Structure of the Lipopolysaccharide Side Chain of Pseudomonas syringae pv. tabaci Strain NCPPB 79 (=CFBP 1615), in Relation to O-serogroup

The lipopolysaccharide (LPS) side chain from Pseudomonas syringae pv. tabaci strain NCPPB 79 (=CFBP 1615) contained l ‐ and d ‐rhamnose, and GlcNAc. Using methylation analysis, periodate oxidation, Smith degradation and ¹H‐ and ¹³C‐nuclear magnetic resonance spectroscopy, the repeat unit was found to have the structure: This structure is correlated with a previously proposed serogrouping system. The involvement of LPS generally in plant disease is briefly discussed.     

Rpa1 mediates an immune response to avrRpm1Psa and confers resistance against Pseudomonas syringae pv. actinidiae

The type three effector AvrRpm1_Pma from Pseudomonas syringae pv. maculicola (Pma) triggers an RPM1‐mediated immune response linked to phosphorylation of RIN4 (RPM1‐interacting protein 4) in Arabidopsis. However, the effector–resistance (R) gene interaction is not well established with different AvrRpm1 effectors from other pathovars. We investigated the AvrRpm1‐triggered immune responses in Nicotiana species and isolated Rpa1 (R esistance to P seudomonas syringae pv. a ctinidiae 1) via a reverse genetic screen in Nicotiana tabacum. Transient expression and gene silencing were performed in combination with co‐immunoprecipitation and growth assays to investigate the specificity of interactions that lead to inhibition of pathogen growth. Two closely related AvrRpm1 effectors derived from Pseudomonas syringae pv. actinidiae biovar 3 (AvrRpm1_Psa) and Pseudomonas syringae pv. syringae strain B728a (AvrRpm1_Psy) trigger immune responses mediated by RPA1, a nucleotide‐binding leucine‐rich repeat protein with an N‐terminal coiled‐coil domain. In a display of contrasting specificities, RPA1 does not respond to AvrRpm1_Pma, and correspondingly AvrRpm1_Psa and AvrRpm1_Psy do not trigger the RPM1‐mediated response, demonstrating that separate R genes mediate specific immune responses to different AvrRpm1 effectors. AvrRpm1_Psa co‐immunoprecipitates with RPA1, and both proteins co‐immunoprecipitate with RIN4. In contrast with RPM1, however, RPA1 was not activated by the phosphomimic RIN4^T166D and silencing of RIN4 did not affect the RPA1 activity. Delivery of AvrRpm1_Psa by Pseudomonas syringae pv. tomato (Pto) in combination with transient expression of Rpa1 resulted in inhibition of the pathogen growth in N. benthamiana. Psa growth was also inhibited by RPA1 in N. tabacum.     

Bactericidal Compounds Controlling Growth of the Plant Pathogen Pseudomonas syringae pv. actinidiae,Which Forms Biofilms Composed of a Novel Exopolysaccharide

Pseudomonas syringae pv. actinidiae is the major cause of bacterial canker and is a severe threat to kiwifruit production worldwide. Many aspects of the disease caused by P. syringae pv. actinidiae, such as the pathogenicity-relevant formation of a biofilm composed of extracellular polymeric substances (EPSs), are still unknown. Here, a highly virulent strain of P. syringae pv. actinidiae, NZ V-13, was studied with respect to biofilm formation and architecture using a flow cell system combined with confocal laser scanning microscopy. The biofilm formed by P. syringae pv. actinidiae NZ V-13 was heterogeneous, consisting of a thin cellular base layer 5 μm thick and microcolonies with irregular structures. The major component of the EPSs produced by P. syringae pv. actinidiae NZ V-13 bacteria was isolated and identified to be an exopolysaccharide. Extensive compositional and structural analysis showed that rhamnose, fucose, and glucose were the major constituents, present at a ratio of 5:1.5:2. Experimental evidence that P. syringae pv. actinidiae NZ V-13 produces two polysaccharides, a branched α-d-rhamnan with side chains of terminal α-d-Fucf and an α-d-1,4-linked glucan, was obtained. The susceptibility of the cells in biofilms to kasugamycin and chlorine dioxide was assessed. About 64 and 73% of P. syringae pv. actinidiae NZ V-13 cells in biofilms were killed when kasugamycin and chlorine dioxide were used at 5 and 10 ppm, respectively. Kasugamycin inhibited the attachment of P. syringae pv. actinidiae NZ V-13 to solid surfaces at concentrations of 80 and 100 ppm. Kasugamycin was bacteriostatic against P. syringae pv. actinidiae NZ V-13 growth in the planktonic mode, with the MIC being 40 to 60 ppm and a bactericidal effect being found at 100 ppm. Here we studied the formation, architecture, and composition of P. syringae pv. actinidiae biofilms as well as used the biofilm as a model to assess the efficacies of bactericidal compounds.     

Identification and expression of the Pseudomonas syringae pv. aptata hrpZPsa gene which encodes an harpin elicitor

A sequence homologous to an internal fragment 0.75 kb BstXI of the Pseudomonas syringae pv. syringae hrpZ gene was identified in Pseudomonas syringae pv. aptata NCPPB 2664, the causal agent of bacterial blight in sugar beet, lettuce and other plants, and in E. coli DH10B (pCCP1069) containing the P. syringae pv. aptata hrp gene cluster. PCR with oligonucleotides, based on the hrpZ_Pss gene and used as primers with the total genomic DNA of P. syringae pv. aptata, amplified a 1 kb fragment that hybridized with the probe in highly stringent conditions. The amplicon was cloned into the pGEM-T® plasmid vector, amplified in E. coli DH5 and sequenced. The sequence showed 95%, 83% and 61% identity with those of hrpZ_Pss, hrpZ_Psg and hrpZ_Pst genes encoding the harpins of the P. syringae pv. syringae, glycinea and tomato, respectively. The amplicon was cloned into the pMAL® expression system. The expressed protein, fused with maltose-binding protein, was cleaved with a specific protease factor Xa, and purified using affinity chromatography. On the basis of the amino acid sequence and its ability to induce HR in tobacco leaves, it was identified as a P. syringae pv. aptata harpin.     

Comparative analysis of the lipopolysaccharides of a rough and a smooth strain of Pseudomonas syringae pv. phaseolicola

The lipopolysaccharides (LPS) of a rough (R) and a smooth (S) strain of Pseudomonas syringae pv. phaseolicola were analysed. The S-LPS revealed markedly more rhamnose and fucose, but less glucose, than the R-LPS. The presence of 3-O-methyl-rhamnose (acofriose) in the S-LPS was confirmed by cochromatography with authentic acofriose. SDS polyacrylamide gel electrophoresis of the S-LPS demonstrated a cluster of regularly spaced high molecular weight fractions, which was almost lacking in the R-LPS. The main fatty acids of the lipid A of both LPS species were 3-OH-10:0,3-OH-12:0,2-OH-12:0, and 12:0. Two N-linked diesters were demonstrated: 3-O(12:0)-12:0 and 3-O(2-OH-12:0)-12:0. S-LPS was subjected to mild hydrolysis and the degraded polysaccharide separated into three fractions by gel permeation chromatography on a Fractogel TSK HW-50 column. Fraction I, representing nearly only the O-specific side chain, consisted of rhamnose and fucose in a molar ratio of 4:1, with 4% of the rhamnose being 3-O-methylated (acofriose). Fraction II, representing mostly core material, was composed of glucose, rhamnose, heptose, glucosamine, galactosamine, alanine, and a still unidentified amino compound, in an approximate molar ratio of 3:1:1:1:1:1:1, and KDO. Fraction III consisted of released monomers and salts. The LPS was highly phosphorylated (3.28% phosphorus in the core fraction). The thus characterized composition of the LPS O-chain seems to be unique for the pathovar phaseolicola of P. syringae, although many similarities exist to other pathovars as well as to other bacterial species.Abbreviations LPS lipopolysacchairdes - GC/MS combined gas liquid chromatography-mass spectrometry - HVE high voltage electrophoresis - KDO 2-keto-3-deoxyoctonic acid - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate P.s. pv. phaseolicola is termed P. phaseolicola in the text     

Identification of the Biosynthetic Gene Cluster for 3-Methylarginine,a Toxin Produced by Pseudomonas syringae pv. syringae 22d/93

The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (K_m, 7 mM; k_cat, 85 min⁻¹). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.Microbial plant pathogens cause severe losses in agriculture each year (1). For example, the plant pathogen Pseudomonas syringae pv. glycinea is responsible for bacterial blight of soybean, a leaf spot disease of great economic impact. Besides chemical treatment, biocontrol agents that antagonize microbial plant pathogens are gaining increasing importance in fighting plant diseases (6, 11, 27). In a screening for possible biocontrol strains, an epiphytic bacterium showing a strong and selective activity against the pathogen P. syringae pv. glycinea was isolated from soybean leaves (29). The strain was characterized as Pseudomonas syringae pv. syringae 22d/93 (Pss22d). The antagonism of Pss22d against P. syringae pv. glycinea has been demonstrated successfully in vitro and in planta under greenhouse and field conditions (19, 29). In order to identify the molecular basis of the antagonism of Pss22d against P. syringae pv. glycinea, we focused on its secondary metabolites. Besides the well-known lipodepsipeptides syringomycin and syringopeptin (3), Pss22d produces the rare amino acid 3-methylarginine (MeArg) (5). As little as 20 nmol of MeArg strongly and selectively inhibits P. syringae pv. glycinea but no other pseudomonads in vitro (29). Since the inhibition can be compensated for by l-arginine supplementation but not by any other essential amino acid, it is likely that the toxin acts as an inhibitor of the arginine biosynthesis pathway or an arginine-dependent pathway, such as nitric oxide formation (13, 16). Feeding experiments and Tn5 transposon mutagenesis suggested that MeArg is produced by an S-adenosyl methionine (SAM)-dependent methyltransferase (5) converting the enol of 5-guanidino-2-oxo-pentanoic acid to 5-guanidino-3-methyl-2-oxo-pentanoic acid. An analogous reaction is known to occur with the methyltransferases GlmT, DptI, and LptI, which form 3-methylglutamate from α-ketoglutarate (18). On the way to MeArg, only a transaminase catalyzing the formation of MeArg from 5-guanidino-3-methyl-2-oxo-pentanoic acid and an amino acid exporter to secrete the toxin would be needed.Here, we describe the identification and functional characterization of the MeArg biosynthesis gene cluster from the epiphyte Pss22d.     

Conjugal transfer of E. coli F''lac from Erwinia chrysanthemi to Pseudomonas syringae pv. glycinea and the apparent stable incorporation of the plasmid into the pv. glycinea chromosome

Summary The E. coli Flac plasmid was transferred from an Erwinia chrysanthemi Hfr8 donor to a multiply-auxotrophic, rifampicin-resistant Pseudomonas syringae pv. glycinea recipient. Transfer occurred at a frequency of approximately 10^-5/donor. Stable transconjugants which were able to utilize lactose as the sole carbon source after several transfers would not donate the Flac plasmid in detectable frequency to other pv. glycinea or E. coli recipients. The plasmid DNA was shown to be integrated into the pv. glycinea chromosome (Fig. 1).     

Pathogenicity and identification of the lilac pathogen, Pseudomonas syringae pv. syringae van Hall 1902

Comparative in planta studies with Pseudomonas syringae pv. syringae have established optimum conditions for disease expression in lilac in terms of inoculum concentration, host age and post-inoculation conditions (temperature and day-length). Reproducible disease reactions required an inoculum concentration exceeding the ED⁵⁰, 5 × 10⁶ cfu/ml, and a temperature for post-inoculation incubation not exceeding 19°C. A revised host range of P. syringae pv. syringae, proposed on the basis of confirmation of pathogenicity of strains to lilac, comprises 44 species from monocotyledonous and dicotyledonous plants. Nine new hosts Abelmoschus esculentus, Bromus willdenowii, Camellia sinensis, Centrosema pubescens, Citrullus lanatus, Cotoneaster sp., Cucumis melo, Populus×euramericana and Triticum aestivum, are recorded. A comparative laboratory study was made of strains of P. syringae pv. syringae using more than 30 selected biochemical and nutritional tests. The pathovar could be characterised on the basis of 11 of these which may prove to be useful determinative tests.     

Cloning of genes required for hypersensitivity and pathogenicity inPseudomonas syringae pv.aptata

A genomic library ofPseudomonas syringae pv.aptata strain NCPPB 2664, which causes bacterial blight of sugar beet, lettuce and other plants, was constructed in the cosmid vector pCPP31. The 13.4 kbEcoRI fragment of the cosmid pHIR11, containing thehrp (hypersensitiveresponse andpathogenicity) gene cluster of the closely related bacteriumPseudomonas syringae pv.syringae strain 61, was used as a probe to identify a homologoushrp gene cluster inP. syringae pv.aptata. Thirty of 2500 cosmid clones, screened by colony hybridization, gave a strong hybridization signal with the probe, but none of these conferred to the non-pathogenic bacterium,Pseudomonas fluorescens, the ability to elicit the hypersensitive response (HR) in tobacco. Southern blot analysis ofEcoRI-digested genomic DNA ofP. syringae pv.aptata showed hybridizing bands of 12 kb and 4.4 kb. Only a 12 kb fragment hybridized in digests of the cosmids. Cosmid clone pCPP1069 was mutagenized with Tn10-minitet and marker-exchanged into the genome ofP. syringae pv.aptata. Three resulting prototrophic mutant strains failed to elicit the HR in tobacco and to cause disease in lettuce. The DNA flanking the Tn10-minitet insertions from mutated derivatives of pCPP1069 hybridized with the 10.6 kbBglII fragment of pHIR11. These results indicate thatP. syringae pv.aptata harbourshrp genes that are similar to, but arranged differently from, homologoushrp genes ofP. syringae pv.syringae.Abbreviations HR hypersensitive response - Hrp mutant unable to induce HR and pathogenicity - Psa Pseudomonas syringae pv.aptata - Pss Pseudomonas syringae pv.syringae - Ea Erwinia amylovora     

Characterization of two ornithine carbamoyltransferases from Pseudomonas syringae pv. phaseolicola,the producer of phaseolotoxin

Two ornithine carbamoyltransferases (OCT 1 and OCT 2) were isolated from Pseudomonas syringae pv. phaseolicola and purified by precipitation with ammonium sulfate, heat denaturation, chromatography on DEAE-Sephadex A-50 and Sephadex G-200. Molecular weights of both enzymes: 110,000; optimal activity: pH 8.5 to 9.5 (OCT 1), pH 8.4 (OCT 2); apparent K _m for ornithine: 7·10^-4 (both enzymes); apparent K _m for carbamoylphosphate: 7·10^-4 (OCT 1), 2.8·10^-3 (OCT 2). Both enzymes possess only an anabolic function. OCT 1 is highly inhibited by low concentrations of phaseolotoxin and Orn-P(O)(NH₂)-NH-SO₃H, OCT 2 is insensitive to both compounds. The inhibition of OCT 1 is reversible.Non-common abbreviation PNSOrn Ornithine--P(O)(NH₂)-NH-SO₃H     

Cloning,characterization and phenotypic expression in Escherichia coli of catF,which encodes the catalytic subunit of catalase isozyme CatF of Pseudomonas syringae

The phytopathogenic, gram-negative bacterium Pseudomonas syringae pv. syringae 61 contains three isozymes of catalase (EC 1.11.1.6), which have been proposed to play a role in the bacterium's responses to various environmental stresses. To study the role of individual isozymes, the gene coding for the catalytic subunit of one catalase isozyme was cloned from a cosmid library hosted in Escherichia coli DH5 by using a designed catalase-specific DNA probe for the screening. One out of four clones with a catalase-positive genotype was subcloned and a pUC19-based 2.7 × 10³-base (2.7-kb) insert subclone, pMK3E5, was used to transform catalase-deficient E. coli strain UM255 (HPI^–, HPII^–). The transformants contained a single isozyme of catalase that had electrophoretic and enzymic properties similar to catalase isozyme CatF from P. syringae pv. syringae 61. Analysis of the sequenced 2.7-kb insert DNA revealed six putative open-reading frames (ORF). The 1542-base-pair DNA sequence of ORF2, called catF, encodes a peptide of 513 amino acid residues with a calculated molecular mass of 66.6 kDa. The amino acid sequence deduced from catF had homology to the primary structure of true catalases from mammals, plants, yeasts and bacteria. The activity of the recombinant catalase was inhibited by 3-amino-1,2,4-triazole and azide and stimulated by chloramphenicol. The N terminus contained a signal sequence of 26 amino acids necessary for secretion into the periplasm, a so-far unique property of Pseudomonas catalases.Paper no. 4552 of the Utah Agricultural Experiment station     

Non contiguous-finished genome sequence of Pseudomonas syringae pathovar syringae strain B64 isolated from wheat

The Gram-negative gammaproteobacterium Pseudomonas syringae is one of the most wide-spread plant pathogens and has been repeatedly reported to cause significant damage to crop plantations. Research on this pathogen is very intensive, but most of it is done on isolates that are pathogenic to Arabidopsis, tomato, and bean. Here, we announce a high-quality draft genome sequence of Pseudomonas syringae pv. syringae B64 which is the first published genome of a P. syringae strain isolated from wheat up to date. The genome sequence will assist in gaining insights into basic virulence mechanisms of this pathogen which has a relatively small complement of type III effectors.     

Transgenic tobacco cultivars resistant to Pseudomonas syringae pv. tabaci

 Six oriental cultivars of tobacco (Nicotiana tabacum L.) were evaluated for transformation and foreign gene expression. Leaf-disc explant tissue was transformed with Agrobacterium tumefaciens strain LBA4404 carrying the plasmid pARK21, which contains NPTII gene and ttr (tabtoxin resistance) gene conferring the resistance to Pseudomonas syringae pv. tabaci. The disease resistance of regenerated plants and segregation of this trait up to R₇ progeny were investigated in a greenhouse and under field conditions. Our results indicated that the resistance to Pseudomonas syringae pv. tabaci introduced by transformation is heritable. Received: 10 June 1997 / Accepted: 31 March 1998     

Systemic Invasion of Immature Sweet Cherry Fruit by Pseudomonas syringae pv. morsprunorum Through Blossoms1

Cherry blossoms inoculated with a rifampicin-resistant strain of Pseudomonas syringae pv. morsprunorum died or gave rise to fruits containing necrotic spots at or near the blossom ends. Scanning electron microscopy of developing fruits indicated that the pathogen had invaded the entire pericarp, including the endocarp. Bacteria also spread to the fruit stalk and, to a lesser extent, to the spurs. Mesocarp cells below the lesion collapsed. Infected fruit, stalks, and spurs contained, respectively, ca. 10⁹, 10⁷, and 10² colony forming units of P. syringae pv. morsprunorum as determined by a dilution plate method on an agar medium supplemented with 50 μg/ml rifampicin. This is the first report of systemic spread of P. syringae from blossoms to developing fruit of a deciduous crop.     

Novel succinylated and large-sized osmoregulated periplasmic glucans of Pseudomonas syringae pv. syringae

Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the Gram-negative bacterial envelope and are important for bacterial-host interactions. The OPGs of Pseudomonas syringae pv. syringae have been known to be highly branched linear glucans ranging from 6 to 13 glucose residues devoid of any substituents, while having backbone structure similar to those of Escherichia coli and Erwinia chrysanthemi. Here, we report for the first time succinylated and large-sized OPGs from P. syringae pv. syringae. The glucans were isolated with trichloroacetic acid treatment and various chromatographic techniques. These were further characterized by thin-layer chromatography, matrix-assisted laser desorption/ionization time of flight mass spectrometer, and 1D ¹H nuclear magnetic resonance spectroscopy. The results demonstrate that novel anionic glucans with one succinyl residue at the C-6 position of the glucose unit as well as neutral glucans including large-sized glucans with up to 28 degrees of polymerization are produced in P. syringae pv. syringae. Furthermore, the succinylated and large-sized OPGs of P. syringae pv. syringae are necessary for hypoosmotic adaptation.     

Polysaccharides of <Emphasis Type="Italic">Pseudomonas</Emphasis> Pathovar Strains That Infect Pea,Tomato, and Soya Bean

In order to understand the mode of action of taxonomically related Pseudomonas syringae pathovar strains that infect pea, tomato, and soya bean, we examined their extracellular polysaccharides (EPS). Maximum production of polysaccharide in shake culture of these pathogens was observed between 24 and 60 h. P. syringae pv. pisi 519, the bacterial blight pathogen of pea, produced a higher amount of polysaccharide (34.87 g/mL) at 60 h compared with 32.67 g/mL produced by P. syringae pv. glycinea NCPPB 1783, the bacterial blight pathogen of soya bean, and 30.03 g/mL produced by P. syringae pv. tomato NCPPB 269, the bacterial speck pathogen of tomato. EPS produced by P. syringae pv. pisi 519, P. syringae pv. tomato NCPPB 269, and P. syringae pv. glycinea NCPPB 1783 was characterized with infrared (FTIR), nuclear magnetic resonance (NMR), high performance thin layer chromatography, (HPTLC), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. HPTLC profiles revealed the presence of glucose and glucuronic acid in all bacteria and mannose only in P. syringae pv. tomato. Molecular mass of EPS of P. syringae pv. pisi (m/z 933.8), P. syringae pv. tomato (m/z 950.4), and P. syringae pv. glycinea (m/z 933.5) was confirmed by MALDI-TOF mass spectrometry.     

Effectiveness of alkyl dimethyl benzyl ammonium chloride in reducing the population of Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. syringae in tomatoes,beans, and peppers

Abstract

Microbial contamination of fruits and vegetables during growth, processing, and post-harvest is a serious problem in agricultural sectors. A study was undertaken to investigate the efficacy of alkyl dimethyl benzyl ammonium chloride (ADBAC) in reducing the population of Xanthomonas campestris pv. vesicatoria, and Pseudomonas syringae pv. syringae on tomatoes, beans, and peppers. Tomatoes, beans, and peppers were inoculated by dipping in bacteria for 15 min then fruits were dried for 2 hour at ambient temperature before they were treated with 0.1, 1, 10, 100, and 1000 ppm of ADBAC. Treatments with 10, 100, and 1000 ppm ADBAC caused an 8-log CFU/ml reduction of X. campestris pv. vesicatoria on surfaces of tomatoes. Treatments with 100 and 1000 ppm ADBAC caused an 8-log CFU/ml reduction of P. syringae pv. syringae and X. campestris pv. vesicatoria on surfaces of tomatoes and peppers, respectively. However, treatment of surfaces of beans with 1000 ppm of ADBAC caused an 8-log CFU/ml reduction of P. syringae pv. syringae. Overall, a 50% reduction on population counts of both pathogens was achieved with 100 and 1000 ppm ADBAC. No X. campestris pv vesicatoria, P. syringae pv. syringae, or other bacteria were detected on the control fruits inoculated with sterile distilled water. This study's findings suggest that ADBAC has good bactericidal and sanitizing activities and could potentially be useful as a new sanitizer for food safety.     

Effect of Iron Concentration on the Growth Rate of Pseudomonas syringae and the Expression of Virulence Factors in hrp-Inducing Minimal Medium

Although chemically defined media have been developed and widely used to study the expression of virulence factors in the model plant pathogen Pseudomonas syringae, it has been difficult to link specific medium components to the induction response. Using a chemostat system, we found that iron is the limiting nutrient for growth in the standard hrp-inducing minimal medium and plays an important role in inducing several virulence-related genes in Pseudomonas syringae pv. tomato DC3000. With various concentrations of iron oxalate, growth was found to follow Monod-type kinetics for low to moderate iron concentrations. Observable toxicity due to iron began at 400 μM Fe³⁺. The kinetics of virulence factor gene induction can be expressed mathematically in terms of supplemented-iron concentration. We conclude that studies of induction of virulence-related genes in P. syringae should control iron levels carefully to reduce variations in the availability of this essential nutrient.The type III secretion system (T3SS) is used by diverse plant and animal pathogens to invade and colonize their hosts (1). This secretion system translocates bacterial proteins (effectors) from the bacterial cytoplasm directly into the eukaryotic host cell cytosol, where the effectors subvert host cell processes to the advantage of the pathogen. In Pseudomonas syringae pv. tomato DC3000, the T3SS is responsible for the elicitation of hypersensitive reactions of nonhost plants and is essential for disease on host plants (14). Many T3SS genes in plant pathogens are denoted hrp, for hypersensitive response and pathogenicity. We know of several regulatory elements that control T3SS genes in P. syringae pv. tomato DC3000 (7, 27), including HrpL, an alternative sigma factor. However, the exact environmental signals that the bacteria respond to are unknown.The expression of avrB, a T3SS effector, varies depending on the carbon source in Pseudomonas syringae pv. glycinea race 0 (9). Other environmental factors affecting the expression of virulence-related genes have also been studied. Nitrogen and osmolarity are important for the expression of the Pseudomonas syringae pv. syringae 61 hrp genes (28). Osmotic strength, pH, and carbon source differentially affected the expression of T3SS genes in Pseudomonas syringae pv. phaseolicola (18). These results imply that catabolite repression by the tricarboxylic acid cycle intermediates may be involved in the induction process. With other pathogenic bacteria, nutritional conditions are reported to be an important factor for the induction of virulence. For example, the Xanthomonas hrp genes are induced by sucrose and sulfur-containing amino acids (21). The optimal condition for hrp gene expression may simulate leaf apoplast environmental factors, including hypo-osmotic pressure, low pH, and limited nutrient concentration (18).Iron is a micronutrient (required in concentrations less than 10⁻⁴ M) for in vitro cultures (22), and the typical concentration needed for optimal bacterial growth is 0.3 to 1.8 μM (24). Iron is an essential element for bacteria due to its participation in the tricarboxylic acid cycle, electron transport, amino acid and pyrimidine biosynthesis, DNA synthesis, and other critical functions (3). Iron uptake must also be regulated due to its lethal effect through the Fenton reaction (2). The effect of iron limitation on bacterial growth has been documented for Escherichia coli cultures (6, 19, 20). Two studies have shown that production of the phytotoxins, syringomycin, and syringotoxin from P. syringae responds in batch culture to iron supplementation (5, 15). Iron is known to alter the physiology of other pseudomonads in both batch and chemostat cultures (11, 16). Although iron is the fourth most abundant element in the earth''s crust, its availability is very low due to its low solubility in aqueous solution ([Fe³⁺] at pH 7, 10⁻¹⁸ μM) (24). Bacteria have evolved complex mechanisms to ensure that iron requirements are met but not exceeded. Siderophore-mediated transport of iron is one of the mechanisms used by bacteria to uptake iron from their environment (17).In this study, medium components in hrp-inducing minimal medium were evaluated systematically with a chemostat culture. Iron was found to be both a growth-limiting nutrient in hrp-inducing minimal medium and a mediator of virulence gene expression in the model plant pathogen P. syringae pv. tomato DC3000.     

Multilocus enzyme electrophoresis analysis ofPseudomonas syringae pv.phaseolicola

A multilocus enzyme electrophoresis technique was developed to detect variation in seven enzyme loci among isolates ofPseudomonas syringae pv.phaseolicola, representing three races from different geographical locations, the causal agent of the halo blight disease of beans. Cellulose acetate gel electrophoresis of seven enzymes revealed 19 electrotypes (ET) among 21Pseudomonas syringae pv.phaseolicola isolates. One of the pathovarsyringae and one of the pathovartomato isolates were represented by two different ET. The population of Turkish isolates and three races of the pathovarphaseolicola appeared to be genetically diverse.     

Characterization of Pyoverdinpss, the Fluorescent Siderophore Produced by Pseudomonas syringae pv. syringae

Pseudomonas syringae pv. syringae B301D produces a yellow-green, fluorescent siderophore, pyoverdin_pss, in large quantities under iron-limited growth conditions. Maximum yields of pyoverdin_pss of approximately 50 μg/ml occurred after 24 h of incubation in a deferrated synthetic medium. Increasing increments of Fe(III) coordinately repressed siderophore production until repression was complete at concentrations of ≥ 10 μM. Pyoverdin_pss was isolated, chemically characterized, and found to resemble previously characterized pyoverdins in spectral traits (absorbance maxima of 365 and 410 nm for pyoverdin_pss and its ferric chelate, respectively), size (1,175 molecular weight), and amino acid composition. Nevertheless, pyoverdin_pss was structurally unique since amino acid analysis of reductive hydrolysates yielded β-hydroxyaspartic acid, serine, threonine, and lysine in a 2:2:2:1 ratio. Pyoverdin_pss exhibited a relatively high affinity constant for Fe(III), with values of 10²⁵ at pH 7.0 and 10³² at pH 10.0. Iron uptake assays with [⁵⁵Fe]pyoverdin_pss demonstrated rapid active uptake of ⁵⁵Fe(III) by P. syringae pv. syringae B301D, while no uptake was observed for a mutant strain unable to acquire Fe(III) from ferric pyoverdin_pss. The chemical and biological properties of pyoverdin_pss are discussed in relation to virulence and iron uptake during plant pathogenesis.