Deciphering the assembly pathway of type IV pili in Myxococcus xanthus

Type IV pili (T4P) are hairlike surface structures, present on a variety of different bacteria. They are polymers involved in diverse functions such as motility, adherence, protein secretion, DNA uptake and in many pathogens they are found to be the primary colonization factor. Especially their role in virulence makes T4P particularly relevant for studying pilus function and assembly. The T4P machinery consists of 12 conserved proteins building an envelope-spanning macromolecular machinery, which localizes polarly in Myxococcus xanthus. Although most of the proteins have been known and studied for a long time, the precise mechanism of how and in which order the individual components are assembled to generate a macromolecular machinery remain largely unknown. Here we uncovered a sequential, outside-in assembly pathway starting with the outer membrane (OM) PilQ secretin, and proceeding inwards over the periplasm and inner membrane (IM) to the cytoplasm. Specifically, by taking advantage of the cell biology tools for studying T4P in M. xanthus, we carried out one of the largest screens comprising 11 of the 12 proteins of the T4P machinery by systematically profiling the stability and localization of T4P proteins in the absence of each individual other T4P protein in combination with mapping direct protein-protein interactions. Using these approaches, we show that assembly of the T4P machinery initiates with the formation of the PilQ secretin ring, assisted by its pilotin Tgl, in the OM. Oligomeric PilQ serves as an assembly platform for further T4P components. PilQ recruits TsaP, a peptidoglycan binding protein, as well as PilP by direct interactions with PilP. PilP, in turn, recruits the IM proteins PilN and PilO. PilP/PilO/PilN likely make up a complex aligning IM and OM components of the T4P machinery. The PilP/PilO/PilN complex recruits cytoplasmic PilM by direct interaction between PilN and PilM and recruits PilC, presumably by direct interaction between PilC and PilO. Finally, the ATPases PilB and PilT that power extension and retraction of T4P, localize independently of other T4P machinery proteins. In this study, we elucidate the assembly process and functional interactions between T4P proteins. This work lays the basis for further understanding of these functionally highly versatile surface structures. Interestingly, the assembly of the type II and III secretion systems also initiates from the OM secretin and proceeds inwards. Thus, an outside-in assembly pathway is emerging as a conserved feature in secretin-containing trans-envelope export machines

Regulation of peptidoglycan biosynthesis in Hyphomonas neptunium

Abstract The spatial and temporal regulation of peptidoglycan biosynthesis and its role in cell morphology has been studied intensively in well-characterized model organisms such as Escherichia coli, Bacillus subtilis, and Caulobacter crescentus, which divide either by symmetric or asymmetric binary fission. To broaden our knowledge of the mechanisms governing bacterial morphogenesis, we started to investigate the dimorphic marine α-proteobacterium Hyphomonas neptunium as a new model organism. This Gram-negative species is characterized by a unique mode of proliferation, whereby the new offspring is generated by the formation of a bud at the tip of a stalk that emanates from the mother cell body. The main focus of our previous studies was the identification of cell wall biosynthetic enzymes and regulatory factors that are critically involved in stalk and bud biogenesis. These studies revealed that peptidoglycan biosynthesis in H. neptunium is a complex process mediated by an intricate interplay of various factors. Among the open questions, it is still unknown how the generation of the daughter cell is regulated and how the mother cell orchestrates the localization of peptidoglycan remodeling enzymes at specific site of action during the cell cycle. Consequently, that includes the initial localization of enzymes at the stalked pole. At a certain point, they have to diffuse through the stalk into the growing bud. There, they center at the junction between the bud and the stalk to separate the mother cell from the bud. The main goal of the present study our current research is a deeper and more thorough characterization of previously investigated peptidoglycan remodeling enzymes, and especially the lytic enzymes, that cleave the peptidoglycan mesh. We particularly focused on two classes, the M23 metallopeptidases and the amidases. In doing so, we compre-hensively analyzed the six M23 endopeptidases of H. neptunium with localization studies and genetic approaches. Our results revealed a high degree of redundancy among these enzymes, which combined with the absence of a distinct localization pattern, indicated a generalized role in cell wall maintenance. We also investigated the role of the only amidase in H. neptunium in cell separation and bud formation. A deletion of the amidase gene led to an aberrant morphology and a mild chaining phenotype. Importantly, we showed that one of the M23 endopeptidases (LmdE) acts as a regulator of AmiC. Using biochemical approaches, we proved an interaction between AmiC and LmdE, where LmdE stimulates the catalytic activity of AmiC and thus regulates peptidoglycan hydrolysis. A further crucial player in this system is the inner membrane-embedded FtsEX complex. A deletion of the whole complex resulted in cells with very elongated and misshapen stalks. Probably, FtsEX plays a role in the regulation of amidase activity by interacting with LmdE. These results are similar between α- and γ-proteobacteria indicating that the mechanism of amidase regulation is conserved. A further goal of our work was the identification of novel factors that are specifically involved in the regulation of budding in H. neptunium. To this end, we started to establish a transposon mutagenesis system to identify all essential genes in this species. In the future, we will be able to investigate these novel factors and their contribution to cell morphology. Taken together, these results provide insight into the mechanisms of morphogenesis in stalked budding bacteria, thus setting the stage for an in-depth analysis of the regulatory mechanisms that control the spatiotemporal dynamics of the peptidoglycan biosynthetic machinery in these organisms

The Type IV Pilus Secretin BfpB: Structural Analysis and Binding Interactions

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. The type IV pilus (T4P) of EPEC, known as the bundle-forming pilus (BFP), plays an important role in EPEC pathogenesis. T4Ps are a family of surface appendages that are important for adhesion, colonization, biofilm formation, virulence, twitching motility and many other functions. One essential component of the BFP system is the secretin, BfpB. Secretins are a large family of integral outer membrane proteins found in T4Ps as well as type II and type III secretion systems, and filamentous phages. Details of the secretin structure have been limited to the overall shape, with atomic resolution of only the soluble amino-terminus domains, which impede our understanding of T4P biogenesis. The goals of this project are: 1) determine the structure of BfpB via cryo-electron microscopy; 2) define the amino-terminus domains of BfpB and its interactions with BfpU, an essential periplasmic protein of the system. We present a 7.1 Å resolution cryo-EM structure of BfpB, the first of a type IVb pilus secretin. Internal features suggest that BfpB is composed of 17 subunits with C17 symmetry. Structural and bioinformatic analyses suggests that monomeric BfpB possesses two amino-terminal domains, N0 and N3, which allowed us to successfully purify BfpB N0 and N0+N3 domains for interaction studies. Additionally, we have successfully purified BfpU for structure determination and interaction studies. Also, a random mutagenesis approach was used for further characterization of BfpU. Furthermore, surface plasmon resonance suggests the possibility that BfpB and BfpU interact with affinity in the micromolar range, but this result must be interpreted cautiously in light of similar interactions between BfpU and proteins chosen as negative controls. Results from these studies will not only further our understanding of BFP biogenesis, but also enhance research for understanding other T4Ps and secretion systems that are confirmed virulence factors

Insights into assembly of the type IVa pilus machine in Myxococcus xanthus

Typ IVa pili (T4aP) sind weiteverbreitete und vielseitige, bakterielle Zelloberflächen Strukturen, die zu Motilität, Adhesion, Biofilm Bildung und Virulenz beitragen. Ihrer Funktionsweise basiert auf der Eigenschaft Zyklen von Verlängerung/Adhäsion/Rückzug zu durchlaufen, die von einer Zellhülle umspannenden T4aP Maschine angetrieben werden. Der Aufbau der T4aP Maschine in Myxococcus xanthus erfolgt von außen nach innen und beginnt mit dem Einbau des secretins PilQ in die äußere Membran, welches dann periplasmatische Protein und Komponenten aus der inneren Membran und dem Zytoplasma rekrutiert. Zusätzlich initiiert ein Komplex aus Minor Pilinen und PilY1 die Verlängerung von T4aP und befindet sich ebenfalls an der Spitze des Pilus, wo der Komplex Adhäsion ermöglicht. Hier fokussieren wir uns auf den Aufbau der bipolaren T4aP Maschine in dem stäbchenförmigen Bakterium M. xanthus. Das Genom von M. xanthus codiert für drei Sätze von Minor Pilinen und PilY1. Hierbei haben wir festgestellt, dass eines dieser Gen Cluster ein nicht-kanonisches Cytochrom c enthält, welches wir TfcP genannt haben. Während TfcP ein unüblich niedriges Redox-Potential besitzt, welches eine Funktion im Stoffwechsel unwahrscheinlich macht, ist TfcP bedingt essenziell für T4aP abhängige Motilität, weil es die Akkumulation von PilY1.1 in Gegenwart von niedrigen Calcium Konzentrationen erlaubt. Wir schlagen vor, dass TfcP die Spanne an Calcium Konzentrationen, in denen PilY1.1 funktional ist, erweitert und dadurch dessen Funktion robuster gegeben über Änderungen in der Umwelt macht. Als nächstes untersuchen wir den Aufbau neuer T4aP Maschinen an den neuen Zellpolen nach der Zellteilung. Wir zeigen, dass PilQ während der Zytokinese an den entstehenden Polen rekrutiert wird, aber hauptsächlich nach Abschluss der Zytokinese an den neuen Polen rekrutiert wird. Diese Rekrutierung hängt von den peptidoglykanbindenden AMIN-Domänen von PilQ ab, und wir schlagen vor, dass dieser Mechanismus für Secretins mit AMIN-Domänen im Allgemeinen gilt. Darüber hinaus rekrutiert PilQ vorübergehend das Pilotin Tgl an den entstehenden und neuen Polen, was dann die Multimerisierung von PilQ in der äußeren Membran induziert. Wir vermuten, dass die vorübergehende Interaktion zwischen PilQ und Tgl durch die ungefaltete β-Lippe von PilQ vermittelt wird, die in die äußere Membran integriert ist. Zusätzlich haben wir herausgefunden, dass die Diguanyletcyclase DmxA für den symmetrischen Aufbau des T4aP-Mechanismus an den neuen Zellpolen nach der Zytokinese wichtig ist. In Abwesenheit von DmxA zeigen die Zellen eine fehlerhafte Zellpolarität und eine sehr heterogene polare Lokalisierung des T4aP-Machine. DmxA wird kurz vor Abschluss der Zytokinese von Komponenten des Divisoms an der Teilungsstelle rekrutiert und erhöht rasch den zellulären c-di-GMP-Spiegel. Wir vermuten, dass dieser Anstieg von c-di-GMP die symmetrische Bildung/Verteilung von polaren Landmarken an den neuen Zellpolen reguliert. Schließlich haben wir ein detailliertes Protokoll für die Anwendung des auf MiniTurboID basierenden Proximity Labelings in M. xanthus erstellt. Wir wenden dieses Protokoll auf den Regulator MglA an und nutzen das Proximity Labeling, um das bedingte Interaktom von MglA zu vergleichen

Characterization of CETA and CETB, Energy Taxis Regulators in Campylobacter jejuni.

Energy taxis is the ability of microbes to alter their direction of motility in response to changes in their local environment that affect energy-generating processes. The energy taxis receptor Aer, in Escherichia coli, senses changes in the electron transport system via an FAD cofactor bound to a PAS domain. The PAS domain is thought to interact directly with another Aer domain, the HAMP domain, to transmit a signal to the conserved signaling domain found in chemotaxis receptors. This work focused on an energy taxis system in Campylobacter jejuni composed of two proteins, CetA and CetB, each sharing specific domains with Aer. CetB has a PAS domain; CetA has a predicted transmembrane region, HAMP domain and signaling domain. We examined the expression of cetA and cetB, as well as the biochemical properties of the proteins they encode. cetA and cetB are co-transcribed independently of the flagellar regulon. Both CetA and CetB localize to the membrane and participate in complexes, including a likely CetB dimer and a complex that may include both CetA and CetB. HAMP domains, found in many bacterial signal transduction proteins, generally transmit an intramolecular signal between an extracellular sensory domain and an intracellular signaling domain. Studies of HAMP domains in proteins where both the input and output signals occur intracellularly are limited to Aer. The CetA HAMP domain differs significantly from that of Aer in predicted secondary structure. Similarity searches identified 55 pairs of HAMP/PAS proteins encoded by adjacent genes in a diverse group of microorganisms. We propose that these HAMP/PAS pairs form a new family of bipartite energy taxis receptors. Additionally, CetA contributes to C. jejuni invasion of human epithelial cells, while CetB does not, suggesting that members of HAMP/PAS pairs may act independently of each other. This work provides a framework for future studies into the molecular mechanism of signal transduction within CetA and CetB. Further, these studies suggest that the CetA/CetB system may be a widespread alternative to Aer with additional functional flexibility arising from a capacity for each protein to act independently to regulate traits other than energy taxis.Ph.D.Microbiology & ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/60684/1/ktyoung_1.pd

Activator-induced conformational changes regulate division-associated peptidoglycan amidases

AmiA and AmiB are peptidoglycan-hydrolyzing enzymes from Escherichia coli that are required to break the peptidoglycan layer during bacterial cell division and maintain integrity of the cell envelope. In vivo, the activity of AmiA and AmiB is tightly controlled through their interactions with the membrane-bound FtsEX–EnvC complex. Activation of AmiA and AmiB requires access to a groove in the amidase-activating LytM domain of EnvC which is gated by ATP-driven conformational changes in FtsEX–EnvC complex. Here, we present a high-resolution structure of the isolated AmiA protein, confirming that it is autoinhibited in the same manner as AmiB and AmiC, and a complex of the AmiB enzymatic domain bound to the activating EnvC LytM domain. In isolation, the active site of AmiA is blocked by an autoinhibitory helix that binds directly to the catalytic zinc and fills the volume expected to accommodate peptidoglycan binding. In the complex, binding of the EnvC LytM domain induces a conformational change that displaces the amidase autoinhibitory helix and reorganizes the active site for activity. Our structures, together with complementary mutagenesis work, defines the conformational changes required to activate AmiA and/or AmiB through their interaction with their cognate activator EnvC

The protein-conducting channel SecYEG

In bacteria, the translocase mediates the translocation of proteins into or across the cytosolic membrane. It consists of a membrane embedded protein-conducting channel and a peripherally associated motor domain, the ATPase SecA. The channel is formed by SecYEG, a multimeric protein complex that assembles into oligomeric forms. The structure and subunit composition of this protein-conducting channel is evolutionary conserved and a similar system is found in the endoplasmic reticulum of eukaryotes and cytoplasmic membrane of archaea. The ribosome and other membrane proteins can associate with the protein-conducting channel complex and affect its activity or functionality. (C) 2004 Elsevier B.V. All rights reserved

The conjugative system encoded by the integrative conjugative element ICEclc in Pseudomonas putida

Integrative and conjugative elements (ICE) are a widespread class of mobile genetic elements, which play an important role for bacterial evolution and adaptation to new niches. ICE transfer between cells requires a multi-subunit protein complex known as type IV secretion system (T4SS) which is usually self-encoded. Although several T4SS have been discovered and characterized in the years, they mostly belong to mobile plasmids and ICE-encoded T4SS remain severely understudied. Here we focused on the T4SS encoded by the ICEclc element first found in Pseudomonas knackmussii B13 in two identical copies. The core region of ICEclc, which includes the genetic locus encoding for the T4SS, is conserved among several β- and γ-proteobacteria, thus understanding peculiarities about this conjugation system would be representative for a large number of mobile elements. In this work we mainly focused on the extensive characterization of the ICEclc T4SS genetic locus with a combination of ‘in silico’, molecular biology and microscopy approaches. The first chapter introduces the theme of horizontal gene transfer with a closer look at integrative and conjugative elements characteristics, evolutionary importance and DNA conjugation mechanism with in depth description of the up to date knowledge in T4SS classification and structural composition. In the second chapter we present the general characterization of the 24 genes of the ICEclc T4SS locus. With bioinformatics analysis we inferred homologies between ICE encoded and known T4SS components. Single gene knockouts of 22 out of the 24 open reading frames were used to understand their essentiality for ICE transfer. To better understand T4SS localization at single cell level, we fused nine predicted T4SS subunits to fluorescent proteins and studied their cellular localization. By coupling fluorescent labelling and gene deletions we showed possible interactions between several T4SS subunits and we proposed a dynamic model of T4SS assembly. The third chapter focuses on the characterization of the conjugative pilus, one of the four major protein subassemblies of T4SS. With protein 3D structure prediction, we identified the gene encoding the pilin in ICEclc, being orf66625. With a microscopy technique based on chemical labelling of cysteine residues we could observe the conjugative pilus in vivo. In addition, we used cryo-CLEM to gain detailed morphological information on this cellular appendage. The fourth chapter presents a study on the dynamic behaviour of two ICEclc T4SS subunits. Here we focused on IceB7 and IceB4 located in the outer and inner-membrane respectively. With high resolution confocal microscopy we followed the localization of the two labelled subunits overtime both in presence and absence of recipient cells and we inferred their dynamics with custom made image analysis pipelines. Our results suggest a highly stable IceB7, which is one of the core members of the outer membrane assembly and a highly dynamic IceB4, suggesting that the ATPase of the T4SS are not constitutively docked to the conjugative machinery and that they might do so only when ICE DNA is about to be transferred. Finally, we conclude with a general discussion on the outcomes and relevance of this work and the perspective for future studies. -- Les éléments intégratifs et conjugatifs (ICE) constituent une classe répandue d'éléments génétiques mobiles qui jouent un rôle important dans l'évolution bactérienne et l'adaptation à de nouvelles niches. Le transfert des ICE nécessite le contact entre deux bactéries et la mise en place d’un complexe protéique reliant les deux cellules appelé système de sécrétion de type IV (T4SS). Bien que plusieurs T4SS appartenant à des familles distinctes aient été découverts et caractérisés au cours des dernières décennies, la plupart appartiennent à des plasmides mobiles et les T4SS codés par les ICE restent gravement sous-étudiés. Dans cette étude, nous nous sommes concentrés sur le T4SS codé par l'élément ICEclc, initialement découvert en deux copies identiques intégrées dans le chromosome de Pseudomonas knackmussii B13. La région centrale d’ICEclc, qui inclut le locus génétique codant le T4SS, est conservée chez plusieurs éléments apparentés présents dans le génome de protéobactéries des groupes β et γ. Ainsi, la compréhension des particularités de ce système de conjugaison serait représentatif d'un grand nombre d'éléments mobiles. Dans ce travail, nous avons focalisé notre attention sur la caractérisation approfondie du locus génétique du T4SS d’ICEclc en utilisant une combinaison d'approches bioinformatiques, génétiques, et microscopiques. Le premier chapitre introduit le thème du transfert horizontal de gènes en se penchant sur les caractéristiques des éléments intégratifs et conjugatifs, leur importance évolutive et le mécanisme de transfert d'ADN par conjugaison, avec une description approfondie des connaissances actuelles sur la classification et la composition structurelle du T4SS. Dans le deuxième chapitre, nous présentons la caractérisation générale des 24 gènes du locus T4SS d'ICEclc. Grâce à une analyse bioinformatique des homologies entre les composants du T4SS codés par ICEclc et d’autres déjà connus, nous avons inféré des fonctions aux produits de la majorité des gènes du locus T4SS. 22 variants d’ICEclc délétés pour 1 des 24 gènes codant des sous-unités putatives du T4SS ont été utilisés pour comprendre la nécessité de chaque sous-unité pour le transfert de l'ICE. Afin de mieux comprendre la dynamique de mise en place et la localisation des différents parties du T4SS au niveau de la cellule individuelle, nous avons fusionné neuf sous-unités prédites du T4SS à des protéines fluorescentes, et étudié leur localisation cellulaire par microscopie à épifluorescence. En associant le marquage fluorescent et les délétions géniques, nous avons montré des interactions possibles entre plusieurs sous-unités du T4SS et avons proposé un modèle dynamique de l'assemblage du T4SS. Le troisième chapitre se concentre sur la caractérisation du pilus de conjugaison, l'un des quatre sous-ensembles protéiques du T4SS. Une analyse bioinformatique reposant sur la prédiction de la structure tridimensionnelle des protéines a permis d’identifier le gène orf66625 codant la piline (sous unités protéique majeure du pilus) chez ICEclc. Grâce à une technique de microscopie basée sur le marquage chimique des cystéines, nous avons pu observer le pilus conjugatif in vivo. De plus, nous avons obtenu des informations morphologiques détaillées sur cet appendice cellulaire grâce à une technique spécifique de microscopie électronique, appelée cryo-CLEM. Le quatrième chapitre présente une étude sur la dynamique d’IceB7 et IceB4, deux sous-unités du T4SS d'ICEclc situées dans la membrane externe et la membrane interne respectivement. Grace à la microscopie confocale à haute résolution, nous avons suivi la localisation des deux sous-unités au fil du temps, en présence et en absence de cellules receveuses, et analysé leur dynamique à l'aide de « pipelines » d'analyse d'images développés par nos soins. D’une part, nos résultats suggèrent qu’IceB7 est hautement stable, et est l'un des membres centraux de l'assemblage du complexe dans la membrane externe. D’autre part, IceB4 est hautement dynamique, ce qui suggère que cette protéine n'est pas constitutivement ancrée à la machinerie de transfert, et pourrait l’être uniquement lorsque l'ADN de l'ICE est sur le point d'être transféré. Enfin, ce manuscrit se termine par une discussion générale sur les résultats et la pertinence de ce travail, ainsi que les perspectives pour des études futures

Investigating the protein subcomplexes from a conjugative Type IV Secretion System

Type IV secretion system (T4SS) are versatile nanomachines that enable the efficient transport of substrates in bacteria. In general, they are formed from two major membrane embedded subassemblies: an outer membrane core complex (OMCC) and an inner membrane complex (IMC). The conjugative T4SS encoded by the F plasmid is of particular interest due to its clinical relevance as it facilitates the spread of antibiotic resistance amongst bacterial population. Despite its importance, atomic details of the F-T4SS structure and protein-protein interactions were rudimentary which in turn precludes thorough understanding of how conjugation is orchestrated. Therefore, this thesis aimed to improve knowledge on the F-T4SS by studying the structure of the F-OMCC and investigating other proteins the complex may interact with. After optimising the detergent solubilisation of the F-OMCC expressed from the pED208 F-like plasmid, and improving the purification of the complex, a cryo-EM dataset was collected. Using single particle analysis, the structure was solved with an overall resolution of 3.3 Å. The F-OMCC is formed from two concentric rings which have two distinct symmetries. The outer ring adopts 13-fold symmetry whereas the inner ring showed 17-fold symmetry, together they form a 2.1 MDa complex. The atomic models of TraB, TraK and TraV were built into the structure, and they revealed a unique stoichiometric arrangement. Interestingly, TraV and TraK proteins were found to adopt two different conformations within the outer ring. TraV and TraB were found to accommodate the symmetry mismatch by existing in both F-OMCC rings, and also appeared to confer flexibility. This makes the F-OMCC a dynamic complex which is likely to have important implications in the pilus and T4SS activity during conjugation. The interactions between the F-OMCC and other Tra/Trb proteins were also investigated to decipher how the concerted dynamics of the pilus may be connected to the complex. A potential interaction between F-OMCC and the proteins TraH and TraN was observed by pull-down assays. Furthermore, initial work on TraG found that it seems to assemble as a high order oligomer in solution. The results are reminiscent of a hexameric protein which may be functionally important. Together, the findings of this thesis reveal novel insights into the F-T4SS and its subassemblies. The approach used to purify the F-OMCC and study the interactions will act as the basis of future work on the F-T4SS and is directly applicable to the other protein complexes within the conjugative nanomachine.Open Acces

Comprehensive analysis of peptidoglycan hydrolases in Caulobacter crescentus

The peptidoglycan (PG) sacculus is a large macromolecule enclosing most bacterial cells. During progression of the cell cycle, it needs to be continuously remodelled to enable elongation of the cell body and, finally, cell division. This process requires a delicate balance between synthetic and hydrolytic reactions, which are executed by an array of different enzymes. However, the roles of individual components in this complex machinery and the mechanisms underlying their temporal and spatial regulation are still incompletely understood. In particular, the functional significance of many PG hydrolases is still unclear. While acting at all stages of cell wall biogenesis, PG hydrolases have a particularly important role during cell division, facilitating the coordinated invagination of the PG layer as constriction proceeds. Previous work has identified the putative PG hydrolase DipM as a key component of the Caulobacter crescentus divisome. However, the extent to which other hydrolases contribute to PG remodelling during the constriction process in this species has remained unknown. To identify the major players and elucidate their function, I have analysed the full set of putative amidases, lytic transglycosylases (LTs), endopeptidases (EPases) and LD-transpeptidases (LD-TPases) encoded in the C. crescentus genome. For this purpose, I generated a variety of fluorescent fusions and deletion mutants and characterized them using microscopic and biochemical approaches. The results obtained indicate that the PG hydrolases of C. crescentus have highly redundant functions. Based on the observation of changes in cell morphology, localization dynamics, stress and antibiotic resistance I have identified a particularly important role of EPases in maintaining cell wall integrity. Deletion of the amiC and chap genes encoding proteins with PG amidase activity had no detectable effect. In contrast, mutants lacking multiple EPases of the NlpC/P60 or LytM subgroups formed either long smooth filaments or occasionally cell chains. Furthermore, the presence of either NlpC/P60 domain containing EPase NlpA, or the Chap amidase is essential to maintain proper growth. Inactivation of all soluble lytic transglycosylases (SLTs), led to cell filamentation accompanied by outer-membrane blebbing. Depletion of DipM, an EPase homologue lacking catalytic activity in strains lacking either all SLTs or all of the remaining LytM factors (a subgroup of EPases), led to a complete block in cell division and finally to cell death. Interestingly, a similar morphological defect was observed upon depletion of a DipM in a strain lacking another LytM factor with degenerated active site, LdpF. Cultivation in stress conditions revealed critical functions of LdpF, SdpA (falling into the SLTs group) and Chap in cell envelope biogenesis, since single deletions of the respective genes led to either osmo- or antibiotic sensitivity. Moreover, SdpA displayed a dynamic localization pattern, characteristic for the divisome components implicated in the final stages of cell wall remodelling, indication a role in the cell division. Collectively, this work provides the first comprehensive analysis of PG hydrolases in C. crescentus and underscores the key role of the catalytically inactive EPase homologues DipM and LdpF in the autolytic system of this species