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].sup.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.