Chloramphenicol acetyltransferase specified by cat-86: relationship between the gene and the protein

Gene cat-86 is chloramphenicol (Cm)-inducible and specifies Cm acetyltransferase, CAT-86. The gene was previously cloned from the DNA of a strain of Bacillus pumilus. In the present study we report the construction of a constitutively expressed version of cat-86 that permits high-level expression of the gene on a plasmid in B. subtilis. A method is described that allows very rapid purification of CAT-86 protein to homogeneity. The sequence of 13 N-terminal amino acids of purified CAT-86, as well as the 26.6-kDa size of the subunit protein, agree with predictions made based on the nucleotide sequence of the gene. The Mr of the native enzyme suggests that CAT-86 is a trimer consisting of three identical protein subunits. Our studies demonstrate that cat-86 provides a convenient system for analyzing relationships between a gene and a multimeric enzyme in the B. subtilis background.