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.

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

By a combination of 1D and 2D 1H-and ~3C-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 lipodepsipcptide. Its amino acid sequence is Ser-Ser-Dab-Dab-Arg-Phe-Dhb-4(Cl)Thr-3(OH)Asp with the fl-carboxy group of the C-terminal residue closing a macrocyclic ring on the OH group of the N-terminal Scr, which in turn is N-acylated by 3-hydroxydodecanoic acid. Syringomycins A~ 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.

With this work we have completed the characterization of the syringomycin synthetase gene cluster. In particular, by sequencing additional 28.5 kilobase pairs we show that the nine modules involved in the binding of the nine amino acids of syringomycin are localized on SyrB and SyrE, with SyrE carrying eight modules. The recombinant SyrB and the first and second modules of SyrE (SyrE1 and SyrE2) have been expressed in Escherichia coli and purified. The biochemical data indicate that SyrB binds threonine, the putative precursor of the last amino acid of syringomycin, whereas SyrE1 and SyrE2 bind serine, the first and the second amino acids of syringomycin, respectively. On the basis of the sequence analysis and the biochemical data presented here, it appears that syringomycin synthetase is unique among peptide synthetases in that its genetic organization does not respect the "colinearity rule" according to which the order of the amino acid binding modules along the chromosome parallels the order of the amino acids on the peptide. This feature, together with the absence of a single transcription unit and the absence of epimerase-like domains make syringomycin synthetase more related to the eukaryotic peptide synthetases than to the bacterial counterparts.Most phytopathogenic strains of Pseudomonas syringae pv. syringae secrete cyclic lipodepsipeptide toxins with phytotoxic activity and a wide spectrum of antimicrobial and antifungal properties. The cyclic lipodepsinonapeptide syringomycin is a key virulence determinant of P. syringae pv. syringae strain B301D and contributes to disease symptom development (1, 2) by disrupting ion transport and the electrical potential of host plasma membranes (3). Syringomycin is composed of a polar peptide head, having the sequence Ser-D-Ser 1 -D-Dab-Dab-ArgPhe-Dhb-(3-OH)Asp-(4-Cl)Thr, linked to a hydrophobic 3-hydroxy fatty acid tail with 10, 12, or 14 carbon atoms (4).Transposon mutagenesis has revealed that insertions in a chromosomal region larger than 25 kb result in the loss of syringomycin production probably as a consequence of the inactivation of one or more large proteins proposed to be part of the syringomycin synthetase complex (1, 5).Four independently transcribed genes, syrB, syrC, syrD, and syrP, covering approximately a 7-kb region, have been sequenced and partially characterized (5-7). syrB shares strong similarities with the amino acid binding domains of peptide synthetases (5), while syrC encodes a thioesterase-like enzyme, a protein often found at one end of bacterial peptide synthetase operons (5, 8). Interestingly, syrD, transcribed in the opposite orientation with respect to syrB and syrC, codes for a protein remarkably similar to the superfamily of ATP binding cassette transporter proteins involved in target-specific secretion. It has been proposed that the syrD product might be involved in the transport of syringomycin across the cytoplasmic membrane (6). Finally, syrP (7), which is located between syrB and syrD, exhibits similarity to the phosphotransfer...

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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 ␣,␤-unsaturated amino acids and a longer ␤-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.Syringopeptins (SPs) are bacterial secondary metabolites belonging to a class of cyclic lipodepsipeptides produced by certain pathovars of the plant bacterium Pseudomonas syringae (1,2,25,36). Their peptide portions contain either 22 (SP22) or 25 (SP25) amino acids that are predominantly hydrophobic, valine and alanine in particular. About 70% of the chiral residues are of the D configuration, and there are four ␣,␤-unsaturated and two 2,4-diaminobutyric acid residues (2,17,21,30). An N-terminal residue dehydroaminobutyric acid (Dhb) is N acylated by a 3-hydroxylated fatty acid chain containing either 10 or 12 carbon atoms; these two types of chains are designated A and B homologs and are typically the more abundant and less abundant forms, respectively. The C-terminal carboxyl group is esterified by the hydroxyl group of the allo-Thr residue positioned at the distance of 7 residues, thus forming an eightmembered lactone macrocycle. So far, two SP25 and three SP22 forms have been identified. SP25 is produced by P. syringae pv. syringae strains that have been isolated from infected millet (B359), citrus (B427) (2), and wheat (M1) (1) as well as from the wheat pathogen P. syringae pv. atrofaciens (36). An isoform differing in the C-terminal residue, SP25-Phe, was detected in a laurel-infecting strain (30). SP22 is produced by a P. syringae pv. syringae strain isolated from pear (B301) (2), and variants are produced by P. syringae pv. syringae strains from sugar cane (SP[SC]) (21) and bean (SPPhv) (17). Each SP-producing P. syringae strain produces one type of SP together with a smaller, nine-amino-acid-containing cyclic lipodepsipeptide-either syringomycin (13, 32), syringotoxin (3), syringostatin (13), or pseudomycin (5).The SPs are produced in infected plant tissues (12, 15), and they play roles as virulence factors in plant diseases (31). The phytotoxic physiological effects of the SPs were demonstrated with isolated plant mitochondria (10) and tobacco protoplasts (18). Lipid bilayer studies have revealed that the probable mechanism of action involves insertion into target membranes with formation of ion channels and consequent ion imbalances that lead to cell death (9, 18). In addition to their phyto...

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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 against Rhodotorula pilimanae. The C-terminal residue was shown to be important for the activity.