Biomedical subjects

B J Wilkinson

Publications and source records attributed to B J Wilkinson.

At least 37 records · Page 2Linked to original sources

PubMed1994

Characterization of cell surface material removed by water and NaCl washing of Paracoccus denitrificans grown under conditions of divalent cation deficiency and sufficiency.

Paracoccus denitrificans grown on complex medium deficient in Mg2+ and Ca2+ are rendered lysozyme susceptible by washing with NaCl, whereas cells grown in a succinate-salts medium (Mg2+ and Ca2+ sufficient) or complex medium supplemented with Mg(2+)+Ca2+ are not. The material released by water washing of cells grown on complex medium and complex medium supplemented with Mg2+ and Ca2+ was characterized by a high protein content. There was a high lipid: protein ratio and an appreciable amount of 3-deoxyoctulosonic acid in the material released by NaCl washing of cells grown under all conditions, indicating release of outer membrane material. The lipid ornithine: lipid phosphorous ratios of NaCl wash from cells grown on complex medium and complex medium supplemented with Mg2+ and Ca2+ were 0.54 and 0.34, respectively. Although NaCl washing removed outer membrane material from cells grown under all conditions, only divalent cation deficient cells were rendered lysozyme susceptible. This might be explained by the increased outer membrane ornithine-containing lipid to phospholipid ratio in these cells yielding a more permeable outer membrane.

Cations, Divalent↗

PubMed1994

Evidence for feedback (trans) regulation of, and two systems for, glycine betaine transport by Staphylococcus aureus.

Previous reports are in conflict as to the number of transport systems for glycine betaine in Staphylococcus aureus. Cells grown in complex medium exhibited a single transport system of moderate affinity. Cells grown in defined medium in the absence of glycine betaine showed a high affinity and a low affinity transport system. Cells grown in the presence of glycine betaine in the presence of osmotic stress in either complex or defined media accumulated large pools of internal glycine betaine. Smaller, but still significant, amounts of glycine betaine were accumulated by cells grown in its presence in either complex or defined media in the absence of osmotic stress. Cells grown in defined medium in the presence of glycine betaine in the presence or absence of osmotic stress showed lower rates of glycine betaine transport than cells grown in its absence. This suggests that glycine betaine transport is subject to feedback or trans inhibition by internal glycine betaine. This can explain the difference in observed kinetics in cells grown in complex or defined media, the high affinity system being predominantly inhibited in cells grown in complex medium.

Betaine↗

PubMed1993

Antibodies to a range of Staphylococcus aureus and Escherichia coli heat shock proteins in sera from patients with S. aureus endocarditis.

Antibodies to a range of Staphylococcus aureus and Escherichia coli heat shock proteins were present in sera from patients with S. aureus endocarditis. This suggests the highly immunoreactive nature of a range of heat shock proteins in addition to the GroEL equivalent (common antigen) protein. In one case, antibodies to three proteins unique to the infecting S. aureus strain, which were more prominent in heat-shocked cells, were also detected.

Antibodies, Bacterial↗

PubMed1993

Choline transport activity in Staphylococcus aureus induced by osmotic stress and low phosphate concentrations.

Uptake of [14C]choline upon hyperosmotic stress of exponential-phase Staphylococcus aureus cultures in a complex medium occurred after a delay of 2.5 to 3.5 h. This uptake could be prevented by chloramphenicol, suggesting that it occurred via an inducible transport system. Radioactivity from [14C]choline was accumulated as [14C]glycine betaine. However, neither choline nor glycine betaine could act as the major carbon and energy source for the organism, suggesting that choline was not metabolized beyond glycine betaine. Assay of choline transport activity in cells grown under different conditions in defined media revealed that osmotic stress was mainly responsible for the induction, but choline gave a further increase in induction. The system was not induced in anaerobically grown cells. Choline transport activity was repressed by glycine betaine and proline betaine, suggesting that these compounds are corepressors. Choline transport activity was not induced in cells osmotically stressed by 1 M potassium phosphate or 0.5 M sodium phosphate, but was induced in cells grown in low-phosphate medium in the absence of osmotic stress. This suggests that there is a connection between the phosphate and osmotic stress regulons. Choline transport was energy and Na+ dependent and had a Km of 46 microM and a maximum rate of transport (Vmax) of 54 nmol/min/mg (dry weight). The results of competition studies suggested that N-methyl and an alcohol group or aldehyde groups at the ends of the molecule were important in its recognition by the system. Glycine betaine was not a highly effective competitor, suggesting that its transport system and the choline transport system were distinct from each other. Choline transport was highly susceptible to a variety of inhibitors, which may be related to the greater dependence on respiratory metabolism of cells grown in the presence of high NaC1 concentrations.

Biological Transport↗

PubMed1992

Autolysis of methicillin-resistant and -susceptible Staphylococcus aureus.

The autolytic activities, including unstimulated, Triton X-100-stimulated, and daptomycin-induced, of various sets of methicillin-resistant and related methicillin-susceptible strains were compared. Faster rates of autolysis were noted in two heterogeneous methicillin-resistant transductants than in their methicillin-susceptible parental recipients, in a heterogeneous resistant strain than in a susceptible derivative created by chemical mutagenesis, and in a homogeneous resistant strain than in a derivative that had decreased methicillin resistance and was created by transposon Tn551 mutagenesis. These results suggest that the presence of the methicillin resistance region, mec, either directly or indirectly through an interaction with other host genes, confers a faster rate of autolysis on strains. Various auxilliary genes are known to affect methicillin resistance expression, and one of these genes, femA, was necessary for the expression of this faster rate of autolysis. These differences in autolytic activities were not observed in isolated crude cell walls retaining autolytic activities, suggesting different modes of regulation of autolysins in intact cells and isolated walls. In contrast, one homogeneous, highly resistant strain, DU4916, had a lower autolytic activity than did derived heterogeneous resistant and susceptible strains created by chemical mutagenesis and a strain that had decreased resistance and was created by transposon mutagenesis. Our observations suggest that methicillin resistance expression is associated with an enhanced rate of autolysis, in heterogeneous resistant strains at least.

Autolysis↗

PubMed1992

Analysis of a peptidoglycan hydrolase gene from Staphylococcus aureus NCTC 8325.

We have investigated the expression of the peptidoglycan hydrolase gene (lytA) of Staphylococcus aureus NCTC 8325. Results from in vitro transcription-translation analysis, maxicell experiments, and Northern (RNA) blot analysis suggest that the lytA gene encodes a polypeptide of M(r) approximately 50,000. Physical mapping data indicate that the lytA gene originated from prophage 11 in the NCTC 8325 strain.

Chromosome Mapping↗

PubMed1992

Proline transport in Staphylococcus aureus: a high-affinity system and a low-affinity system involved in osmoregulation.

L-Proline enhanced the growth of Staphylococcus aureus in high-osmotic-strength medium, i.e., it acted as an osmoprotectant. Study of the kinetics of L-[14C]proline uptake by S. aureus NCTC 8325 revealed high-affinity (Km = 1.7 microM; maximum rate of transport [Vmax] = 1.1 nmol/min/mg [dry weight]) and low-affinity (Km = 132 microM; Vmax = 22 nmol/min/mg [dry weight]) transport systems. Both systems were present in a proline prototrophic variant grown in the absence of proline, although the Vmax of the high-affinity system was three to five times higher than that of the high-affinity system in strain 8325. Both systems were dependent on Na+ for activity, and the high-affinity system was stimulated by lower concentrations of Na+ more than the low-affinity system. The proline transport activity of the low-affinity system was stimulated by increased osmotic strength. The high-affinity system was highly specific for L-proline, whereas the low-affinity system showed a broader substrate specificity. Glycine betaine did not compete with proline for uptake through either system. Inhibitor studies confirmed that proline uptake occurred via Na(+)-dependent systems and suggested the involvement of the proton motive force in creating an Na+ gradient. Hyperosmotic stress (upshock) of growing cultures led to a rapid and large uptake of L-[14C]proline that was not dependent on new protein synthesis. It is suggested that the low-affinity system is involved in adjusting to increased environmental osmolarity and that the high-affinity system may be involved in scavenging low concentrations of proline.

Biological Transport↗

PubMed1992

Staphylococcus aureus osmoregulation: roles for choline, glycine betaine, proline, and taurine.

Choline, glycine betaine, and L-proline enhanced the growth of Staphylococcus aureus at high osmolarity (i.e., they acted as osmoprotectants) on various liquid and solid defined media, while an osmoprotective effect of taurine was shown only for cells growing on high-NaCl solid medium that lacked other osmoprotectants. Potassium pool levels were high, and there was little difference in levels in cells grown at different osmolarities. Glycine betaine accumulated to high levels in osmotically stressed cells, and choline was converted to glycine betaine. Proline and taurine also accumulated in response to osmotic stress but to lower levels than glycine betaine.

Amino Acids↗

PubMed1991

Sequence analysis of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity.

The nucleotide (nt) sequence of a 2.0-kb NheI-XbaI DNA fragment containing a peptidoglycan hydrolase-encoding gene, lytA, tentatively identified as encoding an N-acetylmuramyl-L-alanine amidase, from Staphylococcus aureus, was determined. The nt sequencing revealed an open reading frame (ORF) of 1443 bp with a consensus ribosome-binding site located 7 nt upstream from the ATG start codon. The primary amino acid (aa) sequence deduced from the nt sequence revealed a putative protein of 481 aa residues with an Mr of 53815. Comparison of the aa sequence of the ORF with aa sequences in the GenBank data base (version 63, March 1990) revealed that the C-terminal sequence showed significant homology to the C-terminal sequence of lysostaphin from Staphylococcus simulans biovar staphylolyticus.

Amino Acid Sequence↗

PubMed1991

Antibacterial activity and penicillin-binding protein affinity of new cephalosporin derivatives in Staphylococcus aureus and Escherichia coli.

A series of C3-substituted cephalosporins with different levels of oxidation was synthesized and the activities of eight compounds, including affinity for penicillin-binding proteins (PBPs), in Staphylococcus aureus and Escherichia coli were studied. The more hydrophobic cephalosporins, with a thiophene moiety at the C7 position, were more active than C7-methoxyiminoaminothiazole derivatives against S. aureus H (beta-lactam-susceptible), and compounds with a thioether function at C3 were more active than those with a sulphonyl function at this position. In contrast, the most active compounds against E. coli were hydrophilic, zwitterionic C7-methoxyimino-aminothiazole C3-thioether cephalosporins. The physicochemical properties of the most active compounds were in accord with those predicted for rapid outer membrane penetration. However, among the thiophene cephalosporins the dianionic sulphonyl compounds were more active than the monoanionic thioether derivatives. There was a reasonably good correlation between MICs and PBP affinities for S. aureus H. The compounds had most affinity for PBP 1 and least for PBP 4. The two most potent compounds had high affinities for PBPs 1, 2 and 3. The compounds had low affinities for the beta-lactam-insensitive PBP 2a of methicillin-resistant S. aureus strain DU4916-K7. The novel cephalosporins had the highest affinities for PBPs 1a/1b and 3 of E. coli DCO. The major morphological response of E. coli to the compounds was cell elongation.

Bacterial Proteins↗

PubMed1990

Basic features of the staphylococcal heat shock response.

The major heat shock proteins of Staphylococcus aureus had apparent Mrs of 84,000, 76,000, and 60,000, and other prominent proteins of Mrs 66,000, 51,000, 43,000 and 24,000 were also induced. Staphylococcus epidermidis showed a similar response. These proteins were also induced by CdCl2, ethanol and apparently osmotic stress (1.71 M NaCl or 2.25 M sucrose). Most of the proteins sedimented with the membrane fraction, but the Mr 60,000 protein remained in the cytoplasm.

Cesium↗

PubMed1990

Cloning and expression of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity.

A gene of Staphylococcus aureus PS47 encoding lytic activity was cloned and expressed in Escherichia coli. Deletion analysis of a recombinant plasmid carrying a 7.4-kilobase-pair fragment (kbp) of S. aureus DNA suggested that the gene was located within a 2.5-kbp EcoRI-XbaI fragment. Analysis of extracts of E. coli harboring recombinant plasmids on denaturing polyacrylamide gels containing purified cell walls of S. aureus showed a clearing zone by a polypeptide of apparent Mr 23,000. The release of dinitrophenylalanine but not reducing groups from purified cell walls by a cell extract of recombinant E. coli suggested that we had cloned an N-acetylmuramyl-L-alanine amidase.

Cell Wall↗

PubMed1990

Staphylococcal slime: a cautionary tale.

Slime production by Staphylococcus epidermidis may be important in the adherence to and colonization of biomedical devices, and slime has been proposed to have various effects on the immune system. Attempts were made to isolate, purify, and chemically characterize slime from S. epidermidis cultivated under fluid on tryptic soy broth-agar medium. "Crude slime" from slime-producing strain RP-12 was characterized by a high galactose content. Similar materials in similar yields were isolated from slime-producing strain Kaplan, a non-slime-producing mutant, Kaplan-6A, and sterile medium controls, suggesting that crude slime was derived mainly from the medium. The occurrence of D- and L-galactose and pyruvate and sulfate residues and methylation analysis of these crude slime preparations, monitored by gas-liquid chromatography and mass spectrometry, showed that the agar was the main source of crude slime, suggesting that the preparation was largely an artifact of the growth and isolation procedures. Similar high-galactose-content preparations from both S. epidermidis and Staphylococcus aureus, assumed to be bacterial products and with a variety of biological activities, have been described by other investigators. Growth attached to a solid surface appears to be important for slime production. An accumulation of turned-over cell surface molecules and released macromolecules such as DNA may contribute to slime production. Avoidance of agar and development of a chemically defined medium for slime production are recommended for further studies.

Culture Media↗

PubMed1990

Neutrophil chemotactic activity of peptidoglycan. A comparison between Staphylococcus aureus and Staphylococcus epidermidis.

The ability of peptidoglycan from Staphylococcus epidermidis and Staphylococcus aureus to generate in human serum chemoattractant for peripheral blood neutrophils was studied. It was shown that PG from the two bacteria was able to induce chemotactic activity in normal human serum. Sonication of PG was required to generate this activity. Very little or no activity was generated in heat-treated or C5-deficient human serum by PG, indicating that PG treatment of serum resulted in generation of chemoattractants by activation of complement. Kinetics studies employing C2-deficient or MgEGTA-chelated serum revealed that S. epidermidis induced chemotactic activity by activating the alternative complement pathway. The alternative complement activation induced by S. epidermidis occurred rapidly and was completed after 15 min, whereas S. aureus activated the alternative pathway much more slowly, with activation reaching a maximum at 60 min. The rapid activation of the alternative complement pathway by S. epidermidis PG may partly explain why this bacterium does not normally cause infections in healthy individuals.

Antigens, Bacterial↗

PubMed1990

Attachment of staphylococci to silicone catheters in vitro.

The adherence of radiolabeled staphylococci to silicone catheters was investigated in vitro. Staphylococcus aureus and Staphylococcus epidermidis strains bound to the same extent to the catheters. Also, S. epidermidis strains isolated from patients with plastic-related infections showed binding similar to that of other S. epidermidis strains. By preincubation of catheters the influence of purified staphylococcal cell surface components on the binding was evaluated. The most potent inhibitors of the binding of S. aureus were the two surface proteins, clumping factor and protein A, and the cytoplasmic membrane. Surface proteins and the cell membrane of S. epidermidis also blocked the binding. Only protein-containing surface proteins inhibited the binding. The production of slime correlated with the degree of S. epidermidis binding. Human plasma and serum, as well as purified albumin and IgG, inhibited the binding of both staphylococcal species. Fibrinogen, and to a certain extent fibronectin, inhibited the binding of S. epidermidis, while both these purified plasma proteins enhanced the binding of S. aureus.

Bacterial Adhesion↗

PubMed1989

Lower autolytic activity in a homogeneous methicillin-resistant Staphylococcus aureus strain compared to derived heterogeneous-resistant and susceptible strains.

It has been proposed that in addition to production of a penicillin-binding protein with low affinity for beta-lactam antibiotics, control of autolysin activity is involved in the mechanism of staphylococcal methicillin resistance. A homogeneous methicillin-resistant Staphylococcus aureus strain (DU4916) had lower rates of unstimulated, NaCl- and Triton X-100-stimulated autolysis, and daptomycin (LY146032)-induced lysis than a heterogeneous methicillin-resistant strain (DU4916-K7) and a methicillin-susceptible strain (DU4916S) derived from DU4916.

Bacteriolysis↗

PubMed1988

Increased outer membrane ornithine-containing lipid and lysozyme penetrability of Paracoccus denitrificans grown in a complex medium deficient in divalent cations.

Paracoccus denitrificans grown in a complex medium was highly susceptible to lysozyme, in contrast to cells grown in a complex medium supplemented with Mg2+ and Ca2+ or in a succinate-salts medium. The complex medium was deficient in divalent cations needed for optimum outer membrane stability. The major change in molecular compositions of outer membranes isolated from cells grown under the different conditions was a higher ratio of ornithine-containing lipid to phospholipid in complex-medium-grown cells (0.63) than in cells grown in complex medium with Mg2+ and Ca2+ (0.22) or in succinate-salts medium (0.14). We suggest that the dipolarionic ornithine-containing lipid is less dependent than acidic phospholipids on divalent cations for its incorporation into the outer membrane.

Calcium↗

PubMed1987

Transport, nutritional and metabolic studies of taurine in staphylococci.

A specific, Na+-dependent, energy-requiring transport system for taurine has been reported recently in the Staphylococcus aureus M strain. Taurine was taken up vigorously by all S. aureus strains tested. The system was Na+-dependent, and Na+ decreased the Km but had no effect on the Vmax of the transport system. Among coagulase-negative staphylococci, the Staphylococcus epidermidis group (a taxonomically related group of species associated with humans or other primates) and the free-living, wide-ranging species Staphylococcus sciuri showed vigorous taurine uptake. Somewhat lower rates were found in the Staphylococcus saprophyticus group. Low or barely detectable uptake rates were noted in other staphylococcal species that were primarily of animal origin. No taurine uptake was detected in a variety of other bacterial species tested. Taurine uptake, which was not Na+-dependent, occurred in a Pseudomonas aeruginosa strain grown on taurine as sole energy, carbon, nitrogen, and sulphur source, but not when it was grown in a gluconate/salts medium. In nutritional studies we were unable to demonstrate a role for taurine as a sulphur source for S. aureus. [1,2-14C]- and [35S]taurine were taken up during overnight growth of cells, and radioactivity was distributed similarly among cellular fractions, indicating that the carbon and sulphur atoms of taurine were not cleaved and had the same fate. We were unable to demonstrate any catabolism of taurine in radiorespirometric experiments to detect evolution of 14CO2 by cells incubated with [1,2-14C]taurine. Thus, we found no evidence for a role of taurine in the energy, carbon and sulphur metabolism of S. aureus.

Biological Transport↗