Diguanylate Cyclases Control Magnesium-Dependent Motility ofVibrio fischeri

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Abstract
Flagellar biogenesis and hence motility ofVibrio fischeridepends upon the presence of magnesium. In the absence of magnesium, cells contain few or no flagella and are poorly motile or nonmotile. To dissect the mechanism by which this regulation occurs, we screened transposon insertion mutants for those that could migrate through soft agar medium lacking added magnesium. We identified mutants with insertions in two distinct genes, VF0989 and VFA0959, which we termedmifAandmifB, respectively, formagnesium-dependentinduction offlagellation. Each gene encodes a predicted membrane-associated protein with diguanylate cyclase activity. Consistent with that activity, introduction intoV. fischeriof medium-copy plasmids carrying these genes inhibited motility. Furthermore, multicopy expression ofmifAinduced other phenotypes known to be correlated with diguanylate cyclase activity, including cellulose biosynthesis and biofilm formation. To directly test their function, we introduced the wild-type genes on high-copy plasmids intoEscherichia coli. We assayed for the production of cyclic di-GMP using two-dimensional thin-layer chromatography and found that strains carrying these plasmids produced a small but reproducible spot that migrated with anRfvalue consistent with cyclic di-GMP that was not produced by strains carrying the vector control. Disruptions ofmifAormifBincreased flagellin levels, while multicopy expression decreased them. Semiquantitative reverse transcription-PCR experiments revealed no significant difference in the amount of flagellin transcripts produced in either the presence or absence of Mg2+by either vector control ormifA-overexpressing cells, indicating that the impact of magnesium and cyclic-di-GMP primarily acts following transcription. Finally, we present a model for the roles of magnesium and cyclic di-GMP in the control of motility ofV. fischeri.