Dataset Information

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.

SUBMITTER: Chen S

PROVIDER: S-EPMC1951883 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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Mutational analysis of the ompA promoter from Flavobacterium johnsoniae.

Chen Shicheng S Bagdasarian Michael M Kaufman Michael G MG Bates Adam K AK Walker Edward D ED

Journal of bacteriology 20070504 14

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 m ...[more]

PMID: 17483221

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Project description:Flavobacteriumjohnsoniae and many related bacteria secrete proteins across the outer membrane using the type IX secretion system (T9SS). Proteins secreted by T9SSs have amino-terminal signal peptides for export across the cytoplasmic membrane by the Sec system and carboxy-terminal domains (CTDs) targeting them for secretion across the outer membrane by the T9SS. Most but not all T9SS CTDs belong to the family TIGR04183 (type A CTDs). We functionally characterized diverse CTDs for secretion by the F. johnsoniae T9SS. Attachment of the CTDs from F. johnsoniae RemA, AmyB, and ChiA to the foreign superfolder green fluorescent protein (sfGFP) that had a signal peptide at the amino terminus resulted in secretion across the outer membrane. In each case, approximately 80 to 100 amino acids from the extreme carboxy termini were needed for efficient secretion. Several type A CTDs from distantly related members of the phylum Bacteroidetes functioned in F. johnsoniae, supporting the secretion of sfGFP by the F. johnsoniae T9SS. F. johnsoniae SprB requires the T9SS for secretion but lacks a type A CTD. It has a conserved C-terminal domain belonging to the family TIGR04131, which we refer to as a type B CTD. The CTD of SprB was required for its secretion, but attachment of C-terminal regions of SprB of up to 1,182 amino acids to sfGFP failed to result in secretion. Additional features outside the C-terminal region of SprB may be required for its secretion.IMPORTANCE Type IX protein secretion systems (T9SSs) are common in but limited to members of the phylum Bacteroidetes Most proteins that are secreted by T9SSs have conserved carboxy-terminal domains that belong to the protein domain family TIGR04183 (type A CTDs) or TIGR04131 (type B CTDs). Here, we identify features of T9SS CTDs of F. johnsoniae that are required for protein secretion and demonstrate that type A CTDs from distantly related members of the phylum function with the F. johnsoniae T9SS to secrete the foreign protein sfGFP. In contrast, type B CTDs failed to target sfGFP for secretion, suggesting a more complex association with the T9SS.

Dataset Information

Mutational analysis of the ompA promoter from Flavobacterium johnsoniae.

Publications

Mutational analysis of the ompA promoter from Flavobacterium johnsoniae.

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