Characterization of a gene locus from Erwinia amylovora with regulatory functions in exopolysaccharide synthesis of Erwinia spp.

Abstract

In a genomic library of Erwinia amylovora, a locus has been identified that can suppress an Erwinia stewartii rcsA mutant. In addition, the locus induced a mucoid sticky phenotype of colonies in a wild-type strain of Erwinia stewartii and increased exopolysaccharide synthesis in several species of bacteria belonging to the genus Erwinia. An open reading frame was identified at this locus encoding a 225 amino acid protein that contained a helix-turn-helix motif typical of transcriptional regulators. The corresponding gene was subsequently named rcsV (regulator of capsular synthesis affecting viscosity). A mutant of rcsV in wild-type Erwinia amylovora had no detectable phenotype and produced typical levels of amylovoran under laboratory conditions. The rcsV gene on a high copy number plasmid under the control of its own promoter did not alter amylovoran production, in contrast to in-frame fusions of the structural gene in expression vectors. Since even the lac promoter was inert in the expression of rcsV, a DNA-binding protein could inhibit transcription of the gene in Erwinia amylovora. On the other hand, an Erwinia amylovora rcsA mutant was suppressed by rcsV when its promoter was replaced and the structural gene fused in-frame with lacZ' or malE. Northern blots, with total RNA from Erwinia amylovora, or promoter analysis using the GUS reporter gene did not show expression of rcsV in Erwinia amylovora, although primer extension analysis did. RcsV could be a component involved in the regulation of amylovoran synthesis, and gene expression may require an unknown external signal during the life cycle or pathogenesis of Erwinia amylovora. Key words: amylovoran, fire blight, rcsA-like activator, fusion protein.