Molecular cloning, expression and nucleotide sequence of the rcsA gene of Erwinia amylovora, encoding a positive regulator of capsule expression: evidence for a family of related capsule activator proteins

Abstract

A gene encoding a positive activator of the expression of extracellular polysaccharide (EPS) synthesis in the phytopathogen Erwinia amylovora has been isolated from a genomic library in Escherichia coli. The presence of the cloned gene in E. coli stimulated transcription of the genes encoding colanic acid biosynthesis and could complement rcsA mutations. Introduction of the gene on a multicopy plasmid into Er. amylovora caused a threefold increase in EPS expression. The nucleotide sequence of the gene (designated rcsA) was determined. This revealed a single open reading frame encoding an RcsA protein of 23-7 kDa. This was confirmed by minicell analysis in E. coli. The predicted amino acid sequence of this RcsA protein showed a high degree of homology to the RcsA protein of Klebsiella aerogenes, demonstrating the existence of a family of related RcsA activator proteins capable of stimulating EPS expression. The protein had no significant homology to known DNA-binding activator proteins, indicating, for the first time, that the RcsA family of activator proteins may stimulate expression of EPS synthesis indirectly by acting on other regulatory proteins.