Cyclic-di-GMP Regulates Autoaggregation Through the Putative Peptidoglycan Hydrolase, EagA, and Regulates Transcription of the znuABC Zinc Uptake Gene Cluster in Erwinia amylovora.

Roshni R Kharadi,George W Sundin

doi:10.3389/fmicb.2020.605265

Roshni R Kharadi, George W Sundin

Open Access

https://doi.org/10.3389/fmicb.2020.605265

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Journal: Frontiers in Microbiology	Publication Date: Nov 17, 2020
Citations: 10	License type: CC BY 4.0

Affiliation: Michigan State University

Abstract

Erwinia amylovora is the causal agent of fire blight, an economically impactful disease that affects apple and pear production worldwide. E. amylovora pathogenesis is comprised of distinct type III secretion-dependent and biofilm-dependent stages. Alterations in the intracellular levels of cyclic-di-GMP (c-di-GMP) regulate the transition between the different stages of infection in E. amylovora. We previously reported that hyper-elevation of c-di-GMP levels in E. amylovora Ea1189, resulting from the deletion of all three c-di-GMP specific phosphodiesterase genes (Ea1189ΔpdeABC), resulted in an autoaggregation phenotype. The two major exopolysaccharides, amylovoran and cellulose, were also shown to partially contribute to autoaggregation. In this study, we aimed to identify the c-di-GMP dependent factor(s) that contributes to autoaggregation. We conducted a transposon mutant screen in Ea1189ΔpdeABC and selected for loss of autoaggregation. Our search identified a peptidoglycan hydrolase, specifically, a D, D-endopeptidase of the metallopeptidase class, EagA (Erwinia aggregation factor A), that was found to physiologically contribute to autoaggregation in a c-di-GMP dependent manner. The production of amylovoran was also positively affected by EagA levels. An eagA deletion mutant (Ea1189ΔeagA) was significantly reduced in virulence compared to the wild type E. amylovora Ea1189. eagA is part of the znuABC zinc uptake gene cluster and is located within an operon downstream of znuA. The znuAeagA/znuCB gene cluster was transcriptionally regulated by elevated levels of c-di-GMP as well as by the zinc-dependent transcriptional repressor Zur. We also observed that with an influx of Zn2+ in the environment, the transcription of the znuAeagA/znuBC gene cluster is regulated by both Zur and a yet to be characterized c-di-GMP dependent pathway.

Highlights

Fire blight is a bacterial disease that affects apple production with severe economic implications in the major apple growing regions around the world (Smits et al, 2017)
From our transposon mutant screen, we evaluated 4048 independent Tn5-B30 insertion mutants in E. amylovora Ea1189 pdeABC, and identified 17 mutants (0.42%) that displayed a loss of the autoaggregation phenotype
Our discovery of c-di-GMP-mediated regulation of zinc uptake in E. amylovora was dependent on our initial finding that eagA suppressed the autoaggregation phenotype in the Ea1189 pdeABC mutant

Summary

Introduction

Fire blight is a bacterial disease that affects apple production with severe economic implications in the major apple growing regions around the world (Smits et al, 2017). Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger molecule (Romling et al, 2013) that signals the transition between the type III secretion and biofilm phases of infection in E. amylovora (Edmunds et al, 2013; Kharadi et al, 2019). Biofilm formation, and the production of the major biofilm exopolysaccharides amylovoran and cellulose are positively regulated through c-di-GMP signaling in E. amylovora (Edmunds et al, 2013; Romling et al, 2013; Castiblanco and Sundin, 2018; Kharadi et al, 2019)

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