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C R Harwood

Publications and source records attributed to C R Harwood.

At least 19 recordsLinked to original sources
PubMed1997

An internal FK506-binding domain is the catalytic core of the prolyl isomerase activity associated with the Bacillus subtilis trigger factor.

Two major families of peptidylprolyl cis-trans-isomerases, the cyclophilins and the structurally unrelated FK506-binding proteins (FKBPs), have been identified as cellular factors involved in protein folding in vitro. Here we report on the biochemical characterization of a second prolyl isomerase of Bacillus subtilis that was purified from a cyclophilin-negative (ppiB null) mutant and was shown to be the trigger factor (TigBS). N-terminal sequencing of 27 amino acid residues of the purified protein revealed 100% identity to the deduced sequence encoded by the tig gene, sequenced as a part of the B. subtilis genome project. The tigBS gene, located at 246 degrees on the genetic map upstream of the clpX and lonA,B genes, encodes an acidic protein (pI 4.3) of 47.5 kDa. Purified and recombinant TigBS-His proteins share the same substrate specificity and catalytic activity (Kcat/K(m) of 1.5 microM-1 s-1); both are inhibited by the macrolide FK506 with IC50 the range of 500 nM. We also demonstrate that the prolyl isomerase activity of TigBS is mediated by an internal domain of about 13 kDa (homologous to FKPB12) that represents the catalytic core of the trigger factor.

Amino Acid Isomerases↗
PubMed1996

Sequence and transcriptional analysis of clpX, a class-III heat-shock gene of Bacillus subtilis.

The nucleotide sequence of clpX, which is localized between the tig (trigger factor) and the lon (ATP-dependent protease) genes at 245 degrees on the standard Bacillus subtilis (Bs) genetic map, was determined. The putative clpX gene codes for a 46-kDa protein of 421 amino acid (aa) residues. A comparison of the deduced aa sequence with those of the recently described bacterial clpX gene products from Synechocystis sp., Escherichia coli (Ec), Haemophilus influenzae and Azotobacter vinelandii revealed strong similarities. However, in contrast to Ec, clpX and clpP of Bs are located at different loci on the chromosome and are transcribed as monocistronic genes. A heat-inducible sigma A-like promoter was mapped upstream of the clpX structural gene, but no CIRCE element, characteristic of class-I heat-shock genes (e.g., groESL and dnaK), was found between the transcriptional and translational start sites. Although the majority of the heat-inducible general stress genes in Bs are under the control of the alternative sigma factor, sigma B, the heat induction of clpX appears to be sigma B-independent. The latter indicates that clpX belongs to class-III heat-inducible genes.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1996

Sequencing and functional analysis of the genome of Bacillus subtilis strain 168.

The international programme to sequence the 4.2 Mb genome of Bacillus subtilis, a model Gram-positive bacterium, is a joint project involving European, Japanese and US research groups. To date ca. 3.0 Mb of the genome has been sequenced, with the remaining 1.2 Mb expected to be completed in 1997. The amenability of B.subtilis to genetic manipulation, combined with the availability of extensive expertise on its biochemistry and physiology, makes this bacterium a valuable organism in which to investigate the properties of genes for which functions cannot be readily ascribed by standard methods.

Bacillus subtilis↗
PubMed1996

The dnaB-pheA (256 degrees-240 degrees) region of the Bacillus subtilis chromosome containing genes responsible for stress responses, the utilization of plant cell walls and primary metabolism.

Within the framework of the international programme to sequence the genome of Bacillus subtilis strain 168, we were allocated the region between dnaB (256 degrees) and pheA (240 degrees). The sequencing of this region is now complete and we report our primary analysis of the 114 kb region containing 114 ORFs. In addition to previously characterized genes, we have identified genes involved in the utilization of plant cell wall polysaccharides, stress responses and the metabolism of amino acids, cell walls, DNA and fatty acids. We also discuss various structural and physical features, including the orientation of genes with respect to replication, putative start and stop codons, ribosome binding sites and rho-independent transcription terminators.

Bacillus subtilis↗
PubMed1996

Phosphate-starvation-inducible proteins in Bacillus subtilis: a two-dimensional gel electrophoresis study.

A two-dimensional (2-D) gel electrophoresis study of Bacillus subtilis strain 168 identified 20 proteins that are strongly induced in response to phosphate starvation. The induction of nine of these phosphate-starvation-induced (Psi) proteins was dependent on a functional PhoR protein. PhoR is the histidine sensor-kinase component of a phosphate-concentration-sensing two-component regulatory system which, together with its partner response regulator PhoP, controls the expression of genes in the Pho regulon. Genes encoding PhoR-dependent Psi proteins are therefore likely to be members of the Pho regulon. Spo0A approximately P, the response regulator of the signal transduction pathway required for the induction of sporulation, has previously been shown to negatively affect the induction of the Pho regulon by repressing the phoP-phoR operon. The induction pattern of some PhoR-dependent Psi proteins was altered in a spo0A mutant such that their synthesis continued for longer than was found with the wild-type. The most abundant Psi protein, Psi1-3, was characterized by N-terminal sequencing of internal peptide fragments and shown to have a high similarity to an Escherichia coli protein which is involved in phosphate uptake during phosphate starvation.

Alkaline Phosphatase↗
PubMed1995

Detection of induced beta-galactosidase activity in individual non-culturable cells of pathogenic bacteria by quantitative cytological assay.

One Escherichia coli and two F' lac+ Salmonella strains were carbon and nitrogen stressed at 37 degrees C over 35 days in the presence or absence of chloramphenicol; the number, activity and culturability of cells in the resultant populations were studied. Active cells were enumerated by fluorescence microscopy after treatment with the lac inducer IPTG and cytological assay for beta-galactosidase. In all experiments, active and total cell counts remained within a three-fold range of each other and their initial values, while culturability fell by > 10(8)-fold and 10(3)-fold in chloramphenicol-treated and untreated preparations, respectively. Quantitative image analysis revealed different distributions of cell-specific fluorescence and indicated a progressive decline in the levels of induced enzyme activity in both E. coli and Salmonella enteritidis. It was concluded that the non-culturable cells studied retained inducible enzyme activity and that this activity did not result from a starvation-induced programme of gene expression. Whether or not such active but non-culturable cells are viable, they are clearly responsive and have the potential to influence their environment. The assay described can be applied to heterogeneous populations and environments and shows considerable potential for the study of gene expression at the single cell level.

Bacteriological Techniques↗
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↗
PubMed1992

Cloning and sequencing of a plasmid-mediated erythromycin resistance determinant from Staphylococcus xylosus.

A 2.3-kb DNA fragment cloned from plasmid pCH200, the largest (52 kb) of four plasmids detected in Staphylococcus xylosus, was found to confer resistance to 14-membered ring macrolides in Bacillus subtilis and Staphylococcus aureus. DNA-sequence analysis of the fragment revealed the presence of an open-reading frame, the deduced product of which was identical to one of the two ATP-binding domains encoded by the macrolide/streptogramin-B-resistance gene msrA of Staphylococcus epidermidis. The observation that a polypeptide homologous to the C-terminus of MsrA is capable of mediating erythromycin resistance in the absence of the N-terminal region is of significance both to the evolution and functional activity of members of the ATP-binding transport super-gene family.

Amino Acid Sequence↗
PubMed1992

Bacillus subtilis and its relatives: molecular biological and industrial workhorses.

The non-pathogenic bacterium Bacillus subtilis, since its first reported genetic transformation in 1959, has become a model system for the study of many aspects of the biochemistry, genetics and physiology of Gram-positive bacteria, and particularly of sporulation and associated metabolism. Extensive knowledge of the molecular biology of B. subtilis has led to the recent development of this bacterium as a host for the industrial production of heterologous proteins. Although difficulties have been encountered, these are being systematically addressed and overcome.

Bacillus subtilis↗
PubMed1991

Cell wall assembly in Bacillus megaterium: incorporation of new peptidoglycan by a monomer addition process.

The pattern of cross-linking in the peptidoglycan of Bacillus megaterium has been studied by the pulsed addition of radiolabeled diaminopimelic acid. The distribution of label in muropeptides, generated by digestion with Chalaropsis muramidase and separated by high-performance liquid chromatography, stabilized after 0.15 of a generation time. The proportion of label in the acceptor and donor positions of isolated muropeptide dimers stabilized over the same period of time. The results have led to the formulation a new model for the assembly of peptidoglycan into the cylindrical wall of B. megaterium by a monomer addition process. Single nascent glycan peptide strands form cross-linkages only with material at the inner surface of the wall. Maturation is a direct consequence of subsequent incorporation of further new glycan peptide strands, and there is no secondary cross-linking process. The initial distribution of muropeptides is constant. It follows that the final pattern of cross-linking in the wall is determined solely by, and can be forecast from, this repetitive pattern of incorporation. In a modified form, this model can also be applied to assembly of cell walls in rod-shaped gram-negative bacteria.

Bacillus megaterium↗
PubMed1989

Cell wall assembly in Bacillus subtilis: visualization of old and new wall material by electron microscopic examination of samples stained selectively for teichoic acid and teichuronic acid.

Uranyl acetate staining of thin sections allowed a distinction to be made between cell wall material that contains teichoic acid and that which contains teichuronic acid. The stain was used to study the pattern of wall assembly in Bacillus subtilis undergoing transitions between growth conditions leading to incorporation of the different anionic polymers. The results showed that new material is incorporated along the inner surface of the cylindrical region of the wall confirming, by a more direct method, results obtained earlier with teichoic acid specific phages. New material appears to be evenly distributed along the inner surface and no evidence was obtained for the presence of specific zones of incorporation.

Bacillus subtilis↗
PubMed1989

Cell wall assembly in Bacillus subtilis: partial conservation of polar wall material and the effect of growth conditions on the pattern of incorporation of new material at the polar caps.

The use of phage SP50 as marker for cell wall containing teichoic acid in Bacillus subtilis showed clear differences in the rates at which new wall material becomes exposed at polar and cylindrical regions of the wall, though the poles were not completely conserved. Following transition from phosphate limitation to conditions that permitted synthesis of teichoic acid, old polar caps fairly rapidly incorporated enough teichoic acid to permit phage binding. Electron microscopy suggested that the new receptor material spread towards the tip of the pole from cylindrical wall so that phages bound to an increasing proportion of the pole area until only the tip lacked receptor. Eventually, receptor was present over the whole polar surface. Direct electron microscopic staining of bacteria collected during transitions between magnesium and phosphorus limitations showed that new material was incorporated at the inner surface of polar wall and later became exposed at the outer surface by removal of overlying older wall. The apparent partial conservation of the pole reflected a slower degradation of the overlying outer wall at the pole than at the cylindrical surface, the rate being graded towards the tip of the pole. The relative proportions of the new wall material incorporated into polar and cylindrical regions differed in bacteria undergoing transitions that were accompanied by upshift or downshift in growth rate. These differences can be explained on the basis that growth rate affected the rate of synthesis of cylindrical but not septal wall.

Bacillus subtilis↗
PubMed1988

Isolation of Acanthamoeba from a cerebral abscess.

A 55-year-old diabetic aboriginal woman presented with a two-week history of fever, altered mental state and convulsions. On the basis of computed tomographic scanning a diagnosis of cerebral abscess was made. The pus that was drained produced no bacterial growth but, on microscopy, amoebic cysts were observed. Special cultures produced a growth of Acanthamoeba. The patient appeared to respond to drainage of the abscess and antiprotozoal therapy. Unfortunately, she developed necrotizing enteritis which led ultimately to her death. Antibiotic sensitivity and pathogenicity testing suggest that the Acanthamoeba were unusually virulent. The problems of diagnosis and management are discussed.

Acanthamoeba↗
PubMed1987

The effects of deletions in the leader sequence of cat-86, a chloramphenicol-resistance gene isolated from Bacillus pumilus.

The cat-86 gene of Bacillus pumilus, specifying a Cm-inducible CAT enzyme, was cloned previously into B. subtilis on plasmid pUB110. Various lines of evidence suggest that control of expression of this gene is at the level of translation and involves inverted complementary repeat sequences 5' to the initiation codon. A series of deletions have been generated in this region and their effects on the induction of cat-86 observed in B. subtilis, Escherichia coli and a number of ribosomal mutant strains of B. subtilis. The results indicate that the inverted complementary repeat sequences, which are capable of forming a stable stem-loop structure in the mRNA (delta G = -24.4 kcal/mol), form a barrier to translation in E. coli and B. subtilis.

Acetyltransferases↗
PubMed1987

Impact of plasmids and genetic change on the numerical classification of staphylococci.

Newly isolated bacterial strains often contain extrachromosomal DNA as plasmid DNA. These accessory components of the DNA gene pool confer additional phenotypic properties on their host but, despite this, little attention has been paid to the impact of plasmid-mediated characters on bacterial classification. In the present study, the effect of antibiotic resistance plasmids on the classification of representative staphylococci was determined using numerical phenetic techniques. Over sixty percent of the eighty-one test strains contained one or more plasmids which varied in molecular weight from 1.4 to 36 Mdal. Antibiotic resistance phenotypes were eliminated from strains of S. aureus, S. chromogenes, S. cohnii, S. hyicus and S. xylosus, and from a laboratory isolate, to give sixteen derivative strains. Fourteen had lost one or more plasmids and two had deleted plasmids. In addition three further derivative strains were isolated which showed no plasmid loss but exhibited gross phenotypic changes. The test and derivative strains were the subject of numerical phenetic analyses based on seventy-eight unit characters. Data were examined using the simple matching, Jaccard and pattern coefficients and clustering achieved using the unweighted pair group method with arithmetic averages algorithm. Cluster composition was not markedly affected by the statistics used or by test error, estimated at 1.02%. Numerically circumscribed clusters and subclusters were equated with the established species S. aureus, S. chromogenes, S. cohnii, S. hyicus, S. lentus, S. intermedius, S. sciuri and S. xylosus. The sixteen derivative strains with either lost or delected plasmids were recovered in the same cluster or subcluster as their corresponding parent indicating that the removal of plasmid-expressed characters had little effect on the structure of the numerical classification. In contrast, two of the three strains of S. xylosus with genomically-derived phenotypic variation formed a cluster that separated from their parent strain at the 70% similarity level in the SSM, UPGMA analysis.

Animals↗
PubMed1983

Nucleotide sequence of a Bacillus pumilus gene specifying chloramphenicol acetyltransferase.

Gene cat-86 of Bacillus pumilus, specifying chloramphenicol-inducible chloramphenicol acetyltransferase, was previously cloned in Bacillus subtilis on plasmid pUB110. The nucleotide sequence of cat-86 indicates that the gene encodes a protein of 220 amino acids and contains TTG as the translations-initiation codon. The proteins specified by cat-86 and the cat genes present on pC194, pC221 and Tn9 appear to share regions of amino acid sequence similarity. cat-86 is a structural gene on the B. subtilis expression plasmid pPL608. Restriction sites exist within the gene that should permit the product of inserted heterologous coding sequences to be synthesized in B. subtilis as fusion proteins.

Acetyltransferases↗
PubMed1981

Mecillinam susceptibility of Escherichia coli K-12 mutants deficient in the adenosine 3',5'-monophosphate system.

The mecillinam resistance of Escherichia coli K-12 mutants deficient in the enzyme responsible for the synthesis of adenosine 3',5'-monophosphate, adenylate cyclase, has been investigated. The results suggest that resistance to this antibiotic may be a consequence of the slow growth rate of these mutants rather than an intrinsic property of their genetic lesion.

Amdinocillin↗