Gene transfer in rumen Bacteroides species
Obligately anaerobic bacteria of the genus Bacteroides are important and abundant inhabitants of the rumen and hind gut of mammals. They are the most numerous group in the rumen and play a major role in fibre degradation with the rumen. They are phylogenetically remote from the better studied groups of facultatively anaerobic gut bacteria (eg. enterobacteria), but are closely related to the colonic Bacteroides. Interstrain conjugal transfer of a plasmid, pRRI4 (coding for tetracycline (Tc) resistance), from the multiple plasmid bearing B.ruminicola strain 223/M2/7 to F101, a rifampicin resistant mutant of B.ruminicola B 14, was demonstrated. pRRI4 was demonstrated to be self transmissible and carried the genes coding for TcR in B.ruminicola. Transformation of B.ruminicola F101 to Tc R with pRRI4 was achieved using electroporation at frequencies up to 106 per mug DNA. Four other B.ruminicola strains were not transformed with this plasmid nor was a strain of B.uniformis. Similar procedures gave transformation of B.uniformis strains, but not B.ruminicola strains, with the E.coli:Bacteroides shuttle vectors pDP1 and pE5-2 at frequencies up to 107 per mug DNA. A nuclease assay was developed to determine the nuclease activity of a number of rumen bacteria and high nuclease activity in all B.succinogenes and five B.ruminicola strains was demonstrated. E.coli and B.uniformis strains were also transformed using electroporation by the shuttle vector, pRRI207, which has been constructed from a cryptic B.ruminicola plasmid (pRRI2, 3.4kbp) cut with EcoRI *, an E.coli vector plasmid (pHG165, 3.37kbp) carrying the pUC8 multiple cloning site, and the 4.2kbp Cc-EmRTc R* EcoRI region of pDP1. pRRI207 is capable of transforming B.uniformis, B.distasonis and B.ruminicola to clindamycin (Cc) resistance and E.coli to TcR (only expressed aerobically), and was the only construct from eleven different constructs obtained based on pRRI2 able to do so.