Transfer Protein TraY of Plasmid R1 Stimulates TraI-Catalyzed oriT Cleavage In Vivo

Abstract

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.The traM and traY genes of IncF conjugation systems are essential for transfer proficiency (19,24,25,35). Numerous studies have established a role for traM and traY in regulation of transfer gene expression (6,7,34,35,41). Thus the transferdeficient phenotype of traY and traM mutant derivatives is certain to reflect disruption of positive gene regulation as a minimum and may further reflect the loss of additional functions.The TraM and TraY proteins of different IncF plasmids exhibit sequence-specific DNA binding activity at oriT (1,8,18,21,23,30,38,39), and they have been implicated in the initiation stage of conjugative DNA transfer. During initiation of conjugative plasmid transfer an enzyme known as relaxase cleaves a defined strand of oriT DNA at a specific position (nic) in a transesterification reaction. Cleaving activity at oriT is exhibited as part of a nucleoprotein complex called the relaxosome. This complex includes the relaxase and auxiliary proteins, which can be host or plasmid encoded (12). These accessory factors impart specificity and stability to the complex and enhance the efficiency of the cleavage reaction. In many cases specific interactions between the auxiliary proteins and oriT DNA promote localized melting of the duplex and facilitate access of relaxase to its recognition site (31-33, 37, 40, 42, 46-49).For the IncF system, extensive biochemical analysis has been dedicated to characterizing the relaxase-catalyzed cleavage ...

Plant-Derived Transfer DNAs

“…In the case of pTiC58, this AT-rich DNA contains the sequence 5#-TTCAGTACATTA, which resembles the putative binding site for integration host factor (IHF) binding, 5#-[A/T]-TCAANNNNTTA (Friedman, 1988;McGuire et al, 2000). Because IHF is involved in virtually all forms of nucleoid manipulation including DNA unwinding (Ryan et al, 2004), it is possible that this protein supports DNA cleavage at left borders in a similar way as reported previously for oriT (Karl et al, 2001).…”

Conjugative coupling proteins interact with cognate and heterologous VirB10-like proteins while exhibiting specificity for cognate relaxosomes

“…1). In the case of the related F-like conjugative plasmids, the roles of TrwA seem to be performed by the conjunction of the oriT-binding proteins TraY and TraM, which are functionally redundant to some extent (44). Both proteins, like R388 TrwA, are involved in transcriptional regulation of transfer genes and in activating the relaxase nicking activity (29,(45)(46)(47).…”

Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

“…2 and data not shown), which limited the extent of single-stranded region adjacent to the duplex in a stepwise manner (67,40,30,27,20,16, and 12 nt). The relative efficiency of DNA unwinding activity catalyzed by TraI on the series of substrates was compared using the standard helicase reaction and increasing amounts of purified protein (Fig.…”

“…The molecular mechanisms of these early steps of plasmid strand transfer remain poorly understood. Genetic and biochemical approaches have characterized the role of auxiliary factors in assisting the nic-specific cleavage reaction as well as the contribution of origin DNA sequence and topology (23)(24)(25)(26)(27)(42)(43)(44). Nonetheless, the product of that reaction, the open circular form, is not directly accessible to the duplex unwinding activity of the physically linked DNA helicase domain.…”

“…IncF, IncW, IncP, and IncQ systems offer well studied examples (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Among IncF plasmids factors known to stimulate nic cleavage include E. coli integration host factor and plasmid proteins TraM and TraY (22)(23)(24)(25)(26)(27). In the case of IncW plasmid R388, TrwA protein performs this role (13,14).…”