Identification of volatiles from six marine Celeribacter strains

The volatiles emitted from six marine Rhodobacteraceae species of the genus Celeribacter were investigated by GC–MS. Besides several known compounds including dimethyl trisulfide and S-methyl methanethiosulfonate, the sulfur-containing compounds ethyl (E)-3-(methylsulfanyl)acrylate and 2-(methyldisulfanyl)benzothiazole were identified and their structures were verified by synthesis. Feeding experiments with [methyl-2H3]methionine, [methyl-13C]methionine and [34S]-3-(dimethylsulfonio)propanoate (DMSP) resulted in the high incorporation into dimethyl trisulfide and S-methyl methanethiosulfonate, and revealed the origin of the methylsulfanyl group of 2-(methyldisulfanyl)benzothiazole from methionine or DMSP, while the biosynthetic origin of the benzothiazol-2-ylsulfanyl portion could not be traced. The heterocyclic moiety of this compound is likely of anthropogenic origin, because 2-mercaptobenzothiazole is used in the sulfur vulcanization of rubber. Also in none of the feeding experiments incorporation into ethyl (E)-3-(methylsulfanyl)acrylate could be observed, questioning its bacterial origin. Our results demonstrate that the Celeribacter strains are capable of methionine and DMSP degradation to widespread sulfur volatiles, but the analysis of trace compounds in natural samples must be taken with care.


Introduction
Bacteria from the roseobacter group belong to the most abundant microbial species in marine ecosystems [1,2]. They are present from polar to tropical regions, in marine sediments, in estuarine and open ocean environments in different pelagic zones ranging from surface waters to depths of >2,000 m [3,4]. Some species are associated with other marine organisms, e.g., Thalassococcus halodurans DSM 26915 T has been isolated from the marine sponge Halichondria panicea [5], and Phaeobacter gallaeciensis DSM 26640 T is an isolate from the scallop Pecten maximus [6]. Important interactions are also observed between bacteria from the roseobacter group and various types of marine algae, e.g., the first described organisms Roseobacter litoralis DSM 6996 T and R. denitrificans DSM 7001 T were obtained from seaweed [7], while Dinoroseobacter shibae DSM 16493 T and Marinovum algicola DSM 10251 T are both isolates from the dinoflagellate Prorocentrum lima [8,9]. Especially in algal blooms bacteria of the roseobacter group are highly abundant [10], and here they belong to the main players involved in the enzymatic degradation of the algal sulfur metabolite 3-(dimethylsulfonio)propanoate (DMSP, Scheme 1) [11]. Its catabolism leads either through the demethylation pathway by action of the enzymes DmdABCD to methanethiol (MeSH, Scheme 1A) [12] or through lysis by DddD [13] or hydrolytic cleavage by one of the known DMSP lyases (DddW [14], DddP [15], DddQ [16], DddL [17], DddY [18] or DddK [19]) to dimethyl sulfide (DMS, Scheme 1B).
It has already been pointed out in the 1970s and 1980s that atmospheric DMS is important for the global sulfur cycle [20] and influences the climate on Earth, known as CLAW hypoth-esis according to the authors' initials (Carlson, Lovelock, Andreae, Warren) [21], which underpins the relevance of this algal-bacterial interaction. Isotopic labeling experiments demonstrated that also in laboratory cultures roseobacter group bacteria efficiently degrade DMSP into sulfur volatiles [22,23], but also from other sulfur sources including 2,3-dihydroxypropane-1-sulfonic acid (DHPS, Scheme 1C) labeling was efficiently incorporated into sulfur volatiles [24,25]. Notably, DHPS is produced in large quantities by the marine diatom Thalassiosira pseudonana [26], and diatoms from this genus live in symbiotic relationship with bacteria of the roseobacter group [27]. Another interesting aspect of sulfur metabolism in marine bacteria from the roseobacter group is the production of the sulfur-containing antibiotic tropodithietic acid (TDA) in Phaeobacter piscinae DSM 103509 T [28], a compound that is in equilibrium with its tautomer thiotropocin [29] that was first described from Pseudomonas sp. CB-104 [30]. Its biosynthesis depends on the clustered tda genes [31] and has been studied by feeding experiments with labeled precursors to the wildtype and gene knockout strains of P. inhibens DSM 17395 T , demonstrating the formation of TDA from phenylalanine through phenylacetyl-CoA and the phenylacetyl-CoA catabolon [32,33]. These experiments also led to a suggestion for the mechanism for sulfur incorporation, but further research is required for a deep understanding of TDA biosynthesis. Besides its function as an antibiotic, TDA acts as a signaling molecule, similar to N-acylhomoserine lactones, at concentrations 100 times lower than required for a significant antibiotic activity [34]. The biosynthesis of tropone [35] and of the algicidal sulfur-containing roseobacticides [36] are most likely connected to the TDA pathway. Interestingly, in the interaction with marine algae P. inhibens can change its lifestyle from a symbiotic relationship during which the antibiotic TDA and growth stimulants are produced to a pathogenic interaction promoted by lignin degradation products in fading algal blooms that induce roseobacticide biosynthesis [36]. All these examples demonstrate the importance of sulfur metabolism for marine bacteria from the roseobacter group. Here we report on the volatiles emitted by six Celeribacter species with a special focus on sulfur volatiles. The results from feeding studies with labeled precursors demonstrate that the Celeribacter strains can form sulfur volatiles from methionine and DMSP, but also showed that some of the detected sulfur compounds are not or only partly of bacterial origin.

Headspace analysis
The volatiles released by six marine Celeribacter type strains, including C. marinus DSM 100036 T , C. neptunius DSM 26471 T , C. manganoxidans DSM 27541 T , C. baekdonensis DSM 27375 T , C. halophilus DSM 26270 T and C. indicus DSM 27257 T , were collected through a closed-loop stripping apparatus (CLSA) on charcoal [37]. After extraction with dichloromethane the obtained extracts were analyzed by GC-MS ( Figure 1). The compounds were identified by the comparison of the recorded EI mass spectra to library spectra and of retention indices [38] to tabulated literature data (Table 1), or by a direct comparison to authentic standards. The structures of the identified compounds are shown in Figure 2.

Synthesis of reference compounds
The mass spectrum of the component 41 showed strong similarities to the library mass spectrum of 2-mercaptobenzothiazole that has a molecular weight of 167 Da. The isotope pattern of the molecular ion at m/z = 213 indicated the presence of three sulfur atoms. The strong base peak at m/z = 167 in the mass spectrum of 41 suggested a benzothiazol-2-ylsulfanyl moiety, while the mass difference to the molecular ion pointed to the connection to a methylsulfanyl group. Taken together, this analysis resulted in the structural proposal of 2-(methyldisulfanyl)benzothiazole for 41. For the structural verification a synthesis was performed by a BF 3 ·OEt 2 -catalyzed reaction of bis(benzothiazol-2-yl)disulfane with dimethyl disulfide, giving     access to 41 with a yield of 64% (Scheme 2). The synthetic compound 41 showed an identical mass spectrum and retention index compared to the volatile in the Celeribacter extracts. The Z and E stereoisomers of 42 were obtained by the Michael addition of NaSMe to ethyl propiolate (45), yielding a mixture of stereoisomers inseparable by silica gel column chromatography (92%). The major stereoisomer was found to be (Z)-42 (dr 94:6), whose preferred formation may be a result of a chalcogen-chalcogen interaction between the sulfur and an ester oxygen. This phenomenon was first described in supramolecular structures by Gleiter [59] and later also used to explain the outcome of organocatalytic reactions [60]. The pure stereoisomers of 42 were isolated by preparative HPLC, for which the best separation was achieved using a YMC ChiralART Cellulose-SC column. This yielded 70% of (Z)-42 and 6% of (E

Feeding experiments with isotopically labeled precursors
The biosynthesis of sulfur volatiles in C. marinus was investigated in a series of feeding experiments with isotopically labeled precursors. Feeding of (methyl-2 H 3 )methionine resulted in the efficient incorporation of labeling into 37 (79% incorporation rate, Figure S1B in Supporting Information File 1), 38 (78%, Figure S1F in Supporting Information File 1) and the S-methyl group of 41 (84%), as indicated by a shift of the molecular ion from m/z = 213 to 216 ( Figure 3B, deuterated compounds can be separated from their non-deuterated analogs by gas chromatography [61]). The base peak appears at m/z = 168, demonstrating its formation with participation of one deuterium from the S-methyl group. Analogous results were obtained by feeding of (methyl-13 C)methionine, showing incorporation into 37 (74%, Figure S1C in Supporting Information File 1), 38 (71%, Figure S1G in Supporting Information), and the MeS group of 41 (71%, Figure 3C; the signal at m/z = 213 represents unlabeled 41 that, in contrast to a deuterated compound, cannot be separated from 13 C-labeled 41 by gas chromatography). Furthermore, feeding of [ 34 S]DMSP gave an incorporation into the MeS groups of 37 (50%, Figure S1D), into both sulfur atoms of 38 (47%, Figure S1H in Supporting Information File 1), but only into one sulfur atom of 41 (46%), as indicated by the molecular ion at m/z = 215, while no signals at m/z = 217 and 219 were visible that would account for the incorporation of labeling into two or three of the sulfur atoms in 41 ( Figure 3D; also here the signal at m/z = 213 represents inseparable unlabeled 41). In this experiment, the base peak did not change which allowed the localization of labeling specifically in the MeS group of 41.
The fact that no incorporation was observed for the other two sulfur atoms of 41 prompted us to further investigate the biosynthetic origin of the benzothiazol-2-ylsulfanyl portion of 41 to establish its natural origin. Several feeding experiments with central primary metabolites including ( 13 C 6 )glucose, ( 13 C 5 )ribose and (indole-2 H 5 )tryptophan were performed, but none of these experiments resulted in a detectable incorporation of labeling. Conclusively, a non-biological origin of this part of the molecule seems likely, which may also explain why the detection of 41 in Celeribacter was not always reproducible. Notably, 2-mercaptobenzothiazole is used in the sulfur vulcanization of rubber and could react spontaneously with MeSH of bacterial origin in the presence of oxygen to form 41, giving a reasonable explanation for its formation.
Also none of the feeding experiments with the various labeled precursors resulted in an incorporation of labeling into the sulfur volatiles 39, 40, and 42, which also questioned their natural origin. This finding is rather surprising for 42, especially regarding the feeding experiment with ( 34 S)DMSP, because its formation would be explainable by a DMSP degradation through the demethylation pathway, for which all relevant enzymes are encoded in the six Celeribacter strains (only a DmdA homolog is missing in C. indicus, Table S1 in Supporting Information File 1), and e.g., transesterification of the DmdC product with EtOH (Scheme 1A). Compound 42 is not a widespread sulfur volatile, but has been reported before from pineapples [53], pears [62], passion fruits [63], and apples [64].

Conclusion
Six marine Celeribacter strains were investigated for their volatiles, leading to the identification of 42 compounds from different classes, including several sulfur volatiles. However, feeding experiments with isotopically labeled precursors suggested that only the widespread compounds dimethyl trisulfide (37) and S-methyl methanethiosulfonate (38) are of natural origin, while no labeling from any of the fed precursors was incorporated into 2-acetylthiazole (39), benzothiazole (40), and ethyl (E)-3-(methylsulfanyl)acrylate (42), thus questioning their natural source from Celeribacter. These results demonstrate that the six Celeribacter strains are able to degrade methionine and DMSP with formation of MeSH as a source for the likely non-enzymatic oxidation in the presence of air to 37 and 38, opening possibilities for future studies on methionine and DMSP degrading enzymes and pathways in Celeribacter. Our study also shows that the results from trace compound analyses must be taken with care and contaminations from other sources must always be taken into consideration. For the unusual compound 2-(methyldisulfanyl)benzothiazole (41) the incorporation of labeling was observed only into the MeS group, while the benzothiazol-2-ylsulfanyl portion is likely of anthropogenic origin from the rubber vulcanization agent 2-mercaptobenzothiazole that reacts with MeSH from the bacterial metabolism.

Experimental
Strains, culture conditions, and feeding experiments All six Celeribacter type strains were cultivated at 28 °C on marine broth agar plates. In case of feeding experiments, the isotopically labeled compound (1 mM) was added to the agar medium before inoculation.

Collection of volatiles
The volatiles emitted by Celeribacter spp. agar plate cultures were collected on charcoal filters (Chromtech, Idstein, Germany, precision charcoal filters charged with 5 mg of charcoal) by use of a closed-loop stripping apparatus as developed by Grob and Zürcher [37]. After a collection time of 24 h the charcoal was extracted with CH 2 Cl 2 (50 μL) and the extract was analyzed by GC-MS.

GC-MS
GC-MS analyses were carried out through a 7890B GC -

General synthetic and analytical methods
Reactions were carried out in oven-dried flasks under Ar atmosphere and using distilled and dried solvents.

Supporting Information
Supporting Information File 1 DMSP demethylation pathway in Celeribacter spp. and copies of spectra.