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J Errington

Publications and source records attributed to J Errington.

At least 19 recordsLinked to original sources
PubMed1992

A novel Bacillus subtilis expression vector based on bacteriophage phi 105.

We have developed a novel expression vector based on the bacteriophage phi 105, and employed it for the production of mutant beta-lactamases in Bacillus subtilis. Expression of the beta-lactamase-encoding gene was low when cloned into the prophage under the control of its own promoter. However, expression was considerably elevated when the gene was inserted into the phage genome in the same orientation as phage transcription. A defective phi 105 vector was constructed with a deletion removing a region needed for cell lysis, and with a mutation in the immunity repressor, rendering it temperature sensitive. Production of beta-lactamase could then be induced by a shift in temperature and without concomitant cell lysis, facilitating purification of the protein from the culture supernatant. This phage has considerable potential for development as a vector for controllable production of heterologous proteins in B. subtilis.

Bacillus subtilis↗
PubMed1992

Structure and function of the spoIIIJ gene of Bacillus subtilis: a vegetatively expressed gene that is essential for sigma G activity at an intermediate stage of sporulation.

The spo-87 mutation is one of two sporulation mutations originally used to define the spo0J locus of Bacillus subtilis. We now show that it blocks sporulation after completion of prespore engulfment (stage III). Surprisingly, the operon is expressed vegetatively, probably from a sigma A-dependent promoter, and its expression is shut down at the transcriptional level at about the onset of sporulation. DNA sequencing reveals that the locus defined by spo-87, which we now designate spoIIIJ, consists of a bicistronic operon. However, only the first gene is essential for sporulation; the function of the second cistron is cryptic. The predicted SpoIIIJ product has an M(r) of 29,409. It probably forms a lipoprotein and is rich in basic and hydrophobic amino acids. Mutations in spoIIIJ abolish the transcription of prespore-specific genes transcribed by the sigma G form of RNA polymerase but not transcription of the spoIIIG gene encoding sigma G. The SpoIIIJ product could be involved in a signal transduction pathway coupling gene expression in the prespore to events in the mother cell, or it could be necessary for essential metabolic interactions between the two cells.

Amino Acid Sequence↗
PubMed1992

Establishment of cell-specific transcription during sporulation in Bacillus subtilis.

One of the most intriguing questions posed by bacterial spore formation concerns the establishment of cell-specific gene expression in the prespore and mother cell. Recent results now suggest that sigma factors, in addition to their temporal roles in the control of gene expression, may also be the key determinants of differential gene expression during sporulation in Bacillus subtilis. The genes encoding two sporulation-specific sigma factors, sigma E and sigma F, are expressed soon after the initiation of sporulation, before the formation of the spore septum that separates the prespore and mother cell compartments. It now appears that sigma E and sigma F direct transcription only after septation and then in a specific cell type, suggesting that the segregation of the sigma activities after septation is a key event in the establishment of differential gene expression. The mechanism responsible for this segregation is complex, involving at least seven other gene products. We discuss possible models for the interactions between the sigma factors and the establishment of cell-specific transcription.

Bacillus subtilis↗
PubMed1992

Characterization of a sporulation gene, spoIVA, involved in spore coat morphogenesis in Bacillus subtilis.

Mutations in the spoIVA locus of Bacillus subtilis abolish cortex synthesis and interfere with the synthesis and assembly of the spore coat. We have characterized the cloned spoIVA locus in terms of its physical structure and regulation during sporulation. The locus contains a single gene capable of encoding an acidic protein of 492 amino acids (molecular weight, 55,174). The gene is transcribed from a sigma E-dependent promoter soon after the formation of the spore septum. A genetic test indicated that expression of spoIVA is only necessary in the mother cell compartment for the formation of a mature spore. This, together with the phenotypic properties of spoIVA mutations, would be in accord with the hypothesis that sigma E is only active after septation and in the mother cell compartment.

Amino Acid Sequence↗
PubMed1991

Possible intermediate steps in the evolution of a prokaryotic developmental system.

Sigma factors (sigma) are transcription factors that operate global switches in gene expression in prokaryotes. They work by directing core RNA polymerase to specific cis-acting promoter sequences; each sigma has a cognate class of promoters with specific sequence characteristics. In Bacillus subtilis four different sigma factors have been implicated in the regulation of gene expression during spore formation, which is a simple differentiation system involving two cell types. In this review I show how the modern developmental system may have arisen from a primitive organism that used only two sigma factors, by a series of steps involving gene duplication and divergence. The increasing sophistication of eukaryotic developmental systems may reflect similar evolutionary processes.

Amino Acid Sequence↗
PubMed1991

The role of sigma F in prespore-specific transcription in Bacillus subtilis.

Sporulation in Bacillus subtilis is a simple developmental system in which a single cell undergoes differentiation to two 'sister' cells, namely the prespore and the sporangium. Prespore-specific gene expression is largely dependent on the synthesis of a transcription factor, sigma G. Transcription of spolllG, the gene encoding sigma G, is under precise temporal and spatial control, requiring the products of at least eight genes that are expressed in the pre-divisional cell. Here we show that the product of one of these genes, another sigma factor, sigma F, is by itself sufficient to direct transcription of spolllG in non-sporulating cells. The results indicate that the cell-specificity of prespore gene expression is determined by a mechanism that exerts temporal and spatial control over the activity of sigma F.

Bacillus subtilis↗
PubMed1991

A model for asymmetric septum formation during sporulation in Bacillus subtilis.

Many differentiation processes in both prokaryotes and eukaryotes begin with an asymmetric division, producing 'daughter' cells that differ in size and developmental fate. This is particularly obvious in the well-studied prokaryotic life cycles of Caulobacter and Bacillus. In no system, however, is the mechanism of asymmetric division understood. Here I propose a model for the mechanism of asymmetric division during sporulation in Bacillus subtilis. The model explains both the timing and asymmetric localization of spore-septum formation. It also explains the morphological phenotypes of various asporogenous (spo) mutants.

Bacillus subtilis↗
PubMed1991

Sequential activation of dual promoters by different sigma factors maintains spoVJ expression during successive developmental stages of Bacillus subtilis.

The spoVJ gene of Bacillus subtilis encodes a 36 kDa protein and is expressed only in the mother cell. spoVJ has an interesting pattern of regulation during sporulation because it is expressed from sequentially activated promoters. These promoters, designated P1 and P2, are under the control of different sigma factors, sigma E and sigma K, which become active at separate times during sporulation. Removal of promoter P1, leaving promoter P2 active, resulted in about a 30-minute delay in the formation of heat-resistant spores and demonstrated that the expression of spoVJ from both promoters is essential for normal sporulation. A comparison is made between the sequences of the spoVJ promoters and the promoters of other genes dependent upon sigma E and sigma K.

Alkaline Phosphatase↗
PubMed1991

The spoIIIA operon of Bacillus subtilis defines a new temporal class of mother-cell-specific sporulation genes under the control of the sigma E form of RNA polymerase.

We have cloned and characterized a 5 kbp region of the Bacillus subtilis chromosome and show that it contains the promoter-proximal part of the spoIIIA locus. The locus consists of a polycistronic operon containing at least three genes. We show that the operon is regulated at the transcriptional level, from a promoter that is first activated about 80 minutes after the induction of sporulation, immediately after septation. Expression of spoIIIA in different spo mutant backgrounds correlates with the ability of each strain to synthesize the sporulation-specific sigma factor, sigma E. Moreover, synthesis of sigma E in vegetative cells by use of an inducible promoter causes expression of mother-cell-specific genes spoIID, spoIIIA, and spoIIID, but not the prespore-specific genes, spoIIIG and spoVA. We suggest that sigma E may be the primary determinant of mother-cell-specific gene expression and that the SpoIIID protein exerts an additional level of regulation on spoIIIA, apparently by acting as a transcriptional repressor. Since the onset of spoIIID expression occurs about 10 minutes after that of spoIIIA, spoIIIA expression is transient. Thus spoIIIA defines a third temporal class of gene controlled by the sigma E form of RNA polymerase.

Amino Acid Sequence↗
PubMed1991

Chromosome strand segregation during sporulation in Bacillus subtilis.

After the initiation of spore formation in Bacillus subtilis, the products of the final round of DNA replication segregate into two cells, i.e. the prespore and the mother cell. The prespore, which is known to contain a single completed chromosome, develops into a mature endospore which can be readily separated from mother cells and non-sporulating cells on the basis of its resistance properties. We have used a procedure originally developed to label the terminus region of the B. subtilis chromosome to specifically label the newly synthesized strands of DNA during the final round of DNA replication before sporulation. We have purified prespore DNA and used strand-specific probes to measure the radioactivity incorporated. The results show that the sister chromosomes segregate at random into the prespore. This result has implications for the segregation of chromosomes during vegetative growth and for the generation of cellular asymmetry during sporulation.

Bacillus subtilis↗
PubMed1991

Genetic regulation of morphogenesis in Bacillus subtilis: roles of sigma E and sigma F in prespore engulfment.

Electron microscopic examination of sporulating cultures of wild-type Bacillus subtilis revealed that the morphological events previously characterized as stages II and III can be divided into four substages, namely, stages IIi, IIii, IIiii, and III. The ultrastructural phenotypes of several stage II mutant strains indicate that each of the four substages has a biochemical and genetic basis. Two of the genes needed for the transition from stage II to stage III encode transcription factors sigma E and sigma F. Their roles during spore morphogenesis have been the subject of much speculation. We now show that sigma E controls genes involved in the morphological transition from stage IIi to stage IIii and then stage IIiii, while the transition to stage III may be determined by genes controlled by sigma F. The results also indicate the existence of at least two undiscovered sporulation genes involved in B. subtilis spore morphogenesis.

Bacillus subtilis↗
PubMed1991

The Bacillus subtilis spo0J gene: evidence for involvement in catabolite repression of sporulation.

Previous observations concerning the ability of the Bacillus subtilis bacteriophages SP10 and PMB12 to suppress mutations in spo0J and to make wild-type sporulation catabolite resistant suggested that spo0J had a role in catabolite repression of sporulation. This suggestion was supported in the present report by the ability of the catabolite-resistant sporulation mutation crsF4 to suppress a Tn917 insertion mutation of the B. subtilis spo0J locus (spo0J::Tn917 omega HU261) in medium without glucose. Although crsF4 and SP10 made wild-type B. subtilis sporulation catabolite resistant, neither crsF4 nor SP10 caused a mutant with spo0J::Tn917 omega HU261 to sporulate in medium with glucose. Sequencing the spo0J locus revealed an open reading frame that was 179 codons in length. Disruption of the open reading frame resulted in a sporulation-negative (Spo-) phenotype that was similar to those of other spo0J mutations. Analysis of the deduced amino acid sequence of the spo0J locus indicated that the spo0J gene product contains an alpha-helix-turn-alpha-helix unit similar to the motif found in lambda Cro-like DNA-binding proteins.

Amino Acid Sequence↗
PubMed1990

Differential gene expression during sporulation in Bacillus subtilis: structure and regulation of the spoIIID gene.

The gene spoIIID, which is essential for spore formation in Bacillus subtilis, was cloned and sequenced. It consists of one open reading frame which would encode a 93-amino-acid protein with a classic helix-turn-helix motif, characteristic of sequence-specific DNA-binding proteins. SpoIIID protein is a previously identified transcription factor, capable of altering the specificity of RNA polymerase containing sigma K in vitro (Kroos et al., 1989). The spoIIID83 mutation (by which the locus was originally identified), was sequenced and found to be a single base substitution in the ribosome binding site upstream of the spoIIID open reading frame. A transcriptional fusion to lacZ was constructed and used to examine the regulation of spoIIID. Expression of spoIIID occurred only during sporulation, beginning 1.5 to 2 hours after the initiation of sporulation. The dependence of spoIIID expression on other spo loci suggests that it is mother-cell-specific, and that it is transcribed by sigma E-containing RNA polymerase.

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of mutations in the gene encoding an endogenous Bacillus subtilis beta-galactosidase and its regulator.

We have isolated mutations that appear to inactivate the gene (lacA) encoding an endogenous beta-galactosidase activity in Bacillus subtilis and in a closely linked negative regulatory element (lacR). Both genes map to the hisA-thrA region. The lacA mutations may help to avoid some of the problems arising from the use of the Escherichia coli lacZ gene as a reporter gene in B. subtilis.

Bacillus subtilis↗
PubMed1990

The spoIIIA locus is not a major determinant of prespore-specific gene expression during sporulation in Bacillus subtilis.

During sporulation in Bacillus subtilis, expression of several prespore-specific genes is strongly dependent on the spoIIIE and spoIIIG gene products. Previous reports have also indicated a requirement for the products of the spoIIIA locus. However, we have now systematically studied six different well-defined spoIIIA mutations and find that, relative to spoIIIE and spoIIIG mutations, they have only a minor effect on the expression of two different prespore-specific genes, spoVA and sspA. Moreover, we have shown that strain IS37, which has been used as a spoIIIA mutant in several previous studies, actually contains a lesion in the spo0A gene. We conclude that spoIIIA has a relatively minor or indirect role in the regulation of prespore-specific gene expression.

Bacillus subtilis↗
PubMed1990

Use of integrational plasmid excision to identify cellular localization of gene expression during sporulation in Bacillus subtilis.

Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two sister cells, the prespore and the mother cell. Many of the genes that regulate sporulation (spo genes) are thought to be expressed differentially. However, direct demonstration of differential gene expression, by fractionation of prespore and mother cell proteins, is possible only at a relatively late stage of development. H. De Lencastre and P. J. Piggot (J. Gen. Microbiol. 114:377-389, 1979) have described a genetic method for determining the cellular location of the requirement for spo gene expression. Here we describe a similar method based on the use of integrational plasmids that can insertionally inactivate any given spo gene. Loss of the integrated plasmid by homologous recombination leads to the restoration of spo gene function. If this occurs just before sporulation begins, the phenotypes of the progeny of heat-resistant spores should depend on whether the gene is required in the prespore or the mother cell. Thus, we show that for known prespore-specific genes, such as spoIIIG and spoVA, only phenotypically Spo+ progeny that have lost the integrated plasmid are produced. In contrast, for mother-cell-specific genes, such as spoIIIC and spoVJ, a substantial proportion of the progeny are asporogenous, having retained the integrated plasmid. On the basis of our results, the spoIID and spoIIIA genes, which are expressed soon after division, appear to be required only in the mother cell compartment.

Bacillus subtilis↗
PubMed1989

A new bacteriophage vector for cloning in Bacillus subtilis and the use of phi 105 for protein synthesis in maxicells.

Zabarovsky and Allikmets [Gene 42 (1986) 119-123] have described a cloning procedure based on partial filling-in of vector and target DNA cohesive ends, which strongly enriches for recombinant molecules with single insertions. Improved Bacillus subtilis bacteriophage phi 105 vectors containing unique cloning sites for SalI have been constructed to take advantage of the partial fill-in method. The new vectors have been used to construct B. subtilis genomic libraries from which several sporulation loci have been isolated, including five not previously cloned. On inserting a promoterless lacZ gene into the cloning site, beta-galactosidase (beta Gal) was detected at a late stage in lytic phage growth, indicating that phage transcription is directed through the cloning site. When UV-irradiated cells ('maxicells') were infected with the recombinant phage containing the lacZ gene, in the presence of labelled amino acids, a protein of the expected Mr for beta Gal was visualised, in addition to the phage proteins. This system should provide a useful general approach for the identification of the products of cloned genes from B. subtilis and other Gram-positive organisms.

Bacillus subtilis↗