Biomedical subjects

Brian J Wilkinson

Publications and source records attributed to Brian J Wilkinson.

8 recordsLinked to original sources

PubMed2003

NaCl-sensitive mutant of Staphylococcus aureus has a Tn917-lacZ insertion in its ars operon.

Staphylococcus aureus is a Gram-positive bacterium that is extremely halotolerant. To investigate the molecular mechanisms by which S. aureus can cope with osmotic stress, Tn917-lacZ-induced NaCl-sensitive mutants were isolated. An NaCl-sensitive mutant showed a longer lag period, slower growth rate, and lower final culture turbidity than the parent strain in liquid medium containing 1.5 M NaCl. Electron microscopic observation of the NaCl-sensitive mutant under NaCl stress conditions revealed large, pseudo-multicellular cells. Addition of exogenous osmoprotectants, such as glycine betaine, choline, L-proline, and proline betaine, did not relieve the NaCl sensitivity of the mutant. The region flanking the transposon insertion site in the NaCl-sensitive S. aureus chromosome was sequenced. The mutated gene was 99% identical to arsR, the arsenic operon regulatory protein present on the pI258 plasmid of S. aureus. The ars operon from pI258 was subcloned into the shuttle vector pLI50 and transferred into the NaCl-sensitive mutant. The ars operon in trans restored NaCl tolerance in the mutant, suggesting that NaCl sensitivity is due to the mutation in arsR.

Bacterial Proteins↗

PubMed2003

Impact of sigB mutation on Staphylococcus aureus oxacillin and vancomycin resistance varies with parental background and method of assessment.

Previous studies of Staphylococcus aureus transposon insertion mutants showing decreased methicillin or teicoplanin resistance have suggested a role for the RNA polymerase alternative sigma factor SigB in the expression of resistance to these antibiotics. A knockout mutation was created in the S. aureus strain COL sigB gene and its influence on oxacillin and vancomycin resistance was studied in a variety of parental backgrounds. Typically, sigB mutants of methicillin-resistant strains had oxacillin minimum inhibitory concentrations (MICs) one-half of their parent strains. The effect of the sigB mutation appeared to be more dramatic when assessed by population analysis profiles or by growth in liquid culture in shaking flasks than by MIC determinations. Oxacillin MICs of COL and the COLDeltasigB mutant were 400 and 200 mg/l, respectively, by conventional determination and 800 and 100-200 mg/l from population analysis profiles. The COLDeltasigB mutant strain was significantly more inhibited by a range of oxacillin concentrations in a shake flask culture than strain COL. Mutation of sigB caused a decrease in vancomycin resistance in two laboratory derived glycopeptide-intermediate S. aureus strains. The results suggest that some protein products whose expression is controlled by SigB play a role in resistance to cell wall-active antibiotics.

Bacterial Proteins↗

PubMed2003

Alterations in phage-typing patterns in vancomycin-intermediate Staphylococcus aureus.

The ability of phage-typing and SmaI chromosomal RFLPs to conclude appropriate strain relatedness between a collection of 12 well-characterized in vitro-selected vancomycin-intermediate Staphylococcus aureus (VISA) strains and their seven vancomycin-susceptible parent strains is reported. Generally, no SmaI RFLP alterations were observed in VISA strains when they were compared with their respective parent strains, and clonal relationships between isogenic strains were clearly evident. Unlike the SmaI RFLP results, parent strains and VISA derivatives generally did not share similar phage-typing profiles. Depending on the phage set investigated, some VISA strains even became untypable by this method. Loss of phage infectivity is probably due to cell wall (phage receptor) alterations that are expressed by the VISA strains investigated. Collectively, these findings indicate that inappropriate relationships between VISA and vancomycin-susceptible parents might be drawn if only phage-typing and antibiotic susceptibility are utilized to determine epidemiological relationships.

Anti-Bacterial Agents↗

PubMed2002

Purification and characterization of methionine sulfoxide reductases from mouse and Staphylococcus aureus and their substrate stereospecificity.

Many organisms have been shown to possess a methionine sulfoxide reductase (MsrA), exhibiting high specificity for reduction the S form of free and protein-bound methionine sulfoxide to methionine. Recently, a different form of the reductase (referred to as MsrB) has been detected in several organisms. We show here that MsrB is a selenoprotein that exhibits high specificity for reduction of the R forms of free and protein-bound methionine sulfoxide. The enzyme was partially purified from mouse liver and a derivative of the mouse MsrB gene, in which the codon specifying selenocystein incorporation was replaced by the cystein codon, was prepared, cloned, and overexpressed in Escherichia coli. The properties of the modified MsrB protein were compared directly with those of MsrA. Also, we have shown that in Staphylococcus aureus there are two MsrA and one nonselenoprotein MsrB, which demonstrates the same substrate stereospecificity as the mouse MsrB.

Animals↗

PubMed2002

Pine oil cleaner-resistant Staphylococcus aureus: reduced susceptibility to vancomycin and oxacillin and involvement of SigB.

Mutants of Staphylococcus aureus strain COL resistant to a household pine oil cleaner (POC) were isolated on laboratory media containing POC. S. aureus mutants expressing the POC resistance (POC(r)) phenotype also demonstrate reduced susceptibility to the cell wall-active antibiotics vancomycin and oxacillin. The POC(r) phenotype is reliant on the S. aureus alternative transcription factor SigB, since inactivation of sigB abolished expression of elevated POC resistance and the reductions in vancomycin and oxacillin susceptibilities. The isolation of suppressor mutants of COLsigB::kan, which maintain the sigB::kan allele, indicates that the POC(r) phenotype can also be expressed to a lesser degree via a sigB-independent mechanism. These results bolster a growing body of reports suggesting that common disinfectants can select for bacteria with reduced susceptibilities to antibiotics. A series of in vitro-selected glycopeptide-intermediate S. aureus (GISA) isolates also expressed reductions in POC susceptibility compared to parent strains. Viewed collectively, our evidence suggests that mutations leading to the POC(r) phenotype may also be involved with the mechanism that leads to the GISA phenotype.

Anti-Bacterial Agents↗

PubMed2002

Identification of Listeria monocytogenes genes expressed in response to growth at low temperature.

Listeria monocytogenes is a food-borne bacterial pathogen that is able to grow at refrigeration temperatures. To investigate microbial gene expression associated with cold acclimation, we used a differential cDNA cloning procedure known as selective capture of transcribed sequences (SCOTS) to identify bacterial RNAs that were expressed at elevated levels in bacteria grown at 10 degrees C compared to those grown at 37 degrees C. A total of 24 different cDNA clones corresponding to open reading frames in the L. monocytogenes strain EGD-e genome were obtained by SCOTS. These included cDNAs for L. monocytogenes genes involved in previously described cold-adaptive responses (flaA and flp), regulatory adaptive responses (rpoN, lhkA, yycJ, bglG, adaB, and psr), general microbial stress responses (groEL, clpP, clpB, flp, and trxB), amino acid metabolism (hisJ, trpG, cysS, and aroA), cell surface alterations (fbp, psr, and flaA), and degradative metabolism (eutB, celD, and mleA). Four additional cDNAs were obtained corresponding to genes potentially unique to L. monocytogenes and showing no significant similarity to any other previously described genes. Northern blot analyses confirmed increased steady-state levels of RNA for all members of a subset of genes examined during growth at a low temperature. These results indicated that L. monocytogenes acclimation to growth at 10 degrees C likely involves amino acid starvation, oxidative stress, aberrant protein synthesis, cell surface remodeling, alterations in degradative metabolism, and induction of global regulatory responses.

Bacterial Proteins↗

PubMed2001

Correlation of long-range membrane order with temperature-dependent growth characteristics of parent and a cold-sensitive, branched-chain-fatty-acid-deficient mutant of Listeria monocytogenes.

Listeria monocytogenes is a food-borne, pathogenic, psychrotolerant bacterium that grows at refrigeration temperatures. Long-range membrane order of the parent (10403S) and of a cold-sensitive mutant ( cld-1) deficient in odd-numbered, branched-chain fatty acids was measured using the width of the central line of spectra of an electron paramagnetic resonance probe, 4,4-dimethyl-2-heptyl-2-hexyloxazolidine- N-oxyl (7N14), that locates deep in the hydrocarbon region of the membranes. The line width decreased from 0.9 to 0.5 milliTesla (mT) over the temperature range of 0-10 degrees for strain 10403S and -5 to 32 degrees C for strain cld-1 independent of protein state (heat denatured or intact). This provided new evidence for phase transitions in the membranes. When strain cld-1 was grown in medium supplemented with 2-methylbutyric acid, which restores anteiso fatty acids and the ability to grow at low temperature, the change in central line width as a function of temperature resembled that of strain 10403S. The temperatures at which the central line width became 0.8 mT corresponded to those at which growth became very slow in both strains (3-5 degrees C for 10403S, 15 degrees C for cld-1) as determined by Arrhenius plots. These data underscore the critical role of odd-numbered anteiso fatty acids in influencing the lower temperature limits of growth through their effects on long-range membrane fluidity.

Butyric Acid↗

PubMed1996

Proline is biosynthesized from arginine in Staphylococcus aureus.

Staphylococcus aureus NCTC 8325 exhibited a long lag phase (11 h) when inoculated into defined medium lacking proline, that could be shortened by increasing the concentration of arginine in the medium, or by supplying ornithine. Radioactivity from L-[14C]arginine, but not L-[14C]glutamate was incorporated into a spot with the chromatographic mobility of [14C]proline in the pool metabolites fraction. Selection for transposon Tn917-lacZ mutants impaired in arginine catabolism yielded four proline auxotrophs. Enzyme assays and precursor feeding experiments suggested that the major pathway for proline biosynthesis in S. aureus was from arginine via ornithine and delta'-pyrroline 5-carboxylate, rather than from glutamate. Strain 8325 Pro+, a proline prototrophic variant obtained by cultivation of 8325 in the absence of proline, accumulated L-[14C]arginine from the medium at about eight times the rate of strain 8325, suggesting its response to proline starvation was to increase arginine uptake.

Arginine↗