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R A Skurray

Publications and source records attributed to R A Skurray.

At least 19 recordsLinked to original sources
PubMed1992

Substrate specificity and energetics of antiseptic and disinfectant resistance in Staphylococcus aureus.

Clinical isolates of Staphylococcus aureus carry various antiseptic and disinfectant resistance determinants (qac genes) on a variety of plasmids. The biochemistry and specificity of these resistance genes in S. aureus is the subject of this report. The qac genes were separated into two families on the basis of resistance profiles and DNA homology. Isotopic and fluorimetric assays demonstrated that the qac genes encode efflux systems that rely on proton motive force.

Anti-Infective Agents, Local↗
PubMed1992

Membrane transport proteins: implications of sequence comparisons.

Analyses of the sequences and structures of many transport proteins that differ in substrate specificity, direction of transport and mechanism of transport suggest that they form a family of related proteins. Their sequence similarities imply a common mechanism of action. This hypothesis provides an objective basis for examining their mechanisms of action and relationships to other transporters.

Amino Acid Sequence↗
PubMed1991

Sequence and conservation of genes at the distal end of the transfer region on plasmids F and R6-5.

The nucleotide sequence of the region downstream of transfer gene traI, including fertility inhibition gene finO, on the conjugative plasmids F and R6-5, has been determined. Analysis of the F sequence revealed two open reading frames (ORF's), ORF248 and ORF186; ORF186 (finO) is interrupted by the insertion of IS3. The R6-5 sequence also contained ORF248 and an intact ORF186, although an additional ORF (ORF286) was located between the two genes. ORF248, which we have designated traX, and ORF186 (finO) are highly conserved on both plasmids. The organisation of these genes indicates that traI and traX on F, and traI, traX and ORF286 on R6-5 are co-transcribed from their respective promoters upstream of traI. Sequences homologous to traX were detected on a range of conjugative F-like plasmids, whereas sequences homologous to ORF286 were only found on plasmids R6-5, R100 and R1. The conservation of traX sequences suggests a functional importance for that gene and/or its product.

Amino Acid Sequence↗
PubMed1991

Structure and evolution of a family of genes encoding antiseptic and disinfectant resistance in Staphylococcus aureus.

Resistance to antiseptics and disinfectants in Staphylococcus aureus, encoded by the qacC/qacD gene family, is associated with genetically dissimilar small, nontransmissible (pSK89) and large conjugative (pSK41) plasmids. The qacC and qacD genes were analysed in detail through deletion mapping and nucleotide sequence analysis, and shown to encode the same polypeptide, predicted to be 107 aa in size. Direct repeat elements flank the qacD gene, elements which also flank the qacC gene in truncated forms. These elements contain palA sequences, regions of DNA required for replication of some plasmids in S. aureus. The qacC gene is predicted to have evolved from the qacD gene, and in the process to have become reliant on new promoter sequences for its expression. The entire sequence of the 2.4-kb plasmid pSK89 (which contains qacC) was determined, and is compared with other plasmids from Gram + bacteria.

Amino Acid Sequence↗
PubMed1991

4',4'' adenyltransferase activity on conjugative plasmids isolated from Staphylococcus aureus is encoded on an integrated copy of pUB110.

In staphylococci, linked resistance to the aminoglycosides kanamycin, neomycin, paromomycin, and tobramycin (KmNmPmTmr) is generally mediated by an aadD determinant which encodes production of an adenyltransferase aminoglycoside modifying enzyme, AAD(4',4''). The aadD resistance determinant is located on small multicopy plasmids such as pUB110, and has also been found on large multiresistance plasmids and on the chromosome in some strains. Examination of two conjugative plasmids from strains of Staphylococcus aureus isolated in North America indicated that the aadD determinant on these plasmids is located on an integrated copy of pUB110. The integrated pUB110 is flanked by direct repeats of the staphylococcal insertion sequence IS257. Analysis of the conjugative plasmid pSK41 showed an 8-bp duplication of the pUB110 sequence immediately adjacent to flanking IS257 elements, suggesting that integration of pUB110 was mediated by IS257.

Aminoglycosides↗
PubMed1990

Typing of methicillin-resistant Staphylococcus aureus by antibiotic resistance phenotypes.

The identification of new epidemic strains of methicillin-resistant Staphylococcus aureus is essential for rapid, effective infection control. We have developed a typing method which uses antibiotic sensitivity patterns to differentiate methicillin-resistant S. aureus and which is faster and more cost-effective than biochemical analysis or bacteriophage typing. Characterisation of phenotypes which are chromosomally-encoded, plasmid- or chromosomally-encoded or exclusively plasmid-mediated has enabled us to separate Australian strains of methicillin-resistant S. aureus into 11 classes, representatives of which were indistinguishable by bacteriophage type, or plasmid profile alone. The value of this procedure is thus clearly shown.

Acriflavine↗
PubMed1990

Efflux-mediated antiseptic resistance gene qacA from Staphylococcus aureus: common ancestry with tetracycline- and sugar-transport proteins.

Resistance to intercalating dyes (ethidium, acriflavine) and other organic cations, such as quaternary ammonium-type antiseptic compounds, mediated by the Staphylococcus aureus plasmid pSK1 is specified by an energy-dependent export mechanism encoded by the qacA gene. From nucleotide sequence analysis, qacA is predicted to encode a protein of Mr 55017 containing 514 amino acids. The gene is likely to initiate with a CUG codon, and a 36 bp palindrome immediately preceding qacA, along with an upstream reading frame with homology to the TetR repressors, may be components of a regulatory circuit. The putative polypeptide specified by qacA has properties typical of a cytoplasmic membrane protein, and is indicated to be a member of a transport protein family that includes proteins responsible for export-mediated resistance to tetracycline and methylenomycin, and uptake of sugars and quinate. The analysis suggests that N- and C-terminal regions of these proteins are involved in energy coupling (proton translocation) and substrate transport, respectively. The last common ancestor of the qacA and related tet (tetracycline resistance) lineages is inferred to have been repressor controlled, as occurs for modern tet determinants from Gram-negative, but not those from Gram-positive, bacteria.

Amino Acid Sequence↗
PubMed1990

Molecular analysis of a gentamicin resistance transposonlike element on plasmids isolated from North American Staphylococcus aureus strains.

Plasmid-encoded resistance to the aminoglycosides gentamicin (Gm), tobramycin (Tm), and kanamycin (Km) (GmTmKmr) in strains of Staphylococcus aureus isolated in Australia and North America appears to be mediated by one resistance determinant. In Australian isolates, this determinant is flanked by inverted copies of a 1.3-kb insertion sequence, IS256, thereby forming a composite transposon, Tn4001. Analysis of two conjugative plasmids and a related nonconjugative plasmid from strains of S. aureus isolated in North America showed that the GmTmKmr determinant on these plasmids is also flanked by inverted repeats. In the nonconjugative plasmid, these repeats include only 425 bp of IS256 immediately adjacent to the GmTmKmr region and identical to that on Tn4001. This truncated Tn4001 element is flanked by copies of the insertion element IS257, and together these elements form a truncated Tn4001-IS257 hybrid transposonlike structure. A third copy of IS257 was located 418 bp from the hybrid structure. The truncated Tn4001 and three repeats of IS257 were present at a conserved site on the plasmids studied. Four additional copies of IS257 were identified on the two conjugative plasmids. These elements flank determinants for resistance to the aminoglycosides neomycin and paromomycin and to ethidium bromide and quaternary ammonium compounds, as well as the region involved in conjugative plasmid transfer.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence analysis of IS256 from the Staphylococcus aureus gentamicin-tobramycin-kanamycin-resistance transposon Tn4001.

Resistance to the aminoglycosides gentamicin, tobramycin and kanamycin (GmTmKmR) in Australian clinical strains of Staphylococcus aureus is commonly carried on the composite transposon Tn4001. The resistance gene aacA-aphD of Tn4001, which encodes a bifunctional AAC(6')-APH(2") modifying enzyme, is flanked by two 1324-bp inverted repeats, IS256L and IS256R, that are identical in sequence. Analysis of the IS256 sequence revealed structural features characteristic of IS elements including 26-bp imperfect terminal inverted repeats and a single open reading frame with coding capacity for a 45.6 kDa protein. The nucleotide sequence of IS256 described here, together with the sequence of the aacA-aphD gene reported previously [Rouch et al., J. Gen. Microbiol. 133 (1987) 3039-3052], completes the entire sequence of Tn4001, which totals 4566 bp.

Amino Acid Sequence↗
PubMed1989

IS257 from Staphylococcus aureus: member of an insertion sequence superfamily prevalent among gram-positive and gram-negative bacteria.

The nucleotide sequences for the IS257 family of insertion sequences from Staphylococcus aureus were compared with those of the ISS1 family from Streptococcus lactis and the IS15 family which is widespread amongst Gram-negative bacteria. These elements have a striking degree of similarity in both their putative transposase polypeptide sequences and their nucleotide sequences (40 to 64% between pairs), including 12 out of 14 bp conservation in their terminal inverted repeats. The evolutionary distance between the IS15 family and the IS257 and ISS1 families of Gram-positive origin is approximately twice that between the IS257 and ISS1 families. Analysis of base substitutions in the three sequences has provided insights into the effect of selection for the G + C content of immigrant genes to conform to that of their hosts, and into the evolution of biases in overall amino acid composition of cellular proteins in prokaryotes and eukaryotes. The IS257, ISS1, IS15 families form a superfamily of insertion sequences that has been involved in the spread of a number of antimicrobial resistance determinants in Gram-positive and Gram-negative pathogens.

Amino Acid Sequence↗
PubMed1989

Physical and biochemical characterization of the qacA gene encoding antiseptic and disinfectant resistance in Staphylococcus aureus.

We have previously cloned a 3.5 kb fragment from the Staphylococcus aureus multiresistance plasmid pSK1 which carries the qacA determinant responsible for linked resistance to acriflavine (Acr), ethidium bromide (Ebr), quaternary ammonium compounds (Qar), propamidine isethionate (Pir), and diamidinodiphenylamine dihydrochloride (Ddr). This report presents a biochemical and physical analysis of qacA and shows the widespread carriage of this gene on S. aureus resistance plasmids. Tn5 insertion mutagenesis defined the extent of qacA to within 2.40 kb of pSK1 DNA. Examination of the expression of insertion and deletion mutants of the cloned qacA sequences in both maxicells and minicells led to the association of a 50 kDa protein, designated QacA, with the AcrEbrQarPirDdr phenotype. Based on fluorimetric and isotopic assays used to determine the extent of accumulation of ethidium bromide by S. aureus strains harbouring pSK1, we propose that the basis of AcrEbrQarPirDdr in S. aureus is a qacA-mediated efflux system.

Anti-Infective Agents, Local↗
PubMed1989

Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257.

Trimethoprim resistance mediated by the Staphylococcus aureus multi-resistance plasmid pSK1 is encoded by a structure with characteristics of a composite transposon which we have designated Tn4003. Nucleotide sequence analysis of Tn4003 revealed it to be 4717 bp in length and to contain three copies of the insertion element IS257 (789-790 bp), the outside two of which are flanked by directly repeated 8-bp target sequences. IS257 has imperfect terminal inverted repeats of 27-28 bp and encodes for a putative transposase with two potential alpha-helix-turn-alpha-helix DNA recognition motifs. IS257 shares sequence similarities with members of the IS15 family of insertion sequences from Gram-negative bacteria and with ISS1 from Streptococcus lactis. The central region of the transposon contains the dfrA gene that specifies the S1 dihydrofolate reductase (DHFR) responsible for trimethoprim resistance. The S1 enzyme shows sequence homology with type I and V trimethoprim-resistant DHFRs from Gram-negative bacteria and with chromosomally encoded DHFRs from Gram-positive and Gram-negative bacteria. 5' to dfrA is a thymidylate synthetase gene, designated thyE.

Amino Acid Sequence↗
PubMed1989

Genetic analysis of Staphylococcus aureus with Tn4001.

Tn4001, a 4.5-kilobase composite transposon with IS256 ends that confers resistance to gentamicin (Gmr), tobramycin, and kanamycin in Staphylococcus aureus, can transpose to diverse chromosomal sites in S. aureus. Chromosomal insertions of Tn4001 were isolated either after UV irradiation of transducing lysates carrying pII147::Tn4001 or by selection for thermoresistant Gmr isolates with strains containing thermosensitive derivatives of plasmids pI258 and pII147 carrying Tn4001. Frequent integration of the entire delivery plasmid occurred under these selective conditions in recombination-proficient hosts. When selection for thermoresistant Gmr isolates was done with these plasmids in recombination-deficient hosts, 99% or more of the Gmr isolates resulted from transposition of Tn4001 in the absence of plasmid integration. Efficient isolation of Tn4001 insertions near markers of interest and the isolation of insertional auxotrophs were achieved. Reversion frequencies of insertional auxotrophs were between 10(-6) and 10(-7) (higher than those observed with Tn551 and Tn917). About 50% of the prototrophic revertants were Gms, and these are attributed to precise excision of Tn4001. The Gmr prototrophic revertants were due to intergenic suppression.

Chromosome Mapping↗
PubMed1988

Nucleotide sequence and transcriptional analysis of a third function (Flm) involved in F-plasmid maintenance.

The leading region of the conjugative F plasmid encodes for a function, Flm, capable of extending the maintenance of normally unstable plasmids. Nucleotide sequencing and functional studies of flm locus have shown that it consists of at least two genes, flmA and flmB, which are physically and functionally homologous to hok and sok of parB in plasmid R1. The 52-amino acid flmA-coded polypeptide is almost identical to the hok product which has been shown to be a membrane-associated lethal protein [Gerdes et al., EMBO J. 5 (1986) 2023-2029]. Gene flmB codes for a 100 nucleotide, non-translated, complementary RNA which overlaps the 5' leader sequence of the flmA RNA. The flmA RNA also encodes an open reading frame (ORF70) which overlaps the flmA-coding sequence and may be a third gene involved in the Flm function. S1 analysis and functional studies suggest that the antisense flmB RNA binds to the flmA RNA and suppresses the expression of the lethal product, presumably by blocking coupled translation of ORF70 and flmA. Secondary structure analysis predicts that the flmA RNA is extremely stable compared to the regulatory flmB RNA. We suggest that when these RNA species are retained by cells which have lost the F plasmid, the more stable flmA RNA will eventually be translated thus leading to cell death. This phenomenon provides a third mechanism, additional to ParFIA and Ccd functions, to ensure maintenance of the F plasmid in a growing bacterial population.

Base Sequence↗
PubMed1988

Multiresistant Staphylococcus aureus: genetics and evolution of epidemic Australian strains.

Molecular and genetic analysis of multiresistant isolates of Staphylococcus aureus from widely separated hospitals in Australia has demonstrated that these are clearly related, and that the predominant strains possess up to three different plasmids, which fall into the following classes: (i) small 1.6 kb plasmids, such as pSK3, which are phenotypically cryptic, (ii) 4.5 kb chloramphenicol resistance plasmids, such as pSK2, and (iii) the pSK1 family of multiresistance plasmids, which range in size from 20 to 42 kb and variously encode resistance to antiseptics and disinfectants, trimethoprim (Tpr), penicillin (Pcr) and the aminoglycosides gentamicin, tobramycin and kanamycin (Gmr Tmr Kmr). Gmr Tmr Kmr is encoded on the pSK1 family plasmids by transposon Tn4001, which was also detected on the chromosomes of some clinical isolates. Tn4001 is composed of inverted repeats of the insertion sequence IS256; these repeats flank a Gmr Tmr Kmr sequence encoding for a 57,000 dalton bifunctional protein with aminoglycoside acetyltransferase [AAC(6')] and phosphotransferase [APH(2")] activities. A Tn4001-like structure, which is defective in transposition but encodes for a Gmr Tmr Kmr determinant homologous with that on Tn4001, occurs on conjugative plasmids from strains isolated in North America. Physical studies indicate that Pcr, via a beta-lactamase, and Tpr, via a trimethoprim-insensitive dihydrofolate reductase (DHFR), are also encoded on the pSK1 family by transposons; these transposons have been designated Tn4002 and Tn4003, respectively. Tn4003 is flanked by direct repeats of the insertion sequence IS257. The evolution of the pSK1 family of multiresistance plasmids is traced through the transposition and genetic rearrangement of resistance determinants. Transposition and genetic rearrangement have also contributed to the evolution of a multiresistant chromosome in Staph. aureus. In the majority of contemporary multiply resistant Staph. aureus strains the determinants for resistance to erythromycin (Emr), fusidic acid, methicillin (Mcr), minocycline, rifampicin, spectinomycin, streptomycin, sulphonamides, tetracycline (Tcr), cadmium (Cdr), and mercury (Hgr) are chromosomally encoded; these strains also possess chromosomally encoded Pcr, via a beta-lactamase. Evidence indicates that some of these determinants, Pcr, Cdr, Hgr, and Tcr, were plasmid encoded in isolates collected from Australian hospitals prior to 1970. Through transposition and site-specific integration, they have since been acquired by the chromosome in more recent Staph. aureus strains.(ABSTRACT TRUNCATED AT 400 WORDS)

Australia↗
PubMed1988

Structural and evolutionary relationships of beta-lactamase transposons from Staphylococcus aureus.

A comparison of the beta-lactamase elements detected on three classes of large plasmids together with the chromosomes of penicillin-resistant Staphylococcus aureus revealed substantial physical and genetic relatedness. In most cases, beta-lactamase production could be associated with the presence of a DNA segment of approximately 6.7 kb. Analysis showed that the plasmid-borne determinants constitute nearly identical transposons or transposon-like elements. An element indistinguishable from one of these, Tn4002, which is carried by the pSK1 family of plasmids in clinical isolates from Australian hospitals, was also identified on the staphylococcal chromosome and is implicated in an evolutionary cycle of transposition between chromosomal and extrachromosomal sites in Australian strains of multiresistant S. aureus.

Biological Evolution↗