Identification And Structure-Function Analyses Of Bacterial C-Di-Gmp Receptors

Abstract

In recent years a novel, nucleotide-based small molecule, c-di-GMP, has emerged in the spotlight of scientific investigation as a second messenger unique to the bacterial world. The discovery that its intracellular levels strictly regulate cell adhesion and persistence of bacterial biofilms on one hand, and motility and virulence of planktonic cells on the other, has related this RNA molecule to a variety of disease states including both chronic and acute bacterial infections. Interestingly, intracellular signaling mechanisms involving c-di-GMP appear to be spatially restricted, yet cellular targets for this nucleotide remain mostly unknown. Here we set out to identify and provide comprehensive structure-function analyses of putative or known c-di-GMP receptors. By using structural biology methods we would first determine the atomic resolution structures and conformational states of appropriate targets and then use these molecular blueprints to guide our research into their mechanistic role in the big picture of intracellular signaling networks. We identified VpsT of V.cholerae as a novel c-di-GMP receptor and solved the crystal structures of the nucleotide-free and c-di-GMP-bound states. Our studies identified two biologically relevant dimerization interfaces and the potential formation of higher order oligomeric species assembling upon nucleotide recognition. VpsT defines a novel class of response regulators that utilize a characteristic structural feature to dimerize upon a signaling input regardless of concurrent phosphorylation events. The relative orientation of the DNA-binding domains of VpsT favors a model in which gene regulation is likely accompanied by major changes in DNA architecture. We showed that VpsT is a master regulator of biofilm formation, integrating c-di-GMP signaling events to inversely control exopolysaccharide production and flagellar motility. In a separate study, we provide a complete mechanistic analysis of the structure and function of LapD, a transmembrane c-di-GMP receptor in P. fluorescens which directly translates intracellular c-di-GMP levels in a signal for biofilm dispersal or initiation. We solved three novel crystal structures capturing distinct intermediates in the inside-out signaling process. Most importantly, our structural and functional analyses helped us identify homologous systems in a number of free-living and pathogenic species, likely controlling biofilm formation or toxin expression in a largely similar manner.