| Cyclic nucleotides are often used as second messengers to link environmental or intracellular stimuli with the output regulation of cell metabolism and physiology in all kingdoms of life. Recently, a novel signal transduction network involving cyclic diguanylate (c-di-GMP) was recognized and it appears to be restricted to bacteria. C-di-GMP plays a role in switching of bacterial lifestyle, between motile single cells and sedentary multicellular biofilm. In addition, c-di-GMP regulates virulence factors in plant and animal pathogens. Synthesis and degradation of c-di-GMP has been characterized in some enterobacteria. However, little is known about the regulatory cascade in c-di-GMP signaling, including the signal perception, the downstream effectors, and targets that interact with c-di-GMP. In this work, the c-di-GMP signaling network was investigated using the model plant pathogen Dickeya dadantii 3937.;In Chapter 2, a panel of GGDEF and EAL proteins from D. dadantii was characterized. Two EAL proteins, EcpB and EcpC, regulate c-di-GMP-associated phenotypes, including biofilm formation and motility. EcpB and EcpC are also required for the synthesis and/or expression of virulence determinants, exoenzyme production and type III secretion system. Genetic analysis showed that two sigma factors, RpoN and HrpL, are involved in c-di-GMP regulation of the type III secretion system. Biochemical analysis identified EcpC as a c-di-GMP phosphodiesterase. C-di-GMP was shown for the first time to repress a type III secretion system. C-di-GMP effects on cell physiology and virulence factor synthesis make this second messenger a potential target for drug design.;In Chapter 3, the GGDEF and EAL proteins were further analyzed and the molecular mechanism of c-di-GMP signaling was explored. GcpA, a GGDEF protein, and EcpA, an EAL protein, positively regulate biofilm formation and negatively control the type III secretion system, processes related to high c-di-GMP levels. YcgR, a PiIZ-domain protein, was shown to be involved in c-di-GMP signaling and may function as a c-di-GMP-binding effector. From these results, a c-di-GMP signaling model with dynamic interaction of c-di-GMP and its effectors, YcgR and EcpA, was developed.;In addition to the contribution in understanding the c-di-GMP signaling system, the role of lipopolysaccharide in pathogenesis in D. dadantii was evaluated. In Chapter 4, the deletion of waaC, which encodes a heptosyltransferase I ortholog, causes pleiotropic effects including a reduction in virulence. The waaC mutantation led to upregulation of two major virulence factors, exoenzyme production and expression of the type III secretion system. This work indicates that lipopolysaccharide is an important surface-associated virulence factor, and suggests a sophisticated coordination among virulence factors. |
