bctA: a novel pBF4 gene necessary for conjugal transfer in Bacteroides spp.
pBF4 is a 41 kb conjugative R-plasmid that confers MLS (macrolide-lincosamide-streptogramin B) resistance in Bacteroides spp. To identify pBF4 genes governing conjugation, recombinational mutagenesis using a suicide vector carrying fragments of the pBF4 plasmid was employed. One of the six independent insertion mutants of pBF4 isolated using this method was found to be conjugation-deficient. Nucleotide sequence analysis around the insertion site on this plasmid revealed a 2.8 kb ORF that encoded a putative 110 kDa protein. A corresponding protein was observed when a 12 kb DNA fragment containing this ORF was used to program an in vitro transcription-translation system. Both the ORF and the predicted protein were novel when compared to available database sequences. This gene was designated bctA (Bacteroides conjugal transfer). Polyclonal rabbit antibodies that recognized a sub-sequence polypeptide of BctA reacted with a 55 kDa protein in Western blot analysis using a total protein extract from Bacteroides fragilis containing pBF4. The protein 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 work demonstrates the existence of a heretofore unrecognized gene in bacterial conjugation, and the mutagenesis system used provides the means to isolate and characterize other genes involved in conjugal transfer in Bacteroides spp.
Morgan
Roderick M
RM
Department of Microbiology and Immunology. Box 980678 MCV Station, Virginia Commonwealth University, Richmond, VA 23298-0678, USA.
Macrina
Francis L
FL
Department of Microbiology and Immunology. Box 980678 MCV Station, Virginia Commonwealth University, Richmond, VA 23298-0678, USA.
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001439:Bacteroides / Q000235:genetics*; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011817:Rabbits; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; C107797:BctA protein, Bacteroides fragilis; D004269:DNA, Bacterial