Cyclic di-GMP signaling and the Type IV macromolecule secretion system in Xanthomonas citri

Abstract

In this Project we propose to develop two lines of research into the biology of the phytopathogen Xanthomonas citri (Xac), the causative agent of citrus canker. i) Metabolism of the second messenger c-di-GMP: synthesis, degradation and receptors. Bacteria can adopt multiple lifestyles. At one extreme, individual cells can undergo planktonic growth in liquid medium in which they use flagella to swim in the direction of chemo-attractants or away from chemo-repellents. At the other extreme, another lifestyle involves growth on a surface as part of a static community, often called a biofilm. The ability to switch between these and intermediate lifestyles requires the coordination of a series of developmental events and complex physiological behaviors such as quorum sensing, several types of motility, adhesion to surfaces, secretion of exopolysaccharides, surfactants and extracellular enzymes and the production of virulence factors. There is a wealth of evidence that the dinucleotide bis(3´à5´) cyclic di-GMP (c-di-GMP) plays a central role in these processes in bacteria. C-di-GMP is synthesized by diguanylate cyclases (GGDEF domains), degraded by two classes of phosphodiesterases (EAL and HD-GYP domains), and many different c-di-GMP receptor families have been characterized (PilZ, FleQ, CLP, non-catalystic GGDEF and EAL domains as well as riboswitches). The phytopathogen Xanthomonas citri (Xac), that causes citrus canker, will be used as a model to study the complexity of c-di-GMP mediated bacterial signaling networks. The Xac genome codes for several dozen potential cyclases, phosphodiesterases and c-di-GMP receptors, the majority of which are covalently or non-covalently associated with other protein domains involved in a large variety of molecular signaling activities. Our long-term challenge is to understand the structure-function relationships that control cyclase and phosphodiesterase enzymatic activity as well as the principles that regulate receptor affinity for this second messenger. We are also interested in understanding the variety of interactions that these domains undergo with other domain families and how these interactions modulate cyclase and phosphodiesterase activities and receptor affinities. ii) Type IV secretion systems. Many pathogenic bacteria use macromolecular secretion systems to transfer virulence factors into host cells. In this project we plan to continue our studies into the structure and function of the Xac Type IV secretion system (T4SS). Its large size (multiple copies of at least a dozen different subunits and a molecular mass of > 1 megaDalton) creates significant challenges for structural studies. Another major challenge is to identify and characterize specific substrates secreted by this system. In our studies, we propose to employ a variety of experimental methods including X-ray crystallography, nuclear magnetic resonance (NMR), SAXS, molecular dynamics simulations, statistical coupling analysis (SCA), the production and characterization of gene knock-outs, enzymology and gene expression studies (AU)