AUTHOR=Kharadi Roshni R. , Sundin George W. 

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

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.605265

DOI=10.3389/fmicb.2020.605265

ISSN=1664-302X

ABSTRACT=<p><italic>Erwinia amylovora</italic> is the causal agent of fire blight, an economically impactful disease that affects apple and pear production worldwide. <italic>E. amylovora</italic> 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 <italic>E. amylovora</italic>. We previously reported that hyper-elevation of c-di-GMP levels in <italic>E. amylovora</italic> Ea1189, resulting from the deletion of all three c-di-GMP specific phosphodiesterase genes (Ea1189Δ<italic>pdeABC</italic>), 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 Ea1189Δ<italic>pdeABC</italic> and selected for loss of autoaggregation. Our search identified a peptidoglycan hydrolase, specifically, a D, D-endopeptidase of the metallopeptidase class, EagA (<italic><underline>E</underline>rwinia</italic><underline>ag</underline>gregation 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 <italic>eagA</italic> deletion mutant (Ea1189Δ<italic>eagA</italic>) was significantly reduced in virulence compared to the wild type <italic>E. amylovora</italic> Ea1189. <italic>eagA</italic> is part of the <italic>znuABC</italic> zinc uptake gene cluster and is located within an operon downstream of <italic>znuA.</italic> The <italic>znuAeagA</italic>/<italic>znuCB</italic> 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 Zn<sup>2+</sup> in the environment, the transcription of the <italic>znuAeagA/znuBC</italic> gene cluster is regulated by both Zur and a yet to be characterized c-di-GMP dependent pathway.</p>