Isotopically labeled sulfur compounds and synthetic selenium and tellurium analogues to study sulfur metabolism in marine bacteria

Abstract

Members of the marine Roseobacter clade can degrade dimethylsulfoniopropionate (DMSP) via competing pathways releasing either methanethiol (MeSH) or dimethyl sulfide (DMS). Deuterium-labeled [(2)H6]DMSP and the synthetic DMSP analogue dimethyltelluriopropionate (DMTeP) were used in feeding experiments with the Roseobacter clade members Phaeobacter gallaeciensis DSM 17395 and Ruegeria pomeroyi DSS-3, and their volatile metabolites were analyzed by closed-loop stripping and solid-phase microextraction coupled to GC-MS. Feeding experiments with [(2)H6]DMSP resulted in the incorporation of a deuterium label into MeSH and DMS. Knockout of relevant genes from the known DMSP demethylation pathway to MeSH showed in both species a residual production of [(2)H3]MeSH, suggesting that a second demethylation pathway is active. The role of DMSP degradation pathways for MeSH and DMS formation was further investigated by using the synthetic analogue DMTeP as a probe in feeding experiments with the wild-type strain and knockout mutants. Feeding of DMTeP to the R. pomeroyi knockout mutant resulted in a diminished, but not abolished production of demethylation pathway products. These results further corroborated the proposed second demethylation activity in R. pomeroyi. Isotopically labeled [(2)H3]methionine and (34)SO4 (2-), synthesized from elemental (34)S8, were tested to identify alternative sulfur sources besides DMSP for the MeSH production in P. gallaeciensis. Methionine proved to be a viable sulfur source for the MeSH volatiles, whereas incorporation of labeling from sulfate was not observed. Moreover, the utilization of selenite and selenate salts by marine alphaproteobacteria for the production of methylated selenium volatiles was explored and resulted in the production of numerous methaneselenol-derived volatiles via reduction and methylation. The pathway of selenate/selenite reduction, however, proved to be strictly separated from sulfate reduction.

Keywords: Roseobacter clade; dimethylsulfoniopropionate; selenium metabolism; sulfur metabolism; volatiles.

Figure 1

Roseobacter clade metabolites.

Scheme 1

Degradation of DMSP via (A) demethylation pathway and (B) cleavage pathways. FH₄: tetrahydrofolate.

Scheme 2

Sulfate reduction pathway and incorporation of sulfur into the amino acid pool. PAP: adenosine 3’,5’-bisphosphate.

Figure 2

Volatiles from P. gallaeciensis DSM 17395 and R. pomeroyi DSS-3. Feeding of [²H₆]DMSP results in deuterium incorporation into compounds 1–3, feeding of DMTeP results in the formation of compounds 10–12.

Figure 3

Chromatograms of headspace extracts from P. gallaeciensis DSM 17395 after feeding of DMTeP by the use of (A) CLSA (black) and (B) SPME (orange).

Figure 4

Chromatograms of headspace extracts obtained after feeding of [²H₆]DMSP by the use of SPME from (A) R. pomeroyi DSS-3 wild type, (B) R. pomeroyi DSS-3 dmdA⁻, and (C) R. pomeroyi DSS-3 dddQ⁻. Trace amounts of [²H₆]-2 and [²H₆]-3 were also found (not shown).

Figure 5

Chromatograms of headspace extracts from (A) R. pomeroyi DSS-3 wild type, (B) R. pomeroyi DSS-3 dmdA⁻, and (C) R. pomeroyi DSS-3 dddQ⁻ after feeding of DMTeP by use of CLSA (black) and SPME (orange).

Scheme 3

Synthesis of ³⁴S-labeled thiosulfate and sulfate.

Figure 6

Volatiles from P. gallaeciensis after feeding of selenate and selenite.

Figure 7

Chromatograms of headspace extracts from P. gallaeciensis grown on (A) 50% MB2216, (B) 50% MB2216 + 1 mmol/l Na₂SeO₄, (C) 50% MB2216 + 1 mmol/l Na₂SeO₃.

Figure 8

Additional sulfur volatiles.