Roseochelin B, an Algaecidal Natural Product Synthesized by the

Letter pubs.acs.org/OrgLett

Roseochelin B, an Algaecidal Natural Product Synthesized by the Roseobacter Phaeobacter inhibens in Response to Algal Sinapic Acid Rurun Wang† and Mohammad R. Seyedsayamdost*,†,‡ †

Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, United States

‡

S Supporting Information *

ABSTRACT: The secondary metabolome of the representative Roseobacter, Phaeobacter inhibens, was examined in response to algal sinapic acid. In addition to roseobacticides, sinapic acid induced the production of two new natural products, roseochelin A and B, which were characterized by NMR and X-ray crystallography. Functional assays showed that roseochelin B binds iron and is algaecidal against the algal host Emiliania huxleyi. It appears to be produced by a rarely observed combination of nonenzymatic and enzymatic transformations.

A

lgal-bacterial symbioses provide sources of new secondary metabolites and models for more complex eukaryote− bacterial associations.1−3 Discovery of natural products in these contexts allows identification of not only the molecules involved but also their potential functions. We have been interested in the intermittent symbiosis between microalgal species, represented by the haptophyte Emiliania huxleyi, and members of the marine Roseobacter clade within the αProteobacteria, represented by Phaeobacter inhibens DSM 17395 (hereafter, P. inhibens). Our studies have uncovered a biphasic symbiosis model,4 which has been seen in other algal− bacterial interactions as well.5−8 In this model, beneficial metabolites are exchanged under conditions of mutualism:4 the algal host provides food in the form of dimethylsulfoniopropionate (DMSP) and the bacteria produce the auxins phenylacetic acid and indoleacetic acid,9−14 as well as the broad-spectrum antibiotic tropodithietic acid (TDA, Figure 1A, 1), which protects the algal−bacterial microassembly.15−17 However, the symbiosis changes into a parasitic mode, when the host produces p-coumaric acid (pCA), possibly as a result of senescence.4 Under these conditions, the bacteria combine phenylacetic acid with pCA and fragments of DMSP to form the roseobacticides, potent algaecides that kill the algal host.4,18 Because Roseobacter are opportunistic symbionts, they interact with and respond to phenylpropanoids produced by other microalgae as well to create a diverse array of roseobacticides, with one variant shown (2).19−22 In an effort to identify additional, so-called “cryptic” metabolites that modulate this interaction, we have herein examined the secondary metabolome of P. inhibens in response to sinapic acid (3), which is naturally produced by some microalgae.20−22 We report the structure and biological evaluation of roseochelins, cryptic, ironbinding, and algaecidal molecules that are produced only in response to algal sinapic acid. Studies addressing the biosynthesis of one analog, roseochelin B, show that it is generated by © 2017 American Chemical Society

Figure 1. Induction of cryptic metabolites by sinapic acid. (A) Structures of TDA (1), roseobacticide B (2), and sinapic acid (3). (B) HPLC-MS analysis of P. inhibens cultures grown in the absence (blue trace) or presence (red trace) of 3. Molecules identified by HR-HPLCMS are marked (see below).

a combination of nonenzymatic and enzymatic biosynthetic steps. Microalgae produce phenylpropanoids that act as elicitors of cryptic metabolites from Roseobacter, which in turn modulate the algal−bacterial interaction.4,19−22 We previously showed that, among five phenylpropanoids examined, 3 was the most effective elicitor of roseobacticide production, delivering the highest yields and diversity of these algaecides.19 To assess the Received: August 7, 2017 Published: September 18, 2017 5138

DOI: 10.1021/acs.orglett.7b02424 Org. Lett. 2017, 19, 5138−5141

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Organic Letters response to 3 further, we examined in detail the secreted metabolome of P. inhibens in the presence and absence of the elicitor using differential metabolomics. A typical HPLC-MS profile from these studies is shown (Figure 1B). Aside from the roseobacticides reported before, we found additional compound families that were elicited by 3. One of the most abundant of these (Figure 1B), apparently aromatic based on UV−vis spectra (see Supporting Information, Figure S1), consisted of two compounds that were related to each other but not to the roseobacticides. High-resolution (HR) MS data and comparison with a known natural products database indicated they were new metabolites, which we call roseochelins. Additional experiments revealed that their production was strictly dependent on 3, and that they were not synthesized under a variety of conditions lacking the inducer (Figure S2). Thus, aside from roseobacticides, 3 also elicits production of other cryptic metabolites from P. inhibens. To elucidate the structure and function of roseochelins, we generated large-scale production cultures, which allowed isolation of two variants, roseochelin A and B, at yields of 2.3 and 3 mg/L, respectively. A series of NMR spectra were acquired in an effort to solve their structures. However, we observed only six unique 1H NMR signals for the more abundant variant (Figure 2A). The HR-MS data, [M + H]+obs = 401.1237 and 433.0953 for roseochelin A and B, were consistent with molecular formulas of C 21 H 20 O 8 and C21H20O8S, respectively. Given the low H/C ratios, we reasoned that the roseochelin structures could not be elucidated by NMR alone. We subsequently succeeded in growing single crystals of both compounds by slow evaporation from a concentrated MeOH solution, and in solving their structures by X-ray crystallography (Figure 2B). Roseochelins are a new group of natural products. They contain a 2naphthoic acid skeleton that is substituted by dimethoxyphenol at the 4-position (Figure 2C). Variant A bears a 5,7dimethoxy-6-hydroxy group (4, Figure 2), while roseochelin B (5) contains a 8-thiomethyl substituent and is demethylated at the C7-substituent, giving it a 6,7-dihydroxy functionality. As expected, the naphthoic acid moiety is planar in both structures, and its plane is rotated 50.2° and 61.7° round the C4−C11 bond, relative to the plane of the dimethoxy-phenol substituent in 4 and 5, respectively. With the crystal structures at hand, the 1D/2D NMR data could be fully assigned (Figures S3−S7, Tables S1−S2). Aside from roseobacticides, roseochelins are the second group of new, cryptic metabolites that are elicited from P. inhibens by algal molecules. The elicited compounds share with TDA (1)15,16 an aromatic thiol substituent, which is methylated in 2 and 5.19 What, if any, are the roles of roseochelins in the algal− bacterial interaction? To begin to answer this question, we focused on the iron-binding capability of 4 and 5 as well as on possible growth−inhibitory roles. Not surprisingly, 5 showed obvious Fe-binding ability, as judged by the chrome azurol S assay, consistent with its catechol substructure, which is present in many siderophores (Figure S8).23,24 On the other hand, 4 did not bind iron, as expected from its 5,7-dimethoxyphenol moiety. Whether 5 is a bona fide siderophore remains to be determined. Initial assays under Fe-limiting and Fe-saturated conditions (in the presence of 3) result in roseochelin production, suggesting that they are not synthesized in response to Fe-limitation. They may, however, be produced to scavenge iron released from dying algal cells in the parasitic phase of the association.

Figure 2. Characterization of roseochelins. (A) 1H NMR spectrum of 5 in DMSO. The solvent residual and residual acetone peaks are crossed out. (B) Single molecule X-ray crystal structures of roseochelin A (4) and B (5) solved to ∼0.84 Å resolution. (C) Roseochelin structures and numbering schemes.

We also examined potential antibiotic activities of roseochelins against marine bacteria of the Vibrio genus, which are known to compete with Roseobacter for algal surfaces,25,26 as well as other representative bacteria and yeast (Table S3). Roseochelins did not exhibit significant antibacterial activities in standard assays. A similar result was previously obtained for the roseobacticides, which turned out to be algaecides.4 We therefore tested 4 and 5 against two forms of the E. huxleyi hostthe B11 variant which builds coccoliths from CaCO3 and the coccolith-free CCMP 374 strainas well as against the diatom Chaetoceros muelleri and the green algal strain Dunaliella tertiolecta. No significant algaecidal activity was detected against the diatom and the green microalgae. Interestingly, however, we observed weak but clear antialgal activity against the E. huxleyi host (Figure 3). While 4 did not affect the algae, 5 had a visible impact on cell morphology: after a 12 h treatment, we observed morphologically altered, compacted cells (Figure 3B), followed by cell lysis after 24 h (Figure 3C). Quantification of these effects by hemocytometry, to distinguish live and dead cells, gave IC50 values of 51 and 64 μM against E. huxleyi CCMP 374 and B11, respectively. These IC50 values are close to the bulk concentration of 5 (∼25 μM), though the limited diffusion in a bacterial−microalgal assembly, in which the bacteria are attached to the algal cells, may enhance the 5139

DOI: 10.1021/acs.orglett.7b02424 Org. Lett. 2017, 19, 5138−5141

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Figure 3. Effect of roseochelin B on E. huxleyi. Shown are contrast phase micrographs upon exposure of E. huxleyi CCMP 374 to DMSO control after 24 h (A) or to 100 μM of roseochelin B after 12 h (B) or 24 h (C). Scale bar, 2 μm.

“effective” concentration that the algae sense. As such, the bulk and effective concentrations of 5 are likely to be vastly different. Much like the roseobacticides, 5 acts as an algaecide hinting at a role in the parasitic phase of the algal−bacterial symbiosis, when the bacterial symbiont turns against its host. We suggest that against some algae that produce 3, P. inhibens generates a more diverse set of algaecidal molecules in the parasitic phase of the symbiosis. Production of the algaecidal roseobacticides is accompanied by synthesis of 5, which together lead to the demise of the algal host. With the structure and bioactivity of roseochelins characterized, we next focused on their biogenesis. A retrobiosynthetic analysis indicates that roseochelins could be generated from sinapic acid (Figure 4A). 4 appears to be the formal [4 + 2] cycloaddition product of two molecules of sinapic acid, while 5 is a modified version thereof. Indeed, it has previously been reported that 3 can spontaneously combine under weakly basic conditions to give thomasidioic acid (6, Figure 4B), which then decomposes to 4,6-dimethoxy-5hydroxy-naphthoic acid (7) and 2,6-dimethoxy-benzoquinone (8).27 While we did not observe 6, we could see its degradation product 7 (Figure 1B). We thus considered that the first reaction in our biosynthetic pathway, formation of 4 from 3, may be spontaneous, whereas the remaining onesaddition of a thiomethyl group to an unactivated carbon position and demethylation of the methoxy substituentwere likely enzyme-catalyzed. To test this idea, we incubated sinapic acid under the same culture conditions as above, but in the absence of P. inhibens. Somewhat surprisingly, we detected formation of 4, but not 5 (Figure S9). Further experiments, in which the incubation time, sinapic acid concentration, and temperature were varied, failed to deliver 5. Lastly, addition of roseochelin A to P. inhibens cultures in the absence of sinapic acid also did not result in roseochelin B production, consistent with the idea that its synthesis requires biosynthetic enzymes that are induced by 3 (Figure S2). Together, these data support a biosynthetic model, in which 4 is generated spontaneously from inducer 3, though we cannot exclude a scenario in which this process is facilitated by an enzyme in vivo. Upon its formation, 4 is then further modified by P. inhibens to generate 5 (Figure 4C). This model suggests that the biosynthetic pathway for 5 includes both spontaneous and enzyme-catalyzed steps, a phenomenon rarely seen in natural products biogenesis. Further studies are necessary to test this model, including identification of the enzymes involved. We gauged the prevalence of 5 by examining the metabolic response of additional Roseobacter strains to 3. Roseochelin B was not detected in cultures of Phaeobacter arcticus, Silicibacter pomeroyi DSS-3, and Ruegeria sp. R11. These strains also do not produce TDA or roseobacticides. Production of 5 was observed with Ruegeria sp. 27-4, Phaeobacter inhibens DSM 16374, Silicibacter sp. TM1040, and Phaeobacter inhibens 2.10 (Table 1,

Figure 4. Biosynthesis of 5. (A) Retrobiosynthetic analysis of roseochelin A shows that it could be generated by a formal [4 + 2] cycloaddition of two sinapic acid molecules. (B) Spontaneous transformation of 3 at alkaline pH leads to formation of thomasidioic acid (6), followed by O2-mediated decomposition to 7 and 8. (C) Proposed biosynthetic pathway for 5. Spontaneous cycloaddition of 3 leads to 4, which is enzymatically modified by P. inhibens, via removal of a methyl group and substitution by a methylthio moiety, to give 5.

Table 1. Synthesis of TDA, Roseochelin B, and Roseobacticides by Selected Roseobacter Strains

a

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Another algaecide (m/z 226) was also detected in this strain.28

DOI: 10.1021/acs.orglett.7b02424 Org. Lett. 2017, 19, 5138−5141

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ACKNOWLEDGMENTS We thank Dr. P. Jeffrey (Princeton University) for assistance with single crystal X-ray crystallography data acquisition and interpretation, Darcy McRose and Prof. Francois Morel (Department of Geosciences, Princeton University) for providing algal strains, and the National Institutes of Health (Grant GM098299) and the Pew Biomedical Scholars Program for support of this work.

Figure S10). Silicibacter sp. TM1040, which generates TDA and roseochelin B, has been shown to synthesize an additional algaecidal molecule with yet-unknown structure.28 Interestingly, all producers of 5 also synthesized TDA, an important metabolite in the beneficial phase of algal−bacterial symbioses (Table 1).17 With the exception of P. inhibens 2.10 and P. inhibens DSM 17395, all other roseochelin B producers are deficient in roseobacticide production, suggesting perhaps that they can engage in dynamic symbioses with microalgae using TDA in the mutualistic phase and roseochelin B in the parasitic phase of the interaction (Table 1). While this proposal needs to be investigated, the data above show that a number of Roseobacter strains make use of spontaneously generated products of sinapic acid, in this case roseochelin A, to build the Fe-binding, mild algaecide, roseochelin B. In summary, we report that algal sinapic acid induces production of a new family of compounds, the roseochelins, which we have characterized structurally by X-ray crystallography. These molecules may potentiate dynamic interactions with microalgae in a wide range of Roseobacter. In the natural context of P. inhibens, 3 may be produced by senescing algae. Our results suggest that, in addition to eliciting roseobacticides, 3 also condenses spontaneously to form 4, which the bacteria can modify to generate 5. This compound then joins forces with roseobacticides to alter the symbiotic interaction with the algal host, from a mutualistic one to a parasitic association. Spontaneous combination of 3 to roseochelin A is a bimolecular process and thus occurs more rapidly at higher concentrations. As such, at low concentrations of 3, the metabolomic response of P. inhibens may be dominated by roseobacticides, while, at higher concentrations, both roseobacticides and roseochelins will be produced to pathogenize the microalgae. Our results show that sinapic acid acts as an elicitor of at least two groups of cryptic metabolites and that P. inhibens has evolved to create bioactive natural products by a combination of biotic and abiotic processes to modulate an existing symbiotic interaction.

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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.7b02424. Detailed description of the methods used for culturing P. inhibens and other bacteria; induction, purification, characterization, and structural elucidation of 4 and 5; bioactivity assays against various test strains; evaluation of E. huxleyi by microscopy; and biosynthetic studies of roseochelins (PDF) Crystallographic data for 4 (CIF) Crystallographic data for 5 (CIF)

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Letter

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Mohammad R. Seyedsayamdost: 0000-0003-2707-4854 Notes

The authors declare no competing financial interest. 5141

DOI: 10.1021/acs.orglett.7b02424 Org. Lett. 2017, 19, 5138−5141