title: Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence.
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.
authors.affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Shi-qi
authors.initials: SQ
authors.lastname: An
authors.affiliation: School of Microbiology, Biosciences Institute, University College Cork, Cork, Ireland.
authors.forename: Delphine L
authors.initials: DL
authors.lastname: Caly
authors.affiliation: School of Microbiology, Biosciences Institute, University College Cork, Cork, Ireland.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: McCarthy
authors.affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Murdoch
authors.affiliation: Genomic Sequencing Unit, College of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Joseph
authors.initials: J
authors.lastname: Ward
authors.affiliation: Genomic Sequencing Unit, College of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Melanie
authors.initials: M
authors.lastname: Febrer
authors.affiliation: School of Microbiology, Biosciences Institute, University College Cork, Cork, Ireland.
authors.forename: J Maxwell
authors.initials: JM
authors.lastname: Dow
authors.affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Ryan
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D009154:Mutation / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism*; D015290:Second Messenger Systems / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014774:Virulence; D016959:Xanthomonas campestris / Q000472:pathogenicity*
chemical_list: D001426:Bacterial Proteins; D016601:RNA-Binding Proteins; D014157:Transcription Factors; C062025:bis(3',5')-cyclic diguanylic acid; D006152:Cyclic GMP