Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence

PLoS Pathog. 2014 Oct 16;10(10):e1004429. doi: 10.1371/journal.ppat.1004429. eCollection 2014 Oct.

Abstract

Bis-(3',5') cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K(d)∼2 µM). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / metabolism*
  • Cyclic GMP / analogs & derivatives*
  • Cyclic GMP / genetics
  • Cyclic GMP / metabolism
  • Gene Expression Regulation, Bacterial
  • Humans
  • Mutation / genetics*
  • Promoter Regions, Genetic / genetics
  • RNA-Binding Proteins / metabolism*
  • Second Messenger Systems / genetics*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Virulence
  • Xanthomonas campestris / pathogenicity*

Substances

  • Bacterial Proteins
  • RNA-Binding Proteins
  • Transcription Factors
  • bis(3',5')-cyclic diguanylic acid
  • Cyclic GMP