ftsW is an essential cell-division gene in Escherichia coli

In the absence of exogenous promoters, plasmid-mediated complementation of the temperature-sensitive ftsW201 allele requires the presence of the full coding sequence of ftsW plus upstream DNA encompassing the C-terminus of mraY and the full coding sequence of murD . We used molecular and genetic techniques to introduce an insertional inactivation into the chromosomal copy of ftsW , in the presence of the plasmid-borne wild-type ftsW gene under the control of P_BAD. In the absence of arabinose, the ftsW -null strain is not viable, and a shift from arabinose- to glucose-containing liquid medium resulted in a block in division, followed by cell lysis. Immunofluorescence microscopy revealed that in ftsW -null filaments, the FtsZ ring is absent in 50–60% of filaments, whilst between one and three Z-rings per filament can be detected in the remainder of the population, with the majority of these containing only one Z-ring per filament. We also demonstrated that the expression of only ftsWS (the smaller of two ftsW open reading frames) from P_BAD is sufficient for complementation of the ftsW -null allele. We conclude that FtsW is an essential cell-division protein in Escherichia coli , and that it plays a role in the stabilization of the FtsZ ring during cell division.     

Interaction between FtsW and penicillin-binding protein 3 (PBP3) directs PBP3 to mid-cell, controls cell septation and mediates the formation of a trimeric complex involving FtsZ, FtsW and PBP3 in mycobacteria

In bacteria, biogenesis of cell wall at the division site requires penicillin-binding protein 3 (PBP3) (or Ftsl). Using pull-down, bacterial two-hybrid, and peptide-based interaction assays, we provide evidence that FtsW of Mycobacterium tuberculosis (FtsWMTB) interacts with PBP3 through two extracytoplasmic loops. Pro306 in the larger loop and Pro386 in the smaller loop of FtsW are crucial for these interactions. Fluorescence microscopy shows that conditional silencing of ftsW in Mycobacterium smegmatis prevents cell septation and positioning of PBP3 at mid-cell. Pull-down assays and conditional depletion of FtsW in M. smegmatis provide evidence that FtsZ, FtsW and PBP3 of mycobacteria are capable of forming a ternary complex, with FtsW acting as a bridging molecule. Bacterial three-hybrid analysis suggests that in M. tuberculosis, the interaction (unique to mycobacteria) of FtsZ with the cytosolic C-tail of FtsW strengthens the interaction of FtsW with PBP3. ftsW of M. smegmatis could be replaced by ftsW of M. tuberculosis. FtsWMTB could support formation of the FtsZ-FtsW-PBP3 ternary complex in M. smegmatis. Our findings raise the possibility that in the genus Mycobacterium binding of FtsZ to the C-tail of FtsW may modulate its interactions with PBP3, thereby potentially regulating septal peptidoglycan biogenesis.     

The Escherichia coli Cell Division Protein FtsW Is Required To Recruit Its Cognate Transpeptidase, FtsI (PBP3), to the Division Site

The bacterial cell division protein FtsW has been suggested to perform two functions: stabilize the FtsZ cytokinetic ring, and facilitate septal peptidoglycan synthesis by the transpeptidase FtsI (penicillin-binding protein 3). We show here that depleting Escherichia coli cells of FtsW had little effect on the abundance of FtsZ rings but abrogated recruitment of FtsI to potential division sites. Analysis of FtsW localization confirmed and extended these results; septal localization of FtsW required FtsZ, FtsA, FtsQ, and FtsL but not FtsI. Thus, FtsW is a late recruit to the division site and is essential for subsequent recruitment of its cognate transpeptidase FtsI but not for stabilization of FtsZ rings. We suggest that a primary function of FtsW homologues--which are found in almost all bacteria and appear to work in conjunction with dedicated transpeptidases involved in division, elongation, or sporulation--is to recruit their cognate transpeptidases to the correct subcellular location.     

Agrobacterium tumefaciens divisome proteins regulate the transition from polar growth to cell division

The mechanisms that restrict peptidoglycan biosynthesis to the pole during elongation and re‐direct peptidoglycan biosynthesis to mid‐cell during cell division in polar‐growing Alphaproteobacteria are largely unknown. Here, we explore the role of early division proteins of Agrobacterium tumefaciens including three FtsZ homologs, FtsA and FtsW in the transition from polar growth to mid‐cell growth and ultimately cell division. Although two of the three FtsZ homologs localize to mid‐cell, exhibit GTPase activity and form co‐polymers, only one, FtsZ_AT, is required for cell division. We find that FtsZ_AT is required not only for constriction and cell separation, but also for initiation of peptidoglycan synthesis at mid‐cell and cessation of polar peptidoglycan biosynthesis. Depletion of FtsZ_AT in A. tumefaciens causes a striking phenotype: cells are extensively branched and accumulate growth active poles through tip splitting events. When cell division is blocked at a later stage by depletion of FtsA or FtsW, polar growth is terminated and ectopic growth poles emerge from mid‐cell. Overall, this work suggests that A. tumefaciens FtsZ makes distinct contributions to the regulation of polar growth and cell division.     

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.     

Inhibition of cell division initiation by an imbalance in the ratio of FtsA to FtsZ.

Elevated levels of FtsA protein block cell division at a very early stage, similar to that caused by inhibition of the action of FtsZ. In contrast, overexpression of FtsA and FtsZ together does not block division. A specific ratio of FtsA to FtsZ protein, therefore, is required for cell division.     

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.     

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 D,D-carboxypeptidase PBP3 organizes the division process of Streptococcus pneumoniae

Bacterial division requires the co-ordination of membrane invagination, driven by the constriction of the FtsZ-ring, and concomitant cell wall synthesis, performed by the high-molecular-weight penicillin-binding proteins (HMW PBPs). Using immunofluorescence techniques, we show in Streptococcus pneumoniae that this co-ordination requires PBP3, a D,D-carboxypeptidase that degrades the substrate of the HMW PBPs. In a mutant deprived of PBP3, the apparent rings of HMW PBPs and that of FtsZ are no longer co-localized. In wild-type cells, PBP3 is absent at the future division site and present over the rest of the cell surface, implying that the localization of the HMW PBPs at mid-cell depends on the availability of their substrate. FtsW, a putative translocase of the substrate of the PBPs, forms an apparent ring that is co-localized with the septal HMW PBPs throughout the cell cycle of wild-type cells. In particular, the constriction of the FtsW-ring occurs after that of the FtsZ-ring, with the same delay as the constriction of the septal PBP-rings. However, in the absence of PBP3, FtsW remains co-localized with FtsZ in contrast to the HMW PBPs. Our work reveals an unexpected complexity in the relationships between the division proteins. The consequences of the absence of PBP3 indicate that the peptidoglycan composition is central to the co-ordination of the division process.     

Trapping of a spiral-like intermediate of the bacterial cytokinetic protein FtsZ

The earliest stage in bacterial cell division is the formation of a ring, composed of the tubulin-like protein FtsZ, at the division site. Tight spatial and temporal regulation of Z-ring formation is required to ensure that division occurs precisely at midcell between two replicated chromosomes. However, the mechanism of Z-ring formation and its regulation in vivo remain unresolved. Here we identify the defect of an interesting temperature-sensitive ftsZ mutant (ts1) of Bacillus subtilis. At the nonpermissive temperature, the mutant protein, FtsZ(Ts1), assembles into spiral-like structures between chromosomes. When shifted back down to the permissive temperature, functional Z rings form and division resumes. Our observations support a model in which Z-ring formation at the division site arises from reorganization of a long cytoskeletal spiral form of FtsZ and suggest that the FtsZ(Ts1) protein is captured as a shorter spiral-forming intermediate that is unable to complete this reorganization step. The ts1 mutant is likely to be very valuable in revealing how FtsZ assembles into a ring and how this occurs precisely at the division site.     

Probing the catalytic activity of a cell division-specific transpeptidase in vivo with beta-lactams

Penicillin-binding protein 3 (PBP3; also called FtsI) is a transpeptidase that catalyzes cross-linking of the peptidoglycan cell wall in the division septum of Escherichia coli. To determine whether the catalytic activity of PBP3 is activated during division, we assayed acylation of PBP3 with three beta-lactams (cephalexin, aztreonam, and piperacillin) in growing cells. Acylation of PBP3 with cephalexin, but not aztreonam or piperacillin, appeared to be stimulated by cell division. Specifically, cephalexin acylated PBP3 about 50% faster in a population of dividing cells than in a population of filamentous cells in which division was inhibited by inactivation or depletion of FtsZ, FtsA, FtsQ, FtsW, or FtsN. However, in a simpler in vitro system using isolated membranes, acylation with cephalexin was not impaired by depletion of FtsW or FtsN. A conflicting previous report that the ftsA3(Ts) allele interferes with acylation of PBP3 was found to be due to the presence of a thermolabile PBP3 in the strain used in that study. The new findings presented here are discussed in light of the hypothesis that the catalytic activity of PBP3 is stimulated by interaction(s) with other division proteins. We suggest that there might be allosteric activation of substrate binding.     

ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4-phosphate 5-kinase

The arc3 (accumulation and replication of chloroplast) mutant of Arabidopsis thaliana has a small number of abnormally large chloroplasts in the cell, suggesting that chloroplast division is arrested in the mutant and ARC3 has an important role in the initiation of chloroplast division. To elucidate the role of ARC3, first we identified the ARC3 gene, and determined the location of ARC3 protein during chloroplast division because the localization and spatial orientation of such division factors are vital for correct chloroplast division. Sequencing analysis showed that ARC3 was a fusion of the prokaryotic FtsZ and part of the eukaryotic phosphatidylinositol-4-phosphate 5-kinase (PIP5K) genes. The PIP5K-homologous region of ARC3 had no catalytic domain but a membrane-occupation-and-recognition-nexus (MORN) repeat motif. Immunofluorescence microscopy, Western blotting analysis and in vitro chloroplast import and protease protection assays revealed that ARC3 protein was soluble, and located on the outer surface of the chloroplast in a ring-like structure at the early stage of chloroplast division. Prokaryotes have one FtsZ as a gene for division but have no ARC3 counterparts, the chimera of FtsZ and PIP5K, suggesting that the ARC3 gene might have been generated from FtsZ as another division factor during the evolution of chloroplast by endosymbiosis.     

FtsW is a dispensable cell division protein required for Z-ring stabilization during sporulation septation in Streptomyces coelicolor

The conserved rodA and ftsW genes encode polytopic membrane proteins that are essential for bacterial cell elongation and division, respectively, and each gene is invariably linked with a cognate class B high-molecular-weight penicillin-binding protein (HMW PBP) gene. Filamentous differentiating Streptomyces coelicolor possesses four such gene pairs. Whereas rodA, although not its cognate HMW PBP gene, is essential in these bacteria, mutation of SCO5302 or SCO2607 (sfr) caused no gross changes to growth and septation. In contrast, disruption of either ftsW or the cognate ftsI gene blocked the formation of sporulation septa in aerial hyphae. The inability of spiral polymers of FtsZ to reorganize into rings in aerial hyphae of these mutants indicates an early pivotal role of an FtsW-FtsI complex in cell division. Concerted assembly of the complete divisome was unnecessary for Z-ring stabilization in aerial hyphae as ftsQ mutants were found to be blocked at a later stage in cell division, during septum closure. Complete cross wall formation occurred in vegetative hyphae in all three fts mutants, indicating that the typical bacterial divisome functions specifically during nonessential sporulation septation, providing a unique opportunity to interrogate the function and dependencies of individual components of the divisome in vivo.     

Thermoinducible filamentation in Escherichia coli due to an altered RNA polymerase beta subunit is suppressed by high levels of ppGpp.

The Escherichia coli strain known as GC2553, FB8, UTH1038, or K12S (Luria), considered an F- lambda- wild-type strain, is shown here to carry a cryptic mutation, ftsR1, causing nonlethal filamentation during exponential growth in Luria-Bertani (LB) broth at 42 degrees C and the inability to grow in salt-free LB broth at 42 degrees C. The ftsR1 mutation is completely suppressed in genetic backgrounds which increase RelA-dependent synthesis of the nucleotide ppGpp, i.e., argS201 (Mecr) and alaS21 (Mecr) mutations, affecting aminoacyl-tRNA synthetases, or the presence of a plac-relA' plasmid. These backgrounds also confer resistance in LB broth to the beta-lactam mecillinam, an antibiotic which specifically inhibits penicillin-binding protein 2 and, in wild-type cells, causes an indirect block in cell division. Furthermore, the ftsR1 mutant (but not an isogenic ftsR+ strain) is sensitive to mecillinam in minimal glucose medium at 37 degrees C. Since the division block caused by mecillinam can be overcome by overproduction of the cell division protein FtsZ, we tested the effect of plasmid pZAQ (carrying the ftsZ, ftsA, and ftsQ genes) on the ftsR1 mutant; it suppressed the filamentation in LB broth and the mecillinam sensitivity on minimal glucose medium at 37 degrees C but not the growth defect in salt-free LB broth at 42 degrees C. Genetic analysis indicated that the full phenotype of the ftsR1 mutant is due to a single mutation in the rpoB gene (90 min), coding for the beta subunit of RNA polymerase; we call this allele rpoB369(Fts). We propose that the rpoB369(Fts) mutation alters the specificity of the polymerase and that the mutant enzyme can recover normal activity in the presence of high salt concentrations or via interaction with the nucleotide ppGpp.     

A set of ftsZ mutants blocked at different stages of cell division in Caulobacter

FtsZ is required throughout the cell division process in eubacteria and in archaea. We report the isolation of novel mutants of the FtsZ gene in Caulobacter crescentus. Clusters of charged amino acids were changed to alanine to minimize mutations that affect protein folding. Molecular modelling indicated that all the clustered-charged-to-alanine mutations had altered amino acids at the surface of the protein. Of 13 such mutants, four were recessive-lethal, three were dominant-lethal, and six had no discernible phenotype. An FtsZ depletion strain of Caulobacter was constructed to analyse the phenotype of the recessive-lethal mutations and used to show that they blocked cell division at distinct stages. One mutation blocked the initiation of cell division, two mutations blocked cell division randomly, and one mutation blocked both early and late stages of cell division. The effect of the recessive mutations on the subcellular localization of FtsZ was determined. Models to explain the various mutant phenotypes are discussed. This is the first set of recessive alleles of ftsZ blocked at different stages of cell division.     

Overproduction of FtsZ induces minicell formation in E. coli

The ftsZ gene in E. coli K-12 is an essential cell division gene. We report that a two to sevenfold increase in the level of the FtsZ protein resulted in induction of the minicell phenotype. An increase in the level of FtsZ beyond this range resulted in an inhibition of all cell division. Unlike the classical minicell mutant, the formation of minicells induced by increased levels of FtsZ did not occur at the expense of normal divisions, indicating that increasing FtsZ resulted in additional division events per cell cycle. In addition, increased FtsZ caused cell division to be initiated earlier in the cell cycle. These results are consistent with the level or activity of FtsZ controlling the frequency of cell division in E. coli.     

A temperature-sensitive mutation in the dnaE gene of Caulobacter crescentus that prevents initiation of DNA replication but not ongoing elongation of DNA

A genetic screen for cell division cycle mutants of Caulobacter crescentus identified a temperature-sensitive DNA replication mutant. Genetic complementation experiments revealed a mutation within the dnaE gene, encoding the alpha-catalytic subunit of DNA polymerase III holoenzyme. Sequencing of the temperature-sensitive dnaE allele indicated a single base pair substitution resulting in a change from valine to glutamic acid within the C-terminal portion of the protein. This mutation lies in a region of the DnaE protein shown in Escherichia coli, to be important in interactions with other essential DNA replication proteins. Using DNA replication assays and fluorescence flow cytometry, we show that the observed block in DNA synthesis in the Caulobacter dnaE mutant strain occurs at the initiation stage of replication and that there is also a partial block of DNA elongation.     

ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2

Replication of chloroplasts is essential for achieving and maintaining optimal plastid numbers in plant cells. The plastid division machinery contains components of both endosymbiotic and host cell origin, but little is known about the regulation and molecular mechanisms that govern the division process. The Arabidopsis mutant arc6 is defective in plastid division, and its leaf mesophyll cells contain only one or two grossly enlarged chloroplasts. We show here that arc6 chloroplasts also exhibit abnormal localization of the key plastid division proteins FtsZ1 and FtsZ2. Whereas in wild-type plants, the FtsZ proteins assemble into a ring at the plastid division site, chloroplasts in the arc6 mutant contain numerous short, disorganized FtsZ filament fragments. We identified the mutation in arc6 and show that the ARC6 gene encodes a chloroplast-targeted DnaJ-like protein localized to the plastid envelope membrane. An ARC6-green fluorescent protein fusion protein was localized to a ring at the center of the chloroplasts and rescued the chloroplast division defect in the arc6 mutant. The ARC6 gene product is related closely to Ftn2, a prokaryotic cell division protein unique to cyanobacteria. Based on the FtsZ filament morphology observed in the arc6 mutant and in plants that overexpress ARC6, we hypothesize that ARC6 functions in the assembly and/or stabilization of the plastid-dividing FtsZ ring. We also analyzed FtsZ localization patterns in transgenic plants in which plastid division was blocked by altered expression of the division site-determining factor AtMinD. Our results indicate that MinD and ARC6 act in opposite directions: ARC6 promotes and MinD inhibits FtsZ filament formation in the chloroplast.     

Divisome‐dependent subcellular localization of cell–cell joining protein SepJ in the filamentous cyanobacterium Anabaena

Heterocyst‐forming cyanobacteria are multicellular organisms that grow as filaments that can be hundreds of cells long. Septal junction complexes, of which SepJ is a possible component, appear to join the cells in the filament. SepJ is a cytoplasmic membrane protein that contains a long predicted periplasmic section and localizes not only to the cell poles in the intercellular septa but also to a position similar to a Z ring when cell division starts suggesting a relation with the divisome. Here, we created a mutant of Anabaena sp. strain PCC 7120 in which the essential divisome gene ftsZ is expressed from a synthetic NtcA‐dependent promoter, whose activity depends on the nitrogen source. In the presence of ammonium, low levels of FtsZ were produced, and the subcellular localization of SepJ, which was investigated by immunofluorescence, was impaired. Possible interactions of SepJ with itself and with divisome proteins FtsZ, FtsQ and FtsW were investigated using the bacterial two‐hybrid system. We found SepJ self‐interaction and a specific interaction with FtsQ, confirmed by co‐purification and involving parts of the SepJ and FtsQ periplasmic sections. Therefore, SepJ can form multimers, and in Anabaena, the divisome has a role beyond cell division, localizing a septal protein essential for multicellularity.