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.     

Involvement of N-acetylmuramyl-l-alanine amidases in cell separation and antibiotic-induced autolysis of Escherichia coli

N-acetylmuramyl-L-alanine amidases are widely distributed among bacteria. However, in Escherichia coli, only one periplasmic amidase has been described until now, which is suggested to play a role in murein recycling. Here, we report that three amidases, named AmiA, B and C, exist in E. coli and that they are involved in splitting of the murein septum during cell division. Moreover, the amidases were shown to act as powerful autolytic enzymes in the presence of antibiotics. Deletion mutants in amiA, B and C were growing in long chains of unseparated cells and displayed a tolerant response to the normally lytic combination of aztreonam and bulgecin. Isolated murein sacculi of these chain-forming mutants showed rings of thickened murein at the site of blocked septation. In vitro, these murein ring structures were digested more slowly by muramidases than the surrounding murein. In contrast, when treated with the amidase AmiC or the endopeptidase MepA, the rings disappeared, and gaps developed at these sites in the murein sacculi. These results are taken as evidence that highly stressed murein cross-bridges are concentrated at the site of blocked cell division, which, when cleaved, result in cracking of the sacculus at this site. As amidase deletion mutants accumulate trimeric and tetrameric cross-links in their murein, it is suggested that these structures mark the division site before cleavage of the septum.     

FtsQ, FtsL and FtsI require FtsK, but not FtsN, for co-localization with FtsZ during Escherichia coli cell division

During cell division in Gram-negative bacteria, the cell envelope invaginates and constricts at the septum, eventually severing the cell into two compartments, and separating the replicated genetic materials. In Escherichia coli, at least nine essential gene products participate directly in septum formation: FtsA, FtsI, FtsL, FtsK, FtsN, FtsQ, FtsW, FtsZ and ZipA. All nine proteins have been localized to the septal ring, an equatorial ring structure at the division site. We used translational fusions to green fluorescent protein (GFP) to demonstrate that FtsQ, FtsL and FtsI localize to potential division sites in filamentous cells depleted of FtsN, but not in those depleted of FtsK. We also constructed translational fusions of FtsZ, FtsA, FtsQ, FtsL and FtsI to enhanced cyan or yellow fluorescent protein (ECFP or EYFP respectively), GFP variants with different fluorescence spectra. Examination of cells expressing different combinations of the fusions indicated that FtsA, FtsQ, FtsL and FtsI co-localize with FtsZ in filaments depleted of FtsN. These localization results support the model that E. coli cell division proteins assemble sequentially as a multimeric complex at the division site: first FtsZ, then FtsA and ZipA independently of each other, followed successively by FtsK, FtsQ, FtsL, FtsW, FtsI and FtsN.     

Murein (peptidoglycan) binding property of the essential cell division protein FtsN from Escherichia coli

The binding of the essential cell division protein FtsN of Escherichia coli to the murein (peptidoglycan) sacculus was studied. Soluble truncated variants of FtsN, including the complete periplasmic part of the protein as well as a variant containing only the C-terminal 77 amino acids, did bind to purified murein sacculi isolated from wild-type cells. FtsN variants lacking this C-terminal region showed reduced or no binding to murein. Binding of FtsN was severely reduced when tested against sacculi isolated either from filamentous cells with blocked cell division or from chain-forming cells of a triple amidase mutant. Binding experiments with radioactively labeled murein digestion products revealed that the longer murein glycan strands (>25 disaccharide units) showed a specific affinity to FtsN, but neither muropeptides, peptides, nor short glycan fragments bound to FtsN. In vivo FtsN could be cross-linked to murein with the soluble disulfide bridge containing cross-linker DTSSP. Less FtsN, but similar amounts of OmpA, was cross-linked to murein of filamentous or of chain-forming cells compared to levels in wild-type cells. Expression of truncated FtsN variants in cells depleted in full-length FtsN revealed that the presence of the C-terminal murein-binding domain was not required for cell division under laboratory conditions. FtsN was present in 3,000 to 6,000 copies per cell in exponentially growing wild-type E. coli MC1061. We discuss the possibilities that the binding of FtsN to murein during cell division might either stabilize the septal region or might have a function unrelated to cell division.     

Translocation of jellyfish green fluorescent protein via the Tat system of Escherichia coli and change of its periplasmic localization in response to osmotic up-shock

The bacterial twin arginine translocation (Tat) pathway is capable of exporting cofactor-containing enzymes into the periplasm. To assess the capacity of the Tat pathway to export heterologous proteins and to gain information about the property of the periplasm, we fused the twin arginine signal peptide of the trimethylamine N-oxide reductase to the jellyfish green fluorescent protein (GFP). Unlike the Sec pathway, the Tat system successfully exported correctly folded GFP into the periplasm of Escherichia coli. Interestingly, GFP appeared as a halo in most cells and occasionally showed a polar localization in wild type strains. When subjected to a mild osmotic up-shock, GFP relocalized very quickly at the two poles of the cells. The conversion from the halo structure to a periplasmic gathering at particular locations was also observed with spherical cells of the DeltarodA-pbpA mutant or of the wild type strain treated with lysozyme. Therefore, the periplasm is not a uniform compartment and the polarization of GFP is unlikely to be caused by simple invagination of the cytoplasmic membrane at the poles. Moreover, the polar gathering of GFP is reversible; the reversion was accelerated by glucose and inhibited by azide and carbonyl cyanide m-chlorophenylhydrazone, indicating an active adaptation of the bacteria to the osmolarity in the medium. These results strongly suggest a relocalization of periplasmic substances in response to environmental changes. The polar area might be the preferential zone where bacteria sense the change in the environment.     

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.     

Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli

The twin-arginine translocation (Tat) system targets cofactor-containing proteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin-arginine motif. In this study, we have analysed the mechanism and capabilities of the E. coli Tat system using green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active. Export is almost totally blocked in tat deletion mutants, indicating that the observed export in wild-type cells occurs predominantly, if not exclusively, by the Tat pathway. Imaging studies reveal a halo of fluorescence in wild-type cells corresponding to the exported periplasmic form; the GFP is distributed uniformly throughout the cytoplasm in a tat mutant. Because previous work has shown GFP to be incapable of folding in the periplasm, we propose that GFP is exported in a fully folded, active state. These data also show for the first time that heterologous proteins can be exported in an active form by the Tat pathway.     

Role of the Escherichia coli Tat pathway in outer membrane integrity

The Escherichia coli Tat system serves to export folded proteins harbouring an N-terminal twin-arginine signal peptide across the cytoplasmic membrane. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are highly defective in the integrity of their cell envelope. Here, we report the isolation, by transposon mutagenesis, of tat mutant strains that have their outer membrane integrity restored. This outer membrane repair of the tat mutant arises as a result of upregulation of the amiB gene, which encodes a cell wall amidase. Overexpression of the genes encoding the two additional amidases, amiA and amiC, does not compensate for the outer membrane defect of the tatC strain. Analysis of the amiA and amiC coding sequences indicates that the proteins may be synthesized with plausible twin-arginine signal sequences, and we demonstrate that they are translocated to the periplasm by the Tat pathway. A Tat+ strain that has mislocalized AmiA and AmiC proteins because of deletion of their signal peptides displays an identical defective cell envelope phenotype. The presence of genes encoding amidases with twin-arginine signal sequences in the genomes of other Gram-negative bacteria suggests that a similar cell envelope defect may be a common feature of tat mutant strains.     

Self-Enhanced Accumulation of FtsN at Division Sites and Roles for Other Proteins with a SPOR Domain (DamX,DedD, and RlpA) in Escherichia coli Cell Constriction

Of the known essential division proteins in Escherichia coli, FtsN is the last to join the septal ring organelle. FtsN is a bitopic membrane protein with a small cytoplasmic portion and a large periplasmic one. The latter is thought to form an α-helical juxtamembrane region, an unstructured linker, and a C-terminal, globular, murein-binding SPOR domain. We found that the essential function of FtsN is accomplished by a surprisingly small essential domain (^EFtsN) of at most 35 residues that is centered about helix H2 in the periplasm. ^EFtsN contributed little, if any, to the accumulation of FtsN at constriction sites. However, the isolated SPOR domain (^SFtsN) localized sharply to these sites, while SPOR-less FtsN derivatives localized poorly. Interestingly, localization of ^SFtsN depended on the ability of cells to constrict and, thus, on the activity of ^EFtsN. This and other results suggest that, compatible with a triggering function, FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation and that its SPOR domain specifically recognizes some form of septal murein that is only transiently available during the constriction process. SPOR domains are widely distributed in bacteria. The isolated SPOR domains of three additional E. coli proteins of unknown function, DamX, DedD, and RlpA, as well as that of Bacillus subtilis CwlC, also accumulated sharply at constriction sites in E. coli, suggesting that septal targeting is a common property of SPORs. Further analyses showed that DamX and, especially, DedD are genuine division proteins that contribute significantly to the cell constriction process.Bacterial cytokinesis is mediated by a ring-shaped apparatus. Assembly of this septal ring (SR; also called the divisome or septasome) begins at the future site of fission, well before cell constriction initiates, and it remains associated with the leading edge of the invaginating cell envelope until fission is completed. The mature ring in Escherichia coli is made up of at least 10 essential division proteins (FtsA, -B, -I, -K, -L, -N, -Q, -W, and -Z and ZipA), which are each needed to prevent a lethal filamentation phenotype. The first known step in assembly of the division apparatus is polymerization of FtsZ just underneath the cytoplasmic membrane. These polymers are joined by FtsA and ZipA via direct interactions with FtsZ, resulting in an intermediate ring structure (the Z ring), onto which the remaining components assemble in a specific order to form a constriction-competent complex.In addition to the essential SR proteins, a growing number of nonessential proteins that associate with the organelle are being identified. Some of the latter are likely to serve redundant functions, while some may be required only under particular conditions (for reviews on the topic, see references 15, 19, and 25).FtsN belongs to the essential SR proteins and is thought to be the last of this class to join the organelle before the onset of cell constriction (1, 9, 11, 57, 59). It is a type II bitopic transmembrane species of 319 residues with a small cytoplasmic domain (residues 1 to 30), a single transmembrane domain (residues 31 to 54), and a large periplasmic domain (residues 55 to 319) (12) (Fig. ​(Fig.1).1). The periplasmic domain comprises three short regions with an α-helical character that are centered around residues 62 to 67 (H1), 80 to 93 (H2), and 117 to 123 (H3), an unstructured glutamine-rich linker (residues 124 to 242), and a C-terminal globular SPOR domain (residues 243 to 319) that has an affinity for peptidoglycan (55, 60) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.E. coli ftsN locus, FtsN domains, and properties of genetic constructs. Shown are the EZTnKan-2 insertion site in ftsN^slm117 strains and the deletion-replacement in ftsN<>aph (ΔftsN) strains. Numbers refer to the site of insertion (black triangle) or to the base pairs that were replaced with an aph cassette (doubleheaded arrow), counting from the start of ftsN. The domain structure of FtsN is illustrated below the ftsN gene. Indicated are the transmembrane domain (TM; light gray), helices H1, H2, and H3 (black) in the periplasmic juxtamembrane region, and the C-terminal SPOR domain (^SFtsN; dark gray). The small periplasmic peptide that is sufficient for FtsN′s essential function in cell division (^EFtsN [see text]) is indicated with the doubleheaded arrow below the domain structure diagram. Also shown are inserts present on plasmids that produce fusions of various portions of FtsN to GFP or ^TTGFP under the control of the P_lac regulatory region. ^TTGFP-fusions contain the TorA signal peptide (hatched box) that is cleaved upon export to the periplasm via the twin arginine transport (Tat) system. Columns indicate the FtsN residues present in each fusion, whether the fusion could (+) or could not (−) compensate for the absence of native FtsN, and whether it accumulated at constriction sites sharply (+++) or poorly (−−+) or appeared evenly distibuted along the periphery of the cell (−−−).As with most SR proteins, it is unclear what the essential role of FtsN is. The ftsN gene was first identified as a multicopy suppressor of a Ts allele in essential division gene ftsA (11). Elevated levels of FtsN were subsequently found to also suppress some Ts alleles in ftsI, ftsK, and ftsQ (11, 18), and even to allow the propagation of cells with a complete lack of FtsK (22, 26) or of FtsEX (48). Depletion of FtsN allows assembly of all the other known essential components into nonconstricting SRs, but the number of ring structures per unit of cell length in FtsN⁻ filaments is two- to threefold lower than in wild-type (WT) cells (9). Bacterial two-hybrid studies suggest that FtsN interacts with several other SR proteins, including FtsA, FtsI (penicillin-binding protein 3 [PBP3]), FtsQ, FtsW, and MtgA (10, 16, 17, 38). Moreover, it was recently shown that the requirement for FtsN itself can be bypassed in cells producing certain mutant forms of FtsA, which are thought to stabilize the SR to a greater degree than native FtsA (5). These observations are all compatible with a general role of FtsN in stabilizing the ring structure. In addition, it was recently found that FtsN interacts directly with PBP1B, one of the major bifunctional murein synthases in E. coli, and that it can stimulate both its transglycosylase and transpeptidase activities in vitro (46). Thus, in addition to stabilizing the SR, FtsN may have a more specific role in modulating septal murein synthesis. Lastly, based on the fact that FtsN is the last known essential protein to join the SR, it is attractive to speculate the protein plays a role in triggering the constriction phase (10, 25). To what degree any of these proposed functions contribute to the essentiality of FtsN remains unclear.What does seem clear is that the essential activity of FtsN takes place in the periplasm and that residues 139 to 319 are dispensable for its essential function (12, 55). In addition, as residues 1 to 45 are also dispensable for targeting of FtsN to division sites, some portion of the periplasmic domain must also be sufficient to direct the protein to the division apparatus (1).In a genetic screen for synthetic lethality with min (slm) (6, 7), we isolated a mutant strain carrying a transposon insertion in codon 119 of ftsN. The viability of cells containing this severely truncated ftsN^slm117 allele prompted us to better define the functional domains of FtsN, and we did so by studying the properties of fusions between various portions of FtsN to green fluorescent protein (GFP). To sublocalize a subset of these, we took advantage of the ability of the twin arginine transport system (Tat) to export functional and fluorescent GFP fusions into the periplasm, such that their periplasmic localization could be determined in live cells by fluorescence microscopy (6, 8, 50, 54).We show that the essential function of FtsN can be performed by a surprisingly small periplasmic peptide of at most 35 residues that is centered around helix H2 but that this essential domain (^EFtsN) itself is unlikely to contribute much, if anything, to the accumulation of FtsN at constriction sites. On the other hand, the nonessential periplasmic SPOR domain (^SFtsN) localized sharply to these sites by itself, while SPOR-less FtsN derivatives localized poorly, at best. Notably, septal localization of ^SFtsN depended on coproduction of ^EFtsN, in cis or in trans, unless cells were provided with the FtsA^E124A protein (5) to allow constriction to ensue in the complete absence of ^EFtsN. Localization of ^SFtsN also depended on the activity of FtsI (PBP3) and the presence of at least one of the periplasmic murein amidases, AmiA, -B, or -C. The results suggest that FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation, which is compatible with a role of the protein in triggering the constriction phase of the division process. In addition, the results, taken together with earlier biochemical work (44, 46, 55), suggest that ^SFtsN is recruited to some form of septal murein that accumulates only transiently at sites of active constriction.In addition to FtsN, E. coli produces three proteins of unknown function that also bear a C-terminal SPOR domain (PF05036; Pfam 23) (20). Two of these, DamX and DedD, are inner membrane proteins with the same topology as FtsN, while the third, RlpA, is an outer membrane lipoprotein (43, 47, 53). We found that all three also accumulate at septal rings and that each of their SPOR domains act as autonomous septal targeting determinants. Moreover, phenotypes of the mutants indicate that both DamX and DedD contribute to the cell constriction process, leading to classification of these proteins as new nonessential division proteins.A SPOR domain is predicted to be present in at least 1,650 (putative) proteins from over 500 bacterial species (PF05036; Pfam 23) (20), raising the question as to how far SPOR properties have been conserved. We find that the SPOR domain of CwlC, a Bacillus subtilis murein amidase that is active during late stages of sporulation (39, 44), also accumulates sharply at division sites in E. coli.Our results predict that many other bacterial SPOR domain proteins specifically recognize the same or closely related target molecule(s) that accumulates transiently at sites of cell constriction. This is supported by a very recent study showing that SPOR domain proteins from Burkholderia thailandensis, Caulobacter crescentus, and Myxococcus xanthus accumulate at cell constriction sites as well (45).     

Daughter cell separation by penicillin-binding proteins and peptidoglycan amidases in Escherichia coli

As one of the final steps in the bacterial growth cycle, daughter cells must be released from one another by cutting the shared peptidoglycan wall that separates them. In Escherichia coli, this delicate operation is performed by several peptidoglycan hydrolases, consisting of multiple amidases, lytic transglycosylases, and endopeptidases. The interactions among these enzymes and the molecular mechanics of how separation occurs without lysis are unknown. We show here that deleting the endopeptidase PBP 4 from strains lacking AmiC produces long chains of unseparated cells, indicating that PBP 4 collaborates with the major peptidoglycan amidases during cell separation. Another endopeptidase, PBP 7, fulfills a secondary role. These functions may be responsible for the contributions of PBPs 4 and 7 to the generation of regular cell shape and the production of normal biofilms. In addition, we find that the E. coli peptidoglycan amidases may have different substrate preferences. When the dd-carboxypeptidase PBP 5 was deleted, thereby producing cells with higher levels of pentapeptides, mutants carrying only AmiC produced a higher percentage of cells in chains, while mutants with active AmiA or AmiB were unaffected. The results suggest that AmiC prefers to remove tetrapeptides from peptidoglycan and that AmiA and AmiB either have no preference or prefer pentapeptides. Muropeptide compositions of the mutants corroborated this latter conclusion. Unexpectedly, amidase mutants lacking PBP 5 grew in long twisted chains instead of straight filaments, indicating that overall septal morphology was also defective in these strains.     

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.     

Bacterial cell division and the septal ring

Cell division in bacteria is mediated by the septal ring, a collection of about a dozen (known) proteins that localize to the division site, where they direct assembly of the division septum. The foundation of the septal ring is a polymer of the tubulin-like protein FtsZ. Recently, experiments using fluorescence recovery after photobleaching have revealed that the Z ring is extremely dynamic. FtsZ subunits exchange in and out of the ring on a time scale of seconds even while the overall morphology of the ring appears static. These findings, together with in vitro studies of purified FtsZ, suggest that the rate-limiting step in turnover of FtsZ polymers is GTP hydrolysis. Another component of the septal ring, FtsK, is involved in coordinating chromosome segregation with cell division. Recent studies have revealed that FtsK is a DNA translocase that facilitates decatenation of sister chromosomes by TopIV and resolution of chromosome dimers by the XerCD recombinase. Finally, two murein hydrolases, AmiC and EnvC, have been shown to localize to the septal ring of Escherichia coli, where they play an important role in separation of daughter cells.     

Role of FtsEX in cell division of Escherichia coli: viability of ftsEX mutants is dependent on functional SufI or high osmotic strength

In Escherichia coli, at least 12 proteins, FtsZ, ZipA, FtsA, FtsE/X, FtsK, FtsQ, FtsL, FtsB, FtsW, FtsI, FtsN, and AmiC, are known to localize to the septal ring in an interdependent and sequential pathway to coordinate the septum formation at the midcell. The FtsEX complex is the latest recruit of this pathway, and unlike other division proteins, it is shown to be essential only on low-salt media. In this study, it is shown that ftsEX null mutations are not only salt remedial but also osmoremedial, which suggests that FtsEX may not be involved in salt transport as previously thought. Increased coexpression of cell division proteins FtsQ-FtsA-FtsZ or FtsN alone restored the growth defects of ftsEX mutants. ftsEX deletion exacerbated the defects of most of the mutants affected in Z ring localization and septal assembly; however, the ftsZ84 allele was a weak suppressor of ftsEX. The viability of ftsEX mutants in high-osmolarity conditions was shown to be dependent on the presence of a periplasmic protein, SufI, a substrate of twin-arginine translocase. In addition, SufI in multiple copies could substitute for the functions of FtsEX. Taken together, these results suggest that FtsE and FtsX are absolutely required for the process of cell division in conditions of low osmotic strength for the stability of the septal ring assembly and that, during high-osmolarity conditions, the FtsEX and SufI functions are redundant for this essential process.     

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.     

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.     

Twin‐arginine translocation system (tat) mutants of Salmonella are attenuated due to envelope defects,not respiratory defects

The twin‐arginine translocation system (Tat) transports folded proteins across the cytoplasmic membrane and is critical to virulence in Salmonella and other pathogens. Experimental and bioinformatic data indicate that 30 proteins are exported via Tat in Salmonella Typhimurium. However, there are no data linking specific Tat substrates with virulence. We inactivated every Tat‐exported protein and determined the virulence phenotype of mutant strains. Although a tat mutant is highly attenuated, no single Tat‐exported substrate accounts for this virulence phenotype. Rather, the attenuation is due primarily to envelope defects caused by failure to translocate three Tat substrates, the N‐acetylmuramoyl‐l ‐alanine amidases, AmiA and AmiC, and the cell division protein, SufI. Strikingly, neither the amiA amiC nor the sufI mutations alone conferred any virulence defect. Although AmiC and SufI have previously been localized to the divisome, the synthetic phenotypes observed are the first to suggest functional overlap. Many Tat substrates are involved in anaerobic respiration, but we show that a mutant completely deficient in anaerobic respiration retains full virulence in both the oral and systemic phases of infection. Similarly, an obligately aerobic mutant is fully virulent. These results suggest that in the classic mouse model of infection, S. Typhimurium is replicating only in aerobic environments.     

A deficiency in S-adenosylmethionine synthetase interrupts assembly of the septal ring in Escherichia coli K-12

A mutant in which S-adenosylmethionine synthetase is underexpressed makes filaments with no visible septa. Examination with GFP fusions to various septal proteins shows that FtsZ, ZipA and FtsA localize to the septal ring, but FtsQ, FtsW, FtsI or FtsN do not. The requirement for S-adenosylmethionine suggests that some methylation reaction is required before a complete septal ring can be assembled.     

Last but not least: new insights into how FtsN triggers constriction during Escherichia coli cell division

The arrival of FtsN at the division site triggers synthesis of septal peptidoglycan and constriction of the cell envelope. New findings are changing our view of how this happens. Binding of FtsN's cytoplasmic domain to a protein named FtsA recruits a small amount of FtsN to the division site earlier than previously recognized. The ability of FtsA to interact with FtsN is regulated by the ZipA protein. The FtsN–FtsA interaction pushes FtsA into an ‘on’ conformation that activates the machinery for peptidoglycan synthesis. In addition, a small region of FtsN's periplasmic domain appears to interact with the FtsQLB complex, pushing it into an ‘on’ state that also triggers synthesis of peptidoglycan. Thus, FtsN allosterically activates peptidoglycan synthesis by two pathways, one in the cytoplasm and involving FtsA, and the other in the periplasm and involving FtsQLB.     

High-yield export of a native heterologous protein to the periplasm by the tat translocation pathway in Escherichia coli

Numerous high‐value recombinant proteins that are produced in bacteria are exported to the periplasm as this approach offers relatively easy downstream processing and purification. Most recombinant proteins are exported by the Sec pathway, which transports them across the plasma membrane in an unfolded state. The twin‐arginine translocation (Tat) system operates in parallel with the Sec pathway but transports substrate proteins in a folded state; it therefore has potential to export proteins that are difficult to produce using the Sec pathway. In this study, we have produced a heterologous protein (green fluorescent protein; GFP) in Escherichia coli and have used batch and fed‐batch fermentation systems to test the ability of the newly engineered Tat system to export this protein into the periplasm under industrial‐type production conditions. GFP cannot be exported by the Sec pathway in an active form. We first tested the ability of five different Tat signal peptides to export GFP, and showed that the TorA signal peptide directed most efficient export. Under batch fermentation conditions, it was found that TorA‐GFP was exported efficiently in wild type cells, but a twofold increase in periplasmic GFP was obtained when the TatABC components were co‐expressed. In both cases, periplasmic GFP peaked at about the 12 h point during fermentation but decreased thereafter, suggesting that proteolysis was occurring. Typical yields were 60 mg periplasmic GFP per liter culture. The cells over‐expressed the tat operon throughout the fermentation process and the Tat system was shown to be highly active over a 48 h induction period. Fed‐batch fermentation generated much greater yields: using glycerol feed rates of 0.4, 0.8, and 1.2 mL h^?1, the cultures reached OD₆₀₀ values of 180 and periplasmic GFP levels of 0.4, 0.85, and 1.1 g L^?1 culture, respectively. Most or all of the periplasmic GFP was shown to be active. These export values are in line with those obtained in industrial production processes using Sec‐dependent export approaches. Biotechnol. Bioeng. 2012; 109: 2533–2542. © 2012 Wiley Periodicals, Inc.     

A role for FtsA in SPOR‐independent localization of the essential Escherichia coli cell division protein FtsN

FtsN is a bitopic membrane protein and the last essential component to localize to the Escherichia coli cell division machinery, or divisome. The periplasmic SPOR domain of FtsN was previously shown to localize to the divisome in a self‐enhancing manner, relying on the essential activity of FtsN and the peptidoglycan synthesis and degradation activities of FtsI and amidases respectively. Because FtsN has a known role in recruiting amidases and is predicted to stimulate the activity of FtsI, it follows that FtsN initially localizes to division sites in a SPOR‐independent manner. Here, we show that the cytoplasmic and transmembrane domains of FtsN (FtsN_Cyto‐TM) facilitated localization of FtsN independently of its SPOR domain but dependent on the early cell division protein FtsA. In addition, SPOR‐independent localization preceded SPOR‐dependent localization, providing a mechanism for the initial localization of FtsN. In support of the role of FtsN_Cyto‐TM in FtsN function, a variant of FtsN lacking the cytoplasmic domain localized to the divisome but failed to complement an ftsN deletion unless it was overproduced. Simultaneous removal of the cytoplasmic and SPOR domains abolished localization and complementation. These data support a model in which FtsA–FtsN interaction recruits FtsN to the divisome, where it can then stimulate the peptidoglycan remodelling activities required for SPOR‐dependent localization.