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P R Stewart

Publications and source records attributed to P R Stewart.

At least 19 recordsLinked to original sources
PubMed1992

Lethal and mutational effects of solar and UV radiation on Staphylococcus aureus.

Strains of Staphylococcus aureus, an opportunistic pathogen commonly found on human skin, were exposed to sunlight and UV C radiation, and the lethal and mutational effects measured. Sunlight killed cells with an inactivation constant of 3 x 10(-5) per joule per square metre; UV C was much more lethal, giving an inactivation constant of approximately 0.1 per joule per square metre. Some strains tested showed a sensitivity to sunlight that was dependent on the growth phase of the cells, exponentially growing cells showing a greater sensitivity. Mutational effects of irradiation were measured by the appearance of mutants sensitive to methicillin following irradiation of a multiresistant strain. Mutants appeared at a frequency of 10(-3); this high frequency of mutation in the region of the mec gene has also been observed when multiresistant strains are subjected to nutritional or thermal stress. Mutants showed the same chromosomal alteration (seen in pulse-field gel electrophoresis of SmaI-digested DNA) whether induced by solar or UV C irradiation.

DNA Damage↗
PubMed1992

Physical mapping of the mec region of an Australian methicillin-resistant Staphylococcus aureus lineage and a closely related American strain.

Methicillin-resistant (Mcr) staphylococci contain chromosomal DNA that is absent from Mcs cells. This extra DNA harbours the methicillin resistance determinant mec and often other resistance determinants. The mec region can differ substantially in structure among different isolates. We present studies on the mec region of a group of Staphylococcus aureus isolates prevalent in Australia and London. Southern hybridization analyses of a prototype Australian isolate, ANS46, and an isogenic Mcs deletion mutant, ANS62, allowed the physical map of the region to be extended to 55 kb. The DNA corresponding to the deletion, which includes mec and resistance determinants for mercury, cadmium (Cd) and tetracycline, amounted to 41 kb. It was bounded precisely at one end by the macrolides-lincosamides-streptogramin B (MLS)-resistance transposon, Tn554. Near the other end was an element with homology to Tn554, psi Tn554, which carried the Cdr determinant. The mec region of an American Mcr isolate, R35, was found to be virtually the same as that of ANS46, except that it lacked Tn554. Another class of American Mcr isolates, prevalent since 1987, differs markedly from ANS46 in mec region organization. However, this other American class also contains an insertion of Tn554 in the mec region, and the attachment site for this insertion was found to have significant homology to attachment sites for the Tn554 and psi Tn554 insertions in the mec region of the Australian strain. These results suggest possible roles of Tn554 and Tn554-like elements in the evolutionary variation of the mec region.

Australia↗
PubMed1991

Epidemiological analysis of a methicillin-resistant Staphylococcus aureus outbreak using restriction fragment length polymorphisms of genomic DNA.

The genomic DNA of 58 isolates of methicillin-resistant Staphylococcus aureus (MRSA) obtained during an infection outbreak at two major Canberra hospitals was analysed for restriction fragment length polymorphism (RFLP) by digestion with the endonuclease SmaI and resolution of the fragments by pulsed-field gel electrophoresis. Based on the fraction of common fragments generated by the endonuclease, DNA similarities among the isolates were estimated. Distance matrix analysis showed that the MRSA isolates could be divided into two major clusters (RFLP types I and II) and one minor one (type 46). A fourth group of miscellaneous isolates was found to be heterogeneous in terms of DNA sequence similarity. The epidemiological data indicated that RFLP type I was most common in the intensive care units in the two hospitals, with particular subtypes of RFLP type I concentrated in individual units. RFLP type II and the miscellaneous group were more generally distributed. Type 46 isolates appear to be related to a group which was present in epidemics in Melbourne hospitals in the early 1980s. Using the standard phage set, the RFLP type I group was largely untypable. However, type II isolates were all phage typable, with a shared susceptibility to phages 29/85/95/90; type 46 isolates had a shared susceptibility to phages 85/90. The miscellaneous isolates were of variable phage types.

Australia↗
PubMed1991

Tn554 inserts in methicillin-resistant Staphylococcus aureus from Australia and England: comparison with an American methicillin-resistant group.

We have compared methicillin-resistant (Mcr) Staphylococcus aureus isolates from Australia, the UK and the USA with regard to chromosomal inserts of the macrolides-lincosamides-streptogramin B (MLS)-resistance transposon Tn554. The American isolates were known to have a distinctive Tn554 insert, designated insert 6, which was closely associated epidemiologically with the methicillin-resistance phenotype. Southern blots of DNA from Australian and London, UK Mcr isolates were hybridized with a range of probes related to Tn554. The isolates had similar or identical Tn554 inserts, and we consider them to be a single group, designated 'Australondon'. Australondon isolates were compared in detail with a deletion mutant, ANS62, that had lost the methicillin-resistance determinant mec, plus other resistance determinants resident in the mec region of the chromosome, and with an American Mcr isolate containing Tn554 insert 6. The Australondon isolates had three Tn554 inserts. Sequence analysis with the polymerase chain reaction showed that all of these inserts differed from classical Tn554 in that the 3'-terminal residues of the transposons were reverse complements of the usual GATGTA. One of the Australondon inserts, designated 6B, closely resembled Tn554 insert 6 in the sequence of its left flanking chromosomal DNA. This insert was found to abut the deletion from the mec region which results in strain ANS62. We infer that Tn554 insert 6B is part of the mec region of the chromosome in Australondon isolates, supporting the idea that insert 6 of the American isolates is also part of this chromosomal region.

Australia↗
PubMed1991

Physical mapping of the mec region of an American methicillin-resistant Staphylococcus aureus strain.

We mapped part of the mec region of a locally prevalent strain of Staphylococcus aureus. The mec region was found to harbor an insert of the transposon Tn554, which encodes spectinomycin and macrolide-lincosamide-streptogramin B resistance, and a 4.6-kb segment of DNA that contains the kanamycin resistance gene aadD. This 4.6-kb segment appears to be an integrated form of a previously described plasmid, pUB110, and is flanked by copies of the insertion sequence IS257. The integration event may be an example of processes that have led to accretion of resistance determinants in the mec region of S. aureus.

Base Sequence↗
PubMed1990

Bacteriophage 604: a marker phage for multi-resistant Staphylococcus aureus in Australia.

Of 28 multi-resistant isolates of Staphylococcus aureus collected during 1986 from hospitals in major cities around Australia, 27 were found to contain the same prophage (denoted phage 604). Hospital isolates carrying three or fewer resistance markers, and community isolates carrying one or no resistance markers, did not carry this prophage. Phage 604 does not confer antibiotic resistance on its lysogens, nor does it increase virulence in chick embryo assays. Phage 604 appears to be a correlate of antibiotic multi-resistance in S. aureus in Australia, and may provide a molecular marker for incipiently epidemic strains of this bacterium in Australian hospitals.

Animals↗
PubMed1990

Induced deletions within a cluster of resistance genes in the mec region of the chromosome of Staphylococcus aureus.

Variants of a methicillin-resistant Staphylococcus aureus showing loss of or reduced resistance to the antibiotic were isolated at frequencies of 0.1-100% from cultures which had been starved, grown at elevated temperature, or given small doses of UV radiation. Three types of variant were identified on the basis of population distribution of resistance to the antibiotic, and field-inversion gel electrophoresis of digests of the chromosome cut with the rare-cutting restriction endonuclease SmaI. Type I variants are methicillin-sensitive and have a deletion in the mec region of the chromosome. Type II variants have reduced methicillin resistance and rearranged DNA elsewhere in the chromosome. Type II variants show reduced methicillin resistance and no detectable change in the chromosome. Type I deletions were mapped using cloned fragments from the mec region. In 13 of the 16 independently isolated deletion mutants, one of the deletion endpoints appears to correlate with the positions of insertion sequences or transposons found in this region of the staphylococcal chromosome.

Chromosome Deletion↗
PubMed1988

Amplification of a section of chromosomal DNA in methicillin-resistant Staphylococcus aureus following growth in high concentrations of methicillin.

Growth of two independently isolated strains of methicillin-resistant Staphylococcus aureus (MRSA) in increasing concentrations of methicillin (step-selection) resulted in increased resistance in these strains. When chromosomal DNA from the step-selected variants was probed using DNA sequences previously demonstrated to be associated with methicillin resistance in MRSA strains, amplification of the homologous chromosomal sequence was identified. Growth of these step-selected strains in the absence of methicillin resulted in loss of the amplified sequence, while the original sequence remained. There are differences between the two strains in the stability of maintenance of amplified sections. Prolonged storage of the variants on a high concentration of methicillin resulted in loss of amplified sections without concomitant loss of methicillin resistance. Thus amplification may be only one of at least two molecular mechanisms available to S. aureus to increase methicillin resistance in response to step-selection. Probing of cells of the highly resistant sub-population of a heterogeneously resistant MRSA strain showed that duplication of this mec-associated DNA is not involved in the mechanism of heteroresistance.

Chromosome Mapping↗
PubMed1988

The expression in Staphylococcus aureus of cloned DNA encoding methicillin resistance.

A 4 kb fragment of chromosomal DNA was cloned from a clinical strain of methicillin-resistant Staphylococcus aureus. It comprises part of a section of the chromosome that was lost when the strain was cured of resistance to methicillin and to other antimicrobial agents. The fragment mediates an increased level of methicillin resistance when inserted into a shuttle vector and transformed back into the sensitive strain generated when the original DNA was deleted.

Chromosomes, Bacterial↗
PubMed1988

Mercury and tetracycline resistance genes and flanking repeats associated with methicillin resistance on the chromosome of Staphylococcus aureus.

Sections of a cloned 27 kb segment of chromosomal DNA, associated with resistance to four antimicrobial agents in a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA), were tested for their ability to determine resistance when transformed into a sensitive laboratory strain of S. aureus. This was achieved by inserting the sections into a newly constructed shuttle vector, amplifying the recombinant DNA in E. coli, and transforming protoplasts of the sensitive S. aureus strain. Two sections of the cloned DNA were found to determine resistance separately to mercuric ion and to tetracycline, in both S. aureus and Escherichia coli.

Chromosomes, Bacterial↗
PubMed1987

Molecular relatedness of Staphylococcus aureus typing phages measured by DNA hybridization and by high resolution thermal denaturation analysis.

Fifteen bacteriophages representative of the serological and lytic groups of the International Typing Set for Staphylococcus aureus were examined for genomic homology by DNA hybridization and by analysis of high resolution thermal denaturation profiles. Phages 11 and 80 alpha, not part of the set, were also examined. DNA homology measured by filter hybridization showed values ranging from near zero to 88 per cent in pair-wise comparisons. Cluster analysis of these data by standard numerical taxonomical methods yielded clusters which closely reflect the subdivision of the international set on the basis of serological reactions. High resolution thermal denaturation analysis yielded characteristic profiles for each phage DNA, with members closely related by hybridization analysis showing only minor differences. Quantitative analysis of the extent of overlap of these profiles generated relational values which were subjected to the same numerical taxonomic analysis as for the DNA hybridization data. The resultant dendrogram was qualitatively different only in minor respects from that derived from the hybridization analyses, but quantitatively homology was greater by 80 per cent or more for the DNAs which were least related according to the hybridization analyses. This upward shift in measured homology appears to reflect the similar base composition of the DNAs from these phages. Statistical comparison of the homology data obtained by the two methods showed them to be significantly correlated. These results indicate that the International Typing Set consists of phages which all appear to be related to a greater or lesser extent. If, as the history of the collection of these phages indicates, they are a random sample of aureophages, then this group of phages may represent a common genetic pool within which recombination, mutation, and genome rearrangement occur to generate unique individual phages.

DNA, Viral↗
PubMed1987

Polypeptide synthesis during lytic induction of phage 11 of Staphylococcus aureus.

Staphylococcus aureus phage 11 was induced to replicate by treatment of lysogens with mitomycin C. Lysis was complete in 80 min at 37 degrees C in complete synthetic medium. Labelling with L-[3H]lysine showed a stimulation of protein synthesis during phage replication, followed after 30 min by shut-down of host protein synthesis while virion protein synthesis continued. The synthesis of approximately 16 virus-specific proteins was detected during replication, with seven of these recovered in purified virions after lysis. The remainder appeared to be synthesized earlier in phage replication and thus may represent precursors of virion proteins, or regulatory proteins, or enzymes associated with phage replication. Protein synthesis during replication of three suppressible morphological (head, tail) mutants of phage 11 did not differ significantly from that seen in the wild-type lysogen, indicating that the mutation in each case affected a protein whose synthesis was not detectable, or which was synthesized but did not fulfil its role in virion maturation or assembly. However, in a suppressible 'early' mutant which did not lyse when treated with mitomycin C, the synthesis of the 'late' (virion) proteins (with one exception) did not occur. 'Early' proteins were apparently made normally in this mutant, and there was no shut-down of host protein synthesis. This mutant phage presumably encodes a defective 'early' protein involved in the regulation of replication, or a key precursor polypeptide of 'late' protein synthesis. The morphological mutants provided a means to analyse and tentatively to allocate six of the virion proteins to head or tail/baseplate structures.

Mitomycin↗
PubMed1987

Bacteriophages associated with multiresistant Staphylococcus aureus in Australia.

Nineteen multiresistant strains of Staphylococcus aureus from Australian hospitals were examined for lysogenic bacteriophage. Thirteen strains contained prophage inducible with mitomycin C. Three of these lysed completely on induction producing a phage referred to as type 1; this phage plated on S. aureus propagating strains 6, 53 and 77, which are hosts for phages of serogroup-lysogroup A III, B III and F III respectively. Type-1 phage did not plate on other propagating strains representative of the other serogroup-lysogroup combinations in the International Typing Set for S. aureus. Ten strains of S. aureus lysed incompletely when treated with mitomycin C, yielding phage type 2, that plated only on propagating strain 6. The virions of phage types 1 and 2 had isometric heads and flexible tails, and the genome consisted of c. 40 kilobases of double stranded DNA. The DNA from the two phage types was different, as shown by endonuclease digestion and by hybridisation to reference phage DNAs. The remaining six S. aureus strains contained no phage inducible with either mitomycin C or ultraviolet irradiation. However, all contained type 2 DNA, as shown by Southern blotting, present presumably in a defective prophage state. Moreover, the three strains yielding type-1 phage on induction also contained type-2 DNA. Thus, type-2 DNA was found in all 19 strains of multiresistant S. aureus from geographically diverse Australian hospitals.

Australia↗
PubMed1985

The virion proteins and ultrastructure of Staphylococcus aureus bacteriophages.

The number and size of the major virion polypeptides have been determined by SDS-PAGE for the 22 Staphylococcus aureus phages of the International Typing Set, plus phages 11 and 80 alpha. Virion ultrastructure was examined by electron microscopy after negative staining with ammonium molybdate. In addition, serogroup B phages were disrupted and fractionated into head, tail-tube and baseplate components and major polypeptides assigned to these substructures. The number and size of the polypeptides correlated closely with the division of aureophages into four serogroups (A, B, F, L), although serogroup L was represented in the set by only a single phage (187). Apart from serogroup B, however, the polypeptide patterns did not reflect differences between lytic groups. Within serogroup B, polypeptide analysis yielded characteristic patterns for lysogroups I, II and III. Ultrastructural analyses confirm the data provided by polypeptide analysis. Thus, phages from the four serogroups can be identified on the basis of tail-tube length alone, although the differences between phage 187 and members of lysogroups I and III in serogroup B were less than 20 nm, or approximately 12% of the total length. Serogroup A virions differed from those of the other serogroups in that all members of the typing set in this group had elongate, rather than isometric, heads.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Molecular relationships among serogroup B bacteriophages of Staphylococcus aureus.

The typing bacteriophages 55, 80, 83A, and 85 of Staphylococcus aureus, representative of the three major lytic groups of serological group B aureophages, have been examined for relatedness of their genomes and virion proteins. Phages 11 and 80 alpha were also examined to determine the relationship of phage 80 alpha to phages 11 and 80. Total genome hybridization measurements divided the phages into two groups. Phages 55 and 80, in the first group, had DNA homology of 50%. Phages 11, 80 alpha, 83A, and 85 formed a second group with 27 to 65% homology. Homology between the two groups was in the range of 14 to 22%. Phage 80 alpha is more closely related to phage 11 than to phage 80, though it is probably not a simple recombinant of phages 11 and 80. Restriction enzyme digestion and phage [32P]DNA hybridization analysis of the endonuclease-generated fragments from each phage DNA confirmed the findings of the DNA homology measurements. The endonuclease fragment patterns generated by EcoRI and HindIII were distinctive for each phage, confirming that none of the phages are closely related. Common sequences were present in most fragments from the phage DNAs when the labeled probe DNA was from a different phage in the same group. Cross-group probing of endonuclease fragments revealed both a diminished level of homology when similar sequences were present and the probable absence of some sequences. Virion proteins, examined by polyacrylamide gel electrophoresis, were similar in number and molecular weight for phages 11, 80 alpha, 83A, and 85, reflecting the DNA homology analyses. The virion proteins from phages 55 and 80, however, were more distinctive, and both differed from the phages in the other group.

DNA Restriction Enzymes↗
PubMed1979

The isolation of coupled mitochondria from Physarum polycephalum and their response to Ca2+.

A method for the isolation of coupled mitochondria from the acellular slime mould Physarum polycephalum is described. The mitochondria oxidize respiratory substrates at rates comparable to those of mitochondria from other microorganisms and show similar responses to respiratory inhibitors. ADP/O values approach similar values to those obtained with mitochondria from higher organisms: 3 with NAD-linked substrates, 2 with succinate, and 1 with ascorbate-TMPD. Mitochondria actively take up low concentrations of Ca2+ with stimulation of their respiration. With succinate or pyruvate-malate as substrates respiratory responses are depressed by Ca2+ concentrations in excess of 200 micron in the presence or absence of phosphate. Exogenous NADH is unique in supporting the uptake of large amounts of Ca2+ in the presence of phosphate and in showing an unusual 'uncoupled' response in the absence of phosphate. A sigmoidal relationship occurs between initial velocity of Ca2+ uptake and Ca2+ concentration with a maximum velocity of approx. 15 nmol/s per mg protein and half maximum velocity occurring at approx. 50 micron Ca2+.

Biological Transport, Active↗
PubMed1979

Structural states of dictyostelium myosin.

Myosin purified from Dictyostelium amoebae has approximately 10% by weight of RNA associated with it, unless specific steps (DEAE cellulose chromatography or RNase digestion) are taken to remove it. This RNA has significant effects on the structural states formed by the myosin at low ionic strength in the presence of Mg2+. Rapid precipitation of RNA-free myosin by dilution generates bipolar thick filaments (540 nm long, 33 nm thick), often with a bare zone and a 15-nm transverse repeat. Rapid precipitation of myosin with copurified RNA yields linear aggregates of bipolar filaments, showing some lateral association. Slow precipitation of RNA-free myosin by dialysis yields very long filaments or ribbons (greater than 5 micrometer, 30--60 nm wide) in which the myosin may be packed diagonally across the filament, similar to the "side-polar" aggregates formed by other nonmuscle myosins and by smooth muscle myosin (Craig R, Megerman J: J Cell Biol 75:990, 1977; Hinssen H, D'Haese J, Small JV, Sobieszek A: J Ultrastruct Res 64:282, 1978). Slow precipitation of myosin with copurified RNA generates linear filaments with repeat intervals of 290 and 650 nm. Other polyanions were tested for their effects on myosin aggregation. Total RNA and ribosomal RNA from Dictyostelium, when added to RNA-free myosin, also induced the extensive linear aggregation seen with the copurified RNA/myosin complex, although higher concentrations of RNA were required to obtain quantitatively the same effect. DNA and heparin were also effective inducers of linear aggregation, whereas homopolymers of nucleotides and of acidic or basic amino acids were poorly effective.

Dictyostelium↗