The RcsA and RcsB proteins of Erwinia amylovora and Escherichia coli were expressed in E. coli and purified. Their DNA-binding activity was examined using a 1-kb DNA region containing the putative promoter of the ams operon of Ew. amylovora, which is responsible for the biosynthesis of the exopolysaccharide amylovoran. Mobility shift assays indicated specific binding of RcsA and RcsB to a region of 78 bp spanning nucleotide positions -578 to -501 relative to the translational start of the first open reading frame of the operon. This region includes stretches of homology to E. coli sigma 70 promoter consensus sequences and to the E. coli cps promoter region. Binding of the Rcs proteins was not found at a JUMPstart consensus, typical for various promoters of polysaccharide gene clusters. DNA-binding activity was not detected for RcsA alone and only high concentrations of RcsB were able to interact with the ams promoter in our assay. The two proteins bind cooperatively at the indicated region of the ams promoter and further evidence is provided showing that the DNA-protein complex formed involves a heterodimer of RcsA and RcsB. The specific activity of RcsA, but not of RcsB, was enhanced when the protein was expressed in E. coli at 28 degrees C, relative to expression at 37 degrees C. In addition, DNA-protein complex formation is affected by temperature. The E. coli RcsA/RcsB proteins bind to the same region of the ams promoter and are able to interact with the Rcs proteins from Ew. amylovora.

The regulation of capsule synthesis (Rcs) regulatory network is responsible for the induction of exopolysaccharide biosynthesis in many enterobacterial species. We have previously shown that two transcriptional regulators, RcsA and RcsB, do bind as a heterodimer to the promoter of amsG, the first reading frame in the operon for amylovoran biosynthesis in the plant pathogenic bacterium Erwinia amylovora. We now identified a 23-base pair fragment from position ؊555 to ؊533 upstream of the translational start site of amsG as sufficient for the specific binding of the Rcs proteins. In addition, we could detect an RcsA/RcsB-binding site in a corresponding region of the promoter of cpsA, the homologous counterpart to the E. amylovora amsG gene in the operon for stewartan biosynthesis of Pantoea stewartii. The specificity and characteristic parameters of the protein-DNA interaction were analyzed by DNA retardation, protein-DNA cross-linking, and directed mutagenesis. The central core motif TRVGAAWAWTSYG of the amsG promoter was found to be most important for the specific interaction with RcsA/RcsB, as evaluated by mutational analysis and an in vitro selection approach. The wild type P. stewartii Rcs binding motif is degenerated in two positions and an up-mutation according to our consensus motif resulted in about a 5-fold increased affinity of the RcsA/RcsB proteins.The ability to produce capsules or exopolysaccharides (EPS) 1 is characteristic for most bacterial species. General benefits of encapsulation are the prevention of desiccation, advantages in the degradation of substrates by adherence, and the binding of toxins and nutrients (1). EPS is furthermore an essential determinant for the bacterial virulence in several host-pathogen interactions (2), e.g. during infections by the plant pathogenic bacteria Erwinia amylovora and Pantoea stewartii subsp. stewartii (Ref. 3; formerly Erwinia stewartii). The dense layer of EPS is supposed to shield invading microorganisms against host defense systems like the hypersensitive response reaction. It might further prevent cell aggregation by agglutinins, and it has been reported to accelerate the wilting of infected plants by plugging xylem vessels (4).The EPS structure is highly variable, and different types have been classified by molecular weight and structural properties (5). The biosynthesis of the high molecular weight EPS type IA in several enterobacterial species is modulated by the Rcs (regulation of capsule synthesis) regulatory network (6). Prominent examples are the regulation of colanic acid and many K antigens in Escherichia coli (7,8), Klebsiella aerogenes (9 -11), and Salmonella typhi (12). In plant pathogenic bacteria, the regulation of amylovoran synthesis in E. amylovora (13-16) and stewartan synthesis in P. stewartii (17) by Rcs proteins has been reported. EPS biosynthesis is supposed to be induced after perception of external signals by membranelocated sensors like the RcsC protein. The signal might be subsequently transduced by phosphorylation of the resp...