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.     

Agglutination of Erwinia stewartii Strains with a Corn Agglutinin: Correlation with Extracellular Polysaccharide Production and Pathogenicity

A bacterial agglutinin was extracted from ground corn (WI hybrid 64A × W117) seed with phosphate-buffered saline (pH 6.0) and precipitated with (NH₄)₂SO₄ at 70% saturation. The activities of this agglutinin against 22 strains of Erwinia stewartii (agent of bacterial wilt of corn) that varied in virulence were determined. Specific agglutination (agglutination titer per milligram of protein per milliliter) values were correlated negatively with virulence ratings. Strains with high specific agglutination values (15 or higher) were avirulent or weakly virulent; strains with low specific agglutination values (10 or lower) were highly virulent, with two exceptions. Avirulent strains produced butyrous colonies and released only small amounts of extracellular polysaccharide (EPS) into the medium, and the cells lacked capsules; virulent strains produced fluidal colonies and released large amounts of EPS, and the cells were capsulated. There was a strong correlation between the amount of EPS produced by each strain (as determined by increase in viscosity of the medium) and the specific agglutination value; in contrast, lipopolysaccharide compositions were similar in all strains. When cells of six fluidal strains were washed by repeatedly centrifuging and resuspending them in buffer, they were agglutinated more strongly by corn agglutinin than were unwashed cells. When avirulent cells were washed, their specific agglutination values did not increase significantly. Eight EPS-deficient mutants of E. stewartii, selected for resistance to the capsule-dependent bacteriophage K9, had lower virulence but higher specific agglutination than did their corresponding wild-type parents. Production of EPS appears to be essential for virulence; EPS may prevent agglutination of bacteria in the host, thus allowing their multiplication.     

A gene cluster for amylovoran synthesis in Erwinia amylovora: characterization and relationship to cps genes in Erwinia stewartii

A large ams gene cluster required for production of the acidic extracellular polysaccharide (EPS) amylovoran by the fire blight pathogen Erwinia amylovora was cloned. Tn5 mutagenesis and gene replacement were used to construct chromosomal ams mutants. Five complementation groups, essential for amylovoran synthesis and virulence in E. amylovora, were identified and designated amsA-E. The ams gene cluster is about 7 kb in size and functionally equivalent to the cps gene cluster involved in EPS synthesis by the related pathogen Erwinia stewartii. Mucoidy and virulence were restored to E. stewartii mutants in four cps complementation groups by the cloned E. amylovora ams genes. Conversely, the E. stewartii cps gene cluster was able to complement mutations in E. amylovora ams genes. Correspondence was found between the amsA-E complementation groups and the cpsB-D region, but the arrangement of the genes appears to be different. EPS production and virulence were also restored to E. amylovora amsE and E. stewartii cpsD mutants by clones containing the Rhizobium meliloti exoA gene.     

Identification of the Corn Pathogen Pantoea stewartii by Mass Spectrometry of Whole-Cell Extracts and Its Detection with Novel PCR Primers

Pantoea stewartii subsp. stewartii is the causative agent of Stewart''s wilt, a bacterial disease transmitted by the corn flea beetle mainly to sweet corn (Zea mays). In many countries, it is classified as a quarantine organism and must be differentiated from other yellow enteric bacteria frequently occurring with corn. We have created novel primers from the pstS-glmS region of P. stewartii for use in conventional PCR (cPCR) and quantitative PCR (qPCR). To facilitate rapid diagnosis, we applied matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Using whole-cell protein extracts, profiles were generated with a Bruker microflex machine, and the bacteria classified. P. stewartii strains were clearly distinguished from strains of Pantoea agglomerans, Pantoea dispersa, and Pantoea ananatis. Dendrogram analysis of the protein profiles confirmed the score values and showed the formation of separate clades for each species. The identification achieved by MALDI-TOF MS analysis agrees with the diagnosis by specific PCR primers. The combination of both methods allows a rapid and simple identification of the corn pathogen. P. stewartii subsp. stewartii and P. stewartii subsp. indologenes are highly related and can be distinguished not only by virulence assays and indole tests but also by a characteristic pattern in the nucleotide sequence of recA.Stewart''s wilt, caused by Pantoea stewartii subsp. stewartii (synonym Erwinia stewartii) is a serious disease of sweet corn (Zea mays) that was originally described in the United States (17, 18). Its transmission depends on the corn flea beetle (Chaetocnema pulicaria), which ingests the pathogen from infected tissue and transfers the bacteria to healthy plants. The beetle is also the main niche for overwintering of P. stewartii. Direct distribution by seed transmission is also possible (3, 11) but is not considered a major source. Stewart''s wilt is also a problem on certain elite inbred maize lines used for producing hybrid field corn seed in the mideastern United States (2). According to data from the European and Mediterranean Plant Protection Organization (EPPO) about its occurrence in Europe, Stewart''s wilt was reported from but not established in Austria, Greece, Poland, Romania, and Russia. More than 60 countries place import regulations on maize seed imports from affected areas, and surveillance of traded plant material is required to prevent further distribution of the pathogen (14).Several detection methods have been described for P. stewartii, including monoclonal antibodies for enzyme-linked immunosorbent assay (ELISA) (8). For the detection of P. stewartii by PCR analysis, primer pairs derived from rRNA genes and chromosomal markers, such as regions coding for the Hrp type III secretion system (hrp) and capsular exopolysaccharide (EPS) synthesis (cps), have been published (4). These primers were derived from chromosomal regions which are also common to other bacteria. A unique DNA area of P. stewartii might therefore be better suited for the design of specific primers. Another approach, the ligase chain reaction, requires radioactively labeled primers (21). Primers complementary to cpsD (wceL) were applied for quantitative PCR (qPCR) (19). A fingerprinting analysis based on miniprimer PCR and utilizing 10-mer short oligonucleotides combined with modified Taq polymerase has been reported (22). The signal intensity of PCRs is often affected by inhibitory plant components in the extracts. Thus, low levels of P. stewartii may not be detected. A collective drawback of PCR-based identification approaches is the detection of DNA from nonviable cells and traces of residual nucleic acids. This could lead to the rejection of safe seed lots. A method involving culturing of bacteria extracted from plants, lysis, and subsequent PCR analysis and named bio-PCR was established to ensure the detection of only viable bacterial populations (16). Screening of individual colonies from a plate with mixed cultures by PCR to verify reisolation of the pathogen is tedious and needs another fast and reliable method. Strains of Pantoea stewartii subsp. indologenes cause leaf spot on foxtail millet (Setaria italica) and pearl millet (Pennisetum americanum) or rot of Ananas comosus, and one strain was isolated from a diseased cluster bean (Cyamopsis tetragonolobus) (10). It is important to distinguish between P. stewartii subsp. stewartii and P. stewartii subsp. indologenes, since only the stewartii subspecies causes Stewart''s wilt.Furthermore, some bacterial isolates might not be unambiguously identified with PCR and with phytopathological methods. The recent successful identification of Erwinia isolates with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis profiling of protein patterns from whole cells (15) induced us to apply this method for the detection of P. stewartii and its differentiation from Pantoea agglomerans and other Pantoea species.     

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.     

A gene cluster for amylovoran synthesis in Erwinia amylovora: characterization and relationship to cps genes in Erwinia stewartii

A large ams gene cluster required for production of the acidic extracellular polysaccharide (EPS) amylovoran by the fire blight pathogen Erwinia amylovora was cloned. Tn5 mutagenesis and gene replacement were used to construct chromosomal ams mutants. Five complementation groups, essential for amylovoran synthesis and virulence in E. amylovora, were identified and designated amsA-E. The ams gene cluster is about 7 kb in size and functionally equivalent to the cps gene cluster involved in EPS synthesis by the related pathogen Erwinia stewartii. Mucoidy and virulence were restored to E. stewartii mutants in four cps complementation groups by the cloned E. amylovora ams genes. Conversely, the E. stewartii cps gene cluster was able to complement mutations in E. amylovora ams genes. Correspondence was found between the amsA-E complementation groups and the cpsB-D region, but the arrangement of the genes appears to be different. EPS production and virulence were also restored to E. amylovora amsE and E. stewartii cpsD mutants by clones containing the Rhizobium meliloti exoA gene.     

茄科劳尔氏菌复合体毒力基因及调控网络最新研究进展

植物青枯病是一种能造成巨大经济损失的土传病害,其病原茄科劳尔氏菌复合体(Ralstonia solanacearum species complex,RSSC)能通过复杂的毒力调控网络将毒力因子合成并分泌到植物细胞胞质间或细胞质内,从而引起寄主植物发病。本文详细分析了RSSC主要的毒力基因及调控网络,包括其运动性(鞭毛,菌毛)、细菌分泌系统(T2SS、T3SS以及T6SS)、毒力调控系统(Phc、Prh、Vsr、Peh、Sol)、毒力因子(CWDEs、T3Es、EPS)、群体信号因子AHL及植物激素,总结了近年来最新的研究进展并绘制了相关网络调控模式图,以期为进一步研究RSSC的致病机理及防控研究提供参考。