Molecular differentiation of Pantoea stewartii subsp. indologenes from subspecies stewartii and identification of new isolates from maize seeds.

AIMS: Assays to detect Pantoea stewartii from maize seeds should include differentiation of P. stewartii subsp. stewartii and P. stewartii subsp. indologenes. METHODS AND RESULTS: Previously published PCR primers for the identification of P. stewartii subsp. stewartii amplified signals from both subspecies using both conventional and quantitative PCR. In MALDI-TOF mass spectroscopy analysis with the Biotyper software (Bruker), subspecies stewartii and indologenes produced identical score values. Analysis against the Biotyper database produced similar score values for both subspecies. From the subtyping methods provided by the Biotyper software, only composite correlation indexing (CCI) separated both groups. By alignment of 16S rRNA sequences, no subspecies distinction was possible. To develop new techniques for the separation of these subspecies, the partial sequences of several housekeeping genes were compared. The type strains of the two subspecies showed characteristic single-nucleotide polymorphisms (SNPs) in the genes galE, glmS and recA. Other reference strains of P. stewartii subsp. stewartii followed the same nucleotide pattern, whereas known P. stewartii subsp. indologenes strains were different. Based on single-nucleotide polymorphisms in galE and recA, PCR primers were created to separate the subspecies by stepdown PCR analysis. Two putative P. stewartii strains were isolated from imported maize seeds. They were not virulent on maize seedlings, were positive in the indole assay with Kovacs reagent and identified as P. stewartii subsp. indologenes. The subspecies-specific PCR primers confirmed they were subspecies indologenes. CONCLUSIONS: By stepdown PCR, P. stewartii subsp. indologenes can be differentiated from P. stewartii subsp. stewartii. SIGNIFICANCE AND IMPACT OF THE STUDY: A possible detection of P. stewartii subsp. stewartii, the causative agent of Stewart's wilt of maize, in plant material by immunological or molecular assays must exclude contamination with P. stewartii subsp. indologenes, which would create false positives in seed tests and affect quarantine measurements.

Biological activity of harpin produced by Pantoea stewartii subsp. stewartii.

Pantoea stewartii subsp. stewartii causes Stewart's wilt of sweet corn. A hypersensitive response and pathogenicity (Hrp) secretion system is needed to produce water-soaking and wilting symptoms in corn and to cause a hypersensitive response (HR) in tobacco. Sequencing of the hrp cluster revealed a putative harpin gene, hrpN. The product of this gene was overexpressed in Escherichia coli and shown to elicit the HR in tobacco and systemic resistance in radishes. The protein was designated HrpN(Pnss). Like other harpins, it was heat stable and protease sensitive, although it was three- to fourfold less active biologically than Erwinia amylovora harpin. We used antibodies to purified HrpN(Pnss) to verify that hrpN mutants could not produce harpin. This protein was secreted into the culture supernatant and was produced by strains of P. stewartii subsp. indologenes. In order to determine the importance of HrpN(Pnss) in pathogenesis on sweet corn, three hrpN::Tn5 mutants were compared with the wild-type strain with 50% effective dose, disease severity, response time, and growth rate in planta as parameters. In all tests, HrpN(Pnss) was not required for infection, growth, or virulence in corn or endophytic growth in related grasses.

Genetic organization of the Pantoea stewartii subsp. stewartii hrp gene cluster and sequence analysis of the hrpA, hrpC, hrpN, and wtsE operons.

The hrp/wts gene cluster of Pantoea stewartii subsp. stewartii is required for pathogenicity on sweet corn and the ability to elicit a hypersensitive response (HR) in tobacco. Site-directed transposon mutagenesis and nucleotide sequencing were used to identify hrp/wts genes within the left 20 kb of this cluster. Seventeen open reading frames (ORFs) comprise seven genetic complementation groups. These ORFs share homology with hrp and dsp genes from Erwinia amylovora, Erwinia chrysanthemi, and Pseudomonas syringae pathovars and have been designated, in map order, wtsF, wtsE, hrpN, hrpV, hrpT, hrcC, hrpG, hrpF, hrpE, hrpD, hrcJ, hrpB, hrpA, hrpS, hrpY, hrpX, and hrpL. Putative hrp consensus promoter sequences were identified upstream of hrpA, hrpF, hrpN, and wtsE. Expression of the hrpA, hrpC, and wtsE operons was regulated by HrpS. Transposon mutations in all of the hrp operons abolished pathogenicity and HR elicitation, except for the hrpN and hrpV mutants, which were still pathogenic. hrpS, hrpXY, and hrpL regulatory mutations abolished HrpN synthesis, whereas secretory mutations in the hrpC, hrpA, and hrpJ operons permitted intracellular HrpN synthesis. wtsEF mutants were not pathogenic but still produced HrpN and elicited the HR. wtsE encodes a 201-kDa protein that is similar to DspE in E. amylovora and AvrE in P. syringae pv. tomato, suggesting that this protein is a major virulence factor involved in the elicitation of water-soaked lesions.

Genetic organization of the hrp gene cluster and dspAE/BF operon in Erwinia herbicola pv. gypsophilae.

Erwinia herbicola pv. gypsophilae induces gall formation in gypsophila that is dependent on the existence of a pathogenicity plasmid (pPATHEhg). We previously demonstrated the presence of several hrp genes on this plasmid. By employing transposon mutagenesis and sequencing, a functional hrp gene cluster on the pPATHEhg has now been characterized completely. The hrp genes of E. herbicola pv. gypsophilae are remarkably similar to and colinear with those of Erwinia amylovora and Pantoea stewartii and generally showed 60 to 90% nucleotide or deduced amino acid identity. E. herbicola pv. gypsophilae, however, lacks hrpW, which is present in E. amylovora. Additionally, E. herbicola pv. gypsophilae mutants deficient in harpin production retained pathogenicity and were slightly reduced in their ability to elicit a hypersensitive response (HR) in tobacco. The "disease specific" region, dspA/EB/F, exhibited 60 to 74% identity with the dspA/EB/F loci of E. amylovora and P. stewartii, respectively. Mutations in dspA/E abolished pathogenicity of E. herbicola pv. gypsophilae but not HR elicitation on tobacco. Inactivation of HrpL reduced plant-induced transcription of dspA/E by three orders, indicating Hrp-dependent regulation.

A rapid bioassay for screening rhizosphere microorganisms for their ability to induce systemic resistance.

ABSTRACT We developed a rapid and miniaturized bioassay for screening large numbers of rhizosphere microorganisms for their ability to induce systemic resistance to bacterial leaf spot of radish caused by Xanthomonas campestris pv. armoraciae. In this bioassay, Pantoea agglomerans strain E278Ar controlled symptoms of disease as effectively as 2,6-dichloroisonicotinic acid when applied to the roots of seedlings produced in growth pouches in a soilless system. E278Ar essentially did not migrate from seedling roots to the foliage. This suggests that induction of systemic resistance could best explain the observed reduction in disease severity. Three mini-Tn5Km-induced mutants of strain E278Ar were isolated that had lost the ability to induce resistance. The bioassay also was used to demonstrate that the fungal biocontrol agent Trichoderma hamatum strain 382 induces systemic resistance in radish. The bioassay required only 14 to 18 days from seeding until rating for disease severity, which is 10 to 14 days less than earlier bioassays.

Identification of an RcsA/RcsB recognition motif in the promoters of exopolysaccharide biosynthetic operons from Erwinia amylovora and Pantoea stewartii subspecies stewartii.

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.

Characterization of a gene locus from Erwinia amylovora with regulatory functions in exopolysaccharide synthesis of Erwinia spp.

In a genomic library of Erwinia amylovora, a locus has been identified that can suppress an Erwinia stewartii rcsA mutant. In addition, the locus induced a mucoid sticky phenotype of colonies in a wild-type strain of Erwinia stewartii and increased exopolysaccharide synthesis in several species of bacteria belonging to the genus Erwinia. An open reading frame was identified at this locus encoding a 225 amino acid protein that contained a helix-turn-helix motif typical of transcriptional regulators. The corresponding gene was subsequently named rcsV (regulator of capsular synthesis affecting viscosity). A mutant of rcsV in wild-type Erwinia amylovora had no detectable phenotype and produced typical levels of amylovoran under laboratory conditions. The rcsV gene on a high copy number plasmid under the control of its own promoter did not alter amylovoran production, in contrast to in-frame fusions of the structural gene in expression vectors. Since even the lac promoter was inert in the expression of rcsV, a DNA-binding protein could inhibit transcription of the gene in Erwinia amylovora. On the other hand, an Erwinia amylovora rcsA mutant was suppressed by rcsV when its promoter was replaced and the structural gene fused in-frame with lacZ' or malE. Northern blots, with total RNA from Erwinia amylovora, or promoter analysis using the GUS reporter gene did not show expression of rcsV in Erwinia amylovora, although primer extension analysis did. RcsV could be a component involved in the regulation of amylovoran synthesis, and gene expression may require an unknown external signal during the life cycle or pathogenesis of Erwinia amylovora.

A negative regulator mediates quorum-sensing control of exopolysaccharide production in Pantoea stewartii subsp. stewartii.

Classical quorum-sensing (autoinduction) regulation, as exemplified by the lux system of Vibrio fischeri, requires N-acyl homoserine lactone (AHL) signals to stimulate cognate transcriptional activators for the cell density-dependent expression of specific target gene systems. For Pantoea stewartii subsp. stewartii, a bacterial pathogen of sweet corn and maize, the extracellular polysaccharide (EPS) stewartan is a major virulence factor, and its production is controlled by quorum sensing in a population density-dependent manner. Two genes, esaI and esaR, encode essential regulatory proteins for quorum sensing. EsaI is the AHL signal synthase, and EsaR is the cognate gene regulator. esaI, DeltaesaR, and DeltaesaI-esaR mutations were constructed to establish the regulatory role of EsaR. We report here that strains containing an esaR mutation produce high levels of EPS independently of cell density and in the absence of the AHL signal. Our data indicate that quorum-sensing regulation in P. s. subsp. stewartii, in contrast to most other described systems, uses EsaR to repress EPS synthesis at low cell density, and that derepression requires micromolar amounts of AHL. In addition, derepressed esaR strains, which synthesize EPS constitutively at low cell densities, were significantly less virulent than the wild-type parent. This finding suggests that quorum sensing in P. s. subsp. stewartii may be a mechanism to delay the expression of EPS during the early stages of infection so that it does not interfere with other mechanisms of pathogenesis.

Structure of stewartan, the capsular exopolysaccharide from the corn pathogen Erwinia stewartii.

Stewartan, the acidic exopolysaccharide of Erwinia stewartii, was purified from agar grown cells. To facilitate its structural analysis, chemical and enzymatic depolymerizations were carried out using hydrofluoric acid and E. amylovora phage phi-Ea1h, respectively. Structural characterization of the resulting oligosaccharides was performed by a combination of mass spectrometric and one- and two-dimensional (1D/2D) NMR spectroscopic techniques. A branched repeating unit with seven monosaccharide residues, all in their pyranose forms, was found: [Table: see text]

Erwinia stewartii WtsA, a positive regulator of pathogenicity gene expression, is similar to Pseudomonas syringae pv. phaseolicola HrpS.

Erwinia stewartii contains a large cluster of wts genes that are required by this bacterium for pathogenicity on corn plants. Three complementation groups within the right half of this cluster, wtsA, wtsC, and wtsB, were previously identified. In this study, WtsA was found to be a positive activator of wtsB::lacZ expression. The wtsA locus was sequenced and a single open reading frame is present within the wtsA locus, which has the capacity to encode a 323 amino acid polypeptide. A corresponding 38 kDa protein was observed in Escherichia coli minicells containing the cloned wtsA gene. The predicted WtsA polypeptide has significant similarity to HrpS from Pseudomonas syringae pv. phaseolicola, as well as other members of the NtrC class of prokaryotic regulatory proteins. Similar to other genes activated by NtrC regulators, wtsB::lacZ expression in E. coli was dependent upon rpoN.

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 ams A-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 exo A gene.

Exopolysaccharides in plant-bacterial interactions.

Rhizobial plant symbionts and bacterial plant pathogens produce exopolysaccharides that often play essential roles in the plant interaction. Many of these exopolysaccharides are acidic heteropolysaccharides that have repeating subunit structures with carbohydrate and noncarbohydrate substituents, while others are homopolysaccharides such as alginate, levan, cellulose, and glucan. While the homopolysaccharides are synthesized by mechanisms that vary with the particular polysaccharide, the heteropolysaccharides as a rule are synthesized by subunit assembly from nucleotide diphosphate-sugar precursors on a membrane-bound lipid carrier followed by polymerization and secretion. Many mutants in exopolysaccharide synthesis have been isolated, and in several cases this has led to the identification of genes that function in particular steps of biosynthesis, as well as in regulation of exopolysaccharide biosynthesis. The genetic regulation of exopolysaccharide synthesis in many plant pathogens is complex, perhaps reflecting the various niches, free living and in planta, in which exopolysaccharides function. In some cases, exopolysaccharide synthesis is regulated coordinately with other virulence factors, and in other cases separately. Regulatory genes that have homology to the two-component sensor and transcriptional effector systems are a common motif. In Rhizobium species, exopolysaccharide synthesis is regulated by transcriptional as well as posttranslational mechanisms. Exopolysaccharides function differently in the root-nodule symbiosis versus plant pathogenesis. Specific Rhizobium exopolysaccharide structures promote nodule development and invasion in legumes that form indeterminate nodules. In plant pathogenesis, less specific mechanisms of pathogenesis occur: exopolysaccharides cause wilting by blocking xylem vessels, are partly responsible for water-soaked lesions, and may also aid in invasion, growth, and survival in plant tissues.

Structural and functional analysis of the rcsA gene from Erwinia stewartii.

RcsA is a positive regulator of genes for extracellular polysaccharide biosynthesis in the Enterobacteriaceae. The nucleotide sequence of the rcsA gene from Erwinia stewartii was determined and compared to rcsA sequences from E. amylovora, Escherichia coli, and Klebsiella pneumoniae. Three highly conserved regions of the gene were identified. The C-terminal end of the open reading frame (ORF) shared significant amino acid homology to the LuxR class of bacterial activator proteins. Insertion and deletion mutagenesis of the 5' non-coding region indicated that maximal expression of rcsA was dependent upon cis-acting regulatory sequences located more than 300 bp upstream of the translational start site.

The rcsA gene from Erwinia amylovora: identification, nucleotide sequence, and regulation of exopolysaccharide biosynthesis.

RcsA is a positive activator of extracellular polysaccharide synthesis in the Enterobacteriaceae. A cosmid clone containing the rcsA gene from Erwinia amylovora was identified by its ability to restore mucoidy to an E. stewartii rcsA mutant. The rcsA gene was subcloned on a 2.2-kilobase HindIII-PstI fragment that hybridized with an E. stewartii rcsA probe and complemented E. stewartii and Escherichia coli rcsA mutants. In addition, the cloned E. amylovora rcsA gene stimulated expression of cps::lac fusions in E. coli and E. stewartii. The rcsA region was sequenced, and one open reading frame of 211 amino acids was found. The predicted protein sequence specified by this open reading frame was 55% homologous with that of the Klebsiella pneumoniae RcsA protein. Highly conserved regions in the 3' and 5' ends of the two proteins were observed. An E. amylovora rcsA mutant was constructed by Tn5 mutagenesis of the cloned gene followed by recombination of the mutation into the chromosome of wild-type strain Ea1/79. The synthesis of both amylovorin and levan was reduced by more than 90% in this mutant, indicating common regulation of the two polysaccharides by rcsA. Virulence of the rcsA mutant on immature pear fruit was diminished but not completely abolished.

Characterization of a gene cluster for exopolysaccharide biosynthesis and virulence in Erwinia stewartii.

We have previously cloned the genes for synthesis of capsular polysaccharide (cps) and slime from Erwinia stewartii in cosmid pES2144. In this study, pES2144 was shown to complement 14 spontaneous cps mutants. These mutants were characterized by probing Southern blots of mutant genomic DNA with pES2144; insertions were detected in four mutants and deletions in six mutants. Genetic and physical maps of the pES2144 cps region were constructed by subcloning, restriction analysis, and transposon mutagenesis with Tn5, Tn5lac, and Tn3HoHo1. Mutations affecting the ability of pES2144 to restore mucoidy to cps deletion mutants were located in five regions, designated cpsA to cpsE. None of the cps mutants were able to cause systemic wilting of corn plants, and mutations in cps regions B to E further abolished the ability of the bacterium to cause watersoaked lesions on seedlings. The gene for uridine-5'-diphosphogalactose 4-epimerase (galE) was linked to the cps genes on pES2144. In E. stewartii, galE was constitutively expressed, whereas the genes for galactokinase (galK) and galactose-1-phosphate uridyltransferase (galT) were inducible and not linked to galE. Thus, galE does not appear to be part of the gal operon in this species.

Isolation and characterization of Bordetella avium plasmids.

Experiments were conducted to study the plasmids of Bordetella avium, B. avium-like, and B. bronchiseptica isolates from turkeys and the plasmids of the Art-Vax commercial vaccine strain. Plasmids were observed in 6 of 20 B. avium isolates, in 6 of 20 B. avium-like isolates, in all 5 B. bronchiseptica isolates, and in the Art-Vax strain. Plasmids of B. avium correlated with resistance to antibiotics but not with pathogenicity, hemagglutination of guinea pig erythrocytes, or expression of pili.

Control of extracellular polysaccharide synthesis in Erwinia stewartii and Escherichia coli K-12: a common regulatory function.

A primary determinant of pathogenicity in Erwinia stewartii is the production of extracellular polysaccharide (EPS). A single mutation can abolish both EPS synthesis and pathogenicity; both properties are restored by a single cosmid clone. Subcloning and insertion analysis have defined a single positive regulatory function which shares a number of similarities with the rcsA function of Escherichia coli K-12, a positive regulator for capsular polysaccharide synthesis. In E. stewartii, the gene promotes the transcription of at least two operons (cps) involved in EPS synthesis; we have previously demonstrated a similar function for rcsA in E. coli. Both genes code for proteins of 25 to 27 kilodaltons; both proteins are unstable in E. coli. The E. stewartii RcsA protein was stabilized in E. coli lon mutants, as the RcsA product from E. coli is. The E. stewartii function complemented E. coli rcsA mutants, and the E. coli RcsA function increased cps expression and restored virulence in E. stewartii mutants. Therefore, these two gram-negative organisms share a similar component of their regulatory circuitry for the control of capsular polysaccharide synthesis.

Molecular cloning of virulence genes from Erwinia stewartii.

A library of Erwinia stewartii DNA was constructed in cosmid pVK100 and used to complement spontaneous and Mu pf7701-induced (designated by the prefix MU) avirulent mutants. Plasmid pES4507 restored water-soaking ability and extracellular polysaccharide (EPS) synthesis to mutants MU14110 and MU2B70 (group I); pES1044 restored water-soaking ability to MU43, MU51, MU136, MU141, and RDF6011 (group II); and pES2144 complemented four spontaneous EPS- mutants (group III). Hybridization of labeled plasmid DNA to Southern blots of genomic DNA from the mutants revealed that a Mu pf7701 insertion was associated with the respective cloned region in all mutants except MU2B70 and MU223. In these strains, the plasmid may be suppressing the avirulent phenotype rather than complementing the mutation.