Relatedness of Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. antirrhini

The relationships among strains of Pseudomonas syringae pv. tomato, Ps. syr. antirrhini, Ps. syr. maculicola, Ps. syr. apii and a strain isolated from squash were examined by restriction fragment length polymorphism (RFLP) patterns, nutritional characteristics, host of origin and host ranges. All strains tested except for Ps. syr. maculicola 4326 isolated from radish ( Raphanus sativus L.) constitute a closely related group. No polymorphism was seen among strains probed with the 5.7 and 2.3 kb Eco RI fragments which lie adjacent to the hrp cluster of Ps. syr. tomato and the 8.6 kb Eco RI insert of pBG2, a plasmid carrying the β-glucosidase gene(s). All strains tested had overlapping host ranges. In contrast to this, comparison of strains by RFLP patterns of sequences homologous to the 4.5 kb Hind III fragment of pRut2 and nutritional properties distinguished four groups. Group 1, consisting of strains of pathovars maculicola, tomato and apii , had similar RFLP patterns and used homoserine but not sorbitol as carbon sources. Group 2, consisting of strains of pathovars maculicola and tomato , differed from Group 1 in RFLP patterns and did not use either homoserine or sorbitol. Group 3 was similar to Group 2 in RFLP patterns but utilized homoserine and sorbitol. This group included strains of the pathovars tomato and antirrhini , and a strain isolated from squash. Group 4, a single strain of Ps. syr. maculicola isolated from radish, had unique RFLP patterns and resembled Group 3 nutritionally. The evolutionary relationships of these strains are discussed.     

Concomitant induction of systemic resistance to Pseudomonas syringae pv. lachrymans in cucumber by Trichoderma asperellum (T-203) and accumulation of phytoalexins

Most studies on the reduction of disease incidence in soil treated with Trichoderma asperellum have focused on microbial interactions rather than on plant responses. This study presents conclusive evidence for the induction of a systemic response against angular leaf spot of cucumber (Pseudomonas syringae pv. lachrymans) following application of T. asperellum to the root system. To ascertain that T. asperellum was the only microorganism present in the root milieu, plants were grown in an aseptic hydroponic growth system. Disease symptoms were reduced by as much as 80%, corresponding to a reduction of 2 orders of magnitude in bacterial cell densities in leaves of plants pretreated with T. asperellum. As revealed by electron microscopy, bacterial cell proliferation in these plants was halted. The protection afforded by the biocontrol agent was associated with the accumulation of mRNA of two defense genes: the phenylpropanoid pathway gene encoding phenylalanine ammonia lyase (PAL) and the lipoxygenase pathway gene encoding hydroxyperoxide lyase (HPL). This was further supported by the accumulation of secondary metabolites of a phenolic nature that showed an increase of up to sixfold in inhibition capacity of bacterial growth in vitro. The bulk of the antimicrobial activity was found in the acid-hydrolyzed extract containing the phenolics in their aglycone form. High-performance liquid chromatography analysis of phenolic compounds showed a marked change in their profile in the challenged, preelicited plants relative to that in challenged controls. The results suggest that similar to beneficial rhizobacteria, T. asperellum may activate separate metabolic pathways in cucumber that are involved in plant signaling and biosynthesis, eventually leading to the systemic accumulation of phytoalexins.     

Chemotaxis by Pseudomonas syringae pv. tomato

Optimal laboratory conditions for studying chemotaxis by Pseudomonas syringae pv. tomato were determined by using the Adler capillary tube assay. Although they are not an absolute requirement for chemotaxis, the presence of 0.1 mM EDTA and 1 mM MgCl₂ in the chemotaxis buffer (10 mM potassium phosphate [pH 7.2]) significantly enhanced the response to attractant. The addition of mannitol as an energy source had little effect. The optimal temperature for chemotaxis was 23°C, which is 5°C below the optimal growth temperature for this pathogen. The best response occurred when the bacteria were exposed to attractant for 60 min at a concentration of approximately 5 × 10⁶ CFU/ml. P. syringae pv. tomato was strongly attracted to citric and malic acids, which are the predominant organic acids in tomato fruit. With the exception of asparagine, the major amino acids of tomatoes were weak to moderate attractants. Glucose and fructose, which account for approximately 47% of tomato dry matter, also elicited poor responses. In assays with tomato intercellular fluid and leaf surface water, the bacterial speck pathogen could not chemotactically distinguish between a resistant and a susceptible cultivar of tomato.     

Periplasmic glucans of Pseudomonas syringae pv. syringae.

We report the initial characterization of glucans present in the periplasmic space of Pseudomonas syringae pv. syringae (strain R32). These compounds were found to be neutral, unsubstituted, and composed solely of glucose. Their size ranges from 6 to 13 glucose units/mol. Linkage studies and nuclear magnetic resonance analyses demonstrated that the glucans are linked by beta-1,2 and beta-1,6 glycosidic bonds. In contrast to the periplasmic glucans found in other plant pathogenic bacteria, the glucans of P. syringae pv. syringae are not cyclic but are highly branched structures. Acetolysis studies demonstrated that the backbone consists of beta-1,2-linked glucose units to which the branches are attached by beta-1,6 linkages. These periplasmic glucans were more abundant when the osmolarity of the growth medium was lower. Thus, P. syringae pv. syringae appears to synthesize periplasmic glucans in response to the osmolarity of the medium. The structural characteristics of these glucans are very similar to the membrane-derived oligosaccharides of Escherichia coli, apart from the neutral character, which contrasts with the highly anionic E. coli membrane-derived oligosaccharides.     

The Pseudomonas syringae pv. syringae 61 hrpH product, an envelope protein required for elicitation of the hypersensitive response in plants.

Pseudomonas syringae pv. syringae 61 contains a 25-kb cluster of hrp genes that are required for elicitation of the hypersensitive response (HR) in tobacco. TnphoA mutagenesis of cosmid pHIR11, which contains the hrp cluster, revealed two genes encoding exported or inner-membrane-spanning proteins (H.-C. Huang, S. W. Hutcheson, and A. Collmer, Mol. Plant-Microbe Interact. 4:469-476, 1991). The gene in complementation group X, designated hrpH, was subcloned on a 3.1-kb SalI fragment into pCPP30, a broad-host-range, mobilizable vector. The subclone restored the ability of hrpH mutant P. syringae pv. syringae 61-2089 to elicit the HR in tobacco. DNA sequence analysis of the 3.1-kb SalI fragment revealed a single open reading frame encoding an 81,956-Da preprotein with a typical amino-terminal signal peptide and no likely inner-membrane-spanning hydrophobic regions. hrpH was expressed in the presence of [35S]methionine by using the T7 RNA polymerase-promoter system and vector pT7-3 in Escherichia coli and was shown to encode a protein with an apparent molecular weight of 83,000 on sodium dodecyl sulfate-polyacrylamide gels. The HrpH protein in E. coli was located in the membrane fraction and was absent from the periplasm and cytoplasm. The HrpH protein possessed similarity with several outer membrane proteins that are known to be involved in protein or phage secretion, including the Klebsiella oxytoca PulD protein, the Yersinia enterocolitica YscC protein, and the pIV protein of filamentous coliphages. All of these proteins possess a possible secretion motif, GG(X)12VP(L/F)LXXIPXIGXL(F/L), near the carboxyl terminus, and they lack a carboxyl-terminal phenylalanine, in contrast to other outer membrane proteins with no known secretion function. These results suggest that the P. syringae pv. syringae HrpH protein is involved in the secretion of a proteinaceous HR elicitor.     

Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae.

Strains of Pseudomonas syringae pv. syringae resistant to copper, streptomycin, or both compounds were recovered from symptomless and diseased tissue of four woody hosts in three nurseries in Oklahoma. In strains resistant to copper and streptomycin (Cur Smr), resistance to both compounds was cotransferred with a single plasmid which was either 68, 190, or 220 kilobase pairs (kb). All Cus Smr strains contained a 68-kb conjugative plasmid. Cur Sms strains contained one plasmid which varied in size from 60 to 73 kb. All conjugative plasmids which transferred streptomycin resistance contained sequences homologous to the strA and strB Smr genes from the broad-host-range plasmid RSF1010. The Smr determinant was subsequently cloned from a 68-kb Cur Smr plasmid designated pPSR1. A restriction map detailing the organization of the homologous Smr genes from pPSR1 and RSF1010 and cloned Smr genes from P. syringae pv. papulans and Xanthomonas campestris pv. vesicatoria revealed the conservation of all sites studied. The Cur genes cloned from P. syringae pv. tomato PT23 and X. campestris pv. vesicatoria XV10 did not hybridize to the Cur plasmids identified in the present study, indicating that copper resistance in these P. syringae pv. syringae strains may be conferred by a distinct genetic determinant.     

Syringomicin production is stimulated by cysteine in Pseudomonas syringae pv. syringae

Abstract The influence of cysteine and serine in the production of syringomycin by Pseudomonas syringae pv. syringae has been studied. Both amino acids increased toxin synthesis in wild-type strains, although cysteine has a higher stimulatory effect than serine. To corroborate the role of cysteine in the production of syringomycin, a Cys⁻ mutant of P. syringae pv. syringae was isolated by transpositional mutagenesis with Tn5; this Cys⁻ mutant did not produce syringomycin. Nevertheless, and after the addition of high concentrations of cysteine, the cys ∷Tn5 mutant recovered its ability to produce syringomycin. On the other hand, the addition of serine did not return the production of syringomycin to the sys ∷ Tn5 strain: all these data indicated that cysteine modulates the synthesis of syringomycin in P. syringae pv. syringae positively.     

Toxins of Pseudomonas syringae pv. syringae affect H+-transport across the plasma membrane of maize

The activity of some phytotoxic metabolites of Pseudomonas syringae pv. syringae Van Hall strains B359 and B301 on in vivo and in vitro systems of H⁺-transport across the plasma membrane of maize (Zea mays L., hybrid Paolo) was investigated. In particular syringomycin, the first lipodepsinonapeptide isolated from Pss and already studied in plants and yeasts for its effects on several physiological systems, was compared with the recently described lipodepsipeptides with 22 or 25 amino acid residues, so called syringopeptins. The in vivo activity of the phytotoxins was tested on fusicoccin-stimulated H^?-extrusion from cuttings of maize roots, which was inhibited by both types of toxins, with syringomycin more efficient than the syringopeptins. In vitro the H⁺-ATPase activity of predominantly right-side-out plasma membrane vesicles purified by two-phase partitioning was stimulated by 10 μM syringomycin and inhibited by higher levels, in agreement with the results of others with preparations of dicotyledons. Also the inhibition of the phosphohydrolytic activity of inside-out vesicles of mung bean plasma membrane was confirmed for maize. In both types of vesicles the syringopeptirts were better inhibitors than syringomycin. The pH gradient formed on addition of ATP to predominantly (25% latency) inside-out vesicles was immediately and completely collapsed by syringomycin and syringopeptins; H⁺-pumping was prevented if the toxins were added before ATP. The inhibition was concentration dependent, but at very low concentrations the effect was inverted. The results of the present investigation, carried out with maize preparations, confirm and extend the evidence so far obtained with dicotyledons in favour of the plasma membrane as an important site of interaction of syringomycin with the plant cell. They also indicate that, except for some details, the effects of syringopeptins at the level of the plasma membrane are the same as those of syringomycin.     

Pectolytic enzymes produced by Pseudomonas syringae pv. glycinea

Abstract A comparative analysis of 16S ribosomal RNA sequences of all Peptostreptococcus species revealed that most members of the genus Peptostreptococcus should be divided into many different genera. The relationship between clostridia and peptostreptococci was analysed to find the phylogenetic position of peptostreptococci.     

Characterization of alginate lyase from Pseudomonas syringae pv. syringae

The gene encoding alginate lyase (algL) in Pseudomonas syringae pv. syringae was cloned, sequenced, and overexpressed in Escherichia coli. Alginate lyase activity was optimal when the pH was 7.0 and when assays were conducted at 42 degrees C in the presence of 0.2 M NaCl. In substrate specificity studies, AlgL from P. syringae showed a preference for deacetylated polymannuronic acid. Sequence alignment with other alginate lyases revealed conserved regions within AlgL likely to be important for the structure and/or function of the enzyme. Site-directed mutagenesis of histidine and tryptophan residues at positions 204 and 207, respectively, indicated that these amino acids are critical for lyase activity.     

Systemic Induction of Salicylic Acid Accumulation in Cucumber after Inoculation with Pseudomonas syringae pv syringae

Inoculation of one true leaf of cucumber (Cucumis sativus L.) plants with Pseudomonas syringae pathovar syringae results in the systemic appearance of salicylic acid in the phloem exudates from petioles above, below, and at the site of inoculation. Analysis of phloem exudates from the petioles of leaves 1 and 2 demonstrated that the earliest increases in salicylic acid occurred 8 hours after inoculation of leaf 1 in leaf 1 and 12 hours after inoculation of leaf 1 in leaf 2. Detaching leaf 1 at intervals after inoculation demonstrated that leaf 1 must remain attached for only 4 hours after inoculation to result in the systemic accumulation of salicylic acid. Because the levels of salicylic acid in phloem exudates from leaf 1 did not increase to detectable levels until at least 8 hours after inoculation with P. s. pathovar syringae, the induction of increased levels of salicylic acid throughout the plant are presumably the result of another chemical signal generated from leaf 1 within 4 hours after inoculation. Injection of salicylic acid into tissues at concentrations found in the exudates induced resistance to disease and increased peroxidase activity. Our results support a role for salicylic acid as an endogenous inducer of resistance, but our data also suggest that salicylic acid is not the primary systemic signal of induced resistance in cucumber.     

Molecular cloning of copper resistance genes from Pseudomonas syringae pv. tomato.

A cosmid library of copper-resistant (Cur) Pseudomonas syringae pv. tomato PT23 plasmid DNA was constructed and mobilized into the copper-sensitive recipient P. syringae pv. syringae PS61. One resultant cosmid clone, pCOP1 (46 kilobases), conferred copper resistance. The PT23 Cur gene(s) was located on pCOP1 by subcloning PstI restriction endonuclease fragments of pCOP1 in the broad-host-range vector pRK404. A subclone containing a 4.4-kilobase PstI fragment conferred Cur on PS61. The Cur gene(s) was further located by insertional inactivation with Tn5. A subcloned fragment internal to the Cur determinant on pCOP2 was probed to plasmid and chromosomal DNA of four copper-resistant and three copper-sensitive strains of P. syringae pv. tomato. The probe hybridized to plasmids in resistant strains, but showed no detectable homology to copper-sensitive strains.     

Induction of the copper resistance operon from Pseudomonas syringae.

Cupric sulfate induced mRNA specific to the copper resistance gene cluster previously cloned from Pseudomonas syringae pv. tomato PT23. mRNA from each of the four genes of this cluster responded in a similar manner to induction over time and with different concentrations of cupric sulfate. Promoter fusion constructs indicated the presence of a single copper-inducible promoter upstream from the first open reading frame.     

Chromosome mapping in Pseudomonas syringae pv. syringae strain PS224

A conjugation system for mapping the chromosome of Pseudomonas syringae pv. syringae PS224 has been developed using the IncP-10 plasmid R91-5; pMO22, a Tn501-loaded derivative of R91-5; and pMO75, R91-5 loaded with Tn5. Nine different donor origins were identified with R91-5 and pMO22. By insertion of Tn5 into various sites of the chromosome, an additional six donor origins were available using pMO75 as the donor plasmid. In all, 36 markers were located on three linkage groups. Many donor strains were unstable and the limited availability of stable donor strains has limited the extent to which markers have been located. This instability of donor strains is in marked contrast to the highly stable donor strains found in P. putida using the same plasmids. As in P. aeruginosa and P. putida, auxotrophic markers in P. syringae do not show the clustering of related markers found in enterobacteria.     

A new syringopeptin produced by bean strains of Pseudomonas syringae pv. syringae

Two strains (B728a and Y37) of the phytopathogenic bacterium Pseudomonas syringae pv. syringae isolated from bean (Phaseolus vulgaris) plants were shown to produce in culture both syringomycin, a lipodepsinonapeptide secreted by the majority of the strains of the bacterium, and a new form of syringopeptin, SP(22)Phv. The structure of the latter metabolite was elucidated by the combined use of mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy and chemical procedures. Comparative phytotoxic and antimicrobial assays showed that SP(22)Phv did not differ substantially from the previously characterized syringopeptin 22 (SP(22)) as far as toxicity to plants was concerned, but was less active in inhibiting the growth of the test fungi Rhodotorula pilimanae and Geotrichum candidum and of the Gram-positive bacterium Bacillus megaterium.