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Microbiology 153 (2007), 3071-3080; DOI  10.1099/mic.0.2007/006502-0
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Microbiology 153 (2007), 3071-3080; DOI  10.1099/mic.0.2007/006502-0
© 2007 Society for General Microbiology

Isolation and characterization of putative Pseudobutyrivibrio ruminis promoters

Tobias D. Schoep1,{dagger} and Keith Gregg2

1 Murdoch University, Western Australian State Agricultural Biotechnology Centre (SABC), School of Biological Sciences and Biotechnology, South St, Murdoch, 6150 Perth, Australia
2 Curtin University, Biomedical Sciences, Kent Street, Bentley, 6845 Perth, Australia

Correspondence
Tobias Schoep
tdschoep{at}cyllene.uwa.edu.au

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 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.

Abbreviations: GUS, β-glucuronidase; HEX, hexachlorofluorescein; MCS, multiple cloning site; RT-qPCR, reverse transcription quantitative PCR

{dagger}Present address: University of Western Australia, Microbiology and Immunology M502, Helicobacter pylori Research Laboratory, Stirling Highway, Crawley, 6009 Perth, Australia.

The GenBank/EMBL/DDBJ accession numbers for the promoter 6, promoter 10, promoter 18, promoter 46, pBGT, promoter 38 and pBK6 sequences of Pseudobutyrivibrio ruminis are DQ841994–DQ842000.

A supplementary figure showing the relationships between the 16S rDNA sequence of Pseudobutyrivibrio ruminis strain 0/10 and closely related species is available with the online version of this paper.






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