Isolation and characterization of putative Pseudobutyrivibrio ruminis promoters.
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.
Schoep
Tobias D
TD
Murdoch University, Western Australian State Agricultural Biotechnology Centre (SABC), School of Biological Sciences and Biotechnology, South St, Murdoch, 6150 Perth, Australia.
Gregg
Keith
K
Curtin University, Biomedical Sciences, Kent Street, Bentley, 6845 Perth, Australia.
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
D017931:DNA Primers; D004268:DNA-Binding Proteins; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases