Dirk-Jan Scheffers

Cytokinesis in Bacteria

SUMMARY Work on two diverse rod-shaped bacteria, Escherichia coli and Bacillus subtilis, has defined a set of about 10 conserved proteins that are important for cell division in a wide range of eubacteria. These proteins are directed to the division site by the combination of two negative regulatory systems. Nucleoid occlusion is a poorly understood mechanism whereby the nucleoid prevents division in the cylindrical part of the cell, until chromosome segregation has occurred near midcell. The Min proteins prevent division in the nucleoid-free spaces near the cell poles in a manner that is beginning to be understood in cytological and biochemical terms. The hierarchy whereby the essential division proteins assemble at the midcell division site has been worked out for both E. coli and B. subtilis. They can be divided into essentially three classes depending on their position in the hierarchy and, to a certain extent, their subcellular localization. FtsZ is a cytosolic tubulin-like protein that polymerizes into an oligomeric structure that forms the initial ring at midcell. FtsA is another cytosolic protein that is related to actin, but its precise function is unclear. The cytoplasmic proteins are linked to the membrane by putative membrane anchor proteins, such as ZipA of E. coli and possibly EzrA of B. subtilis, which have a single membrane span but a cytoplasmic C-terminal domain. The remaining proteins are either integral membrane proteins or transmembrane proteins with their major domains outside the cell. The functions of most of these proteins are unclear with the exception of at least one penicillin-binding protein, which catalyzes a key step in cell wall synthesis in the division septum.

Bacterial Cell Wall Synthesis: New Insights from Localization Studies

SUMMARY In order to maintain shape and withstand intracellular pressure, most bacteria are surrounded by a cell wall that consists mainly of the cross-linked polymer peptidoglycan (PG). The importance of PG for the maintenance of bacterial cell shape is underscored by the fact that, for various bacteria, several mutations affecting PG synthesis are associated with cell shape defects. In recent years, the application of fluorescence microscopy to the field of PG synthesis has led to an enormous increase in data on the relationship between cell wall synthesis and bacterial cell shape. First, a novel staining method enabled the visualization of PG precursor incorporation in live cells. Second, penicillin-binding proteins (PBPs), which mediate the final stages of PG synthesis, have been localized in various model organisms by means of immunofluorescence microscopy or green fluorescent protein fusions. In this review, we integrate the knowledge on the last stages of PG synthesis obtained in previous studies with the new data available on localization of PG synthesis and PBPs, in both rod-shaped and coccoid cells. We discuss a model in which, at least for a subset of PBPs, the presence of substrate is a major factor in determining PBP localization.

Several distinct localization patterns for penicillin-binding proteins in Bacillus subtilis.

Bacterial cell shape is determined by a rigid external cell wall. In most non‐coccoid bacteria, this shape is also determined by an internal cytoskeleton formed by the actin homologues MreB and/or Mbl. To gain further insights into the topological control of cell wall synthesis in bacteria, we have constructed green fluorescent protein (GFP) fusions to all 11 penicillin‐binding proteins (PBPs) expressed during vegetative growth of Bacillus subtilis. The localization of these fusions was studied in a wild‐type background as well as in strains deficient in FtsZ, MreB or Mbl. PBP3 and PBP4a localized specifically to the lateral wall, in distinct foci, whereas PBP1 and PBP2b localized specifically to the septum. All other PBPs localized to both the septum and the lateral cell wall, sometimes with irregular distribution along the lateral wall or a preference for the septum. This suggests that cell wall synthesis is not dispersed but occurs at specific places along the lateral cell wall. The results implicate PBP3, PBP5 and PBP4a, and possibly PBP4, in lateral wall growth. Localization of PBPs to the septum was found to be dependent on FtsZ, but the GFP–PBP fluorescence patterns were not detectably altered in the absence of MreB or Mbl.

Localization and Interactions of Teichoic Acid Synthetic Enzymes in Bacillus subtilis

The thick wall of gram-positive bacteria is a polymer meshwork composed predominantly of peptidoglycan (PG) and teichoic acids, both of which have a critical function in maintenance of the structural integrity and the shape of the cell. In Bacillus subtilis 168 the major teichoic acid is covalently coupled to PG and is known as wall teichoic acid (WTA). Recently, PG insertion/degradation over the lateral wall has been shown to occur in a helical pattern. However, the spatial organization of WTA assembly and its relationship with cell shape and PG assembly are largely unknown. We have characterized the localization of green fluorescent protein fusions to proteins involved in several steps of WTA synthesis in B. subtilis: TagB, -F, -G, -H, and -O. All of these localized similarly to the inner side of the cytoplasmic membrane, in a pattern strikingly similar to that displayed by probes of nascent PG. Helix-like localization patterns are often attributable to the morphogenic cytoskeletal proteins of the MreB family. However, localization of the Tag proteins did not appear to be substantially affected by single disruption of any of the three MreB homologues of B. subtilis. Bacterial and yeast two-hybrid experiments revealed a complex network of interactions involving TagA, -B, -E, -F, -G, -H, and -O and the cell shape determinants MreC and MreD (encoded by the mreBCD operon and presumably involved in the spatial organization of PG synthesis). Taken together, our results suggest that, in B. subtilis at least, the synthesis and export of WTA precursors are mediated by a large multienzyme complex that may be associated with the PG-synthesizing machinery.

R174 of Escherichia coli FtsZ is involved in membrane interaction and protofilament bundling, and is essential for cell division

We investigated the interaction between FtsZ and the cytoplasmic membrane using inside‐out vesicles. Comparison of the trypsin accessibility of purified FtsZ and cytoplasmic membrane‐bound FtsZ revealed that the protruding loop between helix 6 and helix 7 is protected from trypsin digestion in the latter. This hydrophobic loop contains an arginine residue at position 174. To investigate the role of R174, this residue was replaced by an aspartic acid, and FtsZ‐R174D was fused to green fluorescent protein (GFP). FtsZ‐R174D‐GFP could localize in an FtsZ and in an FtsZ84(Ts) background at both the permissive and the non‐permissive temperature, and it had a reduced affinity for the cytoplasmic membrane compared with wild‐type FtsZ. FtsZ‐R174D could also localize in an FtsZ depletion strain. However, in contrast to wild‐type FtsZ, FtsZ‐R174D was not able to complement the ftsZ84 mutation or the depletion strain and induced filamentation. In vitro polymerization experiments showed that FtsZ‐R174D is able to polymerize, but that these polymers cannot form bundles in the presence of 10 mM CaCl2. This is the first description of an FtsZ mutant that has reduced affinity for the cytoplasmic membrane and does not support cell division, but is still able to localize. The mutant is able to form protofilaments in vitro but fails to bundle. It suggests that neither membrane interaction nor bundling is a requirement for initiation of cell division.

The polymerization mechanism of the bacterial cell division protein FtsZ

Bacteria and archaea usually divide symmetrically by formation of a septum in the middle of the cell. A key event in cell division is the assembly of the FtsZ ring. FtsZ is the prokaryotic homolog of tubulin and forms polymers in the presence of guanine nucleotides. Here, we specifically address the polymerization of FtsZ and the role of nucleotide hydrolysis in polymer formation and stabilization. Recent structural and biochemical results are discussed and a model for FtsZ polymerization, similar to that for tubulin, is presented.

Non-hydrolysable GTP-gamma-S stabilizes the FtsZ polymer in a GDP-bound state

FtsZ, a tubulin homologue, forms a cytokinetic ring at the site of cell division in prokaryotes. The ring is thought to consist of polymers that assemble in a strictly GTP‐dependent way. GTP, but not guanosine‐5′‐O‐(3‐thiotriphosphate) (GTP‐γ‐S), has been shown to induce polymerization of FtsZ, whereas in vitro Ca2+ is known to inhibit the GTP hydrolysis activity of FtsZ. We have studied FtsZ dynamics at limiting GTP concentrations in the presence of 10 mM Ca2+. GTP and its non‐hydrolysable analogue GTP‐γ‐S bind FtsZ with similar affinity, whereas the non‐hydrolysable analogue guanylyl‐imidodiphosphate (GMP‐PNP) is a poor substrate. Preformed FtsZ polymers can be stabilized by GTP‐γ‐S and are destabilized by GDP. As more than 95% of the nucleotide associated with the FtsZ polymer is in the GDP form, it is concluded that GTP hydrolysis by itself does not trigger FtsZ polymer disassembly. Strikingly, GTP‐γ‐S exchanges only a small portion of the FtsZ polymer‐bound GDP. These data suggest that FtsZ polymers are stabilized by a small fraction of GTP‐containing FtsZ subunits. These subunits may be located either throughout the polymer or at the polymer ends, forming a GTP cap similar to tubulin.

The effect of MinC on FtsZ polymerization is pH dependent and can be counteracted by ZapA.

The min system prevents polar cell division in bacteria. Here, the biochemical characterization of the interaction of MinC and FtsZ from a Gram‐positive bacterium, Bacillus subtilis, is reported. B. subtilis MinC inhibits FtsZ polymerization in a pH‐dependent manner by preventing the formation of lateral associations between filaments. The inhibitory effect of MinC on FtsZ polymerization is counteracted by the presence of ZapA, a protein that promotes FtsZ filament bundling.

PBP1 Is a Component of the Bacillus subtilis Cell Division Machinery

Bacillus subtilis penicillin-binding protein PBP1 has been implicated in cell division. We show here that a PBP1 knockout strain is affected in the formation of the asymmetric sporulation septum and that green fluorescent protein-PBP1 localizes to the sporulation septum. Localization of PBP1 to the vegetative septum is dependent on various cell division proteins. This study proves that PBP1 forms part of the B. subtilis cell division machinery.

Immediate GTP hydrolysis upon FtsZ polymerization

To understand the polymerization dynamics of FtsZ, a bacterial cell division protein similar to tubulin, insight is required into the nature of the nucleotide bound to the polymerized protein. In a previous study, we showed that the FtsZ polymers contain mostly GDP. A recent study challenged this result, suggesting that the polymerized FtsZ is in a GTP‐bound state. Here, we show that, when radiolabelled [γ‐32P]‐GTP is used to polymerize FtsZ, GTP is hydrolysed instantaneously. The FtsZ polymer contains both GDP and the radiolabelled inorganic phosphate.