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J. Bacteriol. doi:10.1128/JB.00728-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Diguanylate cyclases control magnesium-dependent motility of Vibrio fischeri

Department of Microbiology and Immunology, 2160 S. First Ave. Bldg. 105, Maywood, IL 60153

^* To whom correspondence should be addressed. Email: kvisick{at}lumc.edu.

	Abstract

Flagellar biogenesis and hence motility of Vibrio fischeri depends upon the presence of magnesium. In the absence of magnesium, cells contain few or no flagella and are poorly or non-motile. 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 termed mifA and mifB, respectively, for magnesium-dependent induction of flagellation. Each gene encodes a predicted membrane-associated protein with diguanylate cyclase activity. Consistent with that activity, introduction into V. fischeri of medium-copy plasmids carrying these genes inhibited motility. Furthermore, multi-copy expression of mifA induced 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 into Escherichia coli. We assayed for the production of c-di-GMP using 2D-TLC and found that strains carrying these plasmids produced a small but reproducible spot that migrated with an Rf value consistent with c-di-GMP that was not produced by strains carrying the vector control. Disruptions of mifA or mifB increased flagellin levels, while multi-copy expression decreased them. Semi-quantitative RT-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 or mifA overexpressing cells, indicating that the impact of magnesium and c-di-GMP primarily acts following transcription. Finally, we present a model for the roles of magnesium and c-di-GMP in the control of motility of V. fischeri.

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