Mutations that affect activity of the Rhizobium meliloti trpE(G) promoter in Rhizobium meliloti and Escherichia coli

Abstract

The cloned Rhizobium meliloti trpE(G) gene is not expressed in Escherichia coli. Oligonucleotide-directed mutagenesis was used to introduce base substitution mutations in the promoter region of this gene. Three separate mutations that increased homology of the putative -10 region of this promoter with the E. coli -10 promoter consensus sequence by 1 bp converted this promoter to an active promoter in E. coli. A deletion extending to position -43 from the 5' side had a minor effect on transcription in R. meliloti. However, transcription was nearly eliminated when a deletion extended to position -33, indicating that the crucial domain of the R. meliloti trpE(G) promoter begins in the region downstream of position -43. The R. meliloti trpE(G) promoter has two regions that show homology with the E. coli -35 and -10 promoter consensus sequences. Mutations in these putative -35 and -10 regions, but not in the spacer region, affected promoter strength in R. meliloti. By comparing four known R. meliloti promoter sequences, we identified a highly conserved trimer near position -35 (5'-TTG-3') but no noticeably conserved sequence near position -10.