Syringomycin Production Research Articles

Pathogenicity and Virulence‐Associated Factors of Pseudomonas syringae pv. syringae and [P. amygdali pv. morsprunorum] Strains From New Zealand Sweet Cherry (Prunus avium) Orchards

ABSTRACTPreviously genetically characterised strains of Pseudomonas syringae. pv. syringae (Pss), [P. amygdali pv. morsprunorum] (Pam, syn. P. s. pv. morsprunorum race 1) and Pseudomonas spp. from New Zealand were characterised for their pathogenicity and aggressiveness in plant tissue and associated virulence factors. Lesions on detached, Pss‐inoculated immature fruit increased rapidly in size and, at 10 days post inoculation (dpi), had larger areas under the disease progress curve (AUDPC) than Pam‐inoculated fruit (48.9 and 22.0, respectively). Detached leaves infiltrated with Pss‐developed symptoms within 1 dpi and from 2 dpi for Pam. Necrosis from most Pss strains extended into the leaf veins by 7 dpi, while Pam strains' necrosis was confined to the inoculation site. On detached 1‐year‐old cherry shoots, Pseudomonas spp. strains exhibited the smallest mean lesion size (2.1–2.4 mm), whereas larger mean lesion sizes were observed with Pss strains (5.7–13.7 mm) and Pam strains (3.9–14.0 mm). A functional T3SS was inferred for Pss and Pam strains based on the hypersensitivity reactions observed on tobacco leaves and symptoms elicited on cherry tissue. Syringomycin production was prevalent (88%) among Pss strains. In contrast, only 1.4% of Pam strains produced coronatine. Most Pss strains (97.0%) were able to catalyse ice formation. The coexistence of strains with varying degrees of virulence and non‐pathogenic strains suggests a complex ecological balance, where multiple factors, including genetic variation, virulence traits and environmental conditions, shape the population dynamics and disease outcomes.

Characterization of Pseudomonas syringae Strains Associated with Shoot Blight of Raspberry and Blackberry in Serbia.

During May 2016, severe blight symptoms were observed in several raspberry and blackberry fields in Serbia. In total, 22 strains were isolated: 16 from symptomatic raspberry shoots, 2 from asymptomatic raspberry leaves, and 4 from symptomatic blackberry shoots. Additionally, eight raspberry strains, isolated earlier from two similar outbreaks, were included in the study. Pathogenicity of the strains was confirmed on detached raspberry and blackberry shoots by reproducing the symptoms of natural infection. The strains were Gram-negative, fluorescent on King's medium B, ice nucleation positive, and utilized glucose oxidatively. All strains were levan positive, oxidase negative, nonpectolytic, arginine dihydrolase negative, and induced hypersensitivity in tobacco leaves (LOPAT + - - - +, Pseudomonas group Ia). Furthermore, all strains liquefied gelatin and hydrolyzed aesculin but did not show tyrosinase activity or utilize tartrate (GATTa + + - -). Tentative identification using morphology, LOPAT, GATTa, and ice-nucleating ability tests suggested that isolated strains belong to Pseudomonas syringae. The syrB gene associated with syringomycin production was detected in all strains. DNA fingerprints with REP, ERIC, and BOX primers generated identical profiles for 29 strains, except for strain KBI 222, which showed a unique genomic fingerprint. In all, 9 of 10 selected strains exhibited identical sequences of four housekeeping genes: gyrB, rpoD, gapA, and gltA. Five nucleotide polymorphisms were found in strain KBI 222 at the rpoD gene locus only. In the phylogenetic tree based on a concatenated sequence of all four housekeeping genes, strains clustered within phylogroup 2 (i.e., genomospecies 1) of the P. syringae species complex, with pathotype strains of P. syringae pv. aceris and P. syringae pv. solidagae as their closest relatives. There was no correlation between genotype and geographic origin, particular outbreak, host, or cultivar.

Autoinduction of Vitreoscilla Hemoglobin Enhanced the Production of Syringomycin from Pseudomonas syringae HS191

Syringomycin is a cyclic lipodepsipeptide produced by strains of Pseudomonas syringae. The potent herbicidal and fungicidal activities of syringomycin make it a promising compound for fungiostasis and weed control. However, the production of syringomycin from the wild-type strains is low. The discoveries that Pseudomonas syringae is aerobic, and the syringomycin synthetase SyrB2 is an O2-dependent halogenase, led us to establish an autoinducible Vitreoscilla hemoglobin expression system for oxygen supply during fermentation, thereby increasing the yield of syringomycin. By employing the quorum sensing system for the expression of Vitreoscilla hemoglobin gene (vgb), we found that Pseudomonas syringae HS191 that expressed vgb, facilitated the cell growth and the general biomass. Furthermore, syringomycin bioassay showed that the fungal inhibition zones increased from 2.5 mm to 3.2 mm, and HPLC analysis confirmed that the expression of vgb resulted in a 71.1% increase in syringomycin production compared to the wild-type strain. The Vitreoscilla hemoglobin has been widely applied to fermentation optimization; however, in the case of Pseudomonas, increased oxygen supply is only beneficial during the stationary phase, while a high concentration of oxygen inhibited the cell propagation during the logarithmic phase. Here we report the autoinduction of Vitreoscilla hemoglobin by engineering the quorum-sensing system. This synthetic circuit significantly improved the syringomycin production. The Vitreoscilla hemoglobin-autoinduction system not only caters to the dynamic oxygen demand but also avoids inducer supplementation.

Genome analysis of the marine bacterium Kiloniella laminariae and first insights into comparative genomics with related Kiloniella species.

Kiloniella laminariae is a true marine bacterium and the first member of the family and order, the Kiloniellaceae and Kiloniellales. K. laminariae LD81T (= DSM 19542T) was isolated from the marine macroalga Saccharina latissima and is a mesophilic, typical marine chemoheterotrophic aerobic bacterium with antifungal activity. Phylogenetic analysis of 16S rRNA gene sequence revealed the similarity of K. laminariae LD81T not only with three validly described species of the genus Kiloniella, but also with undescribed isolates and clone sequences from marine samples in the range of 93.6-96.7%. We report on the analysis of the draft genome of this alphaproteobacterium and describe some selected features. The 4.4Mb genome has a G + C content of 51.4%, contains 4213 coding sequences including 51 RNA genes as well as 4162 protein-coding genes, and is a part of the Genomic Encyclopaedia of Bacteria and Archaea (GEBA) project. The genome provides insights into a number of metabolic properties, such as carbon and sulfur metabolism, and indicates the potential for denitrification and the biosynthesis of secondary metabolites. Comparative genome analysis was performed with K. laminariae LD81T and the animal-associated species Kiloniella majae M56.1T from a spider crab, Kiloniella spongiae MEBiC09566T from a sponge as well as Kiloniella litopenai P1-1 from a white shrimp, which all inhabit quite different marine habitats. The analysis revealed that the K. laminariae LD81T contains 1397 unique genes, more than twice the amount of the other species. Unique among others is a mixed PKS/NRPS biosynthetic gene cluster with similarity to the biosynthetic gene cluster responsible for the production of syringomycin.

Inhibitory effect of Thymus vulgaris and Origanum vulgare essential oils on virulence factors of phytopathogenic Pseudomonas syringae strains

Pseudomonas syringae is a phytopathogenic bacterium that causes lesions in leaves during the colonisation process. The damage is associated with production of many virulence factors, such as biofilm and phytotoxins. The essential oils of Thymus vulgaris (thyme) and Origanum vulgare (oregano) have been demonstrated to inhibit P.syringae. The aim of this study was to investigate the effects of T.vulgaris and O.vulgare essential oils on production of virulence factors of phytopathogenic P.syringae strains, including anti-biofilm and anti-toxins activities. The broth microdilution method was used for determination of MIC and biofilm inhibition assays. Coronatine, syringomycin and tabtoxin were pheno- and genotypically evaluated. Both oils showed good inhibitory activity against P.syringae, with MIC values from 1.43 to 11.5mg·ml-1 for thyme and 5.8 to 11.6mg·ml-1 for oregano. Biofilm formation, production of coronatine, syringomycin and tabtoxin were inhibited by thyme and oregano essential oil in most strains. The results presented here are promising, demonstrating the bactericidal activity and reduction of virulence factor production after treatment with thyme and oregano oil, providing insight into how they exert their antibacterial activity. These natural products could be considered in the future for the control of diseases caused by P. syringae.

Production and Application of Syringomycin E as an Organic Fungicide Seed Protectant against Pythium Damping‐off

AbstractSyringomycin E (SRE) is a cyclic lipodepsinonapeptide with potent antifungal activity and is produced by certain strains of Pseudomonas syringae pv. syringae. In this study, its potential as an organic‐compatible agrofungicide and vegetable seed treatment against the soilborne pathogen Pythium ultimum var. ultimum was examined. A variant of P. syringae pv. syringae strain B301D with enhanced SRE‐producing capabilities was isolated and grown in a bioreactor with SRE yields averaging 50 mg/l in 40 h. SRE was extracted and purified through a large‐scale chromatography system using organic‐compatible processes and reagents. The minimum concentrations of the purified product required to inhibit 50 and 90% of P. ultimum oospore germination were determined as 31.3 and 250 μg/ml, respectively. Drench treatment of cucumber seeds in P. ultimum‐infested potting medium (500 oospores/g) with 50 μg/ml SRE or water with no SRE resulted in 90.2 ± 4.5% and 65.7 ± 4.6% germination rates, respectively. Seed coating with 0.03% (w/w) SRE allowed 65.7 ± 4.6% seedlings to germinate on naturally infested soil while 100.0 ± 0.0% of non‐coated seeds were unable to germinate due to Pythium infection. Organic‐compatible and scalably produced SRE is potentially a novel organic fungicide seed protectant.

CHARACTERIZATION OF PSEUDOMONAS SYRINGAE pv. SYRINGAE ISOLATED FROM MANGO IN SICILY AND OCCURRENCE OF COPPER-RESISTANT STRAINS

Mango (Mangifera indica) is grown throughout a wide range of frost-free climates and is one of the world’s most important fruit crops. During 2010-2014, severe symptoms of bacterial apical necrosis (BAN) caused by Pseudomonas syringae pv. syringae (Pss) were observed on mango plants throughout all mango-growing areas in Sicily (southern Italy). The causal agent was identified based on phenotypic, genotypic and pathogenicity tests. The genetic variability of strains obtained from different areas and cultivars were assessed by rep- PCR and allowed clear differentiation of the Pss strains isolated from mango from other representative strains of the pathovar. In addition, the respective production of syringomycin, syringopeptin and mangotoxin was checked by in vitro tests and PCR detection. All tested strains showed the presence of mgoA and mgoB of the mangotoxin operon. Some copper- resistant Pss strains showing cusCBA genes were also found in some orchards. This feature could explain the failure of the disease control using copper compounds. Among 71 strains tested using in vitro assay, no copper-sensitive strains were detected, whereas 44 strains (62%) had high resistance, 16 strains (22.5%) were resistant and 11 strains (15.5%) had low resistance to copper sulfate. To our knowledge, this is the first report of copper resistance among Pss strains causing BAN on mango in Sicily.

Host specificity, pathogenicity and the presence of virulence genes in Iranian strains of Pseudomonas syringae pv. syringae from different hosts

Pseudomonas syringae pv. syringae (Pss) strains were isolated from almond, apricot, peach, pear, sweet cheery and wheat in Kohgiluye and Boyer-Ahmad, Kordestan, Fras and Chaharmahal and Bakhtiari provinces of Iran. The strains were examined for host specificity, the presence of virulence genes and pathogenicity on different hosts. After inoculation of isolates, in compatible reactions bacterial populations increased within six days of inoculation and final cell numbers increased several-fold over initial inoculum levels, but in incompatible reactions, bacterial populations declined within four days of inoculation. Almond, sweet cherry and wheat isolates induced progressive necrotic symptoms on almond leaves and stems. Apricot, peach and sweet cherry isolates induced necrotic lesions when inoculated on apricot leaves. On pear leaves and stems, only the pear isolate incited pathogenic reaction and isolates from other hosts did not. The syrB gene was detected in all of the tested isolates. Almond and pear isolates did not have the syrD gene. The sypA gene was detected in the almond, peach, pear and sweet cherry isolates while the sypB gene was detected in the apricot, peach, sweet cherry and wheat isolates. Almond, apricot, pear and wheat isolates gave negative results for the detection of nit gene. The gene Ach, was detected only in the peach isolate and gene hrmA, was detected only in the wheat isolate. This study indicates that host specificity exists among different Pss strains, and genes responsible for syringomycin and syringopeptin production contribute to the virulence of Pss strains.

Characterization of Five ECF Sigma Factors in the Genome of Pseudomonas syringae pv. syringae B728a

Pseudomonas syringae pv. syringae B728a, a bacterial pathogen of bean, utilizes large surface populations and extracellular signaling to initiate a fundamental change from an epiphytic to a pathogenic lifestyle. Extracytoplasmic function (ECF) sigma (σ) factors serve as important regulatory factors in responding to various environmental signals. Bioinformatic analysis of the B728a genome revealed 10 ECF sigma factors. This study analyzed deletion mutants of five previously uncharacterized ECF sigma factor genes in B728a, including three FecI-type ECF sigma factors (ECF5, ECF6, and ECF7) and two ECF sigma factors placed in groups ECF11 and ECF18. Transcriptional profiling by qRT-PCR analysis of ECF sigma factor mutants was used to measure expression of their associated anti-sigma and outer membrane receptor proteins, and expression of genes associated with production of extracellular polysaccharides, fimbriae, glycine betaine and syringomycin. Notably, the B728aΔecf7 mutant displayed reduced swarming and had decreased expression of CupC fimbrial genes. Growth and pathogenicity assays, using a susceptible bean host, revealed that none of the tested sigma factor genes are required for in planta growth and lesion formation.

First Report of Bacterial Canker of Kiwifruit Caused by Pseudomonas syringae pv. actinidiae in Turkey.

A new disease was observed during the spring and autumn of 2009 and 2010 on kiwifruit plants (Actinidia deliciosa cv. Hayward) in Rize Province of Turkey. Disease incidence was estimated as 3% in approximately 10 ha. Symptoms were characterized by dark brown spots surrounded by yellow halos on leaves and cankers with reddish exudate production on twigs and stems. Eight representative bacterial strains were isolated from leaf spots and tissues under the bark on King's B medium (KB) and identified as Pseudomonas syringae pv. actinidiae on the basis of biochemical, physiological (1,2), and PCR tests (3). Bacteria were gram negative, rod shaped, and nonfluorescent on KB; positive for levan production, sucrose and inositol utilization, and tobacco (Nicotiana tabacum cv. White Burley) hypersensitivity; and negative for growth at 37°C, oxidase, potato soft rot, arginine dihydrolase, urease, arbutin, erythritol, lactic acid, aesculin hydrolysis, gelatin liquefaction, and syringomycin production. Identity of the eight isolates was confirmed by PCR using P. syringae pv. actinidiae-specific primers PsaF1/R3 to generate a 280-bp DNA fragment (3). P. syringae pv. actinidiae reference strain NCPPB 3739, and CJW7 from Jae Sung Jung, Department of Biology, Sunchon National University, Korea, were employed in all biochemical, physiological, and molecular tests as positive controls. Pathogenicity was confirmed by artificial inoculation of 2-year-old A. deliciosa cv. Hayward. A bacterial suspension (108 CFU ml-1) was injected into kiwifruit twig tips, stems, and leaves with a hypodermic syringe, and the inoculated plants were placed at 25 to 28°C and 80% relative humidity growth chamber for 3 weeks. First symptoms were observed on leaves within 5 days after inoculation and on twigs after 20 days. No symptoms were observed on control plants that were inoculated with sterile water. Reisolation was made from dark brown lesions surrounded by yellow halos on leaves and cankers on twigs and stem and their identities were confirmed using the techniques previously described. All tests were performed three times and pathogenicity tests employed three plants for each strain. To our knowledge, this is the first report of P. syringae pv. actinidiae causing disease on kiwifruit in Turkey. Kiwifruit production in Turkey has expanded rapidly during the last 10 years ( http://www.tuik.gov.tr ) and phytosanitary measures are needed to prevent further spread of the bacterium to other kiwifruit orchards.

Differentiation of Pseudomonas syringae pathovars originating from stone fruits

Due to an overlapping host range, similar symptomatology and many common characteristics, Pseudomonas syringae pathovars originating from stone fruits can easily be misidentified. In order to select tests for rapid and efficient differentiation of P. s. pvs. syringae, morsprunorum and persicae, we studied the suitability and differentiating potential of some standard bacteriological and molecular methods. Differentiation of the strains was performed using LOPAT, GATTa and ice nucleation tests, nutrient sucrose broth growth and utilization of various carbon sources. PCR method enabled the detection of toxin-producing genes: syrB and syrD in P. s. pv. syringae, and cfl gene in P. s. pv. morsprunorum race 1. Syringomycin production by pv. syringae was confirmed in bioassay using Geotrichum candidum, Saccharomyces cerevisiae and Rhodotorula pilimanae as indicator organisms. Pathogenicity test on lemon and immature nectarine fruits, as well as on string bean pods, showed different intensity of reaction of the inoculated material which could separate pv. syringae from the other two pathovars. PCR-based repetitive sequences, Rep-PCR with REP, ERIC and BOX primers revealed different genetic profiles within P. syringae pathovars.

Detection ofPseudomonas syringaepv.actinidiaeusing polymerase chain reaction (PCR) primers based on the 16S–23S rDNA intertranscribed spacer region and comparison with PCR primers based on other gene regions

Several published polymerase chain reaction (PCR) primers to identifyPseudomonas syringaepv.actinidiae, the causal organism of bacterial canker of kiwifruit, were found not to be specific. Two new sets of PCR primers, PsaF1/R2 and PsaF3/R4, were designed to be complementary to a portion of the 16S–23S rDNA intertranscribed spacer (ITS) regions. These primers amplified a DNA fragment from strains ofP. syringaepv.actinidiae, but not from 56 strains of bacteria from six genera and 17 species, except for a strain of the tea pathogen,P. syringaepv.theae. When tested against DNA extracted from a further 20 strains from Japan, Korea, Italy and the USA deposited in culture collections asP. syringaepv.actinidiae, all except six cultures produced the expected product of 280 bp with PsaF1/R2 and 175 bp with PsaF3/R4. Results of multilocus sequence analysis using five housekeeping genes (gyrB,acnB,rpoD,pgiandcts) showed that none of these six strains was phylogenetically similar toP. syringaepv.actinidiae.In contrast to theP. syringaepv.actinidiaetype strain, these strains were positive in the determinative tests for ice nucleation and syringomycin production. It is suggested that these six strains were incorrectly identified asP. syringaepv.actinidiae. It was not possible to distinguishP. syringaepv.actinidiaefrom the phylogenetically similarP. syringaepv.theaeusing the ITS,gyrB,acnB,rpoD,pgiorctsgene regions to design PCR primers. BecauseP. syringaepv.theaeis unlikely to be found on kiwifruit, primers PsaF1/R2 and PsaF3/R4 are recommended for screening bacteria isolated from kiwifruit tissue.

β-Hydroxylation of the Aspartyl Residue in the Phytotoxin Syringomycin E: Characterization of Two Candidate Hydroxylases AspH and SyrP in Pseudomonas syringae

The pseudomonal phytotoxin syringomycin E and related nonribosomal peptides contain an L- threo-beta-hydroxyaspartyl residue at the eighth position of the lipodepsipeptide backbone as part of a conserved nonproteinogenic tripeptide motif. Informatic analysis of the P. syringae genome suggests only one putative non-heme iron hydroxylase, AspH. On heterologous expression in Escherichia coli AspH shows robust catalytic activity with free L-Asp and L-Asp thioesters to make beta-OH-Asp but yields the erythro diastereomer rather than the threo configuration that is found in syringomycin. Further analysis of the Syr gene cluster indicated that SyrP, previously annotated as the gene regulatory protein for the five-gene Syr cluster, is actually homologous to the known non-heme mononuclear iron hydroxylase TauD. Indeed, purified SyrP acts on Asp tethered as the protein-bound S-pantetheinyl thioester on the eighth module of the SyrE megasynthetase. The hydroxylation gives the anticipated L- threo-3-OH-Asp diastereomer found in syringomycin. The knockout of syrP abolishes the production of the mature syringomycin E, while knockout of aspH has no effect on syringomycin production.

Mutational analysis and homology modelling of SyrC, the aminoacyltransferase in the biosynthesis of syringomycin

SyrC, a component of the multienzyme system of syringomycin biosynthesis, has been shown to shuttle Thr/4-Cl-Thr between the thiolation domains SyrB1-T1 and SyrE-T8,9 by transiently linking it to Cys224 in the enzyme active site. We present data on the structure-function relationship in vivo of this protein and an in silico model of its three-dimensional structure. The biosynthetic activity of SyrC was not influenced when either Asp348 or His376 that together with Cys224 form a putative catalytic triad, were replaced with Ala, but it was abolished by the exchange Cys224 with Ser. The presence of the FLAG peptide on either the N- or C-terminus of the protein did not affect activity, whereas the deletion of the first 16 amino acids at the N-terminus or the insertion of Maltose Binding Protein abolished the production of syringomycin. We present the model of the three-dimensional structure of SyrC suggesting a homodimeric structure for the protein and biochemical data that are supportive of this model.

Characterization of the transcriptional activators SalA and SyrF, Which are required for syringomycin and syringopeptin production by Pseudomonas syringae pv. syringae.

Production of the phytotoxins syringomycin and syringopeptin by Pseudomonas syringae pv. syringae is controlled by the regulatory genes salA and syrF. Analysis with 70-mer oligonucleotide microarrays established that the syr-syp genes responsible for synthesis and secretion of syringomycin and syringopeptin belong to the SyrF regulon. Vector pMEKm12 was successfully used to express both SalA and SyrF proteins fused to a maltose-binding protein (MBP) in Escherichia coli and P. syringae pv. syringae. Both the MBP-SalA and MBP-SyrF fusion proteins were purified by maltose affinity chromatography. Gel shift analysis revealed that the purified MBP-SyrF, but not the MBP-SalA fusion protein, bound to a 262-bp fragment of the syrB1 promoter region containing the syr-syp box. Purified MBP-SalA caused a shift of a 324-bp band containing the putative syrF promoter. Gel filtration analysis and cross-linking experiments indicated that both SalA and SyrF form homodimers in vitro. Overexpression of the N-terminal regions of SalA and SyrF resulted in decreased syringomycin production by strain B301D and reduced levels of beta-glucuronidase activities of the sypA::uidA and syrB1::uidA reporters by 59% to 74%. The effect of SalA on the expression of the syr-syp genes is mediated by SyrF, which activates the syr-syp genes by directly binding to the promoter regions. Both SalA and SyrF resemble other LuxR family proteins in dimerization and interaction with promoter regions of target genes.

The Expression of Genes Encoding Lipodepsipeptide Phytotoxins byPseudomonas syringaepv.syringaeIs Coordinated in Response to Plant Signal Molecules

Specific plant signal molecules are known to induce syringomycin production and expression of syrB1, a syringomycin synthetase gene, in Pseudomonas syringae pv. syringae. This report demonstrates that syringopeptin production likewise is activated by plant signal molecules and that the GacS, SalA, and SyrF regulatory pathway mediates transmission of plant signal molecules to the syr-syp biosynthesis apparatus. Syringopeptin production by BR132 was increased two-fold by addition of arbutin (100 microM) and D-fructose (0.1%) to syringomycin minimal medium (SRM). Among 10 plant phenolic compounds tested, only the phenolic glucosides arbutin, salicin, and phenyl-beta-D-glucopyranoside induced substantially the beta-glucuronidase (GUS) activity of a sypA::uidA reporter from 242 U per 10(8) CFU without plant signal molecules up to 419 U per 10(8) CFU with plant signal molecules. Syringopeptin production was found to be controlled by the SalA/SyrF regulon because no toxin was detected from cultures of B301DSL7 (i.e., salA mutant) and B301DSL1 (i.e., syrF mutant), and the expression of sypA::uidA was decreased approximately 99 and 94% in salA (B301DSL30) and syrF (B301DNW31) mutant backgrounds, respectively. Subgenomic analysis of transcriptional expression with a 70-mer oligonucleotide microarray demonstrated that the syr-syp genes are induced 2.5- to 10.5-fold by addition of arbutin and D-fructose to SRM. This study establishes that plant signal molecules are transmitted through the GacS, SalA/SyrF pathway to activate the coordinated transcriptional expression of the syr-syp genes.

Phylogenetic Characterization of Virulence and Resistance Phenotypes of Pseudomonas syringae

Individual strains of the plant pathogenic bacterium Pseudomonas syringae vary in their ability to produce toxins, nucleate ice, and resist antimicrobial compounds. These phenotypes enhance virulence, but it is not clear whether they play a dominant role in specific pathogen-host interactions. To investigate the evolution of these virulence-associated phenotypes, we used functional assays to survey for the distribution of these phenotypes among a collection of 95 P. syringae strains. All of these strains were phylogenetically characterized via multilocus sequence typing (MLST). We surveyed for the production of coronatine, phaseolotoxin, syringomycin, and tabtoxin; for resistance to ampicillin, chloramphenicol, rifampin, streptomycin, tetracycline, kanamycin, and copper; and for the ability to nucleate ice at high temperatures via the ice-nucleating protein INA. We found that fewer than 50% of the strains produced toxins and significantly fewer strains than expected produced multiple toxins, leading to the speculation that there is a cost associated with the production of multiple toxins. None of these toxins was associated with host of isolation, and their distribution, relative to core genome phylogeny, indicated extensive horizontal genetic exchange. Most strains were resistant to ampicillin and copper and had the ability to nucleate ice, and yet very few strains were resistant to the other antibiotics. The distribution of the rare resistance phenotypes was also inconsistent with the clonal history of the species and did not associate with host of isolation. The present study provides a robust phylogenetic foundation for the study of these important virulence-associated phenotypes in P. syringae host colonization and pathogenesis.

Oligonucleotide Microarray Analysis of the SalA Regulon Controlling Phytotoxin Production by Pseudomonas syringae pv. syringae

The salA gene is a key regulatory element for syringomycin production by Pseudomonas syringae pv. syringae and encodes a member of the LuxR regulatory protein family. Previous studies revealed that salA, a member of the GacS/GacA signal transduction system, was required for bacterial virulence, syringomycin production, and expression of the syrB1 synthetase gene. To define the SalA regulon, the spotted oligonucleotide microarray was constructed using gene-specific 70-mer oligonucleotides of all open reading frames (ORFs) predicted in the syringomycin (syr) and syringopeptin (syp) gene clusters along with representative genes important to bacterial virulence, growth, and survival. The microarray containing 95 oligos was used to analyze transcriptional changes in a salA mutant (B301DSL07) and its wild-type strain, B301D. Expression of 16 genes was significantly higher (> twofold) in B301D than in the salA mutant; the maximum change in expression was 15-fold for some toxin biosynthesis genes. Except for the sylD synthetase gene for syringolin production, all ORFs controlled by SalA were located in the syr-syp genomic island and were associated with biosynthesis, secretion, and regulation of syringomycin and syringopeptin. The positive regulatory effect of SalA on transcription of sypA, syrB1, syrC, and sylD was verified by reporter fusions or real-time polymerase chain reaction analysis. None of the genes or ORFs was significantly down-regulated by the salA gene. These results demonstrated that a subgenomic oligonucleotide microarray is a powerful tool for defining the SalA regulon and its relationship to other genes important to plant pathogenesis.

Characterization of the argA gene required for arginine biosynthesis and syringomycin production by Pseudomonas syringae pv. syringae.

Two types of necrosis-inducing lipodepsipeptide toxins, called syringomycin and syringopeptin, are major virulence factors of Pseudomonas syringae pv. syringae strain B301D. A previous study showed that a locus, called syrA, was required for both syringomycin production and plant pathogenicity, and the syrA locus was speculated to encode a regulator of toxin production. In this study, sequence analysis of the 8-kb genomic DNA fragment that complements the syrA phenotype revealed high conservation among a broad spectrum of fluorescent pseudomonads. The putative protein encoded by open reading frame 4 (ORF4) (1,299 bp) in the syrA locus region exhibited 85% identity to ArgA, which is involved in arginine biosynthesis in Pseudomonas aeruginosa. Growth of strain W4S2545, the syrA mutant, required supplementation of N minimal medium with arginine. Similarly, syringomycin production of syrA mutant W4S2545 was restored by the addition of arginine to culture media. Furthermore, the insertion of Tn5 in the genome of the syrA mutant W4S2545 was localized between nucleotides 146 and 147 in ORF4, and syringomycin production was complemented in trans with the wild-type DNA fragment containing intact ORF4. These results demonstrate that the syrA locus is the argA gene of P. syringae pv. syringae and that argA is directly involved in arginine biosynthesis and therefore indirectly affects syringomycin production because of arginine deficiency.

Construction of pMEKm12, an expression vector for protein production in Pseudomonas syringae

Characterization of the biological roles of proteins is essential for functional genomics of pseudomonads. Heterologous proteins overproduced in Escherichia coli frequently fail to exhibit biological function. To circumvent this problem, vector pMEKm12 was constructed and used to overexpress proteins in Pseudomonas. The vector contains the pRO1600 replication origin, the maltose-binding protein (MBP) fusion system, and an inducible tac promoter. The pMEKm12 was successfully used to overexpress the syringomycin synthetase SyrB1 protein fused to MBP in Pseudomonas syringae pv. syringae. Furthermore, expression of the MBP-SyrB1 protein in the syrB1 mutant BR132A1 resulted in the restoration of syringomycin production. This vector will facilitate confirmation of the biochemical roles of nonribosomal peptide synthetase genes in Pseudomonas syringae, and studies of gene function from a wide spectrum of pseudomonads.