Systematic Analysis of c-di-GMP Signaling Mechanisms and Biological Functions in Dickeya zeae EC1.

Dickeya zeae is an important and aggressive bacterial phytopathogen that can cause substantial economic losses in banana and rice plantations. We previously showed that c-di-GMP signaling proteins (cyclases/phosphodiesterases) in D. zeae strain EC1 play a significant role in the bacterial sessile-to-motile transition. To determine whether there is any synergistic effect among these c-di-GMP signaling proteins, we prepared a series of mutant strains by generating consecutive in-frame deletions of the genes encoding diguanylate cyclases (which make c-di-GMP) and phosphodiesterases (which break down c-di-GMP), respectively, using EC1 as a parental strain. The results showed that the complete deletion of all the putative diguanylate cyclases resulted in significantly increased bacterial motility and abrogated biofilm formation but did not appear to affect pathogenicity and virulence factor production. In contrast, the deletion of all the c-di-GMP phosphodiesterase genes disabled motility and prevented the invasion of EC1 into rice seeds. By measuring the c-di-GMP concentrations and swimming motility of all the mutants, we propose that c-di-GMP controlled swimming behavior through a multitiered program in a c-di-GMP concentration-dependent manner, which could be described as an L-shaped regression curve. These features are quite different from those that have been shown for other bacterial species such as Salmonella and Caulobacter crescentus Further analysis identified three c-di-GMP signaling proteins, i.e., PDE10355, DGC14945, and PDE14950, that play dominant roles in influencing the global c-di-GMP pool of strain EC1. The findings from this study highlight the complexity and plasticity of c-di-GMP regulatory circuits in different bacterial species.IMPORTANCE Dickeya zeae is the etiological agent of bacterial foot rot disease, which can cause massive economic losses in banana and rice plantations. Genome sequence analysis showed that D. zeae strain EC1 contains multiple c-di-GMP turnover genes, but their roles and regulatory mechanisms in bacterial physiology and virulence remain vague. By generating consecutive in-frame deletion mutants of the genes encoding c-di-GMP biosynthesis and degradation, respectively, we analyzed the individual and collective impacts of these c-di-GMP metabolic genes on the c-di-GMP global pool, bacterial physiology, and virulence. The significance of our study is in identifying the mechanism of c-di-GMP signaling in strain EC1 more clearly, which expands the c-di-GMP regulating patterns in Gram-negative species. The methods and experimental designs in this research will provide a valuable reference for the exploration of the complex c-di-GMP regulation mechanisms in other bacteria.