ANALYSIS OF THE SEQUENCE OF A NEW CRYPTIC PLASMID, PRJF2, FROM A RUMEN BACTERIUM OF THE GENUS BUTYRIVIBRIO - COMPARISON WITH OTHER BUTYRIVIBRIO PLASMIDS AND APPLICATION IN THE DEVELOPMENT OF A CLONING VECTOR

A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisati on to the previously sequenced Butyrivibrio plasmid, pRJF1; with two o pen reading frames, ORF1 and ORF2, flanking a region tentatively ident ified as the replication origin, and a region of unknown function defi ned by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79 bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved. The functional importance for plasmid repl ication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the ery thromycin resistance gene from pAM beta 1 together with pUC18, to prod uce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and w as stably maintained in both hosts. Both ORF1 and ORF2 were required f or successful transformation of B. fibrisolvens.