Characterization of c-di-GMP signaling in Salmonella typhimurium

Abstract: Signal transduction via cyclic nucleotides is a general mechanism utilized by cells from all kingdoms of life. Identification of cyclic diguanosine monophosphate (c-di-GMP) as an allosteric activator of the cellulose synthase in Gluconacetobacter xylinus 20 years ago, paved the way for the discovery of a novel general signalling system which is unique to bacteria. In this thesis, the c-di-GMP signalling network leading to the formation of a biofilm behavior in Salmonella enterica serovar Typhimurium (S. Typhimurium), the rdar morphotype, is described. The rdar morphotype is characterized by the expression of the extracellular matrix components cellulose and curli, which are controlled by the transcriptional regulator CsgD. Curli production is directly activated by CsgD, whereas cellulose biosynthesis is indirectly activated by CsgD via the expression of the GGDEF domain protein AdrA. AdrA is one of 20 proteins carrying a GGDEF and/or EAL domain. The development of a detection method for c-di-GMP based on high pressure liquid chromatography and mass spectrometry (MS) and structural characterisation of c-di-GMP by MS-MS analysis allowed the characterisation of the c-di-GMP pathway in S. Typhimurium.We demonstrate that the GGDEF domain synthesizes c-di-GMP, whereas the EAL domain is responsible for c-di-GMP degradation. A high c-di-GMP concentration positively regulates the biosynthesis of adhesive matrix components and biofilm formation, whereas it inversely regulates motility in S. Typhimurium, Escherichia coli and Pseudomonas aeruginosa. These findings indicate that c-di-GMP is a general regulator of the transition from motility to sessility in Bacteria. Overexpression of AdrA in the wild type strain S. Typhimurium UMR1 resulted in upregulation of cellulose and curli. The effect of c-di-GMP on curli expression was finally mapped to the level of CsgD transcription or mRNA stability. We further demonstrate that at least two subsets of GGDEF domain proteins are involved in CsgD expression. Chromosomally encoded AdrA controls cellulose production, but is not involved in CsgD expression, whereas the GGDEF-EAL domain proteins STM2123 and STM3388 control CsgD expression, but cannot functionally replace AdrA. Since all three proteins display apparent diguanylate cyclase activity, there are separate c-di-GMP pools dedicated to regulation of CsgD expression and cellulose biosynthesis. Further on, four of 15 EAL domain proteins, STM3611, STM1827, STM1703 and STM 4264, are involved in CsgD expression. All EAL domain proteins displayed apparent phosphodiesterase activity in vivo, which is, however, not directly correlated with the effect on CsgD expression, Therefore, the c-di-GMP pools degraded by the four EAL domain proteins are dedicated to CsgD expression to different extends. Other subsets of EAL domain proteins regulate pellicle formation, biofilm in liquid culture and flagella mediated motility. Although molecular mechanisms of c-di-GMP synthesis and degradation are unraveling, major questions regarding the targets of c-di-GMP signaling are still open. Bioinformatic studies predicted that the PilZ domain may function as a c-di-GMP binding domain. We experimentally demonstrate that c-di-GMP binds to the PilZ domains of the cellulose synthase from G. xylinus and YcgR from Escherichia coli. By inactivation and over expression of YcgR in S. Typhimurium we showed that this protein regulated motility in a c-di-GMP dependent way when c-di-GMP levels were enhanced. In summary, this work shows that c-di-GMP is a central regulator of a biofilm behaviour in S. Typhimurium. Although many details about the c-di-GMP metabolism were unravelled in this work, the molecular mechanisms how c-di-GMP excerts its function remain to be discovered.