The cell poles constitute key subcellular domains that are often critical for motility, chemotaxis, and chromosome segregation in rod-shaped bacteria. However, in nearly all rods, the processes that underlie the formation, recognition, and perpetuation of the polar domains are largely unknown. Here, in Vibrio cholerae, we identified HubP (hub of the pole), a polar transmembrane protein conserved in all vibrios, that anchors three ParA-like ATPases to the cell poles and, through them, controls polar localization of the chromosome origin, the chemotactic machinery, and the flagellum. In the absence of HubP, oriCI is not targeted to the cell poles, chemotaxis is impaired, and a small but increased fraction of cells produces multiple, rather than single, flagella. Distinct cytoplasmic domains within HubP are required for polar targeting of the three ATPases, while a periplasmic portion of HubP is required for its localization. HubP partially relocalizes from the poles to the mid-cell prior to cell division, thereby enabling perpetuation of the polar domain in future daughter cells. Thus, a single polar hub is instrumental for establishing polar identity and organization.

SummaryIn bacteria, cytokinesis is dependent on lytic enzymes that facilitate remodelling of the cell wall during constriction. In this work, we identify a thus far uncharacterized periplasmic protein, DipM, that is required for cell division and polarity in Caulobacter crescentus. DipM is composed of four peptidoglycan binding (LysM) domains and a C-terminal lysostaphin-like (LytM) peptidase domain. It binds to isolated murein sacculi in vitro, and is recruited to the site of constriction through interaction with the cell division protein FtsN. Mutational analyses showed that the LysM domains are necessary and sufficient for localization of DipM, while its peptidase domain is essential for function. Consistent with a role in cell wall hydrolysis, DipM was found to interact with purified murein sacculi in vitro and to induce cell lysis upon overproduction. Its inactivation causes severe defects in outer membrane invagination, resulting in a significant delay between cytoplasmic compartmentalization and final separation of the daughter cells. Overall, these findings indicate that DipM is a periplasmic component of the C. crescentus divisome that facilitates remodelling of the peptidoglycan layer and, thus, coordinated constriction of the cell envelope during the division process.

SummaryIn bacteria, cytokinesis is mediated by a ring-shaped multiprotein complex, called divisome. While some of its components are widely conserved, others are restricted to certain bacterial lineages. FtsN is the last essential cell division protein to localize to the division septum in Escherichia coli and is poorly conserved outside the enteric bacteria. We have identified a homologue of FtsN in the a-proteobacterium Caulobacter crescentus and show that it is essential for cell division. C. crescentus FtsN is recruited to the divisome significantly after cell division initiates and remains associated with the new cell poles after cytokinesis is finished. All determinants necessary for localization and function are located in a largely unstructured periplasmic segment of the protein. Its conserved SPORdomain, by contrast, is dispensable for cytokinesis, although it supports targeting of FtsN to the division site. Interestingly, the SPOR-domain is recruited to the division plane when produced in isolated form and retains its localization potential in a heterologous host background. Searching for proteins that share the characteristic features of FtsN from E. coli and C. crescentus, we identified FtsN-like cell division proteins in b-and d-proteobacteria, suggesting that FtsN is widespread among bacteria, albeit highly variable at the sequence level.

Synthesis and hydrolysis of septal peptidoglycan (PG) are critical processes at the conclusion of cell division that enable separation of daughter cells. Cleavage of septal PG is mediated by PG amidases, hydrolytic enzymes that release peptide side chains from the glycan strand. Most gammaproteobacteria, including Escherichia coli, encode several functionally redundant periplasmic amidases. However, members of the Vibrio genus, including the enteric pathogen Vibrio cholerae, encode only a single PG amidase, AmiB. Here, we show that V. cholerae AmiB is crucial for cell division and growth. Genetic and biochemical analyses indicated that AmiB is regulated by two activators, EnvC and NlpD, at least one of which is required for AmiB's localization to the cell division site. Localization of the activators (and thus of AmiB) is dependent upon the cell division protein FtsN. These factors mediate septal PG cleavage in E. coli as well; however, their precise roles vary between the two organisms in a number of ways. Notably, even though V. cholerae EnvC and NlpD appear to be functionally redundant under most growth conditions tested, NlpD is specifically required for intestinal colonization in the infant mouse model of cholera and for V. cholerae resistance against bile salts, perhaps due to environmental regulation of AmiB or its activators. Collectively, our findings reveal that although the cellular components that enable cleavage of septal PG appear to be generally conserved between E. coli and V. cholerae, they can be combined into diverse functional regulatory networks.

Most bacteria possess a peptidoglycan cell wall that determines their morphology and provides mechanical robustness during osmotic challenges. The biosynthesis of this structure is achieved by a large set of synthetic and lytic enzymes with varying substrate specificities. Although the biochemical functions of these proteins are conserved and well-investigated, the precise roles of individual factors and the regulatory mechanisms coordinating their activities in time and space remain incompletely understood. Here, we comprehensively analyze the autolytic machinery of the alphaproteobacterial model organism Caulobacter crescentus, with a specific focus on LytM-like endopeptidases, soluble transglycosylases, and amidases. Our data reveal a high degree of redundancy within each protein family but also specialized functions for individual family members under stress conditions. In addition, we identify two lytic transglycosylases and an amidase as new divisome components that are recruited to midcell at distinct stages of the cell cycle. The midcell localization of these proteins is affected by two LytM factors with degenerate catalytic domains, DipM and LdpF, which may serve as regulatory hubs coordinating the activities of multiple autolytic enzymes during cell constriction and fission, respectively. These findings set the stage for in-depth studies of the molecular mechanisms that control peptidoglycan remodeling in C. crescentus.This article is protected by copyright. All rights reserved.3

We investigated the roles of the Vibrio cholerae high-molecular-weight bifunctional penicillin binding proteins, PBP1a and PBP1b, in the fitness of this enteric pathogen. Using a screen for synthetic lethality, we found that the V. cholerae PBP1a and PBP1b proteins, like their Escherichia coli homologues, are each essential in the absence of the other and in the absence of the other's putative activator, the outer membrane lipoproteins LpoA and LpoB, respectively. Comparative analyses of V. cholerae mutants suggest that PBP1a/LpoA of V. cholerae play a more prominent role in generating and/or maintaining the pathogen's cell wall than PBP1b/LpoB. V. cholerae lacking PBP1b or LpoB exhibited wild-type growth under all conditions tested. In contrast, V. cholerae lacking PBP1a or LpoA exhibited growth deficiencies in minimal medium, in the presence of deoxycholate and bile, and in competition assays with wild-type cells both in vitro and in the infant mouse small intestine. PBP1a pathway mutants are particularly impaired in stationary phase, which renders them sensitive to a product(s) present in supernatants from stationary-phase wild-type cells. The marked competitive defect of the PBP1a pathway mutants in vivo was largely absent when exponential-phase cells rather than stationary-phase cells were used to inoculate suckling mice. Thus, at least for V. cholerae PBP1a pathway mutants, the growth phase of the inoculum is a key modulator of infectivity.T he main component of the bacterial cell wall, peptidoglycan (PG), is an intricate mesh of polysaccharide chains crosslinked by short peptide bridges. The periplasmic assembly of this complex polymer from [N-acetylglucosamine-N-acetylmuramic acid]pentapeptide subunits is facilitated by extracytoplasmic enzymes known as penicillin binding proteins (PBPs). These enzymes carry out two main reactions: transglycosylation (i.e., polymerization of glycan subunits) and transpeptidation (i.e., cross-linking the peptide side chains between polymerized glycan strands). In Escherichia coli-the focus of most studies of Gram-negative cell wall synthesis-two principal high-molecular-weight (HMW) PBPs with both transglycosylase and transpeptidase activity play a pivotal role in PG synthesis. E. coli PBP1a and PBP1b appear to be largely interchangeable, and mutants lacking one of the two proteins have at most mild phenotypes under standard growth conditions (1-3). However, cells lacking both proteins are not viable, and PBP1a and PBP1b are termed synthetically lethal. The activity of each PBP1 enzyme is strictly dependent on the presence of a specific outer membrane activator, either LpoA (for PBP1a) or LpoB (for PBP1b), and mutations in either lpo locus are consequently also synthetically lethal with a mutation of the noncognate pbp1 locus (4, 5). It has been proposed that PBP1a may contribute preferentially to cell elongation whereas PBP1b may play a more prominent role in cell division (3, 6); however, the viability of the individual mutants clearly demonstrates that each enzyme can ...

Summary The biological roles of low molecular weight penicillin-binding proteins (LMW PBP) have been difficult to discern in Gram-negative organisms. In E. coli, mutants lacking these proteins often have no phenotype, and cells lacking all 7 LMW PBPs remain viable. In contrast, we report here that Vibrio cholerae lacking DacA-1, a PBP5 homolog, displays slow growth, aberrant morphology, and altered peptidoglycan (PG) homeostasis in LB medium, as well as a profound plating defect. DacA-1 alone among V. cholerae's LMW PBPs is critical for bacterial growth; mutants lacking the related protein DacA-2 and/or homologs of PBP4 or PBP7 displayed normal growth and morphology. Remarkably, the growth and morphology of the dacA-1 mutant were unimpaired in LB media containing reduced concentrations of NaCl (100 mM or less), and also within suckling mice, a model host for the study of cholera pathogenesis. PG from the dacA-1 mutant contained elevated pentapeptide levels in standard and low salt media, and comparative analyses suggest that DacA-1 is V. cholerae's principal DD-carboxypeptidase. The basis for the dacA-1 mutant's halosensitivity is unknown; nonetheless, the mutant's survival in biochemically uncharacterized environments (such as the suckling mouse intestine) can be used as a reporter of low Na+ content.

The alphaproteobacterium Hyphomonas neptunium proliferates by a unique budding mechanism in which daughter cells emerge from the end of a stalk-like extension emanating from the mother cell body. Studies of this species so far have been hampered by the lack of a genetic system and of molecular tools allowing the regulated expression of target genes. Based on microarray analyses, this work identifies two H. neptunium promoters that are activated specifically by copper and zinc. Functional analyses show that they have low basal activity and a high dynamic range, meeting the requirements for use as a multipurpose expression system. To facilitate their application, the two promoters were incorporated into a set of integrative plasmids, featuring a choice of two different selection markers and various fluorescent protein genes. These constructs enable the straightforward generation and heavy metal-inducible synthesis of fluorescent protein fusions in H. neptunium, thereby opening the door to an in-depth analysis of polar growth and development in this species. Bacteria are a phylogenetically diverse group of organisms whose study has provided important insights into the mechanisms that mediate the spatiotemporal organization of cells. However, most of our knowledge on bacterial cell biology so far has come from the analysis of only a few well-established model species, such as Escherichia coli, Bacillus subtilis, and Caulobacter crescentus, which typically exhibit a rod-like morphology and divide by symmetric or asymmetric binary fission.To further our understanding of subcellular organization in bacteria, we have started to investigate the marine alphaproteobacterium Hyphomonas neptunium (1), a representative of the stalked budding bacteria (2, 3). Similar to other members of this polyphyletic bacterial group, H. neptunium is characterized by a unique mode of reproduction that involves the formation of buds at the tip of a stalk emanating from the mother cell body (Fig. 1A). During the budding process, the nascent daughter cell is equipped with a single polar flagellum at the pole opposite the stalk. Cytokinesis then gives rise to a motile swarmer cell, which initially is unable to replicate, and an immotile stalked cell, which immediately enters a new round of budding and cell division (4). At a defined time in the cell cycle, the swarmer cell undergoes a differentiation process during which it sheds the flagellum and establishes a stalk at the opposite pole. Subsequently, a bud emerges from the tip of the stalk, setting the stage for the next division event.H. neptunium was isolated from the harbor of Barcelona (Spain). Based on morphological criteria, it was originally described as Hyphomicrobium neptunium (1). Later, DNA-DNA hybridization experiments, 5S rRNA sequence analyses, and metabolic profiling revealed a close phylogenetic relationship to members of the genus Hyphomonas (5, 6). Interestingly, 16S rRNA-based phylogenetic studies identify H. neptunium as a member of the Rhodobacterales (7). However, 23S rR...