Nucleotide sequence analysis and overexpression of the gene encoding a type III chloramphenicol acetyltransferase

Abstract

The gene catIII, encoding a type III enterobacterial chloramphenicol acetyltransferase, was cloned from the transmissible plasmid R387 into pBR322 and bacteriophage M13 mp8. Nucleotide sequence analysis of 1160 bp of DNA identified an open reading frame encoding a protein of 213 amino acid residues and a calculated molecular mass of 24965 Da. The predicted N-terminal sequence is identical with that determined by Edman degradation of chloramphenicol acetyltransferase purified from Escherichia coli harbouring R387. Sequences equivalent to the consensus motifs for initiation and rho-factor-independent termination of transcription in E. coli occur 5′ and 3′ to the catIII open reading frame. In contrast with the catI gene, present on transposon Tn9 and many enterobacterial plasmids, expression of catIII is not subject to cyclic AMP-mediated catabolite repression in vivo and there is no sequence in the 5′ non-coding DNA that resembles that deduced as the consensus for the binding of cyclic AMP receptor protein. Unique restriction-endonuclease cleavage sites were introduced adjacent to the catIII reading frame by using oligonucleotide-directed mutagenesis to facilitate insertion into E. coli expression vectors. Fully active chloramphenicol acetyltransferase represents 30-50% of the soluble protein component of cell-free extracts of E. coli containing the appropriate plasmids.