Cryptic plasmid pBf1 fromButyrivibrio fibrisolvens AR10: Its use as a replicon for recombinant plasmids

Abstract

A cryptic 2.85 kb plasmid (pBf1) was isolated from the rumen bacteriumButyrivibrio fibrisolvens strain AR10, ampped with restriction endonucleases, and cleavage sites suitable for attachment toEscherichia coli plasmids were identified. AR10 was not able to be cured of pBf1 by growth at 42°C or in 0.25 μg ampicillin/ml, but growth in 50 μg acridine orange/ml for three culture passages produced cured colonies at a frequency of <1%. Chimeric plasmids were constructed by combining pBf1 with theE. coli plasmid pUC18, in addition to the clindamycin resistance gene fromBacteroides fragilis plasmid pDP1 (pCW2 and pCW3), or the CAT gene fromE. coli plasmid pKK232-8 (pCK1). For plasmid construction, pBf1 was cleaved at two alternative restriction sites to increase the likelihood that replication control sequences would remain functional in at least one of the plasmids. Electroporation of AR10 yielded transformant populations that clearly maintained the plasmids and that appeared to express the ampicillinase gene of pUC18, although transformants were not readily selectable with any of the three antibiotics. The suitability of pBf1 as a replicon on which to base the construction of shuttle vectors was demonstrated clearly, by persistence of plasmid pCW3 in the absence of selective pressure, and the addition of appropriate selection factors is expected to yield practical transformation vectors.