The rcsA Promoter of Pantoea stewartii subsp. stewartii Features a Low-Level Constitutive Promoter and an EsaR Quorum-Sensing-Regulated Promoter

Abstract

The upstream region of the Pantoea stewartii rcsA gene features two promoters, one for constitutive basallevel expression and a second autoregulated promoter for induced expression. The EsaR quorum-sensing repressor binds to a site centered between the two promoters, blocking transcription elongation from the regulated promoter under noninducing conditions. Pantoea stewartii subsp. stewartii is the etiological agent of Stewart's wilt disease in maize (2, 3). The organism grows in the plant xylem, where it produces large amounts of stewartan capsular polysaccharide/exopolysaccharide (EPS), thereby impeding normal vascular transport (16). A cluster of 12 genes, designated cps, encodes the functions required for stewartan biosynthesis and translocation (5,8). This gene system is related to group 1 cps gene clusters based on the chromosomal linkage to the rfb and his locus and regulation by the RcsC/ YojN/RcsA/B phosphorelay signal transduction system (17,24,32). The RcsAB heterodimeric transcription factor binds to an RcsAB box element generally located some distance (ϳ100 bp) from the regulated promoters (31).In P. stewartii subsp. stewartii, the synthesis of stewartan EPS is cell density dependent and governed by the EsaI/EsaR quorum-sensing system (28). The EsaI protein is an acyl homoserine lactone (AHL) signal synthase (27,30), and EsaR is the cognate AHL-responsive transcription factor (19,28). This system differs from the LuxR paradigm in that EsaR dimerizes and binds DNA in the absence of the AHL coinducer, thereby repressing or activating target genes depending on the location of the esaR box DNA binding site (18,21,26). We recently reported that EsaR controls the expression of the cps gene system indirectly through transcriptional repression and AHLmediated derepression of the rcsA gene (18). Quorum-sensing systems govern the synthesis of EPS in other phytopathogenic bacteria, but the underlying regulatory mechanisms differ (7, 9, 22). To our knowledge, P. stewartii subsp. stewartii represents the only described example of a species in which the rcsA gene is under direct quorum-sensing control (18).In this study, we identify the transcriptional start site(s) and promoter site(s) within the upstream region of the rcsA gene to further define the mode of quorum-sensing regulation at this promoter. We observe that the rcsA gene expresses at significant basal levels from a previously unrecognized constitutive promoter and that the induced expression initiates from a separate, AHL-inducible, RcsA-autoregulated promoter. The previously established EsaR DNA binding site (18, 19) is centered between the two promoters, suggesting that EsaR represses transcription by interfering with transcription elongation rather than by steric exclusion of RNA polymerase from the inducible promoter.Bacterial strains and growth conditions. The P. stewartii subsp. stewartii strains used in this study were ESN10 and ESN51, which are mutant strains derived from the wild-type strain DC283 (8). These strains carry different mutated all...

Probing the Impact of Ligand Binding on the Acyl-Homoserine Lactone-Hindered Transcription Factor EsaR of Pantoea stewartii subsp. stewartii

“…Radiolabeled PesaR28 DNA probe (2 nM) (lane 1) was incubated with various concentrations of HMGE as indicated (lanes 2, 3, and 4) and shown to produce a higher-molecular-size complex. Specificity of binding was verified by adding excess unlabeled PesaR28, which resulted in a downshift of the labeled probe (lanes 5,6).…”

“…In the absence of AHL, EsaR is capable of repressing transcription from the esaR and rcsA promoters (6,14,15) and activating transcription of an sRNA (20) at low cell density. AHL hinders the ability of EsaR to regulate transcription at high cell density.…”

“…At low cell densities, EsaR negatively regulates its own expression and the expression of RcsA, an activator of exopolysaccharide (EPS) biosynthesis genes, which in turn positively regulates EPS production (6,15,29). EsaR also positively regulates expression of a small RNA (sRNA) native to P. stewartii (20).…”

Cell-Cell Communication in Bacteria: United We Stand

“…In this system, the unliganded apo form of EsaR, the LuxR homolog QS regulator of P. stewartii, binds DNA and represses rcsA. RcsA is a transcription factor of the Rcs phosphorelay system that together with RcsB activates EPS biosynthesis in P. stewartii (16,66). Susanne von Bodman, University of Connecticut (S. B. von Bodman et al, CCCB-07, abstr.…”

Structure/Function Analysis of thePantoea stewartiiQuorum-Sensing Regulator EsaR as an Activator of Transcription

“…Like LuxR, which requires the lux box for DNA binding, EsaR also requires a DNA binding site known as the esaR box. Unlike the lux box, which is centered around the Ϫ42.5 region of the promoter in a class II promoter orientation (12,20), the esaR box is centered around the Ϫ10 site and blocks transcription by RNA polymerase at the esaR and rcsA promoters (6,24). The lux box and the P esaR esaR box sequences differ by five bases.…”