Erwinia amylovora is the bacterial phytopathogenic causal agent of fire blight, an economically impactful disease that affects apple and pear production worldwide. The successful orchestration of infection by E. amylovora within the host entails a coordinated implementation of several different virulence strategies. A key step in the disease cycle of E. amylovora is the transition from a primarily Type III secretion system (T3SS) effectors-dependent phase in the leaf apoplast to a biofilm-dependent phase within xylem vessels. Cyclic-di-GMP (c-di-GMP), a ubiquitous bacterial second messenger mediates this phase transition into a sessile, attached lifestyle within biofilms in E. amylovora. This body of work encompasses several aspects of the complex and multifactorial signaling network dependent on c-di-GMP in E. amylovora. Diguanylate cycles enzymes (encoded by edc genes) dimerize GTP subunits to synthesize c-di-GMP and phosphodiesterase enzymes (encoded by pde genes) hydrolyze c-di-GMP. We found that the deletion of the three active pde genes in E. amylovora, singly and in combinations of two and all three genes, led to a measurable increase in intracellular c-di-GMP levels. In addition, the elevated c-di-GMP levels correlated with increased production of amylovoran, which is the most abundant exopolysaccharide (EPS), and, a pathogenicity factor in E. amylovora. The expression of T3SS, quantified by the transcriptional level of hrpL as well as by virulence measurements in the apple and pear models, was found to be negatively regulated by c- di-GMP. While biofilm formation generally increased with elevated levels of c-di-GMP, the total pde deletion mutant, ΔpdeABC, showed a relative depreciation in the ability to form biofilms, owing to the physical autoaggregative characteristic of this strain when grown in liquid media. Autoaggregation also impaired cell separation post division, leading to the presence of filamentous cells within cellular aggregates. In addition to the EPSs amylovoran and cellulose, EagA, a peptidoglycan hydrolase was found to be a major contributor to the facilitation of autoaggregation in E. amylovora. The eagA/znuABC zinc uptake gene cluster, was found to be transcriptionally regulated by c-di-GMP and the zinc-dependent repressor Zur. Further, we evaluated the impact of a systemic deletion of all genetic components involved in c-di-GMP metabolism, including active and degenerate dgc and pde encoding genes. The resulting mutant, Ea1189Δ12 was found to be impaired in surface sensing and attachment, which are key steps in the initiation of biofilm formation both in-vitro and in-planta. The transcriptomic profile of WT Ea1189 and Ea1189Δ12 at various stages of biofilm development revealed marked differences in critical metabolic and signal transduction pathways. The correlational clustering of phenotypic data gathered from single gene complemented strains generated from Ea1189Δ12, enabled the functional categorization of each of the systemic components.
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