The contribution of syringopeptin and syringomycin to virulence of Pseudomonas syringae pv. syringae strain B301D on the basis of sypA and syrB1 biosynthesis mutant analysis

Sequencing of an approximately 3.9-kb fragment downstream of the syrD gene of Pseudomonas syringae pv. syringae strain B301D revealed that this region, designated sypA, codes for a peptide synthetase, a multifunctional enzyme involved in the thiotemplate mechanism of peptide biosynthesis. The translated protein sequence encompasses a complete amino acid activation module containing the conserved domains characteristic of peptide synthetases. Analysis of the substrate specificity region of this module indicates that it incorporates 2,3-dehydroaminobutyric acid into the syringopeptin peptide structure. Bioassay and high performance liquid chromatography data confirmed that disruption of the sypA gene in strain B301D resulted in the loss of syringopeptin production. The contribution of syringopeptin and syringomycin to the virulence of P. syringae pv. syringae strain B301D was examined in immature sweet cherry with sypA and syrB1 synthetase mutants defective in the production of the two toxins, respectively. Syringopeptin (sypA) and syringomycin (syrB1) mutants were reduced in virulence 59 and 26%, respectively, compared with the parental strain in cherry, whereas the syringopeptin-syringomycin double mutant was reduced 76% in virulence. These data demonstrate that syringopeptin and syringomycin are major virulence determinants of P. syringae pv. syringae.     

Structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae

The covalent structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae isolates, pathogenic on various species of citrus trees, has been deduced from 1D and 2D 1H- and 13C-NMR spectra combined with extensive FAB-MS data and results of some chemical reactions. Similarly to syringomicins and syringostatins, produced by other plant pathogenic strains of P. syringae pv. syringae, syringotoxin is a lipodepsinonapeptide. Its peptide moiety corresponds to Ser-Dab-Gly-Hse-Orn-aThr-Dhb-(3-OH)Asp-(4-Cl)Thr with the terminal carboxy group closing a macrocyclic ring on the OH group of the N-terminal Ser, which in turn is N-acetylated by 3-hydroxytetradecanoic acid.     

Characterization of the salA, syrF, and syrG regulatory genes located at the right border of the syringomycin gene cluster of Pseudomonas syringae pv. syringae

Sequence analysis of the right border of the syr gene cluster of Pseudomonas syringae pv. syringae strain B301D revealed the presence of the salA gene 8,113 bp downstream of syrE. The predicted SalA protein of strain B301D differs by one amino acid from that of strain B728a. Two homologs of salA, designated syrF and syrG, were identified between syrE and salA. All three proteins contain helix-turn-helix DNA-binding motifs at their C termini and exhibit homology to regulatory proteins of the LuxR family. A salA mutant failed to produce syringomycin, whereas syrF and syrG mutants produced 12 and 50%, respectively, of syringomycin relative to the wild-type strain. The salA, syrF, and syrG mutants were significantly reduced in virulence, forming small, nonspreading lesions in immature cherry fruits. Translational fusions to the uidA gene were constructed to evaluate expression of syrB1 in regulatory mutant backgrounds and to determine the relationship among the three regulatory loci. Expression of a syrB1::uidA fusion required functional salA and syrF genes and, in series, the expression of a syrF::uidA fusion required a functional salA gene. These results demonstrate that salA is located upstream of syrF in the regulatory hierarchy controlling syringomycin production and virulence in P. syringae pv. syringae.     

Requirement of sphingolipid alpha-hydroxylation for fungicidal action of syringomycin E

Syringomycin E is an antifungal cyclic lipodepsinonapeptide produced by Pseudomonas syringae pv. syringae. To understand the mechanism of action of syringomycin E, a novel resistant Saccharomyces cerevisiae strain, BW7, was isolated and characterized. Lipid analyses revealed that BW7 contained only the hydrophobic subspecies of sphingolipids that are normally minor components in wild type strains. This aberrant sphingolipid composition was the result of lack of alpha-hydroxylation of the amide-linked very long chain fatty acids, suggesting a defective sphingolipid alpha-hydroxylase encoded by the FAH1 gene. A yeast strain that lacks the FAH1 gene was resistant to syringomycin E, and failed to complement BW7. These results demonstrate that BW7 carries a mutation in the FAH1 gene, and that the lack of alpha-hydroxylated very long chain fatty acids in yeast sphingolipids confers resistance to syringomycin E.     

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.     

Role of ergosterol in growth inhibition of Saccharomyces cerevisiae by syringomycin E

The antifungal activity of the lipodepsipeptide syringomycin E from Pseudomonas syringae pv. syringae is modulated by sterols. To study the requirement of the predominant fungal sterol, ergosterol, in syringomycin E action, the sterol composition of Saccharomyces cerevisiae sterol auxotroph strain FY-14 was modified and sensitivity to syringomycin E examined. Cells containing solely ergosterol, cholesterol, β-sitosterol or stigmasterol were sensitive to syringomycin E with the latter two being the most sensitive. Cells containing growth-promoting cholesterol were the most sensitive and those with growth-promoting ergosterol the least sensitive. It is concluded that sensitivity to syringomycin E is modulated by growth-promoting sterols and does not necessarily require ergosterol.     

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.     

Phytotoxin production by Pseudomonas syringae pv. syringae: Syringopeptin production by syr mutants defective in biosynthesis or secretion of syringomycin

Abstract Syringomycin and syringopeptin are lipodepsipeptide phytotoxins produced by Pseudomonas syringae pv. syringae . Four syr genes were identified previously and hypothesized to be involved in the regulation ( syrA ), biosynthesis ( syrB and syrC ), or export ( syrD ) of syringomycin. This study determines the influence of syr mutations on the composition of phytotoxic metabolites produced by P. syringae pv. syringae strain B301D-R. Levels of syringomycin and syringopeptin produced in liquid cultures were estimated by reverse phase HPLC analyses and differential antimicrobial assays. Significant quantities of syringopeptin were produced by both syrB and syrC mutants despite their inability to produce syringomycin. Only trace quantities of both lipodepsipeptides were produced by syrA and syrD mutants of P. syringae pv. syringae . These results indicate that syringomycin and syringopeptin are synthesized by separate pathways, but may share common mechanisms for secretion and regulation.     

Analysis of the syrP gene, which regulates syringomycin synthesis by Pseudomonas syringae pv. syringae.

Syringomycin is a lipodepsinonapeptide phytotoxin synthesized by Pseudomonas syringae pv. syringae on multienzymatic peptide synthetases. Sequence analysis of the interval between the syrB and syrD genes of P. syringae pv. syringae strain B301D revealed a 1,059-bp open reading frame (ORF), designated syrP. The predicted product of this ORF was a 39.6-kDa protein consisting of 353 amino acid residues. Searches of protein sequence databases demonstrated that SyrP was most similar to histidine kinases such as the CheA regulatory protein of Escherichia coli. The predicted SyrP sequence was aligned with the N terminus of CheA, a region corresponding to the phosphotransfer and acceptor domains of CheA. The SyrP region that aligns with the phosphotransfer domain of CheA contained a His at position 101 which is flanked by a weak consensus sequence of the unorthodox sensory kinase subfamily of two-component regulatory systems. Strain B301D-31, obtained by site-directed insertional mutagenesis of the syrP gene, exhibited an unusual pleiotropic phenotype including a failure to produce syringomycin in liquid media in contrast to production of elevated levels of the toxin on agar media. The syrP mutant was relieved of the suppression of toxin production that accompanies inorganic phosphate concentrations of > 1 mM on agar media. Nevertheless, the syrP mutant was substantially less virulent than the wild-type strain in pathogenicity assays in cherry fruits. These results suggest that the syrP gene encodes a regulatory protein that participates in a phosphorylation cascade controlling syringomycin production and virulence in P. syringae pv. syringae.     

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.     

beta-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.     

Physical and functional analyses of the syrA and syrB genes involved in syringomycin production by Pseudomonas syringae pv. syringae.

The syrA and syrB genes involved in syringomycin production in Pseudomonas syringae pv. syringae B301D were identified from an EcoRI-pLAFR3 cosmid library and then physically and functionally analyzed in relation to plant pathogenicity. Homologous recombination of the genes required for syringomycin production from cosmids pGX183 (syrA) and pGX56 (syrB), respectively, introduced into nontoxigenic (Tox-) Tn5 mutants W4S2545 and W4S770 resulted in the concomitant restoration of toxin production and full virulence. The disease indices of the Tox+ strains obtained by recombination of the cloned, homologous DNA into the corresponding Tn5 mutant were essentially equivalent to that of strain B301D-R and significantly higher than those of W4S2545 and W4S770. A 12-kilobase (kb) EcoRI fragment from pGX183 was subcloned (i.e., pGX15) and found to contain the sequences necessary for syringomycin production. A map of pGX15 prepared by a combination of restriction endonuclease digestions and Tn5 mutagenesis showed that the syrA sequence was 2.3 to 2.8 kb. Marker exchange of syrA::Tn5 from pGX15 into B301D-R yielded nonpathogenic phenotypes, indicating that syrA is a regulatory gene since it is necessary for both syringomycin production and pathogenicity. The 4.9-kb EcoRI fragment from pGX56 was subcloned (i.e., pGX4) and shown to carry the syrB sequence which was 2.4 to 3.3 kb. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of protein extracts from B301D-R associated five proteins, ranging from approximately 130,000 to approximately 470,000 in molecular weight, with syringomycin production. The syrA and syrB genes were required for the formation of proteins SR4 (approximately 350,000) and SR5 (approximately 130,000), which are believed to be components of the syringomycin synthetase complex.     

Regulation of syringomycin synthesis in Pseudomonas syringae pv. syringae and defined conditions for its production

Production of the phytotoxin, syringomycin (SR), by Pseudomonas syringae pv. syringae strain B301D was regulated by both iron and inorganic phosphate similar to that of many bacterial secondary metabolites. Iron concentrations of 2 µmol/1 or more in deferrated potato-dextrose broth (PDB) resulted in the production of 1024 SR units/ml, a yield comparable to that produced in non-deferrated PDB. Moreover, production of one SR unit required approximately 0†4 ng of available FeCl₃. No SR was produced by strain B301D in deferrated PDB despite growth nearly identical with that of B301D in deferrated PDB supplemented with 10 µmol/1 FeCl₃. Furthermore, a phosphate concentration of 1 mmol/1 or more was suppressive to SR production. Of the amino acids tested, L-histidine at a concentration of ca 20 mmol/1 was the most effective nitrogen source for SR synthesis under defined conditions. Based on these observations, a synthetic medium, SR minimal, was formulated for SR or syringotoxin production by representative strains of Ps. syringae pv. syringae. The regulation of phytotoxin production is discussed in relation to pathogen survival and virulence.     

Contribution of the Regulatory Gene lemA to Field Fitness of Pseudomonas syringae pv. syringae

In Pseudomonas syringae pv. syringae, lemA is required for brown spot lesion formation on snap bean and for production of syringomycin and extracellular proteases (E. M. Hrabak and D. K. Willis, J. Bacteriol. 174: 3011-3022, 1992; E. M. Hrabak and D. K. Willis, Mol. Plant-Microbe Interact. 6:368-375, 1993; D. K. Willis, E. M. Hrabak, J. J. Rich, T. M. Barta, S. E. Lindow, and N. J. Panopoulos, Mol. Plant-Microbe Interact. 3:149-156, 1990). The lemA mutant NPS3136 (lemA1::Tn5) was previously found to be indistinguishable from its pathogenic parent B728a in its ability to grow when infiltrated into bean leaves of plants maintained under controlled environmental conditions (Willis et al., Mol. Plant-Microbe Interact. 3:149-156, 1990). We compared population sizes of NPS3136 and B728aN (a Nal(supr) clone of wild-type B728a) in two field experiments to determine the effect of inactivation of lemA on the fitness of P. syringae pv. syringae. In one experiment, the bacterial strains were spray inoculated onto the foliage of 25-day-old bean plants. In the other, seeds were inoculated at the time of planting. In both experiments, the strains were inoculated individually and coinoculated in a 1:1 ratio. NPS3136 and B728aN achieved similar large population sizes on germinating seeds. However, in association with leaves, population sizes of NPS3136 were diminished relative to those of B728aN in both experiments. Thus, lemA contributed significantly to the fitness of P. syringae pv. syringae in association with bean leaves but not on germinating seeds under field conditions. When NPS3136 was coinoculated with B728aN, the mutant behaved as it did when inoculated alone. However, population sizes of B728aN in the coinoculation treatment were much lower than those when it was inoculated alone. Inactivation of the lemA gene appeared to have rendered the mutant suppressive to B728aN.     

A physical map of the syringomycin and syringopeptin gene clusters localized to an approximately 145-kb DNA region of Pseudomonas syringae pv. syringae strain B301D.

Genetic and phenotypic mapping of an approximately 145-kb DraI fragment of Pseudomonas syringae pv. syringae strain B301D determined that the syringomycin (syr) and syringopeptin (syp) gene clusters are localized to this fragment. The syr and syp gene clusters encompass approximately 55 kb and approximately 80 kb, respectively. Both phytotoxins are synthesized by a thiotemplate mechanism of biosynthesis, requiring large multienzymatic proteins called peptide synthetases. Genes encoding peptide synthetases were identified within the syr and syp gene clusters, accounting for 90% of the DraI fragment. In addition, genes encoding regulatory and secretion proteins were localized to the DraI fragment. In particular, the salA gene, encoding a regulatory element responsible for syringomycin production and lesion formation in P. syringae pv. syringae strain B728a, was localized to the syr gene cluster. A putative ATP-binding cassette (ABC) transporter homolog was determined to be physically located in the syp gene cluster, but phenotypically affects production of both phytotoxins. Preliminary size estimates of the syr and syp gene clusters indicate that they represent two of the largest nonribosomal peptide synthetase gene clusters. Together, the syr and syp gene clusters encompass approximately 135 kb of DNA and may represent a genomic island in P. syringae pv. syringae that contributes to virulence in plant hosts.     

Biosynthetic origin of syringomycin and syringopeptin 22, toxic secondary metabolites of the phytopathogenic bacterium Pseudomonas syringae pv. syringae

The biosynthesis of syringomycin (SR) and syringopeptin 22 (SP22), bioactive lipodepsipeptides of the phytopathogenic bacterium Pseudomonas syringae pv. syringae, was studied by feeding (14)C-labeled precursors to chloramphenicol-containing bacterial suspensions. The preferential sites of incorporation were determined by comparing the specific activities of the intact radiolabeled metabolites and their single structural elements, obtained by hydrolytic degradation followed by derivatization and isolation by high performance liquid chromatography. The results show that, upon feeding L-[(14)C(U)]-Thr, 35.0 and 31.0% of the SR radioactivity is retained in 2,3-dehydro-2-aminobutyric acid (Dhb) and 4-chlorothreonine (Thr(4-Cl)), respectively. L-[(14)C(U)]-Asp labels the same sites, though less efficiently, and is also incorporated in 2,4-diaminobutyric acid (Dab) and 3-hydroxyaspartic acid (Asp(3-OH)). Dhb is also labeled by Thr and Asp in SP22. These are the first data on the biosynthetic origin of the modified residues in P. syringae lipopeptides.     

Mechanism of action of Pseudomonas syringae phytotoxin, syringomycin. Interaction with the plasma membrane of wild-type and respiratory-deficient strains of Saccharomyces cerevisiae

The effects of the phytotoxin, syringomycin, produced by Pseudomonas syringae pv. syringae, were examined on cells of a wild-type and a respiratory-deficient (rho0) mutant of Saccharomyces cerevisiae. The growth of both strains in liquid culture was inhibited by 0.5 micrograms syringomycin per ml and higher. Uptake rates of tetraphenylphosphonium and dimethyloxazolidine ions in cell suspensions of both strains increased when 1.5 micrograms per ml syringomycin was added. These responses were kinetically and quantitatively similar in the two strains and indicated increases in electrical potential (cell interior negative) and pH differences (cell interior alkaline) across the plasma membrane. Glucose (0.1 M) enhanced the effect on the electrical potential, was required for the pH changes, and increased the cellular ATP levels. These results show that the effects of syringomycin are energy-dependent and are due to alterations of plasma membrane and not to mitochondrial function.