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P Reeves

Publications and source records attributed to P Reeves.

At least 37 records · Page 2Linked to original sources
PubMed1989

Cloning and nucleotide sequence of the Salmonella typhimurium LT2 gnd gene and its homology with the corresponding sequence of Escherichia coli K12.

The complete nucleotide sequence of the Salmonella strain LT2 gnd gene for 6-phosphogluconate dehydrogenase was determined. The gene contains 1404 bases and encodes a 468 amino acid polypeptide, which is the same as for Escherichia coli K12. The DNA sequence shows 14.8% difference between the two and the amino acid sequence 3.6% difference. Changes are mostly in the third codon base and most of the amino acid changes are conservative.

Amino Acid Sequence↗
PubMed1989

Identification and sequence of the gene for abequose synthase, which confers antigenic specificity on group B salmonellae: homology with galactose epimerase.

The O antigen of Salmonella group B strains contains the sugar abequose, whereas those from group A and D strains contain paratose or tyvelose in its place. This is the essential difference between these Salmonella groups. Only the final step in the biosynthesis of abequose differs from that of paratose, and the abequose confers on group B strains their specific O4 antigen. The gene, rfbJ, encoding the enzyme abequose synthase for this last specific step has been cloned, identified, and sequenced and has been shown to function in group A and D strains to make them O4+. This one gene thus differentiates group B from group A or group D salmonellae. The enzyme abequose synthase appears to be related to galactose epimerase, and the significance of this is discussed. The rfbJ gene and adjacent DNA is of much lower G+C content than is usual for salmonellae, indicating that the region did not originate in a salmonella but was transferred from outside.

Amino Acid Sequence↗
PubMed1989

Identification and sequence of rfbS and rfbE, which determine antigenic specificity of group A and group D salmonellae.

Salmonella group A, group B, and group D strains have paratose, abequose, and tyvelose, respectively, as the immunodominant sugar in their O antigens, which are otherwise identical; only the final steps differ in the biosynthetic pathways of these sugars. The gene rfbJ from a group B strain, encoding abequose synthase, the final and only unique step in the biosynthesis of CDP-abequose, has been cloned and sequenced (P. Wyk and P. Reeves, J. Bacteriol. 171:5687-5693, 1989). In this study, we locate and sequence rfbS and rfbE from serovars typhi and paratyphi, representative of groups A and D. Gene rfbS is present in both groups and encodes paratose synthase, which carries out a step parallel to that of abequose synthase, but the product is CDP-paratose. The DNA and inferred amino acid sequences are compared with those of rfbJ. We conclude that the genes are homologous, but the divergence is extremely ancient. Gene rfbE encodes CDP-tyvelose epimerase, which converts CDP-paratose to CDP-tyvelose in group D strains; the gene is active in group D strains, and we find it to be present in a mutant form in group A strains. These two genes encode the steps unique to groups A and D and, like rfbJ of group B, are of low G+C content, suggesting transfer from outside of salmonellae. The evolutionary origin of these genes is discussed.

Amino Acid Sequence↗
PubMed1987

Mediation of serum resistance in Salmonella typhimurium by an 11-kilodalton polypeptide encoded by the cryptic plasmid.

A cosmid bank of the DNA (including cryptic plasmid DNA) of a virulent strain of Salmonella typhimurium was prepared in Escherichia coli K12, and clones that contained cryptic plasmid DNA were detected by probing. Two such clones expressed a new outer membrane protein of 11 kilodaltons (kDa) and were serum resistant (E. coli K12 is serum sensitive). The gene encoding the 11-kDa protein was subcloned in a 2.1-kilobase fragment and shown to mediate serum resistance in both E. coli K12 and a cryptic plasmid-free (serum-sensitive) strain of S. typhimurium. The cryptic plasmid-free S. typhimurium strain did not express normal lipopolysaccharide, but introduction of the 11-kDa protein gene into the strain rendered the strain serum resistant without restoration of normal lipopolysaccharide synthesis. The 11-kDa protein gene was not sufficient to restore either macrophage resistance or virulence to a cryptic plasmid-free strain of S. typhimurium.

Animals↗
PubMed1987

Occupational health and safety: a NSW hospital experience.

This paper outlines the experience of the establishment of an Occupational Health and Safety Committee at the Canterbury Hospital and Area Health Service and its achievements over the past two years. As a priority the committee members reviewed and refined safety reporting systems. The committee also considered the need to complement retrospective reporting with a proactive hazard prevention program. A system of departmental inspections was introduced in order to meet this perceived need. Following identification of hazards, education programmes and control strategies were developed to minimise risks. These strategies included some very innovative approaches to perennial problems.

Accident Prevention↗
PubMed1985

Intermediates in the synthesis of TolC protein include an incomplete peptide stalled at a rare Arg codon.

TolC is a minor outer membrane protein of Escherichia coli K 12 and is initially synthesized as a precursor. A distinct intermediate polypeptide of Mr about 46 000 was consistently observed at the initial stages of biosynthesis. The further elongation of this peptide can be blocked by chloramphenicol. We have investigated the cause of the temporary accumulation of the 46 000-Mr intermediate and we postulate that the presence of a rare codon AGA (Arg) at codon 402 of the tolC mRNA halts translating ribosomes owing to a limiting amount of the tRNAArg (AGA) species in the cell. The translation of tolC mRNA can be increased by providing T4 tRNAArg (AGA), encoded on a plasmid.

Arginine↗
PubMed1985

Molecular characterisation of the Stc- mutation of Escherichia coli K-12.

The previously described Stc- (suppressor of TolC) mutation modifies the phenotype of tolC mutants from OmpF- to OmpF+. Restriction mapping of chromosomal DNA from Stc+ and Stc- strains was performed to investigate the nature of the mutation which was shown to be a deletion, upstream of the ompC gene. DNA from the region of the deletion was cloned into pUC18 and a 650-bp PstI-EcoRI fragment was sequenced. The deletion started 49 bp upstream of the AUG start codon of the ompC gene, thus removing part of the ompC promoter and the whole of the micF gene. We suggest that the deletion of micF gives rise to the Stc- phenotype since the effect of micF expression is assumed to reduce ompF expression, and the Stc- phenotype involves increase in ompF expression.

Bacterial Outer Membrane Proteins↗
PubMed1985

High-level synthesis of the phage lambda outer-membrane protein from the cloned lom gene.

A 2.7-kb KpnI-EcoRI fragment carrying the lom gene of bacteriophage lambda has been cloned into plasmid pPR42 and recloned into the SmaI site of pUC9. Large quantities of Lom were seen in outer-membrane (OM) preparations of strains carrying the latter clone and its derivatives. The reading frame of lom was identified as ORF206a. The protein was not demonstrably associated either covalently or non-covalently with the peptidoglycan layer of the cell envelope.

Bacterial Outer Membrane Proteins↗
PubMed1983

Primary structure of the tolC gene that codes for an outer membrane protein of Escherichia coli K12.

We present the nucleotide sequence of the tolC gene of Escherichia coli K12, and the amino acid sequence of the TolC protein (an outer membrane protein) as deduced from it. The mature TolC protein comprises 467 amino acid residues, and, as previously reported (1), a signal sequence of 22 amino acid residues is attached to the N-terminus. The C-terminus of the gene is followed by a stem-loop structure (8 base pair stem, 4 base loop) which may be a rho-independent termination signal. The codon usage of the gene is nonrandom; the major isoaccepting species of tRNA are preferentially utilised, or, among synonomous codons recognized by the same tRNA, those codons are used which can interact better with the anticodon (2,3). In contrast to the codon usage for other outer membrane proteins of E. coli (4) the rare arginine codons AGA and AGG are used once and twice respectively.

Bacterial Outer Membrane Proteins↗
PubMed1983

The TolC protein of Escherichia coli K12 is synthesised in a precursor form.

We examined the biosynthesis of the TolC protein of Escherichia coli K12 in a pulse-chase experiment, followed by immunoprecipitation with anti-TolC antibody and SDS-PAGE of the immunoprecipitate. This showed that TolC protein was originally synthesised in a precursor form (Mr 54 500) which could be chased into the mature form (Mr 52 000). DNA sequencing of a portion of the cloned tolC gene showed that the N-terminus of the mature rotein was preceded by a typical signal sequence of 22 residues (Mr 2542). The initiator Met was preceded by a Shine-Dalgarno sequence, with the correct spacing.

Bacterial Proteins↗
PubMed1983

A class of ompA mutants of Escherichia coli K12 affected in the interaction of ompA protein and the core region of lipopolysaccharide.

A group of ompA mutants of Escherichia coli K12 are described which were sensitive to bacteriophage K3 in a background wild-type for lipopolysaccharide (LPS). With mutant LPS in vivo (lacking some core sugar residues), however, the ompA mutations gave resistance to K3. Outer membrane levels of OmpA protein were normal or near-normal when the mutations resided in either wild-type or mutant LPS backgrounds. Strains in which the mutations occurred in a wild-type LPS background adsorbed K3 phage at the same initial rate and to the same extent as a wild-type strain, but the efficiency of plaquing of the adsorbed K3 was reduced to 25-50% of wild-type levels. Under conditions where a wild-type strain irreversibly adsorbed over 90% of available phage K3 within 3 min, double mutants (ompA mutant, LPS mutant) left 90% of the phage viable after 1 h. The 10% of inactivated phage did not form plaques.

Adsorption↗
PubMed1983

Identification and characterization of the TolC protein, an outer membrane protein from Escherichia coli.

We used the cloned tolC gene to identify, locate, and purify its gene product. Strains carrying pPR13 or pPR42 overproduced a cell envelope protein (molecular weight, 52,000). A protein of the same molecular weight was identified in radioactively labeled minicells carrying pPR13; this protein was absent in pPR11-carrying minicells. This protein was the tolC gene product, since pPR11 differed from pPR13 in having a Tn10 insertion in the tolC gene. The protein seen in cell envelopes of whole cells (TolC protein) was found to exist in an aggregated state in the outer membrane; under conditions in which OmpC and OmpF were peptidoglycan associated, TolC protein was not likewise associated. Using these properties, we purified the TolC protein and determined the sequence of twelve amino acids from the amino-terminal end. The location of the TolC protein in the outer membrane was consistent with the proposed function for the tolC gene product as a processing protein in the outer membrane.

Amino Acid Sequence↗
PubMed1982

The tolC locus of Escherichia coli affects the expression of three major outer membrane proteins.

tolC mutants, which are resistant to colicin E1 and also highly sensitive to detergents and dyes, were shown to lack the OmpF outer membrane protein. There was little effect on transcription as judged by the use of an ompF-lac operon fusion strain, and the tolC effect was probably due to a post-transcriptional effect. The NmpC protein and protein 2 were also tolC dependent.

Bacterial Outer Membrane Proteins↗
PubMed1981

Molecular cloning of the tolC locus of Escherichia coli K-12 with the use of transposon Tn10.

We have cloned the tolC gene of E. coli K-12 into pSF2124 by using transposon Tn10 as the marker to first isolate the relevant DNA fragment. The gene is on a 10.5 kb EcoRI fragment, and Tn5 insertion mutagenesis locates the gene near one end of this EcoRI fragment. An EcoRI-PstI fragment has been subcloned into pBR322 to facilitate further analysis of the gene.

Cloning, Molecular↗
PubMed1981

The tsx protein of Escherichia coli can act as a pore for amino acids.

The tsx protein is known to be a specific diffusion pathway for nucleosides. The ability of this protein to facilitate the transport of molecules other than nucleosides was examined in strains lacking detectable amounts of porin (ompB mutants). The tsx protein was shown to promote serine, glycine, and phenylalanine transport and to have no effect on either glucose or arginine transport.

Amino Acids↗
PubMed1980

Periplasmic maltose-binding protein confers specificity on the outer membrane maltose pore of Escherichia coli.

ompB mutants of Escherichia coli K-12 are markedly deficient in porin in their outer membrane. This results in a decreased rate of uptake for many substrates: the maltose pore (lambda receptor) can in some circumstances, in the absence of the periplasmic maltose-binding protein, compensate for the consequent defects in permeability to lactose, mannitol, glycylglycyl-L-valine, and tri-L-ornithine. It is postulated that the maltose-binding protein associates with the maltose pore and confers on it the specificity for maltose, and that the absence of the maltose-binding protein leaves the pore open and results in enhanced transmembrane diffusion of molecules other than maltose. This paper presents evidence to support this hypothesis.

Bacteriophage lambda↗