Bacterial Rotary Export ATPases Are Allosterically Regulated by the Nucleotide Second Messenger Cyclic-di-GMP

The widespread second messenger molecule cyclic di-GMP (cdG) regulates the transition from motile and virulent lifestyles to sessile, biofilm-forming ones in a wide range of bacteria. Many pathogenic and commensal bacterial-host interactions are known to be controlled by cdG signaling. Although the biochemistry of cyclic dinucleotide metabolism is well understood, much remains to be discovered about the downstream signaling pathways that induce bacterial responses upon cdG binding. As part of our ongoing research into the role of cdG signaling in plant-associated Pseudomonas species, we carried out an affinity capture screen for cdG binding proteins in the model organism Pseudoinonas fluorescens SBW25. The flagella export AAA + ATPase Flit was identified as a result of this screen and subsequently shown to bind specifically to the cdG molecule, with a K14 in the low micromolar range. The interaction between Flit and crki appears to be very widespread. In addition to Flit homologs from diverse bacterial species, high affinity binding was also observed fir the type III secretion system homolog HrcN and the type VI ATPase CIpB2. The addition of cdG was shown to inhibit Flit and HrcN ATPase activity in vitro. Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silk analysis was used to predict that cdG binds to Flit in a pocket of highly conserved residues at the interface between two Flit subunits. Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.