Dimethylsulfoniopropionate (DMSP) is abundant in marine environments and an important source of reduced carbon and sulfur for marine bacteria. While both Ruegeria pomeroyi and Ruegeria lacuscaerulensis possessed genes encoding the DMSP demethylation and cleavage pathways, their responses to DMSP differed. A glucose-fed, chemostat culture of R. pomeroyi consumed 99% of the DMSP even when fed a high concentration of 5 mM. At the same time, cultures released 19% and 7.1% of the DMSP as dimethylsulfide (DMS) and methanethiol, respectively. Under the same conditions, R. lacuscaerulensis consumed only 28% of the DMSP and formed one-third of the amount of gases. To examine the pathways of sulfur and methyl C assimilation, glucose-fed chemostats of both species were fed 100 μM mixtures of unlabeled and doubly labeled [dimethyl-13C, 34S]DMSP. Both species derived nearly all of their sulfur from DMSP despite high sulfate availability. In addition, only 33% and 50% of the methionine was biosynthesized from the direct capture of methanethiol in R. pomeroyi and R. lacuscaerulensis, respectively. The remaining methionine was biosynthesized by the random assembly of free sulfide and methyl-tetrahydrofolate derived from DMSP. Thus, although the two species possessed similar genes encoding DMSP metabolism, their growth responses were very different. IMPORTANCE Dimethylsulfoniopropionate (DMSP) is abundant in marine environments and an important source of reduced carbon and sulfur for marine bacteria. DMSP is the precursor for the majority of atmospheric dimethylsulfide (DMS), a climatically active gas that connects the marine and terrestrial sulfur cycles. Although research into the assimilation of DMSP has been conducted for over 20 years, the fate of DMSP in microbial biomass is not well understood. In particular, the biosynthesis of methionine from DMSP has been a focal point, and it has been widely believed that most methionine was synthesized via the direct capture of methanethiol. Using an isotopic labeling strategy, we have demonstrated that the direct capture of methanethiol is not the primary pathway used for methionine biosynthesis in two Ruegeria species, a genus comprised primarily of globally abundant marine bacteria. Furthermore, although the catabolism of DMSP by these species varied greatly, the anabolic pathways were highly conserved.

Cog.na.ti.shi'mi.a. L. masc. adj. cognatus relative, related, kindred; N.L. fem. n. Shimia a bacteria generic name; N.L. fem. n. Cognatishimia related to Shimia . Proteobacteria / Alphaproteobacteria / Rhodobacterales / Rhodobacteraceae / Cognatishimia Gram‐negative‐staining, aerobic, and catalase‐ and oxidase‐positive rods. Optimal growth occurs at 30°C, 2% (w/v) NaCl, and pH 7.0–8.0. Capable of degrading a variety of carbon sources including carbohydrates, alcohols, carboxylic acids, and amino acids. The principal fatty acid is C 18 :1 ω7 c . The major respiratory quinone is Q‐10. The type species was isolated from the seawater of the South Sea of Korea. Member of the family Rhodobacteraceae . The type species is Cognatishimia maritima . DNA G + C content (mol%) : 57 (LC). Type species : Cognatishimia maritima (Park, Lee, Lee, Oh and Yoon 2012) Wirth and Whitman 2018, 2403 VP (Park, Lee, Lee, Oh and Yoon 2012, 11).

The acyl-CoA dehydrogenase family enzyme DmdC catalyzes the third step in the dimethylsulfoniopropionate (DMSP) demethylation pathway, the oxidation of 3-methylmercaptopropionyl-CoA (MMPA-CoA) to 3-methylthioacryloyl-CoA (MTA-CoA). To study its substrate specificity, the recombinant DmdC1 from Ruegeria pomeroyi was characterized. In addition to MMPA-CoA, the enzyme was highly active with short chain acyl-CoAs, with K m values for MMPA-CoA, butyryl-CoA, valeryl-CoA, caproyl-CoA, heptanoyl-CoA, caprylyl-CoA and isobutyryl-CoA of 36, 19, 7, 11, 14, 10, and 149 μM, respectively, and k cat values of 1.48, 0.40, 0.48, 0.73, 0.46, 0.23 and 0.01 sec −1 , respectively. Among these compounds, MMPA-CoA was the best substrate. The high affinity of DmdC1 for its substrate supports the model for kinetic regulation of the demethylation pathway. In contrast to DmdB, which catalyzes the formation of MMPA-CoA from MMPA, CoA and ATP, DmdC1 was not affected by physiological concentrations of potential effectors, such as DMSP, MMPA, ATP and ADP. Thus, compared to the other enzymes of the DMSP demethylation pathway, DmdC1 has only minimal adaptations for DMSP metabolism compared to other enzymes in the same family with similar substrates, supporting the hypothesis that it evolved relatively recently from a short chain acyl-CoA dehydrogenase involved in fatty acid oxidation. Importance We report the kinetic properties of DmdC1 from the model organism R. pomeroyi and close an important gap in the literature. While the crystal structure of this enzyme was recently solved and its mechanism of action described (X. Shao, H. Y. Cao, F. Zhao, M. Peng, et al., Mol Microbiol 111:1057-1073, 2019, https://doi.org/10.1111/mmi.14211 ), its substrate specificity and sensitivity to potential effectors was never examined. We show that DmdC1 has a high affinity for other short chain acyl-CoAs in addition to MMPA-CoA, which is the natural substrate in DMSP metabolism and is not affected by the potential effectors tested. This evidence supports the hypothesis that DmdC1 possesses few adaptations to DMSP metabolism and likely evolved relatively recently from a short chain acyl-CoA dehydrogenase involved in fatty acid oxidation. This work is important because it expands our understanding about the adaptation of marine bacteria to the increased availability of DMSP about 250 million years ago.

Dimethylsulfoniopropionate (DMSP) is the most abundant low-molecular-weight organic compound in marine surface water and source of dimethyl sulfide (DMS), a climatically active gas that connects the marine and terrestrial sulfur cycles. Marine bacteria are the major DMSP consumers, either generating DMS or consuming DMSP as a source of reduced carbon and sulfur.

The 2,835-bp open reading frame of ckxgl74A (Locus_tag CALKRO_RS04315) with a natural carbohydrate module (CBM3b) from thermophilic anaerobic microorganism Caldicellulosiruptor kronotskyensis encodes a calculated 104-kDa of GH74 xyloglucanase Ckxgl74A. The purified recombinant Ckxgl74A expressed in Escherichia coli BL21 (DE3) revealed its optimal pH of 4.5 and temperature of 80 °C. The Ckxgl74A was stable over a temperature no more than 70 °C and a pH range of 4.5 to 5.0. Kinetic experiments with xyloglucan as a substrate gave a Km of 2.29 ± 0.04 mg mL-1, Vmax of 22.98 ± 0.02 mol mg-1 min-1, and kcat of 66.98 ± 0.01 s-1. Its activity could be activated by Ca2+ approximately two folds, while being significantly inhibited by Cu2+. These results showed that Ckxgl74A could be utilized in acid condition and possessed a good thermostability.