Enhanced Agrobacterium-mediated transformation revealed attenuation of exogenous plasmid DNA installation in recipient bacteria by exonuclease VII and SbcCD

Abstract

In DNA transfer via type IV secretion system (T4SS), relaxase enzyme releases linear ssDNA in donor cells and recircularizes in recipient cells. Using VirB/D4 T4SS, Agrobacterium cells can transfer an IncQ-type plasmid depending on Mob relaxase and a model T-DNA plasmid depending on VirD2 relaxase. Mobilization to Escherichia coli of the former plasmid is much more efficient than that of the latter, whereas an entirely reverse relationship is observed in transfer to yeast. These data suggest that either plasmid recircularization or conversion of ssDNA to dsDNA in the recipient bacterial cells is a rate-limiting step of the transfer. In this study, we examined involvement of exonuclease genes in the plasmid acceptability. By the VirD2-dependent T-DNA plasmid, E. coli sbcDΔ and sbcCΔ mutant strains produced threefold more exconjugants, and a sbcDΔ xseAΔ mutant strain yielded eightfold more exconjugants than their wild-type strain. In contrast to the enhancing effect on the VirD2-mediated transfer, the mutations exhibited a subtle effect on the Mob-mediated transfer. These results support our working hypothesis that VirD2 can transport its substrate ssDNA efficiently to recipient cells and that recipient nucleases degrade the ssDNA because VirD2 has some defect(s) in the circularization and completion of complementary DNA synthesis.