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Isolation and characterization of putative Pseudobutyrivibrio ruminis promoters

Schoep, T.D. and Gregg, K. (2007) Isolation and characterization of putative Pseudobutyrivibrio ruminis promoters. Microbiology, 153 (9). pp. 3071-3080.

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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 σ 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 β-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.

Item Type: Journal Article
Murdoch Affiliation: Western Australian State Agricultural Biotechnology Centre
School of Biological Sciences and Biotechnology
Publisher: Society for General Microbiology
Copyright: © 2007 SGM.
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