Structural similarity among Escherichia coli FtsW and RodA proteins and Bacillus subtilis SpoVE protein, which function in cell division, cell elongation, and spore formation, respectively.

The Escherichia coli cell division gene ftsW (2 min) was cloned and sequenced. It encodes a hydrophobic protein(s) with 414 and/or 384 amino acid residues. The deduced amino acid sequence and the hydropathy profile of the protein showed high homology with those of the E. coli RodA protein functioning in determination of the cell shape and the Bacillus subtilis SpoVE protein functioning in spore formation. Probably similar functional membrane proteins are involved in these three cell cycle process.     

Sequence encoding ribosomal protein L33 of Lactococcus lactis.

A cloned fragment from Lactococcus lactis chromosome encoding the L33 ribosomal protein was sequenced. Two incomplete open reading frames (ORFs) were also found: the upstream ORF shows similarity to the tetracycline-resistance protein (Tet) of Bacillus stearothermophilus, and the downstream ORF shows homology to a protein of Bacillus subtilis participating in sporulation (SpoVE), and to proteins of Escherichia coli involved in cell division (FtsW) and the maintenance of cell shape (RodA).     

Determinants for the subcellular localization and function of a nonessential SEDS protein

The Bacillus subtilis SpoVE integral membrane protein is essential for the heat resistance of spores, probably because of its involvement in spore peptidoglycan synthesis. We found that an SpoVE-yellow fluorescent protein (YFP) fusion protein becomes localized to the forespore during the earliest stages of engulfment, and this pattern is maintained throughout sporulation. SpoVE belongs to a well-conserved family of proteins that includes the FtsW and RodA proteins of B. subtilis. These proteins are involved in bacterial shape determination, although their function is not known. FtsW is necessary for the formation of the asymmetric septum in sporulation, and we found that an FtsW-YFP fusion localized to this structure prior to the initiation of engulfment in a nonoverlapping pattern with SpoVE-cyan fluorescent protein. Since FtsW and RodA are essential for normal growth, it has not been possible to identify loss-of-function mutations that would greatly facilitate analysis of their function. We took advantage of the fact that SpoVE is not required for growth to obtain point mutations in SpoVE that block the development of spore heat resistance but that allow normal protein expression and targeting to the forespore. These mutant proteins will be invaluable tools for future experiments aimed at elucidating the function of members of the SEDS (“shape, elongation, division, and sporulation”) family of proteins.     

Functional analysis of the cell division protein FtsW of Escherichia coli

Site-directed mutagenesis experiments combined with fluorescence microscopy shed light on the role of Escherichia coli FtsW, a membrane protein belonging to the SEDS family that is involved in peptidoglycan assembly during cell elongation, division, and sporulation. This essential cell division protein has 10 transmembrane segments (TMSs). It is a late recruit to the division site and is required for subsequent recruitment of penicillin-binding protein 3 (PBP3) catalyzing peptide cross-linking. The results allow identification of several domains of the protein with distinct functions. The localization of PBP3 to the septum was found to be dependent on the periplasmic loop located between TMSs 9 and 10. The E240-A249 amphiphilic peptide in the periplasmic loop between TMSs 7 and 8 appears to be a key element in the functioning of FtsW in the septal peptidoglycan assembly machineries. The intracellular loop (containing the R166-F178 amphiphilic peptide) between TMSs 4 and 5 and Gly 311 in TMS 8 are important components of the amino acid sequence-folding information.     

Peptidoglycan synthetic activities in membranes of Escherichia coli caused by overproduction of penicillin-binding protein 2 and rodA protein

Penicillin-binding protein (PBP)-2 and the RodA protein are known to function in determining the rod shape of Escherichia coli cells. Peptidoglycan biosynthetic reactions that required these two proteins were demonstrated in the membrane fraction prepared from an E. coli strain that overproduced both of these two proteins and which lacked PBP-1B activity (the major peptidoglycan synthetase activity in the normal E. coli membranes). The cross-linked peptidoglycan was synthesized from UDP-N-acetylmuramylpentapeptide and UDP-N-acetylglucosamine in the presence of a high concentration of cefmetazole that inhibited all of PBPs except PBP-2. The peptidoglycan was synthesized via a lipid intermediate and showed up to 30% cross-linking. The cross-linking reaction was strongly inhibited by the amidinopenicillin, mecillinam, and by other beta-lactam antibiotics that have a high affinity for PBP-2, but not by beta-lactams that had very low affinity for PBP-2. The formation of peptidoglycan required the presence of high levels of both PBP-2 and the RodA protein in the membranes, but it is unclear which of the two proteins was primarily responsible for the extension of the glycan chains (transglycosylation). However, the sensitivity of the cross-linking reaction to specific beta-lactam antibiotics strongly suggested that it was catalyzed by PBP-2. The transglycosylase activity of the membranes was sensitive to enramycin and vancomycin and was unusual in being stimulated greatly by a high concentration of a chelating agent.     

In vivo labeling of Escherichia coli cell envelope proteins with N-hydroxysuccinimide esters of biotin.

The primary amine coupling reagents succinimidyl-6-biotinamido-hexanoate (NHS-A-biotin) and sulfosuccinimidyl-6-biotinamido-hexanoate (NHS-LC-biotin) were tested for their ability to selectively label Escherichia coli cell envelope proteins in vivo. Probe localization was determined by examining membrane, periplasmic, and cytosolic protein fractions. Both hydrophobic NHS-A-biotin and hydrophilic NHS-LC-biotin were shown to preferentially label outer membrane, periplasmic, and inner membrane proteins. NHS-A- and NHS-LC-biotin were also shown to label a specific inner membrane marker protein (Tet-LacZ). Both probes, however, failed to label a cytosolic marker (the omega fragment of beta-galactosidase). The labeling procedure was also used to label E. coli cells grown in low-salt Luria broth medium supplemented with 0, 10, and 20% sucrose. Outer membrane protein A (OmpA) and OmpC were labeled by both NHS-A- and NHS-LC-biotin at all three sucrose concentrations. In contrast, OmpF was labeled by NHS-A-biotin but not by NHS-LC-biotin in media containing 0 and 10% sucrose. OmpF was not labeled by either NHS-A- or NHS-LC-biotin in E. coli cells grown in medium containing 20% sucrose. Coomassie-stained gels, however, revealed similar quantities of OmpF in E. coli cells grown at all three sucrose concentrations. These data indicate that there was a change in outer membrane structure due to increased osmolarity, which limits accessibility of NHS-A-biotin to OmpF.     

Control of cell shape and elongation by the rodA gene in Bacillus subtilis

The Escherichia coli rodA and ftsW genes and the spoVE gene of Bacillus subtilis encode membrane proteins that control peptidoglycan synthesis during cellular elongation, division and sporulation respectively. While rodA and ftsW are essential genes in E. coli , the B. subtilis spoVE gene is dispensable for growth and is only required for the synthesis of the spore cortex peptidoglycan. In this work, we report on the characterization of a B. subtilis gene, designated rodA , encoding a homologue of E. coli RodA. We found that the growth of a B. subtilis strain carrying a fusion of rodA to the IPTG-inducible P_spac promoter is inducer dependent. Limiting concentrations of inducer caused the formation of spherical cells, which eventually lysed. An increase in the level of IPTG induced a sphere-to-short rod transition that re-established viability. Higher levels of inducer restored normal cell length. Staining of the septal or polar cap peptidoglycan by a fluorescent lectin was unaffected during growth of the mutant under restrictive conditions. Our results suggest that rodA functions in maintaining the rod shape of the cell and that this function is essential for viability. In addition, RodA has an irreplaceable role in the extension of the lateral walls of the cell. Electron microscopy observations support these conclusions. The ultrastructural analysis further suggests that the growth arrest that accompanies loss of the rod shape is caused by the cell's inability to construct a division septum capable of spanning the enlarged cell. RodA is similar over its entire length to members of a large protein family (SEDS, for shape, elongation, division and sporulation). Members of the SEDS family are probably present in all eubacteria that synthesize peptidoglycan as part of their cell envelope.     

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.     

The lipid II flippase RodA determines morphology and growth in Corynebacterium glutamicum

Lipid II flippases play an essential role in cell growth and the maintenance of cell shape in many rod‐shaped bacteria. The putative lipid II flippase RodA is an integral membrane protein and member of the SEDS (shape, elongation, division and sporulation) protein family. In contrast to its homologues in Escherichia coli and Bacillus subtilis little is known about the role of RodA in actinobacteria. In this study, we describe the localization and function of RodA in Corynebacterium glutamicum, a rod‐shaped, apically growing actinobacterium. RodA‐GFP localizes exclusively at the cell poles. Surprisingly, time‐lapse microscopy revealed that apical cell growth is sustained in a rodA deletion strain. However, growth rates and antibiotic susceptibility are altered. In the absence of RodA, it appears that apical growth is driven by lateral diffusion of lipid II that is likely flipped by the septal flippase, FtsW. Furthermore, we applied a previously described synthetic in vivo system in combination with FRET to identify an interaction between C. glutamicum RodA and the polar growth organizing protein DivIVA.     

Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes

SEDS family peptidoglycan (PG) glycosyltransferases, RodA and FtsW, require their cognate transpeptidases PBP2 and FtsI (class B penicillin binding proteins) to synthesize PG along the cell cylinder and at the septum, respectively. The activities of these SEDS-bPBPs complexes are tightly regulated to ensure proper cell elongation and division. In Escherichia coli FtsN switches FtsA and FtsQLB to the active forms that synergize to stimulate FtsWI, but the exact mechanism is not well understood. Previously, we isolated an activation mutation in ftsW (M269I) that allows cell division with reduced FtsN function. To try to understand the basis for activation we isolated additional substitutions at this position and found that only the original substitution produced an active mutant whereas drastic changes resulted in an inactive mutant. In another approach we isolated suppressors of an inactive FtsL mutant and obtained FtsW^E289G and FtsI^K211I and found they bypassed FtsN. Epistatic analysis of these mutations and others confirmed that the FtsN-triggered activation signal goes from FtsQLB to FtsI to FtsW. Mapping these mutations, as well as others affecting the activity of FtsWI, on the RodA-PBP2 structure revealed they are located at the interaction interface between the extracellular loop 4 (ECL4) of FtsW and the pedestal domain of FtsI (PBP3). This supports a model in which the interaction between the ECL4 of SEDS proteins and the pedestal domain of their cognate bPBPs plays a critical role in the activation mechanism.     

Novel bacterial membrane surface display system using cell wall-less L-forms of Proteus mirabilis and Escherichia coli

We describe a novel membrane surface display system that allows the anchoring of foreign proteins in the cytoplasmic membrane (CM) of stable, cell wall-less L-form cells of Escherichia coli and Proteus mirabilis. The reporter protein, staphylokinase (Sak), was fused to transmembrane domains of integral membrane proteins from E. coli (lactose permease LacY, preprotein translocase SecY) and P. mirabilis (curved cell morphology protein CcmA). Both L-form strains overexpressed fusion proteins in amounts of 1 to 100 microg ml(-1), with higher expression for those with homologous anchor motifs. Various experimental approaches, e.g., cell fractionation, Percoll gradient purification, and solubilization of the CM, demonstrated that the fusion proteins are tightly bound to the CM and do not form aggregates. Trypsin digestion, as well as electron microscopy of immunogold-labeled replicas, confirmed that the protein was localized on the outside surface. The displayed Sak showed functional activity, indicating correct folding. This membrane surface display system features endotoxin-poor organisms and can provide a novel platform for numerous applications.     

Interaction between FtsZ and FtsW of Mycobacterium tuberculosis

The recruitment of FtsZ to the septum and its subsequent interaction with other cell division proteins in a spatially and temporally controlled manner are the keys to bacterial cell division. In the present study, we have tested the hypothesis that FtsZ and FtsW of Mycobacterium tuberculosis could be binding partners. Using gel renaturation, pull-down, and solid-phase assays, we confirm that FtsZ and FtsW interact through their C-terminal tails, which carry extensions absent in their Escherichia coli counterparts. Crucial to these interactions is the cluster of aspartate residues Asp(367) to Asp(370) of FtsZ, which most likely interact with a cluster of positively charged residues in the C-terminal tail of FtsW. Mutations of the aspartate residues 367-370 showed that changing three aspartate residues to alanine resulted in complete loss of interaction. This is the first demonstration of the direct interaction between FtsZ and FtsW. We speculate that this interaction between FtsZ and FtsW could serve to anchor FtsZ to the membrane and link septum formation to peptidoglycan synthesis in M. tuberculosis. The findings assume particular significance in view of the global efforts to explore new targets in M. tuberculosis for chemotherapeutic intervention.     

Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane

Bacterial cell growth necessitates synthesis of peptidoglycan. Assembly of this major constituent of the bacterial cell wall is a multistep process starting in the cytoplasm and ending in the exterior cell surface. The intracellular part of the pathway results in the production of the membrane-anchored cell wall precursor, Lipid II. After synthesis this lipid intermediate is translocated across the cell membrane. The translocation (flipping) step of Lipid II was demonstrated to require a specific protein (flippase). Here, we show that the integral membrane protein FtsW, an essential protein of the bacterial division machinery, is a transporter of the lipid-linked peptidoglycan precursors across the cytoplasmic membrane. Using Escherichia coli membrane vesicles we found that transport of Lipid II requires the presence of FtsW, and purified FtsW induced the transbilayer movement of Lipid II in model membranes. This study provides the first biochemical evidence for the involvement of an essential protein in the transport of lipid-linked cell wall precursors across biogenic membranes.     

Identification of FtsW and characterization of a new ftsW division mutant of Escherichia coli.

The product of the ftsW gene has been identified as a polypeptide that, like the related RodA protein, shows anomalous mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FtsW is produced at low levels that can be increased by altering the translation initiation region of the mRNA. Overproduction of FtsW strongly inhibits cell growth. A new mutant allele, ftsW201, causes a temperature-dependent block in the initiation stage of cell division which is similar to the division block in ftsZ mutants. The block in initiation of division in the ftsW201 allele is shown to be independent of FtsZ or the FtsZ inhibitor, SulA. In addition, the ftsW201 mutant is hypersensitive to overproduction of the division initiation protein FtsZ at the permissive temperature. Our results suggest a role for FtsW in an early stage of division which may involve an interaction with FtsZ.     

Topological characterization of the essential Escherichia coli cell division protein FtsW

The membrane topology of Escherichia coli FtsW, a 46-kDa essential protein, was analyzed using a set of 28 ftsW-alkaline phosphatase (ftsW-phoA) and nine ftsW-beta-lactamase (ftsW-bla) gene fusions obtained by in vivo and in vitro methods. The alkaline phosphatase activities or resistance pattern of cells expressing the FtsW-PhoA or FtsW-Bla fusions confirmed only eight out of 10 transmembrane segments predicted by computational methods. After comparison with the recent topology of Streptococcus pneumoniae FtsW, we could identify all the fusions in absolute agreement with the predicted model: N-terminal and C-terminal ends in the cytoplasm, 10 transmembrane segments and one large loop of 67 amino acids (E240-E306) located in the periplasm.     

Ammonia-induced cell envelope injury in Escherichia coli and Enterobacter aerogenes

Ammonia-induced cell envelope injury was examined in pure cultures of Escherichia coli and Enterobacter aerogenes. Cell injury, as determined by the ratio of colony-forming units on m-T7 agar to colony-forming units on m-Endo agar, increased with exposure to increasing concentrations of ammonia. Cell envelopes appeared to be the site of injury as indicated by increasing susceptibility to lysozyme with increasing ammonia concentration. Cells exposed to ammonia also exhibited more cellular leakage than control cells. Leakage from cells exposed to ammonia included proteins, and all leaked substances increased in concentration as ammonia concentrations increased. The concentration of 2-keto-3-deoxyoctonate (KDO) in the outer membrane of E. coli increased with ammonia exposure, while KDO concentration in the outer membrane of E. aerogenes decreased. The results suggest that exposure of E. coli cells to high concentrations of ammonia disrupts the outer membrane and lipopolysaccharide-associated proteins, while E. aerogenes cells are affected through the disruption of bonds between KDO and the outer membrane.     

Maturation of the Escherichia coli divisome occurs in two steps

Cell division proteins FtsZ (FtsA, ZipA, ZapA), FtsE/X, FtsK, FtsQ, FtsL/B, FtsW, PBP3, FtsN and AmiC localize at mid cell in Escherichia coli in an interdependent order as listed. To investigate whether this reflects a time dependent maturation of the divisome, the average cell age at which FtsZ, FtsQ, FtsW, PBP3 and FtsN arrive at their destination was determined by immuno- and GFP-fluorescence microscopy of steady state grown cells at a variety of growth rates. Consistently, a time delay of 14-21 min, depending on the growth rate, between Z-ring formation and the mid cell recruitment of proteins down stream of FtsK was found. We suggest a two-step model for bacterial division in which the Z-ring is involved in the switch from cylindrical to polar peptidoglycan synthesis, whereas the much later localizing cell division proteins are responsible for the modification of the envelope shape into that of two new poles.     

The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex

MreB proteins of Escherichia coli, Bacillus subtilis and Caulobacter crescentus form actin-like cables lying beneath the cell surface. The cables are required to guide longitudinal cell wall synthesis and their absence leads to merodiploid spherical and inflated cells prone to cell lysis. In B. subtilis and C. crescentus, the mreB gene is essential. However, in E. coli, mreB was inferred not to be essential. Using a tight, conditional gene depletion system, we systematically investigated whether the E. coli mreBCD-encoded components were essential. We found that cells depleted of mreBCD became spherical, enlarged and finally lysed. Depletion of each mre gene separately conferred similar gross changes in cell morphology and viability. Thus, the three proteins encoded by mreBCD are all essential and function in the same morphogenetic pathway. Interestingly, the presence of a multicopy plasmid carrying the ftsQAZ genes suppressed the lethality of deletions in the mre operon. Using GFP and cell fractionation methods, we showed that the MreC and MreD proteins were associated with the cell membrane. Using a bacterial two-hybrid system, we found that MreC interacted with both MreB and MreD. In contrast, MreB and MreD did not interact in this assay. Thus, we conclude that the E. coli MreBCD form an essential membrane-bound complex. Curiously, MreB did not form cables in cell depleted for MreC, MreD or RodA, indicating a mutual interdependency between MreB filament morphology and cell shape. Based on these and other observations we propose a model in which the membrane-associated MreBCD complex directs longitudinal cell wall synthesis in a process essential to maintain cell morphology.     

Fractionation of the Providencia stuartii cell envelope

Cell envelopes (i.e. unfractionated inner and outer membranes) were obtained from Providencia stuartii by following procedures previously applied to the isolation of envelopes from Escherichia coli. The P. stuartii envelopes contained known inner membrane enzymes that included a variety of dehydrogenases and ATPase. The catalytic activity of the ATPase depended upon the concentration of magnesium ions, the substrate (ATP) level and the ratio of magnesium ions to ATP. Cell envelopes from P. stuartii were further fractionated to recover inner and outer membrane polypeptides by treatment with the detergent Sarkosyl. Proteins from the periplasmic region were recovered by a simple osmotic shock procedure also previously applied to E. coli. The purity of the various P. stuartii cell envelope fractions was assessed by a combination of techniques that included one- and two-dimensional gel electrophoresis of proteins, enzyme assays and detection of penicillin-binding proteins.