Ingeborg Grgurina

Syringopeptins, new phytotoxic lipodepsipeptides of Pseudomonas syringae pv. syringae

The primary structure of some new lipodepsipeptides named syringopeptins, produced by plant pathogenic strains of Pseudopmonas syringae pv. syringae has been determined by a combination of chemical methods, 1H and 13C NMR spectroscopy and FAB mass spectrometry. Two syringomycin‐producing strains afforded 3‐hydroxydecanoyl‐Dhb‐Pro‐Val‐Val‐Ala‐Ala‐Val‐Val‐Dhb‐Ala‐Val‐Ala‐Ala‐Dhb‐aThr‐Ser‐Ala‐Dhb‐Ala‐Dab‐Dab‐Tyr, with Tyr acylating a Thr to form a macrolactone ring, and smaller amounts of the 3‐hydroxydodecanoyl homologue. Evidence was obtained that a third syringomycin‐producing strain and a syringotoxin‐producing strain synthesize 3‐hydroxydecanoyl‐Dhb‐Pro‐Val‐Ala‐Ala‐Val‐Leu‐Ala‐Ala‐Dhb‐Val‐Dhb‐Ala‐Val‐Ala‐Ala‐Dhb‐aThr‐Ser‐Ala‐Val‐Ala‐Dab‐Dab‐Tyr, with Tyr and aThr forming again the macrolactone ring, and smaller amounts of the 3‐hydroxydodecanoyl homologue.

The structure of syringomycins A1, E and G

By a combination of 1D and 2D 1H‐ and 13C‐NMR, FAB‐MS, and chemical and enzymatic reactions carried out at the milligram level, it has been demonstrated that syringomycin E, the major phytotoxic antibiotic produced by Pseudomonas syringae pv. syringae, is a new lipodepsipeptide. Its amino acid sequence is Ser‐Ser‐Dab‐Dab‐Arg‐Phe‐Dhb‐4(Cl)Thr‐3(OH)Asp with the β‐carboxy group of the C‐terminal residue closing a macrocyclic ring on the OH group of the N‐terminal Ser, which in turn is N‐acylated by 3‐hydroxydodecanoic acid. Syringomycins A1 and G, two other metabolites of the same bacterium, differ from syringomycin E only in their fatty acid moieties corresponding, respectively, to 3‐hydroxydecanoic and 3‐hydroxytetradecanoic acid.

Phytotoxic properties of Pseudomonas syringae pv. syringae toxins

Abstract Syringomycin E, syringomycin G and syringopeptin 25A, the main components of the Pseudomonas syringae pv. syringae toxin mixture, were assayed for their phytotoxicity, determined as electrolyte leakage from carrot tissues, necrosis of tobacco leaves, and death of potato tissues, and for their antimicrobial activity on Rhodotorula pilimanae. p]In the antimicrobial assay, syringomycins were 30 times more active than syringopeptin 25A, but, in the electrolyte leakage assay, they proved to be 40 times less active than the former since a significant effect required concentrations of 16 and 0·4 μ m respectively. A statistically identical effect on electrolyte leakage was obtained with syringopeptin 25A or with an unfractionated toxin mixture equimolar for syringopeptin 25A. A similar pattern of activity was observed in the tobacco leaf and potato disc assays. After a short incubation at pH 10, syringomycins completely lost their antimicrobial and phytotoxic activities, while syringopeptin 25A retained all its antimicrobial activity and most of its phytotoxicity. These findings indicate that syringopeptin 25A and syringomycins are mainly responsible for, respectively, the phytotoxic activity and the antimicrobial activity on R. pilimanae, characteristic of the unfractionated toxin mixture of P. s. pv. syringae. The high phytotoxicity of syringopeptin 25A is a new finding which prompts a careful examination of the role played by the individual metabolites in the disease caused by P. s. pv. syringae ecotypes.

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.

Multiple forms of syringomycin

Abstract Preparations of syringomycin purified from three isolates of Pseudomonas syringae pv. syringae according to published procedures have been shown to contain a group of structurally related peptides which can be resolved by HPLC on a reverse phase column. In the acid hydrolysate of all components serine, phenylalanine, 2,4-diaminobutyric acid and arginine in the ratio 2:1:2:1 have been found. These products account for nearly 50% of the molecular weights determined by FAB mass spectrometry. Most of the antibiotic activity of the unfractionated preparations is recovered in a limited number of peaks.

Relevance of chlorine-substituent for the antifungal activity of syringomycin and syringotoxin, metabolites of the phytopathogenic bacteriumPseudomonas syringae pv.syringae

Structural analogues of syringomycin and syringotoxin were produced by fermentation, characterized by FAB-MS and amino acid analysis and compared to the parent compounds in the antibiosis test againstRhodotorula pilimanae. The C-terminal residue was shown to be important for the activity.

Novel Cyclic Lipodepsipeptide from Pseudomonas syringae pv. lachrymans Strain 508 and Syringopeptin Antimicrobial Activities

ABSTRACT The syringopeptins are a group of antimicrobial cyclic lipodepsipeptides produced by several plant-associated pseudomonads. A novel syringopeptin, SP508, was shown to be produced as two homologs (A and B) by Pseudomonas syringae pv. lachrymans strain 508 from apple and to structurally resemble syringopeptin SP22. SP508 differed from SP22 and other syringopeptins by having three instead of four α,β-unsaturated amino acids and a longer β-hydroxy acyl chain. Both SP508 and SP22 displayed growth-inhibitory activities against Mycobacterium smegmatis, other gram-positive bacteria, and yeasts but not against gram-negative bacteria. Structure-activity analyses of the SP508 and SP22 homologs indicated chemical structural features that lead to enhanced antimycobacterial activity by these pseudomonad cyclic lipodepsipeptides.

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.

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.

Coordinate Transcription and Physical Linkage of Domains in Surfactin Synthetase Are Not Essential for Proper Assembly and Activity of the Multienzyme Complex

Bacterial peptide synthetases have two common features that appear to be strictly conserved. 1) The enzyme subunits are co-regulated at both transcriptional and translational level. 2) The organization of the different enzymatic domains constituting the enzyme fulfills the “colinearity rule” according to which the order of the domains along the chromosome parallels their functional hierarchy. Considering the high degree of conservation of these features, one would expect that mutations such as transcription uncoupling and domain dissociations, deletions, duplications, and reshuffling would result in profound effects on the quality and quantity of synthesized peptides. To start testing this hypothesis, we designed two mutants. In one mutant, the operon structure of surfactin synthetase was destroyed, thus altering the concerted expression of the enzyme subunits. In the other mutant, the thioesterase domain naturally fused to the last amino acid binding domain of surfactin was physically dissociated and independently expressed. When the lipopeptides secreted by the mutant Bacillus subtilis strains were purified and characterized, they appeared to be expressed approximately at the same level of the wild type surfactin and to be identical to it, indicating that specific domain-domain interactions rather than coordinated transcription and translation play the major role in determining the correct assembly and activity of peptide synthetases.