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 tran sfer origin (oriT) in vivo was investigated. As expected, the cleavage reac tion was not detected in Escherichia coli cells expressing tral and the int egration host factor (IHF) in the absence of other transfer proteins, The T raM 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 dete ctable intracellular cleaving activity was exhibited by TraI in an II-IF mu tant strain despite the additional presence of both TraM and TraY. An essen tial role for MF in this reaction in vivo is, therefore, implied. Mobilizat ion experiments employing recombinant R1 oriT constructions and a heterolog ous conjugative helper plasmid were used to investigate the independent con tributions of TraY and TraM to the R1 relaxosome during bacterial conjugati on. In accordance with earlier observations, traY was dispensable for mobil ization in the presence of traM, but mobilization did not occur in the abse nce of both traM and traY, Interestingly, although the cleavage assays demo nstrate that TraM and TraY independently promote strand scission in vivo, T raM remained essential for mobilization of the R1 origin even in the presen ce of TraY, These findings suggest that, whereas TraY and TraM function may overlap to a certain extent in the R1 relaxosome, TraM additionally perfor ms a second function that is essential for successful conjugative transmiss ion of plasmid DNA.