The cyclic di-GMP network is a global regulator of phase-transition and attachment-dependent host colonization in Erwinia amylovora

Abstract

Cyclic-di-GMP (c-di-GMP) is an essential bacterial second messenger that regulates the transition to biofilm formation in the phytopathogen Erwinia amylovora. The c-di-GMP system in E. amylovora is comprised of 12 diguanylate cyclase/Edc (dimerize cyclic-di-GMP) and phosphodiesterase/Pde (hydrolyze cyclic-di-GMP) proteins that are characterized by the presence of GGDEF and/or EAL motifs in their domain architecture. In order to study the global regulatory effect (without the inclusion of systemic regulatory impedance) of the c-di-GMP system in E. amylovora, we eliminated all 12 edc and pde genes in E. amylovora Ea1189Δ12. Comparisons between the representative transcriptomic profiles of Ea1189Δ12 and the combinatorial edc gene knockout mutant (Ea1189Δ5) revealed marked overall distinctions in expression levels for targets in a wide range of regulatory categories, including metabolic pathways involved in the utilization of methionine, isoleucine, histidine, etc. as well as critical signal transduction pathways including the Rcs phosphorelay and PhoPQ system. A complete loss of the cyclic-di-GMP signaling components resulted in the inability of Ea1189Δ12 cells to attach to and form biofilms in vitro and within the xylem vasculature in apple shoots. Using a flow-based in vitro biofilm system, we found that initial surface sensing was primarily dependent on the flagellar filament (FliC), following which the type IV pilus (HofC) was required to anchor cells to the surface to initialize biofilm development. A transcriptomic analysis of WT E. amylovora Ea1189 and Ea1189Δ12 cells in various stages of biofilm development revealed that cyclic-di-GMP based regulation had widespread effects on purine and pyrimidine biosynthesis pathways, amylovoran biosynthesis genes and the EnvZ/OmpR signal transduction system. Additionally, complementing individual eliminated genes back into Ea1189Δ12, and the collective evaluation of several virulence factors, enabled the correlative clustering of the functional effect rendered by each Edc and Pde enzyme in the system. Significance Cyclic-di-GMP dependent regulation, in the context of biofilm formation, has been studied in several bacterial systems. However, the comprehensiveness of the studies exploring the role of individual genetic components related to cyclic-di-GMP is affected by the often large number of diguanylate cyclase and phosphodiesterase enzymes present within individual bacterial systems. To explore the evolutionary dependencies related to cyclic-di-GMP in E. amylovora, we used a collective elimination approach, whereby all of the enzymes involved in cyclic-di-GMP metabolism were eliminated from the system. This approach enabled us to highlight the critical importance of cyclic-di-GMP in plant xylem colonization due to its effect on surface attachment. Additionally, we highlight the global transcriptomic effect of cyclic-di-GMP dependent signaling at various stages of biofilm development. Our approach is aimed at exploring the regulatory role of individual cyclic-di-GMP related enzymes in a background that is free from any redundancy-based feedback.