Isolation and characterization of Pseudobutyrivibrio ruminis gene promoters

Tobias Schoep

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 alpha -70 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 beta-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

Isolation and characterization of Pseudobutyrivibrio ruminis gene promoters

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