked into 30 µg.ml -1 and grow at 37°C for overnight. The final strains harbouring a C-terminal SPA fusion under the control of the gene native promotor are listed at Table S 4.

To obtain N-terminal SPA tagged strains, the whole sequences of ORF corresponding to the genes of interest were amplified by PCR from the BSB1 chromosomal DNA, including the start codon and stop codon. For this, we used a specific pair of primers for each gene, carrying NotI and XhoI restriction sites at their 5' and 3' ends, respectively. The resulting PCR product was digested with NotI and XhoI restriction enzymes and ligated into pSPANter plasmid, previously digested with the same enzymes. E. coli MC1061 cells were transformed with the ligation mixtures and, then, single colonies were selected on LB-agar plates supplemented with 100 µg.ml-1 ampicillin. The plasmids harbouring the N-terminal SPA construction were transformed into BSB1 and integrated into the amyE locus by double crossing over. This integration gives origin to the SPA-tagged protein of interest expressing strains, under the control of the xylose inducible promoter P xyl . The positive clones, were selected in 100 µg.ml -1 spectinomycin, confirmed by PCR from the amyE flanking region, tested by the inability of the strain to produce amylase (starch-iodine test) and sequenced. All the strains constructed are listed in Table S2 and S3 of appendix. The final strains harboring a SPA fusion were confirmed by PCR and sequenced.

The starch-iodine test is used to check the presence of starch, when present the starch reacts with the iodine crystals and turns into an intense blue/dark color [START_REF] Dhar | The Starch-Iodine reaction[END_REF]. As the amyE gene encodes for an amylase, this test allows a quick selection of the positive clones. The insertion of the gene of interest disrupts the amyE gene, consequently the starch is cannot be not degraded, so when the clones are streaked into a plate containing starch the positive ones turn dark in the presence of iodine.

Deletion mutants

The deletion of sppA, yteJ and the sppA yteJ operon at their native locus in the BSB1 strain was performed by sequence replacement with a chloramphenicol resistance cassette (Cm R ) expressed under the control of the native promoter. Therefore, PCR fragments were obtained by amplifying the upstream and downstream regions, approximately 1 Kb each, of the gene sequence, from the BSB1 genomic DNA in two sequential PCR steps using the appropriate pair of primers, KO-P1 plus KO-P2

and KO-P5 plus KO-P6 respectively, listed in Table S1. Then a resistance cassette of chloramphenicol (Cm R ), approximately 1 Kb, was amplified from pUC19::chloramph plasmid using the primers KO-P3

and KO-P4. The primers P2, P3, P4 and P5, of each construction, have an extension containing an overlap sequence between the upstream, chloramphenicol and downstream fragments. These three amplified products were then purified and joined by an overlap PCR reaction. The final PCR product was purified from agarose gel, directly transformed into BSB1 and plated on LB agar supplemented with 5 µg.ml -1 chloramphenicol. The deletion of the genes of interest from the native locus was confirmed by PCR and the final constructs were sequenced, resulting in GHBs023, GHBs025 and GHBs028 strains. Primers sequences and strains are listed inTable S1 and Table S2 of appendix.

Construction of the GFP Fusions

Two types of superfolder green fluorescent protein (sfGFP) [START_REF] Pédelacq | Engineering and characterization of a superfolder green fluorescent protein[END_REF] fusions were constructed during this study: transcriptional fusions with the native σ W and σ A -dependent promoters, of sppA yteJ operon and translation fusions of SppA and YteJ.

The expression of the sppA-yteJ operon is under the control of two adjacent promoters, dependent of σ W and σ A . To study the sppa-yteJ native promoters activity we constructed two strains

His-tag constructions

To obtain E. coli strains overexpressing N-terminal 6xHis tagged proteins construct, the genes of interest were amplified by PCR from the BSB1 chromosomal DNA, excluding the start codon. For this, we used a specific pair of primers for each gene, carrying NdeI and XhoI restriction sites (

Fluorescence Microscopy

B. subtilis strains were grown overnight in LB at 37 ºC with the appropriate antibiotic and, the next day, were diluted (1:200) in 20 ml of fresh LB supplemented with 0.05 mM IPTG and grown until OD600nm 0.3-0.5 -exponential growth phase -and until OD600nm 1-2 -early stationary growth phase. Thereafter at regulated intervals samples were taken and cells were observed by fluorescence microscopy. For that, 2 µl of cell culture were placed onto a thin film of 1.2% agarose. The cell membranes were stained with FM4-64 (Molecular probes) and nucleoids were stained with DAPI.

Fluorescence microscopy was performed using a Leica DMRA2, controlled by Metamorph software (Molecular devices). Processing of fluorescence images was performed using Metamorph and ImageJ software.

Live-cell array (LCA)

The Live Cell Array is a method [START_REF] Botella | PBaSysBioII: an integrative plasmid generating Gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis[END_REF]

Bacteria survival test

The BSB1 and deletion mutant strains, described in section 2.1.2, were grown overnight in LB medium, supplemented with the appropriate antibiotic, at 37°C. On the following day, the overnight cultures were diluted 200-fold into fresh LB or M9 medium and then the cultures grew for 48h at 37 °C while shaking. Samples of 100µl were taken after 18h, 24h and 48h. Serial dilutionswere done and the last two dilutions, 10 -6 and 10 -7 fold, were plated onto LB agar supplemented with the appropriate antibiotic. The number of colonies, corresponding to the number of cells that survived, were counted after overnight incubation at 37°C.

To test if the WT cells could complement or help the growth of the mutants, the WT cells and deletion mutants were mixed and grew for 48h at 37 °C while shaking. The overnight cultures were diluted 200-fold into fresh LB and grown at 37°C until reach the exponential phase, then the cultures were diluted 1000-fold and mixed 1:1 (50% WT culture+50% Deletion mutant). The mixed cultures and the controls grew for 48h at 37 °C while shaking. From this point the remaining steps were done as described above.

Swimming and swarming capacity

The surfaces of agar concentrations above 1% prohibit swarming and swimming of many bacterial species, consequently, the humidity of the media is crucial for conducting these experiments.

Therefore, it is necessary to define a rigorous protocol in order to control the water content of the medium, we tested the ability of the bacteria swim at 0.3% of agar and the capacity of swarm at 0.7% [START_REF] Kearns | A field guide to bacterial swarming motility[END_REF]. All the plates were consistently prepared, 25 ml of the LB media were poured when the agar is approximately 50°C, let to solidify and to dry open for 15 minutes -in a laminar flow chamber. The cells were harvested (100µl) from overnight cultures or at mid exponential phase (OD600nm ~0.5), centrifuged at 2000rpm to avoid breaking the flagella, resuspended in PBS buffer and spotted (5µl) in the middle of the plate. Thereafter, the halo created by the cell movement were measured at time intervals of 1h during 6h.

Biofilm formation

B. subtilis strains in study were grown in LB shaking at 37°C, supplemented with the appropriate antibiotic, to an OD600nm of 1,0. To observe the architectural differences between biofilm from distinct strains, 10 µL cultures were then spotted on a dried MSgg plate (1.5% agar) and incubated for 70 hours at 30 °C. Colonies were measured and photographed.

4.9 Protein analysis 4.9.1 Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis (SDS

PAGE gels)

All the proteins purified in this project were analysed by 10% or 12.5% SDS-PAGE gels.

Samples were mixed with protein loading buffer (0.5 M Tris-HCl pH = 6.8, 1 M β-mercaptoethanol, 20 % (v/v) SDS, 0.2% (v/v) bromophenol blue, 12.5 % (v/v) glycerol), usually incubated 5 min at 95

The detection was performed, after a last wash step, using Clarity™ Western ECL Blotting detection substrates (Biorad), according to the manufacturers guidelines, through the ChemiDoc™ XRS+ System and Image lab imaging software.

Secreted proteins analysis

To check if a protein complex is involved in protein secretion we analyzed the if extracellular amount of a highly secreted reporter protein, as the case of AmyM, is affected. For that purpose, the B.subtilis strains of interest were naturally transformed with a multi copy plasmid containing the gene encoding AmyM (pCS73) and an empty plasmid (pCS74) to use as a control, all carrying a neomycin (Neo) resistance cassette. Dr. Van Rij provided these plasmids from DSM company, our collaborator from the Protein Factory project. The final strains, carrying the pCS73 and pCS74, were pre-cultured overnight at 37°C in LB medium supplemented with 10µg/ml Neo and other antibiotic markers correspondent to the background strains, if needed. The following day the culture was diluted 1000fold (to an OD 600nm of 0.001) into 50ml of LB medium with the same supplements. Grown at 37°C

until the OD 600nm reached 0.5-1 and the cells were harvested by centrifugation at 4500 rpm for 15 min at 4°C. The cell pellet was discarded and the culture media was filtered using a 0.2µm filter, removing possible remaining cells. The clear culture media was precipitated overnight at 4°C by 10% trichloroacetic acid (TCA). After centrifugation at 15000 rpm for 30 min, the supernatant was discarded and the protein pellet was washed two times with 200 µl cold acetone, repeating the centrifuge steps each time. The protein pellets were then dissolved by the addition of 3µl of 10 mM Tris-HCl pH 8, 8 M urea.

Sequential Peptide Affinity (SPA) purification

The sequential peptide affinity (SPA) technique has been developed, from the TAP-tag technique, to study protein-protein interactions [START_REF] Rigaut | A generic protein purification method for protein complex characterization and proteome exploration[END_REF][START_REF] Zeghouf | Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes[END_REF]). The translational fusion of the SPA-tag, composed of three FLAG sequences (3×FLAG) and the calmodulin binding peptide (CBP) separated by a TEV protease cleavage site, to a protein of interest allows its purification together with the protein(s) to which it interacts under native conditions, i.e.

without adding any cross-linking agents [START_REF] Delumeau | The dynamic protein partnership of rna polymerase in Bacillus subtilis[END_REF].

The strains expressing SPA-tagged proteins were pre-cultured overnight in the presence of erythromycin at 37°C. The following day the culture was diluted 1000-fold (to an OD 600nm of 0.001) into 2 liters of LB medium supplemented with 0.6 µg/ml erythromycin and 0.5 mM IPTG. When the OD 600nm reached 0.5 -1, cells were harvested by centrifugation at 6000 rpm for 10 min at 4°C. The pellet was washed with 40 ml of cold resuspension buffer (buffer R: 10 mM Tris-Cl pH 7.5, 150 mM NaCl), centrifuged at 4500 rpm for 10 min at 4°C and instantly frozen in liquid nitrogen. The cells were resuspended in 20 ml of buffer R supplemented with 1 mg/ml of lysozyme, 1 mM EDTA and 5 µl of Benzonase (Invitrogen), and incubated in a water bath at 37 °C for 15-30 min. To remove the cell debris the samples were centrifuged at 4500rpm for 30min at 4°C and the pellet was discarded.

Membrane and cytosolic fractions were then separated by ultracentrifugation at 100,000 g for 1 h at 4°C. The membrane pellet was solubilized using buffer R supplemented with 1% DDM. Identical protein amounts of the membrane and soluble protein fractions were loaded on 10% SDSpolyacrylamide gels for Coomassie staining and Western blotting using anti-FLAG antibodies.

In addition, protein complexes from the membrane fraction were pulled down using a SPA purification method [START_REF] Charbonnier | molecular and physiological logics of the pyruvate-induced response of a novel transporter in Bacillus subtilis[END_REF]. The solubilized membrane fraction, 2 mM CaCl22 and 1%DDM, was mixed with 200 µl of pre-equilibrated calmodulin resin (CaM) in 2 ml tubes, which were placed on a rotating wheel for a minimum of 2 h at 4°C. The resin was transferred in a column, the eluate was discarded and the CaM resin was washed with 2 ml of buffer R supplemented with 1% DDM and 0.1 mM CaCl2. The proteins remaining bound to the CaM resin were eluted with a total volume of 0.75 ml of buffer R supplemented with 1% DDM and 3 mM EGTA. The eluate was collected and mixed with 200 µl of anti-FLAG resin. After its incubation under gentle agitation on the rotating wheel for, at least two hours, the eluate from the anti-FLAG resin was discarded, the resin was washed with 2 ml of buffer R supplemented with 1% DDM. The proteins remaining bound to the anti-FLAG resin were eluted with a total volume of 1ml of 0.1M glycine pH 3.

Proteins from the eluate were precipitated overnight at 4°C by 10% trichloroacetic acid (TCA). After centrifugation at 15000 rpm for 30 min, the supernatant was discarded and the protein

His-tag purification

The E. coli strains harboring a translational 6XHis-tag fusion were pre-cultured overnight in the presence of 30 µg.ml -1 kanamycin at 37°C. The following day the culture was diluted 1000-fold (to an OD 600nm of 0.001) into 2 liters of LB medium supplemented with 30 µg.ml -1 kanamycin and grown until an OD 600nm of 0.8 at 37°C. Thereafter the expression was induced by the addition of 250 µM were analyzed by SDS-PAGE. The samples containing the highest concentration of the His-taged protein were joined and concentrated by Amicon Ultra-15 Centrifugal Filters, mixed with 50%

IPTG
glycerol and stored at -20°C.

Double tag purification

With the objective of increasing the purity of the membrane protein complex, a E. coli stain harboring SppA with a N-terminal His tag and YteJ with a C-terminal Flag ta ( 6xHis-SppA/YteJ-Tev-3xFlag), a double tagged SppA-YteJ complex, was constructed as described above on section 4.3.5.

The membrane fraction was obtained and solubilized as described above. The sample was mixed with 200 µl of anti-FLAG resin and incubated under gentle agitation on the rotating wheel. The eluate from the anti-FLAG resin was discarded and the resin was washed with 2 ml of buffer R supplemented with

GHBs

BSB1 pCS74

This study

GHBs

BSB1 pCS73

This study

GHBs

BSB1 pCS58

This study The observation of some contaminants in the protein sample after the gel filtration raised the question, is the pH buffers used for the anionic exchange chromatography correct?

GHBs

Therefore, to test this hypothesis the protein sample was subjected to a new anion exchange chromatography at 4 °C in a 1 ml Hitrap HP-Q column equilibrated in 20 mM Tris HCl pH 8.5 and 5 mM CaCl2. The protein was eluted with a linear gradient of NaCl from 0 mM up to 1 M. Through the analysis of the UV value measured at 280 nm it was possible to see that the protein did not bind to the resin and was eluted during the flow-through. The flow-through sample was concentrated

(with an Amicon Ultra-15 column) and analysed by SDS-PAGE gel, were we could confirm the presence of AmyM protein at the flow-through. Consequently, it was performed a cationic exchange chromatography at 4 °C in a 1 ml Hitrap HP-S column equilibrated in 20 mM Tris HCl pH 7.5 and 5 mM CaCl2. The protein was eluted with a linear gradient of NaCl from 0 mM up to 1 M. The measurements of the UV values at 280 nm showed that the protein had not bound to the resin and, once more, the protein was eluted with the flow-through.

Then the AmyM sample was reapplied to a Hitrap 26/60 Superdex 200 column equilibrated with TBS and 5 mM CaCl2 and eluted at a flow rate of 2 ml/min for 5 hours. This column allows a better separation of the different fractions of the sample since have a bigger size. The protein was eluted as a single peak with an apparent molecular weight of 75 KDa. However, the sample fractions correspondent to the protein elution showed the contaminants were kept.

Although the theoretically pI be 5.38 [START_REF] Dauter | X-Ray structure of Novamyl, the fivedomain 'maltogenic' α-Amylase from Bacillus Stearothermophilus: Maltose and Acarbose complexes at 1.7 Å resolution[END_REF][START_REF] Cha | molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose[END_REF][START_REF] Park | Molecular cloning and characterization of a thermostable α-amylase exhibiting an unusually high activity[END_REF] it is possible that this is not the real value, what could explain the why the protein is not binding to the anionic or cationic resin of the exchange chromatography process. In 1984, H. Outtrup and B. E.

Norman studied the B. stearothermophilus maltogenic amylase and determined by thin layer gelelectrofocusing that this protein has a pI of 8.5. To confirm the correct pI value we measured the pI of our sample by iCE3 Capillary IEF System. This is a HPLC-like method that performs free solution Isoelectric Focusing (IEF) in a capillary column, in which a pH gradient is created to separate and focus the proteins according to their pI. The all process is monitored in real time by a whole-column UV detector. Before run, the samples are premixed with carrier ampholytes, additives and pI markers.

The pI measured for our sample was 8.35. To complement the pI measurement a molecular weight analysis was performed by time-of-flight mass spectrometry (LC-QTOF). The used technique allows the obtainment of the protein molecular weight thought the determination of an ion mass-to-charge ratio that is measured according to the time the ion takes to reach a detector at a known distance [START_REF] Sigaud | A Novel double-focusing time-of-flight mass spectrometer for absolute recoil ion cross sections measurements[END_REF][START_REF] Kernphysik | 100 Years of Ion Beams : Willy Wien ' s Canal Rays Introduction : How Wien Came to Physics with Canal Rays Wien ' s Experiments with Cathode and Canal Rays in[END_REF][START_REF] Beauchemin | Inductively coupled plasma mass spectrometry[END_REF]. The measure

Figure 5 :

 5 Figure 5 : A : Croissance en milieu LB de la souche sauvage (BSB1) et des souches délétées de sppA, yteJ ou sppA-yteJ. B : survie à long-terme des différentes souches après comptage des cellules viables sur boites gélosées LB-agar.

Figure 6 :

 6 Figure 6 : Analyse par SDS-PAGE de la quantité de AmyM sécrétée dans la souche sauvage comparée aux souches délétées de sppA, yetJ et sppA-yteJ.

Figure 7 :

 7 Figure 7 : Croissance de cellules de B. subtilis mesurée par densité optique à 600 nm dans un lecteur de microplaques en présence de différentes concentrations de subtiline. Chaque courbe représente la moyenne de 6 réplicats.

Figure 8 :

 8 Figure 8 : Croissance des cellules de la souche sauvage (BSB1) et des souches délétées sppA et yetJ complémentées de leurs gènes réciproques sous le contrôle d'un promoteur Phyperspank (Phs) inductible à l'IPTG (notées Phs-sppA et Phs-yteJ). L'effet de la subtiline (30%) est testé en fonction de concentrations croissantes d'IPTG (0 à 100 µM).

  SppA, SppA ∆1-52 , du complexe SppA/YteJ et YteJ chez E. coli. Afin de répondre à l'hypothèse d'une régulation de l'activité de SppA par YteJ dans le complexe SppA/YteJ identifié chez B. subtilis, nous avons exprimé chez E. coli les proteines suivantes : SppA, SppA 1-52 , SppA/YteJ (sous forme d'opéron existant nativement chez B. subtilis) et YteJ. Chaque protéine fut exprimée avec un tag 6xHis fusionné à son extrémité N-terminale (Figure 12).

Figure 12 :

 12 Figure 12 : Schéma représentant les différentes expressions chez E. coli des protéines SppA, SppA 1-52 , SppA/YteJ et YteJ, dont les formes sont étiquetées His en N-terminal pour une purification par colonne d'affinité Ni-agarose.

Figure 14 :

 14 Figure 14 : Test de l'activité protéase de SppA ∆1-52 sur différentes protéines. Après incubation de 16h, les échantillons ont été analysés par SDS-PAGE.

Figure 16 :

 16 Figure 16 : A : Analyse par SDS-PAGE et spectrométrie de masse des différentes formes purifiées de SppA après découpage des bandes sur gel ; B : illustration des peptides (surlignés en vert et jaune) retrouvés par spectrométrie de masse uniquement dans la bande SppA purifiée en complexe avec YteJ.

Figure 17 :

 17 Figure 17 : Schéma représentant les insertions de l'opéron modifié sppA-yteJ avec les différentes troncations de yteJ. Ces constructions ont été insérées dans le chromosome de B. subtilis au locus amyE.

FMFigure 18 :

 18 Figure 18 : Observation au microscope à contraste de phase et fluorescence de l'état morphologique des cellules surexprimant SppA et différentes troncations de YteJ, en absence d'IPTG (1 ère colonne) et en présence de 100 µM d'IPTG (4 dernières colonnes) dans le milieu de culture.

1. 2

 2 .7 Holins ............................................................................................................................. 1.3 A guarantee of a correct protein translocation in B. subtilis ................................................ 1.3.1 A bacterial cytoplasmic quality control system: ClpXP protease .................................. 1.3.2 Membrane-bound proteases, an important quality control step for membrane and secretory proteins. ........................................................................................................................ 1.3.3 Signal peptide peptidase A (SppA) ................................................................................ 1.4 Scope of the Thesis ................................................................................................................2 Results ........................................................................................................................................... 2.1 Identification of membrane protein complexes involved in the B. subtilis Sec pathway ..... 2.1.1 SPA-tag constructions .................................................................................................... 2.1.2 Purifications of the SPA-tagged proteins ...................................................................... 2.1.3 Optimisation of the BN-PAGE technique ....................................................................... 2.2 The SppA/YteJ membrane complex ...................................................................................... 2.2.1 The sppA yteJ operon is regulated by sigma factors σ A and σ W ..................................... 2.2.2 Deletion of the sppA and yteJ genes ............................................................................. 2.2.3 Role of the SppA/YteJ complex in protein secretion ..................................................... 2.2.4 The deletion of sppA yteJ affects the long term survival ..............................................

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .Figure 9 .Figure 11 .Figure 12 .Figure 14 .Figure 15 .Figure 16 .Figure 17 .Figure 18 .Figure 19 .Figure 20 . 51 Figure 21 . 51 Figure 22 .Figure 23 .Figure 24 .Figure 25 .Figure 26 .Figure 27 .Figure 28 .

 12345678911121415161718192051215122232425262728 Figure 1. Schematic representation of the distinct cell fates in Bacillus subtilis biofilms…………....16 Figure 2. Protein export pathways in B. subtilis………………………………...………………….....19 Figure 3. Schematic prediction of the B. subtilis Sec-dependent secretory protein translocase……....21 Figure 4. Components involved in Sec-dependent protein export in B. subtilis………………...…….22 Figure 5. General features of the signal peptides and propeptides of Bacillus secretory proteins…....24 Figure 6. Schematic representation of the different modes of protein translocation across the membrane of B. subtilis……………………………………………………………………...26 Figure 7. Scheme of SecA-mediated protein translocation………..……………………………….....29 Figure 8. Signal peptide cleavage of precursor proteins by signal peptidases….…….……………….31 Figure 9. Tat pathway components………………………………………………………………...….34 Figure 10. Gram-positive bacterial Tat pathway..…………………………………………………….35 Figure 11. Model of the regulation of TatA pore formation by TatBC and substrate……..………….36 Figure 12. Bacterial flagella structure………………………………………………………………....39 Figure 13. B. subtilis flagellum…..……………………………………………………………….…...40 Figure 14. Model for the assembly process of the Gram-negative flagellar type III export apparatus 41 Figure 15. An entropic chain mechanism for flagellum growth outside the cell………….…….…….42 Figure 16. Schematic representation of type IV secretion architecture and functions in bacteria….....44 Figure 17. T4SS of ICEBs1……………………………………….………………………..………....46 Figure 18. Schematic depiction of the proteins involved in Gram-negative bacterial cell envelope disruption by holin-type lysis systems………………………………………………………48 Figure 19. Scanning electron micrographs……………………………………………………………50 Figure 20. Cartoon model of substrate recognition and degradation by the ClpXP protease….….…..51 Figure 21. Interaction of ClpX and ClpP……………………………………………………………...51 Figure 22. Representation of the expansion and contraction of the axial pore by a snake-jaws model………………………………………………………………………………………...52 Figure 23. The B. subtilis SppA protein………………………………………….…………………...56 Figure 24. Comparison between octameric SppABS and tetrameric SppAEC…………….………....57 Figure 25. Cross section representation of the oligomeric dome shape structure of SppAEC and SppABS………………………………………………………………………………….…..58 Figure 26. Growth analysis of strains harbouring C-terminal SPA-tag fusions………………..……..67 Figure 27. Expression of the SPA tag fused proteins…………………………………..….………….68 Figure 28. Purification of SpoIIIJ-SPA and SecDF-SPA protein with different concentrations of DDM………………….………………………………………………………….……….….70

Figure 29 .Figure 30 .Figure 31 .Figure 32 .Figure 33 .Figure 34 .Figure 35 .Figure 36 .Figure 37 .Figure 38 .Figure 39 .Figure 40 .Figure 42 .Figure 43 .Figure 44 .Figure 45 .Figure 46 .Figure 47 .Figure 48 .Figure 49 .Figure 50 .Figure 51 .Figure 52 .Figure 53 .Figure 54 .Figure 57 .Figure 58 .Figure 59 .Figure 60 .Figure 61 .Figure 62 .Figure 63 .Figure 64 .Figure 65 .Figure 66 .Figure 67 .

 293031323334353637383940424344454647484950515253545758596061626364656667 Figure 29. Analysis by Western blot of a Blue native PAGE 4-16% Bis-Tris Gel of SecDF-SPA in the presence of different concentrations of DDM………………………………….……………72

Figure S 1 Figure S 2 .Figure S 3 195 Figure S 4 .Figure S 5 .Figure S 6 .

 123195456 Figure S 1 YteJ predicted structure by Protter…………………………………...…………………..193 Figure S 2. Purification of SppA, YteJ, SppA/YteJ and SppA K199A by Gel Filtration…………….....194 Figure S 3 Pulse chase assays for sppA, yteJ and sppA yteJ deletion mutants and complemented strains……………………………………………………………………………………….195 Figure S 4. The growth of B. subtilis sppA and yteJ deletion mutant strains in the presence of different concentrations of LP9………………………………………………………………...…….196 Figure S 5. The growth of B. subtilis sppA and yteJ overexpressing mutant strains in the presence of different concentrations of LP9…………………………………………………………….197 Figure S 6. AmyM supernatant purification…………………………………...…………………….199 Figure S 7. AmyM Size exclusion purification results……………………………………...……….200

B

  . subtilis is able to survive in or adapt to adverse conditions, to engage various life-styles by triggering different genetic programs, in response to unfavourable environmental signals (Mielich-Süss and Lopez 2015; López and Kolter 2010). B. subtilis responds to different environmental changes or to the lack of an essential nutrient, by differentiating into subpopulations of specialized cells: I.motile cells, give the capacity to the cells to move through liquid and over surfaces;

Figure 1 .

 1 Figure 1. Schematic representation of the distinct cell fates in Bacillus subtilis biofilms. A mature biofilm is composed of distinct subpopulations of cell types that coexist and exhibit different spatiotemporal distribution patterns. The different subpopulations interact and cooperate with each other with the objective of overcome changes of external and environmental factors. Adapted from Mielich-Süss and Lopez 2015.

Figure 2 .

 2 Figure 2. Protein export pathways in B. subtilis. Schematic representation of B. subtilis cell and its export pathways.After the protein synthesis by the ribosomes, proteins undergo different destinations depending on the presence (+SP) or absence (-SP) of an N-terminal signal peptide (SP) and its category. Proteins without a signal peptide remain in the cytoplasm. Proteins that have a typical secretory signal peptide are exported via the Sec pathway, but if the SP contains a twin-arginine motif (+RR) the protein is exported by the Tat pathway. These proteins can be then secreted to the medium, retained at the membrane by a transmembrane segment (TM), anchored to the extra-cytoplasmic side of the membrane by a lipid modification (+lipobox) or anchored to the cell wall, the mature parts of these proteins contain cell wall-binding repeats (+CWB). Pseudopilins are exported by the Com system. ABC transporters can export peptides too, as it is the case of pheromones and lantibiotics.[START_REF] Tjalsma | Proteomics of protein secretion by Bacillus subtilis: separating the 'secrets' of the secretome[END_REF]

Figure 3 .

 3 Figure 3. Schematic prediction of the B. subtilis Sec-dependent secretory protein translocase. A preprotein substrate is pushed, in an unfolded state, through the heterotrimeric pore (SecYEG) by the SecA motor protein. The SecDF-YrbF complex and the main type I signal peptidases (SipS and SipT) are involved in the substrate translocation by helping on the translocation mechanism and cleaving the signal peptide, respectively. After translocation, the preprotein is folded into its correct formation by Sec accessory proteins (folding factors). Adapted from Harwood and Cranenburgh 2008. Briefly, the translocation through the Sec pathway can be divided in to three main steps (Figure 4): I. Targeting: The secreted proteins are synthesised as precursors (pre-pro-protein) with a Nterminal signal peptide (SP), which is recognized by cytoplasmic factors (Signal Recognition Particle-SRP) or chaperones, such as SecB in E. coli and CsaA in B. subtilis (Collinson 2005; H Tjalsma et al.

Figure 4 .

 4 Figure 4. Components involved in Sec-dependent protein export in B. subtilis.Secretory proteins are ribosomally synthesized as precursor proteins with an N-terminal signal peptide (SP). Cytoplasmic chaperones, such as SRP/FtsY, keep the precursors in a translocation-competent state and facilitate their targeting to the translocase in the membrane, consisting of SecA, SecY, SecE, SecG, and SecDF. During or shortly after translocation, the preprotein is cleaved by one of the type I signal peptidases (SipS-W) or lipid modified by the diacylglyceryl-transferase (Lgt) and cleaved by the lipoprotein-specific signal peptidase (Lsp). SppA may be involved in the degradation of cleaved signal peptides, whereas the folding of several secreted proteins depends on the activities of PrsA, BdbBC, and/or SpoIIIJ/YqjG. HtrA, HtrB, and WprA are involved in the quality control of secretory proteins. It should be noted that for reasons of simplicity, HtrAB are depicted in the cell wall, although HtrA is detected in both the membrane and the medium. On passage through the cell wall, the mature protein is released into the environment. Adapted fom[START_REF] Tjalsma | Proteomics of protein secretion by Bacillus subtilis: separating the 'secrets' of the secretome[END_REF].

Figure 5 .

 5 Figure 5. General features of the signal peptides and propeptides of Bacillus secretory proteins. The signal peptides tripartite structure, N-terminal (N) containing lysine and/or arginine residues (indicated by +), hydrophobic (H, indicated by a grey box) and cleavage (C) regions are schematically represented. The export pathways via which the preproteins are exported and the SPases responsible for their cleavage are indicated. The break between the boxes represent cleaved sites and the most frequently occurring first amino acid of the mature protein (+1) is indicated. Tat-dependent signal peptides with twin arginine motif (SRRxFLK) and the Secdependent signal peptides containing an AXA cleavage site, are cleaved by a type I signal peptidase (SPase). Lipoprotein signal peptide cleaved by the type II SP. The pseudopilin-like signal peptides and bacteriocin and pheromone signal peptides have specific SPases. The length of the signal peptides and their subdomains is drawn to the same scale. (H[START_REF] Tjalsma | Proteomics of protein secretion by Bacillus subtilis: separating the 'secrets' of the secretome[END_REF]

Figure 6 .

 6 Figure 6. Schematic representation of the different modes of protein translocation across the membrane of B. subtilis. (A) Different stages of cotranslational translocation. The polypeptide is moved directly from the ribosome tunnel into the SecYEG channel. The ribosome is anchored to the membrane by the SRP-receptor complex. (B) In the model for posttranslational translocation, the preprotein translocation is mediated by the SecA ATPase.

Figure 7 .

 7 Figure 7. Scheme of SecA-mediated protein translocation. (A) SecYEG closed channel; (B) Formation of the SecA-SecY complex; (C and D) At this stage the channel is active and the translocation starts; (E) The final stage where the signal peptide is cleaved and the protein is released. The translocating polypeptide is shown in green and the signal peptide as a green cylinder. The lateral gate of the channel is shown as a broken line on the left. (Alguel et al. 2016) Kakeshita et al. 2010 showed that the SecA C-terminal region is not conserved in eubacteria and by deleting 61 amino acids of this region led to significant enhanced production of heterologous proteins in B. subtilis. The membrane translocase complex itself is of great interest for the optimisation of production strains. In an attempt to solve the problem of jamming of the membrane caused by the overexpression of secretory proteins through the increase of the number of translocon,

Figure 8 .

 8 Figure 8. Signal peptide cleavage of precursor proteins by signal peptidases. Signal peptidase I (SPaseI) employs a Ser-Lys catalytic dyad for signal peptide cleavage from secretory precursor proteins at the extracytoplasmic surface of the membrane. The Protein Data Bank (PDB) structure of the catalytic domain (accession number 1T7D) and the program JMol were used to generate the three-dimensional (3D) structure image of SPI. Signal peptidase II (SPII) is an aspartic acid protease that cleaves signal peptides from bacterial lipoprotein precursors just beneath the extracytoplasmic membrane surface. Signal peptidase IV (SPIV) is an aspartic protease that cleaves signal peptides from prepilins and pseudopilins at the cytoplasmic surface of bacterial membranes. Transmembrane helices of signal peptidases are depicted as blue barrels, and substrate helices are depicted as red barrels. A zoomed-in view of the active site residues of SPI is shown. The locations of the N and C termini of the signal peptidases and their substrates are indicated. Dalbey, Wang, and van Dijl 2012.

Figure 9 .

 9 Figure 9. Tat pathway components. (A) Schematic representation of the Sec signal peptide and Tat signal peptide. The different regions of the respective signal peptides (N-terminal (N), hydrophobic (H) and cleavage (C) domains), mature protein parts, and conserved SP recognition sites are indicated. The black arrow indicate the SPase cleavage site. (B) Illustration of the basic components of the Tat apparatus. The large transmembrane protein TatC is shown in purple and the TatA-like proteins TatA, TatB and TatE are shown in pink, green and orange respectively. Adapted from (Dalbey, Wang, and van Dijl 2012; Goosens, Monteferrante, and Van Dijl 2014).

Figure 10 .

 10 Figure 10. Gram-positive bacterial Tat pathway. Schematic representation of the envelope structure Grampositive bacteria and its Tat translocation machinery. Tat substrates can be targeted to the membrane, the cell wall or the growth medium. Adapted from Goosens 2014. Crosslinking and fluorescent microscopy experiments, showed the dynamic oligomerization of TatA. Particularly, it was possible to visualize directly the dynamics in E. coli through the fusion of

  2013, Rodriguez and colleagues provided the first experimental evidence supporting this model, schematically presented on Figure11. They showed by NMR that E. coli TatA forms a ringlike complex in the presence of the detergent C12E9, where the transmembrane helices form a ring inside the membrane from which the APH extends allowing the formation of complexes with variable subunit numbers. They also showed by molecular dynamics simulation that the periplasm side of the lipid bilayer surrounding the TatA complex was destabilized because of the unusual short TMD[START_REF] Rodriguez | Structural model for the protein-translocating element of the twin-arginine transport system[END_REF].Later in 2018, Hou and colleagues demonstrated in vivo that in the presence of a Tat substrate the N-terminus TMD of TatA can destabilize the membrane. However in the absence of substrate, the full length TatA counterbalances such weakening effects[START_REF] Hou | The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia Coli upon protein substrate binding[END_REF]. Therefore, they proposed a two-state model: (i) the activated state -in which the binding of the substrate to the TatABC complex promotes the position switch of the TatA APH, which locally weakens the membrane, allowing the substrate translocation across the membrane; (ii) the resting state-in the absence of the substrate, the N-terminal membrane part of the APH compensates for distorting effects of the short TatA membrane TMD anchor[START_REF] Hou | The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia Coli upon protein substrate binding[END_REF].

Figure 11 .

 11 Figure 11. Model of the regulation of TatA pore formation by TatBC and substrate. (A) The inactive monomeric form of TatA accommodates the depth of the membrane by tilting the short TMH, increasing the angle between the TMH and the APH, and deeply inserting the APH into the membrane. (B) Interactions with TatBC/substrate complex force an orientation of the TatA TMH roughly parallel to the membrane normally favoring TatA polymerization.The N ter-minus is pulled into the membrane thinning and distorting the bilayer in the pore and sensitizing the membrane to disruption. The TatA APH serves as a platform for the substrate that is tethered to the TatBC complex by a signal peptide. In the drawing the TatA oligomeric ring is represented by two opposing subunits. (C) Pulling of substrate protein into the membrane forces TatA subunits apart and ruptures the membrane[START_REF] Rodriguez | Structural model for the protein-translocating element of the twin-arginine transport system[END_REF].

Figure 12 .

 12 Figure 12. Bacterial flagella structure. Schematic representation of the flagellar motor of Gram-negative (A) and Gram-positive (B) bacteria. Electron micrographic reconstruction of the flagellar basal body side view (C). All the components that compose the flagellar motor are specified on the Figure 13. Adapted from Berg 2003.

Figure 13 .

 13 Figure 13. B. subtilis flagellum. Cartoon depicting the putative structure of the B. subtilis flagellum based on empirical data and similarity to S. enterica. Peptidoglycan is indicated in light gray. Membrane is indicated in dark gray. Flagellar components are colored and labeled. Micrographs are superresolution fluorescence micrographs of the indicated structures in wild type cells: filament (maleimide-stained HagT209C), hook (maleimide-strained FlgET123C) and basal body (FliMGFP fusion protein). Adapted from Mukherjee and Kearns 2014.

Figure 15 .

 15 Figure 15. An entropic chain mechanism for flagellum growth outside the cell. Subunit crystallization beneath the cap foldase provides a strong anchor (force to break anchor, FA). Sequential subunits are linked (force to break link, FL) head-to-tail in a chain by juxtaposed terminal helices forming parallel coiled-coils. Subunits docked at the export ATPase are unfolded and docked by the export machinery. The N-terminal helix of the docked subunit is then captured (force to break docking, FM) into the subunit chain by the free C-terminal helix of an existing subunit in the flagellar channel. Adapted from Evans et al. 2013.

Figure 16 .

 16 Figure 16. Schematic representation of type IV secretion architecture and functions in bacteria. (A) Conjugative T4SSs translocate DNA from the donor bacterium into various recipients, including other bacteria or eukaryotic cells. (B) DNA release systems facilitate an exchange of DNA with the extracellular space as well as biofilm formation. (C) DNA uptake from the environment proceeds by the ComB T4SS. (D) The Xanthomonas citri T4SS can deliver a protein toxin to kill neighboring G -ve bacterial competitors. (E) Various pathogenic bacteria and symbionts have evolved T4SSs to deliver effector proteins or DNA-protein complexes into their host (either eukaryotic target cells or protozoan hosts). The T4SSs can either inject their effectors directly into the host cell or secrete them into the medium, thereby exerting remarkably different effects on host cell functions during infection. (F) To achieve genetic exchange during bacterial conjugation, two relaxase monomers collaborate, adopting distinct structural conformations to provide the two necessary enzymatic activities for processing the DNA. Adapted from Grohmann et al. 2018)

  conjugation. CwlT has two functional domains, a C-terminal domain with DL-endopeptidase function and an N-terminal domain that works as an N-acetylmuramidase. Mutations in the catalytic site of CwlT muramidase domain completely abolish the conjugation, while if the catalytic site of the endopeptidase domain is mutated, it leads to a significant decrease of the conjugation[START_REF] Auchtung | Biology of ICEBs1, an integrative and conjugative element in Bacillus subtilis[END_REF]. The ICEBs1 membrane channel is a membrane protein complex composed of four integral membrane proteins ConB, ConC, ConD, and ConG (Figure17). ConG is the larger component of this complex, predicted to have seven transmembrane helices, and ConB, as the second larger component, together may form the bulk of the translocation channel. In contrast, ConC and ConD, smaller proteins, may work as scaffolding or assembly factors, a role that is still unclear. ConE is a doughnut-shaped hexamer ATPase, homolog to the Gram-negative canonical VirD4, which belongs to the VirB4 clade of the FtsK-HerA superfamily, is essential for the DNA translocation upon the channel[START_REF] Iyer | Comparative genomics of the FtsK-HerA superfamily of pumping ATPases: implications for the origins of chromosome segregation, cell division and viral capsid packaging[END_REF]). The study of its subcellular localization, through fluorescent microscopy using a ConE tagged with the green fluorescent protein (GFP), revealed, the localization of the ICEBs1 apparatus. In ICEBs1 donor cells ConE-GFP had a polar localization, and it is possible to see clear dots in the cell pole in close association with the membrane, while when observed in cells lacking ICEBs1 ConE-GFP was dispersed within the cell cytoplasm[START_REF] Berkmen | Polar positioning of a conjugation protein from the integrative and conjugative element ICEBs1 of Bacillus subtilis[END_REF].[START_REF] Leonetti | Critical components of the conjugation machinery of the integrative and conjugative element ICEBs1 of Bacillus subtilis[END_REF], demonstrated by two-hybrid assays that ConE directly interacts with ConB, proving that ConB is essential for the recruitment and maintenance of ConE[START_REF] Leonetti | Critical components of the conjugation machinery of the integrative and conjugative element ICEBs1 of Bacillus subtilis[END_REF].

Figure 17 .

 17 Figure 17. T4SS of ICEBs1. Working model of the ICEBs1 T4SS. This speculative model relies on data from other T4SSs. The single-stranded conjugative DNA is shown in red, covalently attached to the NicK relaxase. The presumed coupling protein ConQ likely delivers NicK and associated T-DNA to the membrane-associated T4SS. ConB and ConG may make up the bulk of the membrane channel. The ConE ATPase may provide energy for T4SS assembly and/or DNA transfer. Adapted from Auchtung et al. 2016.

  spore morphogenesis and germination in B. subtilis;  biofilm formation and DNA release for S. aureus;  programmed cell death and acetate metabolism in S. aureus;  biofilm formation and oxidative stress adaptation in Streptococcus mutans. Holins are small membrane proteins that play two clear roles in the phage infection cycle. These proteins can be classified in three different membrane topologies-(I) Class I holins that have three TMDs, beginning with the N-terminal outside and ending with the C-terminal inside the cytoplasm; (II) Class II have two TMDs, whereas both ends N-and C-terminal are in the cytoplasm; (III) Class III just have a single highly hydrophilic TMD, with a disposition of the N-terminal in and C-terminal out.

  pathway serves to translocate a highly diverse family of substrates, it possesses the collective capacity to (a) recognize and translocate DNA substrates (conjugation machines) to bacterial recipients, (b) deliver effector proteins to eukaryotic target cells, (c) exchange DNA with the milieu, (d) contribute to biofilm development and (e) deliver a killing toxin to bacterial neighbours. The integrative and conjugative elements (ICEs) are a major form of horizontal gene transfer mechanism present on bacterial host chromosomal DNA, playing a key role in bacterial evolution by spreading antibiotic resistance and moving genes, which can be involved in cell metabolism, symbiosis, degradation of pollutants and pathogenesis. The B. subtilis ICEBs1 T4SS machinery comprises a coupling protein (ConQ), a cell wall hydrolase (CwlT), an ATPase (ConE)and the membrane channel components (ConB, ConC, ConD, and ConG)[START_REF] Leonetti | Critical components of the conjugation machinery of the integrative and conjugative element ICEBs1 of Bacillus subtilis[END_REF].endolysin system are clustered in a ''lysis cassette''(I.-N.[START_REF] Wang | Holins: the protein clocks of bacteriophage infections[END_REF]. Therefore, it is possible to define the holins as "clock" proteins, as they coordinate the timing of the end of the infection cycle (I.-N.[START_REF] Wang | Holins: the protein clocks of bacteriophage infections[END_REF].[START_REF] Rapoport | A gene encoding a holin-like protein involved in spore morphogenesis and spore germination in Bacillus subtilis[END_REF] demonstrated that B. subtilis has a gene, ywcE, which encodes for a holinlike protein. Performing transcriptional analysis, they prove that this protein is involved in B. subtilis sporulation[START_REF] Rapoport | A gene encoding a holin-like protein involved in spore morphogenesis and spore germination in Bacillus subtilis[END_REF]. Furthermore, fluorescent microscopy analysis of a strain bearing an ywcE-gfp translational fusion showed that ywcE localizes at the cell and spore membranes. Its deletion was also shown to affect the spore morphology. By transmission electron microscopy analysis of the ∆ywcE spores, they indeed showed that the ∆ywcE mutant had a reduced outer coat without the characteristic striated pattern, and the outer coat failed to attach to the underlying inner coat[START_REF] Rapoport | A gene encoding a holin-like protein involved in spore morphogenesis and spore germination in Bacillus subtilis[END_REF].

Figure 18 .

 18 Figure 18. Schematic depiction of the proteins involved in Gram-negative bacterial cell envelope disruption by holin-type lysis systems. Pinholins form small heptameric pores that collapse the membrane potential (the PMF) across the inner membrane, while the more conventional holins form large multisubunit pores of variable sizes that allow release of fully folded endolysins from the cytoplasm, which in the periplasm hydrolyze specific bonds in the peptidoglycan cell wall, depending on the lysin type. C + , a cation. Adapted from Saier and Reddy 2015.

Figure 19 .

 19 Figure 19. Scanning electron micrographs of B. subtilis IS58 (A), the clpP (B) and clpX (C) mutant strains grown exponentially. Adapted from Gerth et al. 1998.ClpP exhibits a cylindrical structure composed of two associated heptameric rings and, as a typical serine protease, contains internal (inner surface) proteolytic sites composed of the conserved catalytic triad (Ser-His-Asp) (B. G.[START_REF] Lee | Structural insights into the conformational diversity of ClpP from Bacillus subtilis[END_REF]. The hexameric ClpX ATPase ring binds to the heptameric ClpP ring in a symmetry mismatch, aligning the two axial pores that creates a continuous central channel between them (Figure20). Allowing the translocation, of the denatured and linear protein substrate, from the narrow ATPase pore into the concealed proteolytic chamber[START_REF] Olivares | Mechanistic insights into bacterial AAA+ proteases and protein-remodelling machines[END_REF]. Despite the fact that high-resolution structures of the ClpXP complex do not exist, it was demonstrated, by different studies and techniques, that the stability of this complex relies on interactions involving flexible loops near the axial pores of each ring and another set between peripheral structural elements, as represented on Figure 21[START_REF] Baker | ClpXP, an ATP-powered unfolding and proteindegradation machine[END_REF]. Joshi and colleagues demonstrated that the major determinant of interaction within E. coli ClpXP complex, are surface ClpX loops, which contains a IGF conserved motif. They observed mutations that ClpX IGF loop lead to a weakening or elimination of the ClpX-ClpP interaction without affecting other

Figure 20 .

 20 Figure 20. Cartoon model of substrate recognition and degradation by the ClpXP protease. In an initial recognition step, a peptide tag in a protein substrate binds in the axial pore of the ClpX hexamer. In subsequent ATP-dependent steps, ClpX unfolds the substrate and translocates the unfolded polypeptide into the degradation chamber of ClpP for proteolysis, where it is cleaved into small peptide fragments. (Baker and Sauer 2012).

Figure 21 .

 21 Figure 21. Interaction of ClpX and ClpP. The ClpXP complex is stabilized by peripheral interactions between the IGF loops of ClpX and hydrophobic clefts on ClpP, and by axial interactions between the pore loops of ClpX and the N-terminal stem-loop of ClpP (Baker and Sauer 2012).

Figure 22 .

 22 Figure22. Representation of the expansion and contraction of the axial pore by a snake-jaws model. The main contacts between these jaws are formed by the interfaces between the large and small AAA+ domains of the unloadable (U) ClpX subunits. In which pore size is controlled by the size of the substrates and the conformation of the hinge region and flanking structure of the unloadable (U) ClpX subunits. Opening of these interfaces, as shown in the exaggerated right cartoon, provides a potential mechanism for pore expansion to accommodate large substrates, including those with multiple chains. Each colour represents a ClpX protomer. (L) loadable subunits.[START_REF] Baker | ClpXP, an ATP-powered unfolding and proteindegradation machine[END_REF].

Figure 23 .Figure 24 .

 2324 Figure 23. The B. subtilis SppA protein. (A) A schematic diagram showing the full-length SppA B. subtilis with its predicted transmembrane segment (pink) and the confirmed amino-terminal thermolysin cleavage site. The light-green region is what is observed in the electron density. (B) A topology diagram of SppA B. subtilis showing the full-length protein with β-strand as arrows and αhelices as cylinders. The protein is colored as a gradient from N-terminus (blue) to C-terminus (red). The regions not seen in the electron density are shown as bro-ken lines. (C) A cartoon diagram showing the tertiary structure of the SppA B. subtilis protomer. The βstrands are shown as arrows, and the αhelices are shown as cylinders. The color scheme is the same as in (B). The nucleophile Ser147 (red) and the general base Lys199Ala (blue) are shown as spheres (Nam, Kim, and Paetzel 2012).

Figure 25 .

 25 Figure 25. Cross section representation of the oligomeric dome shape structure of SppAEC and SppABS. (A) A section through the middle of SppAEC revealing the dimensions of the opening to the bowl-shaped structure. The catalytic residues S409 (green) and K209 (magenta) are shown as van der Waals spheres. (B) A crosssection view of the SppABS octamer, revealing the continuous concave groove that encircles the entire inner bowl, with dimensions given. Adapted from Kim, Oliver, and Paetzel 2008 and S. E. Nam, Kim, and Paetzel 2012.

Figure 26 .

 26 Figure 26. Growth analysis of strains harbouring C-terminal SPA-tag fusions. The different strain cultures were grown overnight in LB supplemented with 0.6 µg/ml Erythromycin and 0.5mM IPTG at 37°C, and in the next day diluted 1:200 into a new medium with the same supplements. Compared to the BSB1 WT strain the SPA-tag fusions do not affect the growth of the mutant strains. Samples were taken on specific time points.

Figure 27 .

 27 Figure 27. Expression of the SPA tag fused proteins. (A) Table with the corresponding molecular weights of each SPA tagged protein in kDa. (B), (C) and (D) Western blot analysis, using an anti-FLAG specific antibodies. Cell samples from exponentional, transition and stationary phases were loaded for each strain: SecDF-SPA (a), SecG-SPA (b), SipS-SPA (c), SipT-SPA (d), BdbC-SPA (e), SppA-SPA (f), YetJ-SPA (g), SpoIIIJ-SPA (h) and BdbB-SPA (i).

Figure 28 .

 28 Figure 28. Purification of SpoIIIJ-SPA and SecDF-SPA protein with different concentrations of DDM. (A) Western blot analysis, using an anti-FLAG specific antibodies, of SecDF-SPA and SpoIIIJ-SPA in the presence of a range of n-Dodecyl β-D-maltoside (DDM) concentrations, from 0.5% up to 2%. (B) Identification by LC-MS/MS of the expected protein partner: poitive (٧) negative (-).

  components of a stable complex are not separated and migrate together in the gel. The n-Dodecyl β-Dmaltoside (DDM) was the detergent chosen to solubilize the membrane samples obtained after ultracentrifugation of disrupted B. subtilis cells. After solubilisation (1% DDM) the samples are supplemented with an anionic dye, Coomassie blue G-250, and the protein complexes are separated according to their sizes by electrophoresis on a gradient 4-16% polyacrylamide gel. High molecular weight complexes are running high in the gel. If the concentration of detergent is too high, these complexes dissociate and individual proteins are running lower in the gel.

Figure 29 .

 29 Figure 29. Analysis by Western blot of a Blue native PAGE 4-16% Bis-Tris Gel of SecDF-SPA in the presence of different concentrations of DDM. The triangle on the top represents the increase of DDM concentration, from 0.05% up to 2%. The red circles represent the loaded samples, which with the increase of the DDM concentration decreases its position in the gel.

Figure 30 .

 30 Figure 30. Number of peptides identified, by LC-MS/MS for each of the proteins purified in the presence of 1% DDM. On the top of the figure, the gel lane represents how it is divided for in gel trypsin digestion and posterior LC-MS/MS analysis. These graphs represents the number of peptides identified for SppA, YteJ, SpoIIIJ and SecDF in function of each piece of gel.

  putative structure using structure prediction softwares such as Protter (Figure S 1 of appendix chapter)[START_REF] Omasits | Protter: interactive protein feature visualization and integration with experimental proteomic data[END_REF]. Using BLAST-P to align the sequence of the Bacillus subtilis sppA yteJ operon with different bacterial genomes of different bacterial species, the results showed that yteJ is often present following sppA. The sppA gene is not only present in B. subtilis but also in most of the bacteria from the firmicutes phylum, including pathogenic bacteria as listeria monocytogenes (Figure31).

Figure 31 .

 31 Figure 31. Alignment of sppA and yteJ genes with other bacteria of firmicutes phylum. The red highlighted box indicate the sppA yteJ operon. (Overmars et al. 2013).

  and the sfGFP (superfolder GFP). One construct possessed the sequence of the sppA yteJ σ Wdependent promoter, a fragment of 100 bp upstream the sppA gene and including the natural RBS (Figure32). A second construct possessed both σ A and σ W -dependent promoter sequences, a fragment 85 bp longer than the previous fragment, later denoted σ AW . The promoter positions and lengths were defined based on supplementary data provided in[START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF].

Figure 32 .

 32 Figure 32. Transcriptional fusions of the Pσ AW and Pσ W , promoters and sfGFP. Pσ W strain contain a fusion of a fragment of 100bp upstream sppA and Pσ AW strain a fragment 185bp upstream sppA. Both constructions harbour the sppA yteJ native RBS. These constructs were inserted at the ectopic amyE locus together with a Spectinomycin resistance cassette.

2

 2 OD600nm. OD600nm and fluorescence were recorded at time intervals of 10 minutes. The Figure 33 and Figure 34 display the Pσ AW and Pσ W strains growth curves and the fluorescence/OD600nm ratio of sfGFP after induction (time point 0 min) with nisin or subtilin, respectively. The background signal and the auto-fluorescence intensity were subtracted from all the graphs.

Figure 33 .

 33 Figure 33. Nisin induced increasing expression of sfGFP, regulated by σ A and σ W sigma factors. Transcriptional fusion of sfgfp with the full σ A and σ W dependent promoter sequence (P AW ) or only the σ W dependent promoter sequence (P W ) found upstream of sppA yteJ operon. The sfGFP expression was induced with different concentrations of nisin, from 1µg to 6µg. Each graph line corresponds to a specific nisin concentration, labelled on the left of the panel. The cell growth is shown on the left column of the panel while the sfGFP fluorescence intensity /OD600nm is presented on the right. The dashed black line highlights the intensity peak always found 60min after induction. represents the Pσ AW and Pσ W growth (left panel) and fluorescence/OD600nm (right panel).

Figure 34 .

 34 Figure 34. Subtilin induced increasing expression of sfGFP, regulated by σ A σ W sigma factors. Transcriptional fusion of sfgfp.with the σ A and σ W dependent promoter sequences, native of the sppA yteJ operon. The sfGFP expression was induced with different concentrations of of subtilin, from 5% up to 30%. Each graph line corresponds to a specific subtilin concentration, labelled on the left of the panel. The cell growth is shown on the left column of the panel while the sfGFP fluorescence intensity /OD600nm is presented on the right. The dashed black line highlights the intensity peak always found 60min after induction.represents the Pσ AW and Pσ W growth.

Figure 35 .

 35 Figure 35. Growth of B.subtilis sppA and yteJ deletion mutant strains. The control strain (BSB1) culture was grown overnight in LB and the mutant strains (∆yteJ, ∆sppA and ∆sppA yteJ) grown in LB supplemented with 5 μg.ml -1 chloramphenicol at 37°C, and the next day diluted 1:400 into a new medium with the same conditions.

Figure 36 .

 36 In exponential phase, first line of Figure 36, comparing the cells of the BSB1 strain (on the left side of the panel)

Figure 36 .

 36 Figure36. Cell morphology of the sppA yteJ deletion mutant. The strains, control strain (BSB1) and ∆sppA yteJ, were grown overnight in LB at 37°C, supplemented with 5μg/ml chloramphenicol when required, and in the next day diluted 400-fold into fresh medium with the same conditions. Samples were collected during the exponential (exp), transition (tran) and stationary (stat) phases for microscopy analysis. Phase contrast microscopy pictures and corresponding red fluorescence microscopy (515/640nm) are shown. Membranes (false coloured red) were stained with FM4-64. Scale bar is 10 micron.

Figure 37 .

 37 Figure 37. Analysis of AmyM extracellular amount by SDS PAGE. pCS74 empty plasmid; pCS73 plasmid encoding amyM gene. The BSB1 wild type strain, BSB1 with pCS74 empty plasmid and with the pCS73 AmyM plasmid were used as controls. The black arrow points for the correct band of AmyM.

  5 µg.µl-1. These results are shown at Figure 39.

Figure 39 .

 39 Figure 39. SDS PAGE analysis of AmyM extracellular amount produced by B. subtilis WT and overexpression mutant strains. This picture corresponds to a 10% SDS-PAGE gel, coomassie staining. The BSB1 wild type strain without and with the pCS73 AmyM plasmid were used as controls. BSB1 PhsyteJ, BSB1 PhssppA and BSB1 Phs sppA yteJ stands for B. subtilis yteJ, sppA and sppA yteJ overexpression mutant strains, respectively. pCS73 multi copy plasmid encoding amyM gene. The black arrows highlight the 85 kDa band of the protein standard and the band corresponding AmyM (75.2 kDa).

Figure 40 .

 40 Figure40. Capacity of survival of BSB1 and ∆yteJ, ∆sppA and ∆sppA yteJ deletion mutants. The strains were grown in rich liquid media (LB), at 37°C for 48h. After 18h, 24h and 48h, samples of the cultures were collected and plated out onto LB agar plates. The number of colonies were counted after the plates have been incubated for 18h at 37°C.

Figure 41 .

 41 Figure 41. Long-term survival of BSB1 and ∆sppA yteJ deletion mutant, in LB and M9 liquid media. (A) Strains grown in LB, a rich liquid media; (B) strains grown in M9 minimal medium. Strains were grown at 37°C for 48h. After 24h and 48h, samples of the cultures were collected and plated out onto LB agar plates. The number of colonies were counted after the plates have been incubated for 18h at 37°C.

  Figure 42.

Figure 42 .

 42 Figure 42. Complementation assays in LB with BSB1 and ∆sppA yteJ deletion mutant strains. (A) results obtained after 24h of growth; (B) results obtained after 48h of growth; BSB1 strain was plated in LB agar; ∆sppA yteJ mutant strain was plated into LB agar supplemented with Cm 5µg/ml; Comp-stands for complementation mixed sample. A mixture of 50% BSB1 and 50% ∆sppA yteJ composes this sample. Comp1 shows the number of colonies counted when the Comp sample is plated into LB agar and Comp2 LB agar supplemented with Cm 5 µg/ml.

  with aeration and vigorous shaking in flasks. The overnight cultures were diluted 20-fold into 100µl (into the 96 well plate) of fresh pre-warmed LB medium and grown until beginning of exponential phase. Then the cells were re-diluted 400-fold into a new plate, containing different concentrations of nisin or subtilin. The Figure43shows the results obtained with nisin.

Figure 43 .

 43 Figure 43. The growth of B. subtilis sppA and yteJ deletion mutant strains in the presence of different concentrations of Nisin. The BSB1, ∆sppA and ∆yteJ strains grown in LB in a 96-well plate, where the OD600 was measured at specific time points. The different graphs represent the growth of the strains in the presence of different concentrations (2 µg, 3 µg, 4 µg, 6 µg and 8 µg/ml) of nisin, labelled on the left of the graph.

Figure 44 .

 44 Figure 44. Growth of the B. subtilis sppA and yteJ mutant strains in the presence of different concentrations of subtilin. The BSB1, ∆sppA and ∆yteJ strains were grown in LB in a 96-well plate, and the OD600 was measured at specific time points. The different graphs represent the growth of the strains in the presence of different concentrations (20%, 25%, 30%, 34% and 38%) of subtilin, labelled on the left of the graph.

Figure 45 .

 45 Figure 45. The growth rates of BSB1, ∆sppA and ∆yteJ in the presence of different concentrations of subtilin. The BSB1, ∆sppA and ∆yteJ strains were grown in LB in a 96-well plate, and the OD600 was measured at specific time points. The different columns of the graph represent the growth rates of the strains in the presence of different concentrations (20%, 25%, 30%, 35% and 40%) of subtilin.

Figure 46 .

 46 Figure 46. The growth of B. subtilis sppA and yteJ deletion mutant strains in the presence of different concentrations of vancomycin. The BSB1, ∆sppA and ∆yteJ deletion strains grown in LB in a 96-well plate. The OD600 was measured at specific time points. The different graphs represent the growth of the strains in the presence of different concentrations (0µg, 0.1µg, 0.2µg, 0.5µg and 1µg) of vancomycin, labelled on the left of the graph. Colours, on the right side of the picture, indicate the wild type or mutated strains.

Figure 47 .

 47 Figure 47. The growth of B. subtilis sppA and yteJ deletion mutant strains in the presence of different concentrations of erythromycin. The BSB1, ∆sppA and ∆yteJ deletion strains grown in LB in a 96-well plate. The OD600 was measured at specific time points. The different graphs represent the growth of the strains in the presence of different concentrations (0 µg, 0.1 µg, 0.2 µg, 0.5 µg and 1 µg) of erythromycin, labelled on the left of the graph. Colours, on the right side of the picture, indicate the wild type or mutated strains.

Figure 48 .

 48 Figure 48. The B. subtilis sppA and yteJ deletion mutant strains treated with different concentrations of lysozyme. The BSB1, ∆sppA and ∆yteJ deletion strains grown in LB in a 96-well plate. The OD600 was measured at specific time points. The different graphs represent the growth of the strains in the presence of different concentrations (0 µg, 50 µg, 100 µg, 150 µg, 200 µg and 250 µg) of lysozyme, labelled on the left of the graph. Colours, on the right side of the picture, indicate the wild type or mutated strains.

Figure 49 .

 49 Figure 49. The B. subtilis sppA and yteJ overexpressing strains. The BSB1, PhssppA and PhsyteJ overexpression strains grown in LB in a 96-well plate, and the OD600 was measured on specific time points. The different graphs represent the growth of the strains in different conditions: top left strains growing in LB; top right-LB with 30% subtilin; remaining graphs: growths of the strains in 30% subtilin and different concentrations (10 µM, 25 µM, 50 µM and 100 µM) of IPTG. The conditions are labelled on the top of each graph.

Figure 50 .

 50 Figure 50. The B. subtilis sppA yteJ overexpressing strain. The BSB1 and PhssppA yteJ strains were grown in LB in a 96-well plate, and the OD600 was measured at specific time points. The different graphs represent the growth of the strains in different conditions: top left in LB; top right-30% subtilin; remaining graphs: growth of the strains in 30% subtilin and different concentrations (10 µM, 25 µM, 50 µM and 100 µM) of IPTG. The conditions are labelled on the top of each graph.

Figure 51 .

 51 Figure 51. Cell morphology of the sppA, yteJ or sppA yteJ overexpression mutants. The strains, BSB1, Phs sppA, Phs yteJ and Phs sppA yteJ were grown overnight in LB at 37°C, supplemented with 5 μg/ml Cm and 100 µg/ml Spec when required, and diluted 400-fold into fresh LB medium. Then samples were collected during the early stationary (stat) phase for microscopy analysis. Phase contrast microscopy and fluorescence microscopy pictures are shown. Membranes (false coloured red) were stained with FM4-64 and DNA (false coloured blue) were stained with DAPI. Scale bar is 10 micron.

Figure 52 .

 52 Figure 52. Cell morphology of the sppA and sppA K199A overexpression mutants. The strains, control strain (BSB1), PhssppA and PhssppA K199A were grown overnight in LB at 37°C, supplemented with 100 µg/ml Spec when required. The next day the strains were diluted 400-fold into (A) LB and (B) LB supplemented with 100 µM IPTG. Then samples were collected during the early stationary (stat) phase for microscopy analysis (Phase contrast microscopy pictures). Scale bar is 10 micron.

Figure 53 .

 53 Figure 53. SppA and YteJ cell localization. The strains, YteJ-sfGFP and sfGFP-SppA, contain a sfGFP translational fusion with yteJ and sppA genes, respectively. Strains were grown in LB at 37°C; samples were collected during the early stationary (stat) phase for microscopy analysis. Phase contrast microscopy and fluorescence microscopy pictures are shown. sfGFP is false coloured of green. Scale bar is 10 micron.

Figure 54 .

 54 Figure 54. Scheme representing the different His-tag constructions. The rectangles represents SppA and YteJ proteins, orange represents cytosolic domain, green the transmembrane domains and blue extracellular domain. The numbers represent the length in amino-acids.

  Figure 55B) shows a band of the expected size for YteJ-His, around 20 kDa. When SppA is expressed together with YteJ-His (lane 4, Figure 55A), SppA (highest band ≈36 kDa) was pulled down with

Figure 55 .

 55 Figure 55. SDS PAGE analysis of SppA ∆1-52 , SppA, SppA/YteJ (A) and YteJ His-tagged (B) purified proteins. Analysis of the purified samples on 12.5% SDS-PAGE gels, coomassie blue stained. For both gels, A and B, the first lane corresponds to protein markers, which the bands correspondent to 40, 30, 25 and 20 kDa are highlighted.

Figure 56 .

 56 Figure 56. SDS PAGE analysis of SppA ∆1-52 activity. For both gels, (A) and (B), the first lane corresponds to protein markers, for which the bands corresponding to 40 and 25 kDa are indicated. SppA ∆1-52 alone at the last right lane of gel A.. Protein substrates: bovine serum albumin (BSA), GFP ext , lysozyme, E. coli DNA single strand binding protein (SSB), B. subtilis nucleoid associated protein (Rok), E. coli DNA-binding protein (Ec-HNS) and B. subtilis catabolite control protein A (CcpA). (-) protein substrate alone and (+) SppA ∆1-52 and the substrate protein. The samples were incubated 16 h at 37°C.

Figure 57 .

 57 Figure 57. SDS PAGE analysis of SppA ∆1-52 , SppA and SppA/YteJ activity. A and B corresponds to 12.5% SDS-PAGE gels, coomassie blue stained. A. activity after 16h at 37°C; B. kinetics of 60 minutes of activity at 37°C. C. Graph of protease activity kinetics of the different forms of SppA. The intensity of the product bands were measured by Image Lab (Biorad software).

  YteJ, two peptides belonging to the C-terminal domain of SppA were present (peptides are highlighted in Figure 58B (highlighted by green and yellow). (iii) finally the second major band on lane 4 was YteJ. We therefore concluded that the presence of YteJ inhibits the SppA-mediated self-cleavage of its C-terminal part. The presence of a normally absent C-terminal domain in an active SppA indicated that SppA was inactive in the complex SppA/YteJ, and it confirmed the results obtained with the enzymatic experiments shown in Figure 57.

Figure 58 .

 58 Figure 58. SDS-PAGE and mass spectrometry analysis of SppA and SppA/YteJ complex (A) The first lane corresponds to protein marker, for which the band corresponding to 40 kDa is highlighted. All the proteins contain a his-tag. Expected bands for SppA K199A and SppA≈37 kDa and YteJ 19 kDa. (B) SppA protein sequence. The major bands of SppA and SppA/YteJ were analysed by MS/MS as indicated in the figure with labels i, ii, iii. The MS/MS analysis showed that the band of SppA, in the SppA/YteJ sample, contains two peptides belonging to the C-terminal domain of SppA (highlighted in green and yellow).

Figure 59 .

 59 Figure 59. Scheme of SppA/YteJ protein complex in the different YteJ truncated constructions. (A) sppA yteJ overexpression strain; (B) sppA yteJ∆152-164 overexpression strain, the las 12 amino-acids from YteJ Cterminal were removed; (C) sppA yteJ∆129-164 overexpression strain, truncated YteJ from thirty five amino-acids from its C-terminal region; (D) sppA yteJ∆92-164 overexpression strain, the C-terminal domain was removed and substituted by a SPA-tag; Orange represents cytosolic domain, green the transmembrane domains and blue extracellular domain. The numbers represent the length in amino acids.

Figure 60 .

 60 Figure 60. Cell morphology of the overexpressing strains. All the strains grown in LB until early stationary phase, then a sample was taken for microscopy analysis. The first panel (left side) shows the phase contrast microscopy pictures of un-induced strains, indicated genotypes. The remaining panels (from left to right side) represent the phase contrast and fluorescence microscopy of the indicated strains grown in IPTG presence. sppA, sppA yteJ, sppA yteJ ∆152-164 , sppA yteJ ∆129-164 and sppA yteJ ∆92-164 SPA were expressed from an artificial Phs promoter and induced with 50µM IPTG. Membranes (false coloured red) were stained with FM4-64 and nucleoids (false coloured blue) were stained with DAPI. Scale bar is 10 micron. Black arrows represents the cell size, by indicating the end and begging of the cell; White triangles represents the cell septum or septum formation points.

Figure 60 )

 60 restored the long phenotype observed for the sppA overexpression. Contrarily, the strain containing a swap between the C-terminal region of YteJ by the SPA-tag (sixth line Figure 60), Phs (sppA-yteJ ∆92-164 SPA), show shorter cells similar to the BSB1 and PhssppA yteJ cells (first and third lines Figure 60). However, these cells show cell wall and division problems. These results allowed us to conclude that the C-terminal domain of YteJ is involved and important for the regulation of SppA activity, as the removal of the last 25 amino acids affected the cell growth, and that removing the last 45 amino acids of YteJ led to a a cell phenotype similar to what was observed with the overexpression of SppA alone, i.e. a complete deregulation.of SppA protease activity.

Figure 61 .

 61 Figure61. Growth of BSB1 and ∆sppA in the presence of different subtilin solutions. The BSB1 and ∆sppA strains were grown in LB in a 96-well plate, and the OD600 was measured at specific time points. The different graphs represent the growth of the strains in different conditions: top left in LB without subtilin; top right LB supplemented with 30% subtilin; in the remaining graphs, the strains grown in 30% subtilin treated 16h at 37 °C just in reaction buffer 200mM NaCl) or mixed with SppA ∆1-52 or proteinase K, respectively; The conditions are labelled on the top of each graph.

Figure S 3 )

 3 , in Prof.van Dijl's laboratory (Groningen, The Neterlands). Their results showed no major effect of any deletion (ΔsppA, ΔyteJ or Δsppa yteJ) on the secretion of the model protein AmyM. Taking into account these results, our conclusions are that neither SppA nor YteJ are directly involved in protein secretion. It does not rule out any indirect effect that could explain what was previously observed.

Figure 51

 51 Figure 51 the affected long cells of the PhssppA strain are not just only filamentous but they seem to show a defect on cell division, malformation of the septum and impaired chromosomal segregation. This morphological phenotype defect is reminiscent of the cell defect phenotype described in deletion mutants involving protein regulators of the divisome. The B. subtilis cell division is well coordinated with the elongation, as it requires a switch in the orientation of the cell wall synthesis, and the formation of the mid-cell Z ring for cell constriction and division (Adams and Errington, 2009; Leaver et al., 2009; Errington and Wu, 2017). The Min system prevents the polar division of the cells. It was shown that the deletion of B. subtilis noc combined with the depletion of MinD lead to a defect in the division, absence of division septa formation and elongated filaments phenotype, similar to what we observed on our results obtained for the overexpression of SppA (Figure Figure 51 and Figure 60) (L.

Figure 62 .

 62 Figure 62. Cells mutated for minJ are filamentous. Fluorescence microscopy of wild type (A), ∆minJ (B) were grown to mid-exponential phase and stained with FM 4-64 (membrane, false coloured red) and DAPI (chromosome, false coloured blue). Scale bar is 4 mm. (Patrick and Kearns 2008).

Figure 63 .

 63 Figure 63. Morphology of B. subtilis cells lacking multiple penicillin-binding proteins (Pbp). (A) wild-type strain; (B) cells lacking Pbp1; (C) cells lacking Pbp1 and 4; (D) cells lacking (Adapted from Popham and Setlow 1996)).

  [START_REF] Shao | Characterization of a functionally unknown arginine-aspartateaspartate family protein from halobacillus andaensis and functional analysis of its conserved arginine/aspartate residues[END_REF], it is clearly related to H. andaensis RDD (Figure64). Moreover the corresponding rdd gene was identified adjacent to a putative sppA gene. B. subtilis YteJ shares all six conserved arginine and aspartate residues identified in the homologs of H. andaensis RDD[START_REF] Shao | Characterization of a functionally unknown arginine-aspartateaspartate family protein from halobacillus andaensis and functional analysis of its conserved arginine/aspartate residues[END_REF], and overall the two protein sequences show 39% identity.

Figure 64 .

 64 Figure 64. Amino acid sequence alignment between H. andaensis RDD and B. subtilis YteJ. Conserved amino acids are highlighted in red.

Figure 65 .

 65 Figure 65. Model for the regulation of SppA by YteJ. (A) shows the SppA² inactive state, where YteJ is structurally blocking SppA active site; A cell stress (represented by the black icon) induces a cell response that leads to complex conformational change (represented by a black arrow). This structural modification liberated the SppA active site, passing to its active state (B).

Figure 66 .

 66 Figure 66. LIC integration into B. subtilis. (A)The insert, corresponding to the last 400bp of the gene of interested, was amplified using the forward and reverse primers P1 and P2. These primers contain specific sequences homologous to the pMUTIN-SPA vector allowing the hybridization between the insert and the vector. After mixed the insert with the vector, the sample was transformed into E.coli MC1061 strain, a step that allows the amplification of the plasmid. (B) Thereafter, the final product plasmid was transformed into B. subtilis and inserted into the chromosome by simple cross-over. Then a fragment containing the gene of interest fused to the SPA tag was amplified from the genomic DNA of the final strain and sequenced.

4. 3 . 3

 33 SppA, YteJ and SppA-YteJ overexpressing strainsWith the goal of re-insert sppA and yteJ into the deletion mutants, under the control of inducible promotor, to observe if we could recover the effect on lantibiotics resistance caused by their deletion we have constructed strains overexpressing sppA and yteJ. To obtain strains overexpressing SppA or YteJ, or both, we inserted the gene sequences fused to the Phyperspank promoter in the BSB1 chromosome at the amyE locus. For this, the entire gene sequences of sppA and yteJ and the sppA-yteJ operon, including the RBS sequence, were amplified by PCR from BSB1 genomic DNA , and fused to the Phyperspank promoter sequence. This promoter is Isopropyl-β-D-thiogalactopyranoside (IPTG) inducible and lacI-repressible promoter[START_REF] Overkamp | Benchmarking various green fluorescent protein variants in Bacillus subtilis , Streptococcus pneumoniae , and Lactococcus lactis for live cell[END_REF],. For this, the RBS+genes sequences were amplified by PCR using a specific pair of primers, the Hspank_P1 and Hspank_P2 of each gene, carrying the HindIII and SalI restriction sites. The resulting PCR products were digested with HindIII and SalI fast restriction enzymes and ligated into pDR111 plasmid, previously digested with the same enzymes. HindIII cuts into the sppA sequence, consequently the constructions involving this gene were done with a blunt 5' end and a sticky 3' end created by SalI digestion. The vector and the fragments were ligated and transformed into E. coli MC1061 competent cells. After the selection of transformants on 100 µg.ml-1 ampicillin LB-agar plates, the plasmids were extracted, purified and transformed into BSB1. The positive clones, harboring an integration of the Phyperspank+gene of interest at the amyE locus, were selected on 100 µg.ml -1 spectinomycin LB-agar plates, confirmed by PCR using primers in the amyE flanking regions, tested for the inability of the strains to produce amylase (starch-iodine reaction) and sequenced. With the objective of complementing the deletion mutants by reintroducing the deleted gene at the ectopic amyE locus, under the control of the inducible Phyperspank promoter, the final plasmids were also transformed into deletion mutant strains (GHBs023, GHBs025 and GHBs028).

  harboring a fusion of one and the two promoters with the DNA sequence of the sfgfp introduced at the ectopic locus amyE.. First, a fragment of 100bp upstream of sppA, containing the natural RBS and the σ W promoter sequence, and a 185bp fragment, including the natural RBS, σ W and σ A promoter sequences[START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF], were amplified from BSB1 genomic DNA by PCR, and then ligated upstream of the pSG13 plasmid-borne sequence of the fluorescent protein[START_REF] Guiziou | A part toolbox to tune genetic expression in Bacillus subtilis[END_REF]) by Gibson assembly. The final mix was transformed into E. coli MC1061 competent cells and plated onto 100 µg.ml -1 ampicillin LB-agar. The plasmid was extracted from positive E. coli clones purified and, after transformation of B. subtilis BSB1 competent cells, was integrated into the amyE locus. The positive clones were selected onto 100 µg.ml-1 spectinomycin LB-agar plates, the presence of the promoter-sfGFP sequence fusions were confirmed by PCR, the clones were also tested by starchiodine reaction and, finally, sequenced. The final strains were tested by LCA, as described on section 4.5.To study SppA and YteJ localization, N-terminal tagged sfGFP-SppA and C-terminal tagged YteJ-GFP proteins were conditionally expressed from the IPTG inducible Phyper-spank promoter, at the ectopic amyE locus of B. subtilis chromosome. The appropriate primers, one carrying a HindIII or SalI restriction site and the second carrying an extension with a linker, were used to PCR amplify the sppA and yteJ genes from BSB1 chromosomal DNA and the sfgfp gene from the pSG13 plasmid. These fragments were joined by PCR, digested by HindIII or SalI and ligated into the pDR111 plasmid, previously digested with the same enzymes. E. coli MC1061 cells were transformed by the ligation mixtures and transformants were selected onto 100 µg.ml-1 ampicillin LB-agar. The plasmid was extracted from positive E. coli clones and purified. The sfgfp-sppA and yteJ-sfgfp constructs were then integrated at the amyE locus of BSB1 competent cells by transformation, and transformant clones were selected using 100 µg.ml-1 spectinomycin LB-agar plates. The presence of the integrated sfGFP fusions were verified by PCR amplification from the amyE flanking regions and by the inability of the strain to produce amylase. The final constructs were sequenced.

  , which allows the high throughput acquisition of time-course profiles of bacterial cellular growth and gene expression. It measures both the optical density for recording cell growth and the fluorescence emission of the fluorescent reporter protein, usually the GFP, expressed in specifically modified strains of interest. The B. subtilis strains were grown overnight in 5ml of LB medium at 37°C and then diluted 20-fold in 100µL/well of fresh LB medium (pre-culture) into 96-well microtiter plate. The LB pre-cultures were grown until an OD600nm of 0.3-0.4 and then diluted 400-fold into a fresh medium, LB or CH defined medium. OD600nm and fluorescence (excitation 485, emission 528) were measured at time intervals of 10 minutes. Only the 60 central wells of the 96-well microtiter plates were inoculated since the wells around the edges exhibited evaporation higher than 5 % over 20 hours; therefore these peripheral wells were only filled in with sterile medium.

  pellet was washed two times with 200 µl cold acetone, repeating the centrifuge steps each time. The protein pellets were then dissolved by the addition of 3µl of 10 mM Tris-HCl pH 8, 8 M urea. The volume was then extended up to 25µl with 10 mM Tris-HCl pH 8, decreasing the urea concentration to 1 M. Then to analyze if the SPA tagged protein was captured, with possible interacting partners, the protein concentrations of the samples were measured by the Bradford's method and the same amount of protein was loaded and the samples were separated by native polyacrylamide gel electrophoresis (PAGE), which was coomassie stained after. These samples were analyzed by a second technique, nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). For this the samples were equal loaded into SDS-Page gel, as before, but the samples just run 1 cm into the resolving part of the gel. The gel was stained by Bio-Safe Coomassie G-250 stain solution and the small pieces of the gel containing the samples were cut and sent to be treated and analyzed by mass spectrometry.

Figure 67 .

 67 Figure 67. Sequential peptide affinity purification. (A) The SPA-tag is composed of three FLAG sequences (3×FLAG) and the calmodulin binding peptide (CBP) separated by a TEV protease cleavage site. (B) Schematic representation of the double step SPA purification. This technique allows the purification of protein complexes under native conditions.

  Fig ure 1| Seq ue nti al pe

  1% DDM. The resin was resuspended in 200 ml of TeV buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.5mM EDTA, 1 mM DTT) with 50units of Ac-TeV protease. After incubation at 4°C for overnight, the eluate was collected, diluted up to 2ml (4-fold dilution) and mixed with 200µl Ni-NTA preequilibrated agarose resin. The Ni-NTA columns were washed with 50ml 20mM Tris-HCl pH7,5, 250mM NaCl and 0.01% DDM (Buffer R3) supplemented with 5mM imidazole ,and then with 20 ml Buffer R3 supplemented with 20mM imidazole. After washing the proteins bounded to the Ni-NTA resin were eluted, sequentially, with 1ml of Buffer R3 supplemented with 50 mM imidazole and then 4ml of the same buffer with 200mM imidazole. The eluted proteins were concentrated by Amicon Ultra-15 Centrifugal Filters, mixed with 50% glycerol and storage at -20°C.

Figure S 1 Figure S 2 .

 12 Figure S 1 YteJ predicted structure by Protter. Extra-extracellular compartment; Intra-cytosolic compartment; The structure shows iYteJ amino acid composition, the amino acids are represented by the unique letter symbol (Omasits et al. 2014).

Figure S 3

 3 Figure S 3 Pulse chase assays for sppA, yteJ and sppA yteJ deletion mutants and complemented strains. All the strains were transformed with pHTK10 (AmyQ secretion),as such were grown at MBU supplemented with Kanamycin. (A) pulse chase gel results. P-pre-protein. M-mature protein; (B,C) Graphic representation of the AmyQ processing results.

Figure S 4 .

 4 Figure S 4. The growth of B. subtilis sppA and yteJ deletion mutant strains in the presence of different concentrations of LP9. The BSB1, ∆sppA and ∆yteJ deletion strains grown in LB in a 96-well plate, where the OD600 was measured on specific time points. The different graphs represent the growth of the strains in the presence of different concentrations (0 µg, 50 µg, 75 µg, 100 µg, 150 µg and 200µg) LP9, labelled on the Top of the graph. Colours, on the picture, indicate the wild type or mutated strains; Black arrow represents the time point of induction with LP9. Data from our collaborators at the Prof. Jan Maarten van Dijl.

Figure S 5 .

 5 Figure S 5. The growth of B. subtilis sppA and yteJ overexpressing mutant strains in the presence of different concentrations of LP9. The BSB1, ∆sppA and ∆yteJ deletion strains grown in LB in a 96-well plate, where the OD600 was measured on specific time points. The different graphs represent the growth of the strains in the presence of different concentrations (0 µg, 50 µg, 75 µg, 100 µg, 150 µg and 200µg) LP9, labelled on the Top of the graph. Colours, on the picture, indicate the wild type or mutated strains; Black arrow represents the time point of induction with LP9. Data from our collaborators at the Prof. Jan Maarten van Dijl.

Figure S 6 .

 6 Figure S 6. AmyM supernatant purification. The AmyM sample was purified by anionic exchange (Q-Sepharose resin). The different eluted samples and fractions were analysed by 10% SDS-PAGE gel. The description for each lane shown at the right side of the picture.This experiment was repeated but the result obtained was the same. The expiry date can may be the reason why the AmyM sample is not binding to the Q-rresin. To test this hypothesis, the concentrated enzyme sample was applied to a new Q-resin charged with two columns volumes of 20 mM Tris HCl and 1 M NaCl, washed with and equilibrated with 20 mM Tris HCl pH 7.5 and 5 mM CaCl2. Like the previous experiments the protein was collected from the run-trough. This fraction was reapplied to a Size exclusion Hitrap 16/60 Superdex 200 column, that allows the separation of the protein and contaminants based on their size. The protein was eluted as a single peak with an apparent molecular weight of 75 KDa, according with the molecular weight predicted. Although, the protein has been eluted in a single peak the analysis by SDS-PAGE gel of each fractions correspondent to the protein elution showed that some contaminants were eluted at the same peak (Figure S 7).

Figure S 7 .

 7 Figure S 7. AmyM Size exclusion purification results. The protein sample was size-fractionated at 4 °C using a Hitrap 16/60 Superdex 200 column equilibrated with 20 mM Tris HCl pH 7.5, 5 mM CaCl2 and 100 mM NaCl buffer and eluted at a flow rate of 2 ml/min. The legend of the loaded samples is described at the right side of the figure.

  Characterization of a Functionally Unknown Arginine-Aspartate-Aspartate Family Protein From Halobacillus andaensis and Functional Analysis of Its Conserved Arginine/Aspartate Residues. Frontiers in Microbiology, 2018. 9: p. 807-807.

TABLE OF CONTENT

 OF 1 INTRODUCTION ............................................................................................................................. 1.1 General introduction ............................................................................................................. 1.1.1 Project context .............................................................................................................. Bacillus subtilis, a Gram-positive bacterium ................................................................. Gram-positive model ................................................................................. 1.1.4 B. subtilis: a bacterium of industrial interest ................................................................ 1.2 Protein translocation across cytoplasmic membrane in bacteria ......................................... 1.2.1 The general protein secretion pathway: the Sec system .............................................. 1.2.2 Substrate recognition ....................................................................................................

	1.1.2
	1.1.3	B. subtilis: a

1.2.3 Intracellular chaperoning and targeting to the Sec translocase ................................... 1.2.4 Twin-Arginine Transport (Tat) Pathway ........................................................................ 1.2.5 Flagella Export Apparatus (FEA) .................................................................................... 1.2.6 Type IV Secretion Systems .............................................................................................

Table 1 .

 1 Set of proteins involved in the Sec pathway chosen to be SPA-tagged for tandem affinity purification……………………………….…………………..……………………………...65

Table 2 .

 2 Oligonucleotides used for LIC constructions………………………………………...……..137

Table S 1

 S . Stock and working solutions of growth supplements…………………..………………...180

Table S 2

 S . Stock and working solutions of antibiotics…………………………...…………………..180

Table S 3

 S . List oligonucleotides………………………………………………………...……………181

Table S 4

 S . List of B. subtilis strains………………….………….…………………………………...188

Table S 5

 S List of E. coli strains……………………………………………………………...……….190

Table S 6

 S List of plasmids………………………………………………………..….……………….191

  . subtilis by co-expressing SecB from E. coli together with a B. subtilis hybrid SecA protein, in which the C-terminal 32 amino acids were replaced by the corresponding part of SecA from E. coli[START_REF] Diao | Functional implementation of the posttranslational SecB-SecA protein-targeting pathway in Bacillus subtilis[END_REF]. This data indicated that, in this route, SecA plays part of the role of SecB.

	pathway in BHowever, B. subtilis possibly has another homologous protein of SecB that contains other binding
	domains that might serve as a docking site for the SecB analogue. The cytosolic CsaA protein is the
	possible homologue of SecB in B. subtilis. It was demonstrated, that, a CsaA has a chaperone-like
	activity both in vitro and in vivo, with affinity for SecA and preproteins, as well as a role in the protein
	secretion, as the suppression of the csaA gene leads to a decrease of the secretion efficiency in B.
	. The absence of outer
	membrane proteins in Gram-positive bacteria might explain why a homologue of SecB has not yet
	been found in this group of organisms. However, according to Fekkes et al. (1997) the B. subtilis
	SecA protein contains a highly conserved binding domain that is present in SecB, which suggests that
	SecA has a chaperone-like function (Fekkes, Van der Does, & Driessen, 1997). In 2012, Diao et al.
	showed a significant improvement on the secretion of a maltose binding protein (MalE11) and an
	alkaline phosphatase (PhoA) with the implementation of an artificial posttranslational protein targeting

  Together, the LTAs and the WTAs, create a negative gradient that goes from the bacterial cell surface until the outer most layers of the PG[START_REF] Weidenmaier | Teichoic Acids and related cell-wall glycopolymers in grampositive physiology and host interactions[END_REF][START_REF] Pasquina | Teichoic acid biosynthesis as an antibiotic target[END_REF]. B. subtilis has two distinct phosphate rich WTAs, the poly(glycerol phosphate) and poly(glucosyl N-acetylgalactosamine 1-phosphate),

Table 1 . Set of proteins involved in the Sec pathway chosen to be SPA-tagged for tandem affinity purification.

 1

	Name	Protein	Function	Molecular weight (kDa)
	SecDF	Preprotein translocase subunit (ATPase)	Regulates SecYEG interaction activity of SecA with the and	81.47
	SecG	Sec subunit	tanslocase	Protein secretion	7.86
				Cleavage	of	the
	SipT	Signal peptidase I	signal peptide from	21.71
				the emerging protein

  is found in three other proteins in the B. subtilis genome (Yczc, YckC, YxaI), and the Pfam database indicates that this RDD domain is present in many transmembrane proteins, mainly in bacteria, but also with some occurrences in Archeae and Eukaryota. It could be the main domain of the protein or associated with other domains of various functions. Until recently the molecular function of this RDD domain remained completely elusive. However, looking for novel Na + /H + antiporters, Shao et al (2018) identified a protein named RDD in the moderate halophile Halobacillus andaensis. These authors demonstrated that the RDD protein functions as an Na + (Li + , K + )/H + antiporter, but also that four out of six conserved arginine or aspartate residues were indispensable to the antiporter activity[START_REF] Shao | Characterization of a functionally unknown arginine-aspartateaspartate family protein from halobacillus andaensis and functional analysis of its conserved arginine/aspartate residues[END_REF]. Although B. subtilis YteJ did not appear in the sequence alignment showed by Li

  Bertani medium (LB) was used for general-purpose growth of E. coli and B. supplemented with 30 % (v/v) glycerol and stored at -80 °C in cryovials. Growth media and buffers are described below in the Appendix.

	subtilis. For B. subtilis DNA transformation, MC media was used (described at the transformation of
	B. subtilis section). For specific experiments we used CH defined medium, M9 or Belitsky minimal
	media. In general, the bacterial growth was at 37 °C with orbital shaking at 200 rpm, unless otherwise

stated. Growth supplements (Table S 1) and antibiotics (Table

S

2) were added to the media when appropriate. Growth was monitored by measuring optical density at 600 nm (OD600nm) in a spectrophotometer. For long-term storage of bacterial strains, LB broth cultures, after overnight growth, were

Table 2 Oligonucleotides used for LIC constructions

 2

	(Sequences required for LIC are underlined)
	Sequence	Primer name	Primer sequence
	amplified
	secDF	secDF_P1	GGGTTCCTGGCGCGAGCTTGCTATAGCTTCTATTGG
		secDF_P2	TTGGGCTGGCGCGAGC
			TTGCGCCGAATCTTTTTTCAG
		secDF_seq	ACGGATCTGATCCAAATGTCAGC
	secG	secG_P1	GGGTTCCTGGCGCGAGCCACGCAGTTTTGATTACC
		secG_P2	TTGGGCTGGCGCGAGC TAGGATATAAGCAAGCGC
		secG_seq	TCTGATACAGCGTGACATGC
	spoIIIJ	SpoIIIJ_P1	GGGTTCCTGGCGCGAGC
			AGCTTCAACAGGAAACAATGGC
		SpoIIIJ_P2	TTGGGCTGGCGCGAGC CTTTTTCTTTCCTCCGGC
		SpoIIIJ_seq AGTTCGAAAGCGATGCAGGC
	bdbB	BdbB_P1	GGGTTCCTGGCGCGAGC
			TGGCACAATGGCTAGTTTATTC
		BdbB_P2	TTGGGCTGGCGCGAGC ATTATATACATGTTGATTTTG
		BdbB_seq	TCTTGCCAACTCACTCCAC
	bdbC	BdbC_P1	GGGTTCCTGGCGCGAGCTATGCTTCCTGGGTTGTG

  Table S2 of appendix.

Table S 3

 S ). The resulting PCR product was digested with NotI and XhoI fast restriction enzymes and ligated into pJ411_6His, previously digested with the same enzymes, a plasmid harboring the 6XHis-tag sequence under the control of the P T7 promoter. E. coli MC1061 cells were transformed by the ligation mixtures and transformants were selected onto 30 µg.ml-1 kanamycin LBagar plates. The plasmids were extracted by minipprep and the presence of the right inserts were confirmed by PCR and sequenced. Thereafter, the final plasmids were used to transform into E.coli 2566 overexpressing strains. The final strains obtained, from GHEc 054 up to GHEc 058, is listed on

	Table S 4.

Table S 4. List of B. subtilis strains

 S

	Strain	Genotype	Source
	GHBs	BSB1 (Trp+) strain	Laboratory stock
	GHBs	BSB1 sipT::sipT-SPA lacImC;EryR	This study
	GHBs	BSB1 sipS::sipS-SPA lacImC;EryR	This study
	GHBs	BSB1 secG::secG-SPA lacImC;EryR	This study
	GHBs	BSB1 sppA::sppA-SPA lacImC;EryR	This study
	GHBs	BSB1 bdbC::bdbC-SPA lacImC;EryR	This study
	GHBs	BSB1 secDF::secDF-SPA lacImC;EryR	This study
	GHBs	BSB1 yteJ::yteJ-SPA lacImC;EryR	This study
	GHBs	BSB1 spoIIIJ::spoIIIJ-SPAlacImC;EryR	This study
	GHBs	BSB1 ∆yteJ::cm	This study
	GHBs	BSB1 ∆sppA::cm	This study
	GHBs	BSB1 ∆sppA-yteJ::cm	This study

Table S 5 List of E. coli strains

 S

	GHBs GHBs 075 GHEc E.coli BL21 + pJ411_His-yteJ BSB1 ∆sppA::cm pCS58 BSB1 amyE:: Phyper-spank sppA pCS58 spec neo	This study This study This study
	GHBs GHBs 076 GHEc E.coli BL21 + pJ411_His-sppA BSB1 ∆sppA-yteJ::cm pCS74 BSB1 amyE:: Phyper-spank yteJ pCS74 spec neo	This study This study This study
	GHBs GHBs 077 GHEc E.coli MC1061 pDR111 yteJ BSB1 ∆sppA-yteJ::cm pCS73 BSB1 amyE:: Phyper-spank yteJ pCS73 spec neo	This study This study This study
	GHBs GHBs 078 GHEc E.coli MC1061 pDR111 yteJ-GFP BSB1 ∆sppA-yteJ::cm pCS58 BSB1 amyE:: Phyper-spank yteJ pCS58 spec neo	This study This study This study
	NCIB3610 (wt strain) BSB1 amyE:: Phyper-spank sppA-yteJ pCS74 spec neo GHEc E.coli MC1061 pDR111 sppA GHBs 079 GHBs GHBs 080 BSB1 amyE:: Phyper-spank sppA-yteJ pCS73 spec neo GHEc E.coli MC1061 pDR111 GFP-sppA	Laboratory This study This study stock This study This study
	GHBs GHBs 081 GHEc E.coli MC1061 pDR111 sppA-yteJ NCIB3610 ∆yteJ::cm BSB1 amyE:: Phyper-spank sppA-yteJ pCS58 spec neo	This study This study This study
	GHBs GHBs 085 GHEc E.coli 2566 pJ411 His-sppA BSB1 amyE::Paw-GFP BSB1 amyE:: Phyper-spank sppA-yteJ ∆129-164 spec	This study This study This study
	GHBs GHBs 087 GHEc E.coli 2566 pJ411 His-steJ BSB1 amyE::Pw-GFP BSB1 amyE:: Phyper-spank sppA-yteJ ∆152-164 spec	This study This study This study
	GHBs GHBs 089 GHEc E.coli 2566 pJ411 His-sppA-yteJ BSB1 amyE::SPA-sipS BSB1 amyE:: Phyper-spank sppA-yteJ ∆92-164 SPA spec	This study This study This study
	GHBs GHBs 089 GHEc E.coli 2566 pSMG212 His-sppA-yteJ-TEV_FLAG B.subtilis ATCC6633 (subtilin) BSB1 amyE::Phyper-spank sppA K199A spec	stock Laboratory This study This study
	GHEc E.coli 2566 pJ411 His-∆52SppA	This study
	GHBs	BSB1 amyE::Phyper-spank yteJ spec	This study
	GHBs	BSB1 ∆yteJ::cm amyE::Phyper-spank yteJ spec	This study
	Strain	Genotype	Source
	GHBs	BSB1 ∆sppA-yteJ::cm amyE::Phyper-spank yteJ spec	This study
	GHEc E.coli pSPA_LIC+bdbB	This study
	GHBs	BSB1 amyE::Phyper-spank yteJ-gfp spec	This study
	GHEc E.coli pSPA_LIC+secDF	This study
	GHBs	BSB1 ∆sppA-yteJ::cm amyE::Phyper-spank yteJ-gfp spec	This study
	GHEc E.coli pSPA_LIC+secG	This study
	GHBs	BSB1 amyE::Phyper-spank sppA spec	This study
	GHEc E.coli pSPA_LIC+sdbC	This study
	GHBs	BSB1 ∆sppA::cm amyE::Phyper-spank sppA spec	This study
	GHEc E.coli pSPA_LIC+sipS	This study
	GHBs	BSB1 ∆sppA-yteJ::cm amyE::Phyper-spank sppA spec	This study
	GHEc E.coli pSPA_LIC+sipT	This study
	BSB1 ∆yteJ::cm pCS74 BSB1 amyE::Phyper-spank gfp-sppA spec GHEc E.coli pSPA_LIC+sppA GHBs	This study This study This study
	GHBs GHBs GHEc E.coli pSPA_LIC+spoIIIJ BSB1 ∆yteJ::cm pCS73 BSB1 ∆sppA-yteJ::cm amyE:: Phyper-spank gfp-sppA spec	This study This study This study
	GHBs GHBs GHEc E.coli pSPA_LIC+yteJ BSB1 ∆yteJ::cm pCS58 BSB1 amyE:: Phyper-spank sppA-yteJ spec	This study This study This study
	GHBs GHBs GHEc E.coli MC1061 + pJ411_His-yteJ BSB1 ∆sppA::cm pCS74 BSB1 ∆sppA-yteJ::cm amyE:: Phyper-spank sppA-yteJ spec	This study This study This study
	GHBs GHBs GHEc E.coli MC1061 + pJ411_His-sppA BSB1 ∆sppA::cm pCS73 BSB1 amyE:: Phyper-spank sppA pCS74 spec neo	This study This study This study
	GHBs	BSB1 amyE:: Phyper-spank sppA pCS73 spec neo	This study

Table S 6

 S List of plasmids

	pGH015	pMutin SPA_Lic with sppA gene	Amp R Ery R	This study
	pGH017	pMutin SPA_Lic with spoIIIJ gene Amp R Ery R	This study
	pGH018	pMutin SPA_Lic with yteJ gene	Amp R Ery R	This study
	pGH021	pMutin SPA_Lic with prsA gene	Amp R Ery R	This study
	pGH022	pCS74		Neo R	From DSM
	pGH023	pCS73		Neo R	From DSM
	pGH024	pCS58		Neo R	From DSM
	pGH025	pSG13	Amp R Spec R	Laboratory
					stock
	pGH026	pSG13_PAW-GFP	Amp R Spec R	This study
	pGH029	pSG13_PW-GFP	Amp R Spec R	This study
		pJ411_6His	Kan R		Laboratory
					stock
	pGH032	pJ114_His-YteJ	Kan R		This study
	pGH035	pJ114_His-SppA	Kan R	Resistance	This study
	Plasmid pGH037	Construction pSPANter_Nter SPA-Sips	E.coli Amp R Spec R B.subtilis	Source This study
	pGH001 pGH032	pMutin SPA_Lic pJ114_His-SSppA	Amp R Ery R Kan R	Laboratory This study
		pJ114_His-operon	Kan R		stock This study
	pGH002 pGH042	pSPANter_Nter pDR111	Amp R Spec R Amp R Spec R	Laboratory Laboratory
					stock stock
	pGH004 pGH043	pMutin SPA_Lic with bdbB gene pDR111 yteJ	Amp R Ery R Amp R Spec R	This study This study
	pGH006 pGH046	pMutin SPA_Lic with secDF gene Amp R Ery R pDR111 yteJ-GFP Amp R Spec R	This study This study
	pGH007 pGH047	pMutin SPA_Lic with secG gene pDR111 SppA	Amp R Ery R Amp R Spec R	This study This study
	pGH009 pGH050	pMutin SPA_Lic with bdbC gene pDR111 GFP-SppA	Amp R Ery R Amp R Spec R	This study This study
	pGH011 pGH052	pMutin SPA_Lic with sipS gene pDR111 operon	Amp R Ery R Amp R Spec R	This study This study
	pGH013 pGH053	pMutin SPA_Lic with sipT gene pJ114 His-SppA-YteJ	Amp R Ery R Kan R	This study This study

Etude des mutants de délétion de sppA et/ou yteJ : D'après la littérature, SppA est une signal peptide peptidase, mais est aussi impliquée dans la résistance à la nisin, un peptide antibiotique de la famille des lantibiotiques, supposément grâce à son activité peptidase. Afin d'étudier le rôle de YteJ dans le complexe que cette protéine forme avec SppA, des souches de B. subtilis délétées de sppA et/ou yetJ ont été construites et caractérisées.2.1 Caractérisation des souches mutantes : La croissance de ces souches mutantes n'est nullement affectée dans les premières 24 heures (Figure5A). Toutefois, la survie à 48 heures (nombre de cellules formant colonie) est particulièrement affectée pour les souches ∆sppA et ∆sppA-yteJ, alors que la survie de la souche ∆yteJ était moins affectée. La survie de la souche ∆sppA n'a pas pu être restaurée en co-cultivant celle-ci avec des cellules de la souche sauvage. Il semblerait donc que l'activité de SppA soit importante sur la survie à long terme de B. subtilis. En revanche, le phénotype observé pour la souche ∆yteJ est intermédiaire (Figure5 B).

Acknowledgments

This study was carry out at the Systems Biology for bacterial Engineering and Redesign (SyBER) project was part of the European Marie Skłodowska-Curie ITN programme: ProteinFactory -"Engineering of New-Generation Protein Secretion Systems''. The development of an international PhD project it is not an easy road but with the support, encouragement and generosity of a large number of people, including my family, friends, colleagues and collaborators it has been continued towards its completion.

RESULTS

MATERIAL AND METHODS

°C and electrophoresis was performed in Laemlli 1X buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at 200 V. After staining with Coomassie blue staining solution (0.1% Coomassie Brilliant Blue R-250, 50% ethanol and 10% glacial acetic acid) protein sizes were estimated relative to a protein marker ladder.

Blue Native PAGE

For Blue-Native electrophoresis, the strains of interest were pre-cultured overnight in LB, supplemented with antibiotics if needed, at 37°C. The following day the culture was diluted 1000-fold (to an OD 600nm of 0.001) into 1-2 liters of LB medium, supplemented if needed. When the OD 600nm reached 0.5 -1, cells were harvested by centrifugation at 6000 rpm for 10 min at 4°C. The pellet was washed with 40 ml of cold resuspension buffer (buffer R: 10 mM Tris-Cl pH 7.5, 150 mM NaCl), centrifuged at 4500 rpm for 10 min at 4°C and instantly frozen in liquid nitrogen. The cells were resuspended in 20 ml of buffer R supplemented with 1 mg/ml of lysozyme, 1 mM EDTA and 5 µl of Benzonase (Invitrogen), and incubated in a water bath at 37 °C for 15-30 min. To remove the cell debris the samples were centrifuged at 4500rpm for 30min at 4°C and the pellet was discarded.

Membrane and cytosolic fractions were then separated by ultracentrifugation at 100,000 g for 1 h at

Western blot analysis

To analyze if the tagged proteins were expressed, Western blots were performed using the appropriate primary and secondary antibodies for each specific tag. In the case of FLAG-tag the Monoclonal ANTI-FLAG M2 antibody (F3165 Sigma) as a primary antibody and Anti-Mouse IgG (A2304 Sigma) as a secondary antibody. For the AmyM the primary antibody, we used, was raised during my UCB secondments, this antibody was raised in rabbits, as such we used Anti-Rabbit IgG (A0545 Sigma) (Appendix section 6.2) Equal amounts of protein, from each sample, were heated to 100 ºC for 5 minutes, loaded onto a 10% SDS-PAGE gel and separated at 200V. Proteins were then transferred to a Hybond-P Polyvinylidene fluoride (PVDF) membrane (GE Healthcare) using a semidry transfer cell (Bio-Rad) according to standard Western blotting techniques [START_REF] Burnette | Western Blotting': electrophoresis of protein from sodium dodecyl sulphate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated Protein A[END_REF].

APPENDIX Subtilin preparation-

The subtilin is produced by the B.subtilis ATCC6633 strain, which was supplied by Jan Maarten van Dijl group. To extract it, this strain was grown overnight in LB medium at 37°C. The supernatant was collected by centrifugation at 1400 rpm for 10 min, the pellet was discarded. Aliquots of 25 ml of supernatant were heated at 80°C for 10 min and filtered. These samples were freeze with liquid nitrogen and then freeze dry overnight. Initial experiments were done with 30% m/v subtilin. However, to avoid the effect of volume in the 96 well plate culture we concentrated 10X times the stock solution. The sample powder was weighed and concentrated 10 times by resuspensing in water, by other words the stock powder corresponding to 25 ml of liquid sample was ressuspended into 2.5 ml. This 10X stock solution was then used to induce the strains at 30% v/v or other concentrations when needed for testing. The powder sample was kept at -20°C. DSM with the details of the supernatant sample but is slightly different from those reported previously [START_REF] Cha | molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose[END_REF][START_REF] Outtrup | Properties and Application[END_REF].

The real pI value (8.35) of the AmyM sample can explain the problems that we had with the binding of the protein to the cationic and anionic resins, because the range of pH tested was from 7.5 up to 8.5 and to be possible the exchange of ions the pH value used should be two values above or below of the protein pI value [START_REF] Svensson | Isoelectric fractionation, analysis, and characterization of ampholytes in natural ph gradients. I. The differential equation of solute concentrations at a steady state and its solution for simple cases[END_REF] .

Furthermore, to avoid high pH values in which the protein is denatured we tested a cationic exchange chromatography at pH 6.5. The buffer of a 2 ml supernatant sample was exchange to 50 mM MES pH 6.5 and 50 mM NaCl with the PD-10 Desalting columns GE Health Care. The sample was then diluted up 20 ml and applied to a cationic a 1 ml Hitrap HP-S column equilibrated in 50 mM MES pH 6.5 and 50 mM NaCl. The protein was eluted with a linear gradient of NaCl from 50 mM up to 1 M. During the collection of the flow-through fraction it was possible to observe an increase of the UV values at 280 nm what normally indicates that the protein is not binding to the column resin, but because it is not a high value as the values previous measured it can be some contaminants that did not bound to the resin. Therefore, a 50 mM MES pH 6.5 buffer with 2 M NaCl was loaded into the purification system to create a gradient of NaCl up to 2 M. This additional step has the objective of elute the sample in the case that it binds tightly and it is necessary to have higher concentrations of salt to elute the protein. However, it was not the case and the protein was collected in the flow-through fraction once again.Since the previous purifications were not successful we procedure to a new anionic exchange chromatography with an increase of the pH buffer up to 9,5 [START_REF] Dauter | X-Ray structure of Novamyl, the fivedomain 'maltogenic' α-Amylase from Bacillus Stearothermophilus: Maltose and Acarbose complexes at 1.7 Å resolution[END_REF]. A 2 ml protein sample from the previous experiment Flow-through fractions sample was buffer exchanged to We have identified a membrane protein complex of Bacillus subtilis involving an unknown protein, YteJ, and SppA, a membrane protein first described as a signal peptide peptidase and later shown to be also involved in the resistance to antibacterial peptides of the lantibiotic family. Using deletion mutant strains, we showed that both proteins are involved in this resistance. In the ∆sppA strain, the ectopic overexpression of SppA not only restored the resistance, it also induced the formation of elongated cells, a phenotype suppressed by the simultaneous overexpression of YteJ. Furthermore, the expression of truncated versions of YteJ pinpointed the inhibitory role of a specific domain of YteJ. Finally, in vitro biochemical studies showed that SppA protease activity was strongly reduced by the presence of YteJ, supporting the hypothesis of an inhibition by YteJ. Our in vivo and in vitro studies showed that YteJ, via one of its domain, acts as a negative regulator of the protease activity of SppA in this complex.

In conclusion, we have shown that SppA/YteJ complex is involved in lantibiotic resistance through the protease activity of SppA, which is regulated by YteJ.