The — 16 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli

Abstract

The promoter (amyP) of the Bacillus subtilisα-amylase gene, which is recognized by Eτ^A, has a three out of six match to the consensus promoter in both the −35 and −10 hexamers. Oligonucleotide-directed mutagenesis was used to identify important bases for promoter utilization in the spacer sequence between the hexamers. Mutations in the sequence TGTG extending from positions −18 to −15 (the −16 region) caused a 5–94-fold decrease in α-amylase production. A G-C transversion at position −15 was the most detrimental mutation: it essentially eliminated amyP utilization in B. subtilis and in Escherichia coli. Mutating the −35 and −10 hexamers to the Eτ^A consensus promoter increased α amylase production 56-fold in B. subtilis and fivefold in E. coli Introducing the −15 G to C transversion into the consensus promoter reduced α-amylase production threefold, in contrast to the 94-fold reduction for the wild-type promoter in B. subtilis. The −15 G to C transversion did not reduce α-amylase synthesis directed by the consensus promoter in E. coli. The α amylase gene is subject to two forms of transcriptional regulation: catabolite repression and temporal regulation. None of the mutants constructed in this study had any effect on either type of regulation. The −16 region, especially the G at position −15, appears to be moderately conserved in B. subtilis and in other Gram-postitive organisms and weakly conserved in E. coli. The evidence suggests that the −16 region is an additional region of Eτ^A promoters in B. subtilis and Eτ⁷⁰ promoters in E. coli, essential in some weak promoters such as the α-amylase promoter but, of little benefit to very strong promoters.