PubMed Health⌕ Search

Biomedical subjects

K G Dyke

Publications and source records attributed to K G Dyke.

At least 19 recordsLinked to original sources
PubMed2001

Studies of the operator region of the Staphylococcus aureus beta-lactamase operon.

The repressor proteins BlaI and MecI bind similarly to the bla operator implicated in the regulation of beta-lactamase synthesis in Staphylococcus aureus. BlaI binds to two separate dyads but neither copper-phenanthroline footprinting nor dimethyl sulphate (DMS) methylation protection assays produced any evidence of a change in the geometry of the DNA between the two dyads. It is concluded that BlaI molecules bound at the dyads probably do not cause bending or looping of the intervening DNA. DMS protection assays of BlaI binding to the bla operator in vitro and in vivo gave similar results so that it is tentatively concluded that the in vitro results are an accurate reflection of the in vivo situation. Deletion of the dyad nearest to the blaZ gene resulted in decreased synthesis of the chloramphenicol acetyltransferase reporter protein synthesized from the blaZ promoter/translation initiator. Explanations for this are considered.

Bacterial Proteins↗
PubMed2001

Staphylococcus caprae strains carry determinants known to be involved in pathogenicity: a gene encoding an autolysin-binding fibronectin and the ica operon involved in biofilm formation.

The atlC gene (1,485 bp), encoding an autolysin which binds fibronectin, and the ica operon, involved in biofilm formation, were isolated from the chromosome of an infectious isolate of Staphylococcus caprae and sequenced. AtlC (155 kDa) is similar to the staphylococcal autolysins Atl, AtlE, Aas (48 to 72% amino acid identity) and contains a putative signal peptide of 29 amino acids and two enzymatic centers (N-acetylmuramoyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase) interconnected by three imperfect fibronectin-binding repeats. The glycine-tryptophan (GW) motif found in the central and end part of each repeat may serve for cell surface anchoring of AtlC as they do in Listeria monocytogenes. The S. caprae ica operon contains four genes closely related to S. epidermidis and S. aureus icaA, icaB, icaC, and icaD genes (> or = 68% similarity) and is preceded by a gene similar to icaR (> or =70% similarity). The polypeptides deduced from the S. caprae ica genes exhibit 67 to 88% amino acid identity to those of S. epidermidis and S. aureus ica genes. The ica operon and icaR gene were analyzed in 14 S. caprae strains from human specimens or goats' milk. Some of the strains produced biofilm, and others did not. All strains carry the ica operon and icaR of the same sizes and in the same relative positions, suggesting that the absence of biofilm formation is not related to the insertion of a mobile element such as an insertion sequence or a transposon.

Bacteriolysis↗
PubMed2000

MecI represses synthesis from the beta-lactamase operon of Staphylococcus aureus.

Plasmid diploids were constructed in Staphylococcus aureus to study the effect of the repressor of methicillin resistance (MecI) on the synthesis of both beta-lactamase and the beta-lactamase repressor (BlaI). MecI-mediated repression of the synthesis of beta-lactamase was shown by reduction in the specific activity of nitrocefinase in bacteria containing a plasmid carrying mecI but not when containing the same plasmid deleted for mecI. Antisera prepared against purified MecI and against purified BlaI were used in Western blots which showed that MecI repressed the synthesis of BlaI in these diploids. The interactions between the mec operon and the bla operon are discussed.

Bacterial Proteins↗
PubMed2000

Replication of staphylococcal multiresistance plasmids.

Based on structural and functional properties, three groups of large staphylococcal multiresistance plasmids have been recognized, viz., the pSK1 family, pSK41-like conjugative plasmids, and beta-lactamase-heavy-metal resistance plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiation genes from the Staphylococcus aureus plasmids pSK1, pSK41, and pI9789::Tn552 were found to be related to each other and to the Staphylococcus xylosus plasmid pSX267 and are also related to rep genes of several plasmids from other gram-positive genera. Nucleotide sequence similarity between pSK1 and pI9789::Tn552 extended beyond their rep genes, encompassing upstream divergently transcribed genes, orf245 and orf256, respectively. Our analyses revealed that genes encoding proteins related to the deduced orf245 product are variously represented, in several types of organization, on plasmids possessing six seemingly evolutionarily distinct types of replication initiation genes and including both theta-mode and rolling-circle replicons. Construction of minireplicons and subsequent functional analysis demonstrated that orf245 is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to orf245 is evident on the conjugative plasmid pSK41, and a minireplicon encoding only the pSK41 rep gene was found to exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large multiresistance plasmids that have been identified in clinical staphylococci, which were formerly presumed to be unrelated, actually utilize an evolutionarily related theta-mode replication system.

Amino Acid Sequence↗
PubMed1998

Analysis of two Staphylococcus epidermidis plasmids coding for resistance to streptogramin A.

The two Staphylococcus epidermidis plasmids pIP1629 (7.5 kb) and pIP1630 (14.4 kb) contain the vga gene conferring resistance to streptogramin A. All the sequences of pIP1629, except two of the four 22-nt iterons preceding the replication gene, were found in pIP1630. The additional 6.9-kb fragment of pIP1630 is similar to the mobilizable S. epidermidis plasmid pSK639, carrying the dfrA-thyE-orf140 operon and thought to replicate by an iteron controlled theta-type replication mechanism. The replication-mobilization elements of pIP1629 and pSK639 are very similar despite having been isolated in France and in Australia, respectively, showing that they are geographically widely dispersed in S. epidermidis. The gene thyE encoding thymidylate synthetase carried by pSK639 is not present in pIP1630. pIP1630 probably arose by the recombination of two homologous plasmids carrying distinct resistance determinants.

Australia↗
PubMed1997

Studies of the repressor (BlaI) of beta-lactamase synthesis in Staphylococcus aureus.

Purified BlaI, the putative repressor of the beta-lactamase operon in Staphylococcus aureus, binds specifically to two regions of dyad symmetry (operators) located in the blaZ-blaR1 intergenic region. BlaI binds with similar affinity to the two regions and to the related sequence upstream of the mec gene found in methicillin-resistant strains of S. aureus, providing physical evidence for the cross-talk previously observed between these systems. A change from a lysine in the N-terminus of BlaI to an alanine or deletion of the C-terminal 23 amino acids severely reduces its DNA-binding ability, demonstrating the functional importance of both the N- and C-termini. An operator DNA-protein complex observed with crude cell lysates from repressed cells, indistinguishable from that observed with purified BlaI, was eliminated by induction of the beta-lactamase operon. Furthermore, BlaI is proteolytically cleaved in response to the addition of inducer in a blaR1-dependent manner, providing primary evidence for the molecular basis of induction. Thus, BlaI is shown to be the repressor of the beta-lactamase system.

Bacterial Proteins↗
PubMed1996

Suppression of the thermosensitive replication phenotype of the derivative plasmid of pI9789::Tn552 in Staphylococcus aureus may involve integration of the plasmid into the host chromosome.

Plasmid-chromosome co-integration was found to be the mechanism of choice to overcome thermosensitivity of replication of the plasmid pS1 in PS80d and RN4220 strains of Staphylococcus aureus. The integration of the plasmid was sometimes accompanied by deletion of a specific section of the plasmid pS1 in PS80d. Growth of bacteriophage on strains containing the integrated plasmid and the subsequent use of the phage in transduction gave transductants containing plasmids that had regained their replication thermosensitivity. These plasmids had not acquired any detectable chromosomal DNA. The 16-kb EcoRI fragment of the PS80d chromosome that hybridizes to pS1 is the target for recombination in many cases, but apparently other sites are also used. This fragment contains sequence homologous to parts of the transposon Tn552 and it is probable that site-specific recombination is involved in the integration. The possible mechanisms for the integrations and the deletions are discussed.

Arsenates↗
PubMed1996

Characterization of a Staphylococcus aureus transposon, Tn5405, located within Tn5404 and carrying the aminoglycoside resistance genes, aphA-3 and aadE.

A new staphylococcal composite transposon, designated Tn5405, carrying the genes aphA-3 and aadE, which encode resistance to aminoglycosides, was partially characterized. The transposon is 12 kb long and is flanked by inverted repeated sequences displaying the characteristic features of an insertion sequence, named IS1182. This insertion sequence is 1864 bp long and has 23/33-bp imperfect inverted repeats at its ends. One of the IS1182 copies delimiting Tn5405 contains a copy of IS1181 flanked by 8-bp direct repeats. Tn5405 was found in the chromosome of MRSA clinical isolate BM3121, within a Tn552-related transposon, Tn5404. Tn5404 was previously characterized following its transposition onto a beta-lactamase plasmid harbored by BM3121. Two forms of the recombinant beta-lactamase-encoding plasmid generated by the inversion of Tn5405 within Tn5404 were detected. IS1182 was not detected in the DNA of 4 of the 17 tested MRSA isolates containing aphA-3 and resistant to streptomycin. Thus, aphA-3 and aadE genes are not disseminated only by Tn5405 or related transposons delimited by IS1182.

Amino Acid Sequence↗
PubMed1996

A novel plasmid from Staphylococcus epidermidis specifying resistance to kanamycin, neomycin and tetracycline.

The naturally occurring plasmid pSTS7 from Staphylococcus epidermidis mediated resistance to tetracycline via a tetL gene and to kanamycin and neomycin via an aadD gene. Plasmid pSTS7 showed partial restriction map and sequence homology to the previously described tetracycline resistance plasmid pNS1981 from Bacillus subtilis and to the kanamycin/neomycin/bleomycin resistance plasmid pUB110 from S. aureus. Sequence analysis of the regions flanking the two resistance genes in pSTS7 led to the identification of a novel site for interplasmid recombination which could explain the derivation of pSTS7 from the incompatible pNS1981- and pUB110-like parental plasmids under tetracycline-selective pressure.

Amino Acid Sequence↗
PubMed1995

Sites for co-integration of large staphylococcal plasmids.

Site-specific recombination is thought to play an important role in the evolution of multi-resistant plasmids in bacteria, including the human pathogen Staphylococcus aureus (Sa). A mechanism for site- and orientation-specific recombination between large Sa plasmids was identified in Sa strain 1054. A replication-thermosensitive derivative of plasmid pI9789::Tn552 (called pS1) was found to form stable co-integrates with the large plasmid pOX1054 in the Sa strain 1054. Two closely related recombination sites were identified on these plasmids at which recombination occurred to form co-integrates. The sites (rs9789 from plasmid pI9789::Tn552 and rs1054 from pOX1054) were cloned and studies with them showed that the recombination at these sites occurs by a new method. The site rs1054 (27 bp) is deleted and rs9789 (26 bp) is duplicated during recombination. The data show that plasmid pS1 contributes the site for recombination and that the gene(s) encoding the protein(s) involved in recombination are encoded on either pOX1054 or the 1054 chromosome.

Base Sequence↗
PubMed1995

Interaction of the chromosomal Tn 551 with two thermosensitive derivatives, pS1 and p delta D, of the plasmid pI9789 in Staphylococcus aureus.

The plasmid pI9789::Tn552 carries genes conferring resistance to penicillins and to cadmium, mercury and arsenate ions. The presence of Tn551 at one location in the chromosome of Staphylococcus aureus enhances the frequency of suppression of thermosensitivity of replication of the plasmids pS1 and p delta D which are derivatives of pI9789::Tn552. Bacteriophage propagated on the bacteria in which thermosensitivity of replication had been suppressed was used to transduce cadmium resistance to S. aureus PS80N. The cadmium-resistant transductants obtained carried plasmid pS1 or p delta D with a copy of Tn551 inserted into a specific site on pS1 but into several different sites on p delta D. The possible mechanisms of the suppression are discussed.

Base Sequence↗
PubMed1995

The staphylococcal insertion sequence IS257 is active.

The plasmid pJ3356 confers high-level mupirocin resistance on a strain of Staphylococcus aureus isolated from a hospital. The plasmid also carries two copies of IS257. Recombination of an IS257-containing plasmid conferring erythromycin resistance, pOX7-IS, into either of the IS257s of pJ3356 has been observed. The co-integration of pJ3356 and a small plasmid, pOX7, is also reported and involves duplication of one of the IS257s from pJ3356 together with 8 bp of pOX7 at the site of integration. Thus IS257 has been shown to be an active mobile genetic element.

Base Sequence↗
PubMed1995

Rearrangements in the staphylococcal beta-lactamase-encoding plasmid, pIP1066, including a DNA inversion that generates two alternative transposons.

The plasmid plP1066, harboured by by a methicillin-resistant Staphylococcus aureus strain isolated in France, carries genes specifying beta-lactamase. This plasmid undergoes numerous rearrangements. One of these was insertion, between the genes binR and sin encoding resolvases, of a 16 kb element which displayed the characteristic features of a transposon. This putative transposon, named Tn5404, carried genes encoding proteins involved in its transposition, as well as a resolution system, which were indistinguishable from those of the S. aureus transposon Tn552. These were: p480 encoding a probable transposase, p271 encoding a putative ATP-binding protein, binL encoding a resolvase, and a resolution site, resL. In addition, Tn5404 carried aminoglycoside-resistance genes (aphA, str) and the insertion sequence IS1181. Tn5404 contained at its termini 116 bp imperfect inverted repeats, similar to those of Tn552, and was flanked by 6 bp direct repeats. Insertion of Tn5404 close to resR and to the structural and regulatory beta-lactamase genes (blaZ, blal, blaR1) of pIP1066, generated a 3.5 kb invertible segment flanked by inversely repeated resolution sites (resR, resL). This invertible segment, which carried p480, p271 and binL, generated in Tn552 or Tn5404, depending on its orientation. Thus, these two transposons share their transposition and resolution systems.

Base Sequence↗
PubMed1994

Characterisation of sin, a potential recombinase-encoding gene from Staphylococcus aureus.

The staphylococcal beta-lactamase (Bla) transposon Tn4002 has previously been reported to have a high level of insertional specificity for a 1.8-kb region on the plasmid pSK1. Nucleotide sequences of this region and of a related region on plasmid pI9789 were determined. Sequence analysis revealed that these two plasmids contain sin, a gene whose deduced product shows similarity to a family of DNA recombinases. Southern hybridisation analysis indicated that sin is located on alpha-, beta- and gamma-families of Bla plasmids and on pSK1 family plasmids. A region of dyad symmetry located upstream from sin on pSK1 and pI9789 was identified as the site of insertions of Tn552 and Tn4002 in separate isolates.

Amino Acid Sequence↗
PubMed1994

Isolation and characterization of IS1181, an insertion sequence from Staphylococcus aureus.

The repeated nucleotide sequence isolated from a methicillin-resistant Staphylococcus aureus isolate displays the characteristic features of an insertion sequence and was named IS1181. It has a size of 1512 bp and consists of a 1359-bp open reading frame that encodes a 439-amino-acid protein which is predicted to be highly basic and 23-bp terminal inverted complementary repeated sequences exhibiting six mismatches. The three copies of IS1181 isolated from distinct parts of the chromosome of S. aureus, BM3121, are flanked at their ends by 8-bp direct repeats, suggesting a duplication of the target sequence. IS1181 exhibits similarities with IS1165 from Leuconostoc mesenteroides and IS1001 from Bordetella parapertusis. IS1181 was detected in at least two to eight copies in 41 of the 52 S. aureus isolates tested, whereas none of the 26 coagulase-negative staphylococci, 24 streptococci, or 11 enterococci analyzed carried nucleotide sequences hybridizing with IS1181.

Amino Acid Sequence↗
PubMed1994

An investigation of plasmids from Staphylococcus aureus that mediate resistance to mupirocin and tetracycline.

Plasmids conferring mupirocin resistance were prepared from isolates of Staphylococcus aureus obtained from four patients in the same ward. The plasmids are related and in all of them the gene conferring mupirocin resistance (mupA) is flanked by copies of IS257 in direct repeat. In two plasmids mupA and IS257 have been duplicated and in one of these plasmids (pJ3358) a small pT181-like plasmid conferring tetracycline resistance is present flanked by copies of IS257. Filter mating with a strain containing pJ3358 as donor and selection on tetracycline sometimes resulted in transfer of the pT181-like plasmid containing a copy of IS257. Analysis showed that the pT181-like plasmid with the insertion of IS257 is present in high copy number and that the IS257 element is inserted in the copy number control region of the plasmid.

Base Sequence↗