Alessandro Ballio

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.

Novel bioactive lipodepsipeptides from Pseudomonas syringae: The pseudomycins

The covalent structure and most of the stereochemistry of the pseudomycins, bioactive metabolites of a transposon‐generated mutant of a Pseudomonas syringae wild‐type strain proposed for the biological control of Dutch elm disease, have been determined. While two pseudomycins are identical to the known syringopeptins 25‐A and 25‐B, pseudomycins A, B, C, C′ are new lipodepsinonapeptides. For all of these the peptide moiety corresponds to l‐Ser‐d‐Dab‐l‐Asp‐l‐Lys‐l‐Dab‐l‐aThr‐Z‐Dhb‐l‐Asp(3‐OH) ‐l‐Thr(4‐Cl) with the terminal carboxyl group closing a macrocyclic ring on the OH group of the N‐terminal Ser. This is in turn N‐acylated by 3,4‐dihydroxytetradecanoate in pseudomycin A, by 3‐hydroxytetradecanoate in pseudomycin B, by 3,4‐dihydroxyhexadecanoate in pseudomycin C, and by 3‐hydroxyhexadecanoate in pseudomycin C′. Some preliminary data on the biological activity of pseudomycin A are reported.

The interaction of lipodepsipeptide toxins from Pseudomonas syringae pv. syringae with biological and model membranes : A comparison of syringotoxin, syringomycin, and two syringopeptins

Pseudomonas syringae pv. syringae produces two groups of cyclic lipodepsipeptides (LDPs): the nona-peptides syringomycins, syringostatins, and syringotoxin (ST), and the more complex syringopeptins composed of either 22 or 25 amino acid residues (SP22 and SP25). Both classes of peptides significantly contribute to bacterial pathogenesis and their primary target of action seems to be the plasma membrane. We studied and compared the activity of some members of these two classes of LDPs on red blood cells and on model membranes (monolayers and unilamellar vesicles). All peptides induced red blood cell hemolysis. The mechanism was apparently that of a colloid-osmotic shock caused by the formation of pores, as it could be prevented by osmoticants of adequate size. Application of the Renkin equation indicated a radius of approximately 1 nm for the lesions formed by syringopeptins SP22A and SP25A, whereas those formed by syringomycin E (SRE) had a variable, dose-dependent size ranging from 0.7 up to 1.7 nm. All tested LDPs displayed surface activity, forming peptide monolayers with average molecular areas of 1.2 nm2 (SRE), 1.5 nm2 (SP22A), and 1.3 nm2 (SP25A). They also partitioned into preformed lipid monolayers occupying molecular areas that ranged from 0.6 to 1.7 nm2 depending on the peptide and the lipid composition of the film. These LDPs formed channels in lipid vesicles as indicated by the release of an entrapped fluorescent dye (calcein). The extent of permeabilization was dependent on the concentration of the peptide and the composition of the lipid vesicles, with a preference for those containing a sterol. From the dose dependence of the permeabilization it was inferred that LDPs increased membrane permeability by forming oligomeric channels containing from four to seven monomers. On average, syringopeptin oligomers were smaller than SRE and ST oligomers.

Phytotoxins and Plant Pathogenesis

In a broad sense, “phytotoxins” can be considered as microbial metabolites, other than enzymes, that damage or are harmful to plants at very low concentrations (1–3, 23, 25). Many plant pathogenic bacteria and fungi produce phytotoxins both in culture and in their hosts during the infection process. In several cases — especially if they are produced during the early stages of plant disease development — these compounds have a function in pathogenesis and cause part or even all of the symptoms of the disease (4, 5, 20). For microbial products which are not phytotoxic per se but have a role in pathogenesis, the term aggressions has been proposed (1).

Pseudomonas lipodepsipeptides and fungal cell wall-degrading enzymes act synergistically in biological control.

Pseudomonas syringae pv. syringae strain B359 secreted two main lipodepsipeptides (LDPs), syringomycin E (SRE) and syringopeptin 25A (SP25A), together with at least four types of cell wall-degrading enzymes (CWDEs). In antifungal bioassays, the purified toxins SRE and SP25A interacted synergistically with chitinolytic and glucanolytic enzymes purified from the same bacterial strain or from the biocontrol fungus Trichoderma atroviride strain P1. The synergism between LDPs and CWDEs occurred against all seven different fungal species tested and P. syringae itself, with a level dependent on the enzyme used to permeabilize the microbial cell wall. The antifungal activity of SP25A was much more increased by the CWDE action than was that of the smaller SRE, suggesting a stronger antifungal role for SP25A. In vivo biocontrol assays were performed by using P. syringae alone or in combination with T. atroviride, including a Trichoderma endochitinase knock-out mutant in place of the wild type and a chitinase-specific enzyme inhibitor. These experiments clearly indicate that the synergistic interaction LDPs-CWDEs is involved in the antagonistic mechanism of P. syringae, and they support the concept that a more effective disease control is given by the combined action of the two agents.

Corceptins, new bioactive lipodepsipeptides from cultures of Pseudomonas corrugata

© 1998 Federation of European Biochemical Societies.

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.

Structure of fuscopeptins, phytotoxic metabolites of Pseudomonas fuscovaginae

The structure of the fuscopeptins, bioactive lipodepsipeptides produced in culture by the gramineae pathogen Pseudomonas fuscovaginae, has been determined. The combined use of FAB mass spectrometry, NMR spectroscopy and chemical and enzymatic procedures allowed one to define a peptide moiety corresponding to ZDhb‐DPro‐LLeu‐DAla‐DAla‐DAla‐DAlaDVal‐Gly‐DAla‐DVal‐DAla‐DVal‐ZDhb‐DaThr‐LAla‐LDabDDab‐LPhe with the terminal carboxyl group closing a macrocyclic ring on the hydroxyl group of the allothreonine residue. The N‐terminus is in turn acylated by 3‐hydroxyoctanoate in fuscopeptin A and 3‐hydroxydecanoate in fuscopeptin B. Some preliminary data on the biological activity of fuscopeptins are also reported.