Sequence analysis and characterization of pOM1, a small cryptic plasmid from Butyrivibrio fibrisolvens, and its use in construction of a new family of cloning vectors for Butyrivibrios

Abstract

As a preliminary step in the development of vector systems, we have isolated and begun to characterize small, cryptic plasmids from several strains of the rumen bacterium Butyrivibrio fibrisolvens. We present here the complete nucleotide sequence of Butyrivibrio plasmid pOM1, which was isolated from B. fibrisolvens Bu49. While it is very similar in size to the previously characterized Butyrivibrio plasmids pRJF1 and pRJF2, pOM1 exhibits a restriction pattern which is quite distinct. Analysis of sequence data reveals that pOM1 contains only two open reading frames of significant length (ORF1 and ORF2), both of which are required for self-replication and maintenance. The protein encoded in ORF1 shows homologies with Pre (plasmid recombination enzyme) proteins encoded in plasmids from gram-positive organisms such as Staphylococcus aureus, Streptococcus agalactiae, Lactobacillus plantarum, and Bacillus thuringiensis. The putative translation product of ORF2, on the other hand, resembles Rep (replication) proteins of a different group of gram-positive plasmids, for which the Staphylococcus plasmid pSN2 is a prototype. Unlike the other characterized-Butyrivibrio plasmids, pOM1 appears to replicate via a rolling-circle mechanism. Experimental evidence showing the presence of a single-stranded replication intermediate consistent with this mechanism is presented. pOM1 has been used in the construction of a new Escherichia coli-B. fibrisolvens shuttle vector, pSMerm1, which has been successfully used to introduce a cloned gene into B. fibrisolvens harboring the pRJF1 plasmid.