Letter pubs.acs.org/OrgLett
Ralstonins A and B, Lipopeptides with Chlamydospore-Inducing and Phytotoxic Activities from the Plant Pathogen Ralstonia solanacearum Yuta Murai,† Shoko Mori,‡ Hiroyuki Konno,§ Yasufumi Hikichi,∥ and Kenji Kai*,† †
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan ‡ Bioorganic Research Institute, Suntory Foundation for Life Sciences, 8-1-1 Seikadai, Seika-cho, Soraku-gun, Kyoto 619-0284, Japan § Graduate School of Science and Technology, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992-8510, Japan ∥ Laboratory of Plant Pathology and Biotechnology, Kochi University, 200 Otsu, Monobe, Nanko-ku, Kochi 783-8502, Japan S Supporting Information *
ABSTRACT: Ralstonia solanacearum has an orphan hybrid polyketide synthasenonribosomal peptide synthetase gene cluster. We herein isolate its products (named ralstonins A and B) from R. solanacearum and elucidate their structures and biological activities. Ralstonins are unusual lipodepsipeptides composed of 11 amino acids (containing unique amino acids such as β-hydroxytyrosine and dehydroalanine) and a 3-amino-2-hydroxyoctadecanoic acid, and their production is controlled by quorum sensing, a mechanism of bacterial cell−cell communication. Ralstonins exhibited chlamydospore-inducing activity and phytotoxicity.
Ralstonia solanacearum is a β-proteobacterium that causes a lethal disease, called “bacterial wilt”, in more than 200 plant species in tropical, subtropical, and warm temperature regions of the world.1 When this pathogen invades host vascular tissues, it grows vigorously and produces extracellular polysaccharide (EPS). The accumulation of EPS prevents water flow in vessels, eventually causing severe wilting symptoms in infected plants.1 The expression of EPS biosynthetic genes is controlled by PhcA, a LysR-type transcriptional regulator, the activity of which is regulated by quorum sensing (QS), a cell−cell communication mechanism mediated by diffusible signaling molecules.1 We recently reported that the strains of R. solanacearum employ (R)-methyl 3-hydroxymyristate (3-OH MAME) or (R)-methyl 3-hydroxypalmitate (3-OH PAME) as their QS signals.2 The production of secondary metabolites is also controlled by the QS system. For example, ralfuranones, which were elucidated by comparisons of metabolic profiles between wild-type and QS mutants, contribute to virulence by possibly acting as secondary intercellular signals.3 Therefore, the identification of new QS-controlled metabolites will provide insights into the mechanisms responsible for the virulence of R. solanacearum. In order to discover the as yet unidentified secondary metabolites of R. solanacearum, we conducted a transcriptome analysis between strain OE1-1, a R. solanacearum isolate in Japan, and its QS mutants.3c We found that the expression of the hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) genes, Rsp0641 and Rsp0642, is under the control of QS (Figure 1A). These genes were initially detected © XXXX American Chemical Society
Figure 1. (A) Schematic presentation of Rsp0641 and Rsp0642 genes of R. solanacearum strain OE1-1 and their predicted domain architectures. FAAL, fatty acyl-AMP ligase domain; PKS, polyketide synthase domain; AT, aminotransferase domain; MO, monooxygenase domain; A, adenylation domain; TE, thioesterase domain; gray circles, phosphopantetheine attachment site; black circles, condensation domains. (B) Structures of ralstonins A (1) and B (2).
Received: June 6, 2017
A
DOI: 10.1021/acs.orglett.7b01685 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters in the genome of R. solanacearum strain GMI1000 and expected to produce a derivative of syringomycins, phytotoxic lipopeptides of Pseudomonas syringae.4 Lipopeptides are a class of microbial metabolites composed of fatty acid and peptide moieties and exhibit various biological activities. However, the details of this QS-controlled metabolite in R. solanacearum have remained unclear for more than a decade. Keller et al. recently reported that those genes (named rmyAB) are responsible for the production of a lipopeptide showing an [M + H]+ at m/z 1291.7.5 Although they named the compound ralsolamycin, the suffix “mycin” is typically limited for application to products derived from actinomycetes. Thus, we renamed this lipopeptide as ralstonin. Moreover, Keller et al. found that R. solanacearum enters fungal hyphae and induces chlamydospores in fungi. A mutant strain wherein the rmyA gene is inactive resulted in the complete loss of chlamydospore development in fungi and a significant reduction in bacterial invasion into fungal hyphae. Thus, ralstonin may be a key factor that controls the endofungal stage of R. solanacearum. The endosymbiosis of bacteria in fungi has been attracting increasing attention.6 Interactions between R. solanacearum and soil fungi may represent a model system to investigate how bacteria enter fungal tissues. However, ralstonin has not yet been isolated, and thus, its structure and biological activity remain elusive. Furthermore, although bacterial lipopeptides such as syringomycins are known to act as phytotoxins in host plants,4b it currently remains unclear whether ralstonin contributes to the virulence of R. solanacearum. We herein report, for the first time, the isolation and structural elucidation of ralstonins A (1) and B (2) produced by R. solanacearum strain OE1-1 (Figure 1B). Using isolated ralstonins, we evaluated their activities against fungi and plants. Furthermore, the biosynthesis of ralstonins was discussed based on the chemical structures of ralstonins and modules of RmyAB enzymes. In a previous study, ralstonin was detected by MALDI-TOF imaging MS from a colony of R. solanacearum strain GMI1000, but not from strain K60.5 In order to confirm the production of ralstonin by strain OE1-1, we analyzed its culture extract using LC/MS. A peak showing the same retention time and ESI-MS data as those of ralstonin from strain GMI1000 was detected, indicating that strain OE1-1 also produces ralstonin (Figure S1). Furthermore, we detected an additional peak showing m/z 1306 [M + H]+, 14 Da higher than that of ralstonin. The compound was also found in the culture extract of strain GMI1000. We expected these two peaks to be ralstonin and its derivative and thus named the first peak as ralstonin A (1) and second as ralstonin B (2). In order to isolate ralstonins, we prepared 1200 BG agar plates in which strain OE1-1 was grown for 3 days and then extracted cells and media with acetone. After the evaporation of acetone, the aqueous concentrate was extracted with EtOAc. Ralstonins were only detected in the EtOAc extract by LC/MS. Thus, the EtOAc extract was separated on a silica gel column by eluting with mixtures of n-hexane/EtOAc and thereafter EtOAc/MeOH, and ralstonins A (1) and B (2) were eluted in 60% and 100% MeOH in EtOAc fractions. These fractions were combined and subjected to preparative reversed-phase HPLC with an ODS column, giving almost pure ralstonins. Final purification by reversed-phase HPLC with a naphtylethyl column afforded ralstonins A (1) and B (2). Ralstonin A (1) was obtained as a colorless amorphous solid. Its molecular formula was deduced as C60H98N12O19 by HRESIMS with m/z 1291.7158 [M + H]+, indicating the
presence of 18 degrees of unsaturation in the molecule. The H NMR spectrum (in CD3OH) showed the occurrence of 11 amide protons, one amino proton, one oxymethine proton, ten α-methine/methylene protons, two exo-methylene protons, para-substituted benzene protons, and overlapping protons (Table S1). The 13C NMR spectrum consistently showed 12 carbonyl carbons, two olefin carbons, aromatic carbons, one oxymethine carbon, ten α-methine/methylene carbons, and many aliphatic methylenes (Table S1). These results suggested the structure of a lipopeptide composed of 11 amino acids, such as dehydroalanine (Dha) and β-hydroxytyrosine (β-OH-Tyr, 3), and a β-amino fatty acid. Detailed analyses of DQF-COSY, TOCSY, HSQC, and HMBC spectra revealed the presence of two Gly, Ala, β-Ala, Dha, Val, two homoserine (Hse), Ser, Thr, and β-OH-Tyr, (Figure 2). High-resolution TOCSY and 1
Figure 2. Key COSY/TOCSY, HMBC, and ROESY correlations of ralstonin A (1).
HMBC spectra (in CD3OH and in CD3OD) indicated the presence of an unbranched 3-amino-2-hydroxyfatty acid moiety (Tables S1 and S2). Taking into account the molecular formula, the presence of 3-amino-2-hydroxyoctadecanoic acid (Ahod) in this peptide could be suggested. The connectivity of the 12 partial structures (two Gly, Ala, β-Ala, Dha, Val, two Hse, Ser, Thr, β-OH-Tyr, Ahod) was established by an analysis of HMBC and ROESY correlations (Figure 2). The HMBC correlation from the amide proton of Thr to the carbonyl carbon belonging to Ahod established connectivity between Ahod and Thr. Similarly, HMBC correlations from the amide protons to the neighboring carbonyl carbons revealed their sequence as Ahod-Thr-(β-OH-Tyr)-SerHse-Gly-Hse′-Val-Dha-Gly′-Ala-(β-Ala). The ROESY correlations observed also supported this linkage. The HMBC correlation of the H-4 of Hse′ to the carbonyl carbon of β-Ala secured the ring closure, satisfying the 18 degrees of unsaturation deduced from the molecular formula. The absolute configurations of the stereogenic centers of the amino acids in ralstonin A (1) were elucidated by Marfey’s method with L-FDAA (1-fluoro-2,4-dinitrophenyl-5-L-alaninamide). After the acid hydrolysis of 1, the free amino acids in the hydrolysate were derivatized with L-FDAA. An HPLC analysis of the derivatives revealed the presence of L-Ala, L-Val, L- and D-Hse, L-Ser, and L-Thr (Figure S2). The coupling constant between the C-2 and -3 methine protons of the β-OH-Tyr (3) residue in 1 was 3.9 Hz. In previous studies, this value was between 3.5 and 4.8 Hz for the anti-configuration, while it was between 4.6 and 6.6 Hz for the syn-configuration.7 Thus, the relative configuration of β-OH-Tyr (3) in 1 was assigned as anti. However, since β-OH-Tyr (3) is decomposed during B
DOI: 10.1021/acs.orglett.7b01685 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
showed an identical retention time to synthetic anti-5 (Figure S3). Thus, we prepared (2S,3S)- and (2R,3R)-5, derivatized them with L-FDAA, and compared the retention times of their derivatives with that of the L-FDAA derivative of natural 5 from ralstonin A (1). Natural 5 showed an identical retention time with synthetic (2S,3S)-5 (Figure 3C), and thus, the absolute configuration of natural 5 was assigned as 2S,3S. Ralstonin B (2) was also obtained as a colorless amorphous solid. Its molecular formula was elucidated as C61H100N12O19 by HRESIMS with m/z 1305.7304 [M + H]+, which differed from 1 only by a CH2 group. The 1H NMR shifts for 2 were similar to those for 1, except for the proton signals of L-allo-Ile (Tables S3 and S4). A complete analysis of 1D and 2D NMR data showed that the ten other amino acids and Ahod were identical in 1 and 2. In addition, HMBC and ROESY confirmed that the sequence was identical (Figure S4). The absolute configurations of the amino acid residues in 2 were examined by Marfey’s method combined with acid hydrolysis and Pronase digestion (Figure S5). Similar to 1, the absolute configuration of Ahod in 2 was elucidated as 2S,3S (Figure S6). A previous study reported that coculturing with R. solanacearum strain GMI1000 induced chlamydospores in Aspergillus flavus.5 We found that strain OE1-1 induced more chlamydospores of Fusarium oxysporum when they were cocultured. Thus, by using a disc diffusion assay, the chlamydospore-inducing activities of ralstonins on the fungus were evaluated. The formation of chlamydospores in F. oxysporum was induced by ralstonins (Figure 4A). The induction activity of ralstonin B (2) was stronger
strong acid hydrolysis, there has been no example to elucidate its absolute configuration in lipopeptides. Thus, we performed the enzymatic digestion of ralstonin A (1) with Pronase from Streptomyces griseus. The release of free β-OH-Tyr (3) from 1 was confirmed by the LC/MS analysis of the reaction mixture. Therefore, we compared the LC retention times of the L-FDLA (l-fluoro-2,4-dinitrophenyl-5-L-leucinamide) derivatives of synthetic (2S,3S)- and (2R,3R)-37e with that of 3 prepared from the Pronase digest of 1. The retention time of natural 3 prepared from 1 was identical to that of synthetic (2S,3S)-3 (Figure 3A), and thus, the absolute configuration of the β-OHTyr (3) residue in 1 was elucidated as 2S,3S.
Figure 3. (A) LC/MS analysis of L-FDLA derivatives of natural and synthetic β-OH-Tyr (3). (B) Pronase digestion of ralstonin A (1) and an LC/MS analysis of the L-FDLA derivatives of Hse′ from fragment 4, of Hse from the reaction mixture, and of standards. (C) LC/MS analysis of L-FDAA derivatives of natural and synthetic 5.
Marfey’s analysis described above clarified that two Hse residues in 1 were composed of the L and D forms. A detailed LC/MS analysis of the Pronase digest of ralstonin A (1) indicated that 1 was decomposed to fragment 4, the dipeptide Ahod-Thr, and amino acids (β-OH-Tyr, Ser, and Hse) (Figure 3B). In order to distinguish the absolute configurations of these two Hse residues in 1, fragment 4 was isolated from the reaction mixture and subjected to Marfey’s analysis. The L-FDLA derivative prepared from the hydrolysate of 4 gave a peak showing an identical retention time with D-Hse; however, the L-FDLA derivative of free amino acids in the reaction mixture contained L-Hse (Figure 3B). Thus, we showed that Hse is the L-form and Hse′ is the D-form. A peak possibly attributable to the methyl ester of Ahod, methyl 3-amino-2-hydroxyoctadecanoate (5), was detected in the methylated hydrolysate of ralstonin A (1) by LC/MS. In order to confirm this identity, we synthesized the anti and syn isomers of 5 (Scheme S1). Natural 5 prepared from 1
Figure 4. (A) Induction of chlamydospore formation by ralstonins in F. oxysporum. White arrow heads indicate chlamydospores. (B) Phytotoxic activity of ralstonins on tobacco leaves. Ralstonins induced the necrosis of tobacco cells. (C) Effects of 3-OH MAME on the induction of ralstonin production in ΔphcB (the phcB-deletion mutant of strain OE1-1). The areas of the LC/MS peaks corresponding to ralstonins A and B were used to evaluate the induction levels of ralstonins. Error bars are mean ± SDs (n = 3).
than that of ralstonin A (1) (Figure S7). The phytotoxicity of ralstonins was then evaluated. After the inoculation of ralstonins into tobacco leaves, apparent necrosis was observed in their regions, but not in control regions (Figure 4B). A similar response was observed on the leaves of tobacco after the injection of a lipopeptide produced by Pseudomonas corrugata.8 Thus, we confirmed that ralstonins exhibit strong chlamydospore-inducing activity and moderate phytotoxicity. C
DOI: 10.1021/acs.orglett.7b01685 Org. Lett. XXXX, XXX, XXX−XXX
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We then investigated whether the production of ralstonins is controlled by QS in R. solanacearum. The deletion of phcB, a gene encoding 3-OH MAME synthase, resulted in the loss of ralstonin production (Figure 4C). This ablation was rescued by 3-OH MAME in a dose-dependent manner. Therefore, we concluded that QS controls the production of ralstonins in strain OE1-1. RmyA contains an initiating fatty acyl-AMP ligase domain, followed by a keto-synthase domain and acetyl ornithine aminotransferase domain coupled to luciferase-like monooxygenase (module 1) (Figure 1A). The remainder of the rmyA gene product contains four NRPS modules (modules 2−5). RmyB is an NRPS enzyme composed of five NRPS modules (modules 6−10). The architecture of module 1 is similar to that of MycA responsible for the production of the lipopeptide mycosubtilin generated by Bacillus subtilis.9 The Ahod formed appears to be loaded to those NPRS modules and is then condensed with 11 amino acids to form undecapeptide ralstonins. However, RmyAB only has nine NRPS modules. The substrate predictions by NRPSpredictor2 for each NRPS module were consistent with the sequences of ralstonins except for module 5 (for more discussion, see Supporting Information Discussion). This implies that module 5 may utilize the tripeptide Hse-GlyHse as a substrate. The structures of ralstonins are unique in several aspects. They contain unusual amino acids such as β-OH-Tyr and Dha and the novel β-amino fatty acid Ahod. Several lipopeptides with a β-amino fatty acid have been reported.9,10 In these cases, the amino groups of N-terminal β-amino fatty acids were connected to the carboxyl groups of the C-terminal residues, forming large cyclic peptides. However, the amino groups of Ahod in ralstonins are of the free form and the carboxyl groups in the C-terminal β-Ala were connected to the hydroxy group of Hse′, forming small cyclic depsipeptide moieties. Thus, to the best of our knowledge, ralstonins are a previously undescribed type of lipodepsipeptide in nature. In conclusion, we herein elucidated the structures and absolute configurations of ralstonins produced by R. solanacearum. They are unique lipodepsipeptides containing the new β-amino fatty acid Ahod. This is the first study to examine the absolute configuration of β-OH-Tyr in lipopeptides. Ralstonins exhibited chlamydospore-inducing activity and phytotoxicity, suggesting that these compounds contribute to the entry of R. solanacearum into their hosts (plants and fungi). Since limited information is currently available on the endosymbiotic interactions of R. solanacearum with fungi, we are interested in how to organize this unique symbiosis in nature. A detailed analysis of ralstonindeficient mutants will help to answer these questions.
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Seiya Kato (Yamagata University) for his technical assistance. This work was supported by JSPS KAKENHI Grant Number: 17K19244.
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b01685. All experimental details, additional figures and tables, and copies of NMR spectra (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Kenji Kai: 0000-0002-4036-9959 D
DOI: 10.1021/acs.orglett.7b01685 Org. Lett. XXXX, XXX, XXX−XXX
