Figure 2. Classification of MreB*s based on their phenotypic characterization. (1) Expression of P mreBH based on the color of the colony on LB plates supplemented with Xgal; L, low; H, high. (2) Estimated relative protein levels during exponential growth, based on western blot analysis. (3) Growth curve of cells grown in CH media at 37 °C; WeB stands for "Worst than ∆mreB". (4) Growth curve of cells grown in LB media at 37 °C. (5) Morphology of exponentially grown cells in CH media at 37 °C observed with bright field microscopy; interm. stands for intermediate phenotype between that of WT and ∆mreB strains. (6) GFP-MreB localization and dynamic properties qualitatively analyzed from TIRFM acquisitions; P, patches; Dif, diffusive.

MreB peut jouer un rôle dans

la synthèse de la CW, la morphologie cellulaire et le métabolisme cellulaire Malgré des années d'efforts prolongés, la (les) fonction (s) exacte (s) de MreB restent insaisissables. En créant un criblage génétique qui sélectionne des mutants de MreB de perte de fonctionne dans la bactérie G+ B. subtilis, nous avons pu contourner cette problématique et établir des liens entre la structure et la fonction de MreB. Il est difficile d'extraire des conclusions solides de nos résultats préliminaires, mais nous avons réussi à l'acquisition d'un groupe très intéressant de mutants de MreB qui indiquent que MreB a plus d'une fonction chez B. subtilis. Les corrélations structure-fonctionne nous permettent d'extraire des conclusions. Les mutants MreB G160R et MreB G14E , avec un phénotype similaire a ce du ΔmreB, sont localisés dans le site putatif de liaison nucléotidique de MreB. En combinant la localisation et les résultats qui prouvent la perte de fonctionne de ces mutations, on peut en déduire que cette zone et, éventuellement l'activité de liaison des nucléotides de MreB, sont importantes pour le bon fonctionnement de la protéine. MreB*s MreB G231D , MreB G56R , avec un phénotype similaire a ce du ΔmreB, et MreB L171P , avec un phénotype plus sévère que ce du ΔmreB, se trouvent à proximité immédiate de la zone putatif de formation du protofilament (les monomères de MreB se lient à d'autres monomères de MreB pour former des chaînes ou protofilaments). Ces trois mutants, comme ceux mentionnés ci-dessus, montrent une fonction MreB altérée et on peut donc déduire que cette zone, et probablement la capacité de polymérisation de MreB, sont importantes pour le bon fonctionnement de la protéine. Il est intéressant de noter que certains mutants montrent un désaccouplement des défauts de forme et une déficience de la croissance. Quatre des MreB*s avec un phénotype plus sévère que ce du ΔmreB (MreB S109P , MreB A276G , MreB I279V et MreB V72A ) ont une morphologie WT et une localisation de MreB faiblement perturbées pendant que la croissance cellulaire, à la fois dans les milieux CH et LB, est très fortement perturbée. L'utilisation de E. coli montre que LB a présument des faibles quantités de sucres (estimées <100 μM). Leur épuisement marque la fin de la phase exponentielle de croissance des bactéries, moment où les cellules passent à la consommation d'acides aminés (Sezonov, Joseleau-Petit, & D'Ari, 2007). Lorsque B. subtilis est cultivé dans du LB, un diauxie peut être observé autour de OD 600 nm ~ 0,5, ce qui est vraisemblablement dû à l'épuisement des sucres du milieu aussi. C'est précisément le moment où la croissance des mutants MreB S109P , MreB A276G , MreB I279V et MreB V72A commence à se dégrader. MreB S109P et MreB V72A sont localisés à la surface de MreB, à proximité immédiate de l'interface putatif d'interaction interprotofilament; MreB A276G est localisé près de la zone putative de protofilament. Enfin, MreB I279V est muté dans un résidu interne, près de la zone supposée de polymérisation de MreB. Notre hypothèse est que ces mutations de MreB, d'une certaine manière, empêchent la cellule de passer de la glycolyse à la gluconéogenèse. Une possibilité tentante est que MreB agirait comme un point de contrôle reliant le métabolisme cellulaire et la synthèse de la CW. On sait que MreB interagit avec les protéines impliquées dans la synthèse des précurseurs de la CW (Favini-Stabile, Contreras-Martel, Thielens, & Dessen, 2013; Rueff et al., 2014) et nous pensons qu'il existe un équilibre entre MreB associée à la membrane et polymérisée et MreB qui est cytosolique. Il pourrait être possible que MreB agit comme un senseur du statut métabolique cellulaire pour coordonner la synthèse de la CW avec les besoins de la cellule. Cela pourrait expliquer pourquoi les défauts de forme et de croissance pourraient être désaccouplés.

. YvcK a deux rôles distincts, l'un dans le métabolisme du carbone et un autre dans la synthèse de la CW. La modification de ses niveaux de phosphorilation découpe les deux fonctionnes. Bien que sa fonctionne dans le métabolisme du carbone ne soit pas affectée par ses niveaux de phosphorilation, sa capacité à positionner correctement PBP1 est réduite.

Ce qui est encore plus intéressant, c'est qu'YvcK, lorsqu'il est surproduit, est capable de sauver le mutant ΔmreB.

Compte tenu de toutes ces données, nous supposons que MreB peut servir de point de contrôle entre la synthèse latérale de la CW et le métabolisme cellulaire. MreB pourrait avoir une deuxième fonctionne dans le métabolisme des acides aminés qui est modifié par nos mutations avec un phénotype plus sévère que ce du ΔmreB. L'ajout de sucres permettrait à ces mutants de surmonter les effets négatifs d'un métabolisme des acides aminés altéré. Une vérification plus poussée sera faite pour l'identification du processus du métabolisme spécifiquement lié à MreB. Pour poursuivre la compréhension de MreB, si nous avons découplé le rôle de la protéine dans la morphologie cellulaire et le métabolisme cellulaire, nous pouvons la faire croitre dans des milieux minimaux définis complétés par différentes sources de carbone et monitoriser sa croissance. Nous profiterons également du MreB L171P qui se développe comme la souche WT dans du LB pour élucider ce qui est nécessaire pour que ce mutant récupère la morphologie des cellules WT et la dynamique de la protéine MreB.

Possibilité de connexion entre la suppression de mreB et l'apparition de la mutation dans ydcH

Au début de ce projet, l'induction spécifique de l'opéron d'ydcFGH avait été observée en absence de mreB, appelant à l'élucidation de la fonctionne de cet effecteur potentiel spécifique de MreB, ainsi que son mode d'induction. Le résultat, dans les dernières semaines du doctorat, était inattendu.

 Mercier, 2013).

Bien qu'il ne soit pas possible de suivre complètement la chaîne d'événements menant à l'apparition de tant de mutations dans la souche 3725, elles apparurent toutes dans un temps et un lieu (Oxford) où la forme L et le gène essentiel de MreB étaient étudiés. Il est concevable qu'ils aient été involontairement sélectionnés comme suppresseurs. À l'heure actuelle, on ne sait pas si la mutation dans ydcH était fortuite ou sélectionnée car elle améliore le phénotype du ΔmreB. Mais on peut spéculer que, si YdcH joue un rôle dans la régulation de l'état métabolique cellulaire, facilitant l'adaptation de la cellule aux conditions changeantes, y compris l'appauvrissement du carbone comme dans la transition de phase (agissant sur ~ 60 gènes impliqués dans le métabolisme du carbone) et si MreB relie la croissance cellulaire au métabolisme du carbone (voir les section sur YdcH et la mutagenèse de MreB), cette mutation peut donc augmenter la survie de la souche ΔmreB en modifiant le métabolisme du carbone et / ou des acides aminés et peut ne pas être accidentelle.

Synthèse en français Introduction

La majorité des cellules bactériennes sont entourées d'une enveloppe. Cette enveloppe est une structure complexe qui sépare la cellule de son environnement, agit comme une barrière de diffusion et une interface de communication et qui contrecarre la forte pression de turgescence interne [START_REF] Radeck | The cell envelope stress response of Bacillus subtilis: from static signaling devices to dynamic regulatory network[END_REF]. La structure de l'enveloppe divise la plupart des bactéries en mono derme ou Gram-positives (G+) et di derme ou Gram-négatives (G-) [START_REF] Dufresne | Biology and assembly of the bacterial envelope[END_REF]. Chez les bactéries G+, l'enveloppe est formée par une membrane cytoplasmique et une paroi cellulaire (CW) multicouches. L'enveloppe des G-a une membrane cytoplasmique ou intérieure, une membrane externe supplémentaire et une CW entre elles, plus mince que celles des G+s [START_REF] Dufresne | Biology and assembly of the bacterial envelope[END_REF].

Les parois des G+s et G-s sont principalement formées de peptidoglycan, ainsi que de molécules chargées négativement (acides téichoïques) chez les bactéries G+. Le peptidoglycan (PG) forme un maillage macromoléculaire dont la structure est critique pour le maintien de l'intégrité cellulaire [START_REF] Van Heijenoort | Assembly of the monomer unit of bacterial peptidoglycan[END_REF], dont la synthèse est assurées par des machineries de synthèse du PG. Les protéines cytosquelettiques constituent un élément important de ces machineries de synthèse du PG en permettant lier tous les protéines nécessaires à ce procès. On peut cependant différencier deux types de machinerie de synthèse de fonctions différentes et présentant des protéines cytosquelettiques caractéristiques : celles permettant la synthèse de la paroi latérale lors de l'élongation (élongasome) et celles réalisant la synthèse du septum pendant la division (divisome). L'élongasome comporte un homologue procaryote de l'actine, MreB. Le divisome a comme élément le plus caractéristique FtsZ, un homologue procaryote de la tubulin [START_REF] Egan | Regulation of bacterial cell wall growth[END_REF]. On pense que ces protéines cytosquelettiques bactériennes créent des échafaudages, positionnant les machineries de synthèse du PG pendant l'allongement et la division cellulaire, respectivement [START_REF] Egan | Regulation of bacterial cell wall growth[END_REF].

Les protéines impliquées dans la synthèse du PG ont fait l'objet d'études approfondies depuis longtemps. Néanmoins, il existe encore de nombreuses questions sans réponses. L'absence ou la malformation de cette barrière du PG essentielle provoque la perte de forme et, en fin de compte, la lyse des cellules. L'intégrité de la paroi est donc une question d'importance vitale pour les bactéries. La composition et le fonctionnement précis des mécanismes de synthèse du PG pendant l'allongement ne sont pas complètement compris, mais ils dépendent d'un acteur clé: MreB. J'ai utilisé Bacillus subtilis, le modèle des bactéries G+, pour mieux comprendre le rôle de MreB dans la morphogenèse bactérienne. Dans ce but, j'ai caractérisé un opéron de fonction inconnu dont l'expression est dépendante de MreB et j'ai développé un crible génétique qui m'a permis de sélectionner des mutants de perte de fonction de mreB.

Résultats Étude d'un effecteur potentiel de MreB

Des résultats non publiés de notre groupe ont révélé l'existence d'un opéron non caractérisé (ydcFGH), dont l'expression est fortement induite en absence de mreB. Un certain nombre d'expériences ont été menées afin de caractériser l'opéron ydcFGH, sa régulation et le lien entre cet opéron et MreB. Les conclusions tirées des expériences menées sont:

1. YdcH: un répresseur / activateur régulateur de la transcription du type MarR? L'approche globale que nous avons utilisée a révélé un très grand nombre de gènes affectés, positivement et négativement, par YdcH (Figure 1). Bien que surprenant, plusieurs hypothèses peuvent expliquer ces résultats. Une possibilité simple serait qu'YdcH affecte l'activité d'autres régulateurs. Cela peut être soit par activation ou répression directe des gènes impliqués dans la régulation d'autres procès, soit parce que les modifications physiologiques de l'absence d'ydcH, à leur tour, ont activé ou désactivé ces régulateurs. Nous ne pouvions pas trouver des régulons complets régulés vers le haut ou vers le bas, mais tels régulons peuvent être partiellement cachés par des régulations croisés de leurs gènes. Results from the RNAseq experiment comparing gene expression levels in a WT (ABS2005) and a ∆ydcH strain (ASEC56) show that, in absence of ydcH, during exponential growth (A), there are 180 unrepressed genes (green) and 184 down-regulated genes (red); during stationary growth (B) there are 13 up-regulated (dark green) and 11 down-regulated genes (dark red).

Une autre hypothèse est qu'YdcH pourrait agir à la fois comme un répresseur et un activateur de l'expression des gènes. La plupart des régulateurs de transcription du type MarR (ce qu'YdcH semble être) décrits agissent comme des répresseurs et quelques-uns comme activateurs [START_REF] Grove | MarR family transcription factors[END_REF]. Il n'y a que deux régulateurs de transcription du type MarR connus qui ont les deux activités [START_REF] Oh | Dual role of OhrR as a repressor and an activator in response to organic hydroperoxides in Streptomyces coelicolor[END_REF][START_REF] Tran | Analysis of RovA, a transcriptional regulator of Yersinia pseudotuberculosis virulence that acts through antirepression and direct transcriptional activation[END_REF]. Ils agissent comme des répresseurs en se liant à des séquences d'ADN proches des régions promotrices des gènes réprimés. Lorsque les conditions environnementales sont modifiées, ils subissent une modification structurelle qui entraîne une réduction de leur affinité pour l'ADN et leur permet de lier l'ARN polymérase, ce qui améliore sa liaison à la région promotrice du gène régulé. Par conséquent, c'est une possibilité qui vaut la peine d'être vérifiée dans le futur pour YdcH.

YdcH: un nouveau régulateur de l'état de transition

Plusieurs lignes de preuve préconisent qu'YdcH soit un nouveau régulateur de l'état de transition qui pourrait aider à la cellule à s'adapter aux changements environnementaux, de manière similaire à celle de AbrB ou SigH [START_REF] Britton | Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis[END_REF][START_REF] Phillips | Bacillus subtilis sporulation and stationary phase gene expression[END_REF]. Tout d'abord, le profil d'expression d'ydcFGH montre que le point culminant de l'expression (probablement lorsque YdcH est inactif) coïncide avec la transition entre la phase de croissance exponentielle et stationnaire. Ceci est bien confirmé par la différence dramatique entre les profils globaux d'expression génique pendant la phase de croissance exponentielle et la phase stationnaire. Ceux-ci montrent que l'énorme regulon d'YdcH est dérégulé une fois que les cellules ont entrées en phase stationnaire (presque pas de différences entre WT et ∆ydcH). Il n'est pas surprenant que plusieurs gènes régulés par un autre régulateur de l'état de transition, par AbrB, soient également affectés en l'absence d'YdcH. Deuxièmement, il existe une grande diversité de fonctions affectées par YdcH conduisant à une reprogrammation globale de l'expression des gènes, typique de ces régulateurs [START_REF] Strauch | Transition-state regulators: sentinels of Bacillus subtilis postexponential gene expression[END_REF]. Troisièmement, parmi ces nombreux gènes, un grand sous-ensemble est impliqué dans le métabolisme du carbone ou des acides aminés, tous ces deux étant considérablement affectés à l'entrée en phase stationnaire en raison de l'épuisement de certaines sources de carbone. Fait intéressant, nous avons remarqué de nombreux gènes du regulon d'YdcH liés à la synthèse de plusieurs bacteriotoxins: albABCDEFG (bacteriocine antilistérienne subtilosine), ntdR (antibiotique kanosamine), sdpA, sdpI et yknW (toxine de SdpC) et yydGHIJ (contrôle du système LiaR-LiaS comme réponse à la bacitracine). Cela pourrait être une stratégie pour la cellule pour récupérer de nouvelles ressources à partir d'un environnement appauvri [START_REF] Bibliography Abriouel | Diversity and applications of Bacillus bacteriocins[END_REF]. Tous ensemble, nos résultats suggèrent que, lors de la transition de la phase exponentielle à la phase stationnaire de croissance, la répression d'YdcH est partiellement libérée (comme l'a observé l'étude de l'expression de P ydc1 ) conduisant à l'activation ou répression d'un grand ensemble de gènes, entraînant potentiellement une reprogrammation de la cellule. Nous faisons l'hypothèse qu'YdcH agit comme un régulateur de l'état de transition chez B. subtilis, agissant peut-être à la fois comme activateur et répresseur. Nous pouvons imaginer qu'YdcH soit actif pendant la croissance exponentielle, réprime sa propre expression et celle d'autres régulateurs de transcription. Pendant la phase stationnaire, YdcH est inactive. Cela entraînerait l'activation de ces régulateurs de transcription qui pourraient alors agir positivement ou négativement sur les plus de 300 gènes qui apparaissent différemment exprimés dans la souche ΔydcH par rapport au WT pendant la phase stationnaire.

Pour aller plus loin dans notre compréhension d'YdcH et de ses fonctions et pour vérifier notre hypothèse, nous devrions identifier la séquence d'ADN à laquelle YdcH se lie, la "boîte d'YdcH ", dans P ydc1 . Nous pourrions alors effectuer une prédiction in silico des "boîtes d'YdcH" dans d'autres promoteurs afin d'essayer une caractérisation plus précise du regulon d'YdcH. Si YdcH provoque un effet large dans la cellule, en utilisant l'approche de RNAseq nous récupérerons les données de tous les changements produits dans la cellule: ceux qui sont directement liés à YdcH et ceux qui sont causés indirectement (par des gènes contrôlés par YdcH). Les expériences de démonstration pourraient également nous fournir des informations sur la fonction et les effecteurs d'YdcH.

Etude de structure-fonctionne de MreB 1. Une bibliothèque de mutants de MreB avec affectation de sa fonctionne Nous avons développé une méthodologie pour obtenir des mutants de MreB (MreB*s) et un criblage pour leur sélectionner selon leur perte de fonctionnalité grâce à l'activation de P mreBH . Bien qu'il soit difficile d'obtenir MreB*s avec un seul résidu modifié en raison de l'acquisition spontanée de mutations supplémentaires ou de mutations suppresseurs, nous avons construit avec succès une collection de mutants présentant une variété de phénotypes (Figure 2). À l'avenir, en utilisant différents rapporteurs de la fonctionnalité de MreB, nous pourrions agrandir notre bibliothèque et avoir une vision complète de la manière dont MreB exerce son activité. L'addition de Mg + 2 et de sucres (regarder la prochaine sectionne) pendant le processus de mutagenèse pourrait aider à récupérer des mutants supplémentaires. Enfin, ces mutants seront un atout pour les futures études biochimiques afin de déterminer les propriétés biochimiques de MreB chez B. subtilis. L'interaction des protéines pourrait également être étudiée à travers des expériences de pull down ou du système des doubles hybrides chez la levure. Lysogeny broth (LB) medium has a composition as in [START_REF] Sezonov | Escherichia coli physiology in Luria-Bertani broth[END_REF]. Casein hydrolysate (CH) medium has a composition as in [START_REF] Formstone | Localization and interactions of teichoic acid synthetic enzymes in Bacillus subtilis[END_REF] . coli............................................................................ 33 ................................................................... 88

List of tables

List of figures

Introduction

INTRODUCTION

Introduction

Free-living bacterial cells are surrounded by an envelope. This envelope is a complex structure that separates the cell from its environment, acts as a diffusive barrier and communication interface and counteracts the high internal turgor pressure [START_REF] Radeck | The cell envelope stress response of Bacillus subtilis: from static signaling devices to dynamic regulatory network[END_REF]. The structure of the envelope divides most of the bacteria in monoderm or Gram-positives (G+) and diderm or Gramnegatives (G-) [START_REF] Dufresne | Biology and assembly of the bacterial envelope[END_REF]. In Gram-positive bacteria, the envelope is formed by a cytoplasmic membrane (CM) and a multilayered cell wall (CW). Gram-negative's envelope has a cytoplasmic or inner membrane (IM), an additional outer membrane (OM), and a CW in between, thinner than in Gram-positives (Figure 1) [START_REF] Dufresne | Biology and assembly of the bacterial envelope[END_REF]. Both G+'s and G-'s CW are mainly formed of peptidoglycan and negatively charged molecules, in G+ bacteria (teichoic acids). Peptidoglycan forms a macromolecular mesh whose structure is crucial to maintaining cell integrity (J. [START_REF] Van Heijenoort | Assembly of the monomer unit of bacterial peptidoglycan[END_REF]. Cytoskeletal proteins connect PG synthetic machineries to cytosolic processes. These machineries vary depending on whether the CW is being formed during elongation (elongasome) or division (divisome). Even though the process of PG synthesis follows the same steps, the proteins involved in it vary depending on the moment of the cell cycle [START_REF] Egan | Regulation of bacterial cell wall growth[END_REF].

INTRODUCTION

Bacterial envelope

Cytoplasmic membrane

Gram-positive and Gram-negative bacteria have very similar CM. It is a symmetrical phospholipid bilayer carrying undecaprenyl-phosphate (lipid II carrier; see section 2.2.2), teichoic acid precursor carriers in G+ bacteria, intramembrane α-helice proteins and lipoproteins [START_REF] Dufresne | Biology and assembly of the bacterial envelope[END_REF]. Proteins in the CM are involved in many metabolic processes and look either towards the periplasm in G-(the space between the CM and OM) or the cytoplasm [START_REF] Silhavy | The bacterial cell envelope[END_REF].

Cell wall

The CW is mainly formed of peptidoglycan (PG, also called murein) both in Gram-positive and Gramnegative bacteria. PG forms a polymeric macromolecule of various layers called sacculus. It is thought to be thicker in G+ bacteria than in G- [START_REF] Gan | Molecular organization of Gram-negative peptidoglycan[END_REF][START_REF] Hayhurst | Cell wall peptidoglycan architecture in Bacillus subtilis[END_REF]. It is formed by linear chains of the disaccharide N-acetyl glucosamine-N-acetyl muramic acid (NAG-NAM), cross-linked by pentapeptide bridges (Figure 2; see section 1.2) [START_REF] Vollmer | Peptidoglycan structure and architecture[END_REF]. The flexibility of the sacculus is given by the pentapeptide bond that can expand and shrink, whereas the glycan strand is rigid [START_REF] Cava | Peptidoglycan plasticity in bacteria: emerging variability of the murein sacculus and their associated biological functions[END_REF]. Gram-positive bacteria have an extra CW component that isn't present in Gram-negatives, teichoic acids (TA). They constitute about 30-60 % of the CW mass, the rest being PG (Neuhaus & Baddiley, INTRODUCTION 2003). TA are anionic polymers with a short saccharidic linkage unit and multiple hydroxyl functional groups. They can be divided into wall teichoic acids (WTA) and lipoteichoic acids (LTA) [START_REF] Neuhaus | A continuum of anionic charge: structures and functions of Dalanyl-teichoic acids in Gram-positive bacteria[END_REF]. There is a large variety of WTAs. They bind covalently to PG and go past it towards the exterior of the cell [START_REF] Dufresne | Biology and assembly of the bacterial envelope[END_REF]. The most common WTAs have a disaccharide attached to a polyribitol phosphate (polyRboP) or a polyglycerol phosphate (polyGroP) chain with a maximum of 60 repeats. The hydroxyl groups are modified with different molecules.

These make WTAs such a varied class [START_REF] Silhavy | The bacterial cell envelope[END_REF]. LTAs are shorter than WTAs and have different chirality. They bind to the cytoplasmic membrane and don't stretch beyond the PG mesh [START_REF] Dufresne | Biology and assembly of the bacterial envelope[END_REF]. LTAs are always formed by polyGroP and have modified hydroxyl groups, as WTAs. TAs are essential to determine bacterial surface charge and hydrophobicity [START_REF] Brown | Wall teichoic acids of Gram-positive bacteria[END_REF]. They are also involved in the regulation of cell division, PG synthesis and morphogenesis [START_REF] Brown | Wall teichoic acids of Gram-positive bacteria[END_REF]. LTAs are also implicated in divalent cation homeostasis and membrane physiology [START_REF] Percy | Lipoteichoic acid synthesis and function in Gram-positive bacteria[END_REF].

G-CW is formed of a thin layer of PG. It does not have TA anchored on the CW, but instead has another negatively charged lipidic molecule attached to the OM, the lipopolysaccharides (LPS, see section 1.3). In these organisms, the CW is located in the periplasm, the space between the CM and the OM. PG composition in G-bacteria is similar to that of G+s, but less cross-linked [START_REF] Vollmer | Peptidoglycan structure and architecture[END_REF].

Outer membrane

Gram-negatives have an extra layer, the OM, that is not present in Gram-positives. It is porous to certain substances and is capable of transporting others, an aspect needed to allow cell metabolism and growth [START_REF] Beveridge | Structures of gram-negative cell walls and their derived membrane vesicles[END_REF][START_REF] Dufresne | Biology and assembly of the bacterial envelope[END_REF]. It has intramembrane β-barrel proteins and specific lipoproteins organized in an asymmetrical lipid bilayer [START_REF] Dufresne | Biology and assembly of the bacterial envelope[END_REF].

The inner leaflet is formed by phospholipids while the outer leaflet is composed of anionic glycolipids, principally lipopolysaccharides (LPS) (Figure 1).

LPS are most commonly made of an anchor of lipid A (glucosamine-based phospholipid) that binds to the outer leaflet, a core oligosaccharide and a highly variable chain of oligosaccharides named Oantigen [START_REF] Silhavy | The bacterial cell envelope[END_REF]. The oligosaccharide core is divided into inner and outer core. The inner core links to lipid A and is formed by negatively charged sugars. The outer core is more diverse and links with the O-antigen, which is, again, highly variable. LPS are negatively charged and very abundant in the outer leaflet of the OM. The negative charges are counterbalanced by divalent cations like Mg 2+ and Ca 2+ [START_REF] Dufresne | Biology and assembly of the bacterial envelope[END_REF]. As TA in Gram-positive bacteria, LPS are determinants of surface charge and hydrofobicity in Gram-negative bacteria.

INTRODUCTION

I will now focus on PG, the most important structural element of the bacterial envelope that is common to both G+ and G-bacteria. Its structure and synthesis are key for bacterial survival [START_REF] Typas | From the regulation of peptidoglycan synthesis to bacterial growth and morphology[END_REF].

Peptidoglycan

PG, besides being such an important molecule in bacteria, is unique to bacteria [START_REF] Bern | Towards an automated analysis of bacterial peptidoglycan structure[END_REF]. This makes it the target of many antibiotics. Even though it is common to most freeliving bacteria, it presents some differences depending on the species [START_REF] Bern | Towards an automated analysis of bacterial peptidoglycan structure[END_REF] and in response to environmental conditions [START_REF] Ruiz | Filling holes in peptidoglycan biogenesis of Escherichia coli[END_REF]. There are two moments during the cell cycle in which PG is synthesized: cell division (at the septum) and cell elongation (at the side wall). The machineries in charge of the synthesis share most of their components, but some are specific for either division or elongation [START_REF] Chastanet | The actin-like MreB proteins in Bacillus subtilis: a new turn[END_REF]. For a more detailed description of these machineries see section 1.2.2.4.

Peptidoglycan structural models

The length of the glycan strand varies in different strains and growth conditions. Most Gram-negative bacteria have 20-100 monomers of PG per strand (M/S), but there is a preference for the lower values, 20-40 M/S (de Pedro [START_REF] De Pedro | Structural constraints and dynamics of bacterial cell wall architecture[END_REF]. Entry into stationary phase causes a progressive reduction of PG strand length in both G+ and G-bacteria. This adaptation is due to post-synthesis processing of the macromolecule, rather than modification of the PG termination process [START_REF] Pisabarro | Structural modifications in the peptidoglycan of Escherichia coli associated with changes in the state of growth of the culture[END_REF].

The 3D structural organization of the PG is still unknown, but three main theoretical models have been proposed: 1. the horizontal layer model in which the strands run parallel to the cell surface, forming a circumference, 2. the scaffold model with PG strains running perpendicularly to the membrane and 3.

the coiled-coil model with glycan strands forming a "rope" that surrounds the cell perpendicularly to the cell long axis (Figure 3).

Horizontal layered model:

Electron cryotomography studies performed on Escherichia coli and Caulobacter crescentus sacculi show glycan strands running parallel to the cell surface, wrapping the cell (Figure 3A) [START_REF] Gumbart | Escherichia coli peptidoglycan structure and mechanics as predicted by atomic-scale simulations[END_REF]. In this study, they advocate for a single layered PG CW in G-bacteria where glycan strains have a low level of organization. Another study showed similar results also in E. coli, with new PG being inserted in strands or patches [START_REF] Typas | From the regulation of peptidoglycan synthesis to bacterial growth and morphology[END_REF]. Studies on the 3D structure of PG in Gram-positive bacteria have to overcome the difficulty of a thick PG mesh. Fortunately, innovative techniques are starting to attenuate these difficulties. Recent results from Kim and coworkers [START_REF] Kim | Peptidoglycan architecture of Gram-positive bacteria by solid-state NMR[END_REF] using solid-state NMR have demonstrated that PG strands in Staphylococcus aureus follow the layered model, as Gram-negative bacteria, but are highly ordered and packed, contrary to the data from studies on G-s [START_REF] Gumbart | Escherichia coli peptidoglycan structure and mechanics as predicted by atomic-scale simulations[END_REF][START_REF] Kim | Peptidoglycan architecture of Gram-positive bacteria by solid-state NMR[END_REF].

Scaffold model:

The scaffold model was proposed using Gram-negative CW as a model. It describes glycan strands as perpendicular to the cytoplasmic membrane, growing towards the outside of the cell, and pentapeptide bonds parallel to it (Figure 3B) [START_REF] Dmitriev | Tertiary structure of bacterial murein: the scaffold model[END_REF]. In the scaffold model, the cross-linking rate of glycan strands would be higher closer to the cytoplasmic membrane than towards the OM, contemplating the possibility of the PG tips being free, with no pentapeptide bonds. The authors accept that it is difficult to reconcile this idea with cell division in E. coli and Bacillus subtilis (the G+ bacterium model). However, it is supported by the existence of spheroid bacteria with alternate division planes [START_REF] Dmitriev | Tertiary structure of bacterial murein: the scaffold model[END_REF]. The problematic of cell division is still ongoing and further INTRODUCTION studies will be necessary to fully understand the process. Other results go against the scaffold model in the G-bacterium E. coli [START_REF] Vollmer | The architecture of the murein (peptidoglycan) in Gram-negative bacteria: vertical scaffold or horizontal layer(s)?[END_REF]. According to their results derived from the analysis from multiple measurements including PG thickness, strand length and degree of crosslinkage, the scaffold model is highly unlikely.

Coiled-coil model:

The coiled-coil model advocates that the glycan strands act as a guide for the synthesis of new PG (Figure 3C) [START_REF] Turner | Atomic force microscopy analysis of bacterial cell wall peptidoglycan architecture[END_REF]. AFM studies on B. subtilis have shown helical "cables"

surrounding the cell, parallel to the cytoplasmic membrane [START_REF] Hayhurst | Cell wall peptidoglycan architecture in Bacillus subtilis[END_REF]. According to this hypothesis, during PG synthesis, glycan strands are polymerized and cross-linked in groups to form a "rope" that will then be attached to preexisting PG with a helical pattern [START_REF] Hayhurst | Cell wall peptidoglycan architecture in Bacillus subtilis[END_REF].

There are still many open questions about PG 3D structure. What is accepted by all the scientific community is that the steps taken to synthesize PG are conserved in all bacteria. I will now proceed to describe this process.

Peptidoglycan synthesis

PG synthesis is a highly controlled process as it has to adapt to growth changes like transition from exponential growth to stationary phase or cell division without compromising cellular integrity. This process is very similar between Gram-positive and Gram-negative bacteria. During elongation, PG synthesis takes place along the cell cylinder and is controlled by the elongasome, the cell wall synthetic machinery (CSM) regulating CW formation during cell growth. During division, it takes place at the septum, controlled by the divisome, the CSM that regulates septum formation during division. Even though the synthesis of PG in both cases follows the same process, some of the components of the CSM regulating it vary [START_REF] Bhavsar | Cell wall assembly in Bacillus subtilis: how spirals and spaces challenge paradigms[END_REF]. Broadly, PG synthesis can be divided into three different stages: stage 1. synthesis of the cytoplasmic precursor lipid II, stage 2.

flipping of lipid II across the membrane and stage 3. assembly of PG (Figure 4) [START_REF] Siegel | Biogenesis of the Gram-positive bacterial cell envelope[END_REF].

Stage 1, synthesis of the cytoplasmic precursor lipid II

The synthesis of lipid II is accomplished stepwise (Figure 4). First, MurAA and MurB catalyze the transformation of UDP-N-acetylglucosamine (UDP-GlcNAc) and phosphoenolpyruvate into UDP-Nacetylmuramic acid (UDP-MurNAc) [START_REF] Bhavsar | Cell wall assembly in Bacillus subtilis: how spirals and spaces challenge paradigms[END_REF]. Then, MurC, MurD, MurE and MurF, In stage 2, lipid II is translocated from the inner leaflet of the cytoplasmic membrane to the exterior (Figure 4). It has been estimated that there are 1000-2000 lipid II molecules in exponentially growing E. coli (Y. [START_REF] Van Heijenoort | Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3[END_REF]. According to this study, in order to keep up with growth rate, the flipping of lipid II to the exterior of the cell must be fast. It is accepted that this process requires the action of flippase(s) that would form a channel through which lipid II would cross the cytoplasmic membrane, easing the process. The hydrophilic part of lipid II would cross the membrane while the hydrophobic moiety would remain in the membrane [START_REF] Kramer | Resistance of Gram-positive bacteria to nisin is not determined by lipid II levels[END_REF]. The identity of the flippase(s) has been the focus of a number of studies. There are three identified flippases: FtsW, MurJ and AmJ [START_REF] Meeske | MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis[END_REF][START_REF] Young | 'ils dépendent d'une protéine essentielle : MreB, une protéine procaryote similaire à l'actine. MreB est suspectée de contrôler l'activité et/ou l'assemblage des PGEMs, mais sa fonction exacte comme son mode de régulation sont actuellement inconnus[END_REF].

FtsW, a protein required for cell septation, and its paralog RodA, required for lateral PG synthesis, were the first proteins lipid II flippases in E. coli [START_REF] Ehlert | Role of precursor translocation in coordination of murein and phospholipid synthesis in Escherichia coli[END_REF]. It is only recently that Mohammadi and coworkers performed in vitro experiments with FtsW suggesting that the protein could flip lipid II in liposomes [START_REF] Mohammadi | Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane[END_REF].

A decade ago, another study in E. coli had proposed MurJ to be the lipid II flippase based on an informatic approach [START_REF] Ruiz | Lipid flippases for bacterial peptidoglycan biosynthesis[END_REF]. But its activity was demonstrated in vivo only recently in E. coli and in B. subtilis [START_REF] Meeske | MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis[END_REF]. Recently, they showed that MurJ is needed for PG synthesis and deleting it causes an increase in the accumulation of lipid-linked PG precursors in the Gbacterium model [START_REF] Sham | Bacterial cell wall. MurJ is the flippase of lipid-linked precursors for peptidoglycan biogenesis[END_REF], questioning the role of FtsW/RodA. However, it does not flip lipid II in vitro, as FtsW does, maintaining some suspense on which, between FtsW and MurJ is the flippase.

In B. subtilis, the situation is even more intricate, with the presence of FtsW and RodA, a homolog of MurJ (YtgP), and three paralogs. When deleting the four murJ paralogs in B. subtilis, the strain was still viable, going against the hypothesis of MurJ being the lipid II flippase [START_REF] Sham | Bacterial cell wall. MurJ is the flippase of lipid-linked precursors for peptidoglycan biogenesis[END_REF]. But a recent work from Meeske and collaborators reinforced the hypothesis of MurJ being a candidate for lipid II flippases [START_REF] Meeske | MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis[END_REF]. They identified AmJ ("Alternate to murJ") in B. subtilis, a functionally redundant protein with MurJ. A strain lacking the four murJ paralogs and amj was shown to be lethal. Furthermore, AmJ can substitute native MurJ in E. coli. AmJ is a very different protein to MurJ and its expression is activated in absence of YtgP, which might benefit the organism during MurJ inhibiting conditions [START_REF] Meeske | MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis[END_REF]. It is possible that MurJ and AmJ act in the same process, under different conditions.

Despite all efforts, it is still unclear which of these proteins are the lipid II flippases in G+ and Gbacteria or if all of them flip lipid II across the cytoplasmic membrane depending on growth conditions.

INTRODUCTION

Stage 3, assembly of peptidoglycan

The final step of PG synthesis is stage 3, where the PG precursor is linked to existing glycan strands (Figure 4). Glc-NAc from the lipid II is bound by transglycosilation to the reducing part of the growing strand. A molecule of undecaprenyl-pyrophosphate is liberated and dephosphorylated to form bactoprenol, which becomes available to form lipid I again. This is a critical step as it will limit the formation of PG precursors. The PG subunit is then linked to the preexisting PG mesh by PBPs, penicillin binding proteins. As their name suggests, PBPs are the target of the antibiotic penicillin and its derivatives. Penicillin resembles to the D-ala-D-ala moiety of the PG pentapeptide. Therefore, penicillin can bind the active site of PBPs, inactivating them permanently [START_REF] Tipper | Mechanism of action of penicillins: a proposal based on their structural similarity to acyl-D-alanyl-D-alanine[END_REF]. PBPs have been studied for a long time, and their nomenclature is confusing. In what follows, I

will summarize what is known about PBPs here.

Some PBPs have both transglycosilation (TG) and transpeptidation (TP) activities. It is thought that most of the peptide bonds are created at the same time as TG happens, but some peptide bonds can be formed by other PBPs after TG (see section 1.2.2.3.1) [START_REF] Bhavsar | Cell wall assembly in Bacillus subtilis: how spirals and spaces challenge paradigms[END_REF]. A study showed that

PBP1b from E. coli performs TG and TP simultaneously [START_REF] Bertsche | In vitro murein peptidoglycan synthesis by dimers of the bifunctional transglycosylase-transpeptidase PBP1B from Escherichia coli[END_REF] while, in another study, PBP1a from E. coli showed a preference for peptides from elongating glycan strands [START_REF] Born | In vitro synthesis of cross-linked murein and its attachment to sacculi by PBP1A from Escherichia coli[END_REF]. TP takes place between the pentapeptide moieties of neighboring strands, but not all pentapeptide moieties form a pentapeptide bond and the percentage of TP varies between strains (de Pedro [START_REF] De Pedro | Structural constraints and dynamics of bacterial cell wall architecture[END_REF]. PBPs are multidomain proteins. Most of them have a cytoplasmic tail, a transmembrane domain and two domains looking at the periplasmic area, where the PG synthesis takes place [START_REF] Sauvage | The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis[END_REF]. The amount of PBPs and their activity vary depending on the strain (Table 1). I will continue with a short classification of PBPs and their activity.

Transpeptidases

The C-terminal domain of PBPs, the penicillin-binding (PB) domain, is in charge of the peptide crosslinking of the sacculus, the TP. PBPs that present TP activity are called high molecular mass PBPS or HMM [START_REF] Sauvage | Glycosyltransferases and transpeptidases/penicillin-binding proteins: valuable targets for new antibacterials[END_REF]. In B. subtilis, there are ten HMM PBPs (Table 1). These can be divided into class A (PBP1, PBP2c, PBP2d and PBP4) and class B (PBP2a, PBP2b, PBP3, PBP4b, PbpH and SpoVD). Proteins in class A have both TP and TG activities while those in class B only perform TP.

Table 1 PBPs classification (modified from [START_REF] Sauvage | The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis[END_REF]).

INTRODUCTION

The most common TP reaction is DD-TP. In this reaction the enzyme binds to the D-ala-D-ala moiety of the peptide stem, releases the last D-ala and binds the penultimate one to the terminal DAP of a neighbour acceptor PG strand [START_REF] Sauvage | The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis[END_REF]. The second most extended way of transpeptidation that happens in bacteria is LD-TP (Figure 2D). In this case, it is DAP-D-ala from the donor that binds to the DAP from the acceptor. LD cross-linking isn't universal nor essential for bacteria and, in those bacterial species with both DD-and LD-TP, the relative abundance for each type is variable [START_REF] De Pedro | Structural constraints and dynamics of bacterial cell wall architecture[END_REF].

Transglycosylases

Some PBPs have an N-terminal domain with glycosyltransferase activity (TG). These also have a Cterminal TP domain and are, therefore, bifunctional PBPs. They fall into the HMM class A category and, in B. subtilis, there are four of them: PBP1, PBP2c, PBP2d and PBP4 (Table 1) [START_REF] Sauvage | Glycosyltransferases and transpeptidases/penicillin-binding proteins: valuable targets for new antibacterials[END_REF]. These form glycan strands with varying lengths. PBP1a from E. coli forms glycan strands of about 20 disaccharides while PBP1b would form strands with more than 25 disaccharides on average [START_REF] Sauvage | The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis[END_REF].

Carboxypeptidases and endopeptidases

In B. subtilis, there are six carboxipeptidases (CP) or endopeptidases (EP) PBPs (also called class C or low molecular mass PBPs -LMM): PBP4*, PBP4a, PBP5, PBP5*, DacF and PbpX PBPs (Table 1).

They can be linked to the cytoplasmic membrane or interact directly with either PG or TA. These enzymes modify the peptide stem of existing PG at the CW. Their exact role isn't established, but PG modifications allow the introduction of new PG to the mesh and cell division. They are thought to be involved in cell morphology, competence, cell motility, germination and biofilm formation [START_REF] Frirdich | Peptidoglycan hydrolases, bacterial shape, and pathogenesis[END_REF].

Cell wall synthetic machineries

During the cell cycle, a bacterial cell has to grow and form two daughter cells. To do so, the continuous PG macromolecule (sacculus) has to be enlarged and then divided without compromising cell integrity. CSM are multiprotein complexes that ensure this process. They are formed of PG synthases (most of the PBPs), hydrolases and other morphoproteins. Depending on when they're active, we can divide CSM into elongasome and divisome. Some of the enzymes are present in both complexes but some are specific to each CSM. The most characteristic element of elongasomes is the presence of MreB, an actin-like protein, highly conserved in rod-like bacteria, while in the case of divisomes, it is FtsZ, the first described bacterial tubulin homolog [START_REF] Bhavsar | Cell wall assembly in Bacillus subtilis: how spirals and spaces challenge paradigms[END_REF]Jones, INTRODUCTION Carballido-Lopez, & Errington, 2001). Cytoskeletal proteins are thought to create scaffolds, positioning CSMs during elongation and cell division [START_REF] Egan | Regulation of bacterial cell wall growth[END_REF].

Bacterial cytoskeleton

Eukaryotic cells have a filament system that acts as an organization center for a number of essential cellular processes like cell division, chromosome segregation, cell polarity and intracellular traffic [START_REF] Howard | Dynamics and mechanics of the microtubule plus end[END_REF]. It is a complex and dynamic system formed of three main filaments: actin microfilaments, tubulin microtubules and intermediate filaments (IF). For a long time, it was believed that only eukaryotic cells had a cytoskeleton, but the first prokaryotic homologs of cytoskeletal proteins were discovered two decades ago, and members of all three eukaryotic filament family have now been discovered in bacteria [START_REF] Cabeen | The bacterial cytoskeleton[END_REF]. Furthermore, bacteria contain additional specific cytoskeletal proteins absent from eukaryotes [START_REF] Ingerson-Mahar | A growing family: the expanding universe of the bacterial cytoskeleton[END_REF].

Before that, the bacterial cell was thought to be an unorganized bag, but it has been demonstrated that there is an intricate organization in bacterial cells, forming supramolecular assemblies in the cytoplasm or associated with the membrane [START_REF] Govindarajan | Where are things inside a bacterial cell?[END_REF]. An example of this organization is the localization of specific proteins to the cell poles. Bacteria have different ways to attain this organization, which is thought to enable the integration of extracellular information and optimize producing a correct response [START_REF] Govindarajan | Where are things inside a bacterial cell?[END_REF]. There is a very interesting and well written review covering this aspect from the Jacobs-Wagner group [START_REF] Laloux | How do bacteria localize proteins to the cell pole?[END_REF].

By organizing the cell, the bacterial cytoskeleton is crucial for virulence, cell shape maintenance, growth and motility. Cytoskeletal proteins also help by forming an organized structure that enables molecular transport. I will now make a more detailed description of these prokaryotic cytoskeletal proteins.

Bacterial cytoskeletal proteins

Actin-like proteins

Actin is the most abundant protein in eukaryotic cytoplasms. It presents a characteristic fold, with four domains stabilized in the center by an ADP molecule. Actin polymerizes in the presence of ATP and can be found in two conformations: globular or G-actin and filamentous or F-actin [START_REF] Ingerson-Mahar | A growing family: the expanding universe of the bacterial cytoskeleton[END_REF]. Actin-like proteins are present in every known cell, from eukaryotic cells to prokaryotic, including archaeal cells [START_REF] Petek | Bacterial actin-like proteins: purification and characterization of self-assembly properties[END_REF]. Several bacterial proteins belonging to the actin family have been described, and are involved in many cellular processes. Some examples are FtsA that participates in cell division [START_REF] Pichoff | Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA[END_REF], MreB who is involved in CW synthesis and INTRODUCTION cell morphology [START_REF] Dominguez-Escobar | Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria[END_REF][START_REF] Garner | Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis[END_REF]van Teeffelen & Gitai, 2011), some Alp proteins and ParM that play key roles in plasmid segregation [START_REF] Cabeen | The bacterial cytoskeleton[END_REF] and MamK, closely related with organelle organization [START_REF] Toro-Nahuelpan | Segregation of prokaryotic magnetosomes organelles is driven by treadmilling of a dynamic actin-like MamK filament[END_REF].

ParM and MreB only share ~11% and ~15% similarity, respectively, with eukaryotic actin, but do contain its characteristic 3D fold formed by subdomains IA, IB, IIA and IIB that correspond to subdomains 1, 2, 3 and 4 in actin [START_REF] Carballido-Lopez | The bacterial actin-like cytoskeleton[END_REF][START_REF] Van Den Ent | Prokaryotic origin of the actin cytoskeleton[END_REF] (Figure 5).

INTRODUCTION

Actin microtubules polymerize from both ends, but each end has a different affinity for the addition of monomers. We can distinguish a slow growing end (-or pointed) and a fast growing end (+ or barbed) (Figure 5). Depending on the local concentration of the protein, actin can undergo polymerization, called treadmilling (that is an equilibrium between the loss of monomers at the -end and gain at the + end), steady state or a rapid depolymerization called catastrophe [START_REF] Cleveland | Treadmilling of tubulin and actin[END_REF]. In vivo polymerization studies of MreB and Mbl in B. subtilis ruled out treadmilling as their source of processive movement, which does not exclude the possibility that treadmilling exists at a different timescale [START_REF] Dominguez-Escobar | Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria[END_REF]. ParM polymerization is driven by treadmilling where the stabilization of the polymers by ATP binding and instability due to hydrolysis of the nucleotide give a directionality to ParM polymerization, as in actin.

Tubulin-like proteins

Tubulin microtubules in eukaryotes are formed of α-and β-tubulin. They are dynamic, GTP-dependent and serve as tracks for other motor proteins [START_REF] Lowe | Evolution of cytomotive filaments: the cytoskeleton from prokaryotes to eukaryotes[END_REF]. The first bacterial cytoskeletonlike protein to be described was FtsZ, a tubulin homologue (Figure 6A) [START_REF] Bi | FtsZ and cell division[END_REF]. It is conserved in the majority of free-living bacteria. It forms linear filaments in a GTP-dependent manner that localize at the cell division site forming a structure called the Z-ring [START_REF] Erickson | FtsZ in bacterial cytokinesis: cytoskeleton and force generator all in one[END_REF]. It is used as a cytokinetic scaffold to which other proteins of the cell division complex bind. It has also been showed that FtsZ uses GTP hydrolysis to bend the linear filaments, pulling the membrane inward to help septum formation [START_REF] Li | The structure of FtsZ filaments in vivo suggests a force-generating role in cell division[END_REF]. A recent study from the Xiao group suggests that FtsZ polymerization (highly regulated both by positive and negative regulators) can be fashioned to allow cell division to happen at the correct place and timing [START_REF] Coltharp | Beyond force generation: Why is a dynamic ring of FtsZ polymers essential for bacterial cytokinesis?[END_REF]. According to this study, FtsZ regulators modify the Z-ring structure and dynamics which, in turn, acts as a signal integrator and transduction system for cell wall and cell division.

INTRODUCTION

Using bioinformatic tools, two FtsZ-like protein families were discovered. One of them, the FtsZ1 family, is present in over 120 bacterial and archaeal species, but their function is not well known. They are normally found in operons with genes that share homology with eukaryotic genes involved in vesicle trafficking and membrane remodeling [START_REF] Makarova | Two new families of the FtsZ-tubulin protein superfamily implicated in membrane remodeling in diverse bacteria and archaea[END_REF]. The other family of proteins is even less well understood.

There are also plasmid encoded bacterial tubulin homologs that may play a role in their plasmid partitioning. TubZ is an example from Bacillus thuringiensis, encoded on the virulence plasmid pBToxis (Figure 6B). Interestingly, it forms double-helical filaments that show treadmilling in vivo and are more similar to actin filaments than to tubulin microtubules [START_REF] Larsen | Treadmilling of a prokaryotic tubulin-like protein, TubZ, required for plasmid stability in Bacillus thuringiensis[END_REF]. Another plasmid-encoded tubulin-like protein is RepX, encoded in the plasmid pX01 from Bacillus anthracis.

It has been identified to have a role in plasmid replication [START_REF] Pogliano | The bacterial cytoskeleton[END_REF].

Another family of prokaryotic tubulin homologs was identified in some Prosthecobacter bacteria. It includes the proteins BtubA and BtubB (Figure 6C). They actually have a higher similarity with alpha and beta tubulin than FtsZ or TubZ, but their function remain unknown (Ingerson-Mahar & Gitai, 2012).

IF-like proteins

IF proteins in eukaryotes share a rod-like domain between two variable domains. The most common IF proteins are keratin and lamin. They self-assemble in a nucleotide-independent manner [START_REF] Lowe | Evolution of cytomotive filaments: the cytoskeleton from prokaryotes to eukaryotes[END_REF]. The most studied IF-like bacterial protein is crescentin (CreS), from Caulobacter crescentus (Figure 7A). It was the first IF-like protein to be discovered, in 2003 [START_REF] Ausmees | The bacterial cytoskeleton: an intermediate filament-like function in cell shape[END_REF]. It has mild sequence similarity with eukaryotic IF, but does not depend on nucleotides to form slightly curved filaments in vitro, as eukaryotic IFs. Lack of CreS causes

Caulobacter to lose its typically curved form and to become a straight rod [START_REF] Ingerson-Mahar | A growing family: the expanding universe of the bacterial cytoskeleton[END_REF].

The main similarity between IF and CreS is that they are coiled coil-rich proteins. There are a number of coiled coil-rich proteins in bacteria that self-assemble into filaments in a nucleotide-independent manner. Some act as scaffolds or localization factors of other proteins, but their exact functions are still unknown [START_REF] Lin | Nucleotide-independent cytoskeletal scaffolds in bacteria[END_REF]). FilP is an example of a coiled coil-rich protein from

Streptomyces coelicolor that plays a role in the formation of hyphae (Figure 7B). Deletion of filP induces abnormal hyphal morphology and decreased stability [START_REF] Cabeen | The bacterial cytoskeleton[END_REF]. Other

Walker A Cytoskeletal ATPases (WACAs)

Walker A Cytoskeletal ATPases (WACAs) are a subfamily of the P-loop NTPase family; which includes GTPases, signal recognition particle proteins and eukaryotic cytoskeleton-associated proteins. ParA is a plasmid and chromosome segregation protein that determines the position of other proteins in the cell [START_REF] Pogliano | The bacterial cytoskeleton[END_REF]. In its free state, it is a monomer that dimerizes and then polymerizes when MinD polymerizes at the inner leaflet of the cytoplasmic membrane, attached to it by an amphipatic helix, in an ATP-dependent manner (Figure 8) [START_REF] Lowe | Evolution of cytomotive filaments: the cytoskeleton from prokaryotes to eukaryotes[END_REF]. It associates with MinC and, together, they inhibit FtsZ polymerization, acting as spatial regulators of the site of cell division [START_REF] Lutkenhaus | Assembly dynamics of the bacterial MinCDE system and spatial regulation of the Z ring[END_REF]. In E. directing FtsZ assembly at mid-cell [START_REF] Pogliano | The bacterial cytoskeleton[END_REF].

CtpS

CtpS is an ubiquitous protein that synthesizes CTP from UTP, ATP and glutamine. It has also been

shown that it polymerizes forming linear filaments, which might be used to regulate its enzymatic activity. It is thought to also have a role in C. crescentus' shape determination as it is localized at the inner cell curvature, its overexpression forms straighter cells and the curvature of the cell increases in absence of CtpS [START_REF] Ingerson-Mahar | The metabolic enzyme CTP synthase forms cytoskeletal filaments[END_REF]. In addition, CtpS and CreS co-localize in C. crescentus but can form filaments independently from one another. Also, point mutations of CtpS that abolish its enzymatic activity do not have an effect on C. crescentus cell shape [START_REF] Ingerson-Mahar | The metabolic enzyme CTP synthase forms cytoskeletal filaments[END_REF].

Bacterial actin homologs

MreB

MreB is the first discovered and certainly the better studied member of the family of prokaryotic actinlike proteins (Figure 9A). This protein will be further described in section 1.4. Since its discovery, several other bacterial actin-like proteins have been characterized. I will pursue with their description now.

MamK

Magnetospirillum magneticum has two actin-like genes: mreB and mamK (Figure 9B). MamK forms linear filaments and filament bundles that organize magnetosomes in the bacteria. Magnetosomes are vesicles arranged in chains that enclose a magnetite crystal. Magnetotactic bacteria take advantage of them as a cellular compass to sense the geomagnetic field [START_REF] Bennet | Biologically controlled synthesis and assembly of magnetite nanoparticles[END_REF][START_REF] Pradel | Biogenesis of actin-like bacterial cytoskeletal filaments destined for positioning prokaryotic magnetic organelles[END_REF]. In absence of MamK, magnetosomes are disordered and no filaments are seen [START_REF] Cabeen | The bacterial cytoskeleton[END_REF]. MamK has another key role in magnetosome organization as it is hypothesized that it recruits magnetosomes to the division site and ensures equal inheritance of the organelles [START_REF] Toro-Nahuelpan | Segregation of prokaryotic magnetosomes organelles is driven by treadmilling of a dynamic actin-like MamK filament[END_REF]. and dynamic filament will become straight and stable and plasmid partitioning will be defective [START_REF] Cabeen | The bacterial cytoskeleton[END_REF].

FtsA

One of the elements of the divisome, together with the tubulin-like protein FtsZ, is FtsA. FtsA filaments are polar, dynamic and depend on ATP for their formation. They act as a scaffold for FtsZ,

INTRODUCTION

together with the membrane. At the same time, both the cytoplasmic membrane and FtsZ act as a scaffold for FtsA [START_REF] Mura | Roles of the essential protein FtsA in cell growth and division in Streptococcus pneumoniae[END_REF]. They are called "collaborative filaments" [START_REF] Fink | SnapShot: The bacterial cytoskeleton[END_REF]. In absence of FtsA, septation isn't achieved and cells lyse [START_REF] Mura | Roles of the essential protein FtsA in cell growth and division in Streptococcus pneumoniae[END_REF].

AlfA

AlfA is similar to ParM in that it is another plasmid-encoded actin-like protein linked to plasmid segregation; pLS32 from B. subtilis, specifically (Figure 9D). It also forms left-handed helices along the cell long axis, but they do not present dynamic instability as ParM filaments. AlfA helices group in bundles of mixed polarity. AlfA forms part of a three components system where AlfB acts as a repressor of alfA expression and binds to a three tandem repeat upstream of the alfA promoter, parN [START_REF] Becker | DNA segregation by the bacterial actin AlfA during Bacillus subtilis growth and development[END_REF].

Actin-like protein (Alp) families

After the discovery of AlfA, BLAST studies revealed the existence of 35 actin-like protein (Alp)

families. Some Alps are encoded in chromosome genes, but the majority of them are on plasmids, phage genomes and integrating conjugative elements [START_REF] Cabeen | The bacterial cytoskeleton[END_REF]. The Alp protein that has been studied the most is Alp7A (Figure 9E). It forms filaments that are involved in plasmid segregation in a similar way as ParM, but with in a treadmilling fashion [START_REF] Derman | Phylogenetic analysis identifies many uncharacterized actin-like proteins (Alps) in bacteria: regulated polymerization, dynamic instability and treadmilling in Alp7A[END_REF].

MreB

MreB has been considered as the main prokaryotic actin-like protein for a long time. Nevertheless controversial results from the past few years are raising doubts about how similar these two proteins really are. Actin forms dynamic filaments that are involved in intracellular trafficking, cell shape maintenance, cytokinesis (cell division) and cell movement. MreB is also a dynamic, polymeric protein, but not all of actin's functions are covered by MreB. Furthermore, many of its functions are species-specific.

MreB isoforms

There are three MreB isoforms in B. subtilis: MreB, MreBH (MreB homolog) and Mbl (MreB-like).

They have overlapping functions, even though there are some differences between them that still remain unexplained.

INTRODUCTION

In many bacteria mreB is upstream of the highly conserved morphogenes mreC and mreD. In B.

subtilis, the three genes are co-transcribed and essential [START_REF] Formstone | Localization and interactions of teichoic acid synthetic enzymes in Bacillus subtilis[END_REF]. They are important for cell wall synthesis and cell morphology, although their exact functions remain unclear. The genes encoding MreBH and Mbl are positioned separately from the mreBCD operon.

Deletion of each of the paralogs causes slightly different phenotypes (Figure 10), although bulging and loss of cell form is common to all three (depending on the growth conditions), but the deletion of mreBH causes a much milder phenotype than those linked to the deletion of mreB or mbl. These defects are all rescued by elevated concentration of Mg +2 or (for mreB and mbl mutants) deletion of ponA, the gene encoding for PBP1 [START_REF] Carballido-Lopez | The bacterial actin-like cytoskeleton[END_REF][START_REF] Dominguez-Cuevas | Differentiated roles for MreBactin isologues and autolytic enzymes in Bacillus subtilis morphogenesis[END_REF][START_REF] Kawai | Regulation of cell wall morphogenesis in Bacillus subtilis by recruitment of PBP1 to the MreB helix[END_REF]. The only viable double deletion mutant is ∆mbl ∆mreBH [START_REF] Kawai | Partial functional redundancy of MreB isoforms, MreB, Mbl and MreBH, in cell morphogenesis of Bacillus subtilis[END_REF], even in the presence of Mg +2 . Therefore, under native conditions, MreB is the only of the three isoforms that can support growth and viability on its own, always with high Mg +2 concentrations. In a different study from the Errington group [START_REF] Schirner | The cell wall regulator {sigma}I specifically suppresses the lethal phenotype of mbl mutants in Bacillus subtilis[END_REF], a triple deletion mutant was created, but in a very specific background. The viability of this mutant is possible because of an extragenic suppressor mutation causing the overexpression of SigI (by the deletion of rsgI, gene encoding an anti-sigma factor that controls SigI expression). The resultant strain (∆mreB ∆mbl ∆mreBH ∆rsgI) generates more or less spherical cells that can divide, also Mg +2 -dependent [START_REF] Kawai | Partial functional redundancy of MreB isoforms, MreB, Mbl and MreBH, in cell morphogenesis of Bacillus subtilis[END_REF][START_REF] Schirner | The cell wall regulator {sigma}I specifically suppresses the lethal phenotype of mbl mutants in Bacillus subtilis[END_REF].

INTRODUCTION

Not only the absence of the isoforms is deleterious for the cell, but the overexpression of MreB or MreBH too (although surprisingly Mbl's overproduction does not have deleterious effects on cell growth or shape) [START_REF] Kawai | Partial functional redundancy of MreB isoforms, MreB, Mbl and MreBH, in cell morphogenesis of Bacillus subtilis[END_REF]. The interactions of the actin-like isoforms in B. subtilis with other proteins are also different. All three isoforms interact between them and with RodZ (Defeu Soufo et al., 2010;[START_REF] Dominguez-Escobar | Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria[END_REF][START_REF] Muchova | Control of Bacillus subtilis cell shape by RodZ[END_REF]. MreBH interacts with LytE, a major autolysin in B. subtilis involved both in cell elongation and division [START_REF] Carballido-Lopez | Actin homolog MreBH governs cell morphogenesis by localization of the cell wall hydrolase LytE[END_REF]. As for MreB, there are results suggesting its interaction with TufA, TagT, TagU, PBP1, DapI and ComGA [START_REF] Kawai | Regulation of cell wall morphogenesis in Bacillus subtilis by recruitment of PBP1 to the MreB helix[END_REF][START_REF] Mirouze | MreB-dependent inhibition of cell elongation during the escape from competence in Bacillus subtilis[END_REF][START_REF] Rueff | An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis[END_REF].

On the other hand, there may be an overlap of function as all three proteins, MreB, Mbl and MreBH, colocalize at the cell-side wall together with other elements of the elongasome and present the same velocity and directionality of movement [START_REF] Dominguez-Escobar | Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria[END_REF][START_REF] Formstone | A magnesium-dependent mreB null mutant: implications for the role of mreB in Bacillus subtilis[END_REF]. This suggests that all three are involved in the same process. In line with the common function hypothesis, the results from [START_REF] Kawai | Partial functional redundancy of MreB isoforms, MreB, Mbl and MreBH, in cell morphogenesis of Bacillus subtilis[END_REF] show that, when expressed at the correct level, each MreB isoform is capable of maintaining WT growth and cell shape on their own. They

Biochemical properties of MreB

MreB biochemistry has proven to be a though area of investigation. It is a difficult protein to purify in its soluble, active form. This explains why for a long time MreB's biochemistry was entirely studied on the thermophilic organism T. maritima [START_REF] Bean | Polymerization properties of the Thermotoga maritima actin MreB: roles of temperature, nucleotides, and ions[END_REF][START_REF] Esue | The assembly of MreB, a prokaryotic homolog of actin[END_REF][START_REF] Popp | Filament structure, organization, and dynamics in MreB sheets[END_REF]. With years, more MreBs have been purified in E. coli, Leptospira interrogans, Magnetospirillium gryphiswaldense, Candidatus Magnetobacterium casensis (Mcas),

Chlamydophila pneumoniae [START_REF] Barko | Large-scale purification and in vitro characterization of the assembly of MreB from Leptospira interrogans[END_REF][START_REF] Bean | Polymerization properties of the Thermotoga maritima actin MreB: roles of temperature, nucleotides, and ions[END_REF][START_REF] Deng | In vitro assembly of the bacterial actin protein MamK from ' Candidatus Magnetobacterium casensis' in the phylum Nitrospirae[END_REF][START_REF] Gaballah | Functional analysis of the cytoskeleton protein MreB from Chlamydophila pneumoniae[END_REF][START_REF] Nurse | Purification and characterization of Escherichia coli MreB protein[END_REF][START_REF] Sonkaria | Insight into the assembly properties and functional organisation of the magnetotactic bacterial actin-like homolog, MamK[END_REF]. Still, only a single -and controversial-publication has been released in a Gram-positive organism, B. subtilis [START_REF] Mayer | Assembly properties of the Bacillus subtilis actin, MreB[END_REF]. Table 3 summarizes the biochemical properties of the MreBs studied and mentioned above. 6) [START_REF] Nurse | Purification and characterization of Escherichia coli MreB protein[END_REF]). ( 7) [START_REF] Bean | Polymerization properties of the Thermotoga maritima actin MreB: roles of temperature, nucleotides, and ions[END_REF]. ( 8) [START_REF] Barko | Large-scale purification and in vitro characterization of the assembly of MreB from Leptospira interrogans[END_REF]. ( 9) [START_REF] Gaballah | Functional analysis of the cytoskeleton protein MreB from Chlamydophila pneumoniae[END_REF]. ( 10) [START_REF] Sonkaria | Insight into the assembly properties and functional organisation of the magnetotactic bacterial actin-like homolog, MamK[END_REF]. ( 11) [START_REF] Deng | In vitro assembly of the bacterial actin protein MamK from ' Candidatus Magnetobacterium casensis' in the phylum Nitrospirae[END_REF]. ( 12) [START_REF] Mayer | Assembly properties of the Bacillus subtilis actin, MreB[END_REF].

The only studies that didn't differentiate polymerization from aggregation by further analyzing light scattering and sedimentation results where those performed on B. subtilis MreB (Bs-MreB) and C.

pneumoniae MreB (Cp-MreB) [START_REF] Gaballah | Functional analysis of the cytoskeleton protein MreB from Chlamydophila pneumoniae[END_REF][START_REF] Mayer | Assembly properties of the Bacillus subtilis actin, MreB[END_REF]. What is most striking is that these two studies are the only ones to defend that MreB's polymerization/aggregation is nondependent on ATP. It is very interesting to note that A22, an inhibitor of MreB polymerization, does not affect neither Bs-MreB [START_REF] Noguchi | Anti-infectious effect of S-benzylisothiourea compound A22, which inhibits the actinlike protein, MreB, in Shigella flexneri[END_REF] nor Cp-MreB [START_REF] Gaballah | Functional analysis of the cytoskeleton protein MreB from Chlamydophila pneumoniae[END_REF] while it INTRODUCTION inhibits MreB polymerization in many other organisms [START_REF] Noguchi | Anti-infectious effect of S-benzylisothiourea compound A22, which inhibits the actinlike protein, MreB, in Shigella flexneri[END_REF]. A22 binds to the nucleotide binding (NB) pocket of MreB preventing ATP binding and inhibiting MreB polymerization [START_REF] Bean | A22 disrupts the bacterial actin cytoskeleton by directly binding and inducing a lowaffinity state in MreB[END_REF]. The NB pocket is highly conserved in all MreBs, however, the protein conformation could vary in each MreB in a way that it prevents A22 binding to this NB pocket.

C. pneumoniae is a coccoid, intracellular bacteria and no peptidoglycan has been reliably detected in its CW. It is thought that MreB is needed in these bacteria to maintain the proper functionality of the divisome.

Localization and dynamics of MreB

MreB's localization is one of its most polemic aspects. The subcellular localization of MreB was first performed in 2001 by the Errington group [START_REF] Jones | Control of cell shape in bacteria: helical, actinlike filaments in Bacillus subtilis[END_REF]. This work, together with results from another group on MreB, Mbl and MreBH of B. subtilis [START_REF] Defeu Soufo | Dynamic movement of actin-like proteins within bacterial cells[END_REF], described the MreB proteins to form filaments in the cytoplasm, very close to the membrane. These filaments followed a helical pattern along the cell long axis and were dynamic. Further confirmation came from studies on MreB from E. coli and C. crescentus [START_REF] Figge | MreB, the cell shape-determining bacterial actin homologue, co-ordinates cell wall morphogenesis in Caulobacter crescentus[END_REF][START_REF] Kruse | Dysfunctional MreB inhibits chromosome segregation in Escherichia coli[END_REF]. However, a decade after the first localization study of MreB was published, three independent groups reported results contradicting the helical pattern of MreB and the formation of micrometer long filaments [START_REF] Dominguez-Escobar | Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria[END_REF][START_REF] Garner | Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis[END_REF]van Teeffelen et al., 2011). They observed MreB homologs forming discrete patches that moved along the cell circumference, perpendicularly to the cell long axis, in B. subtilis and in E. coli. These results

were supported by works from the Jensen group who, moreover, demonstrated that the strain used to study MreB in E. coli, MC1000/pLE7, caused artifacts due to the YFP fusion of the protein [START_REF] Swulius | Long helical filaments are not seen encircling cells in electron cryotomograms of rod-shaped bacteria[END_REF][START_REF] Swulius | The helical MreB cytoskeleton in Escherichia coli MC1000/pLE7 is an artifact of the N-Terminal yellow fluorescent protein tag[END_REF]. Since then, new publications have argued against this model, showing high resolution pictures of long structures [START_REF] Olshausen | Superresolution imaging of dynamic MreB filaments in B. subtilis--a multiple-motordriven transport?[END_REF]. Based on these, a new model reviving "the helices" was proposed [START_REF] Errington | Bacterial morphogenesis and the enigmatic MreB helix[END_REF]. There is still an ongoing conflict between helices vs. perpendicular tracks and filaments vs. diffraction-limited clusters. It must be noted that, because of the resolution power of microscopes, a "diffraction limited" object only means that it is the smallest form distinguishable by a microscope. As a consequence, everything smaller than this limit (~ 300 nm), no matter its shape or level of organization, will look like a globular 300 nm patch and smaller filaments will also look as round blobs. Therefore, the debate between filaments and patches is not about the existence of filamentous structures but rather about their size.

-Helix vs. perpendicular tracks:

While original observations made with epifluorescence microscopy suggested helical structures [START_REF] Defeu Soufo | Dynamic movement of actin-like proteins within bacterial cells[END_REF][START_REF] Jones | Control of cell shape in bacteria: helical, actinlike filaments in Bacillus subtilis[END_REF], TIRF and confocal microscopy images showed movements along perpendicular tracks [START_REF] Dominguez-Escobar | Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria[END_REF][START_REF] Garner | Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis[END_REF]van INTRODUCTION Teeffelen et al., 2011). New results using single-molecule tracking experiments have confirmed these observations in E. coli with MreB SW -PAmCherry (PAm-cherry sandwich fusion) [START_REF] Lee | A dynamically assembled cell wall synthesis machinery buffers cell growth[END_REF] and a monomeric Venus sandwich fusion of mreB (MreB mVenus ) [START_REF] Ursell | Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization[END_REF].

Even in new reports claiming the existence of long filaments, those ring shaped, discontinuous structures are oriented perpendicularly to the cell long axis, with a Gaussian distribution around 90° [START_REF] Olshausen | Superresolution imaging of dynamic MreB filaments in B. subtilis--a multiple-motordriven transport?[END_REF][START_REF] Reimold | Motion of variable-length MreB filaments at the bacterial cell membrane influences cell morphology[END_REF].

In conclusion, all recent data agrees about the orientation of the movement, leaving aside the length of the structures.

-Long vs. short structures Graumann's group has showed that GFP (green fluorescent protein)-and YFP (yellow fluorescent protein)-MreB and GFP-Mbl from B. subtilis and MreB SW -RFP (sandwich red fluorescent protein) from E. coli, in exponential growth, form filaments longer than the diffraction limit and up to 1.5 µm (about half a circumference of a typical B. subtilis cell). These were seen with SIM (structured illumination microscopy), STED (stimulated emission depletion) and TIRF-SIM (total internal reflection fluorescent-SIM) superresolution techniques. They also indicate that filaments were disassembled upon cell stress, forming small clusters, and reassembled when optimal growth conditions were renewed [START_REF] Olshausen | Superresolution imaging of dynamic MreB filaments in B. subtilis--a multiple-motordriven transport?[END_REF][START_REF] Reimold | Motion of variable-length MreB filaments at the bacterial cell membrane influences cell morphology[END_REF]. YFP-MreB was shown to be an exception among these fusions because it is non-functional and the YFP tag seems to over-stabilize the MreB polymers, generating artificially long filaments (Figure 11) [START_REF] Swulius | The helical MreB cytoskeleton in Escherichia coli MC1000/pLE7 is an artifact of the N-Terminal yellow fluorescent protein tag[END_REF].

Observations from Olshausen, Reimold and coworkers with GFP and RFP fusions are much puzzling since these fusions have been used in other labs, including ours, producing drastically different results

showing light-diffraction limited structures [START_REF] Dominguez-Escobar | Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria[END_REF][START_REF] Garner | Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis[END_REF].

However, in E. coli, the most recent study using the less perturbing fluorescent MreB fusion to date in this bacterium (a monomeric superfolder GFP sandwiched into MreB) reported filaments of 500 ± 10 nm in minimal media [START_REF] Ouzounov | MreB orientation correlates with cell diameter in Escherichia coli[END_REF].

The ability to form filaments is not the core of the debate since in vitro works support the existence of MreB polymers (see section 1.4.2). The questions are: 1. how long are the structures, 2. what is their orientation relative to their movement and to the cell long axis, 3. is their length correlated with other parameters (speed, direction, density of protein in the cell, growth rate...) and 4. what is their ultrastructure, specially when they are shorter than the diffraction limit (polymer of monomers, polymer of dimers, bundle, parallel or antiparallel sheets...). An interesting result from our lab, to be published, comparing different strains from various labs under different growth conditions, suggests that over-accumulation of MreB above natural levels leads to the formation of long filaments in B. subtilis (Chastanet et al., unpublished results). This suggests that protein expression levels could be key to understand MreB filament length.

INTRODUCTION

Role of MreB in cell shape determination and cell wall synthesis

The most obvious phenotype and probably the primary function of MreB, seems to be the control of rod shape during elongation, although its precise role and way of action are not known. Curiously, most non rod-shaped bacteria lack mreB, but have mreC and mreD. We could hypothesize that MreB is linked to lateral CW elongation in rods and that is why it is absent in non-rods, but there are some exceptions of coccoids with MreB [START_REF] Ouellette | Chlamydia co-opts the rod shapedetermining proteins MreB and Pbp2 for cell division[END_REF]) and this explanation wouldn't fit those cases. Anyway, from pioneering studies on MreB, the shape defect of mreB was reported [START_REF] Doi | Determinations of the DNA sequence of the mreB gene and of the gene products of the mre region that function in formation of the rod shape of Escherichia coli cells[END_REF][START_REF] Wachi | Mutant isolation and molecular cloning of mre genes, which determine cell shape, sensitivity to mecillinam, and amount of penicillin-binding proteins in Escherichia coli[END_REF]. These mutants have a characteristic phenotype, they bulge and curve, even forming telephone cord-like cell chains, and, finally, they lyse (Figure 10). A study from the Shaevitz group [START_REF] Ouzounov | MreB orientation correlates with cell diameter in Escherichia coli[END_REF] uses sub-lethal concentrations of A22 (a drug that inhibits MreB polymerization) and a series of MreB mutants to study the polymerization properties of MreB and its effect in cell shape in E. coli. They obtained a number of MreB point mutations that caused a higher resistance to A22. This allowed them to find two exciting correlations, the strongest being between cell diameter and MreB polymer angle and the second between cell diameter and polymer number. Interestingly, their data shows that a reduction of the helical angle of MreB entails an increase of cell width.

Cell wall integrity is tightly linked to cell morphology. An impressive study from [START_REF] Ursell | Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization[END_REF] measures simultaneously cell shape dynamics, CW insertion and cytoskeletal localization in E. coli.

INTRODUCTION

Their conclusions are that MreB's localization is biased to negatively curved areas along the cell cylinder, directing PG insertion to those areas, causing the straightening of the cell. There are multiple evidences of MreB from different organisms (including B. subtilis) interacting with Mur and DAP proteins as well as with proteins from the CW machinery [START_REF] Divakaruni | The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes[END_REF][START_REF] Favini-Stabile | MreB and MurG as scaffolds for the cytoplasmic steps of peptidoglycan biosynthesis[END_REF][START_REF] Gaballah | Functional analysis of the cytoskeleton protein MreB from Chlamydophila pneumoniae[END_REF][START_REF] Rueff | An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis[END_REF][START_REF] White | Positioning cell wall synthetic complexes by the bacterial morphogenetic proteins MreB and MreD[END_REF]. Kawai and co-workers showed that the localization and correct function of PBP1 is MreB-dependant [START_REF] Kawai | Regulation of cell wall morphogenesis in Bacillus subtilis by recruitment of PBP1 to the MreB helix[END_REF]. Also, in pulldown assays, both

MreB and Mbl associate with PBP1, PBP2a, PBP4 and possibly PBP5, independently [START_REF] Kawai | Partial functional redundancy of MreB isoforms, MreB, Mbl and MreBH, in cell morphogenesis of Bacillus subtilis[END_REF]. Furthermore, PbpH, PBP2a, MreC, MreD and RodA move similarly and colocalize with the MreB/Mbl/MreBH complex [START_REF] Dominguez-Escobar | Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria[END_REF][START_REF] Garner | Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis[END_REF]. Further validation of these results comes from single-molecule experiments that revealed how PBP2 and MreB colocalize transiently to coordinate cell wall synthesis [START_REF] Lee | A dynamically assembled cell wall synthesis machinery buffers cell growth[END_REF]. Their results show how, by PBP2 having a diffusive motion and MreB following directed paths, their transitory interactions are beneficial to buffer growth through variable enzyme abundances and changing environmental conditions. It is possible that MreB acts as the link between PG precursor synthesis and its insertion to the CW.

Other roles of MreB

MreB has been linked to motility in Myxococcus xanthus, spore formation in Streptomyces and chromosome segregation and cell polarity in various bacteria [START_REF] Chastanet | The actin-like MreB proteins in Bacillus subtilis: a new turn[END_REF].

In B. subtilis, it has only been shown to, may be, have a role in chromosome segregation and viral DNA and replication proteins localization.

Chromosome segregation

For some time, MreB was thought to have a role in DNA segregation and that the state of the chromosome affected MreB polymerization [START_REF] Defeu Soufo | Bacillus subtilis actin-like protein MreB influences the positioning of the replication machinery and requires membrane proteins MreC/D and other actin-like proteins for proper localization[END_REF]. Once in-frame deletions of mreB were created, avoiding polar effects on downstream genes, the impact on chromosomal segregation wasn't detectable [START_REF] Formstone | Localization and interactions of teichoic acid synthetic enzymes in Bacillus subtilis[END_REF].

Cell division

Until recently, there was no established link between the different cytoskeletal elements in bacteria. In [START_REF] Fenton | Direct interaction of FtsZ and MreB is required for septum synthesis and cell division in Escherichia coli[END_REF] it is demonstrated that MreB and FtsZ interact and that this interaction is essential for the correct localization of the divisome and septum synthesis. It seems that MreB, besides interacting with FtsZ, recruits PBP1B and PBP2 to the divisome, which is an essential step to pursue with cell division.

Pathogenicity

A very interesting study in Bdellovibrio bacteriovorus, a predatory bacterium with two mreB genes, mreB1 and mreB2, has demonstrated a link between the actin-like protein and predatory rate [START_REF] Fenton | Manipulating each MreB of Bdellovibrio bacteriovorus gives diverse morphological and predatory phenotypes[END_REF]. Both genes are essential in B. bacteriovorus, but they succeeded in creating GFP fusions of each protein with partial functionality that allowed them to study the role of MreB1 and MreB2 independently. MreB1 is implicated in the early differentiation of predatory cells while MreB2 is involved in the late differentiation of those. Addition of A22 helps those cells with partially functional MreB1, most likely because it stabilizes its polymers. In the other hand, addition of A22 during the late stages of WT attack-phase cells didn't have an effect, while it did affect cells with partially functional MreB2. This suggests that, in this condition, MreB2 continued to be active in CW turnover for longer than in the WT strain.

Another example of MreB being involved in pathogenicity comes from a study performed in Pseudomonas aeruginosa [START_REF] Cowles | Surface association and the MreB cytoskeleton regulate pilus production, localization and function in Pseudomonas aeruginosa[END_REF]. They discovered that pilus localization and its correct function are MreB-dependent. They also saw that this is a secondary function of MreB, separate from cell shape maintenance. This is evident through the addition of low concentrations of A22 that don't cause a morphology defect while it alters pilus localization.

Gliding

Bacteria have different motility systems that allow them to change location allowing them to go to more beneficial zones or to develop a different physiological state. Gliding is a type of motility that takes place on surfaces and doesn't involve the development of appendages on the cell [START_REF] Islam | The mysterious nature of bacterial surface (gliding) motility: A focal adhesion-based mechanism in Myxococcus xanthus[END_REF]. Studies performed on M. xanthus have described MglA, a cytoplasmic Ras-like GTPase, as a key factor in the organization of gliding motion [START_REF] Hodgkin | Genetics of gliding motility in Myxococcus xanthus (Myxobacterales): Two gene systems control movement[END_REF][START_REF] Mauriello | Bacterial motility complexes require the actin-like protein, MreB and the Ras homologue, MglA[END_REF]. The interaction of MglA with MreB promotes the assembly of the gliding machinery at the leading cell pole according to movement directionality [START_REF] Islam | The mysterious nature of bacterial surface (gliding) motility: A focal adhesion-based mechanism in Myxococcus xanthus[END_REF].

INTRODUCTION

Aims of the thesis

MreB has been studied for a long time, but it seems that there are more and more open questions than solid answers. No one doubts the role of MreB in cell shape and CW synthesis. The three MreB paralogs in B. subtilis, MreB, Mbl and MreBH, although highly overlapping, have paralog-specific functions that were revealed when cells underwent stress conditions [START_REF] Kawai | Partial functional redundancy of MreB isoforms, MreB, Mbl and MreBH, in cell morphogenesis of Bacillus subtilis[END_REF]. These secondary functions remain unknown.

The main aim of the thesis was to understand the specific function of MreB in B. subtilis that isn't shared with its two other paralogs, Mbl and MmreBH. To do so we took advantage of an operon, ydcFGH, that showed high levels of induction in absence of mreB. The problematic was approached through the characterization of the three genes in the ydcFGH operon, the study of the regulatory link between these and mreB and building a screen to select loss-of-function point mutants of MreB by means of the promoter region of the ydcFGH operon.

Materials and Methods

Media Composition:

Lysogeny broth (LB) medium has a composition as in [START_REF] Sezonov | Escherichia coli physiology in Luria-Bertani broth[END_REF]. Casein hydrolysate (CH) medium has a composition as in [START_REF] Formstone | Localization and interactions of teichoic acid synthetic enzymes in Bacillus subtilis[END_REF].

Media supplements:

Supplements added to the media and the concentration they were used at are in Table 4.

Strains and plasmids

Bacterial strains and plasmids used in this study are given in Table 5 and 6, respectively. This study CCBS170 → RCL422 100 pmol/µL aliquots were stored at -20 °C. Oligonucleotides used in this study are given in Table 7. 50 µL LB cultures were inoculated with the colony of interest and incubated at 37 °C for 30 min.

Reactions were set up on ice using Taq DNA polymerase, following manufacturer's recommendations.

The DNA was introduced by adding 1 µL of the culture to the PCR mix.

E.coli grown under selective pressure and saved as glycerol stock at -80 °C. Checking of the construction was performed by PCR and sequencing (see next section).

2.4.1.9 DNA sequencing:

1. Following sub-cloning in E. coli and cloning into B. subtilis, the complete plasmidic or chromosomal DNA regions that were subject to PCR or isothermal assembly, were sequenced to ensure the complete absence of unwanted mutations. For this, primers upstream and downstream of the inserted area were used. Sequencing of PCR products was performed by the sequence facility of Eurofins MWG and according to their recommendations. 2.4.1.12 Construction of plasmids with transcriptional fusion reporters:

-P ydc1 lacZ: A DNA fragment containing the putative P ydc1 promoter was PCR-amplified (see 2.4.1.3) using oligonucleotides AC1240 and AC1241and purified DNA (2.4.2.4) from WT strain RCL44 as template. The purified product (2.4.1.5) along with plasmid pDG1663 (containing the reporter gene lacZ), were subject to EcoRI/BamHI digestions (according to manufacturer's instructions) prior ligation and transformation in E. coli, generating pAC824.

-P ydc1 luxABCDE: A DNA fragment containing the putative P ydc1 promoter was PCR-amplified (see 2.4.1.3) using oligonucleotides AC1240 and AC1286 and purified DNA (2.4.2.4) from WT strain RCL44 as template. The purified product (2.4.1.5) along with plasmid pAH328 (containing the reporter gene luxABCDE), were subject to SpeI/EcoRI digestions (according to manufacturer's instructions) prior ligation and transformation in E. coli, generating pAC832.

-P ydc2 lacZ: A DNA fragment containing the putative P ydc2 promoter was PCR-amplified (see 2.4.1.3) using oligonucleotides AC1242 and AC1243 and purified DNA (2.4.2.4) from WT strain RCL44 as template. The purified product (2.4.1.5) along with plasmid pDG1728 (containing the reporter gene lacZ), were subject to EcoRI/BamHI digestions (according to manufacturer's directions) prior ligation and transformation in E. coli, generating pAC772.

-P ydc1-2 lacZ: A DNA fragment containing the putative P ydc1-2 promoter was PCR-amplified (see 2.4.1.3) using oligonucleotides AC1240 and AC1242 and purified DNA (2.4.2.4) from WT strain RCL44 as template. The purified product (2.4.1.5) along with plasmid pDG1728 (containing the reporter gene lacZ), were subject to EcoRI/BamHI digestions (according to manufacturer's directions)

prior ligation and transformation in E. coli, generating pAC775.

-P ydc0 lacZ: A DNA fragment containing the putative P ydc0 promoter was PCR-amplified (see 2.4.1.3) using oligonucleotides AC1243 and AC1241 and purified DNA (2.4.2.4) from WT strain RCL44 as template. The purified product (2.4.1.5) along with plasmid pDG1728 (containing the reporter gene lacZ), were subject to EcoRI/BamHI digestions (according to manufacturer's directions) prior ligation and transformation in E. coli, generating pAC778.

-P fruRKA lacZ: A DNA fragment containing the putative P fruRKA promoter was PCR-amplified (see 2.4.1.3) using oligonucleotides AC1248 and AC1249 and purified DNA (2.4.2.4) from WT strain RCL44 as template. The purified product (2.4.1.5) along with plasmid pDG1728 (containing the reporter gene lacZ), were subject to EcoRI/BamHI digestions (according to manufacturer's directions)

prior ligation and transformation in E. coli, generating pAC783.

-P mreBH -lacZ: A DNA fragment containing the putative P mreBH promoter was PCR-amplified (see 2.4.1.3) using oligonucleotides AC1246 and AC1247 and purified DNA (2.4.2.4) from WT strain RCL44 as template. The purified product (2.4.1.5) along with plasmid pDG1729 (containing the reporter gene lacZ), were subject to EcoRI/BamHI digestions (according to manufacturer's directions)

prior ligation and transformation in E. coli, generating pAC783.

Manipulation in B. subtilis

Preparation of B. subtilis competent cells:

To make B. subtilis competent for the uptake of DNA, 500 µL of MC medium per transformation were inoculated with a colony of a freshly streaked strain and incubated at 37 °C until OD 600 nm = 1-1.5.

Preparation of chromosomal DNA:

To isolate genomic DNA, 3 mL of LB medium with the appropriate antibiotic were inoculated with a single colony from a freshly streaked strain and incubated either at 30 °C o/n or at 37 °C for 7 h. Two mL of cell culture were pelleted and resuspended in 450 µL of 50 mM EDTA with 10 µL of lysozyme (100 µg/µL stock). The sample was incubated 20 min at 37 °C. Then 600 µL of Nuclei Lysis Solution (Promega) were added and mixed gently. 200 µL of Protein Precipitation Solution (Promega) were added. The sample was mixed by vortexing and incubated at 4 °C for 5 min. The sample was then centrifuged at 13000 rpm. The supernatant was added to 700 µL of isopropanol and mixed gently.

DNA was pelleted, washed with 600 µL of 70 % (w/v) ethanol and rehydrated with 200 µL of pure water.

2.4.2.5 Creation of marker-less mutants with pop-in, pop-out method:

Marker-less deletion mutants were created mainly as described in [START_REF] Tanaka | Building the repertoire of dispensable chromosome regions in Bacillus subtilis entails major refinement of cognate large-scale metabolic model[END_REF], with minor modifications (Figure 13). In brief the gene of interest is substituted by a phleomycine cassette linked to the c1 gene at a strain containing P λ neo at upp. Selection is possible as C1 inhibits the activation of P λ . Homologous regions at both sides of the pleo-c1 cassette allow a recombination process that exiceses the cassette, producing a clean deletion of the gene of interest leaving no scar.

Creation of marker-less mutants from BKE strains:

BKE knockouts are made by replacing the gene of interest by an erythromycin cassette flanked by loxP sites. The plasmid pDR244 contains a cre gene that catalyzes a site-specific recombination between the lox sites. We can then cure the strain of plasmid pDR244 by growing it at high temperatures. BKE knockout strains were transformed with plasmid pDR244 and selected for spectinomycin resistance at 30 °C, the plasmid replication permissive temperature. Selected colonies were grown it in LB media at 42 °C. Serial dilutions were plated on LB-agar plates and incubated o/n at 42 °C.

Test of lacZ expression:

Strains bearing the lacZ reporter gene were grown in LB cultures at 37 °C for < 8 h and spotted on LB-agar plates supplemented with X-gal. Plates were incubated o/n at 37 °C. If lacZ was expressed, X-gal will have been metabolized and the colony will present a blue coloration. If lacZ is not expressed, the colony will be white.

Growth curves

A single colony from a freshly streaked strain was inoculated in 3 mL of the appropriate medium with supplements and incubated o/n at 30 °C. The precultures were diluted into fresh media to an OD 600 nm 0.005 and incubated at 37 °C until OD 600 nm ≈ 0.2. They were then re-diluted into a 96 well plate to OD 600 nm 0.005 and incubated in a plate reader where OD 600 nm was measured every 5 min for a maximum of 24 h.

This methodology was used to test growth phenotypes in different media, osmotic pressure resistance and resistance to different antibiotics.

Disk diffusion test:

To test the resistance of B. subtilis cells to H 2 O 2 , we performed a disk diffusion assay. The strains were grown in CH media supplemented with the correct selective pressure, o/n, at 30 °C. The cultures were diluted to OD 600 nm = 0.005 in CH medium and grown at 37 °C until OD 600 nm = 0.2. 0.5 mL aliquots of the cultures were plated on LB-agar plates and a sterile disk of filter paper was placed on top of it with 5 µL of 30 % H 2 O 2 . Plates were incubated o/n at 37 °C. H 2 O 2 diffuses from the paper disk creating a gradient. Cells start growing forming a halo around the disk. The size of the halo depends on their resistance to the substance.

Viability test:

Cells were grown in CH at 37 °C and aliquots of the culture were taken at OD 600 nm = 0.3 (exponential phase of growth), OD 600 nm = 1.2 (growth phase transition) and OD 600 nm = 2,1 (stationary phase of growth). Serial dilutions were made in fresh CH media and plated on DSM-agar plates. After an incubation of 12 h at 37 °C, colony forming units (CFU)/mL were calculated.

Transformation efficiency:

Strains were transformed with DNA from strain ABS1990 carrying a erythromycin marker.

Transformation was performed as in [START_REF] Mirouze | MreB-dependent inhibition of cell elongation during the escape from competence in Bacillus subtilis[END_REF].

Sporulation efficiency:

Strains were grown in DSM medium during 30 h at 37 C. Serial dilutions were plated on DSM-agar plates before and after a 20 min heat shock at 80 °C. Sporulation efficiency was calculated as the percentage of the vegetative cells that undergo a complete sporulation process yielding heat resistant spores and was calculated as the ratio between the spore concentration and total cell concentration.

2.4.3

Western blot:

Strains were grown in CH media at 37 °C until OD 600 nm = 0.2 -0.3. 2 mL aliquots of the cultures were spun down at 13000 rpm for 10 min. The pellet was either used to continue with the process or frozen at -20 °C. The pellet as resuspended in 25µL resuspension buffer (50 mM glucose, 1 mM EDTA, 50 mM Tris pH = 8,0, 1 mg/mL lysozyme, protease inhibitors) and incubated for 5 min at RT. Then 25 µL of ice-cold lysis buffer (500 mM NaCl, 1 % NP40, 50 mM tris pH = 8,0, 5 mM MgCl 2 , 0,05 % benzonase) is added and incubated on ice for 20 min. The clear lysate is used for SDS-PAGE (see 2.3.3.1). The proteins on the SDS-PAGE are transfered to a nitrocellulose membrane during 2 h, at 140 mA. The membrane is then blocked in TBST buffer (0,2 M Tris base, 1,5 M NaCl, 0,05 % Tween 20) with 5 % milk for 1 h, at RT. Three rounds of 10 min washing are performed afterwards with TBST buffer and then the incubation of the primary antibody is performed o/n at RT at a ratio of 1/10000 in TBST buffer. Another three rounds of 10 min washing are performed with TBST buffer and then an incubation of the secondary antibody in TBST at a ratio of 1/10000 for 1 h at RT. The membrane was developed using ECL Bio-Rad reagents as per the manufacturer on a Chemidoc imaging system.

RNA procedures:

2.4.

RNAseq

WT strain (ABS2005) and ∆ydcH strain (ASEC56) were grown as in 2.4.4.1 and RNA was extracted as in 2.4.4.2. Samples were processed by the platform Imagif (Gif sur Yvette) to generate a library of RNA fragments that were then sequenced using the NEXT SEQ SR 150 nt method. Treatment of this data was performed by collaboration with C. Guerin from the Maiage team at INRA, Jouy en Josas.

Microscopy methods:

Sample preparation for microscopy

For sample preparation, pre-cultures of B. subtilis were grown o/n in CH medium supplemented with 20 mM MgSO 4 and appropriate antibiotic selection, from -80 °C stocks, at 30 °C. Pre-cultures were diluted in CH media to an OD 600 nm = 0,005 and incubated at 37 °C without antibiotics and without supplementations, except if specified. Samples for microscopic observation were taken during exponential growth (OD 600 nm ≈ 0,2 -0,3) and mounted on CH-1 % agarose pads freshly made.

Total internal fluorescence microscopy

Time-lapse TIRFM movies were acquired at least two different days for each strain and condition. 1 µL of culture was spotted on thin agarose pad (1 % agarose in CH media), topped by a coverslip and immersion oil and mounted immediately in the temperature-controlled microscope stage. For all TIRFM acquisitions, exposure time was 100 ms. Inter-frame intervals were 1 s over 1 min movies (2 min for B30 and B46). Imaging was performed on an inverted microscope (Nikon Ti-E) equipped with an Apo TIRF 100x oil objective (Nikon, NA 1.49), with an iLas2 laser coupling system from Roper Scientific (150 mW, 488 nm). Images were collected with an electron-multiplying charge-coupled device (EMCCD) camera (iXON3 DU-897, Andor) at maximum gain setting (300) attached to a 2.5X magnification lens. Final pixel size was 64 nm. Image acquisition was controlled by a Metamorph v.7 software package.

Results

Previous data from our group (E. Marchadier, unpublished) had revealed in a whole-genome transcriptional analysis that the ydcFGH operon was highly overexpressed in absence of MreB (Figure 12). This characteristic was specific for the deletion of mreB as the operon's expression was not modified in absence of Mbl or MreBH, the two isoforms of MreB present in B. subtilis. It suggested that MreB may have a specific property leading to the repression of ydcFGH that was not shared by the two other actin-like proteins. We decided to undertake a comprehensive analysis aiming to both, reveal the function of this operon and to decipher the regulatory mechanism linking the presence of mreB with the expression of these genes. Using a variety of genetic techniques, we showed i-that

YdcH is a probable transcriptional repressor controlling its own expression, ii-that this regulator may constitute a new transition state regulator in B. subtilis, and iii-that a frameshift mutation in ydcH is probably responsible for the deregulation of the operon in our ∆mreB laboratory strain.

As a second but parallel part of the project, in a first attempt, we used the promoter of ydcFGH, highly activated in absence of mreB, to create a genetic screen of randomly generated loss-of-function MreB mutants. Because we demonstrated that the link between ydcFGH and MreB was not direct, the genetic screen was finally performed by means of a different upregulated gene in absence of mreB (mreBH, Figure 12). MreB's function remains elusive despite two decades of extensive efforts both in Gram-positive and Gram-negative models (see Introduction) therefore the characterization of these mutants through the study of their cell growth, cell morphology and MreB dynamics, revealed very promising results. We show critical residues that uncouple the growth of B. subtilis to its ability to form actively moving directional patches, and suggest that MreB could act as a link between cell metabolism and CW synthesis.

By combining all these data and techniques, we have acquired a better understanding of MreB and some insights on the ydcFGH operon in B. subtilis. Although a subset of the results was, at the time, somewhat disappointing, it led us to very interesting and promising preliminary conclusions that may allow us to pin point the so long searched MreB's function(s).

Functional analysis of ydcF, ydcG and ydcH.

3.1.1 The ydcFGH operon is composed of three genes of unknown functions.

To get clues about the potential function of this operon, we first performed an in silico analysis using the BLAST (looking for sequence similarities) and Phyre2 (searching for protein fold conservation) web tools on each gene of the operon [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF][START_REF] Kelley | The Phyre2 web portal for protein modeling, prediction and analysis[END_REF]. The first gene, ydcF, a small 292 nucleotides long orf (open reading frame), encodes a protein carrying a domain (residues 20-53) similar to the relaxase superfamily as predicted by BLAST. Proteins in this superfamily are involved in secretion (type IV systems), horizontal gene transfer and nicking of ssDNA [START_REF] Balzer | Essential motifs of relaxase (TraI) and TraG proteins involved in conjugative transfer of plasmid RP4[END_REF]. The second orf encodes YdcG, a protein predicted to have an EVE domain: potentially a RNA-binding domain present in proteins with broad types of functions [START_REF] Bertonati | Structural genomics reveals EVE as a new ASCH/PUA-related domain[END_REF]. Finally YdcH was predicted, with a very strong confidence, to be a MarR-type transcription regulator (TR). MarR TRs are usually involved in the response to environmental changes, helping cells to improve their survival. They are frequently linked to multiple antibiotic, salt and aromatic molecules resistance as well as to virulence (Chang, Chen, Ko, [START_REF] Chang | Structural analysis of the antibiotic-recognition mechanism of MarR proteins[END_REF][START_REF] Ellison | Regulation of virulence by members of the MarR/SlyA family[END_REF][START_REF] Grove | MarR family transcription factors[END_REF][START_REF] Vazquez-Torres | Redox active thiol sensors of oxidative and nitrosative stress[END_REF].

Construction of knock-out mutants of ydcF, ydcG and ydcH

To understand what ydcF, ydcG and ydcH are involved in, we created a series of strains inactivated for each of the genes separately. ydcH was inactivated by replacement with a spectinomycin resistance cassette (ABS1381) while ydcF and ydcG, to prevent polar effect on downstream gene(s), were inactivated by marker-less deletions following the pop-in pop-out method (see Materials and Methods), generating strains ASEC24 and ASEC5 respectively (Figure 13).

Phenotypic characterization of ydc genes exposes an inappropriate strain frame

The three mutant strains were used along the WT 168, in a variety of assays in order to fully characterize the ydcFGH operon. These experiments strongly suggested that deletion mutants of ydcF and ydcG were affected in several stationary phase events including cell survival and sporulation (see Appendix 1). But by the end of this study, an ultimate whole genome transcriptomic profiling experiment (section 3.3) revealed, to our surprise, the presence of significant gaps, covering more than 200 genes, in the genome of the two strains constructed via the "pop-in popout" method. This unveiled that the strain NC101 bearing P λ neo required for this approach (and increasingly used in our lab) had been cured of its prophages SPβ, PBSX and the Skin element.

Suspecting that at least some of the phenotypes we had observed could be due to the difference of background between these strain and the WT reference, we generated new marker-less deletion mutants. This time we used mutants constructed as part of a comprehensive knock-out library (BKE) that were recently made available by the Bacillus Genetic Stock Centre. Each of these "BKE" strains has a single orf replaced by an erythromycin cassette that can be evicted easily (see Materials and Methods for details), creating new marker-less mutants for ydcF (ASEC287) and ydcG (ASEC289).

We also took this opportunity to make a marker-less mutant of ydcH (ASEC293).

None of the phenotypes previously observed with the original ydcF and ydcG knock-out mutants could be confirmed in the newly constructed deletion strains (see Appendix 1 for details), and as shown on Figure 14, all three mutants grew as the wild type strain in a variety of media, from exponential to stationary phase of growth.

YdcF, YdcG and YdcH are not involved in stress resistance

YdcH is predicted to be a TR of the MarR family. MarR TRs are DNA-binding proteins that regulate the expression of genes by binding as dimers to a palindromic sequence in their promoters [START_REF] Grove | MarR family transcription factors[END_REF]. Most MarR TRs act as repressors and usually control their own expression in addition to that of other genes. Accordingly, as we will show below (section 3.2.2), YdcH is involved in its own repression, reinforcing this prediction. Since MarR TRs are implicated in the response to multiple environmental adaptations as antibiotic stress, oxidative stress, synthesis of virulence factors and degradation of aromatic compounds [START_REF] Krasper | The MarR-Type Regulator Rdh2R Regulates rdh Gene Transcription in Dehalococcoides mccartyi Strain CBDB1[END_REF], we decided to test the effect of deleting ydcH on the adaptive ability of B. subtilis to various stresses.

We first tested high osmolarity by growing cells in rich CH medium supplemented with NaCl ranging from 1 to 2 M (see Materials and Methods). As seen in Figure 15A, no impact on growth, relative to that of the wild type strain, could be detected with any of the concentrations tested. Similarly, cells were tested for resistance to oxidative stress using H 2 O 2 in a disc diffusion assay (see Materials and Methods; Figure 15E). The halos of both strains presented no differences, suggesting identical resistance to H 2 O 2 between the deletion mutant and the WT. Finally, we tested the resistance of the deletion mutant to a set of antibiotics (vancomycin, methicillin, D-cycloserine and rifampicin) by growing cells in liquid rich CH medium supplemented with increasing concentrations of each antibiotic (see Materials and Methods; Figure 15B-D). Again, no benefits or impairments could be observed in the mutants, relatively to the wild type strain.

Together, these results show no involvement of YdcH in a broad range of stress conditions known to affect other MarR-type TR mutants. This suggests that YdcH is either involved in resistance to more exotic stresses, or in a completely different function.

Transcriptional study of ydcFGH

Fuctional characterization of the genes in the ydcFGH operon did not shed any light on their link with MreB or their function. We therefore decided to focus on undersanding the regulatory relation that caused the increase of expression of the ydcFGH operon in absence of mreB (Marchandier et al., unpublished, Figure 12).

ydcH is under the control of two promoters

The genome-wide transcriptional analysis by tilling array done by Nicolas and coworkers [START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF], suggested the existence of two promoters. One, located in front of ydcF (hereafter named P ydc1 ), that would initiate the expression of a long transcript including the three orfs, and a second (P ydc2 ) lying in the ydcG coding region which would allow only the expression of ydcH (Figure 16).

We initially constructed four reporter transcriptional fusions to lacZ (Figure 16; see Materials and Methods): P ydc1 corresponding to 303 bp in front of the ydcF orf and potentially containing the first promoter; P ydc2 extending from 153 bp upstream ydcF to 56 bp into ydcG, excluding the first and containing the putative second promoter; P ydc1-2 containing both putative promoter regions; and P ydc0 containing none of them. As shown on Figure 16A, in a WT background, no expression could be detected from P ydc0 and P ydc1 , while a weak expression could be observed with P ydc2 and P ydc1-2 .

Therefore, we concluded that the expression of the ydcFGH operon mainly originates from P ydc2 in a WT background, and is maintained at a very low level of expression.

Figure 16 The ydcFGH operon. A. The ydcFGH operon is formed by three genes: ydcF, ydcG and ydcH. The expression of these genes is driven by two promoters: P ydc1 and P ydc2 . Transcriptional fusions with the reporter gene lacZ and the first promoter (P ydc1 lacZ), the second promoter (P ydc2 lacZ), both of them (P ydc1-2 lacZ) or the region in between (P ydc0 lacZ) confirm that the expression of the operon, in WT conditions, is regulated by a low, basal activation of P ydc2 . P ydc1 is activated in the absence of mreB (strain ABS1762) or ydcH (replaced with spc (ABS1820), or in a marker-less strain (ASEC305)) but not in absence of mbl (ABS1769), mreBH (ABS1824), ydcF (ASEC297) or ydcG (ASEC301). B. YdcH may act as a repressor of its own operon's expression through the repression of P ydc1 .

YdcH, but not YdcF nor YdcG, is involved in the control of P ydc1 expression

When ydcH is inactivated, P ydc1 and P ydc1-2 are strongly activated while P ydc2 maintains a low basal level of activation (Figure 16A), suggesting that most of the transcripts initiate from the first promoter in this context. However, inactivation of ydcF or ydcG had no impact on either P ydc1 or P ydc2. Taken together, our results draw a model (Figure 16B) in which YdcH is produced from constitutively low expressed transcripts originating from P ydc2 and represses the expression of the whole ydcFGH operon in a negative regulatory feedback loop acting on P ydc1 . The prediction of YdcH as a TR strongly suggests that the repression occurs directly through the binding of the protein to its own promoter.

The absence of MreB is not responsible for P ydc1 induction

We next tested the impact of the deletion of the mreB paralogs on the expression of the two ydc promoters. Similar results to that obtained with the ydcH deletion are obtained when the reporters are introduced into the ∆mreB 3725 strain (Figure 16A), while deleting mbl or mreBH had no impact.

These results suggest a repressing effect of MreB on the expression of the first promoter of ydcFGH, confirming previous genome wide expression data from E. Marchadier (unpublished).

We next tried to unveil the link between its expression and the absence of the mreB gene. A puzzling result was obtained when mreB was cloned back at an ectopic locus in the genome under a xylose inducible or its natural promoter, in the strain 3725 deleted for mreB. In these trans-complemented strains, the lacZ reporter was still activated suggesting that the sole absence of the MreB protein was not responsible for P ydc1 induction. It should be noted that at this step, many alternative hypotheses were envisioned and tested to explain this puzzling result, which are described in Appendix 2. By the end of our investigation, we suspected that a mutation unlinked to mreB was responsible of the induction of the expression of ydcFGH.

We then decided to realize a complete sequencing of the ∆mreB strain (3725). As shown on table 8, and to our astonishment, this strain bears more than 50 mutations compared to its supposedly parent strain 168. Among them, we noticed a single nucleotide deletion in the ydcH orf. The deletion has occurred in a potentially sliding stretch of A and led to a frameshift. The resulting YdcH protein is predicted to be truncated of more than half its full size. Based on our transcriptional study (see Figure 16), we know that the absence of functional YdcH leads to induction of P ydc1 , which is the most probable cause for the overexpression of the ydcFGH operon in strain 3725 (∆mreB).

Table 8 Mutations found in the ∆mreB strain (3725). Sequencing of strain 3725 revealed the existence of 52 SNPs, 27 introducing an amino-acid change and 4 a frameshift, including one in ydcH (bold).

YdcH, a new regulator for carbon metabolism?

So far, we were not able to infer the function of the ydcFGH operon nor the regulatory link between the operon and mreB or why this operon was upregulated in the ∆mreB strain. Because YdcH is probably a putative TR, a prediction reinforced by our transcriptional analysis of its promoters, we reasoned that clues on its role could emerge from uncovering the genes potentially under its control.

For this, we performed a whole genome expression analysis by an RNAseq analysis, comparing ∆ydcH and WT cells. In order to find the most appropriate time point for this experiment, we first monitored the expression of ydcFGH all along the growth phase, using a transcriptional reporter fusion with luxABCDE [START_REF] Meighen | Bacterial bioluminescence: organization, regulation, and application of the lux genes[END_REF] The two largest functional groups of genes affected by YdcH are prophages (44 down and 23 upregulated) and carbon/amino-acids metabolism (60 upregulated genes) genes. A number of other functions are also affected including sporulation (29), cell wall (19), metal homeostasis ( 16) and translation (19).

(see Materials and Methods). The expression of ydcFGH

The large number of significantly affected genes (> 350, including numerous down-regulated genes) observed in the ∆ydcH strain (in particular during exponential growth) comes a bit as a surprise for a putative transcriptional repressor. This strongly advocates for indirect effects through, potentially, other regulators. Some genes coding for known regulators and three other uncharacterized putative MarR TRs (yisI, ypoP, and ykvN) appear to be affected in absence of YdcH. They may contribute to the extent of the impact of the ydcH deletion. We also imagined that, without affecting the transcription of regulators, their activity could be modified in this context, but a systematic check for known regulations affecting the 350 genes failed to reveal the induction or repression of any complete regulons. The largest affected regulon found was that of the transition state regulator, AbrB, with 37 genes impacted (on a total of 249 genes controlled by AbrB).

Thus, so far, it is unclear how the expression of such a large number of genes can be affected by the absence of ydcH, but a direct control can't be excluded. The most striking characteristics of the YdcH regulon are the fairly large number of genes from the prophages and carbon metabolism categories it contains, and the various functional categories that are affected.

MreB mutagenesis

In order to get new insight into MreB, we used a genetic approach based on previous data from the group (Marchandier et al.,unpublished,Figure 12) that allowed us to identify key residues. We designed a genetic screen to obtain a collection of randomly generated loss-of-function MreB mutants.

After generating fusions of these MreB mutants to the green fluorescent protein (GFP), we performed a full characterization of these strains, including cell growth, cell morphology and MreB dynamics.

The results generated are revealing critical residues uncoupling the growth of B. subtilis to its ability to form actively moving directional patches, and suggest that MreB could act as a link between cell metabolism and CW synthesis.

Setting up a genetic screen for MreB loss-of-function mutants

In order to create a genetic screen that would enable us to identify mreB mutants affected in their function(s), we needed a reporter gene that would be activated in absence of functional MreB. For this purpose, we took advantage of an unpublished investigation performed in our lab (Marchadier et al., unpublished) in which the impact of the absence of each of the actin-like proteins of B. subtilis (MreB, Mbl and MreBH) on gene transcription at the genome-wide level was characterized. To create a reporter gene for MreB functionality, we planned to fuse the promoter region of one of the genes overexpressed in absence of MreB to lacZ. This gene encodes the β-galactosidase enzyme from E. coli, that cleaves lactose into glucose and galactose, or the lactose surrogate Xgal, freeing an insoluble blue product [START_REF] Juers | LacZ beta-galactosidase: structure and function of an enzyme of historical and molecular biological importance[END_REF] (see Materials and Methods for details).

RESULTS

In a first attempt, we created a reporter gene by fusing the promoter region of the operon ydcFGH (P ydc1 ) to lacZ (see Materials and Methods and ydcFGH operon section). ydcF, ydcG and ydcH are the three most overexpressed genes in the absence of mreB (15, 23 and 24 fold induction respectively, Figure 12), and were an obvious first choice as a reporter candidate. While we started investigating the promoter structure and characteristics of the ydcFGH operon (see results from ydcFGH operon above), it quickly appeared that this reporter remained activated in cells deleted for mreB but complemented in trans with a gfp-mreB fusion. Induction of this reporter in the presence of a wild type GFP-MreB fusion made it improper for the screening purpose and other candidate reporters were investigated.

We then turned on other strongly induced genes on the list: mreBH and the fruRKA operon. Our previous transcriptional analysis indicated that fruR, fruK and fruA are induced 4 to 8 times, and mreBH 4 times in absence of mreB (Figure 12). We created two transcriptional fusions with lacZ: one (P fru ) with a region encompassing the three predicted promoters of the fru operon [START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF] and one (P mreBH ) with the promoter region of mreBH. Both fusions were introduced into the WT and ∆mreB backgrounds and compared by plating strains on plates supplemented with Xgal. While differences in P fru lacZ expression between the strains happened to be too subtle for an efficient screen (Figure 19), differences of P mreBH expression in a WT strain and in a ∆mreB strain could be easily perceived. We therefore retained this reporter fusion to screen for MreB loss-of-function mutants. Activation of the promoter region P fru is too high, both in a WT and a ∆mreB background, to easily differentiate between them. However the difference of expression of P mreBH lacZ between WT and ∆mreB allows an easy screening of the colonies.

Random mutagenesis of mreB

Next, we realized a random mutagenesis selectively on the mreB locus (see Materials and Methods).

Briefly, we performed a mutagenic PCR that produced a DNA fragment containing the end of maf, radC, a kanamycin resistance cassette, mreB and the beginning of mreC (Figure 20). The resulting product was transformed and integrated in a single step into a B. subtilis strain containing the abovementioned reporter. Blue colonies, indicating activation of P mreBH , were selected.

We obtained a total of 73 mutants, 24 of which were discarded because they contained a mutation introducing either a frameshift in the mreB orf or a stop codon leading to the expression of a truncated version of MreB. Only 12 false positive clones were not mutants for mreB but carried mutations on surrounding genes (mreC, radC and maf). Mutants in mreC were a fair possibility that we anticipated since MreC and MreB are supposed to act together in a complex [START_REF] Defeu Soufo | Bacillus subtilis actin-like protein MreB influences the positioning of the replication machinery and requires membrane proteins MreC/D and other actin-like proteins for proper localization[END_REF][START_REF] Divakaruni | The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes[END_REF][START_REF] Dominguez-Escobar | Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria[END_REF]. Mutants for maf and radC came more as a surprise since there is, so far, no evidences of their involvement in shape control, CW synthesis or interaction with MreBH [START_REF] Attaiech | RadC, a misleading name?[END_REF][START_REF] Briley | Maf acts downstream of ComGA to arrest cell division in competent cells of B. subtilis[END_REF]. Although these mutants may reveal to be interesting in the future, they were left aside for the present study. The 37 remaining clones presented 51 different amino-acid changes in MreB that were the subject of further investigation. It is worth noting that most clones (28) bear more than 1 SNP (single nucleotide polymorphism) either on mreB or on surrounding genes, preventing us to draw, for these mutants, a direct link between the phenotype observed and the presence of the mutation.

Figure 20 Principle of the mreB mutants screen. 1. A mutation-prone PCR is performed using primers AC1345/AC1335 on a strain containing a kanamycin resistance cassette (km) inserted between radC and mreB (RCL414). 2. The resulting mutated PCR product is transformed and integrated by double cross-over recombination into the recipient B. subtilis strain bearing the reporter gene fusion at the thrC locus (RCL422). Loss-of-function mreB mutants are selected as kanamycin resistant blue clones (activation of P mreBH ) on LB-agar plates supplemented with Xgal and kanamycin. 3. Mutations are mapped by sequencing the region from maf to mreD. PCR product is represented in green; light grey areas highlight regions of homology between the PCR fragment and the chromosome, allowing the recombination events to take place.

Site directed mutagenesis of mreB

In order to fully characterize the behavior of the selected MreB mutants, we needed to create translational fusions to GFP allowing in depth microscopy studies. Since most mutants carried several intra or intergenic SNPs, this engineering step was, in any case, required to individualize the mutations. We thus inserted into B. subtilis gfp fusions to mreB carrying single point mutations. In a first attempt, we tried to clone gfp-mreB fusions carrying SNP ectopically (at the amyE locus) under a xylose inducible promoter, in a strain that was deleted for endogenous mreB. This approach has been the method of choice since the early works from Carballido-López et al. in 2003 on the mbl paralog, until the beginning of our study. The task revealed to be far more difficult than anticipated and only generated a very limited number of clones ( 7) despite numerous attempts. This prompted us to change 83 RESULTS strategy and, as it was revelaed by the results from the sequencing of the ∆mreB strain 3725 (see section 3.2.3), happened to be an astonishingly lucky change. We then cloned mutated gfp-mreB at the natural locus under its own promoter, in place of the wild type mreB gene (Figure 21; see Material and Methods). This strategy of "in locus" cloning is quickly becoming the new paradigm in the field since it theoretically allows to maintain expression conditions as close as possible to those found in the WT strain.

84

RESULTS

Obtaining the mutants proved to be still difficult as they frequently acquired spontaneously extra mutations, a premature stop codon, or reverted to the wild type sequence. In toto, out of the 49 attempted constructions, 32 were successfully cloned and further characterized (Table 9). This suggests the possibility that some aberrant forms of MreB could be more harmful to B. subtilis cells than not having MreB at all. This hypothesis is strengthened by the fact that the mutagenesis was performed in the presence of Mg +2 , a condition known to allow mreB deletion mutant strains to support an almost wild type growth. In the course of this site directed mutagenesis, we noticed the recurrent, independent, appearance of additional mutations that drove two amino-acid changes (MreB I168F I169W ). Without entering into the molecular detail, we could link this to the existence of an unnoticed conserved stretch of amino-acids between MreB (TEVAIISL) and Maf (TEVAFWSL). We then decided to add these mutations to our list and created a MreB I168F I169W mutant (B58; table 9). All these gfp-mreB mutants will be here after called mreB*.

Table 9 List of strains carrying SNP in gfp-mreB, cloned at natural locus, and their controls used for phenotypic characterization. In grey, strains that could not be constructed. RCL numbers correspond to collection number. B* numbering will be used throughout spread all over the protein, on its surface and in the inner core of the protein (Figure 22). There is no apparent reason to attain the goal of creating a particular MreB*, other than perseverance.

Figure 22 3D model of MreB showing the residues that we achieved to construct in green and those that we did not in red. We can see the surface of MreB on A (side 1) and B (side 2), while a ribbon model of MreB is shown in C. There is no apparent trend on the probability of constructing a SNP or not. Both groups of SNPs are localized in big areas of the protein's surface and interior and they are well spread. Some of the SNPs that we did not achieve to construct lay next to others that we did.

Phenotypic characterization reveals different categories of MreB*s

A systematic analysis of phenotypes was undertaken, comparing strains expressing gfp-mreB* with their wild type counterpart (RCL424; gfp-mreB WT ) and the ∆mreB mutant (RCL243). We monitored:

1-growth from exponential to deep stationary phase in two rich complex media (LB and CH), 2-cell shape integrity by bright field microscopy, and 3-MreB dynamics by TIRF microscopy. As controls, the levels of GFP-MreB* proteins were estimated by western blot, and strains were also checked for the expression of the mreBH reporter fusion (see Materials and Methods). Finally the mutations were drawn onto a hypothetical 3D structure of the protein. B. subtilis MreB hasn't been crystallized yet, but a predicted tertiary structure was obtained using the modeling and prediction web tool Phyre2 [START_REF] Kelley | The Phyre2 web portal for protein modeling, prediction and analysis[END_REF].

Based on these, we could regroup MreB*s into four main categories (Table 10): WT-like, ∆mreB-like, mutants with intermediate phenotypes between the 2 previous and mutants with more dramatic growth phenotypes than that of ∆mreB (WeB). For TIRFM acquisitions refer to appendix 4.

Table 10 Classification of MreB*s based on their phenotypic characterization. (1) Expression of P mreBH based on the color of the colony on LB plates supplemented with Xgal; L, low; H, high. (2) Estimated relative protein levels during exponential growth, based on western blot analysis. (3) Growth curve of cells grown in CH media at 37 °C; WeB stands for "Worst than ∆mreB". (4) Growth curve of cells grown in LB media at 37 °C. (5) Morphology of exponentially grown cells in CH media at 37 °C observed with bright field microscopy; interm. stands for intermediate phenotype between that of WT and ∆mreB strains. (6) GFP-MreB localization and dynamic properties qualitatively analyzed from TIRFM acquisitions; P, patches; Dif, diffusive. Mutated residues displayed on a 3D-structure model of MreB showing the surface of the protein, recto (left) and verso (right). Mutated residues in WT-like MreB* mutants (green) are designated by arrows while other colored residues signaled known or presumed aminoacids involved in: monomer:monomer interaction (blue), putative nucleotide binding area (red), putative RodZ interaction area (pink), and putative interfilament bundleing interface (yellow). E. MreB ribbon structure model (recto only) displaying mutated residues embedded in the structure and not visible on the surface display. The color code is the same than in D.

RESULTS

Conversely, the ∆mreB-like mutants B4, B6, B19 and B22 were almost indistinguishable from a deletion mutant of mreB: they presented strong shape defects and poor growth (reversed by the presence of Mg +2 ), combined with strong induction of the P mreBH lacZ reporter (Table 10, Figures 23, 24 and26). In addition, the mutant B58, bearing the 2 amino-acid changes at position 168 169 (mutations that spontaneously and independently arose during the cloning process in many clones), presents an identical ∆mreB-like phenotype. Protein localization shows mostly soluble, diffusive signal instead of the typical patchy and dynamic localization observed with the wild type, strongly suggesting an inability of the mutants to form a complex with the PGEM. Two of the mutations (G14E -B22-, G160R -B6-) are predicted to be active site residues (van den [START_REF] Van Den Ent | Prokaryotic origin of the actin cytoskeleton[END_REF] lying in the putative nucleotide-binding pocket, which advocates for a role of ATP or GTP in MreB's function in vivo. Two other mutated residues (G56R -B4-, G231D -B19-) are present at the surface of domain IB and IIB (corresponding to domain 1 and 4 in G-actin) and were predicted to contact the previous subunit in the polymer (van den [START_REF] Van Den Ent | Prokaryotic origin of the actin cytoskeleton[END_REF]. It suggests that this change may have altered the ability of the MreB*s B4 and B19 to polymerize. Finally, mutations 168-169 of mutant B58 lie deep in the core of MreB and may have simply compromised its tertiary structure. This is supported by the western blot analysis (Figure 24) showing that this strain has (with B32) the lowest levels of MreB of all the mutants. It should be noted that three other "∆mreB-like" mutants described above also have slightly lower levels of MreB ("++" on Table 10) than that of the wild type, although not as low as in B58. Even though we cannot discard the possibility of these mutations slightly destabilizing the protein folding, we could also imagine that it is simply the mislocalization of the protein to the cytosol (instead of the usual membrane association) or their existence as monomers that facilitates their turnover.

RESULTS

Finally, in addition to these WT or completely inactivated mutants, we obtained seven clones (B5, B14, B21, B26, B42, B47 and B53) with a gradation of intermediate phenotypes between these two extremes. In these, increased induction of P mreBH lacZ roughly correlates with degradation of shape integrity and growth defect (Table 10,Figures 23,24 and 27). As expected, the localization of MreB was patchy and dynamic and almost identical to wild type in strains with limited shape defect (B5, B21, B26, B47, B53), while strains B14 and B42 presented dimmer signal and increasing number of cells without MreB dynamics together with increasing shape defects (twisting, uncontrolled width, swelling cells). Mutations A51V, G60R and P151Q (in B26, B53 and B42 respectively) are again located in the surface, potentially responsible for monomer:monomer interaction: residues 60 and 151 were predicted to be "contacting residues" on the -(or pointed) and + (or barbed) side of the monomer, while residue 51 is in the direct vicinity of the contacting residues of the -side and, importantly, it is extremely well conserved from B. subtilis to eukaryotic actin (Figure 27D-F).

Mutation V182A (B21) and V114A (B14) are two very highly conserved residues in MreBs from different organisms (according to an alignment performed using Clustal Omega [START_REF] Goujon | A new bioinformatics analysis tools framework at EMBL-EBI[END_REF][START_REF] Sievers | Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega[END_REF] suggesting that they have an important role in the protein's stability or functionality, although their phenotype is not very severe. Both lie on the surface of the protein, away from the areas important for monomer:monomer interaction, and may be important for bundling of polymers [START_REF] Van Den Ent | Prokaryotic origin of the actin cytoskeleton[END_REF]. Interestingly, the mutation V114A precedes a triplet of charged amino-acid residues shown to be important in Saccharomyces cerevisaie [START_REF] Wertman | Systematic mutational analysis of the yeast ACT1 gene[END_REF]. Unpublished results from our lab had previously shown that a B. subtilis mutant expressing GFP-MreB with a triple substitution (E115A-E116A-R117A), expressed as the only copy of MreB in the cell, showed strong defects both in growth and morphology (unpublished, Dominguez-Escobar et al.). Together, these results strongly suggest an important role of this area in the function of MreB, potentially through protein:protein interactions, either with other protein effectors or with other polymers of MreB.

RESULTS

Some mreB mutants present partial uncoupling of growth and localization defects

The five remaining mutants, hereafter named WeB (worst growth than ∆mreB), are in many ways remarkable. Four (B10, B17, B30 and B46) present a common, very intriguing set of features: they present more dramatic growth defects than a ∆mreB strain, while having normal or mildly affected shape and MreB localization.(Table 10, Figures 23, 24 and28). Indeed, bright field and TIRFM observations show that B46, B17 and B30 present mainly WT shapes with occasional polar swelling, and a majority of directionally moving MreB patches resembling that of the WT (Figure 28B). Only B10 presents a very slightly affected shape and a reduction in the number of patches with a few aberrant movements. However, their growth is severely affected both in CH and LB medium (Figure 28A). In CH, the mutants' optical density grows at a very slow pace, followed (to the exception of B30) by an equivalent decrease during "stationary phase", suggesting an important cell lyses. In LB, growth in exponential phase is identical to that of the control strains, but an abrupt change occurs at the entry of the stationary phase, where the mutants quickly start to lyse. Remarkably these defects were not restored by the addition of magnesium, a trick known to restore ∆mreB growth defects (Figure 28A).

RESULTS

Another very intriguing mutant is B32 (Table 10,Figures 23,24 and 29). This mutant is in all aspects more affected than the mutant deleted for mreB. It causes a stronger activation of the P mreBH lacZ reporter (Figure 23), barely grows and has a dramatic loss of cell shape control in CH medium (with almost no benefits from increasing Mg +2 concentration), and grows similarly to the deletion mutant in LB but with a pronounced lag (Figure 29). What is most surprising is that MreB is completely delocalized to the cytosol with the lowest protein levels of all the strains tested (Figure 24, Table 10), therefore, one could have expected the phenotypes of this strain to be identical to a ∆mreB. Thus, a strong hypothesis is that MreB L171P (B32) may have a dominant effect on other MreB isoforms, preventing them to associate to the membrane, and aggravating the phenotype of a simple ∆mreB under specific conditions present in CH, but not in LB. Growth curves of RCL422 (WT, P mreBH lacZ), RCL424 (P mreB gfp-mreB WT , P mreBH lacZ), RCL423 (∆mreB, P mreBH lacZ) and B32 MreB* grown in CH (upper panels) or LB media (lower panels), supplemented with MgSO 4 (right panels) or not (left panels). B. Bright field microscopy images of control WT strains RCL422 and RCL424 (blue outline), ∆mreB RCL423 strain (red outline) grown in CH media, and acquired during mid-exponential phase of growth (OD ≈ 0,2 -0,3). B32 was grown in CH media and images were acquired after ~ 24 h growth, at an OD ≈ 0,1. Mutant cells display very severe morphological defects. C. Zoomed area of bright field and fluorescent signal of mutant B32. D. 3D-structure model of MreB showing the surface of the protein verso (right). B32 MreB* mutant (orange) is designated by an arrow while other colored residues signaled known or presumed aminoacids involved in: monomer:monomer interaction (blue), putative nucleotide binding area (red), putative RodZ interaction area (pink), and putative interfilament bundleing interface (yellow).

Growth defect of WeB and ∆mreB mutants can be suppressed by addition of fructose

The dramatic growth defect in CH and abrupt lysis at the entry of stationary phase in LB observed with the WeB mutants, pointed toward a defect in carbon source utilization. We had previously seen that fruRKA is overexpressed in ∆mreB (fruK is also overexpressed in ∆mbl). Therefore, we wondered if the addition of fructose could rescue the growth defect of the WeB mutants. As seen on Figure 30, all 5 WeB mutants benefit from the addition of 1,5% fructose and, to the exception of B32 that grew slightly slower, they recover a full WT-like growth. To our surprise, the ∆mreB strain is also completely reverted to WT growth, a phenomenon that was, so far, only observed when Mg +2 was added to the medium. 103 RESULTS Next, we wondered if the improvement in growth was specifically due to the presence of fructose or to the presence of sugars in general, the bacterial preferred carbon source [START_REF] Fondi | Modelling microbial metabolic rewiring during growth in a complex medium[END_REF]. As seen in Figure 30, the growth defect is also bypassed when mutants are grown in CH medium supplemented with 1,5 % glucose.

It is well-established that addition of Mg +2 restores growth and shape defects of ∆mreB cells (although the mechanism behind it is still unclear). We thus wondered if fructose could have the same benefits toward B32, a mutant that presented major morphology defects in CH medium (Figure 31A). Bright field images show that addition of extra Mg +2 to the medium confers a very mild improvement to B32's morphology, with some cells recovering short rod shapes (Figure 31B), while addition of fructose does not (Figure 31C). Thus, in B32, magnesium can partially restore shape without restoring growth while fructose dramatically improves growth without improving cell shape.

Figure 31 Comparative benefits of MgSO 4 or 1,5 % fructose on B3 mutant's shape defect. B32 was grown in CH medium either without supplements (A), supplemented with 20 mM MgSO 4 (B) or 1,5 % fructose (C). Bright field images were acquired at OD 600 nm = 0,1 -0,2. Presence of high concentrations of MgSO 4 , but not 1.5 % fructose, partially restores rod-shape morphology.

Discussion

YdcH: a repressor/activator MarR transcription regulator?

The global approach we used revealed a very large number of genes affected, positively and negatively, by YdcH. Albeit surprising, several hypotheses can explain this result. A simple possibility would be that YdcH affects other regulators activity. This may be either through direct activation/repression of genes involved in other regulators activity or because the physiological alterations of the absence of ydcH, in turn, turned on/off such regulators. We could not find complete up-or down-regulated regulons but such regulons may be partially hidden by cross regulations of their genes.

Another hypothesis is that YdcH could act both as a repressor and activator of gene expression. Most described MarR TRs act as repressors and a few as activators [START_REF] Grove | MarR family transcription factors[END_REF]. There are only two known MarR TRs that have both activities [START_REF] Oh | Dual role of OhrR as a repressor and an activator in response to organic hydroperoxides in Streptomyces coelicolor[END_REF][START_REF] Tran | Analysis of RovA, a transcriptional regulator of Yersinia pseudotuberculosis virulence that acts through antirepression and direct transcriptional activation[END_REF]. They act as repressors by binding to DNA sequences near the promoter regions of the repressed genes. When the environmental conditions are modified, they undergo a structural modification that causes a reduction of their affinity for DNA and makes them bind the RNA polymerase, enhancing its binding to the promoter region of the regulated gene. Hence, this is a possibility that may be worth checking in the future.

YdcH: a new transition state regulator

Several lines of evidence advocate for YdcH being a novel transition state regulator potentially helping the cell to adapt to environmental changes, in a similar way than AbrB or SigH [START_REF] Britton | Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis[END_REF][START_REF] Phillips | Bacillus subtilis sporulation and stationary phase gene expression[END_REF]. First, the pattern of expression of ydcFGH shows that the climax of expression (presumably when YdcH is inactive) coincides with the transition between exponential and stationary phase of growth (Figure 17). This is nicely confirmed by the dramatic difference between exponential and stationary phase global gene expression profiles. These show that the huge YdcH regulon is deregulated once cells have entered stationary phase (almost no more differences between WT and ∆ydcH). Not surprisingly, several genes regulated by another transition state regulator, AbrB, are also affected in absence of YdcH. Second, there is a large diversity of functions affected by YdcH (Figure 18) driving to a global reprogramming of gene expression, typical of such regulators [START_REF] Strauch | Transition-state regulators: sentinels of Bacillus subtilis postexponential gene expression[END_REF]. Third, among these many genes, a large subset is involved in carbon or amino-acids metabolism, both being dramatically affected at the entrance into stationary phase due to the depletion of some carbon sources. Interestingly, we noticed many genes of the YdcH regulon related to the synthesis of several bacteriotoxin: albABCDEFG (antilisterial bacteriocin subtilosin), ntdR (antibiotic kanosamine), sdpA, sdpI and yknW (SdpC toxin) and yydGHIJ (control of LiaR-LiaS system as a response to bacitracin). This could be a strategy for the cell to scavenge new resources from a depleted environment [START_REF] Bibliography Abriouel | Diversity and applications of Bacillus bacteriocins[END_REF]. All together, our results suggest that during transition from exponential to stationary phase of growth, YdcH repression is partially released (as observed with the expression of P ydc1 ) leading to the activation/repression of a large set of genes, potentially leading to a reprogramming of the cell. We hypothesize that YdcH acts as a transition state regulator in B. subtilis, maybe acting both as activator and repressor. We can imagine YdcH being active during exponential growth, repressing its own expression and that of other TRs. This would cause the activation of those TRs that would then be able to act, positively or negatively, upon the more than 300 genes that appear differentially expressed in the ∆ydcH strain compared to the WT during stationary phase.

To go farther in our understanding of YdcH and its function(s) and to verify our hypothesis, we should identify the DNA sequence to which it binds, the "YdcH-box", in P ydc1 . We could then perform an in silico prediction of "YdcH-boxes" in other promoters in order to attempt a more precise characterization of the YdcH regulon. If YdcH causes a broad effect in the cell, by using the genome wide approach of RNAseq, we will recover the data of all the changes produced in the cell: those that are directly linked to YdcH and those that are caused indirectly (by genes controlled by YdcH). Pulldown experiments could also give us information about YdcH's function and effectors.

A library of MreB mutants with impaired functionality

We have developed a methodology to obtain MreB mutants and screen for their loss of functionality by means of the activation of P mreBH . Although it is difficult to obtain MreB*s due to spontaneous acquisition of extra mutations or suppressor mutations, we successfully constructed a collection of mutants displaying a variety of phenotypes. In the future, by using different reporters of MreB functionality, we could enlarge our library and have a complete vision of how MreB performs its activity. Addition of Mg +2 and sugars during the process of mutagenesis could help recovering additional mutants. Finally, these mutants will be an asset for future biochemical studies to pinpoint the biochemical properties of B. subtilis MreB. Protein interaction could also be investigated through pulldown experiments or yeast two hybrid. Structure-function correlations allow us to extract some conclusions. Mutants B6 (G160R) and B22 (G14E), from the ∆mreB-like group, are localized in the putative nucleotide binding site of MreB.

MreB

Combining the localization and the results that prove the loss of function of these mutations (Figure 26) we can infer that this area and possibly the nucleotide binding activity of MreB are important form the correct functioning of the protein. MreB*s B19 (G231D), B4 (G56R), from the ∆mreB-like group, and B32 (L171P), from the WeB group, are in close vicinity of the putative protofilament area, were MreB monomers bind to other MreB monomers to form chains. These three mutants, as the ones mentioned above, show an impaired MreB function (Figure 26 and 29). We can, therefore, deduce that this area and probably the polymerization capability of MreB are important for the proper functioning of the protein.

Interestingly, some mutants show the uncoupling of shape defect and growth impairment. Four of the WeB MreB*s (B10, B17, B30 and B46) have nearly WT morphology and MreB localization while being strongly impaired in cell growth both in CH and LB media (Figure 28). As shown using E. coli, LB has presumably low quantities of sugars (estimated < 100 µM). Their depletion marks the end of the exponential phase of growth, when cells switch to amino-acids consumption [START_REF] Sezonov | Escherichia coli physiology in Luria-Bertani broth[END_REF]. When B. subtilis is grown in LB, a diauxie can be observed around OD~0.5, that is presumably due to the exhaustion of sugars from the medium as well. This is precisely the point at which the growth of mutants B10, B17, B30 and B46 starts to decay. B10 and B46 are localized at the surface of MreB, in close vicinity of the putative interfilament interface; B17 is localized near the putative protofilament area. Lastly, B30 is mutated in an internal residue, near the putative polymerization area.

Our hypothesis is that those MreB mutations are, somehow, preventing the cell to change from glycolysis to gluconeogenesis. A tempting possibility is that MreB would act as a checkpoint linking cell metabolism and CW synthesis. It is known that MreB interacts with proteins involved in the synthesis of CW precursors [START_REF] Favini-Stabile | MreB and MurG as scaffolds for the cytoplasmic steps of peptidoglycan biosynthesis[END_REF][START_REF] Rueff | An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis[END_REF] and we think that there is an equilibrium maintained between cytosolic and polymerized membrane-associated MreB. It could be possible that MreB acts as a sensor of cell metabolic status to coordinate CW synthesis with the needs of the cell. This could explain why the shape and growth defects could be uncoupled.

This hypothesis is further reinforced by the results obtained from mutant B32. In this case we lose both WT morphology and MreB localization (Figure 29). In addition, high concentration of Mg +2 recovers its shape defect, but not its growth anomalies. On the other hand, addition of sugars improves its growth without affecting its morphology. There is previously reported evidence of association between CW synthesis and cell metabolism in bacteria. It has been shown that FtsZ is sensitive to pyruvate levels and that the deletion of a gene encoding a pyruvate kinase (pyk) in B. subtilis affects Zring formation and, consequently, division [START_REF] Monahan | Coordinating bacterial cell division with nutrient availability: a role for glycolysis[END_REF].

Lateral CW synthesis has also been connected to cellular metabolism [START_REF] Foulquier | The YvcK protein is required for morphogenesis via localization of PBP1 under gluconeogenic growth conditions in Bacillus subtilis[END_REF][START_REF] Gorke | YvcK of Bacillus subtilis is required for a normal cell shape and for growth on Krebs cycle intermediates and substrates of the pentose phosphate pathway[END_REF]. YvcK has two distinct roles, one in carbon metabolism and another in CW synthesis. The modification of its phosphorylation levels uncouples both functions. While its function in carbon metabolism is not affected by its phosphorylation levels, its capacity to correctly position PBP1 is. What is even more interesting is that YvcK, when overproduced, is capable of rescuing the ∆mreB mutant.

With all these data in mind, we hypothesize that MreB may act as a checkpoint between lateral CW synthesis and cell metabolism. MreB could have a second function in amino-acids metabolism that is being altered by our WeB mutations. The addition of sugars would allow these mutants to overcome the negative effects of having an altered amino-acids metabolism. Further verification will be done towards the identification of the specific metabolism process linked to MreB. We will grow the WeB MreB*s, were we have uncoupled MreB's role in cell morphology and cell metabolism, in defined minimal media supplemented with different carbon sources. We will also take advantage of the MreB mutant B32 that grows as the WT strain in LB to elucidate what is needed for that mutant to recover WT-like cell morphology and MreB dynamics. When colonies of MreB*s are incubated on LB-agar plates for > 24 h at 37 °C, various colony phenotypes develop. Mutants B6 and B19 (from the ∆mreB-like group) and mutants B30 and B32

Some MreB*s have atypical colony morphologies

(from the WeB group), develop overgrowing colonies that do not turn on the reporter gene, probably through the acquisition of spontaneous mutations (Figure 32). It has been demonstrated that stress conditions increase the appearance of mutations in bacteria [START_REF] Bridges | Microbial genetics. Hypermutation under stress[END_REF][START_REF] Hall | Spontaneous point mutations that occur more often when advantageous than when neutral[END_REF] and that this process is boosted during stationary phase of growth [START_REF] Sung | Adaptive, or stationary-phase, mutagenesis, a component of bacterial differentiation in Bacillus subtilis[END_REF]. Thus, it is tempting to imagine that the stress induced by the presence of those MreB*s may, in turn, increase the mutation rate, favoring the appearance of suppressor mutations. It would be interesting to sequence these overgrowing colonies to verify where the spontaneous mutations occur and why the MreB* benefits from them.

Another observed anomaly is the "wrinkling" of the colony surface of mutants B22, B58 and, most strongly, B4 (all three from the ∆mreB-like group). The wrinkling of a colony surface has been shown to be linked with the formation of areas of increased cell death in the colony [START_REF] Haussler | Biofilms 2012: new discoveries and significant wrinkles in a dynamic field[END_REF] or with biofilm and motility defects [START_REF] Bridier | The spatial architecture of Bacillus subtilis biofilms deciphered using a surface-associated model and in situ imaging[END_REF]. This could be a possibility to investigate with these mutants.

Possible connection between the mreB deletion and the ydcH frame-shift

On the onset of this project was the specific induction of the ydcFGH operon observed in absence of mreB, calling for elucidation of the function of this potentially specific effector of MreB, as well as its mode of induction. The outcome, in the last weeks of the doctorate, was unexpected.

The strain 3725 is supposed to be a direct parent of the wild type B. subtilis 168, and has been used for many years in European labs on the field. Noticing several familiar mutations in the list of SNPs, we realized that one of these mutations, SepF M11T was reported to be required for B. subtilis to form Lforms (Dominguez-Cuevas, 2011), while several others (sigI, walR, accC) affect genes known or suspected to be involved in L-form and/or to be suppressor genes of mreB defects (Dominguez-Cuevas, 2011;[START_REF] Schirner | The cell wall regulator {sigma}I specifically suppresses the lethal phenotype of mbl mutants in Bacillus subtilis[END_REF]Mercier, 2013).

Although it was not possible to completely track the chain of events leading to the appearance of so many mutations in strain 3725, they all appeared in a time and location (Oxford) were L-form and the essential gene mreB were studied. It is then conceivable that they were unwittingly selected as suppressors. At the moment, it is unknown if the frameshift mutation in ydcH was fortuitous or selected because it improves the ∆mreB phenotype. But one can speculate that, if YdcH plays a role in state regulation facilitating the adaptation of the cell to changing conditions including carbon depletion as in phase transition (acting on ~60 genes involved in carbon metabolism) and if MreB is linking cell growth to carbon metabolism (see MreB mutagenesis section), then, this mutation may increase the survival of the ∆mreB strain by modifying carbon and/or amino-acids metabolism and may not be incidental.

A1.2.1 Sporulation

We reasoned that the observed decreased in OD during stationary phase may reflect a change in these strains in their ability to undertake one or several of the processes that take place during stationary phase. Indeed, at the entry into stationary phase, B. subtilis cells can follow different developmental pathways, helping them surviving in changing environments, such as competence for DNA transformation or sporulation [START_REF] Maughan | Stochastic processes influence stationary-phase decisions in Bacillus subtilis[END_REF].

We first focused on sporulation because the process involves at late stages the release of a mature spore and the lysis of its nurturing mother cell, that could impact the OD. As shown on Figure A2.4A, mutants of ydcF and ydcG present a two to three fold increase in sporulation efficiency. WT B. subtilis are known to produce 10-50% spores (strain and medium depending) in optimal conditions due to a complex regulatory pathway allowing a fraction of the population to escape this long and costly process [START_REF] Piggot | Sporulation of Bacillus subtilis[END_REF]. But the spores produced are virtually all viable. Consequently, an increase in spore frequency suggested a more efficient induction of the process, leading to a higher number of cells entering the sporulation pathway. , ∆ydcF (ASEC60), ∆ydcG (ASEC58) and ∆ydcH (ASEC56) strains as described in the method section, using the exhaustion method. Briefly, samples of 30h old cultures were plated before (giving the total number of cells) and after (giving the number of spores) heat kill. Spore frequency (spores/viable cells) and spore counts are calculated from CFU. B. Competence assays were performed on the same strains and as described in the method section. Briefly, samples were grown in competence media and plates in LB plates (giving total number of cells) and in LB plates supplemented with the antibiotic resistance that was introduced (giving the number of transformants). Transformation rate (transformants/total cells) was calculated from CFU.

A1.2.2 Competence

We finally decided to assay our mutants for their proficiency in developing natural competence for DNA transformation. This is a well-known ability of B. subtilis that develops in a subpopulation of cells at the entry of stationary phase in specific conditions (for review, see [START_REF] Dubnau | Genetic competence in Bacillus subtilis[END_REF]). Our lab recently shows that absence of MreB affects the efficiency of transformation, albeit indirectly [START_REF] Mirouze | MreB-dependent inhibition of cell elongation during the escape from competence in Bacillus subtilis[END_REF]. Since we observed a difference in ydcFGH expression in a mreB deletion mutant, we wondered if the operon could play a role in competence development. Competence was tested through the two step method (see material and methods) in each deletion mutant: ∆ydcF, ∆ydcG and ∆ydcH. As presented on Figure A1.4B. The results obtained from these experiments were highly variable, which is demonstrated by the values of the error deviations.

Once we realized that strains mutant for ydcF and ydcG had a different genetic background than our WT B. subtilis strain, ∆ydcF and ∆ydcG were reconstructed (ASEC325 and ASEC327, respectively; see methods). Sporulation experiment assays as well as growth in MSM, CH and LB medium were repeated. No sporulation increase and no stationary phase survival defect could be detected with these strains, strongly suggesting that the differences observed in ASEC58 and ASEC60 were indeed due to differences in the strain background. We noticed that in this alternative background, the absence of several rap genes, notorious sporulation inhibitors [START_REF] Pottathil | The extracellular Phr peptide-Rap phosphatase signaling circuit of Bacillus subtilis[END_REF] whose deletion increase the ratio of sporulating cells. Thus, the absence of such genes is the most probable reason for the phenotypic differences observed.

A2.2. ydcFGH induction is not due to decreased expression of minC

We then considered that the deletion of mreB could affect the expression levels of the downstream minCD genes which, in turn, could induce the overexpression of ydcFGH. However, deleting minC while leaving the mreBCD locus untouched (strain ASEC342) did not activated P ydc1 (Figure A3.1B.III).

A2.3. ydcFGH induction is not caused by the expression of a remnant peptide of MreB

Next, we wondered if the deletion of mreB, that had left a short peptide, could have a dominant effect on the expression of ydcFGH. Indeed, to avoid potential influence on the expression of the downstream essential mreC and mreD genes, mreB deletion mutant had been built without replacing the gene by an antibiotic resistance cassette. Instead, most of mreB was removed and a resistance gene to neomycin (neo) was cloned upstream of the mreB promoter to allow its selection [START_REF] Formstone | A magnesium-dependent mreB null mutant: implications for the role of mreB in Bacillus subtilis[END_REF]. During this process, the 30 N-terminal and 34 C-terminal nucleotides of mreB were kept but we discovered in between a small 21 nucleotide sequence of unknown origin. To test if the resulting DNA fragment (encoding a 28 amino-acid long peptide) was producing a perturbing RNA or peptide, we cloned a DNA fragment from neo up to mreC at the ectopic amyE locus under the control of P xyl in a strain where the mreBCD locus was untouched (amyE::km-P xyl -pep; strain ASEC266). This construction did not activate the expression of ydcFGH (Figure A3.1B.IV).

A2.4. ydcFGH induction is not caused by abnormal levels of MreCD

Intriguingly, we noticed that the repression of P ydc1 was maintained in cells expressing the entire mreBCD operon under its own promoter from the amyE locus, and deleted for the whole native mreBCD operon, while P ydc1 was highly activated in cells deleted for mreB only (Figure A3.1B.V; strains ASEC40 and ASEC20). We therefore hypothesized that an abnormal MreB/CD ratio could be the cause for P ydc1 's induction. To test this, we first created a deletion of mreC (mreD is strictly essential but a mreC deletion can be made in the presence of high Mg +2 concentrations), but again, no induction of the P ydc1 was observed in this construct (Figure A3.1B.VI; strain ASEC7).

Since on our previous construct the cells where P ydc1 was induced carried two copies of mreC and mreD, we then wondered if the overexpression of mreC/mreD rather than the absence of them could cause the induction of P ydc1 . To check this possibility, we looked at the activation of P ydc1 when the expression of mreCD (strain ASEC115) or mreD (strain ASEC168) were under the control of a xylose dependent promoter at natural locus. Under these conditions, the ydcFGH operon wasn't overexpressed (Figure A3.1B.VII). As a second verification, we expressed GFPMreC/GFPMreD with a P xyl promoter at amyE (amyE:: P xyl -gfpmreC, strain ASEC109 and amyE:: P xyl -gfpmreD, strain ASEC111) leaving the mreBCD locus untouched. By having an extra copy of the genes in addition to the gene at locus we were sure to have an excess of MreC/MreD in the cell. Again, P ydc1 wasn't activated in any of these genetic backgrounds either (Figure A3.1B.VII).

A2.5. Absence of the MreB protein is not the cause of ydcFGH induction

Altogether, these results indicated that the deletion in the mreB transcript rather than absence of the MreB protein was responsible for P ydc1 induction. To test this, a mutant was generated in which 3 stop codons were introduced at the beginning of the mreB orf -at the natural locus-(Figure A3.2B), in order to prevent protein synthesis with minimal perturbation of the mreBCD transcript (strain CCBS194).

The absence of MreB synthesis in this strain was confirmed by western blotting using anti-MreB antibodies (Figure A3.2C, see materials and methods). In this construct, where the entire mreBCD transcript remains untouched to the exception of these three added codons, P ydc1 was not induced.

These results apparently confirmed our hypothesis.

Suspecting a regulatory RNA to be the source of this regulation, and to pinpoint the area of the transcript responsible for it, we planned to create deletions of various sizes of the mreB orf.

Surprisingly, the first construct obtained, combining the almost complete mreB deletion (strictly identical to that present in the ∆mreB strain ( 3725)) with the 3 stop codons did not induce P ydc1 ;

indicating that the deletion of mreB was not responsible for the phenotype either (Figure A3.2D; strain CCBS202). In this strain, the 3 stop codons introduced in strain CCBS194 were combined with the deletion present in strain 3725.

A2.6. ydcFGH induction is unlinked to the mreB locus

This result being in complete disagreement with the results so far, we decided to check the genetic link between mreB and the induction of ydcFGH. For this, we either transformed strain ∆mreB (3725) with chromosomal DNA of the strain bearing the reporter fusion (ABS1990) or transformed ABS1990 with chromosomal DNA of strain 3725. When the reporter was passed into 3725, the reporter was systematically induced, indicating the overexpression of ydcFGH, as previously observed. When the reverse transformation was performed, none of the resulting clones displayed any induction of the reporter suggesting that the locus responsible for P ydc1 lacZ induction was not genetically linked to mreB.

We then sequenced the ydcFGH locus, who became an obvious candidate based on our transcriptional study (see results chapter ydcFGH), and found a frameshift mutation lying in the middle of ydcH.

According to our results, inactivation of ydcH leads to induction of the expression of P ydc1 , thus such frameshift has a very high probably to explain the observed induction. Out of curiosity, we sequenced in parallel another locus of interest, walR, in which spontaneous mutations had been selected in the ∆mreB strain 3725 (Dominguez-Cuevas et al; Chastanet et al unpublished). To our surprise, strain 3725 carried also a mutation in this gene. We wondered how many mutations this long-used strain could be carrying, and undertook its complete sequencing. This revealed, to our astonishment, the

Thesis abstract

Acquisition and maintenance of the bacterial shape has been conscientiously studied for a long time.

Nevertheless, there are still many unanswered questions. Gram-positive bacteria present a rigid external coating (cell wall) that allows them to preserve internal osmotic pressure and cell morphology. The cell wall (CW) is mainly formed by the peptidoglycan meshwork (PG), that confers its structure to the CW, to which are connected teichoic acids. The absence of this essential barrier causes the loss of shape and, ultimately, lysis of the cells. Integrity of the CW is, therefore, a matter of vital importance for bacteria.

Proper CW synthesis and structure depends on the so-called peptidoglycan elongation machineries (PGEM) in charge of building the PG meshwork. The precise composition and functioning of the PGEM is not completely understood but they rely on a key player: MreB, a conserved prokaryotic actin-like protein. MreB is suspected to control PGEM activity and/or assembly but its precise Characterization of these mutants revealed numerous key residues for the functioning of the protein.

Interestingly, my results indicate that some mutants have kept their dynamic properties (suggesting functional association to the PGEM) together with a wild type shape, while being strongly affected for growth. Preliminary results indicate an impaired ability to use certain carbon sources linking MreB to cellular metabolism. This suggests the existence of either a checkpoint or a coupling between carbon metabolism and CW expansion in B. subtilis.

(ii) Unpublished results from our group revealed the existence of an uncharacterized operon (ydcFGH), whose expression is highly induced in the absence of mreB by comparison to the wild type.

I have 1-deciphered the cause of ydcFGH induction in the absence of MreB, revealing the existence of multiple mutations in the ΔmreB strain and 2-realized a thorough characterization of each gene of the ydcFGH operon. Although the exact link between MreB and ydcFGH is yet unknown, my results suggest a potential role of YdcH in the control of carbon metabolism and adaptation to stationary phase. In light of my mutagenesis screen data (i), these results are pointing towards a strong link between MreB and carbon metabolism.

Figure 1 .

 1 Figure 1. Differentially expressed genes in absence of ydcH.Results from the RNAseq experiment comparing gene expression levels in a WT (ABS2005) and a ∆ydcH strain (ASEC56) show that, in absence of ydcH, during exponential growth (A), there are 180 unrepressed genes (green) and 184 down-regulated genes (red); during stationary growth (B) there are 13 up-regulated (dark green) and 11 down-regulated genes (dark red).

PBP

  Abbreviations .......................................................................................................................................... Table of Contents .................................................................................................................................... List of tables ............................................................................................................................................ List of figures .......................................................................................................................................... 1. Introduction ....................................................................................................................................... 1.1. Bacterial envelope ...................................................................................................................... 1.1.1 Cytoplasmic membrane ........................................................................................................ 1.1.2 Cell wall ............................................................................................................................... 1.1.3 Outer membrane ................................................................................................................... 1.2. Peptidoglycan ............................................................................................................................. 1.2.1 Peptidoglycan structural models ........................................................................................... 1.2.2 Peptidoglycan synthesis........................................................................................................ 1.3. Bacterial cytoskeleton ................................................................................................................ 1.3.1 Bacterial cytoskeletal proteins .............................................................................................. 1.3.2 Bacterial actin homologs ...................................................................................................... 1.4. MreB ........................................................................................................................................... 1.4.1 MreB isoforms ...................................................................................................................... 1.4.2 Biochemical properties of MreB .......................................................................................... 1.4.3 Localization and dynamics of MreB .................................................................................... 1.4.4 Role of MreB in cell shape determination and cell wall synthesis ....................................... 1.4.5 Other roles of MreB.............................................................................................................. 1.5. Aims of the thesis ....................................................................................................................... 2. Materials and Methods ...................................................................................................................... 2.1. Media Composition: ...................................................................................................................

  . ....................... 2.2. Media supplements: .................................................................................................................... 2.3. Strains and plasmids ................................................................................................................... 2.4. Experimental procedures ............................................................................................................ 2.4.1 Cloning procedures:.............................................................................................................. 2.4.2 Manipulation in B. subtilis ................................................................................................... 2.4.3 Protein procedures: ............................................................................................................... 2.4.4 RNA procedures: .................................................................................................................. 2.4.5 Microscopy methods: ........................................................................................................... 3. Results ............................................................................................................................................... 3.1. Functional analysis of ydcF, ydcG and ydcH. ............................................................................ 3.1.1 The ydcFGH operon is composed of three genes of unknown functions. ............................ 3.1.2 Construction of knock-out mutants of ydcF, ydcG and ydcH .............................................. 3.1.3 Phenotypic characterization of ydc genes exposes an inappropriate strain frame ................ 3.1.4 YdcF, YdcG and YdcH are not involved in stress resistance ............................................... 3.2. Transcriptional study of ydcFGH ............................................................................................... 3.2.1 ydcH is under the control of two promoters ......................................................................... 3.2.2 YdcH, but not YdcF nor YdcG, is involved in the control of P ydc1 expression .................... 3.2.3 The absence of MreB is not responsible for P ydc1 induction ................................................. 3.3. YdcH, a new regulator for carbon metabolism? ......................................................................... 3.4. MreB mutagenesis ...................................................................................................................... 3.4.1. Setting up a genetic screen for MreB loss-of-function mutants .......................................... 3.4.2. Random mutagenesis of mreB ............................................................................................. 3.4.3. Site directed mutagenesis of mreB ...................................................................................... 3.4.4. Phenotypic characterization reveals different categories of MreB*s .................................. 3.4.5. Growth defect of WeB and ∆mreB mutants can be suppressed by addition of fructose ... 4. Discussion ....................................................................................................................................... 4.1 YdcH: a repressor/activator MarR transcription regulator? ...................................................... 4.2 YdcH: a new transition state regulator ...................................................................................... 4.3 A library of MreB mutants with impaired functionality ............................................................ 4.4 MreB may play a role in CW synthesis, cell morphology and cell metabolism ....................... 4.5 Some MreB*s have atypical colony morphologies ................................................................... 4.6 Possible connection between the mreB deletion and the ydcH frame-shift ............................... 6. Appendices ...................................................................................................................................... Appendix 1: Phenotypic analysis of ydcFGH ................................................................................. A1.1 ydcF, -G and -H deletion mutants are not impaired for cell morphology .......................... A1.2. Defects during stationary phase ......................................................................................... Appendix 2: The absence of MreB is not responsible for P ydc1 induction ....................................... A2.1. Absence of mreB complementation is not due to chromosomal positioning of the gene .. A2.2. ydcFGH induction is not due to decreased expression of minC ........................................ A2.3. ydcFGH induction is not caused by the expression of a remnant peptide of MreB .......... A2.4. ydcFGH induction is not caused by abnormal levels of MreCD ....................................... A2.5. Absence of the MreB protein is not the cause of ydcFGH induction ................................

FigureFigure 5

 5 Figure Depiction of Gram-positive and Gram-negative cell walls............................................... 17 Figure Peptidoglycan composition................................................................................................ 18 Figure Peptidoglycan structure models......................................................................................... 21 Figure Peptidoglycan synthesis..................................................................................................... 23 Figure 5 Crystal structure of T. maritima MreB monomer and polymer and a scheme of actin treadmilling...................................................................................................................................... 29 Figure Bacterial tubulin-like homologs......................................................................................... 30 Figure Bacterial intermediate filaments-like homologs................................................................ 32 Figure Dynamics of the Min proteins in E. coli............................................................................ 33

Figure 75 Figure

 75 Figure Bacterial actin homologs.................................................................................................... 35 Figure Effects of the deletion of each of the mreB paralogs in B. subtilis.............................. 37 Figure Filaments and diffraction-limited clusters....................................................................... 42 Figure Most overexpressed genes in ∆mreB (A) and ∆mbl (B).................................................. 72 Figure Pop-In Pop-Out ∆ydcF and ∆ydcG.................................................................................. 74 Figure Growth of deletion mutants of ydcF, ydcG and ydcH derived from BKE strains in different media…………………………………………………………………………………………….... 75 Figure The ∆ydcH strain is not affected by salt, oxidative or antibiotic stresses........................ 77 Figure The ydcFGH operon……………………………………………...…………................. 79 Figure Expression of ydcFGH peaks at the transition from exponential to stationary phase..... 82 Figure Differentially expressed genes in absence of ydcH ……………………........................ 83 Figure 19 Expression of P mreBH and P fru transcriptional fusions to lacZ in presence and absence of mreB……………………………………………………………………………………………..... 85 Figure Principle of the mreB mutants screen ………................................................................. 87 Figure Site directed mutagenesis of mreB………….................................................................... 88

Figure

  Figure 3D model of MreB showing the residues that we achieved to construct in green and those that we did not in red………………………………........................................................................ 90 Figure 23 Comparative expression of P mreBH lacZ in strains expressing gfp-mreB*, assayed by colorimetric assay on plate……………………............................................................................... 93 Figure Comparative expression levels of GFP-MreB................................................................. 93

Figure 1

 1 Figure 1 Depiction of Gram-positive and Gram-negative cell walls (modified from (Silhavy, Kahne, & Walker, 2010)). CAP, covalently attached protein; IMP, integral membrane protein; LTA, lipoteichoic acid; WTA, wall teichoic acid; LP, lipoprotein; OMP, outer membrane protein; LPS, lipopolysaccharide.

  Figure 2 Peptidoglycan composition (modified from (Cava & de Pedro, 2014)). A. Chemical composition of the PG subunit. B. Fragment of the PG mesh with the glycan chains interconnected by a pentapeptide bridge. C. Site of action of PG hydrolases. D. Modified from (Lecoq et al., 2012). DD-and LDtranspeptidation. NAG, Nacetyl-glucosamine; NAM, N-acetyl muramic acid; D-Lac, D-lactate; L-Ala, Lalanine; D-Glu-(γ), Dgamma glutamate; L-Dap-D, meso-diamino pimelic acid; D-Ala, D-alanine; R, glycan strand.

INTRODUCTIONFigure 3

 3 Figure 3 Peptidoglycan structure models (modified from (Kim, Chang, & Singh, 2015) and (Hayhurst et al., 2008)). A. Layered model with PG strands running parallel to the cytoplasmic membrane. B. Scaffold model with PG strands perpendicular to the cytoplasmic membrane. C. Coiled coil model with PG strands acting as ropes, surrounding the cell.

INTRODUCTION

Figure 4

 4 Figure 4 Peptidoglycan synthesis (modified from (Bhavsar & Brown, 2006)). Solid arrows denote catalysis of the reactions by the indicated enzyme. Block arrows denote where activated substrate is utilized in the biosynthesis pathway. The distinction between the reactions taking place in the cytosol and the membrane is denoted by a dashed grey line.

Figure 5

 5 Figure 5 Crystal structure of T. maritima MreB monomer and polymer and a scheme of actin treadmilling. A. Ribbon representations of T. maritima MreB monomer. MreB crystallized with AMPPNP and magnesium. The four domains (IA, IB, IIA, IIB) correspond to those in actin (1, 2, 3, 4). AMPPNP binds at the nucleotide binding site formed by the four domains. B. Comparison of yeast F-actin and T. maritima MreB polymers. The tip residues of domains IA and IIA go into the cleft formed by IB and IIB of MreB in the same manner as those from domains 1 and 3 of actin go into the cleft formed by 2 and 4. A and B are adapted from (van den Ent et al., 2001). C. Polymerization of Actin. ATP-bound monomers are in green, and ADP-bound in grey. The process of polymerization is much faster on the + end than in the -end., giving a directionality to the polymerization process (treadmilling).

Figure 6

 6 Figure 6 Bacterial tubulin-like homologs (modified from (Cabeen & Jacobs-Wagner, 2010)). A. FtsZ-GFP in E. coli. Same field is shown as phase contrast (left panel) or fluorescent image (right panel). FtsZ forms a ring-like structure (the Z-ring) at mid-cell marking the division sites. B. TubZ-GFP forming a filament in Bacillus thuringiensis. C. Immunofluorescence images of BtubA/B in E. coli.

Figure 7

 7 Figure 7 Bacterial intermediate filaments-like homologs (modified from (Cabeen & Jacobs-Wagner, 2010)). A. Fluorescence micrograph of FIAsH-stained crescentin-TC in C. crescentus. B. FilP-GFP in S. coelicolor.

  WACAs are found in most bacteria and some archaea. The two main examples of the WACA proteins are ParA and MinD (Ingerson-Mahar & Gitai, 2012).

  bound to ATP. It also associates with additional factors like DNA and ParB, that stimulates ATP hydrolysis and the dissociation of the filament (Ingerson-Mahar & Gitai, 2012). It seems that in Caulobacter and Vibrio cholerae, ParA binds the parS origin-proximal region and its polymerization-INTRODUCTION depolymerization pulls the chromosome through the cell during its segregation. There are other ParA homologs implicated in plasmid segregation (Ingerson-Mahar & Gitai, 2012).

  coli, MinE binds to MinD and promotes ATP hydrolysis, creating a gradient of MinD bound to the membrane and allowing the polymerization of FtsZ at midcell. B. subtilis lacks MinE, but it still recruits MinD to the cell poles and recent division sites. C. crescentus does not have the Min system, but has a different WACA protein, namely MipZ, that disables the mislocalization of FtsZ (Ingerson-Mahar & Gitai, 2012). At the beginning of the cell cycle, MipZ binds the origin of replication (oriC) of the chromosome via ParB. After DNA replication, one of the daughter origins migrates to the opposite cell pole with MipZ, activating the release of FtsZ from the area. This mechanism creates a gradient of proteins from both cell poles,

Figure 8

 8 Figure 8 Dynamics of the Min proteins in E. coli (modified from[START_REF] Lutkenhaus | Assembly dynamics of the bacterial MinCDE system and spatial regulation of the Z ring[END_REF]).

INTRODUCTIONFigure 9

 9 Figure 9 Bacterial actin homologs A. TIRFM image of GFP-MreB B. subtilis cells. B. Composite fluorescence micrograph of M. magneticum cells (3-4 µm in length) producing MamK-GFP. C. Immunofluorescence images of ParM (green) segregating LacI-GFP-labeled plasmids (red) in E. coli. D. AlfA-GFP fluorescence in B. subtilis. E. Fluorescence micrograph of Alp7A-GFP in B. subtilis cells. (A personal data; B-E modified from (Cabeen & Jacobs-Wagner, 2010)).

Figure 10

 10 Figure 10 Effects of the deletion of each of the mreB paralogues in B. subtilis. Fluorescent membrane dye FM1-43 images of wild type B. subtilis (168; RCL44), ∆mreB (RCL423), ∆mbl (RCL78) and ∆mreBH (RCL49).

  constructed strains with only one of each paralog gene under the control of an IPTG inducible promoter: MreB-only, Mbl-only and MreBH-only. Each gene needs a different induction level to compensate for the loss of the other two, demonstrating that, even though all three proteins have overlapping functions, some differences exist. Likewise, each of the resulting strains (MreB-only, Mbl-only or MreBH-only) has different survival rates when grown in different stress conditions. The MreBH-only strain is the most affected, showing lower endurance than WT to alkaline, salt and heat stress; MreB-only is impaired during alkaline and salt stress and Mbl-only has a decreased survival upon salt stress.The amino acid sequence of the three proteins is different, we can see in Table2the percentage of homology between them. This points too towards a differentiation of their function(s). Probably MreB, Mbl and MreBH have a common, main activity linked to CW synthesis and maintenance of cell morphology. Nevertheless, the mode of action might be different between them and they may come in during different conditions. It is also possible that each of them have different secondary function(s) that haven't been identified yet.

Table 3

 3 Summary of biochemical properties of MreB in different organisms. (1) Dependence on ATP for polymerization. (2) Dependence on Mg for polymerization. (3) Dependence on monovalent ion; max., maximal polymerization; Inh., polymerization inhibited. (4) pH at which polymerization was maximal (max.) or inhibited (inh.). (5) Method used to differentiate between aggregation and polymerization; EM, electron microscopy; FL, fluorescent labelling. (

Figure 11

 11 Figure 11 Filaments and diffraction-limited clusters (modified from (Swulius & Jensen, 2012) and (Reimold et al., 2013)). A. Clusters of MreB-RFP SW in E. coli. B. Filaments of YFP-MreB in E. coli.

  1.4.5.2 Viral DNA and replication proteins localization Efficient ϕ29 DNA replication requires functional MreB proteins in B. subtilis. Interestingly, it needs the presence of all three MreB paralogs (Munoz-Espin, Serrano-Heras, & Salas, 2012). This is the first example of non-redundancy of the three MreB paralogs until now. INTRODUCTION 1.4.5.

2.

  Complete genome sequencing of B. subtilis: Chromosomal DNA of exponentially grown B. subtilis was extracted (2.4.2.4). The subsequent steps were subcontracted to GATC Biotech SARL (Mulhouse). For this, 200 ng of DNA was sent for construction of a genomic library. Sequencing was performed using Illumina technology, with paired-ends, 125bp long reads and 5 million read pairs. A pre-analysis (with with semi-automatic detection and mapping) with mapped SNPs and InDels was delivered. We further analyzed the data with the Tablet software (REF:Milne I,.2.4.1.10 Random mutagenesis:Random mutagenesis was performed by amplifying DNA fragments through PCR reactions. The frequency of mutations was favored by the use of a DNA polymerase devoid of proof-reading ability (Taq) and the presence of MnCl 2 .PCRs were performed as in 2.3.1.2 except that 0-0.4 mM MnCl 2 prepared freshly, were added to the mix. Next, PCR products were dialyzed against ddH 2 O and used to transform B. subtilis competent cells (strain RCL422). Transformations were plated on LB-agar plates supplemented with kanamycin, MgSO 4 and X-gal. Mutants with MreB-impaired functionality formed bleu colonies (due to the activation of P mreBH lacZ).2.4.1.11 Directed mutagenesis PCR:Site directed mutagenesis (Figure21) is performed by "Gibson" assembly of PCR fragments generated on DNA from a strain bearing a km resistance cassette before the natural mreB promoter, and the gfp gene fused in 5' of the mreB gene (RCL424). Two intermediate PCR products are generated using CC181/Rv mutation primer and Fw mutation primer/RK14, the mutation to introduce being bear on the overlapping forward and reverse primers. PCRs were performed with Phusion (NEB) DNA polymerase, according to manufacturer's recommendations. Next, the PCR reaction was cooled down at RT and 1 µL of restriction enzyme DpnI was added and incubated at 37 °C for 1 h to digest methylated DNA (used as template and non-mutated). PCR products were purified as in 2.4.1.5 and DNA concentration was measured as in 2.3.1.6. Then, equivalent molecule quantities of the fragments were mixed to a total volume of 5 µL, added to 15 µL of joining mix (5 % w/v PEG-8000, 100 mM Tris-HCl pH = 7.5, 10 mM MgCl 2 , 10 mM DTT, 1 mM NAD, 0.2 mM each dNTP, 25 U/mL T5 exonuclease, 50 U/mL Pfu DNA polymerase, 6667 U/mL Taq DNA ligase) and incubated 20 min at 50 °C to form a single DNA molecule (2.4.1.7). 10 µL of the joining reaction was used to transform competent B. subtilis cells.

Figure 12

 12 Figure 12 Most overexpressed genes in ∆mreB (A) and ∆mbl (B) in LB medium. Unpublished data from Marchadier E. et al.

Figure 13

 13 Figure 13 Pop-In Pop-Out ∆ydcF and ∆ydcG. Described in[START_REF] Tanaka | Building the repertoire of dispensable chromosome regions in Bacillus subtilis entails major refinement of cognate large-scale metabolic model[END_REF]. A. Example of "Pop-In Pop-Out" marker-less deletion procedure on the ydcF gene. Upstream (oligonucleotides ASEC19+ASEC20) and downstream (ASEC22+ASEC20) fragments of ydcF are PCR-amplified, introducing homologous extremities to the extremities of the phleo-c1 cassette, which is also amplified (oligonucleotides Phleo Fw +Phleo Rv). The three fragments were assembled and used to transform strain NC101 (upp::P λ neo). Eviction of phleo-c1 is achieved by recombination of homologous regions at both sides of the cassette, leaving a clean deletion of ydcF (ASEC24). B. Genetic map of the ydcFGH locus after the "Pop-In Pop-Out " deletion of ydcG (in ∆ydcG strain ASEC5).

Figure 15

 15 Figure 15 The ∆ydcH strain is not affected by salt, oxidative or antibiotic stresses. A-D. ydcH mutant resistance to NaCl and antibiotics. WT (RCL44; continuous lines) and ∆ydcH (ABS1381; dotted lines) cells were grown in CH media supplemented with NaCl (A), vancomycin (B), methicillin (C) or rifampicin (D). F. Resistance to H 2 O 2 was tested on LB plates via a disk diffusion assay with 30 % H 2 O 2 .

Figure 18

 18 Figure18Differentially expressed genes in absence of ydcH. Results from the RNAseq experiment comparing gene expression levels in a WT (ABS2005) and a ∆ydcH strain (ASEC56) show that, in absence of ydcH, during exponential growth (A), there are 180 unrepressed genes (green) and 184 down-regulated genes (red); during stationary growth (B) there are 13 up-regulated (dark green) and 11 down-regulated genes (dark red). For more details, see appendix 4.

Figure 19

 19 Figure 19 Expression of P mreBH and P fru transcriptional fusions to lacZ in presence and absence of mreB. The promoter regions of the fruRKA operon and the mreBH gene were cloned as transcriptional fusions with E. coli lacZ. Resulting constructions were independently inserted into wild type (WT) or mutant for mreB (∆mreB) B. subtilis strains. ABS1749 (WT, P fru lacZ), ABS1750 (∆mreB, P fru lacZ), RCL422 (WT, P mreBH lacZ) and RCL423 (∆mreB, P mreBH lacZ) were spotted on LB-agar plates supplemented with Xgal. The promoter regions used for the reporter constructions are drawn as green-shaded areas on the corresponding right hand cartoons.

Figure 21

 21 Figure 21Site directed mutagenesis of mreB. 1. Site directed mutagenesis is performed by "Gibson" assembly of PCR fragments generated on DNA from a strain bearing a km resistance cassette before the natural mreB promoter, and the gfp gene fused in 5' of the mreB orf (RCL424). a. Two intermediate PCR products are generated using CC181/Rv mutation primer and Fw mutation primer/RK14, the mutation to introduce being bear on the overlapping forward and reverse primers. b. The resulting PCR products are assembled by the "Gibson" technique (see method), generating a single DNA fragment. 2. The resulting mutated DNA product is transformed and integrated by double cross-over recombination into the recipient B. subtilis strain bearing the reporter gene fusion at the thrC locus (RCL422). 3. Mutations are mapped by sequencing the region from maf to mreD. Gibson product is represented in green; light grey highlights regions of homology between the PCR fragment and the chromosome, allowing the recombination events to take place.

Figure 23

 23 Figure 23 Comparative expression of P mreBH lacZ in strains expressing gfp-mreB*, assayed by colorimetric assay on plate. Expression of the reporter gene P mreBH lacZ was tested on RCL422 (WT, P mreBH lacZ), RCL424 (P mreB gfp-mreB WT , P mreBH lacZ), RCL423 (∆mreB, P mreBH lacZ ) and all the MreB*s (P mreB gfp-mreB*, P mreBH lacZ), on LB-agar plates supplemented with Xgal after 12 h of incubation at 37 °C.

Figure 24

 24 Figure 24 Comparative expression levels of GFP-MreB. Cell extracts from RCL422 (WT, P mreBH lacZ), RCL424 (P mreB gfp-mreB WT , P mreBH lacZ), RCL423 (∆mreB, P mreBH lacZ) and all the MreB*s (P mreB gfp-mreB*, P mreBH lacZ), grown in CH media until an OD 600 nm ≈ 0,2. *, indicates cultures supplemented with 20 mM MgSO 4 . Western inmunoblots were probed with antibodies raised againt MreB (note the presence of a lower band for RCL422 due to the absence of a GFP tag).

Figure 25

 25 Figure 25 WT-like MreB*s: growth in CH or LB media (supplemented or not with MgSO 4 ), morphology and spatial localization on the protein. A. Growth curves of RCL422 (WT, P mreBH lacZ), RCL424 (P mreB gfp-mreB WT , P mreBH lacZ), RCL423 (∆mreB, P mreBH lacZ) and the 18 WT-like MreB*s, grown in CH (upper panels) or LB media (lower panels), supplemented with MgSO 4 (right panels) or not (left panels). B. Bright field microscopy images of control WT strains RCL422 and RCL424 (blue outline), ∆mreB strain RCL423 (red outline) and the 18 WT-like MreB* strains grown in CH media, and acquired during mid-exponential phase of growth (OD ≈ 0,2 -0,3). C. Example of zoomed are of bright field and fluorescent signal of mutant B18. D. Mutated residues displayed on a 3D-structure model of MreB showing the surface of the protein, recto (left) and verso (right). Mutated residues in WT-like MreB* mutants (green) are designated by arrows while other colored residues signaled known or presumed aminoacids involved in: monomer:monomer interaction (blue), putative nucleotide binding area (red), putative RodZ interaction area (pink), and putative interfilament bundleing interface (yellow). E. MreB ribbon structure model (recto only) displaying mutated residues embedded in the structure and not visible on the surface display. The color code is the same than in D.

Figure 26

 26 Figure 26 ∆mreB-like MreB*s: growth in CH or LB media (supplemented or not with MgSO 4 ), morphology and spatial localization on the protein. A. Growth curves of RCL422 (WT, P mreBH lacZ), RCL424 (P mreB gfp-mreB WT , P mreBH lacZ), RCL423 (∆mreB, P mreBH lacZ) and the 5 ∆mreB-like MreB*s grown in CH (upper panels) or LB media (lower panels), supplemented with MgSO 4 (right panels) or not (left panles). B. Bright field microscopy images of control WT strains RCL422 and RCL424 (blue outline), ∆mreB strain RCL423 (red outline) and the 5 ∆mreB-like MreB* strains grown in CH media, and acquired during mid-exponential phase of growth (OD ≈ 0,2 -0,3). Mutant cells display an essentially ∆mreB shape. C. Example of zoomed are of bright field and fluorescent signal of mutant B4. D. Mutated residues displayed on a 3D-structure model of MreB showing the surface of the protein, recto (left) and verso (right). Mutated residues in ∆mreB-like MreB* mutants (purple) are designated by arrows while other colored residues signaled known or presumed aminoacids involved in: monomer:monomer interaction (blue), putative nucleotide binding area (red), putative RodZ interaction area (pink), and putative interfilament bundleing interface (yellow). E. MreB ribbon structure model (recto only) displaying mutated residues embedded in the structure and not visible on the surface display. The color code is the same than in D.

Figure 27

 27 Figure 27 Intermediate MreB*s: growth in CH or LB media (supplemented or not with MgSO 4 ), morphology and spatial localization on the protein. A. Growth curves of RCL422 (WT, P mreBH lacZ), RCL424 (P mreB gfp-mreB WT , P mreBH lacZ), RCL423 (∆mreB, P mreBH lacZ) and the 7 intermediate MreB*s grown in CH (upper panels) or LB media (lower panels), supplemented with MgSO 4 (right panels) or not (left panels). B. Bright field microscopy images of control WT strains RCL422 and RCL424 (blue outline), ∆mreB strain RCL423 (red outline) and the 7 intermediate MreB* strains grown in CH media, and acquired during midexponential phase of growth (OD ≈ 0,2 -0,3). Mutant cells display a range of morphological defects. C. Example of zoomed are of bright field and fluorescent signal of mutant B42. D. Mutated residues displayed on a 3D-structure model of MreB showing the surface of the protein, recto (left) and verso (right). Mutated residues in interrmediate MreB* mutants (cyan) are designated by arrows while other colored residues signaled known or presumed aminoacids involved in: monomer:monomer interaction (blue), putative nucleotide binding area (red), putative RodZ interaction area (pink), and putative interfilament bundleing interface (yellow). E. MreB ribbon structure model (recto only) displaying mutated residues embedded in the structure and not visible on the surface display. The color code is the same than in D.

Figure 28 4

 28 Figure 28 4 WeB MreB*s: growth in CH or LB media (supplemented or not with MgSO 4 ), morphology and spatial localization on the protein. A. Growth curves of RCL422 (WT, P mreBH lacZ), RCL424 (P mreB gfp-mreB WT , P mreBH lacZ), RCL423 (∆mreB, P mreBH lacZ) and 4 WeB MreB*s grown in CH (upper panels) or LB media (lower panels), supplemented with MgSO 4 (right panels) or not (left panels). B. Bright field microscope images of control WT strains RCL422 and RCL424 (blue outline), ∆mreB strain RCL423 (red outline) and the 4 WeB MreB* strains grown in CH media, and acquired at OD ≈ 0.1 -0.2. Mutant cells display very mild morphological defects or none. C. Zoomed area of bright field and fluorescent signal of mutants B10, B17, B30 and B46. D. Mutated residues displayed on a 3D-structure model of MreB showing the surface of the protein, recto (left) and verso (right). Mutated residues in 4 WeB MreB* mutants (orange) are designated by arrows while other colored residues signaled known or presumed aminoacids involved in: monomer:monomer interaction (blue), putative nucleotide binding area (red), putative RodZ interaction area (pink), and putative interfilament bundleing interface (yellow). E. MreB ribbon structure model (verso only) displaying mutated residues embedded in the structure and not visible on the surface display. The color code is the same than in D.

Figure 29 B32

 29 Figure 29 B32 MreB*: growth in CH or LB media (supplemented or not with MgSO 4 ), morphology and spatial localization on the protein. A. Growth curves of RCL422 (WT, P mreBH lacZ), RCL424 (P mreB gfp-mreB WT , P mreBH lacZ), RCL423 (∆mreB, P mreBH lacZ) and B32 MreB* grown in CH (upper panels) or LB media (lower panels), supplemented with MgSO 4 (right panels) or not (left panels). B. Bright field microscopy images of control WT strains RCL422 and RCL424 (blue outline), ∆mreB RCL423 strain (red outline) grown in CH media, and acquired during mid-exponential phase of growth (OD ≈ 0,2 -0,3). B32 was grown in CH media and images were acquired after ~ 24 h growth, at an OD ≈ 0,1. Mutant cells display very severe morphological defects. C. Zoomed area of bright field and fluorescent signal of mutant B32. D. 3D-structure model of MreB showing the surface of the protein verso (right). B32 MreB* mutant (orange) is designated by an arrow while other colored residues signaled known or presumed aminoacids involved in: monomer:monomer interaction (blue), putative nucleotide binding area (red), putative RodZ interaction area (pink), and putative interfilament bundleing interface (yellow).

Figure 30

 30 Figure 30 Effect on the growth of WeB and B32 MreB*s by the addition of 1,5 % fructose or 1,5 % glucose. Strains were grown in CH media (black lines), CH media supplemented with 1,5 % fructose (red lines) or CH supplemented with 1,5 % glucose (cyan lines). A. RCL424 (P mreB gfp-mreB WT , P mreBH lacZ) in solid lines and RCL423 (∆mreB, P mreBH lacZ) in dashed lines; B. B10; C. B17; D. B30; E. B46; F. B32. All strains grow to a higher OD 600 nm when 1,5 % fructose or 1,5 % glucose is added to CH media. ∆mreB and all the MreB*s tested recover WT-like growth.

  may play a role in CW synthesis, cell morphology and cell metabolism Despite years of extended efforts, MreB's exact function(s) remains elusive. By creating a genetic screen that selects for loss-of-function MreB mutants (MreB*s) in the Gram-positive bacterium model B. subtilis, we were able to circumvent this problem and make links between MreB structure and function. It is difficult to extract strong conclusions from our preliminary results, but we did succeed in the acquisition of some very interesting group of MreB mutants that hint MreB has more than one function in B. subtilis.

Figure 32

 32 Figure32Development of divergent colony morphologies after extended growth. After > 24 h of growth of MreB*s on LB-agar plates, three different phenotypes start to develop: while most colonies keep a smooth and regular appearance, B4, B22 and B58 (white) display a wrinkled surface wnd B6, B19, B30 and B32 (purple) develop white overgrowing colonies that do not activate P mreBH lacZ, suggesting the appearance of suppressor mutations.

Figure A1. 3

 3 Figure A1.3 Viability of deletion mutants of ydcF, ydcG and ydcH in CH. Colony forming units per mL of culture were estimated by plating aliquots of WT (ABS2005), ∆ydcF (ASEC60), ∆ydcG (ASEC58) and ∆ydcH (ASEC56) strains harvested during exponential (A), entry into stationary phase (B) and late stationary phase (C).

Figure A1. 4

 4 Figure A1.4 Stationary phase processes are affected in ydcF, and ydcG mutants. A. Sporulation assays were performed on WT (ABS2005), ∆ydcF (ASEC60), ∆ydcG (ASEC58) and ∆ydcH (ASEC56) strains as described in the method section, using the exhaustion method. Briefly, samples of 30h old cultures were plated before (giving the total number of cells) and after (giving the number of spores) heat kill. Spore frequency (spores/viable cells) and spore counts are calculated from CFU. B. Competence assays were performed on the same strains and as described in the method section. Briefly, samples were grown in competence media and plates in LB plates (giving total number of cells) and in LB plates supplemented with the antibiotic resistance that was introduced (giving the number of transformants). Transformation rate (transformants/total cells) was calculated from CFU.

Figure A2. 1

 1 Figure A2.1 B. subtilis constructs bearing the reporter P ydc1 lacZ. A. Schematic view of the mreB locus and its neighboring genes in the wild type and the 3725 mutant strains. Results from (Nicolas et al., 2012) indicate that transcripts containing mreB originate upstream from mreB and maf, and extend up to minD. B. Various constructs made to link P ydc1 expression to the mreB locus. First column describes changes (deletions or cloning) at the mreB locus; the two middle columns show ectopic clonings at the amyE and bkdR loci; right column indicates the phenotype (induction of P ydc1 ) of each corresponding strain as assayed by X-gal plating test (see materials and methods).

Figure A2. 2

 2 Figure A2.2 Construction of B. subtilis strains inactivated for mreB. A. Genetic map of the mreB locus in strain 3725. mreB was inactivated by removing nucleotides 31 to 977 and addition of 21 extragenic base pairs. B. Genetic map of the mreB locus in strain CCBS194. MreB synthesis was abolished by introducing 3 stop codons after the sixth base-pair of mreB. C. Western blot of strain CCBS194 (mreB-3STOP) showing the absence of MreB synthesis, comparable to that of strain 3725. D. Genetic map of the mreB locus in strain CCBS202 .In this strain, the 3 stop codons introduced in strain CCBS194 were combined with the deletion present in strain 3725.

  function and mode of regulation are currently unknown. I used Bacillus subtilis, the model for Grampositive bacteria, to gain a better understanding of MreB functions via i-the development and use of a genetic screen for loss-of-function mutants of mreB and ii-the study of a potential effector of MreB. (i) MreB has been studied for almost two decades now and still, little is known about its function(s). Since biochemical approaches proved to be difficult so far, most of the studies have focused on cellular localization and dynamics of the protein. Here, I have designed a genetic screen by means of which I have obtained a collection of functionally impaired mreB mutants in B. subtilis.

Table 1

 1 PBPs classification..............................................................................................................

Table 2

 2 Percentage of amino acid identity between the three MreB isoforms in B. subtilis..........

Table 3

 3 Summary of in vitro biochemical properties of MreB in different organisms....................

Table 4

 4 Media supplements............................................................................................................

Table 5

 5 Strains used in this study....................................................................................................

Table 6

 6 Plasmids used in this study.................................................................................................

Table 7

 7 Oligonucleotides used in this study.....................................................................................

Table 8

 8 Mutations found in the ∆mreB strain (3725).............................................................................

Table 9

 9 List of strains carrying SNP in gfp-mreB, cloned at natural locus, and their controls used for phenotypic characterization……………………………………………………………………….................................

Table 10

 10 Classification of MreB*s based on their phenotypic characterization........................................

Table 2 Percentage of amino acid identity between the three MreB isoforms in B.

 2

subtilis: MreB, Mbl and MreBH. Obtained with ClustalO

[START_REF] Sievers | Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega[END_REF]

.

INTRODUCTION

Table 5 Strains used in this study

 5

	Name	Genotype

ydc1 -lacZ (erm)

This study RCL414 → ABS1990 ABS2054 trpC2 ∆mreB::neo thrC::P ydc1 -lacZ (erm)

A. Chastanet not published

Table 5 Strains used in this study (continuation)

 5

	Name	Genotype

xyl -pep thrC::P ydc1 -lacZ (erm) This study Gibson → ABS1990 ABS1400 trpC2 ∆mreB::neo amyE::P N -mreBCD (spc) A. Chastanet not published ASEC20 trpC2 ∆mreB::neo amyE::P N -mreBCD (spc) thrC::P ydc1 -lacZ (erm)

Table 5 Strains used in this study (continuation)

 5

	Name	Genotype

Table 5 Strains used in this study (continuation)

 5

	Name	Genotype

mreBH -lacZ (cat) mreB::km-P N optrbsgfp-mreB

S33T

Table 5 Strains used in this study (continuation)

 5

	Name	Genotype

Table 6 Plasmids used in this study

 6

	Plasmid	Characteristics	Source
			Laboratory
	pDG1730 bla spc amyE	stock
	pDG1730.1 bla spc amyE3'P N mreBamy5'	This study
			Laboratory
	pDR244	bla cre spc cop repF	stock
			Laboratory
	pDG1663 bla thrC 3' erm lacZ thrC 5'	stock
	pAC824	bla thrC 3' erm P ydc1 lacZ thrC 5'	This study
			Laboratory
	pDG1728 bla amyE 3' spc lacZ amyE 5'	stock
	pAC772	bla amyE 3' spc P ydc2 lacZ amyE 5'	This study
	pAC775	bla amyE 3' spc P ydc1-2 lacZ amyE 5'	This study
	pAC778	bla amyE 3' spc P ydc0 lacZ amyE 5'	This study
	pAC783	bla amyE 3' spc P fruRKA lacZ amyE 5'	This study
			Laboratory
	pDG1729 bla thrC 3' spc lacZ thrC 5'	stock
			Laboratory
	pDag32	bla spc::cat	stock
			Laboratory
	pAH328	bla sacA 3' cat luxABCDE sacA 5'	stock
	pAC832	bla sacA 3' cat P ydc1 luxABCDE sacA 5'	This study
	2.4. Experimental procedures
	2.4.1 Cloning procedures:
		2.4.1.1 Oligonucleotides:

Oligonucleotides were designed using the Clone Manager 9 PE and purchased from Eurofins MWG.

Table 7 Oligonucleotides used in this study

 7

	Primer	Descriptio	Sequence 5' → 3'	Restriction	Used for
		n			sites/homology
	AC1345 Rv	at	AAGTCACTCAGTAATAACCGC	-	verifications	at
		mreC				mreBCD locus and
						mreB	random
						mutagenesis
	AC1335 Fw at maf TCGATCAAGCCGTAGCCTTTGCTG	-	verifications	at
						mreBCD locus and
						mreB	random
						mutagenesis
	CC181	Fw at maf GTCATGGGCCTTCCTATATC	-	mreB	directed
						mutagenesis
	RK14	Rv	at	AATTCGAGCAGACAGACAGCCAGAAC	-	mreB	directed
		mreC				mutagenesis
	AC1240 Fw		GTAGAATTCGCTGAAAATGTATACG	EcoRI	amplify P ydc1 and
				ACATCGAG		P ydc1-2
	AC1241 Rv		GGAGGATCCCCGTCGGCATGTCTTT	BamHI	amplify P ydc1 and
				AGACAGT		P ydc0
	AC1242 Rv		GGAGGATCCGGCAGCGCCTTTTAAT	BamHI	amplify P ydc2 and
				ACATGTT		P ydc1-2
	AC1243 Fw		GAAGAATTCACTTATCATTCTGGGA	EcoRI	amplify P ydc1 and
				GCTTATGGG		P ydc0
	AC1248 Fw		GATGAATTCCAGTTTTTAATTG	EcoRI	amplify P fru
				AATCAGTCG
	AC1249 Rv		GGAGGATCCAACGTATTCATTTTGA	BamHI	amplify P fru
				ATACAATTT
	AC1246 Fw		GATGAATTCTTCATCTACTTTTCTCA	EcoRI	amplify P mreBH
				CAACA
	AC1247 Rv		GGAGGATCCCCTAATTTAATATGAT	BamHI	amplify P mreBH
				TCTACATTT
	ASEC19 Fw at ydcF AGCTGTGAAGAAGCTCAGAGAGGC	-	PI-PO ydcF
				CTTGAT
	ASEC20 Rv at ydcH ACACATAAAAAAAGACAGCTGGCG	-	PI-PO ydcF
				CTGCCC
	ASEC21 Rv at ydcF CGACCTGCAGGCATGCAAGCTCCTT	cassette	PI-PO ydcF
				GCAGAAGCAATGTTCCTTCCGTTCC	phleomycin
				A
	ASEC22 Fw at ydcF CGACGTCGAATTCACTGGCCGTCTG	C1 and ydcF 5' PI-PO ydcF
				GAACGGAAGGAACATTGCTTCTGCA
				AGGGCCGACGGCTGTCAGCAGGCTT
				GTCCATCT
	ASEC23 Fw	at	GTAATCAGGCGATGAAAAACAAAA	-	PI-PO ydcG
		ydcG		GAGGCG
	ASEC48 Rv at ydcG GTCAGGATATAGTCGGCAAGCGGCT	-	PI-PO ydcG
				CAAGG
	ASEC25 Rv at ydcG CGACCTGCAGGCATGCAAGCTTCTG	cassette	PI-PO ydcG
				ACACAACGCCGATCCAGTAATTT	phleomycin

Table 7 Oligonucleotides used in this study (continuation)

 7

	Primer	Descriptio	Sequence 5' → 3'	Restriction	Used for
		n			sites/homology
	ASEC47	Fw	at	CGACGTCGAATTCACTGGCCGTCGAAA	C1 and ydcG 5' PI-PO ydcG
		ydcG		TTACTGGATCGGCGTTGTGTCAGAAAA
				AAAGATTTTTTGACAATAGCACAAGCG
				ATGGG
	ASEC69	Fw	at	TAAGACAGAAGTTGCGTTTTGGTCCCT		∆mreBCD
		radC		CAG
	ASEC70	Rv at 5'	AGCCCAAGCTCTAGACCAAGGTCTCTA	5' neomycin	∆mreBCD
		mreB		GCACCAATTCCAAACATA	cassette
	ASEC71	Fw		TCTAGAGCTTGGGCTGCAGGTCG	5' neomycin	∆mreBCD
		neomycine		cassette
	ASEC72	Rv		GTAACCAACATGATTAACAATTATTAG	3' neomycin	∆mreBCD
		neomycine	AGGTCATCGTTCAAA	cassette
	ASEC73	Fw	3'	TTAATCATGTTGGTTACGTAAAAAGGA	3' neomycin	∆mreBCD
		mreD		TTTTATCTTTTTTTGACGAAATGAGTAT	cassette
				GTTGTTGAG
	ASEC74	Rv minC		CTCCAGCAGTCCTATAATACGGTCAGC		∆mreBCD
				ATC
	ASEC87	Rv mreB		TTTTTTATGGCCTGAATGATGTAATATT	MreB* N88I B2
				TCATCATCG
	ASEC88	Fw mreB GAAATATTACATCATTCAGGCCATAAA	MreB* N88I B2
				AAATAAAGG
	ASEC91	Rv mreB		GCCCGGTGTCCGTCTAATCATATTTTTC	MreB* G56R B4
				GC
	ASEC92	Fw mreB ATATGATTAGACGGACACCGGGCAAC	MreB* G56R B4
				GTGG
	ASEC95	Rv mreB		GTACCGCCCCTGATATCAACAACCATG	MreB* G160R B6
				CTTCC
	ASEC96	Fw mreB TGTTGATATCAGGGGCGGTACGACAGA	MreB* G160R B6
				AGTTGC
	ASEC97	Rv mreB		TTCCTGTAATTCCAATTGTTTTCGGCAA	MreB* E243G B7
				ACCTG
	ASEC98	Fw mreB AAAACAATTGGAATTACAGGAAAAGA	MreB* E243G B7
				GATTTCTAACGCTCTACGCGACAC
	ASEC154 Rv mreB		CAGCTGCTGAAGAACGCGCTGTTATCG	MreB* V114A B14
				ATGCGACAAGACAGG
	ASEC155 Fw mreB CGCGTTCTTCAACAGCTGTAATGCCTG	MreB* V114A B14
				ATGGG
	ASEC156 Rv mreB		GATTGGCTCCGGTTGCTGCGGCAAAAG	MreB* I142T B15
				GCTCTTC
	ASEC157 Fw mreB GCCGCAGCAACCGGAGCCAATCTGCCA	MreB* I142T B15
				GTTTGGG
	ASEC160 Rv mreB		TCAGCGATAATGCCGTCTTTCATCGGG	MreB* G216R B16
				CGAAGAG
	ASEC161 Fw mreB GAAAGACGGCATTATCGCTGATTATGA	MreB* G216R B16
				AA
	ASEC170 Rv mreB		GTAATGCCTGGTGGGACACATACCATT	MreB* S109P B10
				AC
	ASEC171 Fw mreB ATGTGTCCCACCAGGCATTACAGC	MreB* S109P B10

Table 7 Oligonucleotides used in this study (continuation) Primer Description Sequence 5' → 3' Restriction sites/homology Used for

 7

	ASEC172 Rv mreB	ATCGATTTCGCATCCGTCTGCAAAGC	MreB* T41A B11
	ASEC173 Fw mreB	TTGTCGCTTTGCAGACGGATGCGAAAT	MreB* T41A B11
	ASEC174 Fw mreB	CCTGAGCTTGGAGCAGATATCATGGACA	MreB* A276G B17
		GAGGTA
	ASEC175 Rv mreB	GATATCTGCTCCAAGCTCAGGCGGTGTTT	MreB* A276G B17
		TTTCG
	ASEC176 Fw mreB	GAAATCCGCGACCGCGATTTGCTCACAG	MreB* G231D B19
		GTTTGC
	ASEC177 Rv mreB	CGCGGTCGCGGATTTCCATGTTG	MreB* G231D B19
	ASEC178 Rv mreB	CTGACGGCCCTCTCACAACAATTCCTTTT	MreB* E31G B20
		CCTTTT
	ASEC179 Fw mreB	GTTGTGAGAGGGCCGTCAGTTGTCGCTTT	MreB* E31G B20
		GCAG
	ASEC180 Fw mreB	TCAATCCGTGCAGCCGGTGATGAGATGG	MreB* V182A B21
		ATGACGC
	ASEC181 Rv mreB	ATCACCGGCTGCACGGATTGACTGAGAC	MreB* V182A B21
		GTTACGA
	ASEC182 Fw mreB	ATAGATCTTGAAACTGCGAATACGCTTGT	MreB* G14E B22
		TTTTGT
	ASEC183 Rv mreB	TTCGCAGTTTCAAGATCTATACCAAGGTC	MreB* G14E B22
		TC
	ASEC184 Rv mreB	GGTTTGCCGAAAACAAATGAAATTACAG	MreB* I242N B1
		GAAAAGAGATTTCTA
	ASEC185 Fw mreB	CTTTTCCTGTAATTTCATTTGTTTTCGGCA	MreB* I242N B1
		AACCTGTGAGC
	ASEC186 Rv mreB	ATCAGATTGTACGTTTCTCTGATGTAGTT MreB* K197E B5
	ASEC187 Fw mreB	ATTATCAACTACATCAGATAAACGTACA MreB* K197E B5
	ASEC257 Fw mreB	GAAAGACGGCGCTATCGCTGATTATG	MreB* V72I B18
	ASEC258 Rv mreB	TCAGCGATAGCGCCGTCTTTCATCGGG	MreB* V72I B18
	ASEC211 Fw mreB	AATGATGCGAGAAATATGATTGG	MreB* K52R B23
	ASEC212 Rv mreB	ATCATATTTCTCGCATCATTTCCGAC	MreB* K52R B23
	ASEC215 Fw mreB	TGAGAGAGCCGACAGTTGTCGC	MreB* S33T B25
	ASEC216 Rv mreB	GCGACAACTGTCGGCTCTCTCAC	MreB* S33T B25
	ASEC217 Fw mreB	GAAATGATGTGAAAAATATGATTG	MreB* A51V B26
	ASEC218 Rv mreB	CATATTTTTCACATCATTTCCGAC	MreB* A51V B26
	ASEC219 Fw mreB	CGGAGGCATGGTAACGTCTCAG	MreB* I174M B27
	ASEC220 Rv mreB	GAGACGTTACCATGCCTCCGAGGGAAAT	MreB* I174M B27
		A
	ASEC221 Fw mreB	AGATGGATGGCGCGATTATCAACTACA	MreB* D189G B28
	ASEC222 Rv mreB	GATAATCGCGCCATCCATCTCATCAC	MreB* D189G B28
	ASEC223 Fw mreB	GTGGCTCTTTGCCCGATGAAAGAC	MreB* R66C B29
	ASEC224 Rv mreB	TTCATCGGGCAAAGAGCCACCAC	MreB* R66C B29
	ASEC225 Fw mreB	GCAGCAGATGTCATGGACAGAG	MreB* I279V B30
	ASEC226 Rv mreB	CTGTCCATGACATCTGCTGCAAGCTC	MreB* I279V B30
	ASEC229 Fw mreB	TTATTTCCCCCGGAGGCATCGTAACGTC	MreB* L171P B32
	ASEC230 Rv mreB	GATGCCTCCGGGGGAAATAATC	MreB* L171P B32

Table 7 Oligonucleotides used in this study (continuation) Primer Description Sequence 5' → 3' Restriction sites/homology Used for

 7

	ASEC239 Fw mreB	ATGAAACAATGGCGACGATGATG	MreB* T79M B37
	ASEC240 Rv mreB	CATCGTCGCCATTGTTTCAT	MreB* T79M B37
	ASEC243 Fw mreB	TGTTATCGAAGCGACAAGACAGGCG	MreB* D121E B39
	ASEC244 Rv mreB	GTCTTGTCGCTTCGATAACAGCGCGT	MreB* D121E B39
	ASEC247 Fw mreB	GACGCGTATCCGGTTGAAGAGCCTTT	MreB* I134V B41
	ASEC248 Rv mreB	GGCTCTTCAACCGGATACGCGTCAC	MreB* I134V B41
	ASEC249 Fw mreB	TTTGGGAACAGACTGGAAGCATGG	MreB* P151Q B42
	ASEC250 Rv mreB	GCTTCCAGTCTGTTCCCAAACTGGC	MreB* P151Q B42
	ASEC253 Fw mreB	TGAGAGAGCTGTCAGTTGTCGCTT	MreB* P32L B44
	ASEC254 Rv mreB	GACAACTGACAGCTCTCTCACAAC	MreB* P32L B44
	ASEC257 Fw mreB	GAAAGACGGCGCTATCGCTGATTATG	MreB* V72A B46
	ASEC258 Rv mreB	TCAGCGATAGCGCCGTCTTTCATCGGG	MreB* V72A B46
	ASEC259 Fw mreB	CCGACTGGAAGCGTGGTTGTTGATATCG	MreB* M155V B47
		G
	ASEC260 Rv mreB	TCAACAACCACGCTTCCAGTCGG	MreB* M155V B47
	ASEC269 Fw mreB	CGCTGTCGGAAGTGATGCGAAAAAT	MreB* N49S B52
	ASEC270 Rv mreB	TTTCGCATCACTTCCGACAGCGACAA	MreB* N49S B52
	ASEC271 Fw mreB	ACGGACACCGCGCAACGTGGTGG	MreB* G60R B53
	ASEC272 Rv mreB	ACCACGTTGCGCGGTGTCCGTC	MreB* G60R B53
	ASEC196 Rv	TTCCAAACATCCTAGGAATCTCCTTTCT	5' ∆mreB	amyE::P xyl pep
	ASEC197 Fw	GATTCCTAGGATGTTTGGAATTGGTGCT	3' P xyl	amyE::P xyl pep
		AGA
	ASEC198 Rv	TATCAAGCTTTTATCTAGTTTTCCCTTTG	5' amyE	amyE::P xyl pep
		AAAAGATG
	ASEC199 Fw	AACTAGATAAAAGCTTGATATCGAATT	3' ∆mreB	amyE::P xyl pep
		CTAGTT
	2.4.1.2 Polymerase chain reaction (PCR):

PCR reactions were set up on ice and performed according to manufacturer's recommendations. Taq DNA polymerase was used to confirm insertions or deletions and for mutagenesis; Phusion (NEB) DNA polymerase was used for cloning in B. subtilis and sub-cloning in E. coli.

2.4.1.3 Polymerase chain reaction on B. subtilis colonies (cPCR):

Figure 14 Growth of deletion mutants of ydcF, ydcG and ydcH derived from BKE strains in different

	media. WT cells
	(ABS2005), the deletion-
	replacement mutant of
	ydcH (ASEC56) and the
	marker-less		deletion
	mutants	of	ydcF
	(ASEC317),		ydcG
	(ASEC319), and ydcH
	(ASEC321) were grown in
	MSM media (A), CH
	media (B) and LB media
	(C). ∆ydcF, ∆ydcG and
	both ∆ydcH strains have a
	WT growth phenotype in
	all media tested.

Differentially expressed genes in the ∆ydcH strain Overexpressed genes in ∆ydcH during exponential growth

	Expression 71,73 4,12 3,57 3,16 2,82 2,58 difference Expression difference 4,13 Expression difference 3,57 Expression difference 3,17 Expression difference 2,83 Expression difference 2,58 Appendix 2: Gene ydcF yopO ykzT secE yonV yozV Gene pyrP Gene ykvS Gene yopP Gene S903/rpmGC Gene yorB Overexpressed genes in ∆ydcH during exponential growth (continuation) Unknown SP-beta prophage Unknown Protein secretion Protein translocase depletion SP-beta prophage Unknown Function1 Function2 Function1 Function2 Transporters uracil permease Function1 Function2 Unknown Function1 Function2 SP-beta prophage Function1 Function2 Replaces L33 under conditions of zinc Function1 Function2 SP-beta prophage Overexpressed genes in ∆ydcH during exponential growth (continuation) Overexpressed genes in ∆ydcH during exponential growth (continuation) Overexpressed genes in ∆ydcH during exponential growth (continuation) Overexpressed genes in ∆ydcH during exponential growth (continuation) Down-	Regulated by Regulated by PyrR Regulated by Regulated by Regulated by Zur Regulated by
	ydcG yozL ygzA S717/SR5 pyrR yorC yncB trnD-Trp/trnR yoeC yceK yydD yotM/yodV	17,56 14,32 3,56 4,10 3,99 3,54 3,16 3,16 2,81 2,56 2,80 2,56	Unknown Unknown Unknown Anti-toxin Biosynthesis/ acquisition of pyrimidine nucleotides SP-beta prophage Unknown tRNA transcription factors and their similar to transcriptional regulator Antisense RNA to bsrE Transcriptional antiterminator of the pyr operon Unknown control (ArsR family) Transfer RNA-Trp Unknown SP-beta prophage	LexA PyrR LexA
	yosU yjcM sacV/ydzC/xis yonI yxjA/nupG yubF	8,08 3,54 3,91 3,14 2,78 2,54		SP-beta prophage Unknown Prophages and mobile genetic SP-beta prophage Transporters Unknown	Excision of the conjugative Purine nucleosides transporter	SigD, AbrB ImmR G-box
	ykoL bhlB/yomA yckD cotC cotX yozH yocC ydjM/yzvA yonR lrpA/yddO ybeF yknT/cse15 yozF cotM/yneL yydI immR/ydcN ybzH tagB xre ydcP yopE yobL yorS yrzM yopM lrpB/yddP ydjO yopA yrdC yrkD sigX/ypuM yorJ yomO yqaS yoqL yxaC rpmGB/rpmG ynaE phrE pbuO/ytiP mntR/yqhN yobK tnrA/scgR sinR/sin/flaD yoyH yraH yvkN yokB yhcN comC yopQ ywnA	6,46 6,45 6,38 6,28 5,96 5,77 5,47 5,39 5,29 5,26 2,78 3,90 3,88 3,87 3,87 3,86 3,86 3,84 3,84 3,83 3,83 3,10 2,54 3,53 3,52 3,51 3,09 3,08 2,78 2,53 3,07 2,78 2,49 2,78 2,49 3,50 3,49 3,49 3,48 3,06 2,77 2,48 2,45 2,76 2,34 3,05 2,33 3,47 3,05 2,74 3,04 2,29	Unknown SP-beta prophage Sporulation Sporulation Sporulation Unknown Unknown Cell wall Regulation of gene expression Regulation of gene expression Unknown Sporulation Protein Unknown Sporulation proteins ABC transporters Regulation of gene expression Prophage 1 Biosynthesis of teichoic acid Regulation of gene expression Prophages elements SP-beta prophage Toxins, antitoxins and immunity SP-beta prophage Unknown SP-beta prophage Regulation of gene expression Cell envelope stress proteins against toxins/based on similarity Unknown similar to TR copper sensor spore coat protein spore crust assembly May be involved in cell wall metabolism (WalR regulon) Xre family TR Repression of glyA transcription and KinB-SigE, SpoIIID PhoP and TnrA SigF and SigG GerE, SigK and SpoIIID GerE, GerR, SigE and SigK WalR and PhoP resistance of the spore SigE, SigK, GerE control of LiaR-LiaS activity AbrB and Rok Control of transfer of the mobile genetic element ICEBs1 PhoP and WalR regulation of PBSX prophage gene expression (Xre family) Conjugation and replication of ICEBs1, helicase transposon ICEBs1 from the trnS-leu2 locus Repression of glyA SigW Unknown SP-beta prophage Sigma factors Resistance to cationic antimcrobial peptides, RNA SP-beta prophage SP-beta prophage SigX and YvrHb reg; induced in Skin element SP-beta prophage SigD transcription and KinB-dependent sporulation Unknown Holin-like auxiliary protein Ribosomal proteins Ribosomal protein L33b Unknown Regulation of gene expression Phosphatase (RapE) regulator polymerase ECF-type sigma factor SigX stationary phase due to the production of toxic peptides (SdpC, SkfA) Transporters Paralog of PbuG PurR Trace metal homeostasis (Cu, Zn, Ni, Mn, Mo) Regulation of manganese Toxins, antitoxins and immunity Inhibition of the cytotoxic activity MntR against toxins based on similarity of YobL transport, transcriptional Biosynthesis, acquisition of amino Transcriptional pleiotropic regulator (DtxR family) Transcription factors and their control Transcriptional regulator (Xre family) of post-exponential-acids regulator (MerR family) involved AbrB, ScoC and in global nitrogen regulation Spo0A SP-beta prophage phase responses genes, AbrB, CcpA, Unknown control of biofilm formation Unknown RAB11 family (control of CodY, ComA and SigH SP-beta prophage Sporulation proteins Genetic competence Late competence gene ComK membrane traficking) SigF and SigG translocation of ComGC, SP-beta prophage required for processing and Unknown
	rpmB/yloT yqaE/sknR	3,45 3,04		Ribosomal proteins Skin element	dependent sporulation processivity factor ribosomal protein L28 transcriptional repressor (Xre ComGD, ComGE and ComGG
	presence of > 50 mutations. sdpA/yvaW 5,21 ykzB 4,99 yqbG 4,94 yjdG 4,87 yybL 3,82 yugI 3,81 rpsT/yqeO 3,81 yddK 3,76 trnD-Ser/trnR 3,44 yxkC 3,43 ydcO 3,42 yybN 3,40 yfmI 2,74 Down-regulated genes in ∆ydcH during exponential growth Toxins, antitoxins and immunity against toxins maturation of the SdpC toxin Unknown Skin element Unknown ABC transporters Permease Unknown Ribosomal proteins ribosomal prot S20 (BS20) Unknown tRNA Transfer RNA-Ser Unknown Unknown Unknown family); essential in WT due to Transporters AbrB and Spo0A AbrB, Rok and Spo0A TnrA Rok RelA SigD and TnrA Rok overexpression of the toxic proteins YqaH and YqaM yqkE 3,03 Unknown sdpI/yvaZ 3,02 Toxins, antitoxins and immunity AbrB and SdpR comK 2,74 Genetic competence Competence transcription factor (CTF) AbrB, CodY, Gene Expression Function1 Function2 Regulated by ComK, DegU, Rok difference and Spo0A yopC 2,72 gltX 2,09 Aminoacyl-tRNA synthetases glutamyl-tRNA synthetase SigA SP-beta prophage AbrB yydH 4,86 ABC transporter Control of LiaR-LiaS activity, processing of YydF AbrB and Rok ggaA 4,64 Biosynthesis of teichoic acid Galactosamine-containing minor teichoic acid biosynthesis yabE 4,50 Cell wall Similar to CW binding prot yotE 3,75 SP-beta prophage yhjM/ntdR 3,73 Biosynthesis of antibacterial compounds immA/ydcM 3,40 Proteolysis Ccontrol of ImmR activity, site-specific protease against toxins yydJ 3,00 ABC transporters Control of LiaR-LiaS activity yonT 2,72 toxins, antitoxins and immunity yhcY 2,13 Protein kinases two component sensor kinase LiaR, SigA toxin AbrB and Rok against toxins/ based on similarity yhfJ/lplJ 2,14 Biosynthesis of lipoic acid Lipoate:protein ligase AbrB yopN 3,73 SP-beta prophage yneF/yoxG 3,73 Unknown SpoOA yoyI 3,72 SP-beta prophage ylbN 3,39 Unknown may be required for accumulation of 23S rRNA gerD 3,37 Unknown Scaffold of the germinosome SigF and SigG ybfE 3,33 Unknown ypoP 3,00 Transcription factors and their yvzA 2,72 Unknown yvlA 2,14 Cell envelope stress proteins SigW, AbrB control yopK 2,99 yqgW 2,71 Unknown (controlled by SigM, V, W, X, Y) SP-beta prophage trnD-Cys/trnR 2,97 tRNA translation, transfer RNA-Cys ybfF 2,70 deaD/yxiN 2,16 RNA synthesis and degradation, Important for adaptation to low Unknown DEAD-box RNA helicases temperatures Cis-acting RNA dependent on SigX and SigM yydG 4,50 Regulation of gene expression control of LiaR-LiaS activity AbrB yjzI 4,47 Unknown abrB/cpsX/tolB 4,34 Regulation of gene expression yopF 4,33 SP-beta prophage yxlA 4,32 Biosynthesis/acquisition of nucleotides yotD 4,25 SP-beta prophage CsoR tagA 4,24 Biosynthesis of teichoic acid PhoP and WalR yonS 4,23 SP-beta prophage ydzP 4,22 Unknown yisI 4,16 Protein phosphatase Spo0A-P phosphatase, control of the phosphorelay PchR yoaQ 4,16 Unknown ypzA 4,13 Sporulation proteins SigG yokC 3,72 SP-beta prophage yobD 3,69 Regulation of gene expression similar to TR Xre family yoyA 3,68 Unknown ymzD 3,68 Unknown yqbC 3,68 Skin element yybM 3,66 Unknown Rok albG/ywhM 3,64 Biosynthesis of antibacterial compounds Production of subtilosin AbrB, ResD and Rok yjzD 3,62 Unknown putative toxin-antitoxin trnB-Met3/trnB-Ile1/trnE 3,62 tRNA Transfer RNA-Met yozQ 3,61 Sporulation proteins SigG ypzI 3,61 Unknown degR/prtR 3,58 Regulation of degradative enzyme production Positive effector of DegU-phosphate stability SigD yjiA 3,57 Unknown ydcL/int 3,32 Prophages Excision of the conjugative transposon ICEBs1 ggaB 3,31 Biosynthesis of teichoic acid Galactosamine-containing yvnA 2,97 Unknown ywpF 2,70 Unknown mutSB/yshD 2,17 DNA repair, recombination Probable DNA mismatch repair AbrB and CcpA yjfB 2,97 Unknown ydeI 2,70 Unknown protein SigD yezA 2,96 Unknown yosT 2,70 ywcD/gtcA/ip 2,21 Biosynthesis of teichoic acid Teichoic acid glycosylation protein SP-beta prophage a-34d/gtaC minor teichoic acid biosynthesis ykyB/kre 3,31 Genetic competence ComK repressor ComK ydfL 3,30 Regulation of gene expression yaaC 3,28 Sporulation protein yeeD 3,27 ligB/yoqV 2,96 SP-beta phage replication yhfM 2,70 sporulation proteins murF/ydbQ 2,22 Biosynthesis of peptidoglycan Peptidoglycan precursor SigM DNA ligase (ATP dependent) rapI/yddL 2,96 Protein phosphatases Control of transfer of the mobile genetic element ICEBs1 ComA yfzA 2,96 Unknown rpsO 2,95 Ribosomal proteins Ribosomal protein S15 yqzF 2,68 biosynthesis Unknown sda 2,68 Phosphorelay deoC/dra 2,24 Utilization of nucleotides Deoxyribose-phosphate aldolase CcpA, DeoR Sporulation inhibitor by dhbB 2,24 Acquisition of iron Isochorismatase; biosynthesis of AbrB, Fur, Kre preventing Spo0A-P ycxB 2,67 the siderophore bacillibactin Unknown ytlR 2,25 Unknown SigD Unknown yopR 3,26 SP-beta prophage ydeB 3,26 Regulation of gene expression yoyJ 3,26 SP-beta prophage dps/ytkB 3,21 Acquisition of iron Miniferritin SigB tagD 3,17 Biosynthesis of teichoic acid Glycerol-3-phosphate hbs/dbpA/hu bA 2,92 DNA condensation/segregation Non-specific DNA-binding protein Hbsu, DNA packaging LexA and SigH yxxD 2,92 Toxins, antitoxins and immunity Antitoxin yfnC 2,65 Resistance against toxins based on similarity similar to fosfomycin moaE 2,29 Biosynthesis of molybdopterin resistance protein ykvN 2,64 Transcription factors and their control (cofactor) MarR/DUF24 family cpgA/yloQ 2,29 Cell envelope and cell division GTPase transcription regulator against toxins based on similarity ydeH 2,89 Unknown yorM 2,64 SP-beta prophage cheR 2,30 Protein modification MCPs methyltransferase; motility, AbrB mgsR/yqgZ 2,86 Transcription factors and their Transcriptional regulator of a SigB and MgsR ynaB 2,63 chemotaxis Unknown yhaN/sbcE 2,30 DNA repair/ recombination DNA double-strand break repair LexA PhoP and WalR control subset of the SigB general yorE 2,61 SP-beta prophage and competence; SMC-like protein cytidylyltransferase tagC/dinC 3,17 Biosynthesis of teichoic acid LexA stress regulon, required for stress yurQ 2,60 Unknown protection against oxidative ypbS 2,60 Unknown yqeK 2,31 Unknown
						123

regulated genes in ∆ydcH during exponential growth (continuation)

		Down-
	Gene	Expression	Function1	Function2	Regulated by
		difference
	yprA	2,33	DNA repair/ recombination/ based
			on similarity
	yloV	2,34	Phosphoproteins
	fmt/yloL	2,34	tRNA modification and maturation Methionyl-tRNA formyltransferase;
				formylation of Met-tRNA(fMet)
	dnaI/ytxA/dn	2,34	DNA replication	Primosome component (helicase
	aY			loader)
	ykuO	2,34	Unknown		Fur, NsrR, ResD,
					Kre
	aroF	2,34	Biosynthesis, acquisition of amino	Chorismate synthase
			acids
	xtmB/ykxG	2,35	DNA replication	Phage DNA replication, PBSX	Xpf
				terminase (large subunit)
	bglA	2,35	Utilization of beta-glucosides	6-phospho-beta-glucosidase
	motA/mot	2,36	Flagellar proteins	Paralog of MotP	SigD
	resB/ypxB	2,36	Respiration	Part of the ResB-ResC haem	CcpA, PhoP,
				translocase, cytochrome c	ResD
				biogenesis
	rsmB/sun/ylo	2,36	rRNA modification and	S-adenosyl-L-methionine-dependent	SigA
	M		maturation, based on similarity	16S rRNA m5C967
				methyltransferase
	yusY	2,37	Unknown
	spoVT/yabL	2,37	Transcription factors and their	Transcription activator and	SigF, SigG
			control	repressor of SigG-dependent genes
	citZ/citA2	2,38	TCA cycle	Citrate synthase II	CcpA, CcpC
	galK/ipa-35d	2,38	Utilization of galactose	Galactose utilization, galactokinase
	ypgQ	2,38	Unknown	Degradation of excessive or
				abnormal nucleotides
	yukB	2,39	Export of YukE	Membrane FtsK/SpoIIIE-like ATPase,	DegU
				part of the type VII protein secretion
				system
	panB	2,40	Biosynthesis of coenzyme A	3-methyl-2-oxobutanoate
				hydroxymethyltransferase
	yvcQ/psdS	2,41	Protein kinases	Control of psdA-psdB in response to
				lipid II-binding lantibiotics, such as
				nisin and gallidermin
	engA/yphC	2,41	Ribosome assembly	GTPase essential for ribosome 50S
				subunit assembly
	gcvT/yqhI	2,41	Utilization of threonine/ glycine	Glycine utilization	Gly-box
					cooperative-
					riboswitch-
	yqjZ	2,42	Unknown
	ycnE	2,42	Unknown
	crh/yvcM	2,43	Control of transcription factor	Regulating HPr
			(other than two-component
			system)
	yyaE	2,43	Electron transport, based on	Paralogue of YoaE
			similarity
	yuaG/floT/yu	2,44	Membrane dynamics	Membrane-associated scaffold	SigW
	aH			protein, orchestration of
				physiological processes in lipid
				microdomains, involved in the
				control of membrane fluidity,
				confers (together with YuaF)
				resistance to cefuroxime

regulated genes in ∆ydcH during exponential growth (continuation)

		Down-
	Gene	Expression	Function1	Function2	Regulated by
		difference
	queA	2,45	tRNA modification and maturation	S-adenosylmethionine tRNA
				ribosyltransferase
	mdh/citH	2,47	TCA cycle	malate dehydrogenase	CcpA, CcpC
	pdxK/ywdB/i	2,49	Biosynthesis of pyridoxal	Pyridoxine, pyridoxal, and
	pa-52r/thiD		phosphate (cofactor)	pyridoxamine kinase
	yyzE	2,49	Unknown
	tepA/ymfB/yl	2,49	Protein secretion	Degradation of SASPs,orphan	SigG, SpoVT
	xI			ClpP-like germination protease
	tyrA	2,50	Biosynthesis, acquisition of	Prephenate dehydrogenase	TRAP
			aromatic amino acids
	birA	2,50	Biosynthesis of fatty acids	Transcriptional repressor (BirA	Spx
				family)/ biotin-protein ligase
	gmuE/ydhR	2,52	Utilization of glucomannan	Mannose kinase	AbrB, CcpA,
					GmuR
	proB	2,53	Biosynthesis/ acquisition of	Glutamate 5-kinase	T-box
			proline
	gmuD/ydhP	2,53	Utilization of glucomannan	Phospho-beta-mannosidase	AbrB, CcpA,
					GmuR
	ykuN	2,54	Electron transport	Replaces ferredoxin under	Fur, NsrR, ResD,
				conditions of iron limitation,	Kre
				probably involved in electron
				transfer to nitric oxide synthase
	xynD	2,54	Utilization of other polymeric	arabinoxylan degradation	AbrB
			carbohydrates
	ylmE	2,54	biofilm formation	control of the CoA pool	Spo0A
	parE/grlB	2,55	DNA condensation/ segregation	subunit of DNA topoisomerase IV	LexA
	atpF	2,56	ATP synthesis	part of the Fo complex (subunit b)	RelA
	sigF/spoIIAC	2,56	sigma factors	RNA polymerase forespore-	AbrB, SigF, SigG,
				specific (early) sigma factor SigF	SigH, SinR, Spo0A
	yloN	2,57	rRNA modification and	Adenosine methyltransferase for
			maturation, based on similarity	modification of 23S rRNA
	iscU/sufU/yur	2,58	biosynthesis of iron-sulfur clusters iron-sulfur cluster scaffold protein,
	V			receives iron from SufS, synthesis
				of Fe-S clusters
	sucC	2,58	ATP synthesis	TCA cycle	CcpA, RoxS
	valS	2,58	aminoacyl-tRNA synthetases	valyl-tRNA synthetase	T-box, Efp-
					dependent
					proteins
	ytpQ	2,59	Unknown		Spx
	yuaF/NfeD2	2,59	membrane dynamics	NfeD2, role in maintaining	SigW
				membrane integrity during
				conditions of cellular stress,
				confers (together with FloT)
				resistance to cefuroxime
	ylxH/flhG	2,59	motility and chemotaxis/ other	GTPase activating protein,	CodY, DegU,
				activates FlhF, activates assembly	SigD, Spo0A
				of the flagellar C ring, control of
				flagellar basal body position
	yknW	2,60	ABC transporters	modulates assembly of the YknX-	AbrB, SigW
				YknY-YknZ ABC transporter for the
				export of the SdpC toxin
	cca/papS/ypjI	2,63	tRNA modification and maturation	tRNA nucleotidyltransferase,	Spx
				maturation of the single-copy
				tRNACys, which lacks an encoded
				CCA 3 end
	icd/citC	2,64	TCA cycle	isocitrate dehydrogenase	CcpA, CcpC

regulated genes in ∆ydcH during exponential growth (continuation)

		Down-
	Gene	Expression	Function1	Function2	Regulated by
		difference
	mobB	2,66	biosynthesis of molybdopterin	nitrate respiration,
			(cofactor)	molybdopterin-guanine
				dinucleotide cofactor synthesis
				protein
	trpD	2,66	biosynthesis/ acquisition of	biosynthesis of tryptophan,	TRAP
			aromatic amino acids	anthranilate
				phosphoribosyltransferase
	yhbB/ygaQ	2,67	Unknown	spore coat protein, amidase	SigE
	cysH	2,67	sulfur metabolism	sulfate reduction,	CymR, S-box
				phosphoadenosine
				phosphosulfate sulfotransferase
	rseP/rasP/ylu	2,67	cell division	intramembrane protease, cleaves
	C			FtsL, RsiV and RsiW as well as
				signal peptides after release of the
				secreted proteins, control of SigV
				and SigW activity
	gcvPA/yqhJ/g	2,68	utilization of threonine/ glycine	glycine decarboxylase (subunit 1)	Gly-box
	cvP
	yvcR/psdA	2,69	ABC transporters	export of lipid II-binding	PsdR, activated
				lantibiotics, such as nisin and	stat by internal
				gallidermin	toxic peptides
					(Sdp, SkfA)
	xkdE	2,69	PBSX prophage		Xpf
	pfkA/pfk	2,69	glycolysis	6-phosphofructokinase
	pheS	2,70	aminoacyl-tRNA synthetases	phenylalanyl-tRNA synthetase	T-box
				(alpha subunit)
	ycbM	2,71	protein kinases	two-component sensor kinase
	acuB	2,71	Unknown		CcpA
	artQ/yqiY	2,72	ABC transporters	high affinity arginine ABC
				transporter (permease)
	ytaF	2,72	Unknown		SigE
	aroB	2,74	biosynthesis/ acquisition of	3-dehydroquinate synthase
			aromatic amino acids
	trmK/yqfN	2,74	tRNA modification and maturation tRNA:m1A22 methyl transferase
	yszB/pheB/th	2,75	biosynthesis/ acquisition of	control of threonine biosynthesis,
	rR		aromatic amino acids	transcription repressor of
				threonine biosynthetic gene
	yqfA/floA	2,76	cell envelope stress proteins	flottilin-like protein (in addition to	SigW
			(controlled by SigM, V, W, X, Y)	FloT), resistence protein (against
				sublancin), accessory role in
				resistance to cefuroxime
	xtmA/ykxF	2,77	DNA replication prophage	PBSX terminase (small subunit),	Xpf
				phage DNA replication
	xkdQ	2,78	PBSX prophage
	sdhA/citF	2,78	TCA cycle	succinate dehydrogenase	FsrA
				(flavoprotein subunit)
	ynbB	2,79	Unknown
	malL/yvdL	2,80	utilization of starch/ maltodextrin	alpha-glucosidase
	ysmA	2,82	Unknown
	xtrA	2,83	PBSX prophage		Xre
	yrhE	2,84	Unknown
	yxaB	2,85	biofilm formation	biofilm formation, survival of salt	AbrB, SigB
				and ethanol stress

regulated genes in ∆ydcH during exponential growth (continuation)

		Down-
	Gene	Expression	Function1	Function2	Regulated by
		difference
	gmuF/ydhS	2,86	utilization of glucomannan	mannose-6-P-isomerase	AbrB, CcpA,
					GmuR
	exuM/yjmB	2,86	transporters/ other		CcpA, ExuR
	bioW	2,86	biosynthesis/ acquisition of biotin	pimeloyl-CoA synthase	BirA
	xepA/xkdY	2,86	cell wall degradation/ turnover *	phage release, PBSX prophage
			autolysis	lytic exoenzyme
	thiU/ykoF	2,88	ABC transporters	thiamine transporter (binding	Thi-box
				protein), uptake
	truA/ybaH	2,88	tRNA modification and maturation	pseudouridylate synthase I	stringent
					response, RelA
	purH/purJ	2,90	biosynthesis/ acquisition of purine	phosphoribosylaminoimidazole	G-box, PurR
			nucleotides	carboxamide formyltransferase
				and inosine-monophosphate
				cyclohydrolase
	gmuA/ydhN	2,91	phosphotransferase systems	glucomannan-specific	AbrB, CcpA,
				phosphotransferase system, EIIA	GmuR
				component
	folC	2,91	biosynthesis of folate	folyl-polyglutamate synthetase	T-box
	sucD	2,91	ATP synthesis	succinyl-CoA synthetase (alpha	CcpA
				subunit)
	hisH	2,93	biosynthesis/ acquisition of	imidazole glycerol phosphate
			histidine	synthase (glutaminase subunit)
	prpC/yloO	2,95	protein phosphatases	antagonist of PrkC-dependent
				phosphorylation
	atpH	2,96	ATP synthesis	ATP synthase (subunit delta)	stringent
					response, RelA
	yvrO	2,98	ABC transporters		AbrB, ComK
	treA/treC	2,98	utilization of trehalose	phospho-alpha-glucosidase	CcpA, PhoP, TreR
	xkdG	2,98	PBSX prophage		Xpf
	nupC	3,02	transporters/ other	pyrimidine nucleoside transport	CcpA, DeoR
				protein
	mutM/ytaE	3,04	DNA repair/ recombination	formamidopyrimidine-DNA
				glycosidase
	msmE	3,05	ABC transporters		CcpA
	hisA	3,07	biosynthesis/ acquisition of	phosphoribosylformimino-5-
			histidine	aminoimidazole carboxamide
				ribotide isomerase
	xkdF	3,07	PBSX prophage		Xpf
	xkdJ	3,08	PBSX prophage
	gmuC/ydhO	3,08	phosphotransferase systems	glucomannan-specific	AbrB, CcpA,
				phosphotransferase system, EIIC	GmuR
				component
	rbsA	3,09	ABC transporters	ribose ABC transporter (ATP-	AbrB, CcpA
				binding protein), uptake
	fadF/ywjF	3,10	utilization of fatty acids	FA degradation	FadR
	iolE/yxdE	3,11	utilization of inositol	myo-inositol catabolism, 2-keto-	CcpA, IolR
				myo-inositol dehydratase,
				dehydration of 2-keto-myo-
				inositol (2nd reaction)
	xkdH	3,12	PBSX prophage		Xpf
	xhlA	3,13	PBSX prophage	host cell lysis upon induction of
				PBSX
	xkdS	3,13	PBSX prophage

regulated genes in ∆ydcH during exponential growth (continuation)

		Down-
	Gene	Expression	Function1	Function2	Regulated by
		difference
	thiQ/ylmB	3,14	biosynthesis/ acquisition of	thiamine salvage, N-formyl-4-	Thi-box
			thiamine (cofactor)	amino-5-aminomethyl-2-
				methylpyrimidine deformylase
	glcK/yqgR	3,14	utilization of trehalose	phosphorylation of the free
				glucose moiety resulting from
				cleavage of di-and
				oligosaccharides, glucose kinase
				(D-glucose:ATP)
	xkdI	3,14	PBSX prophage		Xpf
	acuA	3,19	utilization of organic acids	protein acetylase for the control	CcpA
				of AcsA activity, Gcn5-related N-
				acetyltransferase
	yxiI	3,20	Unknown
	xkdX	3,21	PBSX prophage
	etfB	3,22	electron transport/ other	fatty acid degradation,	CcpA, FadR
				flavoprotein (beta subunit)
	yhcF	3,26	transcription factor/ other/ based
			on similarity
	trpF	3,26	biosynthesis/ acquisition of	synthesis of tryptophan,	TRAP
			aromatic amino acids	phosphoribosylanthranilate
				isomerase
	xkdR	3,30	PBSX prophage
	iolD/yxdD	3,42	utilization of inositol	myo-inositol catabolism,	CcpA, IolR
				formation of 5-deoxy-D-glucuronic
				acid (3rd reaction)
	thiS/yjbS	3,43	biosynthesis/ acquisition of	sulfur carrier protein	Thi-box
			thiamine (cofactor)
	rocG	3,43	utilization of arginine/ ornithine	arginine utilization, controls the	AbrB, AhrC,
				activity of GltC, trigger enzyme:	CcpA, RocR, SigL
				glutamate dehydrogenase (major)
	xkdC/ykxC	3,43	PBSX prophage		Xre
	lplA	3,44	ABC transporters
	xhlB/xpaB	3,45	PBSX prophage	host cell lysis
	mtlD/mtlB	3,49	utilization of mannitol	mannitol-1-phosphate 5-	MtlR
				dehydrogenase
	pyrF	3,63	biosynthesis/ acquisition of	pyrimidine biosynthesis, orotidine-	PyrR
			pyrimidine nucleotides	5-phosphate decarboxylase
	xkdW	3,64	PBSX prophage
	xkdK	3,65	PBSX prophage		Xpf
	yulD/rhaM	3,68	Unknown	utilization of rhamnose,	CcpA, RhaR
				mutarotase
	hisB	3,70	biosynthesis/ acquisition of	imidazoleglycerol-phosphate
			histidine	dehydratase
	bioF	3,70	biosynthesis/ acquisition of biotin	8-amino-7-oxononanoate	BirA
			(cofactor)	synthase
	iolC/yxdC	3,74	utilization of inositol	myo-inositol catabolism,	CcpA, IolR
				formation of 2-deoxy-5-keto-
				gluconic acid-6-phosphate (5th
				reaction)
	xkdB/ykxB	3,77	PBSX prophage		Xre
	iolG/idh/iol	3,79	utilization of inositol	myo-inositol catabolism, inositol	CcpA, IolR
				2-dehydrogenase

regulated genes in ∆ydcH during exponential growth (continuation)

		Down-
	Gene	Expression	Function1	Function2	Regulated by
		difference
	lrgA/ysbA	3,79	Unknown		Kre, CcpA, LytT
	yxiM	3,80	Unknown
	kdgK	3,84	utilization of hexuronate	utilization of galacturonic acid, 2-	CcpA, KdgR
				dehydro-3-deoxygluconokinase
	tenI	3,97	biosynthesis/ acquisition of	thiazole tautomerase	Thi-box
			thiamine (cofactor)
	bglH	3,98	utilization of beta-glucosides	salicin utilization, phospho-beta-	CcpA, LicT
				glucosidase
	citT/yflQ	3,99	utilization of organic acids	two-component response	CitT
				regulator, regulation of citrate
				uptake
	xkdD/ykxD	4,07	PBSX prophage		Xre
	yjmC	4,10	may be involved in galacturonate		CcpA, ExuR, SigE
			utilization
	mtlA	4,18	phosphotransferase systems	trigger enzyme: mannitol-specific	MtlR
				phosphotransferase system, EIICB
				of the PTS; mannitol uptake and
				phosphorylation, control of MtlR
				activity
	licB/celA	4,30	phosphotransferase systems	trigger enzyme: lichenan-specific	CcpA, LicR
				phosphotransferase system, EIIB
				component of the PTS; lichenan
				uptake and phosphorylation,
				control of LicR activity
	yesM	4,38	protein kinases	two-component sensor kinase	CcpA
	mccB/yrhB	4,43	biosynthesis/ acquisition of	cystathionine lyase/ homocysteine	CymR, Spx
			cysteine	gamma-lyase; methionine-to-
				cysteine conversion
	mccA/yrhA	4,51	biosynthesis/ acquisition of	O-acetylserine-thiol-lyase,	CymR, Spx
			cysteine	methionine-to-cysteine
				conversion
	araL/yseA	4,58		detoxification of accidental	AraR, CcpA
				accumulation of phosphorylated
				metabolites; sugar phosphate
				phosphatase
	levG	4,59	phosphotransferase systems	fructose-specific	CcpA, LevR, SigL
				phosphotransferase system EIID
				component of the PTS; fructose
				uptake and phosphorylation
	iolB/yxdB	4,64	utilization of inositol	myo-inositol catabolism;	CcpA, IolR
				formation of 2-deoxy-5-keto-
				gluconic acid (4th reaction)
	mdxE/yvdG	4,71	ABC transporters	maltodextrin ABC transporter,
				binding protein
	ykzM	4,73	PBSX prophage
	rbsD	5,13	ABC transporters	ribose uptake	AbrB, CcpA
	ykzK	5,55	PBSX prophage
	levD	6,11	phosphotransferase systems	fructose-specific	CcpA, LevR, SigL
				phosphotransferase system, EIIA
				component of the PTS, fructose
				uptake and phosphorylation
	mtlF	6,38	phosphotransferase systems	phosphotransferase system(PTS)	MtlR
				mannitol-specific enzyme IIA
				component

regulated genes in ∆ydcH during exponential growth (continuation)

			Down-
	Gene	Expression	Function1	Function2	Regulated by
		difference
	sat/ylnB	6,52	sulfur metabolism	sulfate activation, sulfate	CymR, S-box
				adenylyltransferase
	cysC/ylnC	7,16	sulfur metabolism	sulfate reduction and activation,	CymR, S-box
				adenylyl-sulfate kinase
	yxeK	7,19	Unknown	sulphur metabolism	CymR
	levF	7,25	phosphotransferase systems	fructose-specific	CcpA, LevR, SigL
				phosphotransferase system, EIIC
				component of the PTS, fructose
				uptake and phosphorylation
	levE	7,43	phosphotransferase systems	fructose-specific	CcpA, LevR, SigL
				phosphotransferase system, EIIB
				component of the PTS, fructose
				uptake and phosphorylation
	yxeN	8,86	ABC transporters	sulphur metabolism	CymR
	yxeL/snaB	9,47	ABC transporters	sulphur metabolism	CymR
	sumT/ylnD	9,87	biosynthesis of heme/ siroheme	siroheme biosynthesis , sulfite	CymR, S-box
				reduction
	ydcH	13,68	Unknown
	acpK	14,40	biosynthesis of antibacterial	polyketide biosynthesis, acyl	AbrB, CodY
			compounds	carrier protein
			Overexpressed genes in ∆ydcH during stationary growth
	Gene	Expression	Function1	Function2	Regulated by
		difference
	ydcF	6,50	Unknown
	ywzD	6,29	Unknown
	ywdA/ipa-	4,79	Unknown		CcpA, SacT
	51d
	ydfQ	4,19	electron transport/ other/ based
			on similarity
	sacA/ipa-50d	3,26	utilization of sucrose	phosphosucrase
	ydcG	3,18	Unknown
	albB/ywhR	3,18	biosynthesis of antibacterial	antilisterial bacteriocin (subtilosin)	AbrB, ResD, Rok
			compounds	production
	albE/ywhO	2,80	biosynthesis of antibacterial	antilisterial bacteriocin (subtilosin)	AbrB, ResD, Rok
			compounds	production; processing protease
	albC/ywhQ	2,77	biosynthesis of antibacterial	export of antilisterial bacteriocin	AbrB, ResD, Rok
			compounds	(subtilosin); ABC transporter
	albA/ywiA	2,71	biosynthesis of antibacterial	antilisterial bacteriocin (subtilosin)	AbrB, ResD, Rok
			compounds	production; radical S-
				adenosylmethionine enzyme
	albD/ywhP	2,65	biosynthesis of antibacterial	export of antilisterial bacteriocin	AbrB, ResD, Rok
			compounds	(subtilosin); ABC transporter;
				membrane protein
	albF/ywhN	2,57	biosynthesis of antibacterial	antilisterial bacteriocin (subtilosin)	AbrB, ResD, Rok
			compounds	production
	yflI	2,39	Unknown
	rsbRD/yqhA	2,17	control of sigma factors	probably part of the stressosome,
				negative regulator of SigB activity
	ywzB	2,17	Unknown
	albG/ywhM	1,81	biosynthesis of antibacterial	antilisterial bacteriocin (subtilosin)	AbrB, ResD, Rok
			compounds	production; U
	yozB	1,71	Unknown

regulated genes in ∆ydcH during stationary growth

	Gene	Expression	Function1	Function2	Regulated by
		difference
	fadN/yusL	2,79	utilization of fatty acids	fatty acid degradation	CcpA, FadR, SdpR
	yoqL	3,11	SP-beta prophage
	yonT	3,29	toxins, antitoxins and immunity	Toxin	as-yonT
			against toxins/ based on similarity		(antisense RNA)
	yotM/yodV	3,45	SP-beta prophage
	yoqY	3,52	SP-beta prophage
	yoqR	4,21	SP-beta prophage
	iolT/ydjK	4,23	transporters	major transporter of inositol; myo-	IolR
				inositol uptake
	yoqK	5,43	SP-beta prophage
	ydcQ/conQ	5,72	Prophages and mobile genetic	conjugative transfer of ICEBs1,
			elements	coupling protein, part of the type
				IV secretion system for DNA
				transfer
	yfnG	5,93	sporulation		GerE, SigK
	ykzV	57,58	Unknown
	ydcH	114,98	Unknown

* Resistance gene abbreviations: neo, neomycin; kan, kanamycin; spc, spectinomycine; cat, chloramphenicol; erm, erythromycin. Other abbreviations: ∆, deletion; Ω, insertion. ** ΔmreB strain 3725. X → Z depicts construction procedure, where X could be plasmid or chromosomal DNA and Z is the recipient strain transformed by X.

Acknowledgements

I would like to thank all the people whose contribution, advice and support made the realization of this work possible. I would also like to acknowledge the following people, without whom, this adventure wouldn't have been possible:

-Dr. Arnaud Chastanet for his way of making everything seem like a game and his enthusiasm. But

Agarose gel electrophoresis of DNA fragments:

Agarose gel electrophoresis was performed in 1 % (w/v) agarose gels containing 1 % (v/v) ethidium bromide (Euromedex) in 1X TBE buffer. DNA samples were mixed at a 6:1 ratio with DNA loading buffer (30 % (v/v) glycerol, 0.25 % (w/v) bromophenol blue, 0.25 % (w/v) xylene cyanol FF) prior to loading. The voltage used for electrophoresis was 120 V.

Purification of DNA:

-DNA fragments:

Following PCR reactions or subsequent enzymatic reactions, DNA fragments are purified using the QIAquick PCR Purification Kit (QIAGEN), according to manufacturer's recommendations.

-Plasmids: Plasmids are purified from E. coli strains containing it using a QIAprep Spin Miniprep Kit (QIAGEN), according to manufacturer's recommendations.

Estimation of DNA concentration:

Concentration of DNA in solution was determined by the use of a NanoDrop spectrophotometer (Thermo Scientific). The blank used was pure water. DNA solutions were assumed to be largely free of contaminating proteins if the ratio of absorbance at 260:280 nm was > 1.6.

Isothermal assembly of DNA fragments:

Isothermal assembly was performed mainly as described in [START_REF] Gibson | Enzymatic assembly of DNA molecules up to several hundred kilobases[END_REF] with minor modifications. In brief, DNA fragments are amplified adding homologous regions t their extremities, a 5' exonuclease creates long overhangs, a polymerase fills in the gaps of the annealed single strand regions, and a DNA ligase seals the nicks of the annealed and filled-in gaps.

2.4.1.8 General strategy for sub-cloning DNA fragments into plasmids in

E. coli:

PCR-generated DNA fragments were purified as in 2.4.1.5, restriction digests were performed as per the manufacturers recommendations followed by dialysis against ddH 2 O for 30 min.

Dephosphorylation was performed with SAP (shrimp alkaline phosphate) as per manufacturers recommendations to then perform a ligation for 1 h at RT and transformation in competent DH5α increases all along the exponential growth phase. Its maximal expression occurs during the transition from exponential to stationary phases (Figure 17). We decided to compare the expression between both strains when the differences should be maximum, before and after the transition, in exponential (OD 600 nm ≈ 0.2) and stationary (OD 600 nm ≈ 2-2.3) phases of growth. With a threshold of 2x difference in gene expression, we observed a total of 364 affected genes during exponential phase, 180 de-repressed and 184 down-regulated genes in ∆ydcH (Figure 18;

Appendix 3). During stationary phase, a different set composed of only a limited number of genes were differentially expressed (13 overexpressed and 11 down-regulated genes). As expected, ydcH appears as one of the most repressed genes both during stationary and exponential growth.

Appendix 1: Phenotypic analysis of ydcFGH

Note to readers: the following experiments were realized with two marker-less ydcF and ydcG mutants and a deletion-replacement mutant of ydcH. The two marker-less mutants were shown lately to have a different genetic background than the reference wild-type strain. As such, the present results can't be interpreted as the result of the deletions and for this reason were removed from the main text.

They are presented here as purely informational regarding the work produced during the PhD.

Following the construction ydcF, ydcG and ydcH mutants, a phenotypic analysis of these genes was undertaken by studying the effects of their deletion on a number of conditions: growth in different media, sporulation and competence. We also verified if they had morphological defects as they may be related to the morphogene mreB.

A1.1 ydcF, -G and -H deletion mutants are not impaired for cell morphology

MreB is a morphogene; when deleted, cells start to swell and deform until they lyse. As the expression of ydcFGH is activated in absence of MreB, we thought that the three genes might have a role in cell morphology maintenance. Deletion mutants of each of the genes were grown in MSM and CH media at 37 °C. Cell width were measured on phase contrast images acquired during exponential growth of these strains in MSM and both during exponential growth and stationary phase in CH media (Figure A1.1). In poor MSM medium a relatively high variability in cell width was observed and no significant differences between strains could be detected. However, a significantly thinner width was observed for ydcF and ydcG mutants compared to WT and ∆ydcH strains when grown in rich CH medium both in exponential and stationary phase.

A1.2. Defects during stationary phase

We next tested the deletion mutants for potential growth defect in various conditions. Extensive growths were recorded, from exponential to late stationary phase, in a set of complex and defined medium (see materials and methods for details). As shown on Figure A1.2, optical density of ∆ydcF and ∆ydcG cultures decrease during stationary phase in CH and MSM media, suggesting cell lysis.

Note that this pattern is very similar to that observed with ∆mreB strain in CH (see results chapter MreB mutagenesis). Then, we wondered if cell viability could be affected during stationary phase. To test this, we measured colony forming units of samples harvested at different points during cell growth: exponential, entry to stationary phase and late stationary phase (see materials and methods; Figure A1.3). Unfortunately, the accuracy of this approach was not sufficient to detect the difference observed with OD measurement.

Appendix 2: The absence of MreB is not responsible for P ydc1 induction

Note to readers: the study of the ydcFGH operon was initiated because of its induction in the ∆mreB background, suggesting a regulatory link between these two loci. After a long -genetic-hunt, we concluded that the induction of ydcFGH was in fact unlinked to mreB, and caused by one of the many mutations present, to our surprise, in strain 3725, most probably a frameshift in ydcH. We decided to present in the main text only the conclusion to this search. Here are presented in detail the various hypotheses and experiments realized that drove us to this unexpected result.

As described on the results chapter ydcFGH, the reporter P ydc1 lacZ was not expressed on a WT context (strain ABS1990) and showed an increase in absence of mreB (strain ABS2054). As a reminder, the mreB gene is present in a three gene operon with two other essential genes, mreC and mreD. A whole genome transcriptional study from Nicolas et al. [START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF] also suggests the existence of longer transcripts originating from the maf promoter and going to the downstream minCD operon (Figure A3.1A).

A2.1. Absence of mreB complementation is not due to chromosomal positioning of the gene

To our surprise, we first noticed that an ectopic complementation at the amyE locus by either an inducible copy of mreB fused to gfp (amyE::P xyl gfpmreB; strain ASEC9) or the mreB gene under its natural promoter (amyE::P N mreB; strain ASEC16) failed to restore the repression of P ydc1 (Figure A3.1B.I). We then tested the possibility that the failed complementation could be due to the position of the ectopic locus (amyE) on the chromosome, potentially affecting the levels of MreB. Indeed, amyE is situated close to the origin of replication (position 327 618-329 597), oriC, while mreB is located at position 2 861 748-2 860 735. It is known that due to multiple replication forks, genes closer to oriC are in higher copy number than the ones close to the terminus, a property exploited by cells to adjust protein levels [START_REF] Slager | Hard-wired control of bacterial processes by chromosomal gene location[END_REF]. To test this hypothesis, we expressed mreB from another locus, bkdB, a gene positioned very near mreB in the B. subtilis chromosome (position 2 498 070-2 496 796). Again, when mreB was expressed alone, the complementation failed and P ydc1 was overexpressed (Figure A3.1B.II; strains ASEC234 and ASEC236). A preferred target of antibacterial compounds is the bacterial wall. This wall is like a shell, protecting them. But as such, it also constitutes their Achilles heel as they can't survive without it. This project aims to understand how this wall is assembled by studying the molecular machines that makes it, and in particular parts of these machines (proteins), which functions are still unknown. I also focus on how other functions of the cell are altered when these proteins are defective.