Characterization of TraM, a Required Bacteroides Fragilis Conjugal Apparatus Protein That Interacts with the B. Fragilis Putative Coupling Protein Bcta and the B. Fragilis Relaxase Protein BmpH

Bacteroides spp. organisms, the most prominent bacteria of the human colon, are reservoirs of antibiotic resistance genes which promiscuously transfer within and from the genus via conjugation. The B. fragilis clinical isolate LV23 harbors a 37kb conjugative transposon, BTF-37. Two important conjugal apparatus proteins encoded by BTF-37 are BctA, a predicted coupling protein (CP) that powers DNA translocation, and ORF7 (TraM). In this study, we demonstrated that TraM exhibits characteristics of a required conjugal apparatus protein including 1) upregulated expression under conjugation conditions; 2) localization to the bacterial inner membrane; 3) interaction with the putative coupling protein BctA and the relaxase BmpH; and 4) absolute requirement for DNA transfer within and from B. fragilis LV23. Quantitative PCR, localization, protein interaction and RNA antisense studies were performed to examine the above characteristics. Moreover, mutagenesis and protein interaction studies revealed that two amino acids (F66 and L123) in two predicted coiled-coil domains of TraM were required for TraM interaction with BctA, suggesting that these two amino acids may be essential for TraM function in mediating DNA transfer in B. fragilis. In addition, we also identified TraM\u27s L123 amino acid as being important for the interaction of TraM with the relaxase BmpH. These results suggest that there are likely complex mechanisms involved in the interaction between TraM and BctA and/or BmpH, to facilitate DNA transfer efficiently within and from B. fragilis. This study represents the first in-depth characterization of a conjugal apparatus protein in B. fragilis, and will be useful for future studies aimed at developing interventions to prevent dissemination of antibiotic resistance from Bacteroides spp. to other bacteria. Moreover, this is one of very few studies using RNA antisense technology to knock-down target gene expression in anaerobes, and has avoided the known difficulty in genetically manipulating DNA in these organisms