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J Errington

Publications and source records attributed to J Errington.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1989

Differential gene expression during sporulation in Bacillus subtilis: regulation of the spoVJ gene.

The process of spore formation in the Gram-positive bacterium Bacillus subtilis is a simple developmental system controlled by 50 or more genes. The complex pattern of regulatory interactions between these genes is beginning to be elucidated. spoVJ is a poorly characterized locus in which mutations affect spore development at a relatively late stage (Stage V). We have now cloned and physically characterized the spoVJ locus, and analysed its expression by lacZ fusion. Expression of spoVJ is temporally delayed until about two hours after the initiation of sporulation. Its expression is also spatially restricted to the mother cell compartment; as such, it represents the earliest known mother-cell-specific event. Control of spoVJ transcription is complex: expression is dependent upon the products of all of the spoO genes and on some of the spoII genes but it is independent of all later genes except spoIIID. As spoIIID mutations do not affect prespore development, this gene must be an important early determinant of mother-cell-specific gene expression.

Bacillus subtilis↗
PubMed1989

The role of the sporulation gene spoIIIE in the regulation of prespore-specific gene expression in Bacillus subtilis.

The spoIIIG gene encodes a sigma factor that determines prespore-specific gene expression during sporulation in Bacillus subtilis. Correct spatial and temporal expression of the spoIIIG gene depends on a number of other sporulation (spo) genes, but only one of these genes, spoIIIE, has a specific effect on spoIIIG expression and not on gene expression in the other differentiating cell, the mother cell. However, the spoIIIE gene is expressed predominantly before differentiation begins. Thus, its product must play an important role in sensing or determining the spatial localization of prespore-specific gene expression in this system.

Amino Acid Sequence↗
PubMed1989

Do electrode and lead design differences for permanent cardiac pacing translate into clinically demonstrable differences? (Comparison of sintered platinum and activated vitreous and porous carbon electrodes).

A randomized prospective study was undertaken to compare the electrical performances of three permanent, endocardial, tined pacing leads with different electrode designs--sintered platinum, vitreous carbon, and porous carbon. Ninety-nine patients received one of the leads (S80 31; 423S 32; S100 36). Acute R wave amplitude and ST elevation of the native endocardial electrogram, voltage threshold, impedance, and current flow at four pulse durations (0.25-1.0 msec) were measured. Voltage thresholds were measured noninvasively at each of four pulse durations at 2 days and 1, 3, and 6 months after implantation. No significant differences were found in sensing properties, or current flow at threshold at 0.5 msec pulse duration. The 423S lead had a significantly higher impedance at threshold and both a higher impedance and lower current flow at 5 V. No significant differences in threshold voltages were found between the three leads at any pulse duration, at any of the assessed times after implantation. Six-month thresholds for the S80, 423S, and S100 leads were 1.18 +/- 0.35, 1.17 +/- 0.29, and 1.06 +/- 0.38 V respectively at 0.5 msec pulse duration. Differences between 'high performance' pacing leads need to be of a greater order of magnitude before they can be exploited to give any real clinical advantage to patients.

Aged↗
PubMed1988

Branched pattern of regulatory interactions between late sporulation genes in Bacillus subtilis.

We measured the synthesis of dipicolinic acid (DPA) during sporulation in spo mutants of Bacillus subtilis by a sensitive biological assay based on cross-feeding of a spoVF mutant strain and also chemically. Many spo mutations, including several that block sporulation at stage III, did not prevent synthesis of DPA but instead prevented its incorporation into the spore. In general, strains with mutations in loci that are expressed in the spore compartment synthesized DPA, whereas strains with mutations in most of the loci that are expressed in the mother-cell compartment did not. Transcription of the gerE gene, as measured by DNA-RNA hybridization, followed a dependence pattern very similar to that of DPA synthesis. However, the dependence patterns of the two operons show that at about stage IV of sporulation there is a branch in the sequence of operon expression in the mother cell. One branch leads through spoVC to synthesis of DPA synthetase, and the other leads through spoVD to expression of gerE.

Bacillus subtilis↗
PubMed1988

Transcriptional regulation and structure of the Bacillus subtilis sporulation locus spoIIIC.

The spoIIIC locus of Bacillus subtilis has been cloned from the lambda library of Ferrari et al. (E. Ferrari, D. J. HEnner, and J. A. Hoch, J. Bacteriol. 146:430-432, 1981) by using as an assay transformation of the mutant allele spoIIIC94 to the wild type. Regulation of the spoIIIC locus was studied by hybridization of cloned spoIIIC DNA to RNA pulse-labeled at various times during growth and sporulation. The relative rate of transcription of the spoIIIC locus was highest 3 h after the end of growth. The DNA sequence of the spoIIIC transcription unit indicated the coding capacity for a small protein (138 amino acids) having significant similarity with one domain of RNA polymerase sigma factors. Interruption of this coding sequence by an insertion mutation caused cells to become Spo-.

Amino Acid Sequence↗
PubMed1987

Two separable functional domains in the sigma-subunit of RNA polymerase in Bacillus subtilis?

The sigma-subunit of RNA polymerase is responsible for promoter recognition in prokaryotes [(1969) Nature 221, 43-46]. Alterations in the sigma-subunit are thought to be involved in controlling 'global' changes in gene expression, such as those involved in differentiation in the spore-forming bacterium Bacillus subtilis [(1981) Cell 25, 582-584]. Stragier et al. [(1985) FEBS Lett. 195, 3-11] have proposed that sigma-factors are composed of two domains: a C-terminal domain involved in promoter recognition and an N-terminal domain involved in interactions with RNA polymerase. We have sequenced another developmental gene from B. subtilis, spoIIIC, and the strong homology of its predicted product suggests that it too may be a sigma-factor. However, the spoIIIC product is small and lacks completely the conserved N-terminal domain of the sigma-subunits. I propose that the product of the spoIIIC gene may carry out the DNA-recognition functions of a sigma-factor but that it probably requires an auxiliary factor to interact with core RNA polymerase.

Amino Acid Sequence↗
PubMed1987

Upper limit for DNA packaging by Bacillus subtilis bacteriophage phi 105: isolation of phage deletion mutants by induction of oversized prophages.

We have determined the upper size limit for DNA packaging in Bacillus subtilis bacteriophage phi 105 by examining the plaque-forming and transducing capabilities of lysates made from strains containing prophages of various sizes. The upper size limit for efficient packaging of the phage genome appears to be about 40.2 kb, which is about 1 kb larger than the wild-type genome. This places an upper limit of about 5 kb on the size of insertions that can be accommodated in phi 105 transfection cloning vectors, such as phi 105J27. Induction of prophages that exceed that upper limit, followed by selection for plaque formation or transduction, provides a powerful means of isolating phage deletion mutants. A comparison of the location of each deletion with the resultant phenotype has enabled us to identify non-essential regions of the phage genome, and regions that are required for tail biosynthesis and for host cell lysis.

Bacillus subtilis↗
PubMed1987

Construction of improved bacteriophage phi 105 vectors for cloning by transfection in Bacillus subtilis.

A series of improved phage vectors have been constructed, based on Bacillus subtilis bacteriophage phi 105, which can be used to clone genes in B. subtilis by direct transfection of protoplasts. The new vectors, designated phi 105J23, phi 105J24, phi 105J27 and phi 105J28, show frequencies of plaque formation that are equal to those of wild-type phi 105. This represents at least a 10-fold improvement over phi 105J9, the vector used in previous cloning experiments. Two of the new vectors phi 105J27 and phi 105J28 incorporate a mutation, cts-52, that renders the prophage temperature inducible. This has made it possible to devise a rapid small-scale procedure for screening progeny phage for the presence of inserted DNA. The usefulness of the new vectors is illustrated in the accompanying paper by cloning more than 20 B. subtilis sporulation genes.

Bacillus subtilis↗
PubMed1987

Cloning in Bacillus subtilis by transfection with bacteriophage vector phi 105J27: isolation and preliminary characterization of transducing phages for 23 sporulation loci.

Bacteriophage cloning vector phi 105J27, the construction of which is described in an accompanying paper, has been used for shotgun cloning of sporulation genes in Bacillus subtilis. Various genomic libraries have been constructed and screened for the presence of recombinant phages capable of transducing strains containing sporulation (spo) mutations to Spo+. Of a total of 30 spo loci tested, transducing phages have been isolated for 23, more than half of the known spo loci. Included are nine loci (spo0D, spo0J, spoIIIA, spoIIIE, spoIIIF, spoIVF, spoVB, spoVH and spoVJ) that do not appear to have been cloned previously. Preliminary genetic characterization of some of the new clones by a rapid screening procedure has enabled the status of various sporulation loci to be clarified.

Bacillus subtilis↗
PubMed1987

Dependent sequences of gene expression controlling spore formation in Bacillus subtilis.

Sporulation in Bacillus subtilis is a simple form of differentiation that involves the formation of a two-cell organism; mother cell and prospective spore. It is regulated by at least 50 operons, some mono- and some polycistronic. These are expressed in a dependent sequence which branches at an early stage so that there are independent lines of expression in both cells; further ramifications then occur in both cell compartments. Three DNA-binding proteins and two sigma factors have so far been identified as probable regulators of the dependent sequence. Most of the operons of the sequence are expressed in the first four hours and the last two stages occur mainly by self-assembly of proteins.

Bacillus subtilis↗
PubMed1986

A general method for fusion of the Escherichia coli lacZ gene to chromosomal genes in Bacillus subtilis.

A series of plasmids has been constructed that can be used to fuse the beta-galactosidase gene (lacZ) of Escherichia coli to chromosomal genes of Bacillus subtilis. Insertion of the lacZ gene is facilitated by the use of a selectable chloramphenicol acetyl-transferase (cat) gene. The latter is included, along with the lacZ gene, in a single DNA fragment or 'cartridge' that can be removed from the plasmid with a variety of different restriction endonucleases. Methods applicable to any cloned B. subtilis gene are described that enable the lac-cat cartridge to be inserted at specific sites, or at random, directly into the B. subtilis chromosome in a single step. These single-copy chromosomal fusions can be readily transferred, by selection for chloramphenicol resistance, to a temperate phage such as phi 105, to permit a more extensive genetic analysis of the expression of the target gene. Alternatively, the lac-cat cartridge and flanking DNA sequences can be transferred into different genetic backgrounds by transformation. These techniques have been used to construct, in a single step, lac fusions to genes in the sporulation operons spoIIA and spoVA.

Bacillus subtilis↗
PubMed1986

Use of a lacZ gene fusion to determine the dependence pattern of sporulation operon spoIIA in spo mutants of Bacillus subtilis.

A spoIIA::lacZ gene fusion has been used to investigate the dependence pattern of expression of the spoIIA operon during sporulation in Bacillus subtilis. beta-Galactosidase activity, encoded by the hybrid gene, begins to appear about 30 to 60 min after the induction of sporulation. spoIIA expression is dependent upon the products of all of the known spoO loci but on none of the 'later' loci tested. The beta-galactosidase activity falls after 1.5 h in Spo+ cells and in late-blocked mutants, but continued accumulation of the enzyme occurs in certain stage II mutants. Kinetic experiments suggest that the fall in activity may be, in part, the result of regulation at the level of translation. Mutations in several loci, spo0J, spoIIIF and spoVIC, delay expression of the operon by 1-3 h. The significance of these results in terms of models for the control of gene expression during sporulation is discussed.

Bacillus subtilis↗
PubMed1986

Use of a lacZ gene fusion to determine the dependence pattern and the spore compartment expression of sporulation operon spoVA in spo mutants of Bacillus subtilis.

A spoVAA::lacZ gene fusion has been used to study expression of the spoVA operon during sporulation in Bacillus subtilis. beta-Galactosidase activity, encoded by the fusion gene, begins to be produced about 2.5 h after the induction of sporulation, well before the phenotypic consequences of spoVA mutations are manifested. spoVA expression is dependent on all of the known spo0 and spoII loci and on some of the 'early' spoIII loci, but not on 'later' loci. Several lines of evidence suggest that spoVA expression occurs only in the spore compartment. The implications of this observation for models of the overall regulation of gene expression during sporulation are discussed.

Bacillus subtilis↗