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

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.

Pathways and substrate specificity of DMSP catabolism in marine bacteria of the Roseobacter clade.

The volatiles released by Phaeobacter gallaeciensis, Oceanibulbus indolifex and Dinoroseobacter shibae have been investigated by GC-MS, and several MeSH-derived sulfur volatiles have been identified. An important sulfur source in the oceans is the algal metabolite dimethylsulfoniopropionate (DMSP). Labelled [2H6]DMSP was fed to the bacteria to investigate the production of volatiles from this compound through the lysis pathway to [2H6]dimethylsulfide or the demethylation pathway to [2H3]-3-(methylmercapto)propionic acid and lysis to [2H3]MeSH. [2H6]DMSP was efficiently converted to [2H3]MeSH by all three species. Several DMSP derivatives were synthesised and used in feeding experiments. Strong dealkylation activity was observed for the methylated ethyl methyl sulfoniopropionate and dimethylseleniopropionate, as indicated by the formation of EtSH- and MeSeH-derived volatiles, whereas no volatiles were formed from dimethyltelluriopropionate. In contrast, the dealkylation activity for diethylsulfoniopropionate was strongly reduced, resulting in only small amounts of EtSH-derived volatiles accompanied by diethyl sulfide in P. gallaeciensis and O. indolifex, while D. shibae produced the related oxidation product diethyl sulfone. The formation of diethyl sulfide and diethyl sulfone requires the lysis pathway, which is not active for [2H6]DMSP. These observations can be explained by a shifted distribution between the two competing pathways due to a blocked dealkylation of ethylated substrates.

Physiological diversity of Roseobacter clade bacteria co-occurring during a phytoplankton bloom in the North Sea.

Organisms of the Roseobacter clade are an important component in marine ecosystems, partially due to their metabolic variety. Not much is known, however, about the physiological diversity of different roseobacters present within one habitat. By using serial dilution cultures with low-nutrient media seven roseobacter strains, co-occurring during a phytoplankton bloom in the southern North Sea, were obtained in this study. Physiological characterization exhibited distinct substrate spectra of the isolates. Although no isolate showed growth on algal osmolyte dimethylsulfoniopropionate (DMSP), feeding experiments revealed that all new strains converted [²H6]DMSP into a variety of volatile compounds. Six strains mainly decomposed DMSP via the demethylation pathway, but four strains were also capable of cleaving DMSP to DMS and acrylate. It is hypothesized that the great physiological diversity of the roseobacters reflects their ability to inhabit different ecological niches and enables the organisms to cope differently with changing substrate supplies during phytoplankton blooms. Denaturing gradient gel electrophoresis and sequencing of excised bands resulted in detection of five additional roseobacters. Three of these sequences showed affiliation with three of the four major clusters of the Roseobacter clade, consisting predominantly of uncultured organisms (i.e. the Roseobacter clade-affiliated (RCA)), the NAC11-7 and the CHAB-I-5 clusters.

Impact of seasonal hydrological variation on the distributions of tetraether lipids along the Amazon River in the central Amazon basin: implications for the MBT/CBT paleothermometer and the BIT index.

Suspended particulate matter (SPM) was collected along the Amazon River in the central Amazon basin and in three tributaries during the rising water (RW), high water (HW), falling water (FW) and low water (LW) season. Changes in the concentration and the distribution of branched glycerol dialkyl glycerol tetraethers (brGDGTs), i.e., the methylation index of branched tetraethers (MBT) and the cyclization of brGDGTs (CBT), were seen in the Amazon main stem. The highest concentration of core lipid (CL) brGDGTs normalized to particulate organic carbon (POC) was found during the HW season. During the HW season the MBT and CBT in the Amazon main stem was also most similar to that of lowland Amazon (terra firme) soils, indicating that the highest input of soil-derived brGDGTs occurred due to increased water runoff. During the other seasons the MBT and CBT indicated an increased influence of in situ production of brGDGTs even though soils remained the main source of brGDGTs. Our results reveal that the influence of seasonal variation is relatively small, but can be clearly detected. Crenarchaeol was mostly produced in the river. Its concentration was lower during the HW season compared to that of the other seasons. Hence, our study shows the complexity of processes that influence the GDGT distribution during the transport from land to ocean. It emphasizes the importance of a detailed study of a river basin to interpret the MBT/CBT and BIT records for paleo reconstructions in adjacent marine setting.