title: Mutational analysis of the ompA promoter from Flavobacterium johnsoniae.
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 members of the phylum Bacteroidetes, but not of proteobacteria, and they function poorly in Escherichia coli. In order to analyze the Flavobacterium promoter structure systematically, 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 promoter activity. The nonconserved 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 sigma(ABfr) consensus -33/-7 promoter elements but lacked similarity to the E. coli sigma(70) promoter elements. The length of the spacer separating the -33 and -7 motifs of the ompA promoter also had a pronounced effect on promoter activity, with 19 bp being optimal. In addition to the consensus promoter elements and spacer length, the 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 Flavobacterium johnsoniae and B. fragilis genomes for putative promoters, resulting in 188 hits in B. fragilis and 109 hits in F. johnsoniae.
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA. shicheng@msu.edu
authors.forename: Shicheng
authors.initials: S
authors.lastname: Chen
authors.forename: Michael
authors.initials: M
authors.lastname: Bagdasarian
authors.forename: Michael G
authors.initials: MG
authors.lastname: Kaufman
authors.forename: Adam K
authors.initials: AK
authors.lastname: Bates
authors.forename: Edward D
authors.initials: ED
authors.lastname: Walker
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D005417:Flavobacterium / Q000235:genetics*; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016415:Sequence Alignment; D024363:Transcription Initiation Site
chemical_list: D001425:Bacterial Outer Membrane Proteins; C078167:OMPA outer membrane proteins