Identification of an RcsA/RcsB Recognition Motif in the Promoters of Exopolysaccharide Biosynthetic Operons from Erwinia amylovora and Pantoea stewartii Subspeciesstewartii

Abstract

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...

Identification of Erwinia amylovora Genes Induced during Infection of Immature Pear Tissue

“…Previously known E. amylovora transcription factors that were upregulated included RcsA, an activator (along with RcsB) of amylovoran production (73), and RlsA, an activator of levan production (79), along with the capsular polysaccharide export protein KpsC. This further confirms that the production of both amylovoran and levan in E. amylovora is induced during infection.…”

Regulation of osmC Gene Expression by the Two-Component System rcsB-rcsC in Escherichia coli

“…The RcsB-RcsC system has been reported to regulate positively the expression of the genes directing exopolysaccharide synthesis in several bacterial species, as well as that of the cell division genes ftsA and ftsZ in E. coli (1,5,8,14,20,(34)(35)(36). Regulation of the exopolysaccharide synthesis genes by RcsB requires a cofactor, RcsA.…”

A Complex Transcription Network Controls the Early Stages of Biofilm Development byEscherichia coli

“…Many of the target genes encode parts of surface appendages (e.g., flagella and curli), components of the cellular multistress response (e.g., osmB, osmY, and osmC), or proteins involved in cell division (ftsAZ) (22,30,32,33,140). RcsB can bind either as a homodimer to the RcsB box (e.g., at ftsAZ and osmC [22,30,140]) or as a heterodimer in a complex with the auxiliary protein RcsA (e.g., at cps [60,139,152,153]). RcsA is related to the response regulators, except for the lack of the conserved aspartate site that is required for phosphorylation (139).…”