Analysis of FimX, a phosphodiesterase that governs twitching motility in Pseudomonas aeruginosa

Mol Microbiol. 2006 May;60(4):1026-43. doi: 10.1111/j.1365-2958.2006.05156.x.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 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

Substances

  • Bacterial Proteins
  • Escherichia coli Proteins
  • bis(3',5')-cyclic diguanylic acid
  • Phosphoric Diester Hydrolases
  • Phosphorus-Oxygen Lyases
  • diguanylate cyclase
  • Cyclic GMP