Construction of a promoter-rescue plasmid for butyrivibrio fibrisolvens and its use in characterization of a Flagellin promoter

The Butyrivibrio fibrisolvens/Escherichia coli shuttle vector pBHerm has been modified to produce a plasmid (pBHE) that can be used for the identification and characterization of promoters in B. fibrisolvens. pBHE allows the insertion of a test promoter immediately upstream of a promoterless erythromycin resistance gene (ermAM). The efficacy of the pBHE plasmid in isolating and characterizing promoters was tested by inserting the flagellin gene (flaA) promoter from B. fibrisolvens OR77. Transcription of the ermAM gene from the flaA promoter was significantly higher than that observed when the ermAM gene was under the control of its own promoter. The flagelling gene of OR77 appears to be transcribed from two different promoters that produce transcripts initiating approximately 130 bp apart. Two mutant flaA promoter constructs, containing mutations in the −10 and −35 regions of either of the two putative promoter regions, showed drastic alterations in both the origin and amounts of the two transcripts produced. Mutations in either promoter affected transcription from both promoters, indicating that both regions contribute to gene expression

Isolation and characterisation of an unusual bacterium, allied to the soil bacterium Bacillus benzoevorans, from feedlot manure pads in Australia

Strains of bacteria were obtained by anaerobic enrichment from feedlot manure pads and were phenotypically characterized. Colonies had a cotton wool or hairy appearance. When cultures grown in liquid media were exposed to a drying atmosphere, they produced a pellicle comprised of a cross-meshed array of cells. Colonies on agar media also produced spiral sheets of cells held well above the agar surface. The strains were Gram positive, according to ultrastructural features from transmission electron micrographs and KOH solubility, but Gram negative by Gram stain. The 16S rDNA from strain YEO5 was determined and is 99·3% similar to the type strain of Bacillus benzoevorans. The inability of our strains to grow aerobically and lack of endospores differentiated them from previously isolated strains of B. benzoevorans. In freshly broken feedlot pad material, a white, hairy coating of the exposed surface appeared within a few hours. We hypothesize that this is due to the insoluble extracellular matrix material produced by this Bacillus sp. to avoid desiccation and, additionally, the bacterial covering is responsible for retaining odours within the pad material