title: Isolation and characterization of putative Pseudobutyrivibrio ruminis promoters.
abstract: Novel plasmids were constructed for the analysis of DNA fragments from the rumen bacterium Pseudobutyrivibrio ruminis. Five previously unidentified promoters were characterized using a novel primer extension method to identify transcription start sites. The genes downstream of these promoters were not identified, and their activity in expression of genomic traits in wild-type P. ruminis remains putative. Comparison with promoters from this and closely related species revealed a consensus sequence resembling the binding motif for the RNA polymerase sigma(70)-like factor complex. Consensus -35 and -10 sequences within these elements were TTGACA and ATAATATA respectively, interspaced by 15-16 bp. The consensus for the -10 element was extended by one nucleotide upstream and downstream of the standard hexamer (indicated in bold). Promoter strengths were measured by reverse transcription quantitative PCR and beta-glucuronidase assays. No correlation was found between the composition and context of elements within P. ruminis promoters, and promoter strength. However, a mutation within the -35 element of one promoter revealed that transcriptional strength and choice of transcription start site were sensitive to this single nucleotide change.
authors.affiliation: Murdoch University, Western Australian State Agricultural Biotechnology Centre (SABC), School of Biological Sciences and Biotechnology, South St, Murdoch, 6150 Perth, Australia.
authors.forename: Tobias D
authors.initials: TD
authors.lastname: Schoep
authors.affiliation: Curtin University, Biomedical Sciences, Kent Street, Bentley, 6845 Perth, Australia.
authors.forename: Keith
authors.initials: K
authors.lastname: Gregg
mesh_terms: D000818:Animals; D001483:Base Sequence; D045856:Butyrivibrio / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D016384:Consensus Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012417:Rumen / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D012808:Sigma Factor
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases