Jeffery Errington

Control of Cell Shape in Bacteria: Helical, Actin-like Filaments in Bacillus subtilis

In the absence of an overt cytoskeleton, the external cell wall of bacteria has traditionally been assumed to be the primary determinant of cell shape. In the Gram-positive bacterium Bacillus subtilis, two related genes, mreB and mbl, were shown to be required for different aspects of cell morphogenesis. Subcellular localization of the MreB and Mbl proteins revealed that each forms a distinct kind of filamentous helical structure lying close to the cell surface. The distribution of the proteins in different species of bacteria, and the similarity of their sequence to eukaryotic actins, suggest that the MreB-like proteins have a cytoskeletal, actin-like role in bacterial cell morphogenesis.

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.

The Bacillus subtilis DivIVA protein targets to the division septum and controls the site specificity of cell division.

The Bacillus subtilisdivIVA gene, first defined by a mutation giving rise to anucleate minicells, has been cloned and characterized. Depletion of DivIVA leads to inhibition of the initiation of cell division. The residual divisions that do occur are abnormally placed and sometimes misorientated relative to the long axis of the cell. The DivIVA phenotype can be suppressed by disruption of the MinCD division inhibitor, suggesting that DivIVA controls the topological specificity of MinCD action and thus septum positioning. A DivIVA–GFP fusion targets to new and used sites of cell division, consistent with it having a direct role in topological specification.

σF, the first compartment-specific transcription factor of B. subtilis, is regulated by an anti-σ factor that is also a protein kinase

Abstract The establishment of compartment-specific transcription in sporulating cells of B. subtilis is governed at the level of the activity of transcription factor σ F . Genetic experiments have suggested that SpoIIAA and SpoIIAB, the other products of the σ F operon, are involved in regulating σ F activity. This activity is inhibited in the predivisional cell but specifically released from inhibition in the prespore about 1.5 hr after sporulation is induced. We now show that purified SpoIIAB Inhibits transcription directed by σ F in vitro. We note that the amino acid sequence of SpoIIAB shows some similarity to a group of bacterial histidine protein kinases, and we find that SpoIIAB is indeed a protein kinase that phosphorylates SpoIIAA on a serine residue. We suggest that this phosphorylation is responsible for the compartment-specific release of σ F activity, perhaps through the formation of a tight complex between SpoIIAB and phosphorylated SpoIIAA.

Dynamic Movement of the ParA-like Soj Protein of B. subtilis and Its Dual Role in Nucleoid Organization and Developmental Regulation

The Spo0J and Soj proteins of B. subtilis belong to a widespread family of bacterial proteins required for accurate segregation of plasmids and chromosomes. Spo0J binds to several sites around the oriC region of the chromosome, which are organized into compact foci that may play a centromere-like role in active chromosome segregation. We now show that Soj has a role in organization or compaction of Spo0J-oriC complexes and possibly other regions of the nucleoid. This activity is accompanied by a dynamic localization pattern in which Soj protein undergoes assembly and disassembly into large nucleoid-associated patches on a timescale of minutes. The dynamic behavior of Soj, like its previously described transcriptional repression activity, is controlled by Spo0J. These interactions may constitute a checkpoint coupling developmental transcription to cell cycle progression.

Direct evidence for active segregation of oriC regions of the Bacillus subtilis chromosome and co‐localization with the Spo0J partitioning protein

We have developed methods for labelling regions of the Bacillus subtilis chromosome with the nucleotide analogue 5‐bromodeoxyuridine (BrdU) and for subcellular visualization of the labelled DNA. Examination of oriC‐labelled chromosomes in outgrowing spores has provided direct evidence for active segregation of sister chromosomes. Co‐immunodetection of Spo0J and BrdU‐labelled DNA has directly confirmed the expected close association between this chromosome partitioning protein and the oriC region of the chromosome. The results provide further support for the notion that bacterial cells use an active mitotic‐like mechanism to segregate their chromosomes.

An expanded view of bacterial DNA replication

A protein-interaction network centered on the replication machinery of Bacillus subtilis was generated by genome-wide two-hybrid screens and systematic specificity assays. The network consists of 91 specific interactions linking 69 proteins. Over one fourth of the interactions take place between homologues of proteins known to interact in other organisms, indicating the high biological significance of the other interactions we report. These interactions provide insights on the relations of DNA replication with recombination and repair, membrane-bound protein complexes, and signaling pathways. They also lead to the biological role of unknown proteins, as illustrated for the highly conserved YabA, which is shown here to act in initiation control. Thus, our interaction map provides a valuable tool for the discovery of aspects of bacterial DNA replication.

Selection of the midcell division site in Bacillus subtilis through MinD‐dependent polar localization and activation of MinC

Bacterial cell division commences with the assembly of the tubulin‐like protein, FtsZ, at midcell to form a ring. Division site selection in rod‐shaped bacteria is mediated by MinC and MinD, which form a division inhibitor. Bacillus subtilis DivIVA protein ensures that MinCD specifically inhibits division close to the cell poles, while allowing division at midcell. We have examined the localization of MinC protein and show that it is targeted to midcell and retained at the mature cell poles. This localization is reminiscent of the pattern previously described for MinD. Localization of MinC requires both early (FtsZ) and late (PbpB) division proteins, and it is completely dependent on MinD. The effects of a divIVA mutation on localization of MinC now suggest that the main role of DivIVA is to retain MinCD at the cell poles after division, rather than recruitment to nascent division sites. By overexpressing minC or minD, we show that both proteins are required to block division, but that only MinD needs to be in excess of wild‐type levels. The results suggest a mechanism whereby MinD is required both to pilot MinC to the cell poles and to constitute a functional division inhibitor.

The importance of morphological events and intercellular interactions in the regulation of prespore-specific gene expression during sporulation in Bacillus subtilis

We have established a time course for the early morphological events of sporulation in Bacillus subtilis and related this to changes in gene expression, particularly those occurring in the prespore compartment. We have also systematically studied the effects of mutations in various regulatory (spo) genes on prespore‐specific gene expression. On the basis of these results, and those of other laboratories, at least four distinct temporal classes of prespore‐specific gene expression can now be distinguished. The first class begins within 15min of the formation of the spore septum, and requires the σ;F form of RNA polymerase. The second class, also directed by RNA polymerase containing σ;F, begins soon after the completion of prespore engulfment, and depends on an intercellular signal from the mother cell. This transcription results in synthesis of σ;G. However, σ;G activity, directing the third class of gene expression, appears only about 30min later and is dependent on the completion of prespore engulfment and on further interactions with the mother cell. The fourth class of gene expression has been described. The results demonstrate that the prespore programme of gene expression incorporates a series of control points modulated by information from the mother cell and on progress through the morphogenetic process.

A magnesium-dependent mreB null mutant: implications for the role of mreB in Bacillus subtilis

MreB shares a common prokaryotic ancestor with actin and is present in almost all rod‐shaped bacteria. MreB proteins have been implicated in a range of important cell processes, including cell morphogenesis, chromosome segregation and cell polarity. The mreB gene frequently lies at the beginning of a cluster of genes, immediately upstream of the conserved mreC and mreD genes. RNA analysis showed that in Bacillus subtilis mreB is co‐transcribed with mreC and that these genes form part of an operon under the control of a promoter(s) upstream of mreB. Construction of an in‐frame deletion of mreB and its complementation by mreB+ only, in trans, established that the gene is important for maintenance of cell width and cell viability under normal growth conditions, independent of polar effects on downstream genes. Remarkably, virtually normal growth was restored to the mreB null mutant in the presence of high concentrations of magnesium, especially when high concentrations of the osmoprotectant, sucrose were also present. Under these conditions, cells could be maintained in the complete absence of an mreB gene, with almost normal morphology. No detectable effect on chromosome segregation was evident in the mutant, nor was there an effect on the topology of nascent peptidoglycan insertion. A GFP–MreB fusion was used to look at the localization of MreB in live cells. The pattern of localization was similar to that previously described, but no tight linkage to nucleoid positioning was evident. Propagation of the mreB null mutant in the absence of magnesium and sucrose led to a progressive increase in cell width, culminating in cell lysis. Cell division was also perturbed but this effect may be secondary to the disturbance in cell width. These results suggest that the major role of MreB in B. subtilis lies in the control of cell diameter.