Chloramphenicol-inducible Gene Expression in Bacillus Subtilis is Independent of the Chloramphenicol Acetyltransferase Structural Gene and Its Promoter

cat-86 specifies chloramphenicol acetyltransferase and is the indicator gene on the Bacillus subtilis promoter cloning plasmid pPL703. Insertion of promoters from various sources into pPL703 at a site ca. 144 base pairs upstream from cat-86 activates expression of cat-86, and the expression is characteristically inducible by chloramphenicol. Thus, chloramphenicol inducibility of cat-86 is independent of the promoter that is used to activate the gene. To determine whether cat-86 or its products were involved in chloramphenicol inducibility, gene replacement studies were performed. cat-86 consists of 220 codons. The lacZ gene from Escherichia coli was inserted into a promoter-containing derivative of pPL703, plasmid pPL603E, at two locations within cat-86. pPL3lac2 contains lacZ inserted in frame after codon 2 of cat-86. pPL3lac30 contains lacZ inserted in frame after codon 30 of cat-86. In both constructions, all cat coding sequences 3' to the site of the lacZ insertion were deleted. Both plasmids exhibited chloramphenicol inducibility of beta-galactosidase in B. subtilis. These studies provide the first direct demonstration that the transcription and translation products of a chloramphenicol-inducible cat gene are uninvolved in chloramphenicol inducibility of gene expression. The results localize the region essential to inducibility to the 144-base pair segment that intervenes between the site of promoter insertion and the cat-86 gene.