Characterization of the MinD/ParA-type ATPase FlhG in Vibrio alginolyticus and implications for function of its monomeric form.

FlhG is a MinD/ParA-type ATPase that works as a negative regulator for flagellar biogenesis. In Vibrio alginolyticus, FlhG functions antagonistically with the positive regulator FlhF to generate a single polar flagellum. Here we examined the effects of ADP and ATP on the aggregation and dimerization of Vibrio FlhG. Purified FlhG aggregated after exposure to low NaCl conditions, and its aggregation was suppressed in the presence of ADP or ATP. FlhG mutants at putative ATP-binding (K31A) or catalytic (D60A) residues showed similar aggregation profiles to the wild-type, but ATP caused strong aggregation of the ATPase-stimulated D171A mutant although ADP significantly suppressed the aggregation. Results of size exclusion chromatography of purified FlhG or Vibrio cell lysates suggested that FlhG exists as a monomer in solution and ATP does not induce FlhG dimerization. The K31A and D60A mutants eluted at monomer fractions regardless of nucleotides, but ATP shifted the elution peak of the D171A mutant to slightly earlier, presumably due to a subtle conformational change. Our results suggest that monomeric FlhG can function in vivo, whose active conformation aggregates easily.