A small chloramphenicol resistance (Cmr) plasmid of approximately 3.75 kb, designated pSCS5, was isolated from Staphylococcus haemolyticus. This plasmid encoded an inducible chloramphenicol acetyltransferase (CAT; EC 2.3.1.28). The cat gene of pSCS5 was cloned into the Escherichia coli plasmid vector pBluescript SKII+. It differed in its nucleotide sequence and deduced amino acid sequence from the cat genes described previously in staphylococci and other gram-positive bacteria. The CAT enzyme was purified from cell-free lysates by ammonium sulfate precipitation, ion-exchange chromatography, and fast protein liquid chromatography. The native enzyme had an Mr of 70,000 and was composed of three identical subunits, each with an Mr of approximately 23,000. Its isoelectric point was at pH 6.15. CAT from pSCS5 exhibited Km values of 2.81 and 51.8 microM for chloramphenicol and acetyl coenzyme A, respectively. The optimum pH for activity was 7.8. CAT encoded by pSCS5 proved to be relatively heat stable, but sensitive to mercury ions. The observed differences in the nucleotide sequence and the biochemical characteristics of the enzyme allowed the identification of the pSCS5-encoded CAT from S. haemolyticus as a CAT variant different from those described previously in gram-positive bacteria.

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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 pNSl981 from Bacillus subtilis and to the kanamycin/neomycin/bleomycin resistance plasmid PUB1 10 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 pUBl1O-like parental plasmids under tetracycline-selective pressure.

A 5.1-kb plasmid, designated pSCS12, isolated from a naturally occurring Staphylococcus sciuri conferred resistance to chloramphenicol (CmR) and streptomycin (SmR). Restriction endonuclease analyses of pSCS12 revealed partial structural homologies to the CmR-plasmids pC221 from S. aureus and pSCS1 from S. intermedius, to the SmR-plasmids pSAI-1 from S. hyicus and pS194 from S. aureus, as well as to the CmR/SmR plasmid pSK68 from S. aureus. Southern-blot hybridization with specific CmR- and SmR-gene probes confirmed these similarities and allowed the mapping of the CmR- and SmR-determinants in the S. sciuri plasmid pSCS12. These observations lead to the suggestion that CmR/SmR-plasmids, such as pSCS12, may have evolved from CmR- and SmR-plasmids by interplasmidic recombination.

Summary A small chloramphenicol resistance (Cm®) plasmid of 4.65 kB could be detected in an “equine” Staphylococcus sciuri‐culture. This plasmid, designated as pSC3, was identified by interspecific protoplast transformation. On the basis of restriction endonuclease analyses a detailed restriction map of pSC 3 could be constructed. This allowed structural comparisons of pSC 3 with Cm®‐plasmids of other staphylococcal species from infections of humans and animals and identification of pSC 3 as a member of the pC221‐family of staphylococcal Cm®‐plasmids. The pSC3‐plasmid encoded an inducible chloramphenicol acetyltransferase as confirmed by enzymatic assays. This enzyme could be demonstrated in cell‐free lysates of Cm®‐induced pSC 3‐transformants.

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