Biomedical subjects

J Errington

Publications and source records attributed to J Errington.

At least 73 records · Page 4Linked to original sources

PubMed1996

The Bacillus subtilis soj-spo0J locus is required for a centromere-like function involved in prespore chromosome partitioning.

During sporulation in Bacillus subtilis a small prespore cell is formed by an asymmetric cell division. Pre-spore chromosome partitioning occurs by a specialised mechanism in which septation precedes chromosome movement. We show that the spo0J gene is needed to specify the orientation of the chromosome at the time of polar division and to impose directionality on the subsequent transport of the remainder of the chromosome through the septum. Both phenotypes may arise by disruption of a centromere-like apparatus that anchors the or/C region of the prespore chromosome in the pole of the cell.

Bacillus subtilis↗

PubMed1996

A complex four-gene operon containing essential cell division gene pbpB in Bacillus subtilis.

We have cloned and sequenced the promoter-proximal region of the Bacillus subtilis operon containing the pbpB gene, encoding essential penicillin-binding protein PBP2B. The first two genes in the operon, designated yllB and yllC, are significantly similar to genes of unknown function similarly positioned upstream of pbpB in Escherichia coli. Both B. subtilis genes are shown to be nonessential. The third B. subtilis gene, yllD, is essential, as is the correspondingly positioned ftsL gene of E. coli. The predicted product of yllD is similar to FtsL in size and distribution of charged residues but is not significantly related in primary amino acid sequence. The major promoter for the cluster lies upstream of the first gene, yllB, but at least one minor promoter lies within the yllC gene. The operon is transcribed throughout growth at a low level.

Amino Acid Sequence↗

PubMed1995

The Bacillus subtilis cell-division 135-137 degrees region contains an essential orf with significant similarity to murB and a dispensable sbp gene.

Sequence similarity analysis has revealed that orf2, in the cell division 135-137 degrees region of the Bacillus subtilis (Bs) chromosome, is the probable homolog of Escherichia coli murB (encoding a reductase involved in peptidoglycan synthesis). The amino-acid sequences of the two protein products show 24% identity (47% overall similarity), with several regions of higher similarity which may represent functional domains of the proteins. Attempts to insertionally inactivate orf2 were unsuccessful, strongly suggesting that it is an essential Bs gene. A small gene found in the same region as orf2, sbp (encoding the 'small basic protein'), was shown to be non-essential in Bs.

Amino Acid Sequence↗

PubMed1995

Postseptational chromosome partitioning in bacteria.

Mutations in the spoIIIE gene prevent proper partitioning of one chromosome into the developing prespore during sporulation but have no overt effect on partitioning in vegetatively dividing cells. However, the expression of spoIIIE in vegetative cells and the occurrence of genes closely related to spoIIIE in a range of nonsporulating eubacteria suggested a more general function for the protein. Here we show that SpoIIIE protein is needed for optimal chromosome partitioning in vegetative cells of Bacillus subtilis when the normal tight coordination between septation and nucleoid partitioning is perturbed or when septum positioning is altered. A functional SpoIIIE protein allows cells to recover from a state in which their chromosome has been trapped by a closing septum. By analogy to its function during sporulation, we suggest that SpoIIIE facilitates partitioning by actively translocating the chromosome out of the septum. In addition to enhancing the fidelity of nucleoid partitioning, SpoIIIE also seems to be required for maximal resistance to antibiotics that interfere with DNA metabolism. The results have important implications for our understanding of the functions of genes involved in the primary partitioning machinery in bacteria and of how septum placement is controlled.

Bacillus subtilis↗

PubMed1995

A conjugation-like mechanism for prespore chromosome partitioning during sporulation in Bacillus subtilis.

Spore formation in Bacillus subtilis begins with an asymmetric cell division that superficially resembles the division of vegetative cells. Mutations in the spoIIIE gene of B. subtilis partially block partitioning of one chromosome into the smaller (prespore) compartment of the sporulating cell. Point mutations that specifically block prespore chromosome partitioning affect a carboxy-terminal domain of SpoIIIE that shows significant sequence similarity to the DNA transfer (Tra) proteins of several conjugative plasmids of Streptomyces. In wild-type sporulating cells, the prespore chromosome passes through an intermediate stage resembling the state in which spoIIIE mutant cells are blocked. The prespore chromosome is then transferred progressively through the newly formed spore septum. We propose that translocation of the prespore chromosome occurs by a mechanism that is functionally related to the conjugative transfer of plasmid DNA.

Bacillus subtilis↗

PubMed1995

Characterization of an insertion in the phage phi 105 genome that blocks host Bacillus subtilis lysis and provides strong expression of heterologous genes.

A defective prophage vector, phi 105MU331, for high-level protein overproduction in Bacillus subtilis, was derived by random insertion of a lacZ reporter gene. The site of insertion not only provided efficient inducible transcription of heterologous genes, but also prevented lysis of the host cell. The region of the insertion in phi 105MU331 lies close to the right cohesive end of phi 105. DNA sequence analysis revealed that this region of phi 105 somewhat resembles the lysis cassette of various phages, including lambda. The site of insertion lies in a possible 'holin' gene, which could explain the block in host cell lysis. Dual promoters apparently responsible for the strong inducible transcription lie in an untranslated region just upstream from the putative holin gene. This region is probably equivalent to the site of the major late promoter and antiterminator of the lambdoid phages. The sequence features could, thus, account for the useful properties of the phi 105MU331 vector system.

Amino Acid Sequence↗

PubMed1995

Characterization of cell cycle events during the onset of sporulation in Bacillus subtilis.

To elucidate the process of asymmetric division during sporulation of Bacillus subtilis, we have measured changes in cell cycle parameters during the transition from vegetative growth to sporulation. Because the propensity of B. subtilis to grow in chains of cells precludes the use of automated cell-scanning devices, we have developed a fluorescence microscopic method for analyzing cell cycle parameters in individual cells. From the results obtained, and measurements of DNA replication fork elongation rates and the escape time of sporulation from the inhibition of DNA replication, we have derived a detailed time scale for the early morphological events of sporulation which is mainly consistent with the cell cycle changes expected following nutritional downshift. The previously postulated sensitive stage in the DNA replication cycle, beyond which the cell is unable to sporulate without a new cell cycle, could represent a point in the division cycle at which the starved cell cannot avoid attaining the initiation mass for DNA replication and thus embarking on another round of the cell cycle. The final cell cycle event, formation of the asymmetric spore septum, occurs at about the time in the cell cycle at which the uninduced cell would have divided centrally, in keeping with the view that spore septation is a modified version of vegetative division.

Bacillus subtilis↗

PubMed1995

Chromosome partitioning in bacteria.

This review addresses chromosome partitioning in Escherichia coli and Bacillus subtilis. The first part deals with events associated with completion of a round of replication to an extent that yields separable chromosomes. Events more directly involved in chromosome movement are covered in the second part. In the final section, a model for chromosome partitioning based on the information presented in the first two parts is presented.

Bacillus subtilis↗

PubMed1994

Role of interactions between SpoIIAA and SpoIIAB in regulating cell-specific transcription factor sigma F of Bacillus subtilis.

Genetic experiments have suggested that sigma F, the first compartment-specific transcription factor in sporulating B. subtilis, is regulated by an anti-sigma factor SpoIIAB and an anti-anti-sigma factor SpoIIAA. Previously, we reported biochemical results demonstrating that SpoIIAB is both a phosphokinase whose substrate is SpoIIAA and an inhibitor of sigma F-directed transcription. We now show that in the presence of SpoIIAB and ATP or ADP, SpoIIAA can undergo two alternative reactions. When ATP is present, SpoIIAA is phosphorylated rapidly and completely to SpoIIAA-phosphate, and SpoIIAB is immediately released; but in the presence of ADP, SpoIIAA forms a long-lasting complex with SpoIIAB. ADP is an inhibitor of the phosphorylation by ATP. Furthermore, we have mutated SpoIIAA at residue Ser 58, the target for phosphorylation, to aspartate or alanine. SpoIIAAS58D, which apparently resembles SpoIIAA-phosphate, is unable to make a complex with SpoIIAB and is devoid of anti-anti-sigma F activity, whereas SpoIIAAS58A, which cannot be phosphorylated, makes complexes with SpoIIAB in the presence of ADP or ATP and has constitutive anti-anti-sigma F activity both in vivo and in vitro. It seems likely that the alternative reactions of SpoIIAA and SpoIIAB, involving ADP or ATP, regulate the anti-anti-sigma capacity of SpoIIAA and hence the activity of sigma F.

Adenosine Diphosphate↗

PubMed1994

Sigma factors, asymmetry, and the determination of cell fate in Bacillus subtilis.

Soon after the initiation of sporulation, Bacillus subtilis divides asymmetrically to produce sister cells that have very different developmental fates. Recently, it has been proposed that the differential gene expression which begins soon after this division is due to cell-specific activation of the transcription factors sigma F and sigma E in the prespore and the mother cell, respectively. We describe the use of a method for the localization of gene expression in individual sporulating cells that lends strong support to the cell-specific localization of sigma F and sigma E activities. The dependence of sigma E activity on integrity of the gene encoding sigma F has led to the suggestion that activation of sigma F in the prespore leads to a directional signal that triggers activation of sigma E only in the mother cell. Here we show that sigma E actually specifies the fate of the mother cell; in the absence of sigma E, two prespore-like cells are made. The appearance of sigma F activity at both poles of a sigma E-deficient mutant supports the idea that sigma F normally remains latent in the mother cell and that its activation depends on some morphological or physiological feature of the prespore. We present a model for the generation of asymmetry and the establishment of cell fate in B. subtilis.

Bacillus subtilis↗

PubMed1994

Bacillus subtilis SpoIIIE protein required for DNA segregation during asymmetric cell division.

Sporulation in Bacillus subtilis begins with an asymmetric cell division, producing a smaller prespore and a larger mother cell, both of which contain intact copies of the chromosome. The spoIIIE gene is required for chromosome segregation into the prespore compartment. The effects of the spoIIIE36 mutation on sigma F-dependent transcription are an indirect consequence of the failure of certain genes to enter the cellular compartment in which their transcription factor has become active. SpoIIIE may also be required to prevent sigma F from becoming active in the mother cell.

Bacillus subtilis↗

PubMed1994

The Bacillus subtilis spoVD gene encodes a mother-cell-specific penicillin-binding protein required for spore morphogenesis.

The Bacillus subtilis spoVD gene has been cloned and sequenced. It encodes a 71,262 Da protein with extensive sequence similarity to penicillin-binding proteins from various organisms. The context of this gene in the B. subtilis chromosome, immediately upstream of the mur operon, suggests that it is related to the pbpB gene of Escherichia coli, which is involved in the synthesis of septal peptidoglycan during cell division. Expression of spoVD in E. coli leads to the synthesis of a membrane-associated protein of the size expected for SpoVD, which can bind labelled penicillin. However, insertional disruption of the spoVD gene has no effect on vegetative growth or division: a second pbp-like gene immediately upstream of spoVD is probably the functional homologue of E. coli pbpB. spoVD seems instead to have a specialized role in the morphogenesis of the spore cortex, which is a modified form of peptidoglycan. spoVD transcription appears to occur from a promoter recognized by the sigma E form of RNA polymerase. Analysis of the expression of a spoVD'-lacZ reporter gene supports this notion and indicates that a second level of negative regulation is dependent on the SpoIIID protein. SpoVD synthesis probably occurs only in the mother cell since both sigma E and SpoIIID are thought to be specific to this cell type. Such localization of SpoVD synthesis was supported by the results of a genetic test showing that expression of spoVD only in the mother cell is sufficient for spore formation. The results support the proposition that spore cortex formation is determined primarily by the mother cell.

Amino Acid Sequence↗

PubMed1994

Use of digitized video microscopy with a fluorogenic enzyme substrate to demonstrate cell- and compartment-specific gene expression in Salmonella enteritidis and Bacillus subtilis.

A rapid and sensitive method for detection of cell- and compartment-specific gene expression in individual cells of both Gram-negative and Gram-positive microorganisms is described. The method combines the use of gene fusions to lacZ, and a fluorogenic beta-galactosidase substrate, fluorescein-di-(beta-D-galactopyranoside), with digitized video microscopy. All of the reporter constructs tested were successfully detected. Secondary staining of the cells with a nucleic acid-specific dye, propidium iodide, allowed cells devoid of nucleic acid to be identified, while cell nucleoid shape and the morphological stage of development could be correlated with the location of beta-galactosidase activity. The double-staining procedure was used to show that gene expression can be induced in non-culturable cells of Salmonella enteritidis produced by carbon/nitrogen starvation. The resolution was sufficient to distinguish between cells at different morphological stages of sporulation in Bacillus subtilis. This highly sensitive and rapid method may have many other applications in basic and applied microbiology.

Bacillus subtilis↗

PubMed1993

An efficient expression and secretion system based on Bacillus subtilis phage phi 105 and its use for the production of B. cereus beta-lactamase I.

A novel expression system based on the Bacillus subtilis bacteriophage phi 105 has been developed to permit the high-level synthesis and secretion of beta-lactamase I (BlaI) from Bacillus cereus. Shotgun insertion of a promoterless lacZ gene into the phage genome permitted the identification of a clone producing large amounts of beta-galactosidase (beta Gal), indicating the transcription of the reporter gene from a strong phage promoter. The insertion also blocked lysis of the host cell. Although the insertion in the original prophage was complex, plasmid vectors and prophage derivatives have been developed to facilitate the replacement of lacZ with other genes for expression. The new prophages contain two additional mutations: an ind mutation, which greatly enhances the normally poor transformability of phi 105 lysogens, and a cts mutation, which allows thermo-induction of phage development and protein production. Induction of a derivative prophage containing the blaI gene from B. cereus resulted in the production of up to 500 micrograms of secreted BlaI per ml of culture supernatant.

Bacillus Phages↗

PubMed1993

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

The establishment of compartment-specific transcription in sporulating cells of B. subtilis is governed at the level of the activity of transcription factor sigma F. Genetic experiments have suggested that SpoIIAA and SpoIIAB, the other products of the sigma F operon, are involved in regulating sigma 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 sigma 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 sigma F activity, perhaps through the formation of a tight complex between SpoIIAB and phosphorylated SpoIIAA.

Amino Acid Sequence↗

PubMed1993

Cloning, DNA sequence, functional analysis and transcriptional regulation of the genes encoding dipicolinic acid synthetase required for sporulation in Bacillus subtilis.

Dipicolinic acid (DPA) is a small polar molecule that accumulates to high concentrations in bacterial endospores, and is thought to play a role in spore heat resistance, or the maintenance of heat resistance. Previous work has shown that mutations in the spoVF locus of Bacillus subtilis prevent the formation of DPA, and give rise to heat-sensitive spores. Addition of exogenous DPA during spore development led to the restoration of heat resistance. This suggested that the spoVF locus encoded dipicolinic acid synthetase, the enzyme thought to catalyse the single reaction needed to synthesise DPA from dihydroxydipicolinic acid, an intermediate in the lysine biosynthetic pathway. We have now cloned and sequenced the spoVF locus of Bacillus subtilis and show that it comprises two coordinately regulated genes, now designated dpaA and dpaB. Expression of fragments of the dpa operon in Escherichia coli has shown that the two gene products together specify DPA synthetase activity. The promoter of the dpa operon, which lies just upstream of the first gene, has been identified by primer extension analysis. Sequences in this region show strong sequence similarity to several promoters recognized by the sigma K form of RNA polymerase. Transcription from this promoter was detected four hours after the onset of sporulation, at about the same time that sigma K activity is known to appear. Furthermore, transcription was abolished by mutations in a series of genes that are known to be required for the synthesis of active sigma K. These results are in accordance with previous work indicating that DPA synthetase activity was present only during the late stages of sporulation and specifically in the mother cell compartment. Transcription was enhanced by a gerE mutation, indicating that, like the previously described cotA gene, spoVF is negatively regulated by GerE. The mother-cell-specific synthesis of an enzyme responsible for a compound that accumulates to high concentrations in the prespore raises interesting questions about intercellular transport mechanisms.

Amino Acid Sequence↗

PubMed1993

Effects of new mutations in the spoIIAB gene of Bacillus subtilis on the regulation of sigma F and sigma G activities.

The spoIIAB gene of Bacillus subtilis encodes an inhibitor of sigma F, a transcription factor that plays a crucial role in the establishment of prespore-specific gene expression during sporulation. The SpoIIAB protein can probably also inhibit a closely related sigma factor sigma G, which determines the later phase of prespore-specific transcription. We have isolated two new missense mutations in the spoIIAB gene. spoIIAB28 behaves like the previously described spoIIAB1 mutation, in that it mainly affects the activity of sigma G. In contrast, the spoIIAB22 mutation seems to be impaired mainly in its ability to inhibit sigma F. All three missense mutations are clustered in the N-terminal coding region of spoIIAB, suggesting that this region of the protein interacts with the sigma factors. The extreme N-terminal part of SpoIIAB may be specifically concerned with the regulation of sigma G activity.

Alleles↗

PubMed1993

DNA sequence of the murE-murD region of Bacillus subtilis 168.

The sequence of a 4.4 kbp region of DNA from Bacillus subtilis 168, lying between sporulation genes spoVD and spoVE, has been determined as part of the B. subtilis genomic sequencing programme. The region contains three genes with high sequence similarity to the murE, mraY and murD genes of Escherichia coli. The products of these genes are likely to catalyse various steps in the formation of the precursors for peptidoglycan synthesis in B. subtilis. The regions at 133 degrees on the standard genetic map of the B. subtilis chromosome, and in the 2 min region of the E. coli genetic map, are now shown to contain a large cluster of functionally related genes. Although the linear order of the genes in the cluster is conserved, three genes that are present in the E. coli chromosome, and which are likely to be essential for peptidoglycan synthesis in both organisms, are absent from this region of the B. subtilis chromosome. In general, the B. subtilis cluster differs from that of E. coli in having more extensive intergenic regions, with less potential for translational coupling.

Amino Acid Sequence↗