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.

FIG. 1

Runoff DNA synthesis measures intracellular TraI-catalyzed cleavage activity. (A) The tra genes on plasmids maintained in vivo are illustrated schematically. The 1.2-kb BglII-PstI fragment of R1 oriT-traM DNA contained in pBR322-based substrate plasmid pBR111 is shown (top). In the second plasmid, pHP2, a 6.1-kb AsnI fragment carrying the R1 traI gene is placed under P_tac control in expression vector pGZ119EH (bottom) (B) oriT primer 8 and its complementary sequence were annealed, and a single copy of the hybrid was introduced into an unrelated vector. A KpnI fragment from this clone that contained the primer sequence was isolated and added exogenously to the cells in the cleavage assay. Reaction mixtures thus contained three primed templates for in vitro DNA synthesis: cleaved (top) and uncleaved (middle) oriT plasmid DNA released from bacterial cells and the exogenously added linear template (bottom). (C) E. coli AG1 harboring pBR111 and pHP2 was harvested after overnight culture without IPTG (lane 1) or after subculture in fresh medium without (lanes 2 and 3) or with IPTG (lanes 4 to 6). In the cleavage assay equivalent cell masses (as indicated by optical densities at 600 nm) were present in all reaction mixtures in addition to 1 ng of purified KpnI fragment. Reaction products synthesized on the different templates can be readily distinguished according to size on denaturing polyacrylamide gels (nic and std). Dideoxynucleotide-terminated DNA sequence ladders (ddATP and ddCTP) generated on oriT DNA with primer 8 were used to determine polynucleotide chain length (lanes A and C).

FIG. 2

TraY stimulates TraI-catalyzed cleavage at oriT in vivo. Overnight cultures of E. coli AG1 strains carrying plasmids R1-16 (lane 1), pBR111M0, pGNKtraI, and pGZ119EH (lanes 2 and 3), pBR111M0, pGNKtraI, and pGZYM1 (lanes 4 and 5), or pBR111, pGNKtraI, and pGZYM1 (lanes 6 and 7) to provide the indicated combinations of proteins were subcultured in fresh medium with antibiotics and IPTG. The numbers of viable cells present in the reaction mixtures resolved in lanes 1 to 7 were 2.0 × 10⁶, 1.4 × 10⁶, 2.8 × 10⁶, 0.34 × 10⁶, 0.7 × 10⁶, 0.8 × 10⁶, and 1.7 × 10⁶ CFU, respectively.