VirD4 coupling proteins suppress accumulation of conjugative pili produced by type IV secretion systems.

Conjugation machines constitute a large subfamily of the bacterial type IV secretion systems (T4SSs). In the absence of recipient cell contact, conjugation machines of gram-negative bacteria produce conjugative pili by extracting pilin subunits from an inner membrane (IM) pool. Upon pilus-mediated or direct contact with a target cell, conjugation systems transition to the recruitment of a cognate mobile element for intercellular DNA transfer. T4SS subunit requirements for pilus assembly and substrate transfer are nearly the same, except that VirD4 is required only for the latter. VirD4 ATPases (also termed type IV coupling proteins [T4CPs]) stimulate early DNA processing reactions and physically couple the DNA substrate to the T4SS channel. By visualizing fluorescently labeled pili, we identified marked differences in numbers, gross morphologies, and cellular organization of pili encoded by IncF F and IncN pKM101 plasmids. Remarkably, deletion of T4CP genes from both plasmids conferred hyperpiliation, a phenotype suppressed by complementation with the wild-type gene, but not alleles encoding variants defective in nucleotide binding/hydrolysis, or DNA substrate binding. In the pKM101 system, the T4CP plus TraK, a DNA processing factor that directs relaxosome assembly at the plasmid's origin-of-transfer (