BifA, a c-di-GMP phosphodiesterase, Inversely Regulates Biofilm Formation and Swarming Motility by Pseudomonas aeruginosa PA14

Department of Microbiology and Immunology, Dartmouth Medical School, Rm. 505 Vail Building, North College Street, Hanover, NH, 03755; Department of Genetics, Harvard Medical School, Boston, MA 02114; Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114

^* To whom correspondence should be addressed. Email: georgeo{at}dartmouth.edu.

	Abstract

The intracellular signaling molecule, cyclic-di-GMP, has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyper-biofilm phenotype, thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner membrane, and in biochemical studies, purified BifA protein exhibits phosphodiesterase activity in vitro, but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the bifA mutant exhibits increased cellular pools of c-di-GMP relative to wildtype and increased synthesis of a polysaccharide produced by the pel locus. This increased polysaccharide production is required for the enhanced biofilm formed by the bifA mutant but does not contribute to the observed swarming defect. The bifA mutation also results in decreased flagellar reversals. Based on epistasis studies with the previously described sadB gene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming.