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PubMed1998

Molecular characterization and sequence of a methionine biosynthetic locus from Pseudomonas syringae.

Two methionine biosynthetic genes in Pseudomonas syringae pv. syringae, metX and metW, were isolated, sequenced, and evaluated for their roles in methionine biosynthesis and bacterial fitness on leaf surfaces. The metXW locus was isolated on a 1.8-kb DNA fragment that was required for both methionine prototrophy and wild-type epiphytic fitness. Sequence analysis identified two consecutive open reading frames (ORFs), and in vitro transcription-translation experiments provided strong evidence that the ORFs encode proteins with the predicted molecular masses of 39 and 22.5 kDa. The predicted amino acid sequence of MetX (39 kDa) showed homology to several known and putative homoserine O-acetyltransferases. This enzyme is the first enzyme in the methionine biosynthetic pathway of fungi, gram-negative bacteria of the genus Leptospira, and several gram-positive bacterial genera. Both metX and metW were required for methionine biosynthesis, and transcription from both genes was not repressed by methionine. MetW (22.5 kDa) did not show significant homology to any known protein, including prokaryotic and eukaryotic methionine biosynthetic enzymes. Several classes of methionine auxotrophs, including metX and metW mutants, exhibit reduced fitness on leaf surfaces, indicating a requirement for methionine prototrophy in wild-type epiphytic fitness. This requirement is enhanced under environmentally stressful conditions, suggesting a role for methionine prototrophy in bacterial stress tolerance.

Acetyltransferases↗
PubMed1998

Growth phase and temperature influence promoter activity, transcript abundance, and protein stability during biosynthesis of the Pseudomonas syringae phytotoxin coronatine.

The plant-pathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 synthesizes high levels of the polyketide phytotoxin coronatine (COR) at 18 degrees C, whereas no detectable toxin is produced at 28 degrees C. Previously, we reported that the temperature-sensitive activation of three promoters within the COR biosynthetic gene cluster might explain thermoregulation of COR biosynthesis. The present study was aimed at furthering our understanding of the transcriptional as well as the posttranslational effects of temperature on expression of cmaB, which encodes an enzyme involved in COR biosynthesis. Transcriptional fusions using a promoterless glucuronidase gene and Northern blot analyses were used to monitor promoter activities and transcript abundance for the cmaABT operon during bacterial growth at 18 and 28 degrees C. Promoter activity and transcription rates were maximal when cells were incubated at 18 degrees C and sampled at mid-logarithmic phase. Transcription declined moderately during the transition to stationary phase but remained higher at 18 C than at 28 degrees C. Western blot analysis indicated that CmaB accumulated in the late stationary phase of P. syringae cultures grown at 18 degrees C but not in cultures incubated at 28 degrees C. Temperature shift experiments indicated that CmaB stability was more pronounced at 18 degrees C than at 28 degrees C. Although temperature has a strong impact on transcription of COR biosynthetic genes, we propose that thermoregulation of protein stability might also control COR synthesis.

Amino Acids↗
PubMed2000

The alternative sigma factor RpoN is required for hrp activity in Pseudomonas syringae pv. maculicola and acts at the level of hrpL transcription.

beta-Glucuronidase (uidA) reporter gene fusions were constructed for the hrpZ, hrpL, and hrpS genes from the phytopathogen Pseudomonas syringae pv. maculicola strain ES4326. These reporters, as well as an avrRpt2-uidA fusion, were used to measure transcriptional activity in ES4326 and a ES4326 rpoN mutant. rpoN was required for the expression of avrRpt2, hrpZ, and hrpL in vitro in minimal media and in vivo when infiltrated into Arabidopsis thaliana leaves. In contrast, the expression of hrpS was essentially the same in wild-type and rpoN mutant strains. Constitutive expression of hrpL in an rpoN mutant restored hrpZ transcription to wild-type levels, restored the hypersensitive response when infiltrated into tobacco (Nicotiana tobacum), and partially restored the elicitation of virulence-related symptoms but not growth when infiltrated into Arabidopsis leaves. These data indicate that rpoN-mediated control of hrp gene expression acts at the level of hrpL and that in planta growth of P. syringae is not required for the elicitation of disease symptoms.

Bacterial Proteins↗
PubMed1981

Transformation of Pseudomonas syringae with nonconjugative R plasmids.

Transformation of Pseudomonas syringae strains with plasmid DNA occurs at a frequency of 1 x 10(-3) to 4 x 10(-9) per recipient cell, depending on the strain, plasmid, and conditions for transformation. R plasmids used successfully in transformation were pR0161 (26 x 10(6) molecular weight) and RSF1010 (5.5 x 10(6) molecular weight). Transformation involved growing the recipient cells to approximately 8 x 10(8) colony-forming units per millilitre in 50 mL of a nutrient broth. After washes with a 150 mM CaCl2 - 10% (v/v) glycerol mixture, cells were concentrated 20-fold and resuspended in this solution. The cells then were incubated with purified plasmid DNA for 1 h prior to heat pulse at 45 degrees C for 2 min. Transformants were selected by antibiotic resistance and plasmid presence was verified by agarose gel electrophoresis. With plasmid pCG131 (34 x 10(6) molecular weight; putatively associated with syringomycin production), transformation of syringomycin-negative P. syringae strains that contained no detectable plasmid or were cured of pCG131 was unsuccessful, whether the plasmid was used alone or in combination with either pR0161 or RSF1010.

Anti-Bacterial Agents↗
PubMedDate not supplied

The structure of the pyoverdin isolated from various Pseudomonas syringae pathovars.

From seven different pathovars of Pseudomonas syringae representing various genetic subgroups, and one strain of Pseudomonas viridiflava the same pyoverdin siderophore (1) was isolated, probably identical with the pyoverdin whose amino acid composition (but not their sequence) had been reported before. 1 is the first pyoverdin where two of the ligands for Fe3+ are beta-hydroxy Asp units. Its remarkably high complexing constant for Fe3+ at pH 5 as compared with other pyoverdins offers a definite advantage in plant infection. The structure elucidation of 1 will be described and the taxonomical implications regarding pyoverdins with different structures ascribed previously to P. syringae strains will be discussed.

Amino Acid Sequence↗
PubMed2001

Effects of nutritional factors on production of tabtoxin, a phytotoxin, by Pseudomonas syringae pv. tabaci.

Pseudomonas syringae pv. tabaci, the causal agent of the wildfire of tobacco, produces the phytotoxin tabtoxin. The effects of carbon, nitrogen sources and amino acids on growth and tabtoxin production by pv. tabaci, were examined by varying the components of a defined basal medium, which contained the following nutrients per liter: sucrose (10 g), KNO3 (5 g), MgSO(4).7H2O (0.2 g), CaCl(2).2H2O (0.11 g), FeSO(4).7H2O (20 mg), NaH2PO(4).2H2O (0.9 g) and H2PO(4).3HO (1 g). Bacterial growth was determined by cell density, and tabtoxin production was measured by the E. coli bioassay technique. Both growth and quantity of tabtoxin synthesized were significantly affected by carbon source, nitrogen source and amino acid supplements. Sorbitol, xylose and sucrose proved to be the best carbon sources for tabtoxin production. Specific toxin production was very low using glucose as a single carbohydrate source, although bacterial growth was well supported by glucose. Amount and type of nitrogen sources (NH4Cl or KNO3) affected the growth of pv. tabaci and quantities of tabtoxin produced. Nitrate was the best of these two forms of nitrogen for production of tabtoxin. Adding threonine to pv. tabaci grown in batch culture decreased the amount of tabtoxin production. Similar results were obtained with lysine, whereas, serine had no effects on quantities of tabtoxin production. The results of the present study were to formulate a medium which allowed for enhanced tabtoxin production by Pseudomonas syringae pv. tabaci.

Amino Acids↗
PubMedDate not supplied

Gene-for-gene interactions between Pseudomonas syringae pv. phaseolicola and Phaseolus.

The gene for cultivar-specific avirulence to Phaseolus vulgaris cv. Tendergreen in races 3 and 4 of Pseudomonas syringae pv. phaseolicola was isolated and sequenced. Genomic clones from libraries of race 3 in pLAFR1 and race 4 in pLAFR3, which altered the phenotype of race 5 from virulent to avirulent in Tendergreen, were found to possess a common approximately 15-kb region of DNA that contained the determinant of avirulence. Subcloning and insertion mutagenesis with Tn1000 located an avirulence gene within a 1.4-kb BglII/HindIII DNA fragment in races 3 and 4. Comparison of the nucleotide sequences of regions of DNA that confer avirulence confirmed that both races have an identical gene for avirulence (designated avrPph3) comprising 801 base pairs (bp) and predicted to encode a cytoplasmic protein of 28,703 Da. A sequence, TGCAACCGAAT, 91% homologous to the motif found in promoter regions of avrB and avrD from P. s. pv. glycinea was located 89-99 bp upstream of the start of the open-reading frame 1. Hybridization experiments showed that avrPph3 was not plasmid-borne and was absent from isolates of P. s. pv. phaseolicola races 1, 2, 5, 6, 7, and 8, P. cichorii, P. s. pvs. coronafaciens, glycinea, maculicola, pisi, syringae, and tabaci. Cosegregation studies of crosses between cultivars resistant (Tendergreen) and susceptible (Canadian Wonder) to races 3 and 4 and transconjugants of race 5 confirmed that a gene-for-gene relationship controls specificity in the interaction between Tendergreen and races 3 and 4 of P. s. pv. phaseolicola.

Amino Acid Sequence↗
PubMed1990

[Antigenic polysaccharides of bacteria. 37. Structure of the polysaccharide chain of Pseudomonas syringae pv.tabaci (serogroup VII) lipopolysaccharide].

The structure of the O-specific polysaccharide chain of Pseudomonas syringae pv. tabaci strain 223 (serogroup VII) lipopolysaccharide was established on the basis of one- and two-dimensional 1H NMR analysis, 13C NMR analysis and calculation of optical rotation. The structure determined by the non-destructive way was confirmed by acid hydrolysis and methylation. (Sequence: see text). O-Antigen of the strain studied is similar in structure and serological properties to O-antigens of Pseudomonas syringae strains belonging to serogroup I.

Antigens, Bacterial↗
PubMedDate not supplied

[The immunochemical characteristics of the lipopolysaccharides of Pseudomonas syringae (pathovars atrofaciens and phaseolicola) and P. holci (serogroup VI)].

Lipopolysaccharides (LPS) of the representatives of strains of serogroup VI Pseudomonas syringae (P. syringae pv. atrofaciens 2399, pv. phaseolicola 120a, 7842 and P. holci 8299) possessing virulence and confinement to the host-plant are characterized by high serological activity in direct and cross reactions of the binary diffusion in agar, immunoelectrophoresis, passive hemagglutination and inhibition of passive hemagglutination. A supernatant and a sediment obtained after ultracentrifugation of LPS preparations possessed O-antigenic activity. O-specific polysaccharide (PS) is serologically less active than the LPS preparations. Problems of the intergroup and intragroup serological affinity in connection with the structure of O-specific PS. It is proved that the basic chain of O-specific polysaccharide (D-rhamnane) plays definite (but not a single) part in displaying antigenic properties of the whole LPS macromolecule.

Immunochemistry↗
PubMedDate not supplied

[Isolation and partial characteristics of biopolymers in the outer membrane of Pseudomonas syringae].

Conditions are developed for obtaining extracellular biopolymers (EBP), water (W), salt-EDTA (SE) and salt-detergent (SD) extracts of Pseudomonas syringae pv. atrofaciens IMV K-1025. The fraction of unsoluble LPS is effectively precipitated from samples by ultracentrifugation (UC). The fractions enriched by proteins and soluble LPS are salted-out from the UC supernatants by ammonium sulphate, that is confirmed by the chemical analysis, electrophoresis in the PAAG-NDS and in ELISA with the monoclonal antibodies against the homologous LPS, a comparative analysis of electrophoretic spectra of UC supernatants of EBP, W and SE extracts has shown their great similarity in the composition rather than in the specific content of basic and minor polypeptides. Nine polypeptides of the major membrane of Pseudomonas syringae pv. atrofaciens IMV K-1025 having the molecular weights of 15.5, 30.0, 34.0, 36.0, 38.0, 41.0, 42.0, 43.0 and 65.0 kDa are identified as major ones.

Bacterial Outer Membrane Proteins↗
PubMed1988

Phospholipid requirement for expression of ice nuclei in Pseudomonas syringae and in vitro.

Delipidation of partially purified outer membranes of Pseudomonas syringae by various delipidating agents resulted in a significant loss of ice nucleation activity associated with the cell envelopes of this and other ice nucleation active bacteria. Lipopolysaccharide depletion of such membranes caused no reduction in ice nucleation activity. Both phospholipid content and ice nucleation activity of membranes were decreased by a similar fractional amount with time after treatment with phospholipase A2. A proportional quantitative relationship between loss of ice nucleation activity and lipid removal with increasing concentrations of sodium cholate and sodium dodecyl sulfate (SDS) was also observed. Significant linear relationships between the amount of lipid removed by phospholipase A2, sodium cholate, and SDS and the loss of ice nucleation activity in P. syringae outer membranes were observed. However, the slopes of these linear relationships for membranes treated with phospholipase A2 (m = 0.80), SDS (m = 0.94), and sodium cholate (m = 0.53) differed. The lower slope value for cholate-treated membranes indicated a partial substitution of sodium cholate for the phospholipids removed. The ice nucleation activity of delipidated outer membranes was restored by reconstitution with various phospholipids in a cholate dialysis procedure. Lipid classes differed in their ability to restore ice nucleation activity to sodium cholate-treated outer membranes. These results suggest that a hydrophobic environment provided either by lipids or certain detergent micelles is required for proper assembly and structural organization of an oligomeric ice protein complex enabling its expression as an ice nucleus.

Cell Membrane↗
PubMedDate not supplied

[The genetic aspects of the study of Pseudomonas syringae bacteria].

The work is a review of the state-of-art of molecular-genetic examination of bacteria P. syringae. The questions concerning new approaches to the study of determinants of pathogenicity, endogenic plasmids, toxins, resistance to antibiotics, avirulence, genes and insertion sequence elements of P. syringae are stated.

Bacterial Toxins↗
PubMedDate not supplied

Characterization of lipopolysaccharides from Pseudomonas syringae (serogroup II).

Lipopolysaccharides (LPS) from the strains of Pseudomonas syringae pv.syringae 90a, 435, pv. atrofaciens K-1025, pv. morsprunorum CF-4 referred by Pastushenko and Simonovich (1979) to serogroup II have been studied. The strains were shown to be heterogeneous by chemical composition of core and lipid A and structure of O-specific polysaccharide. The preparations heterogeneity in serological cross reactions were also detected. O-specific polysaccharides of the strains having similar structures were not identical in serological tests. The assumption on the lipid A role in serogrouping of P. syringae strains has been advanced.

Lipopolysaccharides↗
PubMed1998

Characterization of the phytopathogen Pseudomonas syringae pathovar ribicola NCPPB 963.

In 1939, a bacterial spot caused severe defoliation of Ribes aureum (Golden Currant) The causal agent is now recognized as Pseudomonas syringae pathovar ribicola. This communication extends the phenotype of the only identified strain of P. syringae pv. ribicola, which is reminiscent of those of other pathovars, and provides a molecular biological characterization. A minimum size of 5.55 Mb for the bacterial genome was obtained using pulsed-field electrophoresis. The SDS-PAGE outer-membrane profile contained seven major bands, and has obvious similarities to that of P. aeruginosa. SDS-PAGE of concentrated mid-log phase culture supernatants revealed large amounts of a single, cryptic 24.0 kD protein. The amino acid composition and 57 residues in the N-terminus of this protein. were determined. The protein sequence was nearly identical to the translation of a region of unknown function in the P. aeruginosa genome. Extensive similarity in N-terminal sequence, composition and subunit size to a secreted hydrophilic Vibrio cholerae protein of unknown function was also found. Neither protein has been directly associated with disease development.

Amino Acid Sequence↗
PubMed2025

Lytic properties and genomic analysis of bacteriophage Brt_Psa3, targeting Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa) is the causative agent of bacterial canker in kiwifruit (Actinidia spp.). Psa biovar 3 is the most prevalent and virulent, causing frequent and severe outbreaks worldwide. While current treatments have low efficacy, bacteriophages emerge as possible environmentally safe alternative biocontrol agents. In this study, bacteriophage Brt_Psa3 was isolated from the soil of a kiwifruit orchard in Portugal. Morphologically, Brt_Psa3 forms clear plaques and has a Podoviral morphotype. The bacteriophage exhibited broad lytic activity against several plant-pathogenic Pseudomonas strains, including Psa isolates. The isolated bacteriophage has a latent period of 100 min, a burst size of 143 particles/cell, and demonstrates stability at different temperatures and pH values found in kiwifruit orchards. In addition, Brt_Psa3 exhibited tolerance to UVA irradiation during 120 min of incubation. Brt_Psa3 belongs to the Autographiviridae family and Ghunavirus genus, based on full-genome nucleotide alignment and supported by phylogenetic analysis of structural proteins. The phage contains 51 open reading frames with no antibiotic resistance genes identified, within a genome of 40.509 base pairs. In vitro experiments with kiwifruit leaves demonstrated significant reduction of Psa levels (40%) on leaf surfaces, highlighting the bacteriophage's therapeutic potential in managing bacterial canker in kiwifruits.

Pseudomonas syringae↗
PubMed2026

Bacteriophages Control Epiphytic Pseudomonas syringae Populations in Highbush Blueberry Leaves.

The Pseudomonas syringae complex (Psc) is a group of globally distributed phytopathogens responsible for substantial agricultural losses. Although bacteriophage-based biocontrol has shown promise against Psc, no studies have examined phages targeting blueberry-tropic Psc lineages. Here, we isolated phages infecting Psc strains from diseased highbush blueberry (Vaccinium corymbosum), and evaluated their suitability for biocontrol using a multi-stage screening pipeline incorporating host-range analysis, comparative genomics, environmental stability testing, in vitro antibacterial efficacy assays and ex planta validation. Twelve of the isolated phages exhibited favourable host-range characteristics. Genomic analyses revealed substantial phylogenetic diversity among these candidates but simultaneously identified multiple clonal groups, reducing the collection to eight non-redundant phages spanning five distinct genera. Candidate phages generally retained infectivity under environmentally relevant conditions and exhibited heterogeneous but largely favourable stability profiles. Planktonic killing assays uncovered considerable variation in antibacterial efficacy, but phage performance appeared to be driven by infection compatibility and host-specific factors rather than properties intrinsic to individual phages. Notably, the jumbo phageCB10 emerged as a particularly promising candidate due to its strong antibacterial activity (median GRC = 0.943), favourable environmental stability and unique genomic features. Cocktails containing the most effective candidates produced substantial and longitudinally sustained reductions in epiphytic colonization of detached blueberry leaves by Psc, exceeding five orders of magnitude at peak efficacy and demonstrating robust activity in a biologically relevant ex planta system. Importantly, in vitro antibacterial efficacy was predictive of performance in our ex planta model (r = 0.67; p = 0.0003), supporting the utility of tiered screening approaches for candidate selection. Taken together, these findings establish a framework for the systematic identification and evaluation of phages targeting Psc, and support the development of phage-based interventions for managing plant diseases.

Pseudomonas syringae↗
PubMed2003

Secoiridoid glucosides with free radical scavenging activity from the leaves of Syringa dilatata.

Activity-guided fractionation of the EtOAc and MeOH extract of the leaves of Syringa dilatata NAKAI furnished one free radical scavenger, the secoiridoid glucoside oleuropein together with ligstroside and an iridoid glucoside, syringopicroside. Oleuropein interacted with the stable free radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and showed an IC(50) value of 40.4 microM. L-Ascorbic acid as a positive control showed an IC(50) value of 50.3 microM.

Biphenyl Compounds↗
PubMed2006

Overexpression of GbERF confers alteration of ethylene-responsive gene expression and enhanced resistance to Pseudomonas syringae in transgenic tobacco.

GbERF belongs to the ERF (ethylene responsive factor) family of transcription factors and regulates the GCC-box containing pathogen-related (PR) genes in the ethylene signal transduction pathway. To study the function of GbERF in the process of biotic stress, transgenic tobacco plants expressing GbERF were generated. Overexpression of GbERF did not change transgenic plant's phenotype and endogenous ethylene level. However, the expression profile of some ethylene-inducible GCC-box and non-GCC-box containing genes was altered, such as PR1b, PR2, PR3, PR4, Osmotin, CHN50, ACC oxidase and ACC synthase genes. These data indicate that the cotton GbERF could act as a transcriptional activator or repressor to regulate the differential expression of ethylene-inducible genes via GCC and non-GCC cis-elements. Moreover, the constitutive expression of GbERF in transgenic tobacco enhanced the plant's resistance to Pseudomonas syringae pv tabaci infection. In conclusion, GbERF mediates the expression of a wide array of PR and ethylene-responsive genes and plays an important role in the plant's response to biotic stress.

Base Sequence↗