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Regulatory proteins involved in cyclic-di-GMP-mediated transcriptional regulation of amylovoran production in Erwinia amylovora

Roshni Kharadi: Michigan State University

<div>Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger critical in signaling the transition from a mobile lifestyle to an attached lifestyle in the form of biofilms. In <em>Erwinia amylovora</em>, c-di-GMP is known to positively regulate the production of amylovoran, which is a pathogenicity factor, the most abundant exopolysaccharide in the cell, and is also required for biofilm formation. In order to evaluate whether this regulation occurs at a transcriptional level, we mapped the promoter region of <em>amsG,</em> the first gene of the amylovoran biosynthetic operon and determined the location of the transcriptional start site (TSS). Through the systematic cloning of various sequence regions of the <em>amsG</em> promoter into a GFP reporter transcriptional fusion vector, we were able to determine that a ca. 170-bp region located upstream of the TSS is critical in the c-di-GMP mediated transcriptional regulation of <em>amsG</em>. In order to identify potential c-di-GMP dependent effectors that transcriptionally regulate the <em>amsG</em> promoter, we screened a library of transposon mutants generated using the transcriptional fusion reporter strains via fluorescence based cell sorting. We identified six potential positive regulators of the <em>amsG</em> promoter. Based on evaluating these candidates for their effect on amylovoran production, biofilm formation as well as <em>amsG</em> expression, we illustrate the role of two novel positive transcriptional regulators of amylovoran production.</div>