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Biomedical subjects

A Moir

Publications and source records attributed to A Moir.

At least 37 records · Page 2Linked to original sources
PubMed1996

Proteome of Salmonella typhimurium SL1344: identification of novel abundant cell envelope proteins and assignment to a two-dimensional reference map.

Forty-nine cell envelope proteins of Salmonella typhimurium SL1344 have been identified by microsequencing and assigned to a two-dimensional reference map. Ten of the sequenced proteins appear to be novel. Several others closely match currently hypothetical proteins or proteins found in other bacteria but not previously reported in salmonellae.

Amino Acid Sequence↗
PubMed1995

The construction of a reporter system and use for the investigation of Clostridium perfringens gene expression.

A reporter system was constructed to enable the study of gene expression in Clostridium perfingens. The system was based on plasmid shuttle vector pJIR410, which contained the C. perfringens erythromycin resistance gene. The vector was modified by the introduction of a DNA fragment comprising the open reading frame of the C. perfringens chloramphenicol acetyltransferase gene and flanking transcriptional terminators. The presence of a unique restriction site, engineered into the extreme 5' end of the open reading frame enabled a promoter region to be inserted to form an in-fram transcriptional fusion with catP. The system was tested by inserting the promoter region of the alpha-toxin gene of C. perfringens. The production of chloramphenicol acetyltransferase in C. perfringens was monitored during growth and the pattern of expression was shown to reflect levels of plc mRNA and alpha-toxin in the parent strain.

Amino Acid Sequence↗
PubMed1995

Salmonella typhimurium responses to a bactericidal protein from human neutrophils.

Bactericidal/permeability-increasing protein [BPI] is a cationic antimicrobial protein from neutrophils that specifically binds to the surfaces of Gram-negative bacteria via the lipid A component of lipopolysaccharide. To obtain information about the responses of Salmonella typhimurium to cell-surface damage by BPI, two-dimensional gel electrophoresis and N-terminal microsequencing were used to identify proteins that were induced or repressed following BPI treatment. The majority of the affected proteins are involved in central metabolic processes. Upon addition of BPI, the beta-subunit of the F1 portion of Escherichia coli ATP synthase was repressed threefold whereas six proteins were induced up to 11-fold. Three of the latter were identified as lipoamide dehydrogenase, enoyl-acyl carrier protein reductase, and the heat-shock protein HtpG. Additionally, a novel protein, BipA, was identified that is induced over sevenfold by BPI; sequence analysis suggests that it belongs to the GTPase superfamily and interacts with ribosomes. A conserved direct-repeat motif is present in the regulatory regions of several BPI-inducible genes, including the bipA gene. Only one of the BPI-responsive proteins was induced when cells were treated with polymyxin B, which also binds to lipid A. We therefore conclude that BPI and polymyxin B affect different global regulatory networks in S. typhimurium even though they bind with high affinity to the same cell-surface component.

Amino Acid Sequence↗
PubMed1994

Molecular genetical and phenotypical analysis of the gerM spore germination gene of Bacillus subtilis 168.

The gerM gene, encoding a single product of 22.5 kDa, has been identified by subcloning and sequencing of DNA recovered from adjacent to a Tn917 insertion. The gene product has a potential lipoprotein signal sequence, but otherwise has no homology to known sequences. Spores of the gerM mutant were more heat sensitive than wild-type, but their dipicolinic acid content was normal. The level of cortical peptidoglycan in mutant spores is also normal but release at germination of hexosamine-containing fragments, the breakdown products of cortex degradation, is less complete than wild-type. The sporulation, resistance and germination phenotypes of the gerM mutant would be consistent with the gene product having a role, either directly or indirectly, in peptidoglycan synthesis during sporulation.

Amino Acid Sequence↗
PubMed1994

Analysis of the expression and regulation of the gerB spore germination operon of Bacillus subtilis 168.

The gerB spore germination operon of Bacillus subtilis 168 is a homologue of the gerA spore germination operon. The expression and regulation of the gerB operon has been examined using a lacZ transcriptional fusion and the transcriptional start defined. The gerB operon is expressed during sporulation under the control of RNA polymerase containing the forespore-specific sigma factor, delta G. This is a further homology to the gerA operon, which is similarly regulated. It is predicted from the localization of expression and the encoded primary sequences that the GerB proteins are located at the inner spore membrane.

Bacillus subtilis↗
PubMed1992

Cloning and characterization of a gene from Bacillus stearothermophilus var. non-diastaticus encoding a glycerol dehydrogenase.

A 4.1-kb EcoRI fragment which includes the gene (gldA) encoding a glycerol dehydrogenase (G1DH; EC 1.1.1.6; glycerol:NAD oxidoreductase) from Bacillus stearothermophilus var. non-diastaticus has been cloned by virtue of its ability to restore glycerol utilisation to Escherichia coli glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD) mutants. Sequencing suggests that the gldA gene is likely to be monocistronic and encodes a protein of 39450 Da. The deduced amino acid composition and sequence of G1DH reveals that the protein is extremely similar to a characterized metal-dependent NAD-dependent G1DH from B. stearothermophilus RS93. The enzyme has limited homology to the iron-activated alcohol dehydrogenase of Zymomonas mobilis and the butanol dehydrogenase of Clostridium acetobutylicum.

Amino Acid Sequence↗
PubMed1992

Selection of a thermostable variant of chloramphenicol acetyltransferase (Cat-86).

The moderate thermophile Bacillus stearothermophilus was used as a host in which to detect more thermostable variants of the B.pumilus chloramphenicol acetyltransferase (Cat-86) protein. Seventeen mutants were isolated and detected by their ability to grow in the presence of chloramphenicol at a previously restrictive temperature (58 degrees C). The genes encoding these proteins were sequenced; all 17 mutants carried the same C to T transition that conferred an amino acid substitution of alanine by valine at position 203 of the protein sequence. The wild-type and one mutant Cat-86 protein were purified to homogeneity using affinity chromatography, and kinetic and thermal stability studies were undertaken. Both enzymes had similar sp. act. in the region of 215 U/mg, with Km values for chloramphenicol in the range 13.8-15.4 microM and for acetyl CoA in the range 13.6-15.5 microM. The A203V mutant shows greater stability than the wild-type Cat-86 protein at temperatures above 50 degrees C and appears to pass through a transition state between 48 and 50 degrees C.

Bacillus↗
PubMed1991

Analysis of transcriptional control of the gerD spore germination gene of Bacillus subtilis 168.

The gerD locus of Bacillus subtilis comprises a single gene whose function is essential for the germination of B. subtilis spores in media containing asparagine, glucose, and fructose. The expression of gerD has been characterized by using a chromosomal lacZ fusion to the gerD promoter. The promoter is switched on at the same time as the synthesis of glucose dehydrogenase, 2.5 h after sporulation has been initiated in the developing forespore. The gerD gene is not expressed in spoIIB or spoIIIA, -IIIB, -EIII, -FIII, or -IIIG mutants, but it is expressed in spoIIIC and -IIID and spoIVA mutant backgrounds. The in vivo transcriptional start point of the gene has been mapped by primer extension analysis, and sequences upstream from the start point show considerable homology with the promoter consensus sequences recognized by RNA polymerase containing the forespore-specific sigma factor sigma G (E sigma G). gerD is transcribed in vitro by E sigma G with a similar if not identical start point to that found in vivo, and expression of the gene can be rapidly induced in vegetative cells following the induction of sigma G synthesis. These results indicate that gerD is another member of the sigma G regulon, which includes a number of genes expressed only in the forespore compartment of sporulating cells of B. subtilis.

Bacillus subtilis↗
PubMed1990

Characterization and cloning of the gerC locus of Bacillus subtilis 168.

A Bacillus subtilis gerC spore germination mutant demonstrating a temperature-sensitive response to L-alanine as germinant has been characterized in detail. The gerC58 mutation is 50% cotransformed with aroB in the gene order gerC-aroB-trpC. The mutation is responsible for a severe growth defect which is manifest at all growth temperatures and is most extreme on rich media. A second, unlinked, mutation in the original strain suppressed this growth defect, but spores of the suppressed strain failed to germinate in alanine at 42 degrees C. As this germination defect is dependent on the presence of the gerC58 allele, it is likely to be the direct result of a mutant gerC protein. The gerC gene therefore appears to have a role in both spore germination and vegetative cell growth. A gene library of BclI-digested B. subtilis chromosomal DNA was constructed in phage vector phi 105J27. A derivative containing the gerC region was obtained by complementation of the growth defect of an unsuppressed gerC58 strain. This phage contained a 6.3 kb insert of bacterial DNA, which is above the reported packaging limit of the phage. It failed to form visible plaques, although it could be handled as a prophage and sufficient phage particles be isolated to allow characterization of the insert. A deletion derivative generated in vitro and carrying only 2.9 kb of insert DNA also complemented the gerC defect. This gerC locus is the second locus to be implicated in alanine-stimulated germination. The first, gerA, is a developmentally controlled operon whose gene products are present only in the spore. This study of gerC, in contrast, reveals a role in spore germination for a normally essential vegetative protein.

Alanine↗
PubMed1990

The regulation of transcription of the gerA spore germination operon of Bacillus subtilis.

The gerA operon of Bacillus subtilis 168 comprises three genes concerned with the triggering of spore germination by L-alanine and its analogues. The expression of this operon has been characterized using chromosomal lacZ fusions to the gerA promoter. The gerA promoter is switched on 2.5-3 hours after the initiation of sporulation, in parallel with glucose dehydrogenase. A high proportion of the gerA-driven beta-galactosidase detected in sporulating cells is found in the mature spore; the gerA promoter is therefore active in the forespore compartment of the sporulating cell. The gerA promoter is not expressed in spoO, spoII or spoIIIA, B, E and G mutant backgrounds, but is expressed in spoIIIC and D and in spoIV and V mutants. The in vivo transcriptional startpoint of the operon has been mapped by primer extension experiments; sequences upstream from this startpoint show significant homology with recognition sequences for RNA polymerase containing sigma G (E sigma G). The gerA operon was transcribed in vitro by E sigma G with a startpoint identical to that used in vivo, and expression of the gerA operon was rapidly induced in vegetative cells by induction of sigma G synthesis. These data indicate that the gerA operon is an additional member of the sigma G regulon, which includes a number of genes expressed in parallel only in the forespore compartment of sporulating B. subtilis cells.

Bacillus subtilis↗
PubMed1989

Sigma H-directed transcription of citG in Bacillus subtilis.

The RNA polymerase sigma factor sigma H is essential for the onset of endospore formation in Bacillus subtilis. sigma H also is required for several additional stationary-phase-specific responses, including the normal expression of several genes that are required for the development of competence for DNA uptake. It is necessary to identify the genes that are transcribed by sigma H RNA polymerase (E sigma H) in order to understand the role of this sigma factor during the transition from exponential growth to stationary phase. Feavers et al. (Mol. Gen. Genet. 211:465-471, 1988) proposed that citG, the structural gene for fumarase, is transcribed from two promoters, one of which (citGp2 [P2]) may be used by E sigma H. It is likely that the citGp2 promoter is used by E sigma H because we found that this promoter was used accurately in vitro by E sigma H and directed expression of xylE in vivo. This xylE expression was dependent on spo0H, the structural gene for sigma H, and was independent of the citGp1 promoter. Comparison of the nucleotide sequences of several sigma H-dependent promoters showed that these sequences were similar at two regions approximately 10 and 35 base pairs upstream from the start points of transcription. These sequences may signal recognition of these promoters by E sigma H. Primer extension analyses were used to examine transcription from three sigma H-dependent promoters during growth and sporulation. The citGp2 promoter appeared to be active during the middle and late stages of exponential growth, whereas activation of the spoIIA promoter was delayed until after the end of exponential growth. Evidently, promoters used by E sigma H can display different temporal patterns of expression.

Bacillus subtilis↗
PubMed1989

Role of sigma H in expression of the fumarase gene (citG) in vegetative cells of Bacillus subtilis 168.

The fumarase gene (citG) of Bacillus subtilis is transcribed from two promoter regions, citGp1 and citGp2 (P1 and P2); the P2 promoter is used by the E sigma H form of RNA polymerase. In order to study the role of P1 and P2 in citG expression, the promoter region and various deletion derivatives that effectively separate P1 and P2 were fused to the Escherichia coli beta-galactosidase gene (lacZ) and introduced into the chromosome in single copy at the amyE locus. P1 functioned to provide a relatively low and stable basal level of fumarase activity throughout growth. In contrast, P2 activity was found to vary over at least a 50-fold range and was responsible for regulating fumarase activity during growth and sporulation in a rich medium and in response to changes in carbon source. To further investigate the role of sigma H in fumarase regulation, citGp2-lacZ fusions were introduced into a strain in which the expression of the chromosomal spoOH gene was under the control of the isopropylthiogalactopyranoside-inducible spac promoter. Induction of pspac did not lead to P2 induction, suggesting that citG expression is not regulated at the level of spoOH transcription.

Amino Acid Sequence↗
PubMed1988

The regulation of the fumarase (citG) gene of Bacillus subtilis 168.

The level of fumarase activity in Bacillus subtilis depends on the nutritional environment; in rich medium low vegetative levels increase towards the end of the exponential phase, whereas in minimal glucose medium levels are relatively high throughout growth. Analysis of the enzyme levels in spoO mutants has revealed that a functional spoOH gene is required for the efficient expression of fumarase in both media. This highlights a regulatory role for the spoOH gene product not only in control of postexponentially expressed genes, but also during vegetative growth in defined medium. S1 transcript mapping reveals three transcriptional startpoints for the fumarase structural gene (citG) in B. subtilis. The upstream promoter region P1, which appears to contain two transcriptional startpoints, is functional in both Escherichia coli and B. subtilis. Promoter P2, which is located closer to the structural gene, is only functional in B. subtilis. Transcription from this promoter is strictly dependent on a functional spoOH gene; this gene has recently been shown to encode a minor sigma factor.

Amino Acid Sequence↗
PubMed1987

The nucleotide sequence and gene organization of the gerA spore germination operon of Bacillus subtilis 168.

The nucleotide sequence of the second and third genes in the Bacillus subtilis spore germination locus, gerA, has been determined and the amino acid (aa) sequence was derived. Two open reading frames (ORFs), corresponding to genes II and III, encode 364-aa residue and 373-aa residue polypeptides, respectively. The gene II product, Mr 41,257, would contain long stretches of hydrophobic aa residues and may be a membrane protein; the gene III product, Mr 42,363, is relatively hydrophilic but possesses an apparent signal peptide for transfer across, and perhaps localisation on, a membrane. The ORFs for genes I and II overlap by eleven codons and the termination codon of gene II overlaps the initiation codon of gene III. Insertional inactivation experiments using integrational plasmids have indicated that the gerA locus is a single transcriptional unit. The expression of the gerA genes has been studied using a lacZ transcriptional fusion; they constitute a developmentally regulated operon.

Amino Acid Sequence↗
PubMed1985

The nucleotide sequence of a spore germination gene (gerA) of Bacillus subtilis 168.

The nucleotide sequence of a 2.1-kb fragment of Bacillus subtilis DNA that contains part of the spore germination locus, gerA, has been determined. An open reading frame (ORF) of 1440 bp (480 codons) has been identified which corresponds to the previously located complementation unit I of the gerA locus. The orientations of transcription of the gerA and of the adjacent fumarase (citG) gene are divergent. The deduced polypeptide product of the gerA gene, of Mr 53 506, contains both hydrophobic and hydrophilic domains and is likely to be membrane-associated.

Amino Acid Sequence↗