Novel Cyclic di-GMP Effectors of the YajQ Protein Family Control Bacterial Virulence

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 (kd∼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. © 2014 an et al.