57] Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria

Abstract

Publisher Summary This chapter discuses the assay method and purification of chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Enzyme activity can be quantitated by either measuring (1) the chloramphenicol (CM)-dependent disappearance of acetyl-S-CoA, (2) the appearance of 3-O-acetoxy derivative of CM, or (3) the formation of reduced (unesterified) CoA. In practice, the choice between these alternatives is determined by the level of sensitivity required and by complications created by interfering substances. It has been suggested that conventional techniques of protein fractionation have been adequate for the purification of chloramphenicol acetyltransferase (CAT) from (1) R factor-bearing strains of E. coli, (2) Staphylococci harboring CM plasmids, (3) CM-resistant mutants of Proteus mirabilis in which the CAT gene is probably chromosomal, and (4) selected strains of Agrobacterium tumefaciens. The procedure described in the chapter gives a homogeneous CAT product from R factor-containing strains of E. coli and isolates of S. aureus harboring a CM plasmid. The ease with which such results can be achieved is because of the fact that fully induced S. aureus cultures and virtually all R + E . coli cultures synthesize CAT to levels approximating 0.5–1% of the soluble cell protein in stationary phase.