Breakdown of 3-(allylsulfonio)propanoates in bacteria from the Roseobacter group yields garlic oil constituents

Two analogues of 3-(dimethylsulfonio)propanoate (DMSP), 3-(diallylsulfonio)propanoate (DAllSP), and 3-(allylmethylsulfonio)propanoate (AllMSP), were synthesized and fed to marine bacteria from the Roseobacter clade. These bacteria are able to degrade DMSP into dimethyl sulfide and methanethiol. The DMSP analogues were also degraded, resulting in the release of allylated sulfur volatiles known from garlic. For unknown compounds, structural suggestions were made based on their mass spectrometric fragmentation pattern and confirmed by the synthesis of reference compounds. The results of the feeding experiments allowed to conclude on the substrate tolerance of DMSP degrading enzymes in marine bacteria.


Introduction
The name of the allyl group has been introduced by Wertheim in 1844 when he investigated the constituents of garlic oil and derives from the botanical name of garlic (Allium sativum) [1]. During that time, the structures of the garlic oil constituents and also of the allyl group remained unknown, but its formula was correctly assigned as C 3 H 5 . Five decades later, Semmler reported on the nature of allyl propyl disulfide (1), diallyl disulfide (2), diallyl trisulfide (3), and diallyl tetrasulfide (4) from garlic oil (Scheme 1A) [2]. The antibacterial principle in garlic was identified in 1944 by Cavallito et al. as allicin (5) [3], a formal oxidation product of disulfide 2. Not only 5, but also several other sulfur compounds from garlic are today known to exhibit diverse biological activities, including inter alia antibacterial, antifungal, antioxidant, anti-inflammatory, and anticancer effects [4]. Later on, also heterocyclic compounds including 2-vinyl-4H-1,3-dithiine (6) and 3-vinyl-3,4-dihydro-1,2-dithiine (7) were discovered [5]. The formation of these volatile sulfur compounds starts from alliin (9) [6], a non-volatile precursor that is stored in garlic and related plants and only degraded into sulfur volatiles upon wounding by the pyridoxal phosphate (PLP) dependent alliinase (Scheme 1B) [7]. This initial enzyme-catalyzed reaction yields one equivalent of allyl-Scheme 1: Volatile allyl sulfides. A) Compounds known from garlic oil, B) mechanism of formation from alliin (9) by the PLP-dependent allinase (PLP: pyridoxalphosphate) and subsequent spontaneous reactions. sulfenic acid (10), pyruvic acid (11), and ammonia from 9, followed by a series of proposed spontaneous reactions [5,8]. Through these transformations, acid 10 can undergo a dimerization with elimination of water to allicin (5). The hydrolysis of 5 results in allylsulfinic acid (12) and allyl thiol (13), the latter of which can react with another molecule of 5 to yield 10 and 2. Alternatively, 5 can decompose to 10 and thioacroleine (14) by a Cope elimination, which explains the formation of the heterocycles 6 and 7 by dimerization through a [4 + 2] cycloaddition [5]. Compounds 6 and 7 were also reported to be formed from 5 during gas chromatographic (GC) analysis by an unknown mechanism [9] (7 was confused with its double bond regioisomer 3-vinyl-3,6-dihydro-1,2-dithiine (8) in this study [5]). Under these conditions the formation of the heterocyclic disulfides 7 and 8 may not involve a dimerization of 14, as a [4 + 2] cycloaddition is not a preferred gas-phase reaction.
The ecology of marine bacteria in their interaction with algae is particularly interesting in which the bacteria can promote the algal growth, but can also kill their host [10,11]. For both processes, the phytohormone indole-3-acetic acid is used as a messenger molecule [10]. For the macroalga Ulva mutabilis the presence of bacteria from the Roseobacter group is even mandatory for proper algal development, and 3-(dimethylsulfonio)propanoate (DMSP) is used as a chemotactic signal by the bacteria attracting them towards the algal host [12]. Many bacteria and fungi also release sulfur volatiles [13,14] that are especially important headspace constituents from marine bacteria of the Roseobacter group [15][16][17]. In these organisms, sulfur volatiles are to a large extent generated from algal (DMSP), a metabolite that is produced in massive amounts by algae [18], thus giving another example for the complex interactions between marine bacteria and algae. Known DMSP degradation pathways include its hydrolysis to dimethyl sulfide Scheme 2: Degradation of DMSP by marine bacteria. A) Hydrolysis or lysis to DMS, B) demethylation pathway leading to MeSH. The color code shows which enzymes are encoded in the genomes of the strains investigated in this study.
Feeding of (methyl-2 H 6 )DMSP to Phaeobacter inhibens DSM 17395 and Ruegeria pomeroyi DSM 15171 resulted in the efficient uptake of labelling into dimethyl disulfide (DMDS), the oxidative dimerization product from MeSH, showing the activity of the demethylation pathway in these bacteria. However, knockout of the dmdA gene in R. pomeroyi still gave a low incorporation of labelling into DMDS, suggesting the presence of another gene responsible for the demethylation activity [28]. Also the labelling from ( 34 S)DMSP was efficiently incorporated into DMDS and dimethyl trisulfide (DMTS) [29]. Our previous investigations have also demonstrated that synthetic, i.e., non-natural DMSP analogues such as 3-(ethylmethyl)sulfoniopropanoate (EMSP), 3-(diethylsulfonio)propanoate (DESP), 3-(dimethylselenio)propanoate (DMSeP; this compound is also formed naturally in Spartina alterniflora in the presence of sodium selenate [30]), and even 3-(dimethyltellurio)propanoate (DMTeP) are converted by the demethylation pathway into ethanethiol, methaneselenol, and methanetellurol, respectively, that further react to various volatiles containing EtS, MeSe, and MeTe groups [31]. The in vitro incubations of these DMSP analogues with recombinant DddQ and DddW from R. pomeroyi and DddP from P. inhibens demonstrated that all substrate analogues can be degraded through the lysis pathway into the corresponding dialkyl chalcogenides; only DMTeP was not cleaved by DddQ [32]. Here we describe the synthesis of the new DMSP analogues 3-(allylmethylsulfonio)propanoate (AllMSP) and 3-(diallylsulfonio)propanoate (DAllSP) and their conversion into typical garlic odor constituents by marine bacteria from the Roseobacter group that do not naturally occur in these organisms.

Results and Discussion
3-(Diallylsulfonio)propanoate (DAllSP) and 3-(allylmethylsulfonio)propanoate (AllMSP) were synthesized by the acid-catalyzed addition of allyl methyl sulfide and diallyl sulfide, respectively, to acrylic acid (Scheme 3). The obtained DMSP analogues were fed to marine broth agar plate cultures of three strains from the Roseobacter group with fully sequenced genomes, including P. inhibens DSM 17395, Dinoroseobacter shibae DSM 16493, and Oceanibulbus indolifex DSM 14862. In all cases the bacterial cultures released a strong garlic-like odor, presumptively due to a degradation of the DMSP derivatives to sulfur-containing volatiles, similar to the compounds known from garlic, through one of the pathways shown in Scheme 2. The emitted volatiles were captured on charcoal filter traps using a closed-loop stripping apparatus (CLSA) [33], followed by the extraction of the filters with CH 2 Cl 2 and analysis by gas chromatography-mass spectrometry (GC-MS) of the resulting extracts. Most of the compounds were readily identified by the comparison of their mass spectra and retention indices to published data. Every experiment was performed in triplicate to check for the reproducibility of the results. For comparison, the volatiles from all three strains grown on marine broth medium without the addition of DMSP or its analogues have been reported before [31].
Feeding of DAllSP to P. inhibens resulted in the production of sulfur volatiles including several allyl derivatives ( Figure 1, Figure 2A, Table 1, and Figure S1 in Supporting Information File 1). Besides the methylated sulfur compounds dimethyl trisulfide (31), dimethyl tetrasulfide (33), and S-methyl methanethiosulfonate (28) that were reported previously from P. inhibens [31], large amounts of diallyl sulfide (29) were observed, pointing to an efficient degradation of DAllSP through the lysis pathway, for which the DMSP lyase DddP can account in this organism (Scheme 2). Furthermore, the compounds allyl methyl disulfide (30), diallyl disulfide (2), allyl methyl trisulfide (32), and traces of diallyl trisulfide (3) and allyl methyl tetrasulfide (34) were observed. The formation of these compounds is explainable by the deallylation of DAllSP to 3-(allylsulfanyl)propanoic acid (37) and further degradation to allyl thiol (13) through the enzymes of the demethylation pathway that is fully established in P. inhibens by genes coding for DmdA-D (Scheme 4A). In the presence of air thiol 13 can then undergo an oxidative dimerization, or react analogously with MeSH to form allyl methyl disulfide (30, Scheme 4B). Similar oxidations requiring one additional unit of hydrogen sulfide can lead to the trisulfides 3 and 32 (Scheme 4C), while higher polysulfides such as 34 can arise through a metathesis reaction of two trisulfides (Scheme 4D). Also traces of methyl 3-(allylsulfanyl)propanoate (24) Figure 2 and Figure S1-S3 in Supporting Information File 1 with the same color. Very similar patterns of volatiles were obtained in the feeding experiments of DAllSP with D. shibae and O. indolifex ( Figure 2B,C, Table 1 and Figures S2 and S3 in Supporting Information File 1). An additionally observed compound in one analysis of O. indolifex was diallyl tetrasulfide (35). Both organisms also encode the DMSP demethylation pathway in their genomes, but with missing dmdD genes in both cases. A possible explanation is, that another enoyl-CoA hydratase, e.g., from fatty acid degradation, may functionally substitute for DmdD. Dinoroseobacter shibae additionally encodes genes for the DMSP hydrolase DddD and the DMSP lyase DddL, explaining the formation of 29, while no DMSP hydrolase or lyase is found in O. indolifex. Still, compound 29 is observed within this organism, but in lower quantities than in P. inhibens or D. shibae, and may point to the presence of another, yet unidentified type of DMSP lyase in this organism, because control experiments with medium plates with DAllSP added did not show a spontaneous degradation to 29 that could explain its observation.
The compound identification was based on a comparison to an authentic standard or of mass spectra to data base spectra in our MS libraries and confirmed for most cases by comparison of the retention indices to literature data, only for the mass spectrum of 26 no data base hit was returned. Therefore, a structural suggestion for this compound was based on the observed fragmentation pattern of the mass spectrum ( Figure 3A).  Figure 3A. In addition, compound 26 was synthesized by a method reported previously for the related compound 25 [40], through dimerization of methyl 3-mercaptopropanoate (39) to dimethyl 3,3'-disulfanediyldipropanoate (40), followed by the BF 3 ·OEt 2 -mediated metathesis with 2 (Scheme 5A). The synthetic compound 26 was identical by mass spectrum and retention index to the unknown volatile.
The feeding of AllMSP to P. inhibens resulted in the formation of large amounts of methyl 3-(methylsulfanyl)propanoate (23) in addition to smaller quantities of methyl 3-(allylsulfanyl)propanoate (24, Figure 4A, Table 2 and Figure S4 Figure 4 and Figures S4-S6 in Supporting Information File 1 with the same color. tive 24 may be formed analogously through intermediate 37 (Scheme 4A and E). The higher production of 23 in comparison to 24 suggests that the deallylation of AllMSP is more efficient than its demethylation, which is surprising, because naturally DmdA catalyzes a methyl-group transfer. This finding may reflect the high reactivity of the allyl group towards nucleophiles. Other compounds originating from AllMSP included the di-and trisulfides 2, 26, 30, and 32 that pointed to a breakdown of AllMSP to 13 through the DMSP demethylation pathway and subsequent oxidative polysulfide formation (Scheme 4A-C), but their formation was lower than from DAllSP, likely because of the discussed efficient deallylation of AllMSP. Small amounts of diallyl sulfide (29) were also detected, which is the formal lysis product of DAllSP, but not of AllMSP. In first instance, its formation from AllMSP was surprising, but it is explainable by a degradation of AllMSP to 13, followed by a nucleophilic attack at the allyl group of another AllMSP molecule (Scheme 4G). For D. shibae and O. indolifex the same pattern of compounds was found ( Figure 4B,C, and Figures S5 and S6 in Supporting Information File 1), only the production of the deallylated compound 23 was lower, while in turn the production of the di-and trisulfides from 13 and of 29 was increased. This suggests that the deallylation of AllMSP by the DmdA variants in these organisms may be less efficient than was observed for P. inhibens. Besides these sulfur compounds, only O. indolifex, but not the other two strains, released another compound, 27, whose mass spectrum was not included in our databases. The analysis of the fragmentation pattern ( Figure 3B) suggested that 27 could be methyl

Conclusion
Bacteria from the Roseobacter group can degrade DMSP analogues with S-allyl groups including AllMSP and DAllSP, likely with the participation of the enzymes for DMSP (hydro)lysis and from the DMSP demethylation pathway. Because MeSH can also originate from other sources, the DMSP derivatives used in this study can lead to products that can indicate which metabolic pathways are used for their conversion. Interestingly, the volatiles formed from AllMSP and DAllSP closely resemble flavoring compounds from garlic. The demethylation pathway with all four enzymes DmdABCD is fully established in P. inhibens, while genes for DmdD are missing in D. shibae and O. indolifex, suggesting that another enzyme with a low sequence homology may substitute for DmdD, leading to allylthiol and several sulfur volatiles derived from it in all three strains. The DMSP hydrolase DddD and the lyase DddL are present in D. shibae, and P. inhibens has a DMSP lyase DddP, which can explain the conversion of DAllSP into diallyl sulfide, while the reason for its formation in O. indolifex is currently unclear and may point to an unknown type of DMSP lyase in this organism. Since the observed patterns of allylated sulfur volatiles in the three investigated strains are different, it seems possible that the DMSP (hydro)lases and the enzymes from the DMSP demethylation pathway have different activities towards AllMSP and DAllSP.
In vitro studies with recombinant purified enzymes and mutational work will be needed for more detailed insights to support our hypotheses regarding the involved enzymes in AllMSP and DAllSP breakdown and will be performed in our laboratories in the future.

Experimental
Strains and culture condition

Feeding experiments and sampling of volatiles
Headspace samplings for each strain were done in triplicates.
For the feeding experiments, DAllSP or AlMSP (1 mM) were added to the full strength marine broth agar medium (MB2216) after autoclavation. The medium was then transferred into glass Petri dishes. The agar plates were inoculated with the precultures (400 μL), incubated for two days at 28 °C and then subjected for headspace extraction to a CLSA [33] for 24 h. The released volatiles were collected on charcoal filters (Chromtech, Idstein, Germany), followed by the extraction of the filters with dichloromethane (50 μL), and analysis of the extracts by GC-MS. For comparison, blank experiments with MB medium alone and with MB agar plates spiked with DAllSP or AlMSP were performed in the same way. All the volatiles mentioned in Table 1 and Table 2 were not observed in the blank experiments.

GC-MS
The GC-MS analyses were carried out on a HP7890A GC system connected to a HP5975C mass selective detector fitted with a HP-5MS fused silica capillary column (

General synthetic methods
All chemicals were purchased from TCI (Deutschland) or Sigma-Aldrich Chemie (Germany), and used without purification. Solvents were distilled and dried by standard methods. NMR spectra were recorded on a Bruker (Billerica, USA) Avance III HD Prodigy (500 MHz) or on an Avance III HD Cryo (700 MHz) NMR spectrometer. The spectra were referenced against solvent signals (

Synthesis of allyl DMSP derivatives
A mixture of acrylic acid (0.72 g, 10 mmol) and diallyl sulfide or allylmethyl sulfide (10 mmol) was treated with 2 N HCl at 80 °C for 4 h. The reaction mixture was concentrated in vacuo and the residue was purified by silica gel column chromatography (CH 2 Cl 2 /MeOH 5:1), followed by recrystallization from methanol/diethyl ether 1:1 to yield the pure compounds.