Physical and functional characterization of the ColS8 plasmid.

The restriction map of the 5.1-kb colicinogenic plasmid ColS8 is reported. Transposon-insertion mutagenesis has been carried out to investigate the location of the various functional regions of this plasmid. Twenty-six independent ColS8::Tn1 insertions and six different deletion mutant plasmids were isolated, and the locations of the insertions and deletions were determined. The mapping of the transposon-insertion sites, together with characterization of the phenotypes of these mutants, permitted the localization of the regions of DNA involved in colicin production, colicin S8 immunity and mobilization by other plasmids. There is a polar effect in some mutants in which single insertions result in the non-expression of all three functions. In minicell preparations, the ColS8 plasmid directed the synthesis of colicin S8 (60 kDa) and a 14-kDa immunity protein. One deletion mutant with a colicin-less phenotype can synthesize colicin S8 in minicells, which placed the DNA region involved in colicin release between the colicin production and immunity regions. Both the 60-kDa and 14-kDa proteins are expressed from pColS8 in maxicell preparations.

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.

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.

Occurrence of various types of penicillinase plasmid among 'hospital' staphylococci.

The type of penicillinase plasmid carried by penicillin-resistant cultures of Staphylococcus aureus isolated from eight London hospitals has been determined. Among ;endemic' hospital staphylococci, cultures resistant to mercury salts and producing large amounts of A-type penicillinase, a high proportion of which is extracellular, are most common. C-type penicillinase is rarely associated with resistance to mercury salts.The majority of strains resistant to penicillin and at least one other antibiotic carried the alpha-plasmid. Possible reasons for the prevalence of the alpha-plasmid among endemic hospital staphylococci are discussed.

A Staphylococcus aureus plasmid that specifies constitutive macrolide-lincosamide-streptogramin B resistance contains a novel deletion in the ermC attenuator.

A 2.5 kb plasmid, pA22, isolated from a naturally occurring S. aureus strain confers constitutive MLS-resistance. By restriction enzyme analysis, pA22 is indistinguishable from the S. aureus inducible MLS-resistance conferring plasmid, pT48, apart froma small deletion. DNA sequencing showed that the deletion, is in the leader/attenuator region of the ermC (MLS-resistance) gene and removes some of the complementary repeat regions required by the translational attenuation model in pT48 for inducible ermC expression. The deletion in plasmid pA22 is different from that found in similar 2.5kb constitutive MLS-resistance plasmids in other Gram-positive bacteria. It is suggested that plasmids conferring the constitutive phenotype have evolved from an inducible ancestor on several independent occasions.

Characterization of a small cryptic plasmid isolated from a methicillin-resistant strain of Staphylococcus aureus.

The nucleotide sequence of a small (1613 bp) plasmid, pOX2000, isolated from a methicillin-resistant strain of Staphylococcus aureus has been determined. The sequence contains only one large ORF and the predicted amino acid sequence shows homology to the REP proteins of some other small staphylococcal plasmids. In addition there are two palindromic sequences, palA and palJ, that are similar to but not identical with the palindromes known from other staphylococcal plasmids to be involved in lagging strand initiation and possibly leading strand termination, respectively. Preliminary functional analysis of pOX2000 has been carried out by assessing the effect of interrupting the sequence at three unique restriction endonuclease sites. The plasmid pOX2000, and its relationship to other small staphylococcal plasmids, is discussed.

The nucleotide sequence of a small cryptic plasmid found in Staphylococcus aureus and its relationship to other plasmids.

The nucleotide sequence of a small (1273 bp) plasmid (pOX1000) of Staphylococcus aureus has been determined and compared with similar plasmids. The sequence includes a single open reading frame; two large palindromes and a 22 bp palindrome that is contiguously repeated three times upstream of the open reading frame. Composite plasmids of pE194ts and pOX1000 were constructed with pUC18 separately inserted into five different sites on pOX1000 and used to analyse the replication functions of the cryptic plasmid.

Replication of staphylococcal plasmid pT48.

Insertion of a synthetic DNA linker into the repL gene of staphylococcal plasmid pT48 inactivates the replication system. This defect can be complemented in trans by the presence of a pT48 repL gene, but not by the rep genes of the related Staphylococcus areus plasmids pSN2 and pOX1000. Comparison of the sequences of the three replication proteins indicates that specificity may be determined by a putative helix-turn-helix region.

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.

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.

Change of a single amino acid in the leader peptide of a staphylococcal beta-lactamase prevents the appearance of the enzyme in the medium.

A plasmid encoded beta-lactamase gene from Staphylococcus aureus (strain 3804) was cloned and sequenced. The nucleotide sequence is very similar to those obtained for other such beta-lactamase genes. The beta-lactamase has an N-terminal region characteristic of an exported protein and assay of activity shows that the enzyme is found extracellularly in S. aureus. A residue in the N-terminal region near to the postulated cleavage site was changed by site-directed mutagenesis from a serine into a proline. Comparison of beta-lactamase activity outside the cell in strains containing the cloned wild-type and mutagenised genes shows that this single amino acid completely prevents the appearance of the enzyme in the medium.

Cloning of the gene conferring resistance to mupirocin in Staphylococcus aureus.

A plasmid known to be associated with mupirocin resistance of Staphylococcus aureus has been isolated and a restriction enzyme map constructed. An EcoRI fragment of 4.05 kb from this plasmid has been cloned into an Escherichia coli-Staphylococcus aureus shuttle vector and shown to carry the gene for resistance to mupirocin. The DNA sequence of a small section of the gene has been determined and the derived amino acid sequence compared with a data bank. The amino acid sequence is identical for eight amino acids with the sequence of isoleucyl tRNA synthetase of E. coli. This finding adds to the evidence that mupirocin resistance is the result of a modified isoleucyl tRNA synthetase.

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

The proteolytic cleavage of BlaI was shown to correlate with beta-lactamase synthesis in Staphylococcus aureus. BlaI was found to be autoregulatory when expressed from the blaZ promoter. Insertion of a 10-bp linker into the SnaBI site of blaRI resulted in constitutive synthesis of beta-lactamase.

Plasmids of phage-Group-II Staphylococcus aureus.

Most phage-Group-II Staphylococcus aureus have been shown to carry at least one plasmid. The proportion of strains that are resistant to tetracycline appears to have increased during the last 17 years. Restriction maps of several of the small plasmids isolated from Group-II strains are presented and compared with those known for staphylococcal plasmids. These small plasmids are similar to previously characterised plasmids from staphylococci of other phage groups.

Probes for the study of mupirocin resistance in staphylococci.

Probes constructed from a 4.05-kb EcoRI digest fragment of a mupirocin resistance plasmid and a 751-bp internal part of this fragment hybridised with DNA from all of 36 independent high-level mupirocin-resistant staphylococci tested from seven centres; most were Staphylococcus aureus. In most instances the probes detected an EcoRI digest fragment of approximately 4 kb. Probes did not hybridise to DNA from low-level resistant strains, nor from strains sensitive to mupirocin.

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.

Substrate-specific inactivation of staphylococcal penicillinase.

1. The rate of hydrolysis of methicillin, cloxacillin and quinacillin by staphylococcal extracellular penicillinase decreases progressively with time. 2. The inactivation is prevented but not reversed by benzylpenicillin. 3. The rate of inactivation produced by quinacillin is minimal when the rate of hydrolysis is at a maximum. 4. Under certain conditions, partially inactivated enzyme can be reactivated. 5. Combination of the enzyme with antiserum, while permitting hydrolysis, prevents inactivation. 6. No evidence for a stable enzyme-substrate complex has been found.

Isolation and transduction analysis of temperature-sensitive mutants of Staphylococcus aureus defective in DNA replication.

Four temperature-sensitive mutants of Staphylococcus aureus with defects affecting DNA synthesis have been isolated and partially characterized. They fall into two groups: three have defects either in elongation of DNA or synthesis of its precursors; the fourth has properties inconsistent with a defect in either elongation or initiation. Transduction analysis indicated that the mutation in this fourth mutant is unlinked to the mutations in the other three, which are all clustered on one side of a gene conferrring resistance to novobiocin.