Probing the impact of ligand binding on the acyl-homoserine lactone-hindered transcription factor EsaR of Pantoea stewartii subsp. stewartii

The quorum-sensing regulator EsaR from Pantoea stewartii subsp. stewartii is a LuxR homologue that is inactivated by acyl-homoserine lactone (AHL). In the corn pathogen P. stewartii, production of exopolysaccharide (EPS) is repressed by EsaR at low cell densities. However, at high cell densities when high concentrations of its cognate AHL signal are present, EsaR is inactivated and derepression of EPS production occurs. Thus, EsaR responds to AHL in a manner opposite to that of most LuxR family members. Depending on the position of its binding site within target promoters, EsaR serves as either a repressor or activator in the absence rather than in the presence of its AHL ligand. The effect of AHL on LuxR homologues has been difficult to study in vitro because AHL is required for purification and stability. EsaR, however, can be purified without AHL enabling an in vitro analysis of the response of the protein to ligand. Western immunoblots and pulse-chase experiments demonstrated that EsaR is stable in vivo in the absence or presence of AHL. Limited in vitro proteolytic digestions of a biologically active His-MBP tagged version of EsaR highlighted intradomain and interdomain conformational changes that occur in the protein in response to AHL. Gel filtration chromatography of the full-length fusion protein and cross-linking of the N-terminal domain both suggest that this conformational change does not impact the multimeric state of the protein. These findings provide greater insight into the diverse mechanisms for AHL responsiveness found within the LuxR family.     

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.     

玉米细菌性枯萎病及其病原菌的检测技术

玉米细菌性枯萎病是影响玉米生产的重要病害 ,该病害通过带菌的种子进行远距离传播 ,对该病菌的检测成为防止和控制该病害的重要手段。介绍现有的检测该病菌的各种方法 ,即黑色素选择培养法、double sandwichELISA以及RAPD PCR ,LCR PCR ,Nested PCR ,multiplePCR和荧光实时PCR等分子生物学检测方法 ,并对这些方法的特性进行比较和探讨。     

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.     

Geometrical Membrane Curvature as an Allosteric Regulator of Membrane Protein Structure and Function

Transmembrane proteins are embedded in cellular membranes of varied lipid composition and geometrical curvature. Here, we studied for the first time the allosteric effect of geometrical membrane curvature on transmembrane protein structure and function. We used single-channel optical analysis of the prototypic transmembrane β-barrel α-hemolysin (α-HL) reconstituted on immobilized single small unilamellar liposomes of different diameter and therefore curvature. Our data demonstrate that physiologically abundant geometrical membrane curvatures can enforce a dramatic allosteric regulation (1000-fold inhibition) of α-HL permeability. High membrane curvatures (1/diameter ∼1/40 nm⁻¹) compressed the effective pore diameter of α-HL from 14.2 ± 0.8 Å to 11.4 ± 0.6 Å. This reduction in effective pore area (∼40%) when combined with the area compressibility of α-HL revealed an effective membrane tension of ∼50 mN/m and a curvature-imposed protein deformation energy of ∼7 k_BT. Such substantial energies have been shown to conformationally activate, or unfold, β-barrel and α-helical transmembrane proteins, suggesting that membrane curvature could likely regulate allosterically the structure and function of transmembrane proteins in general.     

Geometrical Membrane Curvature as an Allosteric Regulator of Membrane Protein Structure and Function

Transmembrane proteins are embedded in cellular membranes of varied lipid composition and geometrical curvature. Here, we studied for the first time the allosteric effect of geometrical membrane curvature on transmembrane protein structure and function. We used single-channel optical analysis of the prototypic transmembrane β-barrel α-hemolysin (α-HL) reconstituted on immobilized single small unilamellar liposomes of different diameter and therefore curvature. Our data demonstrate that physiologically abundant geometrical membrane curvatures can enforce a dramatic allosteric regulation (1000-fold inhibition) of α-HL permeability. High membrane curvatures (1/diameter ∼1/40 nm⁻¹) compressed the effective pore diameter of α-HL from 14.2 ± 0.8 Å to 11.4 ± 0.6 Å. This reduction in effective pore area (∼40%) when combined with the area compressibility of α-HL revealed an effective membrane tension of ∼50 mN/m and a curvature-imposed protein deformation energy of ∼7 k_BT. Such substantial energies have been shown to conformationally activate, or unfold, β-barrel and α-helical transmembrane proteins, suggesting that membrane curvature could likely regulate allosterically the structure and function of transmembrane proteins in general.     

WtsE, an AvrE-family type III effector protein of Pantoea stewartii subsp. stewartii, causes cell death in non-host plants

Pantoea stewartii subsp. stewartii ( Pnss ) causes Stewart's bacterial wilt of sweet corn and leaf blight of maize. The pathogenicity of Pnss depends on synthesis of extracellular polysaccharide and an Hrp type III secretion system. WtsE, a type III secreted effector protein, is essential for the virulence of Pnss on corn. It belongs to the AvrE family of effectors, which includes DspA/E from Erwinia amylovora and AvrE1 from Pseudomonas syringae . Previously, WtsE was shown to cause disease-associated cell death in its host plant, sweet corn. Here, we examine the biological activity of WtsE in several non-host plants. WtsE induced cell death in Nicotiana benthamiana , tobacco, beet and Arabidopsis thaliana when it was transiently produced in plant cells following agroinfiltration or translocated into plant cells from Pnss , Escherichia coli or Pseudomonas syringae pv. phaseolicola ( Pph ). WtsE-induced cell death in N. benthamiana , tobacco and beet resembled a hypersensitive response and in N. benthamiana it was delayed by cycloheximide. Interestingly, WtsE strongly promoted the growth of Pnss in N. benthamiana prior to the onset of cell death. Deletion derivatives of WtsE that failed to induce cell death in N. benthamiana and tobacco also did not complement wtsE mutants of Pnss for virulence in sweet corn, indicating a correlation between the two activities. WtsE also induced cell death in A. thaliana , where it suppressed basal defences induced by Pph . Thus, WtsE has growth-promoting, defence-suppressing and cell death-inducing activities in non-host plants. Expression of WtsE also prevented the growth of yeast, possibly due to an innate toxicity to eukaryotic cells.     

Stabilization of pSW100 from Pantoea stewartii by the F conjugation system

Plasmid pSW100 is 1 of the 13 plasmids from Pantoea stewartii subsp. stewartii SW2 which has a replicon that resembles that of ColE1. This work uses a pSW100 derivative, pSW140K, to study how the pSW100 replicon is stably maintained in its hosts. Our results indicate that although pSW140K is stable in Escherichia coli HB101, the plasmid is rapidly lost in another E. coli strain, DH5alpha, indicating that the genetic background of an E. coli strain affects the stability of pSW140K. Mutagenesis of E. coli HB101 with EZ::TN revealed that mutations in traC, traF, traG, traN, and traV, which encode the components of the sex pilus assembly, reduce plasmid stability. Furthermore, this work identified that a 38-bp region located immediately upstream of the RNAII promoter is critical to the maintenance of plasmid stability in E. coli HB101. TraC binds to the region, and in addition, deleting the region destabilizes the plasmid. Furthermore, inserting this 38-bp fragment into a plasmid that contains the minimal replicon from pSW200 stabilizes the plasmid in E. coli HB101. Fluorescence in situ hybridization and immunofluorescence staining also revealed that derivatives of pSW100, pSW128A, and TraC are colocalized in cells, suggesting that pSW100 may use the sex pilus assembly as a partition apparatus to ensure the even distribution of the plasmid during cell division, which may thus maintain the plasmid's stability.     

Siderophore-Mediated Iron Acquisition Influences Motility and Is Required for Full Virulence of the Xylem-Dwelling Bacterial Phytopathogen Pantoea stewartii subsp. stewartii

Iron is a key micronutrient for microbial growth but is often present in low concentrations or in biologically unavailable forms. Many microorganisms overcome this challenge by producing siderophores, which are ferric-iron chelating compounds that enable the solubilization and acquisition of iron in a bioactive form. Pantoea stewartii subsp. stewartii, the causal agent of Stewart''s wilt of sweet corn, produces a siderophore under iron-limiting conditions. The proteins involved in the biosynthesis and export of this siderophore are encoded by the iucABCD-iutA operon, which is homologous to the aerobactin biosynthetic gene cluster found in a number of enteric pathogens. Mutations in iucA and iutA resulted in a decrease in surface-based motility that P. stewartii utilizes during the early stages of biofilm formation, indicating that active iron acquisition impacts surface motility for P. stewartii. Furthermore, bacterial movement in planta is also dependent on a functional siderophore biosynthesis and uptake pathway. Most notably, siderophore-mediated iron acquisition is required for full virulence in the sweet corn host, indicating that active iron acquisition is essential for pathogenic fitness for this important xylem-dwelling bacterial pathogen.