Despite a recent burst of research, knowledge on c-di-GMP signaling pathways remains largely fragmentary and molecular mechanisms of regulation and even c-di-GMP targets are yet unknown for most bacteria. Besides genomics or bioinformatics, accompanying alternative approaches are necessary to reveal c-di-GMP regulation in bacteria with complex lifestyles. We have approached this study by artificially altering the c-di-GMP economy of diverse pathogenic and mutualistic plant-interacting bacteria and examining the effects on the interaction with their respective host plants. Phytopathogenic Pseudomonas and symbiotic Rhizobium strains with enhanced levels of intracellular c-di-GMP displayed common free-living responses: reduction of motility, increased production of extracellular polysaccharides and enhanced biofilm formation. Regarding the interaction with the host plants, P. savastanoi pv. savastanoi cells containing high c-di-GMP levels formed larger knots on olive plants which, however, displayed reduced necrosis. In contrast, development of disease symptoms in P. syringae-tomato or P. syringae-bean interactions did not seem significantly affected by high c-di-GMP. On the other hand, increasing c-di-GMP levels in symbiotic R. etli and R. leguminosarum strains favoured the early stages of the interaction since enhanced adhesion to plant roots, but decreased symbiotic efficiency as plant growth and nitrogen contents were reduced. Our results remark the importance of c-di-GMP economy for plant-interacting bacteria and show the usefulness of our approach to reveal particular stages during plant-bacteria associations which are sensitive to changes in c-di-GMP levels.

SummaryIn Pseudomonas syringae pv. tomato DC3000, the second messenger c-di-GMP has been previously shown to stimulate pellicle formation and cellulose biosynthesis. A screen for genes involved in cellulose production under high c-di-GMP intracellular levels led to the identification of insertions in two genes, wssB and wssE, belonging to the Pto DC3000 cellulose biosynthesis operon wssABCDEFGHI. Interestingly, beside cellulose-deficient mutants, colonies with a rougher appearance than the wild type also arouse among the transposants. Those mutants carry insertions in amrZ, a gene encoding a transcriptional regulator in different Pseudomonas. Here, we provide evidence that AmrZ is involved in the regulation of bacterial cellulose production at transcriptional level by binding to the promoter region of the wssABCDEF-GHI operon and repressing cellulose biosynthesis genes. Mutation of amrZ promotes wrinkly colony morphology, increased cellulose production and loss of motility in Pto DC3000. AmrZ regulon includes putative c-di-GMP metabolising proteins, like AdcA and MorA, which may also impact those phenotypes. Furthermore, an amrZ but not a cellulose-deficient mutant turned out to be impaired in pathogenesis, indicating that AmrZ is a key regulator of Pto DC3000 virulence probably by controlling bacterial processes other than cellulose production.