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The — 16 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli
Author(s) -
Voskuil Martin I.,
Voepel Kai,
Chambliss Glenn H.
Publication year - 1995
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1995.mmi_17020271.x
Subject(s) - biology , bacillus subtilis , escherichia coli , sequence (biology) , escherichia coli proteins , microbiology and biotechnology , genetics , computational biology , bacillus (shape) , bacteria , gene
The promoter ( amyP ) of the Bacillus subtilis α‐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 Eτ 70 promoters in E. coli , essential in some weak promoters such as the α‐amylase promoter but, of little benefit to very strong promoters.