Use of a Synthetic Oligonucleotide to Identify a Chromosomal Gene for Chloramphenicol Acetyltransferase in a Plasmid-bearing Flavobacterium

Abstract

A micro-organism previously designated Flavobacterium sp. CB60 is resistant to chloramphenicol as a consequence of antibiotic acetylation by the enzyme chloramphenicol acetyltransferase and subsequent degradation of the acetylated product by co-metabolism. Although a 15.6 kb plasmid (pCB60) was demonstrated in this Flavobacterium strain, it did not appear to play a role in chloramphenicol acetylation. DNA hybridization was used to identify a fragment of DNA presumptively carrying the cat gene. The sequence of the synthetic probe was based upon known nucleotide sequences corresponding to the highly-conserved active site region of several chloramphenicol acetyltransferase variants. The structural gene for the enzyme in strain CB60 appeared to be chromosomal since the radioactive probe hybridized with a unique restriction fragment from chromosomal DNA but failed to do so with the DNA of plasmid pCB60.