Localization of PBP3 in Caulobacter crescentus is highly dynamic and largely relies on its functional transpeptidase domain

In rod-shaped bacteria, septal peptidoglycan synthesis involves the late recruitment of the ftsI gene product (PBP3 in Escherichia coli) to the FtsZ ring. We show that in Caulobacter crescentus, PBP3 accumulates at the new pole at the beginning of the cell cycle. Fluorescence recovery after photobleaching experiments reveal that polar PBP3 molecules are, constantly and independently of FtsZ, replaced by those present in the cellular pool, implying that polar PBP3 is not a remnant of the previous division. By the time cell constriction is initiated, all PBP3 polar accumulation has disappeared in favour of an FtsZ-dependent localization near midcell, consistent with PBP3 function in cell division. Kymograph analysis of time-lapse experiments shows that the recruitment of PBP3 to the FtsZ ring is progressive and initiated very early on, shortly after FtsZ ring formation and well before cell constriction starts. Accumulation of PBP3 near midcell is also highly dynamic with a rapid exchange of PBP3 molecules between midcell and cellular pools. Localization of PBP3 at both midcell and pole appears multifactorial, primarily requiring the catalytic site of PBP3. Collectively, our results suggest a role for PBP3 in pole morphogenesis and provide new insights into the process of peptidoglycan assembly during division.     

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.     

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.     

Positioning of chemosensory proteins and FtsZ through the Rhodobacter sphaeroides cell cycle

Bacterial chemotaxis depends on signalling through large protein complexes. Each cell must inherit a complex on division, suggesting some co‐ordination with cell division. In Escherichia coli the membrane‐spanning chemosensory complexes are polar and new static complexes form at pre‐cytokinetic sites, ensuring positioning at the new pole after division and suggesting a role for the bacterial cytoskeleton. Rhodobacter sphaeroides has both membrane‐associated and cytoplasmic, chromosome‐associated chemosensory complexes. We followed the relative positions of the two chemosensory complexes, FtsZ and MreB in aerobic and in photoheterotrophic R. sphaeroides cells using fluorescence microscopy. FtsZ forms polar spots after cytokinesis, which redistribute to the midcell forming nodes from which FtsZ extends circumferentially to form the Z‐ring. Membrane‐associated chemosensory proteins form a number of dynamic unit‐clusters with mature clusters containing about 1000 CheW₃ proteins. Individual clusters diffuse randomly within the membrane, accumulating at new poles after division but not colocalizing with either FtsZ or MreB. The cytoplasmic complex colocalizes with FtsZ at midcells in new‐born cells. Before cytokinesis one complex moves to a daughter cell, followed by the second moving to the other cell. These data indicate that two homologous complexes use different mechanisms to ensure partitioning, and neither complex utilizes FtsZ or MreB for positioning.     

Diverse paths to midcell: assembly of the bacterial cell division machinery

At the heart of bacterial cell division is a dynamic ring-like structure of polymers of the tubulin homologue FtsZ. This ring forms a scaffold for assembly of at least ten additional proteins at midcell, the majority of which are likely to be involved in remodeling the peptidoglycan cell wall at the division site. Together with FtsZ, these proteins are thought to form a cell division complex, or divisome. In Escherichia coli, the components of the divisome are recruited to midcell according to a strikingly linear hierarchy that predicts a step-wise assembly pathway. However, recent studies have revealed unexpected complexity in the assembly steps, indicating that the apparent linearity does not necessarily reflect a temporal order. The signals used to recruit cell division proteins to midcell are diverse and include regulated self-assembly, protein-protein interactions, and the recognition of specific septal peptidoglycan substrates. There is also evidence for a complex web of interactions among these proteins and at least one distinct subcomplex of cell division proteins has been defined, which is conserved among E. coli, Bacillus subtilis and Streptococcus pneumoniae.     

Short FtsZ filaments can drive asymmetric cell envelope constriction at the onset of bacterial cytokinesis

FtsZ, the bacterial homologue of eukaryotic tubulin, plays a central role in cell division in nearly all bacteria and many archaea. It forms filaments under the cytoplasmic membrane at the division site where, together with other proteins it recruits, it drives peptidoglycan synthesis and constricts the cell. Despite extensive study, the arrangement of FtsZ filaments and their role in division continue to be debated. Here, we apply electron cryotomography to image the native structure of intact dividing cells and show that constriction in a variety of Gram‐negative bacterial cells, including Proteus mirabilis and Caulobacter crescentus, initiates asymmetrically, accompanied by asymmetric peptidoglycan incorporation and short FtsZ‐like filament formation. These results show that a complete ring of FtsZ is not required for constriction and lead us to propose a model for FtsZ‐driven division in which short dynamic FtsZ filaments can drive initial peptidoglycan synthesis and envelope constriction at the onset of cytokinesis, later increasing in length and number to encircle the division plane and complete constriction.     

Cell wall growth during elongation and division: one ring to bind them?

The role of the cell division protein FtsZ in bacterial cell wall (CW) synthesis is believed to be restricted to localizing proteins involved in the synthesis of the septal wall. In this issue of Molecular Microbiology, the groups of Christine Jacobs-Wagner and Waldemar Vollmer provide compelling evidence that in Caulobacter crescentus, FtsZ plays an additional role in CW synthesis in non-dividing cells. During elongation (cell growth) FtsZ is responsible for the incorporation of CW material in a zone at the midcell by recruiting MurG, a protein involved in peptidoglycan (PG) precursor synthesis. This resembles earlier findings of FtsZ mediated PG synthesis activity in Escherichia coli. A role of FtsZ in PG synthesis during elongation forces a rethink of the current model of CW synthesis in rod-shaped bacteria.     

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.     

FtsZ dynamics in bacterial division: What,how, and why?

Bacterial cell division is orchestrated by the divisome, a protein complex centered on the tubulin homolog FtsZ. FtsZ polymerizes into a dynamic ring that defines the division site, recruits downstream proteins, and directs peptidoglycan synthesis to drive constriction. Recent studies have documented treadmilling of FtsZ polymer clusters both in cells and in vitro. Emerging evidence suggests that FtsZ dynamics are regulated largely by intrinsic properties of FtsZ itself and by the membrane anchoring protein FtsA. Although FtsZ dynamics are broadly required for Z-ring assembly, their role(s) during constriction may vary among bacterial species. These recent advances set the stage for future studies to investigate how FtsZ dynamics are physically and/or functionally coupled to peptidoglycan metabolic enzymes to direct efficient 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.     

Early targeting of Min proteins to the cell poles in germinated spores of Bacillus subtilis: evidence for division apparatus-independent recruitment of Min proteins to the division site

The earliest event in bacterial cell division is the assembly of a tubulin-like protein, FtsZ, at mid-cell to form a ring. In rod-shaped bacteria, the Min system plays an important role in division site placement by inhibiting FtsZ ring formation specifically at the polar regions of the cell. The Min system comprises MinD and MinC, which form an inhibitor complex and, in Bacillus subtilis, DivIVA, which ensures that division is inhibited only in the polar regions. All three proteins localize to the division site at mid-cell and to cell poles. Their recruitment to the division site is dependent on localization of both 'early' and 'late' division proteins. We have examined the temporal and spatial localization of DivIVA relative to that of FtsZ during the first and second cell division after germination and outgrowth of B. subtilis spores. We show that, although the FtsZ ring assembles at mid-cell about halfway through the cell cycle, DivIVA assembles at this site immediately before cell division and persists there during Z-ring constriction and completion of division. We also show that both DivIVA and MinD localize to the cell poles immediately upon spore germination, well before a Z ring forms at mid-cell. Furthermore, these proteins were found to be present in mature, dormant spores. These results suggest that targeting of Min proteins to division sites does not depend directly on the assembly of the division apparatus, as suggested previously, and that potential polar division sites are blocked at the earliest possible stage in the cell cycle in germinated spores as a mechanism to ensure that equal-sized daughter cells are produced upon cell division.     

PomZ,a ParA‐like protein,regulates Z‐ring formation and cell division in Myxococcus xanthus

Accurate positioning of the division site is essential to generate appropriately sized daughter cells with the correct chromosome number. In bacteria, division generally depends on assembly of the tubulin homologue FtsZ into the Z‐ring at the division site. Here, we show that lack of the ParA‐like protein PomZ in Myxococcus xanthus resulted in division defects with the formation of chromosome‐free minicells and filamentous cells. Lack of PomZ also caused reduced formation of Z‐rings and incorrect positioning of the few Z‐rings formed. PomZ localization is cell cycle regulated, and PomZ accumulates at the division site at midcell after chromosome segregation but prior to FtsZ as well as in the absence of FtsZ. FtsZ displayed cooperative GTP hydrolysis in vitro but did not form detectable filaments in vitro. PomZ interacted with FtsZ in M. xanthus cell extracts. These data show that PomZ is important for Z‐ring formation and is a spatial regulator of Z‐ring formation and cell division. The cell cycle‐dependent localization of PomZ at midcell provides a mechanism for coupling cell cycle progression and Z‐ring formation. Moreover, the data suggest that PomZ is part of a system that recruits FtsZ to midcell, thereby, restricting Z‐ring formation to this position.     

How FtsEX localizes to the Z ring and interacts with FtsA to regulate cell division

In Escherichia coli, FtsEX, a member of the ABC transporter superfamily, is involved in regulating the assembly and activation of the divisome to couple cell wall synthesis to cell wall hydrolysis at the septum. Genetic studies indicate FtsEX acts on FtsA to begin the recruitment of the downstream division proteins but blocks septal PG synthesis until a signal is received that divisome assembly is complete. However, the details of how FtsEX localizes to the Z ring and how it interacts with FtsA are not clear. Our results show that recruitment of FtsE and FtsX is codependent and suggest that the FtsEX complex is recruited through FtsE interacting with the conserved tail of FtsZ (CCTP), thus adding FtsEX to a growing list of proteins that interacts with the CCTP of FtsZ. Furthermore, we find that the N‐terminus of FtsX is not required for FtsEX localization to the Z ring but is required for its functions in cell division indicating that it interacts with FtsA. Taken together, these results suggest that FtsEX first interacts with FtsZ to localize to the Z ring and then interacts with FtsA to promote divisome assembly and regulate septal PG synthesis.     

A new factor stimulating peptidoglycan hydrolysis to separate daughter cells in Caulobacter crescentus

Cell division in Gram‐negative bacteria involves the co‐ordinated invagination of the three cell envelope layers to form two new daughter cell poles. This complex process starts with the polymerization of the tubulin‐like protein FtsZ into a Z‐ring at mid‐cell, which drives cytokinesis and recruits numerous other proteins to the division site. These proteins are involved in Z‐ring constriction, inner‐ and outer‐membrane invagination, peptidoglycan remodelling and daughter cell separation. Three papers in this issue of Molecular Microbiology, from the teams of Lucy Shapiro, Martin Thanbichler and Christine Jacobs‐Wagner, describe a novel protein, called DipM for Division Involved Protein with LysM domains, that is required for cell division in Caulobacter crescentus. DipM localizes to the mid‐cell during cell division, where it is necessary for the hydrolysis of the septal peptidoglycan to remodel the cell wall. Loss of DipM results in severe defects in cell envelope constriction, which is deleterious under fast‐growth conditions. State‐of‐the‐art microscopy experiments reveal that the peptidoglycan is thicker and that the cell wall is incorrectly organized in DipM‐depleted cells compared with wild‐type cells, demonstrating that DipM is essential for reorganizing the cell wall at the division site, for envelope invagination and cell separation in Caulobacter.     

Large ring polymers align FtsZ polymers for normal septum formation

Cytokinesis in bacteria is initiated by polymerization of the tubulin homologue FtsZ into a circular structure at midcell, the Z-ring. This structure functions as a scaffold for all other cell division proteins. Several proteins support assembly of the Z-ring, and one such protein, SepF, is required for normal cell division in Gram-positive bacteria and cyanobacteria. Mutation of sepF results in deformed division septa. It is unclear how SepF contributes to the synthesis of normal septa. We have studied SepF by electron microscopy (EM) and found that the protein assembles into very large (～50 nm diameter) rings. These rings were able to bundle FtsZ protofilaments into strikingly long and regular tubular structures reminiscent of eukaryotic microtubules. SepF mutants that disturb interaction with FtsZ or that impair ring formation are no longer able to align FtsZ filaments in vitro, and fail to support normal cell division in vivo. We propose that SepF rings are required for the regular arrangement of FtsZ filaments. Absence of this ordered state could explain the grossly distorted septal morphologies seen in sepF mutants.     

Conserved relationship between FtsZ and peptidoglycan in the cyanelles of Cyanophora paradoxa similar to that in bacterial cell division

Cyanelles of the biflagellate protist Cyanophora paradoxa have retained the peptidoglycan layer, which is critical for division, as indicated by the inhibitory effects of β-lactam antibiotics. An FtsZ ring is formed at the division site during cyanelle division. We used immunofluorescence microscopy to observe the process of FtsZ ring formation, which is expected to lead cyanelle division, and demonstrated that an FtsZ arc and a split FtsZ ring emerge during the early and late stages of cyanelle division, respectively. We used an anti-FtsZ antibody to observe cyanelle FtsZ rings. We observed bright, ring-shaped fluorescence of FtsZ in cyanelles. Cyanelles were kidney-shaped shortly after division. Fluorescence indicated that FtsZ did not surround the division plane at an early stage of division, but rather formed an FtsZ arc localized at the constriction site. The constriction spread around the cyanelle, which gradually became dumbbell shaped. After the envelope’s invagination, the ring split parallel to the cyanelle division plane without disappearing. Treatment of C. paradoxa cells with ampicillin, a β-lactam antibiotic, resulted in spherical cyanelles with an FtsZ arc or ring on the division plane. Transmission electron microscopy of the ampicillin-treated cyanelle envelope membrane revealed that the surface was not smooth. Thus, the inhibition of peptidoglycan synthesis by ampicillin causes the inhibition of septum formation and a marked delay in constriction development. The formation of the FtsZ arc and FtsZ ring is the earliest sign of cyanelle division, followed by constriction and septum formation.     

Escherichia coli low-molecular-weight penicillin-binding proteins help orient septal FtsZ, and their absence leads to asymmetric cell division and branching

Escherichia coli cells lacking low-molecular-weight penicillin-binding proteins (LMW PBPs) exhibit morphological alterations that also appear when the septal protein FtsZ is mislocalized, suggesting that peptidoglycan modification and division may work together to produce cell shape. We found that in strains lacking PBP5 and other LMW PBPs, higher FtsZ concentrations increased the frequency of branched cells and incorrectly oriented Z rings by 10- to 15-fold. Invagination of these rings produced improperly oriented septa, which in turn gave rise to asymmetric cell poles that eventually elongated into branches. Branches always originated from the remnants of abnormal septation events, cementing the relationship between aberrant cell division and branch formation. In the absence of PBP5, PBP6 and DacD localized to nascent septa, suggesting that these PBPs can partially substitute for the loss of PBP5. We propose that branching begins when mislocalized FtsZ triggers the insertion of inert peptidoglycan at unusual positions during cell division. Only later, after normal cell wall elongation separates the patches, do branches become visible. Thus, a relationship between the LMW PBPs and cytoplasmic FtsZ ultimately affects cell division and overall shape.     

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

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

Recruitment of ZipA to the Septal Ring of Escherichia coli Is Dependent on FtsZ and Independent of FtsA

Cell division in prokaryotes is mediated by the septal ring. In Escherichia coli, this organelle consists of several essential division proteins, including FtsZ, FtsA, and ZipA. To gain more insight into how the structure is assembled, we studied the interdependence of FtsZ, FtsA, and ZipA localization using both immunofluorescence and Gfp tagging techniques. To this end, we constructed a set of strains allowing us to determine the cellular location of each of these three proteins in cells from which one of the other two had been specifically depleted. Our results show that ZipA fails to accumulate in a ring shape in the absence of FtsZ. Conversely, depletion of ZipA does not abolish formation of FtsZ rings but leads to a significant reduction in the number of rings per unit of cell mass. In addition, ZipA does not appear to require FtsA for assembly into the septal ring and vice versa. It is suggested that septal ring formation starts by assembly of the FtsZ ring, after which ZipA and FtsA join this structure in a mutually independent fashion through direct interactions with the FtsZ protein.     

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.