CHARACTERIZATION OF THE CHLORAMPHENICOL ACETYLTRANSFERASE VARIANTS ENCODED BY THE PLASMIDS PSCS6 AND PSCS7 FROM STAPHYLOCOCCUS-AUREUS

The two 4.6 kb chloramphenicol resistance (Cm(R)) plasmids pSCS6 and pSCS7, previously identified in Staphylococcus aureus from subclinical bovine mastitis, both encoded an inducible chloramphenicol acetyltransferase (CAT, EC 2.3.1.28). The pSCS6- and pSCS7-encoded CAT variants were purified by ammonium sulphate precipitation, ion-exchange chromatography and fast protein liquid chromatography (FPLC). Both native enzymes showed M(r) values of 70000 on FPLC and were composed of three identical subunits, each of M(r) approximately 23000. The CAT variants from pSCS6 and pSCS7 differed in their net charges and in their isoelectric points. The isoelectric point of the CAT from pSCS6 was pH 5.7 and that of the CAT from pSCS7 pH 5.2. Both CAT variants exhibited highest enzyme activities at pH 8.0. The K(m) values for chloramphenicol and acetyl-CoA of the CAT from pSCS6 were 2.5-mu-M and 58.8-mu-M, respectively, while those of the CAT from pSCS7 were 2.7-mu-M and 55.5-mu-M. Both CAT variants were relatively thermostable. The CAT from pSCS6 was less sensitive to mercuric ions than the CAT from pSCS7.