ARTICLE https://doi.org/10.1038/s41467-020-18434-4 OPEN Bacteria are important dimethylsulfoniopropionate producers in marine aphotic and high-pressure environments Yanfen Zheng 1,6, Jinyan Wang 1,6, Shun Zhou 1, Yunhui Zhang1, Ji Liu 1, Chun-Xu Xue 1, Beth T. Williams2, Xiuxiu Zhao1, Li Zhao3, Xiao-Yu Zhu1, Chuang Sun 1, Hong-Hai Zhang4, Tian Xiao3, ✉ ✉ Gui-Peng Yang 4, Jonathan D. Todd 2 & Xiao-Hua Zhang 1,5 1234567890():,; Dimethylsulfoniopropionate (DMSP) is an important marine osmolyte. Aphotic environments are only recently being considered as potential contributors to global DMSP production. Here, our Mariana Trench study reveals a typical seawater DMSP/dimethylsulfide (DMS) profile, with highest concentrations in the euphotic zone and decreased but consistent levels below. The genetic potential for bacterial DMSP synthesis via the dsyB gene and its transcription is greater in the deep ocean, and is highest in the sediment.s DMSP catabolic potential is present throughout the trench waters, but is less prominent below 8000 m, perhaps indi- cating a preference to store DMSP in the deep for stress protection. Deep ocean bacterial isolates show enhanced DMSP production under increased hydrostatic pressure. Further- more, bacterial dsyB mutants are less tolerant of deep ocean pressures than wild-type strains. Thus, we propose a physiological function for DMSP in hydrostatic pressure protection, and that bacteria are key DMSP producers in deep seawater and sediment. 1 College of Marine Life Sciences, and Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China. 2 School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK. 3 Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China. 4 MOE Key Laboratory of Marine Chemistry Theory and Technology, College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100, China. 5 Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory ✉ for Marine Science and Technology, Qingdao 266237, China. 6These authors contributed equally: Yanfen Zheng, Jinyan Wang. email: jonathan.todd@uea. ac.uk; [email protected] NATURE COMMUNICATIONS | (2020) 11:4658 | https://doi.org/10.1038/s41467-020-18434-4 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18434-4 etagrams of dimethylsulfoniopropionate (DMSP) are pro- ml−1 DMSP and 4.97 × 10−3 nmol ml−1 DMS) and at lower but duced annually1 by marine algae, corals, plants, and het- relatively stable levels (0.96–2.39 × 10−3 nmol ml−1 DMSP and P 2 −3 −1 erotrophic bacteria . In these organisms, DMSP is proposed 0.15–1.06 × 10 nmol ml DMS) in the aphotic waters below 200 to act as an osmolyte, cryoprotectant, predator deterrent, and/or m(Fig.1b, c and Supplementary Table 1). It should be noted that a antioxidant3–5. DMSP is synthesized from methionine (Met) via small portion of this ‘background DMSP’ (<1 nM) detected through three distinct synthesis pathways (Supplementary Fig. 1)3–5. The alkaline hydrolysis may arise from other organic sulfur compounds homologous dsyB (in bacteria) and DSYB (in algae) genes, and that also release DMS upon chemical lysis29. Heterotrophic bacteria, TpMMT in the diatom Thalassiosira pseudonana encode the key photosynthetic phytoplankton (~1.5% of the total microbial com- methylthiohydroxybutyrate methyltransferase enzyme of the munity data determined by metagenomics and 16S rRNA gene transamination pathway6–8. In bacteria producing DMSP via the amplicon analyses), picoeukaryotes, Prochlorococcus,andSynecho- Met methylation pathway, mmtN encodes the key Met methyl- coccus were most abundant in the surface waters with the highest transferase enzyme9,10. Recent studies suggest bacteria are likely seawater DMSP concentration (Supplementary Figs. 2 and 3). Bac- important DMSP producers in coastal sediments, which have far teria were present consistently at 105 cells ml−1 levels throughout the higher DMSP-standing stocks than surface seawater samples water column, whereas the phototrophs were barely detected below where phytoplankton likely drive DMSP production7,10. the first 200 m (Supplementary Fig. 2). Surface water cyanobacteria DMSP, released into the environment through grazing and/or likely take up DMSP30 but are unlikely to be significant DMSP virus-induced lysis, provides a key source of carbon, reduced producersorcyclers,asfewareproventosynthesizeit(andatvery sulfur, and/or energy for microbial communities11,12. Many low levels)31 and none contain known DMSP synthesis or catabolic bacteria and phytoplankton catabolize DMSP via diverse DMSP genes. Dinoflagellates of the Dinophysis genus, e.g., Dinophysis acu- lyase enzymes to generate the climate-active volatile dimethyl- minata that contains DSYB, has intracellular DMSP levels of 477 mM sulfide (DMS)2,11. DMS is an info-chemical13,14 and the largest and large cells (30–120 µm)32, and were the most abundant surface biogenic source of atmospheric sulfur, with roles in cloud for- water phytoplankton (up to 73% of detected phytoplankton) (Sup- mation and, potentially, climate regulation15,16. Alternatively, plementary Fig. 3). These phytoplankton were likely the major bacteria with the DMSP demethylase enzyme (DmdA) deme- contributors to the DMSP levels detected in the photic waters, thylate DMSP, which is thought to be quantitatively more although Picoeukaryotes, proposed to contain DSYB7,andDMSP- important than lysis17,18. producing bacteria (see below) likely also contributed. However, no Seawater DMSP levels in the photic zone (above 200 m) vary eukaryotic DMSP synthesis genes (DSYB or TpMMT)weredetected from 1 to 100 nM in the oligotrophic ocean19–23 to micromolar in any metagenomes (Table 1), even from the surface waters, perhaps levels in phytoplankton blooms24,25, and are generally highest in reflecting the need for deeper sequencing of these waters where chlorophyll a (Chl-a) maximum layers21. Marine aphotic sea- phytoplanktonarefarlessabundantthanbacteria. waters (below 200 m) have lower DMSP levels (~1.0–3.3 nM) in The deep ocean surface sediment DMSP concentrations comparison26,27, but represent a much larger global volume. (3.15–6.14 nmol g−1) were two to three orders of magnitude There are few analyses of DMSP in deep-ocean sediment and higher than in the corresponding seawater samples per equivalent seawater2, and none investigating bacterial DMSP production and mass (ml vs. g) (Fig. 1c and Supplementary Table 1), consistent cycling. Recently, a 4500 m deep Mariana Trench sediment with previous observations of coastal sediments10,33. Given the sample was shown to have high dsyB transcript levels and far cold, dark, and high-pressure deep-sediment and -water condi- higher DMSP levels than in surface water samples10, highlighting tions where few live phytoplankton are present (Supplementary the need for further surveys of deep ocean organosulfur cycling. Figs. 2 and 3), it is unlikely these phototrophs produced the Following the hypothesis that bacteria are important DMSP observed aphotic DMSP in situ, although some is expected to producers in marine aphotic environments, the microbes syn- arise from sinking particles, e.g., dead algae and/or fecal thesizing and catabolizing DMSP were examined in seawater and pellets34,35. However, considering the high DMSP turnover rates sediment samples (surface water—10,500 m depths) from the in photic seawater samples4,36, it is unlikely that photic-produced Challenger Deep of the Mariana Trench (Fig. 1). The DMSP and DMSP is the source of all aphotic DMSP. We propose that DMS stocks were determined in depth-profiled seawater and bacterial DMSP synthesis is likely an important contributor to sediment samples, together with bacterial DMSP synthesis, cata- deep-sea sediment and seawater DMSP levels. To test this bolic gene, and transcript abundance by metagenomics and hypothesis, the distribution and activity of DMSP-producing quantitative PCR (qPCR) analyses. Furthermore, deep ocean bacteria was investigated in Challenger Deep samples. bacteria were isolated and used to explore a role for DMSP in hydrostatic pressure tolerance. This work provides insights into the bacterial contribution to DMSP production and function in Vertical distribution of DMSP synthesis genes. Bacteria with the deep ocean. DsyB or MmtN (and thus the potential to produce DMSP) were always or mostly present, respectively, in all seawater and sedi- ment samples (Fig. 2 and Table 1), and their environmental DsyB Results and MmtN sequences clustered with ratified enzymes (Supple- Environmental parameters of deep ocean seawater and sedi- mentary Figs. 4 and 5). Similar proportions of free-living (FL; ment. Challenger Deep seawater and surface sediment samples 0.22–3 μm) and particle-associated (PA; >3 μm) bacteria, which were taken from its entire ~11,000 depth profile (Fig. 1aand dominated the metagenomes of both these fractions, contained Supplementary Table 1). The clines in temperature (29.8 °C in DMSP biosynthesis and catabolic genes (Figs. 2a and 3a, and surface waters, decreasing to ~1.0 °C below 3000 m) and pres- Table 1), indicating that size fractionation is not a foolproof sure (0.1 MPa in surface waters to ~104 MPa at the bottom of method of separating DMSP-producing bacteria from phyto- the trench) were recorded. The waters were oxic throughout the plankton. Bacteria with dysB were shown by qPCR and metage-