pGpG-signaling regulates virulence and global transcriptomic targets in Erwinia amylovora

Abstract

Cyclic-di-GMP (c-di-GMP) is a critical bacterial second messenger that enables the physiological phase transition in Erwinia amylovora, the phytopathogenic bacterium that causes fire blight disease. C-di-GMP generation is dependent on diguanylate cyclase enzymes while the degradation of c-di-GMP can occur through the action of phosphodiesterase (PDE) enzymes that contain an active EAL and/or a HD-GYP domain. The HD-GYP-type PDEs, which are absent in E. amylovora, can directly degrade c-di-GMP into two GMP molecules. PDEs that contain an active EAL domain, as found in all active PDEs in E. amylovora, degrade c-di-GMP into pGpG. The signaling function of pGpG is not fully understood in bacterial systems. A transcriptomic approach revealed that elevated levels of pGpG in E. amylovora impacted several genes involved in metabolic and regulatory functions including several type III secretion and extracellular appendage related genes. The heterologous overexpression of an EAL or HD-GYP-type PDE in different background E. amylovora strains with varying c-di-GMP levels revealed that in contrast to the generation of pGpG, the direct breakdown of c-di-GMP into GMP by the HD-GYP-type PDE led to an elevation in amylovoran production and biofilm formation despite a decrease in c-di-GMP levels. The breakdown of c-di-GMP into pGpG (as opposed to GTP) also led to a decrease in virulence in apple shoots. The expression of hrpS was significantly increased in response to the breakdown of c-di-GMP into pGpG. Further, our model suggests that a balance in the intracellular ratio of pGpG and c-di-GMP is essential for biofilm regulation in E. amylovora. Importancec-di-GMP is the keystone molecule for regulating the transition from motility to biofilm formation in most bacteria. Interestingly, there are two distinct enzymatic phosphodiesterase (PDE) domains, termed EAL and HD-GYP, that degrade c-di-GMP. EAL domains cleave one bond of the cyclic ring to generate pGpG while HD-GYP enzymes cleave c-di-GMP to two GMP molecules. A central question regarding c-di-GMP signaling is has whether or not pGpG itself functions as a signaling molecule. Here we demonstrate in the plant pathogen Erwinia amylovora that pGpG specifically regulates genes and contributes to biofilm formation and disease progression.