Transfer protein TraY of plasmid R1 stimulates TraI-catalyzed oriT cleavage in vivo

The effect of TraY protein on TraI-catalyzed strand scission at the R1 transfer origin (oriT) in vivo was investigated. As expected, the cleavage reaction was not detected in Escherichia coli cells expressing tral and the integration host factor (IHF) in the absence of other transfer proteins. The TraM dependence of strand scission was found to be inversely correlated with the presence of TraY. Thus, the TraY and TraM proteins could each enhance cleaving activity at oriT in the absence of the other. In contrast, no detectable intracellular cleaving activity was exhibited by TraI in an IHF mutant strain despite the additional presence of both TraM and TraY. An essential role for IHF in this reaction in vivo is, therefore, implied. Mobilization experiments employing recombinant R1 oriT constructions and a heterologous conjugative helper plasmid were used to investigate the independent contributions of TraY and TraM to the R1 relaxosome during bacterial conjugation. In accordance with earlier observations, traY was dispensable for mobilization in the presence of traM, but mobilization did not occur in the absence of both traM and traY. Interestingly, although the cleavage assays demonstrate that TraM and TraY independently promote strand scission in vivo, TraM remained essential for mobilization of the R1 origin even in the presence of TraY. These findings suggest that, whereas TraY and TraM function may overlap to a certain extent in the R1 relaxosome, TraM additionally performs a second function that is essential for successful conjugative transmission of plasmid DNA.