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bctA: a novel pBF4 gene necessary for conjugal transfer in Bacteroides spp
Roderick M Morgan1, Francis L Macrina1
1Department of Microbiology and Immunology. Box 980678 MCV Station, Virginia Commonwealth University, Richmond, VA 23298-0678, USA.
Abstract:
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.
Insights
Researchers identified a new gene, bctA, essential for bacterial conjugation in Bacteroides species. This discovery advances understanding of how bacteria transfer genetic material, specifically antibiotic resistance genes like those conferring macrolide-lincosamide-streptogramin B resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The R-plasmid pBF4 (41 kb) facilitates macrolide-lincosamide-streptogramin B resistance in Bacteroides species.
- Understanding the genetic basis of plasmid conjugation is crucial for controlling antibiotic resistance spread.
Purpose of the Study:
- To identify genes on the pBF4 plasmid responsible for mediating bacterial conjugation.
- To characterize a novel gene involved in the conjugal transfer process.
Main Methods:
- Recombinational mutagenesis using a suicide vector to create insertion mutants of pBF4.
- Nucleotide sequencing to analyze mutations and identify open reading frames (ORFs).
- In vitro transcription-translation, Western blot analysis, and subcellular fractionation to characterize the gene product.
Main Results:
- A conjugation-deficient mutant was isolated, revealing a novel 2.8 kb ORF encoding a putative 110 kDa protein, designated bctA.
- A corresponding 55 kDa protein product of bctA was detected in Bacteroides fragilis containing pBF4, associated with the membrane fraction.
- This protein was absent in strains with the conjugation-deficient mutant, confirming bctA's role.
Conclusions:
- A previously unrecognized gene, bctA, is involved in bacterial conjugation in Bacteroides.
- The developed mutagenesis system is effective for identifying and characterizing genes essential for conjugal transfer in these bacteria.
- Further research is needed to elucidate the precise mechanism of bctA in conjugation.