Effect of PlzD, a YcgR homologue of c-di-GMP-binding protein, on polar flagellar motility in Vibrio alginolyticus.

YcgR, a cyclic diguanylate (c-di-GMP)-binding protein expressed in Escherichia coli, brakes flagellar rotation by binding to the motor in a c-di-GMP dependent manner and has been implicated in triggering biofilm formation. Vibrio alginolyticus has a single polar flagellum and encodes YcgR homologue, PlzD. When PlzD or PlzD-GFP was highly over-produced in nutrient-poor condition, the polar flagellar motility of V. alginolyticus was reduced. This inhibitory effect is c-di-GMP independent as mutants substituting putative c-di-GMP-binding residues retain the effect. Moderate over-expression of PlzD-GFP allowed its localization at the flagellated cell pole. Truncation of the N-terminal 12 or 35 residues of PlzD abolished the inhibitory effect and polar localization, and no inhibitory effect was observed by deleting plzD or expressing an endogenous level of PlzD-GFP. Subcellular fractionation showed that PlzD, but not its N-terminally truncated variants, was precipitated when over-produced. Moreover, immunoblotting and N-terminal sequencing revealed that endogenous PlzD is synthesized from Met33. These results suggest that an N-terminal extension allows PlzD to localize at the cell pole but causes aggregation and leads to inhibition of motility. In V. alginolyticus, PlzD has a potential property to associate with the polar flagellar motor but this interaction is too weak to inhibit rotation.