Kinetics of Induction and Purification of Chloramphenicol Acetyltransferase from Chloramphenicol-resistant Staphylococcus aureus

Abstract

Plasmid-mediated chloramphenicol resistance in Staphylococcus aureus has been shown to involve acetylation of chloramphenicol by an enzyme induced by growth in the presence of the antibiotic and certain analogues. Analysis of the kinetics of induction has been complicated by (i) the intrinsic inhibitory effects of chloramphenicol on induced enzyme synthesis and (ii) the rapid disappearance of inducer after synthesis of the acetylating enzyme. The compound related to D-threo chloramphenicol which lacks a C3 hydroxyl substituent (3-deoxychloramphenicol) is a potent inducer of chloramphenicol acetyltransferase but is ineffective as an antibiotic and is not a substrate for the enzyme. The availability of such a "gratuitous" inducer has simplified an analysis of the kinetics of induction of chloramphenicol acetyltransferase. The enzyme from induced bacteria has been purified to homogeneity and has been compared with the analogous enzyme present in E. coli which harbors a resistance transfer factor with the chloramphenicol resistance determinant.

Plasmid-mediated Chloramphenicol Resistance in Staphylococcus hyicus

“…These acetylated Cm derivatives are devoid of antibiotic activity (Shaw & Unowsky, 1968) and also of the ability to induce CAT gene expression (Briickner & Matzura, 1985). In contrast to enterobacteria, which produce CAT constitutively (Fitton et al, 1978), CAT expression in staphylococci has to be induced (Winshell & Shaw, 1969) with Cm or its fluorinated derivatives (Bruckner & Matzura, 1985). The inducibility of CAT from S .…”

“…hyicus encoded by pSC2, could also be demonstrated since there were great differences in the levels of CAT expression in uninduced and Cm-induced pSC2 transformants. All CmR plasmids of staphylococci that have been investigated carry the genetic information for the CAT monomer, a polypeptide of varying M , in the range of 22500-26200 (Zaidenzaig et al, 1979;Horinouchi & Weisblum, 1982). Formerly, native CAT was considered to be tetrameric (Shaw, 1975), but recently, X-ray crystallography revealed CAT to be a trimer of identical subunits with a distinct protein fold (Leslie et al, 1988).…”

Transduction Analysis of the Genetic Determinants for Chloramphenicol Resistance in Staphylococci

“…There have appeared many reports that the loci governing resistance to TC [4,12], CM [4,11,21], KM [4] and to SM [17] were irreversively lost by curing experiments using either acridine dyes or elevated tem-…”

Comparative Enzymology of Chloramphenicol Resistance*

“…The compound 3-deoxychloramphenicol has proved to be an effective "gratuitous" inducer with such properties.20 Using this approach, we have observed high and sustained rates of synthesis of the acetylating enzyme throughout the exponential phase of growth and have been able, therefore, to isolate and purify the enzyme from S. aureus in high yield. 20 In spite of the obvious taxonomic differences between S. aureus and S. epidermidis, 22 there is evidence that multiple antibiotic resistance occurs in S. epidermidis= and that chloramphenicol resistance may be plasmid-mediated and the result of antibiotic inactivation." Therefore, we examined a number of such chloramphenicol-resistant clinical isolates of S. epidermidis to determine whether an enzyme analogous to that of S. aureus could be detected.…”