Identification of the Biosynthetic Gene Cluster for 3-Methylarginine, a Toxin Produced by Pseudomonas syringae pv. syringae 22d/93

Abstract

The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (K m , 7 mM; k cat , 85 min ؊1 ). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.Microbial plant pathogens cause severe losses in agriculture each year (1). For example, the plant pathogen Pseudomonas syringae pv. glycinea is responsible for bacterial blight of soybean, a leaf spot disease of great economic impact. Besides chemical treatment, biocontrol agents that antagonize microbial plant pathogens are gaining increasing importance in fighting plant diseases (6,11,27). In a screening for possible biocontrol strains, an epiphytic bacterium showing a strong and selective activity against the pathogen P. syringae pv. glycinea was isolated from soybean leaves (29). The strain was characterized as Pseudomonas syringae pv. syringae 22d/93 (Pss22d). The antagonism of Pss22d against P. syringae pv. glycinea has been demonstrated successfully in vitro and in planta under greenhouse and field conditions (19,29). In order to identify the molecular basis of the antagonism of Pss22d against P. syringae pv. glycinea, we focused on its secondary metabolites. Besides the well-known lipodepsipeptides syringomycin and syringopeptin (3), Pss22d produces the rare amino acid 3-methylarginine (MeArg) (5). As little as 20 nmol of MeArg strongly and selectively inhibits P. syringae pv. glycinea but no other pseudomonads in vitro (29). Since the inhibition can be compensated for by L-arginine supplementation but not by any other essential amino acid, it is likely that the toxin acts as an inhibitor of the arginine biosynthesis pathway or an argininedependent pathway, such as nitric oxide formation (13,16). Feeding experiments and Tn5 transposon mutagenesis suggested that MeArg is produced by an S-adenosyl methionine (SAM)-dependent methyltransferase (5) converting the enol of 5-guanidino-2-oxo-pentanoic acid to 5-guanidino-3-methyl-2-oxo-pentanoic acid. An analogous reaction is known to occur with the methyltransferases GlmT, DptI, and LptI, which form 3-methylglutamate from ␣-ketoglutarate (18). On the way to MeArg, only a transaminase catalyzing the formation of MeArg from 5-guanidino-3-methyl-2-oxo-pentano...

The Antimicrobial Compound Xantholysin Defines a New Group of Pseudomonas Cyclic Lipopeptides

“…While xantholysin does not exert a growth-inhibitory effect on fellow pseudomonads, P. putida BW11M1 also produces a plant lectin-like bacteriocin to target other pseudomonads, including members of the P. syringae group [35], [63], indicating that both ribosomally and non-ribosomally synthesized antimicrobials are part of its armory deployed against competitors. Antagonism among plant-associated γ-proteobacterial relatives has been described for some other types of pseudomonad secondary metabolites: the modified amino acid 3-methylarginine causing inhibition between P. syringae pathovars [64]; the salicylate-containing P. putida antibiotic promysalin, targeting other pseudomonads [65]; oxyvinylglycines active against Erwinia amylovora [66], [67]. In view of the structural similarity of xantholysins to certain pseudomonad lipopeptides with antiviral activity (viz.…”

Biosynthesis of the β-Methylarginine Residue of Peptidyl Nucleoside Arginomycin in Streptomyces arginensis NRRL 15941

“…A biosynthesis study demonstrated that three clustered genes, mrsA, mrsB, and mrsC, can produce ␤-methylarginine in Escherichia coli. A catalytic assay of MrsA, a methyltransferase, can convert 5-guanidino-2-oxo-pentanoic acid into 5-guanidino-3-methyl-2-oxo-pentanoic acid in vitro (8). The resulting product was proposed as a promising substrate for MrsB, an aminotransferase, to give the final product ␤-methylarginine, which was discharged from the cell by the exporter MrsC.…”

Aminotenuazonic Acid: Isolation, Structure Elucidation, Total Synthesis and Herbicidal Activity of a New Tetramic Acid from Fruiting Bodies of Laccaria Species

“…Structure of (5S,6S)-aminotenuazonic acid (1a)a nd (5S,6S)-tenuazonic acid (2). Only the predominant tautomeric form in neutral solution is depicted. the [M+ +H] + ion at m/z 213.12364, which are both in agreement with the molecular formula C 10 H 16 O 3 N 2 for 1a.T he presence of ten carbonsi nt he 13 CNMR spectrum confirms the molecular formula, whereas the 1 HNMR spectrum recorded at 300 Ki nD 2 Oe xhibits twelve nonexchangeable protons in total. Consequently, 1a contains four exchangeable protons.…”

“…(2S,3S)-3-Methylornithine (4)m ight be derived from l-ornithine (3)b yt ransamination, yieldingt he corresponding 2-oxo acid, followed by methylation with S-adenosylmethionine (SAM) and subsequent transamination to compound 4 analogously to the biosynthesis of 3-methylarginine in Pseudomonas syringae. [13] (2S,3S)-3-Methylornithine (4) might react with activateda cetoacetatet ot he intermediate Nacetoacetyl-(2S,3S)-3-methylornithine (5). Subsequentc yclisation would finally yield compound 1a (Scheme 1).…”

“…Melting points were measured with am elting point apparatus 530 from Büchi. Optical rotation values were measured on aP erkinElmer 243 polarimeter.N MR spectra were recorded with a Bruker AvanceD RX-600 spectrometer equipped with aT XI cryoprobe ( 1 Ha t6 00.22 MHz, 13 Ca t1 50.93 MHz), Bruker Avance III spectrometer equipped with aT CI cryo-probe ( 1 Ha t5 00.30 MHz, 13 Ca t1 25.81 MHz). 1 Hc hemical shifts are reported in ppm using the solvent as an internal standard (CDCl 3 at 7.26 ppm and D 2 Oa t 4.75 ppm).…”