Mutational analysis of the ompA promoter from Flavobacterium johnsoniae

Dept of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 USA; Dept of Entomology, Michigan State University, East Lansing, MI 48824 USA

^* To whom correspondence should be addressed. Email: shicheng{at}msu.edu.

	Abstract

Sequences that mediate the initiation of transcription in Flavobacterium species are not well known. The majority of identified Flavobacterium promoter elements show homology to those of other Bacteroidetes, but not of proteobacteria, and they function poorly in E. coli. In order to analyze systematically the Flavobacterium promoter structure, we investigated the -33 consensus element, -7 consensus element, and spacer length of the Flavobacterium ompA promoter by measuring the effects of site-directed mutations on the promoter activity. The non-conserved sequences in the spacer region and in regions close to the consensus motifs were randomized in order to determine their importance for promoter activity. Most of the base substitutions in these regions caused large decreases in promoter activity. The optimal -33/-7 motifs (TTTG/TAnnTTTG) were identical to Bacteroides fragilis ^ABfr consensus -33/-7 promoter elements, but lacked similarity to the E. coli ⁷⁰ promoter elements. The length of the spacer separating the -33 and -7 motifs of ompA promoter also had a pronounced effect on the promoter activity, with 19 bp being optimal. In addition to the consensus promoter elements and spacer length, GC content of the core promoter sequences had a pronounced effect on Flavobacterium promoter activity. This information was used to conduct a scan of the F. johsoniae and Bacterioides fragilis genomes for putative promoters, resulting in 188 hits in B. fragilis and 109 hits in F. johnsoniae.