A Fail-Safe Mechanism in the Septal Ring Assembly Pathway Generated by the Sequential Recruitment of Cell Separation Amidases and Their Activators

During cytokinesis in Escherichia coli, the peptidoglycan (PG) layer produced by the divisome must be split to promote cell separation. Septal PG splitting is mediated by the amidases: AmiA, AmiB, and AmiC. To efficiently hydrolyze PG, the amidases must be activated by LytM domain factors. EnvC specifically activates AmiA and AmiB, while NlpD specifically activates AmiC. Here, we used an exportable, superfolding variant of green fluorescent protein (GFP) to demonstrate that AmiB, like its paralog AmiC, is recruited to the division site by an N-terminal targeting domain. The results of colocalization experiments indicate that EnvC is recruited to the division site well before its cognate amidase AmiB. Moreover, we show that EnvC and AmiB have differential FtsN requirements for their localization. EnvC accumulates at division sites independently of this essential division protein, whereas AmiB localization is FtsN dependent. Interestingly, we also report that AmiB and EnvC are recruited to division sites independently of one another. The same is also true for AmiC and NlpD. However, unlike EnvC, we find that NlpD shares an FtsN-dependent localization with its cognate amidase. Importantly, when septal PG synthesis is blocked by cephalexin, both EnvC and NlpD are recruited to septal rings, whereas the amidases fail to localize. Our results thus suggest that the order in which cell separation amidases and their activators localize to the septal ring relative to other components serves as a fail-safe mechanism to ensure that septal PG synthesis precedes the expected burst of PG hydrolysis at the division site, accompanied by amidase recruitment.     

Daughter cell separation is controlled by cytokinetic ring‐activated cell wall hydrolysis

During bacterial cytokinesis, hydrolytic enzymes are used to split wall material shared by adjacent daughter cells to promote their separation. Precise control over these enzymes is critical to prevent breaches in wall integrity that can cause cell lysis. How these potentially lethal hydrolases are regulated has remained unknown. Here, we investigate the regulation of cell wall turnover at the Escherichia coli division site. We show that two components of the division machinery with LytM domains (EnvC and NlpD) are direct regulators of the cell wall hydrolases (amidases) responsible for cell separation (AmiA, AmiB and AmiC). Using in vitro cell wall cleavage assays, we show that EnvC activates AmiA and AmiB, whereas NlpD activates AmiC. Consistent with these findings, we show that an unregulated EnvC mutant requires functional AmiA or AmiB but not AmiC to induce cell lysis, and that the loss of NlpD phenocopies an AmiC^? defect. Overall, our results suggest that cellular amidase activity is regulated spatially and temporally by coupling their activation to the assembly of the cytokinetic ring.     

Early midcell localization of Escherichia coli PBP4 supports the function of peptidoglycan amidases

Insertion of new material into the Escherichia coli peptidoglycan (PG) sacculus between the cytoplasmic membrane and the outer membrane requires a well-organized balance between synthetic and hydrolytic activities to maintain cell shape and avoid lysis. Since most bacteria carry multiple enzymes carrying the same type of PG hydrolytic activity, we know little about the specific function of given enzymes. Here we show that the DD-carboxy/endopeptidase PBP4 localizes in a PBP1A/LpoA and FtsEX dependent fashion at midcell during septal PG synthesis. Midcell localization of PBP4 requires its non-catalytic domain 3 of unknown function, but not the activity of PBP4 or FtsE. Microscale thermophoresis with isolated proteins shows that PBP4 interacts with NlpI and the FtsEX-interacting protein EnvC, an activator of amidases AmiA and AmiB, which are needed to generate denuded glycan strands to recruit the initiator of septal PG synthesis, FtsN. The domain 3 of PBP4 is needed for the interaction with NlpI and EnvC, but not PBP1A or LpoA. In vivo crosslinking experiments confirm the interaction of PBP4 with PBP1A and LpoA. We propose that the interaction of PBP4 with EnvC, whilst not absolutely necessary for mid-cell recruitment of either protein, coordinates the activities of PBP4 and the amidases, which affects the formation of denuded glycan strands that attract FtsN. Consistent with this model, we found that the divisome assembly at midcell was premature in cells lacking PBP4, illustrating how the complexity of interactions affect the timing of cell division initiation.     

Structure–function analysis of the LytM domain of EnvC,an activator of cell wall remodelling at the Escherichia coli division site

Proteins with LytM (Peptidase_M23) domains are broadly distributed in bacteria and have been implicated in a variety of important processes, including cell division and cell‐shape determination. Most LytM‐like proteins that have been structurally and/or biochemically characterized are metallo‐endopeptidases that cleave cross‐links in the peptidoglycan (PG) cell wall matrix. Notable exceptions are the Escherichia coli cell division proteins EnvC and NlpD. These LytM factors are not hydrolases themselves, but instead serve as activators that stimulate PG cleavage by target enzymes called amidases to promote cell separation. Here we report the structure of the LytM domain from EnvC, the first structure of a LytM factor implicated in the regulation of PG hydrolysis. As expected, the fold is highly similar to that of other LytM proteins. However, consistent with its role as a regulator, the active‐site region is degenerate and lacks a catalytic metal ion. Importantly, genetic analysis indicates that residues in and around this degenerate active site are critical for amidase activation in vivo and in vitro. Thus, in the regulatory LytM factors, the apparent substrate binding pocket conserved in active metallo‐endopeptidases has been adapted to control PG hydrolysis by another set of enzymes.     

The crystal structure of the cell division amidase AmiC reveals the fold of the AMIN domain,a new peptidoglycan binding domain

Binary fission is the ultimate step of the prokaryotic cell cycle. In Gram‐negative bacteria like Escherichia coli, this step implies the invagination of three biological layers (cytoplasmic membrane, peptidoglycan and outer membrane), biosynthesis of the new poles and eventually, daughter cells separation. The latter requires the coordinated action of the N‐acetylmuramyl‐L‐alanine amidases AmiA/B/C and their LytM activators EnvC and NlpD to cleave the septal peptidoglycan. We present here the 2.5 Å crystal structure of AmiC which includes the first report of an AMIN domain structure, a β‐sandwich of two symmetrical four‐stranded β‐sheets exposing highly conserved motifs on the two outer faces. We show that this N‐terminal domain, involved in the localization of AmiC at the division site, is a new peptidoglycan‐binding domain. The C‐terminal catalytic domain shows an auto‐inhibitory alpha helix obstructing the active site. AmiC lacking this helix exhibits by itself an activity comparable to that of the wild type AmiC activated by NlpD. We also demonstrate the interaction between AmiC and NlpD by microscale thermophoresis and confirm the importance of the active site blocking alpha helix in the regulation of the amidase activity.     

Lytic transglycosylases RlpA and MltC assist in Vibrio cholerae daughter cell separation

The cell wall is a crucial structural feature in the vast majority of bacteria and comprises a covalently closed network of peptidoglycan (PG) strands. While PG synthesis is important for survival under many conditions, the cell wall is also a dynamic structure, undergoing degradation and remodeling by ‘autolysins’, enzymes that break down PG. Cell division, for example, requires extensive PG remodeling, especially during separation of daughter cells, which depends heavily upon the activity of amidases. However, in Vibrio cholerae, we demonstrate that amidase activity alone is insufficient for daughter cell separation and that lytic transglycosylases RlpA and MltC both contribute to this process. MltC and RlpA both localize to the septum and are functionally redundant under normal laboratory conditions; however, only RlpA can support normal cell separation in low‐salt media. The division‐specific activity of lytic transglycosylases has implications for the local structure of septal PG, suggesting that there may be glycan bridges between daughter cells that cannot be resolved by amidases. We propose that lytic transglycosylases at the septum cleave PG strands that are crosslinked beyond the reach of the highly regulated activity of the amidase and clear PG debris that may block the completion of outer membrane invagination.     

LytM-Domain Factors Are Required for Daughter Cell Separation and Rapid Ampicillin-Induced Lysis in Escherichia coli

Bacterial cytokinesis is coupled to the localized synthesis of new peptidoglycan (PG) at the division site. This newly generated septal PG is initially shared by the daughter cells. In Escherichia coli and other gram-negative bacteria, it is split shortly after it is made to promote daughter cell separation and allow outer membrane constriction to closely follow that of the inner membrane. We have discovered that the LytM (lysostaphin)-domain containing factors of E. coli (EnvC, NlpD, YgeR, and YebA) are absolutely required for septal PG splitting and daughter cell separation. Mutants lacking all LytM factors form long cell chains with septa containing a layer of unsplit PG. Consistent with these factors playing a direct role in septal PG splitting, both EnvC-mCherry and NlpD-mCherry fusions were found to be specifically recruited to the division site. We also uncovered a role for the LytM-domain factors in the process of β-lactam-induced cell lysis. Compared to wild-type cells, mutants lacking LytM-domain factors were delayed in the onset of cell lysis after treatment with ampicillin. Moreover, rather than lysing from midcell lesions like wild-type cells, LytM⁻ cells appeared to lyse through a gradual loss of cell shape and integrity. Overall, the phenotypes of mutants lacking LytM-domain factors bear a striking resemblance to those of mutants defective for the N-acetylmuramyl-l-alanine amidases: AmiA, AmiB, and AmiC. E. coli thus appears to rely on two distinct sets of putative PG hydrolases to promote proper cell division.Cytokinesis in Escherichia coli and other gram-negative bacteria proceeds via the coordinated constriction of their envelope layers (outer membrane, inner membrane, and peptidoglycan [PG]) (12, 13, 34, 89). This coordination is achieved by a multi-protein division machine referred to as the septal ring or divisome (20). Assembly of the septal ring begins with the polymerization of the bacterial tubulin protein, FtsZ, into a ring structure just underneath the inner membrane at the prospective site of cell division (8). Once formed, this so-called Z-ring facilitates the recruitment of a number of essential and nonessential division proteins to the division site for the assembly of the trans-envelope divisome organelle (20).A major function of the cytokinetic machinery is to promote the synthesis of the PG layer that will eventually fortify the new poles of the developing daughter cells. PG is a polysaccharide polymer composed of repeating units of N-acetyl-glucosamine (GlcNAc) and N-acetyl-muramic acid (MurNAc) linked by a β-1,4-glycosidic bond (46). Attached to the MurNAc sugar is a short peptide that is used to form cross-links between adjacent polysaccharide strands (46). Such cross-links allow for the construction of a cell-shaped PG meshwork that surrounds the cell membrane and protects it from osmotic rupture.A new wave of zonal PG synthesis is initiated at the division site during each cell cycle (23, 25, 72, 77, 91). Several of the major PG synthases called penicillin-binding proteins are components of the divisome organelle and play important roles in the synthesis of PG during division (7, 21, 62, 67, 73, 74, 80, 81, 88, 90). The septal PG layer produced by these and perhaps other components of the divisome is thought to be initially shared by the daughter cells (46). In gram-positive bacteria, this septal PG layer is typically split some time after the daughter cells have been compartmentalized by membrane fusion (11). In gram-negative bacteria, however, the septal PG layer is split shortly after it is formed to allow constriction of the outer membrane to closely follow that of the inner (cytoplasmic) membrane (12, 13, 34, 89). This gives rise to the characteristic constricted appearance of predivisional cells of E. coli and its relatives.PG hydrolysis is required to promote septal PG splitting and eventual daughter cell separation (87). E. coli, like many bacteria, encodes a vast array of factors with known or predicted PG hydrolase activity (at least 30 genes and 11 different protein families) (29, 31, 87). In most cases, the loss of individual PG hydrolase factors has little effect on growth and division, suggesting that there is significant functional overlap between the various hydrolases (87). This dearth of phenotypic information has consequently made it difficult to understand the physiological roles of PG hydrolases and identify the subset of these factors needed for septal PG splitting. An approach that has helped overcome this limitation in E. coli, however, has been the systematic deletion of all members of a particular PG hydrolase family from the genome (22, 44, 45, 63). Thus far, of all the families of PG hydrolases encoded by E. coli, the factors that play the predominant role in cell separation appear to be the LytC-type N-acetylmuramyl-l-alanine amidases: AmiA, AmiB, and AmiC (44, 45, 69). Loss of all three of these amidases results in a severe defect in cell separation and the formation of extremely long cell chains. This chaining phenotype can be exacerbated by the loss of members of other classes of PG hydrolases like the lytic transglycosylases or d,d-endopeptidases (44, 68). However, relative to strains defective for the amidases, mutants lacking multiple lytic transglycosylases or d,d-endopeptidases alone do not display significant chaining phenotypes in E. coli. These PG hydrolases therefore appear to be playing more of an ancillary role in cell separation.The LytM (lysostaphin/peptidase M23)-domain containing factors (referred to as LytM factors for convenience) are a widely distributed class of putative PG hydrolases that have been poorly characterized with regard to their role in PG biogenesis in E. coli and other bacteria (31). The most well-studied members of this family of factors, LytM and lysostaphin, are metallo-endopeptidases that cleave the pentaglycine cross-bridges found in staphylococcal PG (9, 30, 64). Based on this activity, other LytM factors are also likely to be PG hydrolases but with altered cleavage specificity because pentaglycine cross-bridges are only found among the staphylococci (75). Indeed, the LytM protein, gp13, from the Bacillus subtilis phage Φ29 was recently shown to be a d,d-endopeptidase that cleaves the meso-diaminopimelic acid-d-Ala cross-links of B. subtilis PG (17).E. coli encodes four factors with identifiable LytM-domains: EnvC, NlpD, YgeR, and YebA (29) (Fig. ​(Fig.1).1). Of the four, only EnvC has been studied in appreciable detail. EnvC⁻ mutants have a mild cell separation (chaining) defect when grown in medium containing salt and a severe division defect when grown at high temperatures in medium lacking salt (5, 42, 48, 71). In addition, purified EnvC protein was found to possess PG hydrolase activity using a gel-based zymogram assay, and an EnvC-green fluorescent protein (GFP) fusion exported to the periplasm via the Tat system was shown to be recruited to the division site (5). In all, these results support a model in which EnvC is targeted to the division site to participate directly in septal PG splitting and daughter cell separation.Open in a separate windowFIG. 1.Predicted domain structure of the E. coli LytM factors. Shown is a diagram depicting the predicted domain architecture of the four E. coli factors with identifiable LytM domains. Abbreviations: LytM, LytM domain; LysM, LysM PG-binding domain (29); CC, coiled coil; T, transmembrane domain; SS, signal sequence; SS*, lipoprotein signal sequence. The UniProtKB/Swiss-Prot accession numbers are as follows: EnvC (P37690), NlpD (P0ADA3), YebA (P0AFS9), and YgeR (Q46798).In the present study, we investigated the physiological role(s) of the entire set of E. coli LytM factors by generating mutant strains lacking all possible combinations of them. We found that, like the amidases, LytM factors play a critical role in daughter cell separation. Furthermore, studies of their subcellular localization revealed that NlpD is recruited to the division site along with EnvC, indicating that both of these LytM factors are likely to be participating directly in the septal PG splitting process. We also discovered that mutants lacking multiple LytM factors lyse more slowly and display an altered morphological response relative to wild-type (WT) cells when they are treated with ampicillin. This finding suggests that in addition to cell separation, LytM proteins play a role in the lytic mechanism of β-lactam antibiotics.     

Peptidoglycan hydrolysis mediated by the amidase AmiC and its LytM activator NlpD is critical for cell separation and virulence in the phytopathogen Xanthomonas campestris

The essential stages of bacterial cell separation are described as the synthesis and hydrolysis of septal peptidoglycan (PG). The amidase, AmiC, which cleaves the peptide side‐chains linked to the glycan strands, contributes critically to this process and has been studied extensively in model strains of Escherichia coli. However, insights into the contribution of this protein to other processes in the bacterial cell have been limited. Xanthomonas campestris pv. campestris (Xcc) is a phytopathogen that causes black rot disease in many economically important plants. We investigated how AmiC and LytM family regulators, NlpD and EnvC, contribute to virulence and cell separation in this organism. Biochemical analyses of purified AmiC demonstrated that it could hydrolyse PG and its activity could be potentiated by the presence of the regulator NlpD. We also established that deletion of the genes encoding amiC1 or nlpD led to a reduction in virulence as well as effects on colony‐forming units and cell morphology. Moreover, further genetic and biochemical evidence showed that AmiC1 and NlpD affect the secretion of type III effector XC3176 and hypersensitive response (HR) induction in planta. These findings indicate that, in addition to their well‐studied role(s) in cell separation, AmiC and NlpD make an important contribution to the type III secretion (T3S) and virulence regulation in this important plant pathogen.     

A conformational switch controls cell wall-remodelling enzymes required for bacterial cell division

Remodelling of the peptidoglycan (PG) exoskeleton is intimately tied to the growth and division of bacteria. Enzymes that hydrolyse PG are critical for these processes, but their activities must be tightly regulated to prevent the generation of lethal breaches in the PG matrix. Despite their importance, the mechanisms regulating PG hydrolase activity have remained elusive. Here we investigate the control of cell division hydrolases called amidases (AmiA, AmiB and AmiC) required for Escherichia coli cell division. Poorly regulated amiB mutants were isolated encoding lytic AmiB variants with elevated basal PG hydrolase activities in vitro. The structure of an AmiB orthologue was also solved, revealing that the active site of AmiB is occluded by a conserved alpha helix. Strikingly, most of the amino acid substitutions in the lytic AmiB variants mapped to this domain and are predicted to disrupt its interaction with the active site. Our results therefore support a model in which cell separation is stimulated by the reversible relief of amidase autoinhibition governed by conserved subcomplexes within the cytokinetic ring. Analogous conformational control mechanisms are likely to be part of a general strategy used to control PG hydrolases present within multienzyme PG-remodelling machines.     

The cell wall amidase AmiB is essential for Pseudomonas aeruginosa cell division,drug resistance and viability

The physiological function of cell wall amidases has been investigated in several proteobacterial species. In all cases, they have been implicated in the cleavage of cell wall material synthesized by the cytokinetic ring. Although typically non‐essential, this activity is critical for daughter cell separation and outer membrane invagination during division. In Escherichia coli, proteins with LytM domains also participate in cell separation by stimulating amidase activity. Here, we investigated the function of amidases and LytM proteins in the opportunistic pathogen Pseudomonas aeruginosa. In agreement with studies in other organisms, ^PaAmiB and three LytM proteins were found to play crucial roles in P. aeruginosa cell separation, envelope integrity and antibiotic resistance. Importantly, the phenotype of amidase‐defective P. aeruginosa cells also differed in informative ways from the E. coli paradigm; ^PaAmiB was found to be essential for viability and the successful completion of cell constriction. Our results thus reveal a key role for amidase activity in cytokinetic ring contraction. Furthermore, we show that the essential function of ^PaAmiB can be bypassed in mutants activated for a Cpx‐like envelope stress response, suggesting that this signaling system may elicit the repair of division machinery defects in addition to general envelope damage.     

Specific Structural Features of the N-Acetylmuramoyl-l-Alanine Amidase AmiD from Escherichia coli and Mechanistic Implications for Enzymes of This Family

AmiD is the fifth identified N-acetylmuramoyl-l-alanine zinc amidase of Escherichia coli. This periplasmic lipoprotein is anchored in the outer membrane and has a broad specificity. AmiD is capable of cleaving the intact peptidoglycan (PG) as well as soluble fragments containing N-acetylmuramic acid regardless of the presence of an anhydro form or not, unlike the four other amidases, AmiA, AmiB, AmiC, and AmpD, which have some specificity. AmiD function is, however, not clearly established but it could be part of the enzymatic machinery involved in the PG turnover in E. coli. We solved three structures of the E. coli zinc amidase AmiD devoid of its lipidic anchorage: the holoenzyme, the apoenzyme in complex with the substrate anhydro-N-acetylmuramic-acid-l-Ala-γ-d-Glu-l-Lys, and the holoenzyme in complex with the l-Ala-γ-d-Glu-l-Lys peptide, the product of the hydrolysis of this substrate by AmiD. The AmiD structure shows a relatively flexible N-terminal extension that allows an easy reach of the PG by the enzyme inserted into the outer membrane. The C-terminal domain provides a potential extended geometrical complementarity to the substrate. AmiD shares a common fold with AmpD, the bacteriophage T7 lysozyme, and the PG recognition proteins, which are receptor proteins involved in the innate immune responses of a wide range of organisms. Analysis of the different structures reveals the similarity between the catalytic mechanism of zinc amidases of the AmiD family and the thermolysin-related zinc peptidases.     

FtsK-dependent dimer resolution on multiple chromosomes in the pathogen Vibrio cholerae

Unlike most bacteria, Vibrio cholerae harbors two distinct, nonhomologous circular chromosomes (chromosome I and II). Many features of chromosome II are plasmid-like, which raised questions concerning its chromosomal nature. Plasmid replication and segregation are generally not coordinated with the bacterial cell cycle, further calling into question the mechanisms ensuring the synchronous management of chromosome I and II. Maintenance of circular replicons requires the resolution of dimers created by homologous recombination events. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover at a specific site, dif, by two tyrosine recombinases, XerC and XerD. The process is coordinated with cell division through the activity of a DNA translocase, FtsK. Many E. coli plasmids also use XerCD for dimer resolution. However, the process is FtsK-independent. The two chromosomes of the V. cholerae N16961 strain carry divergent dimer resolution sites, dif1 and dif2. Here, we show that V. cholerae FtsK controls the addition of a crossover at dif1 and dif2 by a common pair of Xer recombinases. In addition, we show that specific DNA motifs dictate its orientation of translocation, the distribution of these motifs on chromosome I and chromosome II supporting the idea that FtsK translocation serves to bring together the resolution sites carried by a dimer at the time of cell division. Taken together, these results suggest that the same FtsK-dependent mechanism coordinates dimer resolution with cell division for each of the two V. cholerae chromosomes. Chromosome II dimer resolution thus stands as a bona fide chromosomal process.     

Environmental Determinants of Vibrio cholerae Biofilm Development

Vibrio cholerae is a versatile bacterium that flourishes in diverse environments, including the human intestine, rivers, lakes, estuaries, and the ocean. Surface attachment is believed to be essential for colonization of all of these natural environments. Previous studies have demonstrated that the vps genes, which encode proteins required for exopolysaccharide synthesis and transport, are required for V. cholerae biofilm development in Luria-Bertani broth. In this work, we showed that V. cholerae forms vps-dependent biofilms and vps-independent biofilms. The vps-dependent and -independent biofilms differ in their environmental activators and in architecture. Our results suggest that environmental activators of vps-dependent biofilm development are present in freshwater, while environmental activators of vps-independent biofilm development are present in seawater. The distinct environmental requirements for the two modes of biofilm development suggest that vps-dependent biofilm development and vps-independent biofilm development may play distinct roles in the natural environment.     

A protein critical for cell constriction in the Gram‐negative bacterium Caulobacter crescentus localizes at the division site through its peptidoglycan‐binding LysM domains

During division of Gram‐negative bacteria, invagination of the cytoplasmic membrane and inward growth of the peptidoglycan (PG) are followed by the cleavage of connective septal PG to allow cell separation. This PG splitting process requires temporal and spatial regulation of cell wall hydrolases. In Escherichia coli, LytM factors play an important role in PG splitting. Here we identify and characterize a member of this family (DipM) in Caulobacter crescentus. Unlike its E. coli counterparts, DipM is essential for viability under fast‐growth conditions. Under slow‐growth conditions, the ΔdipM mutant displays severe defects in cell division and FtsZ constriction. Consistent with its function in division, DipM colocalizes with the FtsZ ring during the cell cycle. Mutagenesis suggests that the LytM domain of DipM is essential for protein function, despite being non‐canonical. DipM also carries two tandems of the PG‐binding LysM domain that are sufficient for FtsZ ring localization. Localization and fluorescence recovery after photobleaching microscopy experiments suggest that DipM localization is mediated, at least in part, by the ability of the LysM tandems to distinguish septal, multilayered PG from non‐septal, monolayered PG.     

A Novel Peptidoglycan Binding Protein Crucial for PBP1A-Mediated Cell Wall Biogenesis in Vibrio cholerae

The bacterial cell wall, which is comprised of a mesh of polysaccharide strands crosslinked via peptide bridges (peptidoglycan, PG), is critical for maintenance of cell shape and survival. PG assembly is mediated by a variety of Penicillin Binding Proteins (PBP) whose fundamental activities have been characterized in great detail; however, there is limited knowledge of the factors that modulate their activities in different environments or growth phases. In Vibrio cholerae, the cause of cholera, PG synthesis during the transition into stationary phase is primarily mediated by the bifunctional enzyme PBP1A. Here, we screened an ordered V. cholerae transposon library for mutants that are sensitive to growth inhibition by non-canonical D-amino acids (DAA), which prevent growth and maintenance of cell shape in PBP1A-deficient V. cholerae. In addition to PBP1A and its lipoprotein activator LpoA, we found that CsiV, a small periplasmic protein with no previously described function, is essential for growth in the presence of DAA. Deletion of csiV, like deletion of lpoA or the PBP1A–encoding gene mrcA, causes cells to lose their rod shape in the presence of DAA or the beta-lactam antibiotic cefsulodin, and all three mutations are synthetically lethal with deletion of mrcB, which encodes PBP1B, V. cholerae''s second key bifunctional PBP. CsiV interacts with LpoA and PG but apparently not with PBP1A, supporting the hypothesis that CsiV promotes LpoA''s role as an activator of PBP1A, and thereby modulates V. cholerae PG biogenesis. Finally, the requirement for CsiV in PBP1A-mediated growth of V. cholerae can be overcome either by augmenting PG synthesis or by reducing PG degradation, thereby highlighting the importance of balancing these two processes for bacterial survival.     

Inefficient Tat-Dependent Export of Periplasmic Amidases in an Escherichia coli Strain with Mutations in Two DedA Family Genes

The DedA family genes are found in most bacterial genomes. Two of these proteins are Escherichia coli YqjA and YghB, predicted inner membrane proteins of unknown function sharing 61% amino acid identity. The E. coli single deletion mutants are largely without phenotype, but the double mutant (BC202; ΔyqjA::Tet^r ΔyghB::Kan^r) is characterized by incomplete cell division, temperature sensitivity, and altered phospholipid levels (K. Thompkins et al., J. Bacteriol. 190:4489-4500, 2008). In this report, we have better characterized the cell division chaining defect of BC202. Fluorescence recovery after photobleaching indicates that 58% of the cells in chains are compartmentalized by at least a cytoplasmic membrane. Green fluorescent protein fusions to the cell division proteins FtsZ, ZipA, FtsI, FtsL, and FtsQ are correctly localized to new septation sites in BC202. Periplasmic amidases AmiC and AmiA, secreted by the twin arginine transport (Tat) pathway, are localized to the cytoplasm in BC202. Overexpression of AmiA, AmiC, or AmiB, a periplasmic amidase secreted via the general secretory pathway, restores normal cell division but does not suppress the temperature sensitivity of BC202, indicating that YghB and YqjA may play additional roles in cellular physiology. Strikingly, overexpression of the Tat export machinery (TatABC) results in normal cell division and growth at elevated temperatures. These data collectively suggest that the twin arginine pathway functions inefficiently in BC202, likely due to the altered levels of membrane phospholipids in this mutant. These results underscore the importance of membrane composition in the proper function of the Tat protein export pathway.Roughly 25 to 30% of the genes in sequenced genomes are predicted to encode integral membrane proteins (12). The functions of many of these genes, even in a well-studied organism such as Escherichia coli, remain unknown. We have reported on the functional redundancy of two highly conserved and related E. coli inner membrane proteins, YqjA and YghB (40). These proteins belong to a large family (commonly called the DedA family) found widespread in most sequenced genomes. yghB and yqjA encode predicted inner membrane proteins with multiple membrane-spanning domains and 61% amino acid identity. In addition, E. coli contains three other genes predicted to encode proteins with significant similarity to YqjA and YghB (YabI, YohD, and DedA; amino acid BLAST E value of <1 × 10⁻⁶) and two other proteins with lower degrees of similarity (YdjX and YdjZ). Currently, there are >1,000 genes in the NCBI protein database annotated as either belonging to this family or possessing significant amino acid identity to E. coli DedA/YghB/YqjA (protein BLAST E values of <0.02). No member of this family has a known function, nor is it known whether they possess common functions across phylogenetic groups.Individually, yghB and yqjA are nonessential genes, as each single deletion mutant grows normally (2). However, BC202, an E. coli strain with targeted deletions of both yqjA and yghB, does not grow above 42°C and displays a dramatic cell division phenotype by forming chains of cells when grown at the permissive temperature of 30°C. Phase-contrast and scanning electron microscopy analysis of BC202 suggests that mutants can begin septation but are blocked at a later step in constriction (40). The cause of this phenotype is unclear.BC202 also has alterations in membrane phospholipid composition (40). While BC202 is capable of synthesizing all classes of phospholipids at all growth temperatures, it is depleted of phosphatidylethanolamine (PE), with elevated levels of the acidic phospholipids phosphatidylglycerol (PG) and cardiolipin (CL). In some respects, BC202 resembles phosphatidylserine synthase deletion mutants, such as AD90 (pss93::Kan^r), which produces no membrane PE (14). Mutants deficient in PE are viable, but they require divalent cations for growth (14) and display cell division abnormalities (28, 33). Likewise, normal growth and cell division are restored to BC202 when LB growth medium is supplemented with millimolar concentrations of divalent cations (40). Unlike many mutants defective in cell wall synthesis, BC202 is not hypersensitive to detergents or antibiotics, indicating the presence of an intact outer membrane when grown at the permissive temperature.BC202, therefore, displays several phenotypes: a block at an apparent late stage of cell division, temperature sensitivity, and an imbalance in membrane phospholipid composition. To better understand the functions of YghB and YqjA, we have hypothesized two roles for these genes that are not mutually exclusive to explain the phenotypes of BC202. First, YqjA/YghB may play direct roles in cell division. The phospholipid phenotype may be a consequence secondary to the primary cell division defect in this scenario. Second, YqjA/YghB may play a direct role in efficient PE synthesis or controlling membrane phospholipid composition. The cell division phenotype may be a secondary consequence of the lipid imbalance. Here, we have better characterized the cell division phenotype of BC202 by using green fluorescent protein (GFP) fusions of cell division proteins and fluorescence recovery after photobleaching (FRAP) analysis. We find that while most of the cell division proteins are correctly localized to new septal rings, the periplasmic amidase AmiC is not localized to the septal ring as was reported previously (5), and this may be responsible for the observed cell division phenotype of BC202. AmiC is found mostly in the cytoplasmic compartment in BC202, as is AmiA, both of which are exported to the periplasm by the twin arginine pathway (5). The cell division defect of BC202 can be corrected by overexpression of periplasmic amidases or the TatABC operon, collectively suggesting that the Tat pathway functions inefficiently in BC202.     

Characterization of the small untranslated RNA RyhB and its regulon in Vibrio cholerae

Numerous small untranslated RNAs (sRNAs) have been identified in Escherichia coli in recent years, and their roles are gradually being defined. However, few of these sRNAs appear to be conserved in Vibrio cholerae, and both identification and characterization of sRNAs in V. cholerae remain at a preliminary stage. We have characterized one of the few sRNAs conserved between E. coli and V. cholerae: RyhB. Sequence conservation is limited to the central region of the gene, and RyhB in V. cholerae is significantly larger than in E. coli. As in E. coli, V. cholerae RyhB is regulated by the iron-dependent repressor Fur, and it interacts with the RNA-binding protein Hfq. The regulons controlled by RyhB in V. cholerae and E. coli appear to differ, although some overlap is evident. Analysis of gene expression in V. cholerae in the absence of RyhB suggests that the role of this sRNA is not limited to control of iron utilization. Quantitation of RyhB expression in the suckling mouse intestine suggests that iron availability is not limiting in this environment, and RyhB is not required for colonization of this mammalian host by V. cholerae.     

Ecotin and LamB in Escherichia coli influence the susceptibility to Type VI secretion-mediated interbacterial competition and killing by Vibrio cholerae

BackgroundA prevailing action of the Type VI secretion system (T6SS) in several Gram-negative bacterial species is inter-bacterial competition. In the past several years, many effectors of T6SS were identified in different bacterial species and their involvement in inter-bacterial interactions were described. However, possible defence mechanisms against T6SS attack among prey bacteria were not well clarified yet.MethodsEscherichia coli was assessed for susceptibility to T6SS-mediated killing by Vibrio cholerae. TheT6SS-mediated bacterial killing assays were performed in absence or presence of different protease inhibitors and with different mutant E. coli strains. Expression levels of selected proteins were monitored using SDS-PAGE and immunoblot analyses.ResultsThe T6SS-mediated killing of E. coli by V. cholerae was partly blocked when the serine protease inhibitor Pefabloc was present. E. coli lacking the periplasmic protease inhibitor Ecotin showed enhanced susceptibility to killing by V. cholerae. Mutations affecting E. coli membrane stability also caused increased susceptibility to killing by V. cholerae. E. coli lacking the maltodextrin porin protein LamB showed reduced susceptibility to killing by V. cholerae whereas E. coli with induced high levels of LamB showed reduced survival in inter-bacterial competition.ConclusionsOur study identified two proteins in E. coli, the intrinsic protease inhibitor Ecotin and the outer membrane porin LamB, that influenced E. coli susceptibility to T6SS-mediated killing by V. cholerae.General significanceWe envision that it is feasible to explore these findings to target and modulate their expression to obtain desired changes in inter-bacterial competition in vivo, e.g. in the gastrointestinal microbiome.     

Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport

Homology has been established for members of two families of functionally related bacterial membrane proteins. The first family (the resistance/nodulation/cell division (RND) family) Includes (i) two metal-resistance efflux pumps in Alcaligenes eutrophus (CzcA and CnrA), (ii) three proteins which function together in nodulation of alfalfa roots by Rhizobium meliloti (NoIGHI), and (iii) a cell division protein in Escherichia coli (EnvD). The second family (the putative membrane fusion protein (MFP) family) includes a nodulation protein (NoIF), a cell division protein (EnvC), and a multidrug resistance transport protein (EmrA). We propose that an MFP functions co-operatively with an RND protein to transport large or hydrophobic molecules across the two membranes of the Gram-negative bacterial cell envelope.     

Screening for synthetic lethal mutants in Escherichia coli and identification of EnvC (YibP) as a periplasmic septal ring factor with murein hydrolase activity

Bacterial cytokinesis is driven by the septal ring apparatus, the assembly of which in Escherichia coli is directed to mid-cell by the Min system. Despite suffering aberrant divisions at the poles, cells lacking the minCDE operon (Min(-)) have an almost normal growth rate. We developed a generally applicable screening method for synthetic lethality in E. coli, and used it to select for transposon mutations (slm) that are synthetically lethal (or sick) in combination with DeltaminCDE. One of the slm insertions mapped to envC (yibP), proposed to encode a lysostaphin-like, metallo-endopeptidase that is exported to the periplasm by the general secretory (Sec) pathway. Min(-) EnvC(-) cells showed a severe division defect, supporting a role for EnvC in septal ring function. Accordingly, we show that an EnvC-green fluorescent protein fusion, when directed to the periplasm via the twin-arginine export system, is both functional and part of the septal ring apparatus. Using an in-gel assay, we also present evidence that EnvC possesses murein hydrolytic activity. Our results suggest that EnvC plays a direct role in septal murein cleavage to allow outer membrane constriction and daughter cell separation. By uncovering genetic interactions, the synthetic lethal screen described here provides an attractive new tool for studying gene function in E. coli.