Tandem receiver domain response regulator DrdR modulates swarming by controlling PilB/PilT activity in Xanthomonas...

Like many other bacterial organisms, Xanthomonas campestris pv. campestris (Xcc) employs two motility appendages, a single polar flagellum and type IV pili, for sensing environmental cues and traversing liquids and solid surfaces. However, compared to the flagellum, the molecular mechanisms underlying the function and regulation of the pilus in Xcc and other xanthomonads are not fully understood. In this study, we demonstrated that a special conserved orphan response regulator DrdR, which contains double receiver (REC) domains but lacks an output domain, modulates the activities of motor proteins of pili and regulates bacterial motilities, chemotaxis, and virulence in Xcc. Deletion of drdR in Xcc enhanced the flagellum-dependent swimming motility, inhibited pilus-dependent swarming motility, and altered swimming-chemotaxis. In addition, loss of DrdR led to reduced virulence and hydathode colonization. Protein-protein interaction and enzymatic assays demonstrated that DrdR physically interacts with the pili motor proteins PilB and PilT, enhancing their ATPase activity. Moreover, in silico analysis combined with site-directed mutagenesis revealed that the C-terminal REC domain is a functional receiver, whereas the N-terminal REC domain is a catalytically inactive pseudo-receiver. Taken together, our findings suggest that Xcc DrdR most likely controls pilus-dependent swarming motility through modulating the ATPase activities of two key motor proteins, PilT and PilB, and this motility appears to be vital for the early invasion of Xcc.