Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulence

As reported previously for Ralstonia solanacearum strain GMI1000, wild-type strains AW1 and K60 were shown to produce Hrp pili. AW1 and K60 mutants lacking Hrp pili still exhibited twitching motility, which requires type 4 pili (Tfp), and electron microscopy revealed that they still made flexuous polar pili. Twitching-positive cells had an extracellular 17 kDa protein that was associated with piliation, and an internal 43-amino-acid sequence of this protein was typical of type 4 pilins. This amino acid sequence is encoded by an open reading frame, designated pilA, in the genomic sequence of GMI1000. PilA is 46% identical to a Pseudomonas aeruginosa type 4 pilin over its entire length and has all the conserved residues and motifs characteristic of type 4 group A pilins. pilA mutants did not make the 17 kDa PilA protein and did not exhibit twitching motility. When compared with its parent, an AW1 pilA mutant was reduced in virulence on tomato plants and in autoaggregation and biofilm formation in broth culture. Unlike AW1, a pilA mutant did not exhibit polar attachment to tobacco suspension culture cells or to tomato roots; it was also not naturally competent for transformation. We reported previously that twitching motility ceases in maturing AW1 colonies and that inactivation of PhcA, a global transcriptional regulator, results in colonies that continue to exhibit twitching motility. Similarly, in broth culture, expression of a pilA::lacZ fusion in AW1 decreased 10-fold at high cell density, but expression remained high in a phcA mutant. In addition, pilA::lacZ expression was positively regulated 10-fold by PehR, a response regulator that is known to be repressed by PhcA. This signal cascade is sufficient to explain why pilA expression, and thus twitching motility, decreases at high cell densities.