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

Markus Wehland,Clemens Kiecker,David L Coplin,Olaf Kelm,Wolfram Saenger,Frank Bernhard

doi:10.1074/jbc.274.6.3300

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.

Highlights

The ability to produce capsules or exopolysaccharides (EPS)1 is characteristic for most bacterial species
We recently reported the binding of RcsA and RcsB proteins from E. amylovora and E. coli to a putative promoter region located between Ϫ578 and Ϫ501 relative to the translational start site of amsG, the first reading frame in the ams operon [24]
Location of an RcsA/RcsB-binding Site in the amsG Promoter—The binding region for the RcsA/RcsB heterodimer was located by making deletions from each terminus of the previously identified 183-bp fragment F183 and testing the retardation of each in Electrophoretical Gel Mobility Shift Assay (EMSA)

Summary

Introduction

The ability to produce capsules or exopolysaccharides (EPS) is characteristic for most bacterial species. 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]. We recently reported the binding of RcsA and RcsB proteins from E. amylovora and E. coli to a putative promoter region located between Ϫ578 and Ϫ501 relative to the translational start site of amsG, the first reading frame in the ams operon [24]. We could confine the essential Rcs-binding region, and we present the recognition motif for the RcsA/RcsB dimer at the amsG promoter of E. amylovora. Our results give evidence that the binding of RcsA/RcsB to promoters in EPS operons could be a common principle for triggering the capsule synthesis in the Rcs regulatory pathway

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Conclusion