BCTA - A NOVEL PBF4 GENE NECESSARY FOR CONJUGAL TRANSFER IN BACTEROIDES SPP

pBF4 is a 41 kb conjugative R-plasmid that confers MLS (macrolide-linc osamide-streptogramin B) resistance in Bacteroides spp. To identify pB F4 genes governing conjugation, recombinational mutagenesis using a su icide vector carrying fragments of the pBF4 plasmid was employed. One of six independent insertion mutants of pBF4 isolated using this metho d was found to be conjugation-deficient. Nucleotide sequence analysis around the insertion site on this plasmid revealed a 2.8 kb ORF that e ncoded a putative 110 kDa protein. A corresponding protein was observe d when a 12 kb DNA fragment containing this ORF was used to program an in vitro transcription-translation system. Both the ORF and the predi cted protein were novel when compared to available database sequences. This gene was designated bctA (Bacteroides conjugal transfer). polycl onal rabbit antibodies that recognized a sub-sequence polypeptide of B ctA reacted with a 55 kDa protein in Western blot analysis using a tot al protein extract from Bacteroides fragilis containing pBF4. The prot ein was not present in a B. fragilis strain containing the conjugation -deficient insertion mutant of pBF4. The 55 kDa protein was associated with the membrane fraction of B. fragilis. Although the cellular and biochemical basis of bctA-promoted conjugation remains unknown, this w ork demonstrates the existence of a heretofore unrecognized gene in ba cterial conjugation, and the mutagenesis system used provides the mean s to isolate and characterize other genes involved in conjugal transfe r in Bacteroides spp.