Biomedical subjects

J Errington

Publications and source records attributed to J Errington.

At least 55 records · Page 3Linked to original sources

PubMed1998

A fixed distance for separation of newly replicated copies of oriC in Bacillus subtilis: implications for co-ordination of chromosome segregation and cell division.

The Spo0J protein of Bacillus subtilis is required for normal chromosome segregation and forms discrete subcellular assemblies closely associated with the oriC region of the chromosome. Here we show that duplication of Spo0J foci occurs early in the DNA replication cycle and that this requires the initiation of DNA replication at oriC but not elongation beyond the nearby STer sites. Soon after duplication, sister oriC/Spo0J foci move rapidly apart to achieve a fixed separation of about 0.7 microm, reminiscent of the segregation of eukaryotic chromosomes on the mitotic spindle. The magnitude of the fixed separation distance may explain how chromosome segregation is kept in close register with cell growth and the initiation mass for DNA replication. It could also explain how segregation can proceed accurately in the absence of cell division. The kinetics of focal separation suggest that one role of Spo0J protein may be to facilitate formation of separate sister oriC complexes that can be segregated.

Bacillus subtilis↗

PubMed1998

Characterization of the essential cell division gene ftsL(yIID) of Bacillus subtilis and its role in the assembly of the division apparatus.

We have identified the Bacillus subtilis homologue of the essential cell division gene, ftsL, of Escherichia coli. Repression of ftsL in a strain engineered to carry a conditional promoter results in cell filamentation, with a near immediate arrest of cell division. The filaments show no sign of invagination, indicating that division is blocked at an early stage. FtsL is also shown to be required for septation during sporulation, and depletion of FtsL blocks the activation but not the synthesis of the prespore-specific sigma factor, sigmaF. Immunofluorescence microscopy shows that depletion of FtsL has little or no effect on FtsZ ring formation, but the assembly of other division proteins, DivIB and DivIC, at the site of division is prevented. Repression of FtsL also results in a rapid loss of DivIC protein, indicating that DivIC stability is dependent on the presence of FtsL, in turn suggesting that FtsL is intrinsically unstable. The instability of one or more components of the division apparatus may be important for the cyclic assembly/disassembly of the division apparatus.

Bacillus subtilis↗

PubMed1998

Establishment of prespore-specific gene expression in Bacillus subtilis: localization of SpoIIE phosphatase and initiation of compartment-specific proteolysis.

Immunofluorescence microscopy was used to study the establishment of compartment-specific transcription during sporulation in Bacillus subtilis. Analysis of the distribution of the anti-anti-sigma factor, SpoIIAA, in a variety of mutant backgrounds supports a model in which the SpoIIE phosphatase, which activates SpoIIAA by dephosphorylation, is sequestered onto the prespore face of the asymmetric septum. Thus, prespore-specific gene expression apparently arises as a result of the compartmentalization of SpoIIE protein. The results also suggest the existence of at least two compartment-specific programs of proteolysis, one dependent on the mother cell-specific sigma factor sigma E and the other dependent on the prespore-specific sigma factor sigma F.

Bacillus subtilis↗

PubMed1998

Bacillus subtilis cell cycle as studied by fluorescence microscopy: constancy of cell length at initiation of DNA replication and evidence for active nucleoid partitioning.

Fluorescence microscopic methods have been used to characterize the cell cycle of Bacillus subtilis at four different growth rates. The data obtained have been used to derive models for cell cycle progression. Like that of Escherichia coli, the period required by B. subtilis for chromosome replication at 37 degrees C was found to be fairly constant (although a little longer, at about 55 min), as was the cell mass at initiation of DNA replication. The cell cycle of B. subtilis differed from that of E. coli in that changes in growth rate affected the average cell length but not the width and also in the relative variability of period between termination of DNA replication and septation. Overall movement of the nucleoid was found to occur smoothly, as in E. coli, but other aspects of nucleoid behavior were consistent with an underlying active partitioning machinery. The models for cell cycle progression in B. subtilis should facilitate the interpretation of data obtained from the recently introduced cytological methods for imaging the assembly and movement of proteins involved in cell cycle dynamics.

Bacillus subtilis↗

PubMed1997

The complete genome sequence of the gram-positive bacterium Bacillus subtilis.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Bacillus subtilis↗

PubMed1997

Dynamic, mitotic-like behavior of a bacterial protein required for accurate chromosome partitioning.

The Bacillus subtilis spo0J gene is required for accurate chromosome partitioning during growth and sporulation. We have characterized the subcellular localization of Spo0J protein by immunofluorescence and, in living cells, by use of a spo0J-gfp fusion. We show that the Spo0J protein forms discrete stable foci usually located close to the cell poles. The foci replicate in concert with the initiation of new rounds of DNA replication, after which the daughter foci migrate apart inside the cell. This migration is independent of cell length extension, and presumably serves to direct the daughter chromosomes toward opposite poles of the cell, ready for division. During sporulation, the foci move to the extreme poles of the cell, where they function to position the oriC region of the chromosome ready for polar septation. These observations provide strong evidence for the existence of a dynamic, mitotic-like apparatus responsible for chromosome partitioning in bacteria.

Amino Acid Sequence↗

PubMed1997

Septal localization of the SpoIIIE chromosome partitioning protein in Bacillus subtilis.

The 787 amino acid SpoIIIE protein of Bacillus subtilis is required for chromosome partitioning during sporulation. This process differs from vegetative chromosome partitioning in that it occurs after formation of the septum, apparently by transfer of the chromosome through the nascent septum in a manner reminiscent of plasmid conjugation. Here we show that SpoIIIE is associated with the cell membrane, with its soluble C-terminal domain located inside the cell. Immunofluorescence microscopy using affinity-purified anti-SpoIIIE antibodies shows that SpoIIIE is targeted near the centre of the asymmetric septum, in support of a direct role for SpoIIIE in transport of DNA through the septum. We also report on the isolation of a mutation affecting the N-terminal hydrophobic domain of SpoIIIE that interferes with targeting to the septum and blocks DNA transfer. This mutation also causes de-localization of the activity of the normally prespore-specific sigma factor, sigmaF, consistent with the notion that SpoIIIE can form a seal between the chromosomal DNA and the leading edge of the division septum.

Bacillus subtilis↗

PubMed1997

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

The Bacillus subtilis divIVA 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.

Adenosine Triphosphatases↗

PubMed1997

Direct evidence for active segregation of oriC regions of the Bacillus subtilis chromosome and co-localization with the SpoOJ 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.

Bacillus subtilis↗

PubMed1997

Isolation and characterization of the lacA gene encoding beta-galactosidase in Bacillus subtilis and a regulator gene, lacR.

We have isolated transposon insertions in the lacA gene encoding an endogenous beta-galactosidase of Bacillus subtilis. Upstream of the putative operon containing lacA is a negative regulator, lacR, which encodes a product related to a family of regulators that includes the lactose repressor, lacI, of Escherichia coli. New strains with insertions in the lacA gene should be of use in studies using lacZ fusions in B. subtilis.

Bacillus subtilis↗

PubMed1997

Bacillus subtilis SpoIIID protein binds to two sites in the spoVD promoter and represses transcription by sigmaE RNA polymerase.

The Bacillus subtilis spoVD gene encodes a penicillin-binding protein required for spore morphogenesis. SpoIIID is a sequence-specific DNA-binding protein that activates or represses the transcription of many different genes. We have defined the spoVD promoter region and demonstrated that it is recognized by sigmaE RNA polymerase in vitro and that SpoIIID represses spoVD transcription. Two strong SpoIIID-binding sites were mapped in the spoVD promoter region, one overlapping the -35 region and the other encompassing the -10 region and the transcriptional start site.

Bacillus subtilis↗

PubMed1996

Control of the cell-specificity of sigma F activity in Bacillus subtilis.

Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two cell types that are formed by an asymmetric cell division. Major changes in the pattern of transcription during sporulation are brought about by the synthesis of new sigma factors (sigma), which are subunits of RNA polymerase that determine promoter specificity. Transcription in the smaller prespore cell type is initiated by a sigma factor called sigma F, the activity of which is subject to tight spatial and temporal control. It is negatively regulated by an anti-sigma factor, SpoIIAB, which is in turn controlled by an anti-anti-sigma factor, SpoIIAA. SpoIIAA and SpoIIAB participate in two contrasting reactions in vitro. In the presence of ATP, the proteins interact transiently and SpoIIAA is inactivated by phosphorylation on a specific serine residue; SpoIIAA then remains free to inhibit sigma F. In the presence of ADP, SpoIIAA binds tightly to SpoIIAB, and sigma F is set free. Release of sigma F activity in vivo might thus be effected by a prespore-specific reduction in the ATP/ADP ratio. Genetic experiments have implicated a fourth protein, called SpoIIE, in this system. It now appears that SpoIIE has two important and independent functions in the establishment of the prespore-specific transcription by sigma F. First it regulates sigma F activity, probably acting as a phosphatase to regenerate the active, non-phosphorylated form of SpoIIAA. Second it controls the formation of the septum that generates the prespore compartment. Combination of these two functions in a single polypeptide may provide a means of coupling gene expression with morphogenesis.

Amino Acid Sequence↗

PubMed1996

Bifunctional protein required for asymmetric cell division and cell-specific transcription in Bacillus subtilis.

During sporulation in Bacillus subtilis an asymmetric cell division gives rise to unequal progeny called the prepore and the mother cell. Gene expression in the prespore is initiated by cell-specific activation of the transcription factor sigma(F). Three proteins participate in the regulation of sigma(F) activity. The first, SpoIIAB, is an inhibitor of sigma(F), that is, an anti-sigma factor. SpoIIAB is also a protein kinase that catalyzes phosphorylation of the second regulatory protein SpoIIAA (the anti-anti-sigma factor), and thus inactivates it. A third protein, SpoIIE, was shown recently to be able to dephosphorylate SpoIIAA-P in vitro. Here we show that SpoIIE is a bifunctional protein with two critical roles in the establishment of cell fate. First, we confirm by the use of in vivo experiments that it regulates the release of sigma(F) activity by dephosphorylating SpoIIAA-P. Second, we show that SpoIIE is needed for normal formation of the asymmetric septum that separates the prespore from the mother cell. Combination of these two functions in a single polypeptide may serve to couple the release of the cell-specific transcription factors with the formation of the differentiating cells.

Bacillus subtilis↗

PubMed1996

Determination of cell fate in Bacillus subtilis.

On starvation, the soil bacterium Bacillus subtilis stops dividing and initiates sporulation, a simple developmental process involving the differentiation of two cell types. Sporulation begins with a reorganization of the cell cycle, to produce cells with the size and chromosome content appropriate for the developmental process. The central division that would normally occur, to produce a pair of identical daughter cells, is blocked and the cell divides asymmetrically to produce a small, polar prespore cell and a much larger mother cell. The developmental fates of the two cells are dictated by the localized activation of cell-specific transcription factors, which are controlled by mechanisms that respond to the cellular asymmetry.

Bacillus subtilis↗

PubMed1996

Compartmentalized distribution of the proteins controlling the prespore-specific transcription factor sigmaF of Bacillus subtilis.

BACKGROUND: Differential gene expression during sporulation in the prespore and mother cell of Bacillus subtilis is dependent on the correct timing and localization of the activity of specific transcription (sigma) factors. The first sigma factor activated is sigmaF, which directs gene expression specifically in the prespore compartment. Release of sigmaF activity is tightly controlled through a series of complex interactions involving an anti-sigma factor, SpoIIAB, an anti-anti-sigma factor SpoIIAA and a phosphoprotein phosphatase SpoIIE. In vitro studies have shown that SpoIIAB binds to sigmaF, preventing transcription of the sigmaF regulon, and that it can also phosphorylate SpoIIAA, thereby inactivating it. However, non-phosphorylated SpoIIAA can displace sigmaF from SpoIIAB. The SpoIIE phosphatase provides a means of reactivating SpoIIAA-P. RESULTS: We have directly determined the cellular distributions of sigmaF, SpoIIAB, SpoIIAA-P and SpoIIAA during sporulation, using recently developed immunofluorescence methods. While sigmaF activity is restricted to the prespore, the protein is present in both compartments. As development proceeds the sigmaF signal disappears. The anti-sigma factor SpoIIAB is also distributed throughout both cells and rapidly disappears from both cellular compartments soon after sigmaF becomes active. Disappearance of SpoIIAB seems to be closely associated with the activation of the second prespore-specific sigma factor sigmaF. The distribution of phosphorylated SpoIIAA closely mimics that of SpoIIAB, being non-compartmentalized and disappearing soon after sigmaF activation occurs. Significantly, the active, non-phosphorylated form of the anti-anti-sigma factor, SpoIIAA, accumulates in the prespore just before sigmaF becomes active. CONCLUSION: These results support the hypothesis that the accumulation of SpoIIAA within the prespore is the single most important requirement for activation of sigmaF.

Bacillus subtilis↗

PubMed1996

Establishing differential gene expression in sporulating Bacillus subtilis: phosphorylation of SpoIIAA (anti-anti-sigmaF) alters its conformation and prevents formation of a SpoIIAA/SpoIIAB/ADP complex.

Sigma-factor F (sigmaF) is a key transcription factor that initiates prespore development in Bacillus subtilis. Its activity is controlled by an anti-sigma factor, SpoIIAB, which is also a protein kinase that phosphorylates the anti-anti-sigma factor SpoIIAA. We have examined our earlier prediction that SpoIIAA must undergo a major change in its properties when phosphorylated. Upon gel filtration in the presence of ADP, SpoIIAA-P was eluted from a Superdex column much later than SpoIIAB, whereas SpoIIAA was coeluted with SpoIIAB, indicating the formation of a protein/protein complex. The complex contained ADP, and had two monomers of SpoIIAA to each SpoIIAB dimer. Its dissociation constant was 13 mu M. Gel permeation on high-performance liquid chromatography (HPLC) suggested an apparent molecular mass for SpoIIAA-P which was much higher (23.5 kDa) than that of SpoIIAA (15.8 kDa), but Ferguson plots showed that SpoIIAA-P was not a phosphorylated dimer of SpoIIAA. Our tentative conclusion, that SpoIIAA and SpoIIAA-P differ markedly in conformation, was confirmed by the results of partial digestion with chymotrypsin.

Adenosine Diphosphate↗

PubMed1996

Timing and genetic regulation of commitment to sporulation in Bacillus subtilis.

Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two cell types called the prespore and the mother cell. The process is induced by nutrient deprivation and culminates with the formation of a mature spore, which is released by lysis of the mother cell. We have studied commitment to sporulation with several different assays. The results indicate that commitment occurs soon after the formation of the asymmetrically positioned division septum that separates the prespore and the mother cell. This is earlier than the previously postulated point of commitment, prespore engulfment by the mother cell. Commitment coincides approximately with activation of the early prespore- and mother-cell-specific sigma factors, sigma(E) and sigma(F).

Bacillus subtilis↗

PubMed1996

Structure, function and controls in microbial division.

Several crucial genes required for bacterial division lie close together in a region called the dcw cluster. Within the cluster, gene expression is subject to complex transcriptional regulation, which serves to adjust the cell cycle in response to growth rate. The pivotally important FtsZ protein, which is needed to initiate division, is now known to interact with many other components of the division machinery in Escherichia coli. Some biochemical properties of FtsZ, and of another division protein called FtsA, suggest that they are similar to the eukaryotic proteins tubulin and actin respectively. Cell division needs to be closely co-ordinated with chromosome partitioning. The mechanism of partitioning is poorly understood, though several genes involved in this process, including several muk genes, have been identified. The min genes may participate in both septum positioning and chromosome partitioning. Coupled transcription and translation of membrane-associated proteins might also be important for partitioning. In the event of a failure in the normal partitioning process, Bacillus subtilis, at least, has a mechanism for removing a bisected nucleoid from the division septum.

Bacillus subtilis↗