AbstractThe full virulence of Xanthomonas campestris pathovar campestris (Xcc) for its host plant is dependent on many different factors (Chan and Goodwin, 1999). A pivotal role is played by the second messenger cyclic di-GMP. C-di-GMP is essential for successful infection of the host not only by Xcc but also by many other pathogens. Roles for c-di-GMP include the regulation of motility, production of virulence determinants and the switch from a motile to a biofilm forming lifestyle for example (Hengge, 2009; Jenal and Malone, 2006; Römling et al., 2005; Wolfe and Visick, 2008). In Xcc, 37 genes were found to encode proteins with one of the three domains that are involved in c-di-GMP turnover, named GGDEF, EAL and HD-GYP (Ryan et al., 2007). GGDEF domain proteins play a role in the synthesis of c-di-GMP whereas EAL and HD GYP domain proteins are part of the c-di-GMP degradation system (Christen et al., 2005; Paul et al., 2004; Ryan, 2013). Some of these proteins have already been well examined but the functionality of most of the enzymes has yet to be determined.
The basis for this study was a mutant library composed of 37 Tn5 or pk18mob insertion strains for these c-di-GMP pathway genes constructed in Nanning, China. These mutations were constructed using a non-directed approach. A data set for two different virulence assays that were performed using these 37 mutants was provided by Dr Robert Ryan and was analysed in this study. These mutants were further characterised based on their motility, EPS production and protease activity. RpfG, the first characterised HD-GYP domain protein, operates as an active phosphodiesterase and is part of a two component system that is involved in the quorum sensing pathway of the signal molecule DSF (diffusible signal factor). The deletion mutant of the corresponding gene was used to verify the functionality of these phenotypic assays. The phenotypic screen revealed differences between each mutant and the wild type. Based on these results and the analysis of the virulence assays, five genes were chosen for the further work: XC0362, XC1755, XXC2459, XC2793 and XC3962. After successful construction of either unmarked deletion mutants or directed insertion mutants for four of the five selected genes, the phenotypic assays were repeated comparing the new strains and the library mutants. Previously observed phenotypes including reduced production of protease or reduced motility was reproducibly observed for the library mutants, but could not be confirmed using the newly constructed strains. Therefore, no confident conclusions could be drawn to date regarding the activity and role of these proteins in c-di-GMP signalling. A domain analysis of the selected proteins was used to gain more information about their role regarding the sensing of environmental signals.
A mass spectrometry analysis was performed to determine differences in the c-di-GMP levels between the wild type strain and a subset of the mutants. So far, only two mutants, ΔrpfG and XC2459::pK18mob, have been tested, with one replicate each, revealing an increased level of c-di-GMP for both mutants compared to the wild type. This result leads to the suggestion that these genes do play a role in c-di-GMP turnover. Additional replicates are required to confirm this conclusion.
|Date of Award||2016|
|Supervisor||Robert Ryan (Supervisor) & Tracy Palmer (Supervisor)|