Characterization by deletion and localized mutagenesis in vitro of the promoter region of the Escherichia coli ompC gene and importance of the upstream DNA domain in positive regulation by the OmpR protein

Abstract

The ompC gene codes for a major outer membrane protein whose expression is regulated by the ompR and envZ genes. Two sets of promoter deletion mutants, with upstream and downstream deletions, were constructed on a plasmid in vitro, and their promoter activity was studied by connecting them with the lacZ gene. The DNA sequence for the ompC promoter, including the -35 and -10 regions and the mRNA start site, was defined at the region about 100 base pairs upstream from the ATG initiation codon for the pro-OmpC protein. An additional 61-base-pair sequence extending upstream from the -35 region was required for the ompC promoter to function fully. After targeting the upstream region of the ompC promoter fused to the lacZ gene on a plasmid, in vitro-localized mutagenesis was performed to isolate cis-dominant mutations that affect ompC transcription. Four mutant groups, each of which had common phenotypes for expression and regulation of the gene, were identified. The individual groups also had common base substitutions. In two of the groups, the common base substitutions were localized in the upstream region of the ompC promoter, whereas in the other two they were localized in the -35 region. From these results, the upstream region of the ompC promoter was considered to be the domain responsible for activation by the ompR gene product.