PMID:16677312

Citation	Kazmierczak, BI, Lebron, MB and Murray, TS (2006) Analysis of FimX, a phosphodiesterase that governs twitching motility in Pseudomonas aeruginosa. Mol. Microbiol. 60:1026-43
Abstract	Type IV pili (Tfp) are polar surface structures of Pseudomonas aeruginosa required for twitching motility, biofilm formation and adherence. One protein required for the assembly of tfp is FimX, which possesses both GGDEF and EAL domains characteristic of diguanylate cyclases and phosphodiesterases respectively. In this work we demonstrate that FimX has phosphodiesterase activity towards bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), but does not show diguanylate cyclase activity. Instead, the imperfect GGDEF domain of FimX likely serves to activate phosphodiesterase activity when bound to GTP, as has recently been described for the Caulobacter crescentus composite GGDEF-EAL protein, CC3396. Bacteria expressing FimX in which either the GGDEF or EAL domain is deleted or mutated have phenotypes indistinguishable from a DeltafimX strain, demonstrating the importance of both domains to function. Previous work has shown that FimX localizes to the bacterial pole. In this work we show that restriction of FimX to a single pole requires intact GGDEF and EAL domains. Deletion of the amino-terminal REC domain of FimX, which contains a putative polar localization signal, results in a protein that still supports intermediate levels of pilus assembly and function. RFP-FimXDeltaREC, unlike RFP-FimX, is no longer localized to the bacterial pole, while transmission electron microscopy shows that surface pili can originate from non-polar sites in this mutant. Although DeltafimX mutants show limited in vitro cytotoxicity, they are as virulent as the wild-type strain in a murine model of acute pneumonia.
Links	PubMed PMC3609419 Online version:10.1111/j.1365-2958.2006.05156.x
Keywords	Animals; Bacterial Proteins/analysis; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Cell Movement; Cyclic GMP/analogs & derivatives; Cyclic GMP/metabolism; Escherichia coli Proteins; Female; Fimbriae, Bacterial/metabolism; Fimbriae, Bacterial/physiology; Fimbriae, Bacterial/ultrastructure; HeLa Cells; Humans; Mice; Mice, Inbred C57BL; Phosphoric Diester Hydrolases/analysis; Phosphoric Diester Hydrolases/genetics; Phosphoric Diester Hydrolases/metabolism; Phosphorus-Oxygen Lyases/genetics; Phosphorus-Oxygen Lyases/metabolism; Pneumonia, Bacterial/microbiology; Point Mutation; Protein Structure, Tertiary/genetics; Pseudomonas aeruginosa/enzymology; Pseudomonas aeruginosa/pathogenicity; Pseudomonas aeruginosa/ultrastructure; Sequence Deletion; Virulence
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
PSEAI:V6AMP2		GO:0043107: type IV pilus-dependent motility	ECO:0000315: mutant phenotype evidence used in manual assertion		P		FimX is required for twitching motility, as seen in Table 1. Organism: Pseudomonas aeruginosa	complete CACAO 13004
PSEAI:V6AMP2		GO:0071111: cyclic-guanylate-specific phosphodiesterase activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		FimX exhibits phosphodiesterase activity which is stimulated by GTP, as seen in Figure 5. Organism: Pseudomonas aeruginosa	complete CACAO 13005
PSEAI:V6AMP2		GO:0043683: type IV pilus biogenesis	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Determination of surface pili by enzyme-linked immunosorbent assay (ELISA) showed that bacteria expressing either point mutant allele of FimX failed to assemble surface pili (Fig. 4B). Thus the GGDEF and EAL domains appear to be critical for FimX function in vivo, and both the EVL and non-canonical GDSIF motifs are functionally essential. Organism: Pseudomonas aeruginosa	complete CACAO 13014

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