Interaction of Bacteroides fragilis pLV22a relaxase and transfer DNA with Escherichia coli RP4-TraG coupling protein

Many Bacteroides transfer factors are mobilizable in Escherichia coli when coresident with the IncP conjugative plasmid RP4, but not F. To begin characterization and potential interaction between Bacteroides mobilizable transfer factors and the RP4 mating channel, both mutants and deletions of the DNA processing (dtr), mating pair formation (mpf) and traG coupling genes of RP4 were tested for mobilization of Bacteroides plasmid pLV22a. All 10 mpf but none of the four dtr genes were required for mobilization of pLV22a. The RP4 TraG coupling protein (CP) was also required for mobilization of pLV22a, but could be substituted by a C-terminal deletion mutant of the F TraD CP. Potential interactions of the TraG CP with relaxase protein(s) and transfer DNA of both RP4 and pLV22a were assessed. Overlay assays identified productive interactions between TraG and the relaxase proteins of both MbpB and TraI from pLV22a and RP4 respectively. The Agrobacterium Transfer-ImmunoPrecipitation (TrIP) assay also identified an interaction between TraG and both RP4 and pLV22a transfer DNA. Thus, mobilization of the Bacteroides pLV22a in E. coli utilizes both RP4 Mpf and CP functions including an interaction between the relaxosome and the RP4 CP similar to that of cognate RP4 plasmid.