The autoregulatory role of EsaR,a quorum-sensing regulator in Pantoea stewartii ssp. stewartii: evidence for a repressor function

Capsular polysaccharide synthesis and virulence in the plant pathogenic bacterium Pantoea stewartii ssp. stewartii requires the quorum-sensing regulatory proteins, EsaR and EsaI, and the diffusible inducer N-(3-oxo-hexanoyl)-L-homoserine lactone. Prior mutational studies suggested that EsaR might function as a repressor of quorum sensing in the control of capsular polysaccharide synthesis. Further, a lux box-like palindromic sequence coinciding with the putative -10 element of the esaR promoter suggested a possible negative autoregulatory role for EsaR. This report presents genetic evidence that EsaR represses the esaR gene under inducer-limiting conditions, and that addition of inducer promotes rapid, dose-dependent derepression. DNA mobility-shift assays and analyses by surface plasmon resonance refractometry show that EsaR binds target DNAs in a ligand-free state, and that inducer alters the binding characteristics of EsaR. Physical measurements indicate that the EsaR protein binds N-(3-oxo-hexanoyl)-L-homoserine lactone, in a 1:1 protein:ligand ratio, and that inducer binding enhances the thermal stability of the EsaR protein. These combined genetic and biochemical data establish that EsaR regulates its own expression by signal-independent repression and signal-dependent derepression. Additionally, we provide evidence that EsaR does not govern the expression of the linked esaI gene, thus EsaR has no role in controlling coinducer synthesis.     

Biological Role of Pigment Production for the Bacterial Phytopathogen Pantoea stewartii subsp. stewartii

Pantoea stewartii subsp. stewartii, the causal agent of Stewart's wilt of sweet corn, produces a yellow carotenoid pigment. A nonpigmented mutant was selected from a bank of mutants generated by random transposon mutagenesis. The transposon insertion site was mapped to the crtB gene, encoding a putative phytoene synthase, an enzyme involved in the early steps of carotenoid biosynthesis. We demonstrate here that the carotenoid pigment imparts protection against UV radiation and also contributes to the complete antioxidant pathway of P. stewartii. Moreover, production of this pigment is regulated by the EsaI/EsaR quorum-sensing system and significantly contributes to the virulence of the pathogen in planta.     

Pantoea stewartii subsp. stewartii: lessons learned from a xylem-dwelling pathogen of sweet corn

Pantoea stewartii subsp. stewartii is a Gram-negative enteric bacterium that primarily infects sweet corn. Studies of this bacterium have provided useful insight into how xylem-dwelling bacteria establish themselves and incite disease in their hosts. Pantoea stewartii subsp. stewartii is a remarkable bacterial system for laboratory studies because of its relative ease of propagation and genetic manipulation, and the fact that it appears to employ a minimal number of pathogenicity mechanisms. In addition, P. stewartii subsp. stewartii produces copious amounts of its quorum sensing (QS) signal, acyl-homoserine lactone (AHL), making it an excellent organism for studying QS-controlled gene regulation in a plant-pathogenic bacterium. In fact, P. stewartii subsp. stewartii has become the microbial paradigm for QS control of gene expression by both repression and activation via a QS regulator that binds DNA in the absence and dissociates in the presence of the signal ligand. Moreover, P. stewartii subsp. stewartii is a member of the Enterobacteriaceae, and lessons learned from its interaction with plants may be extrapolated to other plant-associated enterics, such as Erwinia, Dickeya and Pectobacterium spp., or enteric human pathogens associated with plants, such as Escherichia coli and Salmonella spp. TAXONOMY: Bacteria; Gammaproteobacteria; family Enterobacteriaceae; genus Pantoea; species stewartii (Mergaert et al., 1993). MICROBIOLOGICAL PROPERTIES: Gram-negative, motile, yellow pigmented, mucoid, facultative anaerobe. HOST RANGE: Pantoea stewartii subsp. stewartii (Smith, 1898) Dye causes Stewart's wilt of corn (Zea mays). Early-maturing sweet corn varieties and some elite inbred maize lines are particularly susceptible. DISEASE SYMPTOMS: There are two major phases of Stewart's wilt disease: (i) wilt and (ii) leaf blight. The wilt phase occurs when young seedlings are infected with P. stewartii subsp. stewartii (Fig. 1A). Water-soaked lesions first appear on the young expanding leaves and, later, seedlings may become severely wilted (Fig. 1B). The plants usually die when infected at the seedling stage. The leaf blight phase occurs when mature plants are infected (Fig. 1C). The bacteria enter the xylem and cause long linear yellow-grey lesions with a wavy margin that run parallel to the leaf veins. These lesions later turn necrotic and dark in colour. The leaf blight phase is most apparent after tasselling and does not generally cause death of the plant. In addition, the bacteria can sometimes break out of the xylem and cause pith rot in mature sweet corn plants. In resistant varieties, lesions are usually limited to only a few centimetres depending on the level of resistance of the particular hybrid (Claflin, 2000; Pataky, 2003). USEFUL WEBSITES: http://www.apsnet.org/publications/apsnetfeatures/Pages/StewartsWilt.aspx.     

A LuxR homologue of Xanthomonas oryzae pv. oryzae is required for optimal rice virulence

In Gram-negative bacteria a typical quorum sensing (QS) system usually involves the production and response to acylated homoserine lactones (AHLs). An AHL QS system is most commonly mediated by a LuxI family AHL synthase and a LuxR family AHL response regulator. This study reports for the first time the presence of a LuxR family-type regulator in Xanthomonas oryzae pv. oryzae ( Xoo ), which has been designated as OryR. The primary structure of OryR contains the typical signature domains of AHL QS LuxR family response regulators: an AHL-binding and a HTH DNA binding motif. The oryR gene is conserved among 26 Xoo strains and is also present in the genomes of close relatives X. campestris pv. campestris and X. axonopodis pv. citri . Disrupting oryR in three Xoo strains resulted in a significant reduction of rice virulence. The wild-type Xoo strains do not seem to produce AHLs and analysis of the Xoo sequenced genomes did not reveal the presence of a LuxI-family AHL synthase. The OryR protein was shown to be induced by macerated rice and affected the production of two secreted proteins: a cell-wall-degrading cellobiosidase and a 20-kDa protein of unknown function. By expressing and purifying OryR it was then observed that it was solubilized when grown in the presence of rice extract indicating that there could be a molecule(s) in rice which binds OryR. The role of OryR as a possible in planta induced LuxR family regulator is discussed.     

Structure-activity relationships of Erwinia carotovora quorum sensing signaling molecules

Production of virulence factors and secondary metabolites is regulated in the phytopathogen Erwinia carotovora by quorum sensing involving N-acylated homoserine lactone (AHL) signaling molecules. Non-hydrolyzable AHL analogues were synthesized and screened in vivo. The biological activity of each compound was correlated with its ability to bind Erwinia AHL receptor proteins (LuxR homologues) in vitro. There is an excellent correlation between carbapenem production in vivo and in vitro binding to CarR. However, no such correlation could be found between exoprotease production and analogue binding to EccR. Our data are consistent with the involvement of a third, as yet uncharacterized LuxR homologue.     

The plant pathogen Pantoea ananatis produces N-acylhomoserine lactone and causes center rot disease of onion by quorum sensing

A number of gram-negative bacteria have a quorum-sensing system and produce N-acyl-l-homoserine lactone (AHL) that they use them as a quorum-sensing signal molecule. Pantoea ananatis is reported as a common colonist of wheat heads at ripening and causes center rot of onion. In this study, we demonstrated that P. ananatis SK-1 produced two AHLs, N-hexanoyl-l-homoserine lactone (C6-HSL) and N-(3-oxohexanoyl)-l-homoserine lactone (3-oxo-C6-HSL). We cloned the AHL-synthase gene (eanI) and AHL-receptor gene (eanR) and revealed that the deduced amino acid sequence of EanI/EanR showed high identity to those of EsaI/EsaR from P. stewartii. EanR repressed the ean box sequence and the addition of AHLs resulted in derepression of ean box. Inactivation of the chromosomal eanI gene in SK-1 caused disruption of exopolysaccharide (EPS) biosynthesis, biofilm formation, and infection of onion leaves, which were recovered by adding exogenous 3-oxo-C6-HSL. These results demonstrated that the quorum-sensing system involved the biosynthesis of EPS, biofilm formation, and infection of onion leaves in P. ananatis SK-1.