Abstract
In this study, we attempted to understand the role of an orphan gene amyR in Erwinia amylovora, a functionally conserved ortholog of ybjN in Escherichia coli, which has recently been characterized. Amylovoran, a high molecular weight acidic heteropolymer exopolysaccharide, is a virulent factor of E. amylovora. As reported earlier, amylovoran production in an amyR knockout mutant was about eight-fold higher than that in the wild type (WT) strain of E. amylovora. When a multicopy plasmid containing the amyR gene was introduced into the amyR mutant or WT strains, amylovoran production was strongly inhibited. Furthermore, amylovoran production was also suppressed in various amylovoran-over-producing mutants, such as grrSA containing multicopies of the amyR gene. Consistent with amylovoran production, an inverse correlation was observed between in vitro expression of amyR and that of amylovoran biosynthetic genes. However, both the amyR knockout mutant and over-expression strains showed reduced levan production, another exopolysaccharide produced by E. amylovora. Virulence assays demonstrated that while the amyR mutant was capable of inducing slightly greater disease severity than that of the WT strain, strains over-expressing the amyR gene did not incite disease on apple shoots or leaves, and only caused reduced disease on immature pear fruits. Microarray studies revealed that amylovoran biosynthesis and related membrane protein-encoding genes were highly expressed in the amyR mutant, but down-regulated in the amyR over-expression strains in vitro. Down-regulation of amylovoran biosynthesis genes in the amyR over-expression strain partially explained why over-expression of amyR led to non-pathogenic or reduced virulence in vivo. These results suggest that AmyR plays an important role in regulating exopolysaccharide production, and thus virulence in E. amylovora.
Highlights
Fire blight, caused by the bacterium Erwinia amylovora, is the most devastating bacterial disease on apples and pears, which results in annual economic losses of around $100 million in the United States alone [1]
The hypersensitive response (HR) and pathogenicity-T3SS gene cluster is essential for E. amylovora to elicit an HR in non-host plants and cause disease in host plants
Our findings indicated that amyR from E. amylovora is functionally conserved with E. coli ybjN, suggesting similar evolution of the YbjN family proteins in enterobacteria
Summary
Fire blight, caused by the bacterium Erwinia amylovora, is the most devastating bacterial disease on apples and pears, which results in annual economic losses of around $100 million in the United States alone [1]. As a member of the family Enterobacteriacae, E. amylovora is closely related to many important human and animal pathogens such as Escherichia coli, Salmonella enterica, Shigella flexineri and Yersinia pestis. Like many other Gram-negative pathogenic bacteria, E. amylovora utilizes a type III secretion system (T3SS) to cause disease [2,3,4]. The hypersensitive response (HR) and pathogenicity (hrp)-T3SS gene cluster is essential for E. amylovora to elicit an HR in non-host plants and cause disease in host plants. Most genes in the hrp cluster are controlled by HrpL, a member of the ECF subfamily of sigma factors [5]. The expression of hrpL is activated by the HrpS sigma 54 enhancer-binding protein. HrpL recognizes promoters (hrp boxes) of genes such as hrpA, encoding a major component of the needle structure, as well as effector genes such as avrRpt, dspE, hrpW, and hrpN [6,7]
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