CHARACTERIZATION OF THE RCSB GENE FROM ERWINIA-AMYLOVORA AND ITS INFLUENCE ON EXOPOLYSACCHARIDE SYNTHESIS AND VIRULENCE OF THE FIRE BLIGHT PATHOGEN

RcsB belongs to a family of positive regulators of exopolysaccharide s ynthesis in various enterobacteria. The rcsB gene of the fire blight p athogen Erwinia amylovora was cloned by PCR amplification with consens us primers, and its role in exopolysaccharide (EPS) synthesis was inve stigated, Its overexpression from high-copy-number plasmids stimulated the synthesis of the acidic EPS amylovoran and suppressed expression of the levan-forming enzyme levansucrase. Inactivation of rcsB by site directed mutagenesis created mutants that were deficient in amylovora n synthesis and avirulent on host plants, In addition, a cosmid which complemented rcsB mutants was selected from a genomic library. The spo ntaneous E. amylovora mutant E8 has a similar phenotype and was comple mented by the cloned rcsB gene, The rcsB region of strain E8 was also amplified by PCR, and the mutation was characterized as a nine-nucleot ide deletion at the start of the rcsB gene, Nucleotide sequence analys is of the E. amylovora rcsB region and the predicted amino acid sequen ce of RcsB revealed extensive homology to rcsB and the encoded protein of other bacteria such as Escherichia coli and Erwinia stewartii. In all three organisms, rcsB is localized adjacent to the rcsC gene, whic h is transcribed in the opposite direction of rcsB. The E. amylovora r csB gene has now been shown to strongly affect the formation of diseas e symptoms of a plant pathogen.