A family of E. coli - P. ruminis shuttle-plasmids was constructed to allow the isolation and characterization of gene promoters from the rumen bacterium P. ruminis. The promoter rescue plasmid pBK was used to isolate a total of 4 genomic DNA fragments that promoted transcription in P. ruminis strains 0/10. These promoters, and an additional promoter, previously isolated from P. ruminis strain OR38 (Schoep, 1999), were identified by their ability to initiate expression of a promoterless ermAM gene in P. ruminis. Within 4 of the fragments, a total of 5 transcription start sites were identified in P. ruminis using a novel, fluorescent-primer extension analysis protocol. Comparison of promoters isolated in this and previous studies revealed a strong consensus RNA polymerase DNA-binding motif, including the well characterized –35 and –10 elements. Consensus sequences established for these elements were: TTgacA and AtAATAta respectively, where bold upper-case font, regular upper-case, and lower-case fonts represent conservation in 100%, 80%, and 70% of promoters respectively. The −10 and −35 motifs were interspaced by 16 – 18 nt. Among the newly identified promoters, the consensus for the –10 element was extended one nucleotide upstream and downstream of the standard hexamer (boxed). These motifs were similar to those recognized by eubacterial RNA polymerase containing the σ70-like factor. Promoters also contained possible UP elements, and were significantly more curved than protein-coding regions. Additional plasmid vectors were constructed, to allow the use of both the quantitative SYBR green real time PCR and ß-glucuronidase assays, to examine 4 promoters in depth. This showed a wide range of promoter strengths within the group. However, no correlation was found between the composition and context of elements within P. ruminis promoters, and promoter strength. A mutation within the –35 element of one promoter revealed that promoter strength, and the choice of transcription start site were both sensitive to single nucleotide