1. Ecology
  2. Microbiology and Infectious Disease
Download icon

A novel ATP dependent dimethylsulfoniopropionate lyase in bacteria that releases dimethyl sulfide and acryloyl-CoA

  1. Chun-Yang Li
  2. Xiu-Juan Wang
  3. Xiu-Lan Chen
  4. Qi Sheng
  5. Shan Zhang
  6. Peng Wang
  7. Mussa Quareshy
  8. Branko Rihtman
  9. Xuan Shao
  10. Chao Gao
  11. Fuchuan Li
  12. Shengying Li
  13. Weipeng Zhang
  14. Xiao-Hua Zhang
  15. Gui-Peng Yang
  16. Jonathan D Todd
  17. Yin Chen
  18. Yu-Zhong Zhang  Is a corresponding author
  1. State Key Lab of Microbial Technology, Marine Biotechnology Research Center, Shandong University, China
  2. College of Marine Life Sciences, Ocean University of China, China
  3. Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, China
  4. School of Life Sciences, University of Warwick, United Kingdom
  5. National Glycoengineering Research Center and Shandong Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, China
  6. Frontiers Science Center for Deep Ocean Multispheres and Earth System, Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, China
  7. School of Biological Sciences, University of East Anglia, Norwich Research Park, United Kingdom
  8. Marine Biotechnology Research Center, State Key Laboratory of Microbial Technology, Shandong University, China
Research Article
  • Cited 0
  • Views 824
  • Annotations
Cite this article as: eLife 2021;10:e64045 doi: 10.7554/eLife.64045

Abstract

Dimethylsulfoniopropionate (DMSP) is an abundant and ubiquitous organosulfur molecule in marine environments with important roles in global sulfur and nutrient cycling. Diverse DMSP lyases in some algae, bacteria, and fungi cleave DMSP to yield gaseous dimethyl sulfide (DMS), an infochemical with important roles in atmospheric chemistry. Here, we identified a novel ATP-dependent DMSP lyase, DddX. DddX belongs to the acyl-CoA synthetase superfamily and is distinct from the eight other known DMSP lyases. DddX catalyses the conversion of DMSP to DMS via a two-step reaction: the ligation of DMSP with CoA to form the intermediate DMSP-CoA, which is then cleaved to DMS and acryloyl-CoA. The novel catalytic mechanism was elucidated by structural and biochemical analyses. DddX is found in several Alphaproteobacteria, Gammaproteobacteria, and Firmicutes, suggesting that this new DMSP lyase may play an overlooked role in DMSP/DMS cycles.

eLife digest

The global sulfur cycle is a collection of geological and biological processes that circulate sulfur-containing compounds through the oceans, rocks and atmosphere. Sulfur itself is essential for life and important for plant growth, hence its widespread use in fertilizers.

Marine organisms such as bacteria, algae and phytoplankton produce one particular sulfur compound, called dimethylsulfoniopropionate, or DMSP, in massive amounts. DMSP made in the oceans gets readily converted into a gas called dimethyl sulfide (DMS), which is the largest natural source of sulfur entering the atmosphere. In the air, DMS is converted to sulfate and other by-products that can act as cloud condensation nuclei, which, as the name suggests, are involved in cloud formation. In this way, DMS can influence weather and climate, so it is often referred to as ‘climate-active’ gas.

At least eight enzymes are known to cleave DMSP into DMS gas with a few by-products. These enzymes are found in algae, bacteria and fungi, and are referred to as lyases, for the way they breakdown their target compounds (DMSP, in this case). Recently, researchers have identified some bacteria that produce DMS from DMSP without using known DMSP lyases. This suggests there are other, unidentified enzymes that act on DMSP in nature, and likely contribute to global sulfur cycling.

Li, Wang et al. set out to uncover new enzymes responsible for converting the DMSP that marine bacteria produce into gaseous DMS. One new enzyme called DddX was identified and found to belong to a superfamily of enzymes quite separate to other known DMSP lyases. Li, Wang et al. also showed how DddX drives the conversion of DMSP to DMS in a two-step reaction, and that the enzyme is found across several classes of bacteria. Further experiments to characterise the protein structure of DddX also revealed the molecular mechanism for its catalytic action.

This study offers important insights into how marine bacteria generate the climatically important gas DMS from DMSP, leading to a better understanding of the global sulfur cycle. It gives microbial ecologists a more comprehensive perspective of these environmental processes, and provides biochemists with data on a family of enzymes not previously known to act on sulfur-containing compounds.

Introduction

The organosulfur molecule dimethylsulfoniopropionate (DMSP) is produced in massive amounts by many marine phytoplankton, macroalgae, angiosperms, bacteria, and animals (Curson et al., 2018; Stefels, 2000; Otte et al., 2004; Curson et al., 2017; Raina et al., 2013). DMSP can function as an antioxidant, osmoprotectant, predator deterrent, cryoprotectant, protectant against hydrostatic pressure, chemoattractant and may enhance the production of quorum-sensing molecules (Sunda et al., 2002; Cosquer et al., 1999; Wolfe et al., 1997; Karsten et al., 1996; Zheng et al., 2020; Seymour et al., 2010; Johnson et al., 2016). DMSP also has important roles in global sulfur and nutrient cycling (Kiene et al., 2000; Charlson et al., 1987). Environmental DMSP can be taken up and catabolised as a carbon and/or sulfur source by diverse microbes, particularly bacteria (Curson et al., 2011b). DMSP catabolism can release volatile dimethyl sulfide (DMS) and/or methanethiol (MeSH) (Reisch et al., 2011a). DMS is a potent foraging cue for diverse organisms (Nevitt, 2011) and the primary biological source of sulfur transferred from oceans to the atmosphere (Andreae, 1990), which may participate in the formation of cloud condensation nuclei, and influence the global climate (Vallina and Simó, 2007).

Bacteria can metabolize DMSP via three known pathways, the demethylation pathway (Howard et al., 2006), the recently reported oxidation pathway (Thume et al., 2018), and the lysis pathway (Curson et al., 2011b; Figure 1). The nomenclature of these pathways is based on the reaction type of the enzyme catalyzing the first step of DMSP catabolism. In the demethylation pathway, DMSP demethylase DmdA first demethylates DMSP to produce methylmercaptopropionate (MMPA) (Howard et al., 2006; Reisch et al., 2008), which can be further catabolized to MeSH and acetaldehyde (Figure 1; Reisch et al., 2011b; Bullock et al., 2017; Shao et al., 2019). In the oxidation pathway, DMSP is oxidized to dimethylsulfoxonium propionate (DMSOP), which is further metabolized to dimethylsulfoxide (DMSO) and acrylate; however, enzymes involved in this pathway are unknown (Thume et al., 2018; Figure 1).

Figure 1 with 2 supplements see all
Metabolic pathways for DMSP degradation.

Different pathways are shown in different colors. The demethylation of DMSP by DmdA produces MMPA (in purple). The oxidation of DMSP produces DMSOP (in yellow). In the lysis pathway (in blue), DMSP lyase DddP, DddL, DddQ, DddW, DddK, DddY, or Alma1 converts DMSP to acrylate and DMS, DddD converts DMSP to 3-HP-CoA and DMS, using acetyl-CoA as a CoA donor, and the newly identified DddX in this study converts DMSP to acryloyl-CoA and DMS, with ATP and CoA as co-substrates. Dotted lines represent unconfirmed steps of the DddX DMSP lysis pathway that we propose in this study. The protein families of enzymes involved in the first step of each pathway are indicated. The protein family of DddX and the products of its catalysis are highlighted in red color. THF, tetrahydrofolate; MMPA, methylmercaptopropionate; 3-HP, 3-hydroxypropionate; DMSOP, dimethylsulfoxonium propionate; DMSO, dimethylsulfoxide.

In the lysis pathway, diverse lyases cleave DMSP to produce DMS and acrylate or 3-hydroxypropionate-CoA (3-HP-CoA), which are further metabolized by ancillary enzymes (Curson et al., 2011b; Johnston et al., 2016; Figure 1). There is large biodiversity in DMSP lysis, with eight different known DMSP lyases that encompass four distinct protein families (DddD a CoA-transferase; DddP a metallopeptidase; cupin containing DddL, DddQ, DddW, DddK, and DddY; and Alma1 an aspartate racemase) functioning in diverse marine bacteria, algae, and fungi (Figure 1; Curson et al., 2011b; Johnston et al., 2016). With the exception of DddD, which catalyzes an acetyl-CoA-dependent CoA transfer reaction, all other DMSP lyases directly cleave DMSP (Bullock et al., 2017; Todd et al., 2007; Alcolombri et al., 2014; Lei et al., 2018; Li et al., 2014). Recently, several bacterial isolates were reported to produce DMS from DMSP but lack known DMSP lyases in their genomes (Liu et al., 2018; Zhang et al., 2019), suggesting the presence of novel enzyme(s) for DMSP degradation in nature.

A common feature of previously characterized DMSP metabolic pathways is that the metabolites (i.e. MMPA, acrylate) need to be ligated with CoA for further catabolism (Figure 1; Curson et al., 2011b; Reisch et al., 2011b). Currently, there is no known pathway whereby DMSP is ligated with free CoA, and it is tempting to speculate that there may be such a novel DMSP metabolic pathway. In this study, we screened DMSP-catabolizing bacteria from Antarctic samples, and obtained a strain Psychrobacter sp. D2 that grew on DMSP and produced DMS. Genetic and biochemical work showed that Psychrobacter sp. D2 possesses a novel DMSP lyase termed DddX for DMSP catabolism (Figure 1). DddX is an ATP-dependent DMSP lyase which catalyzes a two-step reaction: the ligation of DMSP and CoA, and the cleavage of DMSP-CoA to produce DMS and acryloyl-CoA. We further solved the crystal structure of DddX and elucidated the molecular mechanism for its catalysis based on structural and biochemical analyses. DddX is found in both Gram-negative and Gram-positive bacteria. Our results provide novel insights into the microbial metabolism of DMSP by this novel enzyme.

Results

A potentially novel DMSP lyase in a conventional DMSP catabolic gene cluster

Using DMSP (5 mM) as the sole carbon source, DMSP-catabolizing bacteria were isolated from five Antarctic samples including alga, sediments, and seawaters (Figure 2—figure supplement 1, Supplementary file 1a). In total, 175 bacterial strains were obtained (Figure 2—figure supplement 1B). Among these bacterial strains, Psychrobacter sp. D2, a marine gammaproteobacterium, grew well in the medium containing DMSP as the sole carbon source, but not acrylate (Figure 2A). Moreover, gas chromatography (GC) analysis showed that Psychrobacter sp. D2 could catabolize DMSP and produce DMS (44.8 ± 1.8 nmol DMS min–1 mg protein–1) (Figure 2B).

Figure 2 with 3 supplements see all
The utilization of DMSP by Psychrobacter sp. D2 and the putative DMSP-catabolizing gene cluster in its genome.

(A) The growth curve of Psychrobacter sp. D2 on DMSP, sodium pyruvate or acrylate as sole carbon source (5 mM) at 15°C. The error bar represents standard deviation of triplicate experiments. (B), GC detection of DMS production from DMSP by strain D2. The culture medium without bacteria was used as the control. The DMS standard was used as a positive control. Psychrobacter sp. D2 could catabolize DMSP and produce DMS (44.8 ± 1.8 nmol DMS min–1 mg protein–1). (C), RT-qPCR assay of the transcriptions of the genes 1696, 1697, 1,698, and 1,699 in Psychrobacter sp. D2 in response to DMSP in the marine broth 2,216 medium. The bacterium cultured without DMSP in the same medium was used as the control. The recA gene was used as an internal reference. The error bar represents standard deviation of triplicate experiments. The locus tags of 1696, 1697, 1,698, and 1,699 are H0262_08195, H0262_08200, H0262_08205, and H0262_08210, respectively. (D), Genetic organization of the putative DMSP-catabolizing gene cluster. Reported DMSP catabolic/transport gene clusters from Psychrobacter sp. J466, Pseudomonas sp. J465, Marinomonas sp. MWYL1, and Halomonas sp. HTNK1 are shown (Todd et al., 2007; Todd et al., 2010; Curson et al., 2010; Curson et al., 2011b). The dashed vertical line indicates a breakpoint in dddB in the cosmid library of Pseudomonas sp. J466 (Curson et al., 2010).

Figure 2—source data 1

The growth curve of Psychrobacter sp. D2 on DMSP, sodium pyruvate or acrylate as sole carbon source.

https://cdn.elifesciences.org/articles/64045/elife-64045-fig2-data1-v2.xlsx
Figure 2—source data 2

GC detection of DMS production from DMSP by strain D2.

https://cdn.elifesciences.org/articles/64045/elife-64045-fig2-data2-v2.xlsx
Figure 2—source data 3

RT-qPCR assay of the transcriptions of the genes 1696, 1697, 1,698, and 1,699 in Psychrobacter sp. D2.

https://cdn.elifesciences.org/articles/64045/elife-64045-fig2-data3-v2.xlsx

To identify the genes involved in DMSP degradation in Psychrobacter sp. D2, we sequenced its genome and searched homologs of known DMSP lyases. However, no homologs of known DMSP lyases with amino acid sequence identity higher than 30% were found in its genome (Supplementary file 1b), implying that this strain may possess a novel enzyme or a novel pathway for DMSP catabolism. We then sequenced the transcriptomes of this strain when grown with and without DMSP as the sole carbon source. Transcriptional data analyses showed that the transcripts of four genes (1696, 1697, 1,698, and 1699) that compose a gene cluster were all highly upregulated (Figure 2—figure supplement 2) when DMSP was supplied as the sole carbon source, which was further confirmed by RT-qPCR analysis (Figure 2C). These results suggest that this gene cluster may participate in DMSP catabolism within Psychrobacter sp. D2.

In the gene cluster, 1696 is annotated as a betaine-carnitine-choline transporter (BCCT), sharing 32% amino acid identity with DddT, the predicted DMSP transporter in Marinomonas sp. MWYL1 (Sun et al., 2012; Todd et al., 2007); 1,697 is annotated as an acetate-CoA ligase, and shares 26% sequence identity with the acetyl-CoA synthetase (ACS) in Giardia lamblia (Sánchez et al., 2000); 1,698 is annotated as an aldehyde dehydrogenase, sharing 72% sequence identity with DddC in Marinomonas sp. MWYL1 (Todd et al., 2007); and 1,699 is annotated as an alcohol dehydrogenase, sharing 65% sequence identity with DddB in Marinomonas sp. MWYL1 (Todd et al., 2007). DddT, DddC, and DddB have been reported to be involved in DMSP import and catabolism (Sun et al., 2012; Todd et al., 2007; Todd et al., 2010). The pattern of the identified gene cluster 1696–1699 in Psychrobacter sp. D2 is similar to the patterns of those DMSP-catabolizing clusters reported in Pseudomonas, Marinomonas, and Halomonas, in which dddT, dddB and dddC are clustered with the DMSP lyase gene dddD¸ but which is missing in 1696–1699 and is replaced by 1,697 (Todd et al., 2007; Todd et al., 2010; Curson et al., 2010; Figure 2D). These data further support that the 1696–1699 gene cluster is involved in Psychrobacter sp. D2 DMSP catabolism and 1697 encodes a DMSP lyase equivalent to DddD. However, the sequence identity between 1,697 and DddD is less than 15%, suggesting that 1,697 is unlikely a DddD homolog. With these data we predicted that 1,697 encodes a novel DMSP lyase in Psychrobacter sp. D2, which we term as DddX hereafter.

The essential role of DddX in DMSP degradation in Psychrobacter Sp. D2

To identify the possible function of dddX in DMSP catabolism, we first deleted the majority of the dddX gene within the Psychrobacter sp. D2 genome to generate a ΔdddX mutant strain (Figure 2—figure supplement 3). The ΔdddX mutant was unable to grow on DMSP as the sole carbon source, but its ability to utilize DMSP was fully restored to wild type levels by cloned of dddX (in pBBR1MCS-dddX) (Figure 3A), indicating that dddX is essential for strain D2 to utilize DMSP. Furthermore, the ΔdddX mutant lost DMSP lyase activity, that is it no longer produced DMS when cultured in marine broth 2,216 medium with DMSP. DMSP lyase activity was fully restored to wild type levels in the complemented strain (ΔdddX/pBBR1MCS-dddX) (Figure 3B), indicating that dddX encodes a functional DMSP lyase enzyme degrading DMSP to DMS.

Figure 3 with 6 supplements see all
The function of Psychrobacter sp. D2 dddX in DMSP metabolism.

(A) Growth curves of the wild-type strain D2, the ΔdddX mutant, the complemented mutant (ΔdddX/pBBR1MCS-dddX), and the ΔdddX mutant complemented with an empty vector (ΔdddX/pBBR1MCS). All strains were grown with DMSP (5 mM) as the sole carbon source. The error bar represents standard deviation of triplicate experiments. (B), Detection of DMS production from DMSP degradation by the wild-type strain D2, the ΔdddX mutant, the complemented mutant ΔdddX/pBBR1MCS-dddX, and the mutant complimented with an empty vector ΔdddX/pBBR1MCS. The error bar represents standard deviation of triplicate experiments. (C), GC detection of DMS production from DMSP lysis catalyzed by the recombinant DddX. The reaction system without DddX was used as the control. DddX maintained a specific activity of ~8.0 μmol min–1 mg protein–1 at 20°C, pH 8.0.( D), HPLC analysis of the enzymatic activity of the recombinant DddX on DMSP at 260 nm. The peak of the unknown product is indicated with a red arrow. The reaction system without DddX was used as the control. (E), LC-MS analysis of the unknown product. (F), HPLC analysis of the intermediate of DddX catalysis at 260 nm. The HPLC system was coupled to a mass spectrometer for m/z determination. The reaction system without DddX was used as the control.

Figure 3—source data 1

Growth curves of the wild-type strain D2, the ΔdddX mutant, the complemented mutant (ΔdddX/pBBR1MCS-dddX), and the ΔdddX mutant complemented with an empty vector (ΔdddX/pBBR1MCS).

https://cdn.elifesciences.org/articles/64045/elife-64045-fig3-data1-v2.xlsx
Figure 3—source data 2

Detection of DMS production from DMSP degradation by the wild-type strain D2, the ΔdddX mutant, the complemented mutant ΔdddX/pBBR1MCS-dddX, and the mutant complimented with an empty vector ΔdddX/pBBR1MCS.

https://cdn.elifesciences.org/articles/64045/elife-64045-fig3-data2-v2.xlsx
Figure 3—source data 3

GC detection of DMS production from DMSP lysis catalyzed by the recombinant DddX.

https://cdn.elifesciences.org/articles/64045/elife-64045-fig3-data3-v2.xlsx
Figure 3—source data 4

HPLC analysis of the enzymatic activity of the recombinant DddX on DMSP.

https://cdn.elifesciences.org/articles/64045/elife-64045-fig3-data4-v2.xlsx

DddX is an ATP-dependent DMSP lyase and its kinetic analysis

To verify the enzymatic activity of DddX on DMSP, we cloned the dddX gene, overexpressed it in Escherichia coli BL21 (DE3), and purified the recombinant DddX (Figure 3—figure supplement 1). Sequence analysis suggests that DddX is an acetate-CoA ligase, which belongs to the acyl-CoA synthetase (ACD) superfamily and requires CoA and ATP as co-substrates for catalysis (Musfeldt and Schonheit, 2002; Mai and Adams, 1996). Thus, we added CoA and ATP into the reaction system when measuring the enzymatic activity of the recombinant DddX on DMSP. GC analysis showed that the recombinant DddX directly acted on DMSP and produce DMS (Figure 3C). HPLC analysis uncovered ADP and an unknown product as DMS co-products (Figure 3D). The chromatographic retention time of the unknown product was consistent with it being acryloyl-CoA (Wang et al., 2017; Cao et al., 2017). Indeed, liquid chromatography-mass spectrometry (LC-MS) analysis found the molecular weight (MW) of the unknown product to be 822.1317, exactly matching acryloyl-CoA (Figure 3E). These data demonstrate that DddX is a functional ATP-dependent DMSP lyase that can catalyze DMSP degradation to DMS and acryloyl-CoA.

The biochemical results above suggest that DddX catalyzes a two-step degradation of DMSP, a CoA ligation reaction and a cleavage reaction. To perform this two-step reaction, there are two alternative pathways: (i), DMSP is first cleaved to form DMS and acrylate, and subsequently CoA is ligated with acrylate (Figure 3—figure supplement 2A). In this case, the intermediate acrylate is produced. (ii), CoA is primarily ligated with DMSP to form DMSP-CoA. Then, DMSP-CoA is cleaved, producing DMS and acryloyl-CoA (Figure 3—figure supplement 2B). In this scenario, the intermediate DMSP-CoA is produced. To determine the catalytic process of DddX, we monitored the occurrence of acrylate and/or DMSP-CoA in the reaction system via LC-MS. While acrylate was not detectable in the reaction system, a small peak of DMSP-CoA emerged after a 2 min reaction (Figure 3F), indicating that DMSP-CoA is primarily formed in the catalytic reaction of DddX, which is then cleaved to generate DMS and acryloyl-CoA.

Knowing the DddX enzyme activity, we examined its in vitro properties. The DddX enzyme had an optimal temperature and pH of 40°C and 8.5, respectively (Figure 3—figure supplement 3A and B). The apparent KM of DddX for ATP and CoA was 2.5 mM (Figure 3—figure supplement 3C) and 0.4 mM (Figure 3—figure supplement 3D), respectively. DddX had an apparent KM value of 0.4 mM for DMSP (Figure 3—figure supplement 3E), which is lower than that of most other reported DMSP lyases and the DMSP demethylase DmdA (Supplementary file 1c). The kcat of DddX for DMSP was 0.7 s–1, with an apparent kcat/KM of 1.6 × 103 M–1 s–1. The catalytic efficiency of DddX toward DMSP is higher than known DMSP lyases DddK, DddP, DddD, but lower than DddY and Alma1 (Supplementary file 1c).

Despite DddX belongs to the ACD superfamily, the amino acid identity between DddX and known ACD enzymes is relatively low, with the highest being 26 % between DddX and the Giardia lamblia ACS (Sánchez et al., 2000). The kcat/KM value of DddX towards DMSP is lower than several reported ACS enzymes towards acetate (Chan et al., 2011; You et al., 2017). Because ACS enzymes were reported to have promiscuous activity toward different short chain fatty acids, such as acetate and propionate (Patel and Walt, 1987), we tested the substrate specificity of DddX. The recombinant DddX exhibited no activity towards acetate or propionate (Figure 3—figure supplement 4), and the presence of acetate or propionate had little effects on the enzymatic activity of DddX toward DMSP (Figure 3—figure supplement 5), indicating that DddX cannot utilize acetate or propionate as a substrate. Furthermore, we tested the ability of the strain D2 to grow with acetate or propionate as the sole carbon source. The wild-type strain D2 could use acetate or propionate as sole carbon source but deletion of dddX has little effect on the growth of strain D2 on these substrates (Figure 3—figure supplement 6), suggesting that dddX is unlikely to be involved in acetate and propionate catabolism. Together, these results indicate that DddX does not function as an acetate-CoA ligase.

The crystal structure and the catalytic mechanism of DddX

To elucidate the structural basis of DddX catalysis, we solved the crystal structure of DddX in complex with ATP by the single-wavelength anomalous dispersion method using a selenomethionine derivative (Se-derivative) (Supplementary file 1d). Although there are four DddX monomers arranged as a tetramer in an asymmetric unit (Figure 4—figure supplement 1A), gel filtration analysis indicated that DddX maintains a dimer in solution (Figure 4—figure supplement 1B). Each DddX monomer contains a CoA-binding domain and an ATP-grasp domain (Figure 4A), with one loop (Gly280-Tyr300) of the CoA-binding domain inserting into the ATP-grasp domain. ATP is bound in DddX mainly via hydrophilic interactions, including hydrogen bonds and salt bridges (Figure 4B). The overall structure of DddX is similar to that of NDP-forming acetyl-CoA synthetase ACD1 (Weiße et al., 2016; Figure 4—figure supplement 2), with a root mean square deviation (RMSD) between these two structures of 4.6 Å over 581 Cα atoms. ACD1 consists of separate α- and β-subunits (Weiße et al., 2016), which corresponds to the CoA-binding domain and the ATP-grasp domain of DddX, respectively.

Figure 4 with 5 supplements see all
Structural and mutational analyses of DddX.

(A) The overall structure of the DddX monomer. The DddX molecule contains a CoA-binding domain (colored in pink) and an ATP-grasp domain (colored in wheat). The loop region from the CoA-binding domain inserting into the ATP-grasp domain is colored in cyan. The ATP molecule is shown as sticks. (B) Residues of DddX involved in binding ATP. The 2Fo - Fc densities for ATP are contoured in blue at 2.0σ. Residues of DddX involved in binding ATP are colored in green. (C) Enzymatic activities of DddX and its mutants. The activity of WT DddX was taken as 100%. (D) Structural analysis of the possible catalytic residues for the cleavage of DMSP-CoA. The docked DMSP-CoA molecule and the probable catalytic residues of DddX are shown as sticks.

Both DddX and ACS belong to the ACD superfamily, which also contains the well-studied ATP citrate lyases (ACLY) (Weiße et al., 2016; Verschueren et al., 2019; Hu et al., 2017). The biochemistry of DddX catalysis is similar to that of ACLY, which converts citrate to acetyl-CoA and oxaloacetate with ATP and CoA as co-substrates (Verschueren et al., 2019; Hu et al., 2017). The catalytic processes of enzymes in the ACD superfamily involve a conformational change of a ‘swinging loop’ or ‘phosphohistidine segment’, in which a conserved histidine is phosphorylated (Weiße et al., 2016; Verschueren et al., 2019; Hu et al., 2017). Sequence alignment indicated that His292 of DddX is likely the conserved histidine residue to be phosphorylated, and Gly280-Tyr300 is likely the ‘swinging loop’ (Figure 4—figure supplement 3). In the crystal structure of DddX, His292 from loop Gly280-Tyr300 directly forms a hydrogen bond with the γ-phosphate of ATP (Figure 4B), suggesting a potential for phosphorylation, which is further supported by mutational analysis. Mutation of His292 to alanine abolished the activity of DddX (Figure 4C), indicating the key role of His292 during catalysis. Circular-dichroism (CD) spectroscopy analysis showed that the secondary structure of His292Ala exhibits little deviation from that of wild-type (WT) DddX (Figure 4—figure supplement 4), indicating that the enzymatic activity loss was caused by amino acid replacement rather than by structural change. Altogether, these data suggest that His292 is phosphorylated in the catalysis of DddX on DMSP.

Having solved the crystal structure of the DddX-ATP complex, we next sought to determine the crystal structures of DddX in complex with CoA and DMSP. However, the diffractions of these crystals were poor and all attempts to solve the structures failed. Thus, we docked DMSP and CoA into the structure of DddX. In the docked structure, the CoA molecule is bound in the CoA-binding domain, while the DMSP molecule is bound in the interface between two DddX monomers (Figure 4—figure supplement 5A). Because our biochemical results demonstrated that DMSP-CoA is an intermediate of DddX catalysis (Figure 3F), we further docked DMSP-CoA into DddX. DMSP-CoA also locates between two DddX monomers (Figure 4—figure supplement 5B), and two aromatic residues (Trp391 and Phe435) form cation-π interactions with the sulfonium group of DMSP-CoA (Figure 4—figure supplement 5C). Mutations of these two residues significantly decreased the enzymatic activities of DddX (Figure 4C), suggesting that these residues play important roles in DddX catalysis. To cleave DMSP-CoA into DMS and acryloyl-CoA, a catalytic base is necessary to deprotonate DMSP-CoA. Structure analysis showed that Tyr181, Asp208, and Glu432 are close to the DMSP moiety (Figure 4D) and may function as the general base. Mutational analysis showed that the mutation of Glu432 to alanine abolished the enzymatic activity of DddX, while mutants Tyr181Ala and Asp208Ala still maintained ~40% activities (Figure 4C), indicating that Glu432 is the most probable catalytic residue for the final cleavage of DMSP-CoA. CD spectra of these mutants were indistinguishable from that of WT DddX (Figure 4—figure supplement 4), suggesting that the decrease in the enzymatic activities of the mutants were caused by residue replacement rather than structural alteration of the enzyme.

Based on structural and mutational analyses of DddX, and the reported molecular mechanisms of the ACD superfamily (Weiße et al., 2016; Verschueren et al., 2019; Hu et al., 2017), we proposed the molecular mechanism of DddX catalysis on DMSP (Figure 5). Firstly, His292 is phosphorylated by ATP, forming phosphohistidine (Figure 5A), which will be brought to the CoA-binding domain through the conformational change of the swinging loop Gly280-Tyr300. Next, the phosphoryl group is most likely transferred to DMSP to generate DMSP-phosphate (Figure 5B), which is subsequently attacked by CoA to form DMSP-CoA intermediate (Figure 5C). The last step is the cleavage of DMSP-CoA probably initiated by the base-catalyzed deprotonation of Glu432 (Figure 5D). Finally, acryloyl-CoA and DMS are generated (Figure 5E) and released from the catalytic pocket of DddX.

A proposed mechanism for DMSP cleavage to generate DMS and acryloyl-CoA catalyzed by DddX.

(A) The residue His292 attacks the γ-phosphate of ATP. (B), The phosphoryl group is transferred from phosphohistidine to the DMSP molecule. (C), DMSP-phosphate is attacked by CoA. (D), The residue Glu432 acts as a general base to attack DMSP-CoA. (E), DMS and acryloyl-CoA are generated.

Distribution of DddX in bacteria

We next set out to determine the diversity and distribution of DddX in bacteria with sequenced genomes. We searched the NCBI Reference Sequence Database using the DddX sequence of Psychrobacter sp. D2 as the query. The data presented in Figure 6 showed that DddX homologs are present in several diverse groups of bacteria, including Alphaproteobacteria, Gammaproteobacteria, and Firmicutes. Multiple sequence alignment showed the presence of the key residues involved in phosphorylation (H292), co-ordination of the substrate (e.g. W391) and catalysis (D432), suggesting that these DddX homologs are likely functional in bacterial DMSP catabolism. To further validate that these DddX homologs are indeed functional DMSP degrading enzymes, we chemically synthesized representative dddX sequences from Alphaproteobacteria (Pelagicola sp. LXJ1103), Gammaproteobacteria (Psychrobacter sp. P11G5; Marinobacterium jannaschii), and Firmicutes (Sporosarcina sp. P33). These candidate DddX enzymes were purified and all were shown to degrade DMSP and produce acryloyl-CoA confirming their predicted activity (Figure 6—figure supplement 1). We predict that bacteria containing DddX will have DMSP lyase activity, but this will depend on the expression of this enzyme in the host and substrate availability.

Figure 6 with 1 supplement see all
Distribution of DddX in bacterial genomes.

The phylogenetic tree was constructed using neighbor-joining method in MEGA7. The acetyl-coenzyme A synthetase (ACS) (Weiße et al., 2016) was used as the outgroup. Sequence alignment was inspected for the presence of the key histidine residue (His292) involved in histidine phosphorylation that is known to be important for enzyme activity. A conserved Tyr391 is also found which is involved in cation-pi interaction with DMSP. The BCCT-type or ABC-type transporters for betaine-carnitine-choline-DMSP were found in the neighborhood of DddX in several genomes. Those DddX homologs that are functionally characterized (Figure 6—figure supplement 1) are highlighted in bold.

Discussion

The cleavage of DMSP to produce DMS is a globally important biogeochemical reaction. Although all known DMSP lyases liberate DMS, they belong to different families, and likely evolved independently (Bullock et al., 2017). DddD belongs to the type III acyl CoA transferase family (Todd et al., 2007), DddP to the M24 metallopeptidase family enzyme (Todd et al., 2009), DddL/Q/W/K/Y to the cupin superfamily enzymes (Lei et al., 2018; Li et al., 2017) and Alma1 to the aspartate racemase superfamily (Alcolombri et al., 2015). To the best of our knowledge, DddX represents the first DMSP lyase of the ACD superfamily.

Of the reported DMSP lyases, only DddD catalyzes a two-step reaction which comprises a CoA transfer reaction and a cleavage reaction (Alcolombri et al., 2014). It is deduced that DMSP-CoA will be generated in the catalytic process of DddD (Alcolombri et al., 2014; Curson et al., 2011b; Todd et al., 2007). Despite this similarity, DddX is fundamentally different to DddD. Firstly, the co-substrates of DddX and DddD are different. ATP and CoA are essential co-substrates for the enzymatic activity of DddX, while for DddD catalysis, acetyl-CoA is used as a CoA donor, and ATP is not required (Johnston et al., 2016; Alcolombri et al., 2014). When CoA was replaced by acetyl-CoA in the reaction system, DddX failed to catalyze the cleavage of DMSP (Figure 1—figure supplement 1). Secondly, the products of DddD and DddX are different. DddD converts DMSP to DMS and 3-HP-CoA, whereas DddX produces DMS and acryloyl-CoA from DMSP. Except for DddD and DddX, all the other DMSP lyases cleave DMSP to DMS and acrylate.

It has been reported that accumulation of acryloyl-CoA is toxic to bacteria (Reisch et al., 2013; Wang et al., 2017; Cao et al., 2017; Todd et al., 2012). Thus, Psychrobacter sp. D2 requires an efficient system to metabolize the acryloyl-CoA produced from DMSP lysis by DddX. With the transcription of genes 1,698 and 1699,, directly downstream of dddX and likely co-transcribed with dddX, being significantly enhanced by growth on DMSP, their enzyme products (DddC and DddB) likely participate in the metabolism and detoxification of acryloyl-CoA or downstream metabolites. However, the recombinant 1698 and However, 1699 exhibited no enzymatic activity on acryloyl-CoA.

The Psychrobacter sp. D2 genome also contains acuI and acuH homologs (2674, 0105, 1810, 1,692, and 1695) (Supplementary file 1e), which may directly act on acryloyl-CoA to produce propionate-CoA or 3-HP-CoA (Reisch et al., 2013; Wang et al., 2017; Cao et al., 2017; Todd et al., 2012). If Psychrobacter sp. D2 employs its AcuH homolog to convert acryloyl-CoA to 3-HP-CoA (Cao et al., 2017), then, given the high-sequence identity of 1,698 to DddC and 1699 to DddB, it is possible that these enzymes further catabolize 3-HP-CoA to acetyl-CoA (Alcolombri et al., 2014; Curson et al., 2011b). Furthermore, we showed that the recombinant 0105, an AcuI homolog, could act on acryloyl-CoA to produce propionate-CoA with NADPH as a cofactor (Figure 1—figure supplement 2). Thus, Psychrobacter sp. D2 may also employ an AcuI (i.ei.e. 0105) to convert acryloyl-CoA to propionate-CoA (Figure 1), which would be metabolized through the methylmalonyl-CoA pathway (Reisch et al., 2013).

Several DMSP catabolizing bacteria, e.g. Halomonas HTNK1 with DddD, are reported to utilize acrylate as the carbon source for growth via e.g. acuN, acuK, acuI, acuH, and prpE gene products (Curson et al., 2011a; Reisch et al., 2013; Todd et al., 2010). Despite the presence of several acuN, acuK, acuI, acuH and prpE homologs in its genome (Supplementary file 1e), Psychrobacter sp. D2 could not use acrylate as a sole carbon source (Figure 2A). Thus, Psychrobacter sp. D2 either (i), lacks a functional acrylate transporter; (ii), these homologs that are predicted to be involved in acrylate metabolism are not functional in vivo; or (iii), these genes are not induced by acrylate. Clearly further biochemical and genetic experiments are required to establish the how acryloyl-CoA is catabolized in this bacterium.

Many marine bacteria, especially roseobacters, are reported to metabolize DMSP via more than one pathway (Curson et al., 2011b; Bullock et al., 2017). For example, Ruegeria pomeroyi DSS-3, one of the type strains of the marine Roseobacter clade, possesses both the demethylation and the lysis pathway for DMSP metabolism (Reisch et al., 2013). Moreover, it contains multiple ddd genes (dddQ, dddP and dddW) (Reisch et al., 2013; Todd et al., 2011). DmdA homologs were not identified in the genome of Psychrobacter sp. D2, indicating that the demethylation pathway is absent in strain D2. The fact that the mutant ΔdddX could not produce DMS from DMSP and was unable to grow on DMSP as the sole carbon source suggests that Psychrobacter sp. D2 only possesses one DMSP lysis pathway for DMSP degradation. Why some bacteria have evolved multiple DMSP utilization pathways and some bacteria only possess one pathway awaits further investigation.

Here, we demonstrate that DddX is a functional DMSP lyase present in several isolates of Gammaproteobacteria, Alphaproteobacteria and, notably, Gram-positive Firmicutes, for example in Sporosarcina sp. P33. The distribution of DddX in these bacterial lineages points to the role of horizontal gene transfer (HGT) in the dissemination of dddX in environmental bacteria and this certainly warrants further investigation. Interestingly, DddX is found in several bacterial isolates which were isolated from soil or plant roots, suggesting that DMSP may also be produced in these ecosystems. Finally, it has been reported that many other Gram-positive actinobacteria can make DMS from DMSP (Liu et al., 2018). Interestingly, these Actinobacteria lack dddX and any other known DMSP lyase genes. Thus, there is still more biodviversity in microbial DMSP lyases to be uncovered.

Conclusion

DMSP is widespread in nature and cleavage of DMSP produces DMS, an important mediator in the global sulfur cycle. In this study, we report the identification of a novel ATP-dependent DMSP lyase DddX from marine bacteria. DddX belongs to the ACD superfamily, and catalyzes the conversion of DMSP to DMS and acryloyl-CoA, with CoA and ATP as co-substrates. DddX homologs are found in both Gram-positive and Gram-negative bacterial lineages. This study offers new insights into how diverse bacteria cleave DMSP to generate the climatically important gas DMS.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Psychrobacter sp.)D2This study;Zhang LaboratoryWild-type isolate; Available from Zhang lab
Strain, strain background (Psychrobacter sp.)ΔdddXThis study;Zhang Laboratorythe dddX gene deletion mutant of Psychrobacter sp. D2; Available from Zhang lab
Strain, strain background (Psychrobacter sp.)ΔdddX/pBBR1MCS-dddXThis study;Zhang LaboratoryΔdddX containing pBBR1MCS-dddX plasmid; Available from Zhang lab
Strain, strain background (Psychrobacter sp.)ΔdddX/pBBR1MCSThis study;Zhang LaboratoryΔdddX containing pBBR1MCS plasmid; Available from Zhang lab
Strain, strain background (Escherichia coli)WM3064Dehio and Meyer, 1997Conjugation donor strain
Strain, strain background (Escherichia coli)DH5αVazyme Biotech company (China)Transformed cells for gene cloning
Strain, strain background (Escherichia coli)BL21(DE3)Vazyme Biotech company (China)Transformed cells for gene expression
Recombinant DNA reagentpK18mobsacB-EryWang et al., 2015bGene knockout vector
Recombinant DNA reagentpK18Ery-dddXThis study;Zhang LaboratorypK18mobsacB-Ery containing the homologous arms of the dddX gene of Psychrobacter. sp. D2; Available from Zhang lab
Recombinant DNA reagentpBBR1MCSKovach et al., 1995Broad-host-range cloning vector
Recombinant DNA reagentpBBR1MCS-dddXThis study;Zhang LaboratorypBBR1MCS containing the dddX gene and its promoter of Psychrobacter. sp. D2; Available from Zhang lab
Recombinant DNA reagentpET-22b-dddXThis study;Zhang LaboratoryUsed for dddX expression; Available from Zhang lab
Commercial assay or kitPierce BCA Protein Assay KitThermo, USAProtein assay
Commercial assay or kitBacterial genomic DNA isolation kitBioTeke Corporation, ChinaDNA extraction
Commercial assay or kitRNeasy Mini KitQIAGEN, AmericaRNA extraction
Commercial assay or kitPrimeScript RT reagent KitTakara, JapanReverse transcription
Commercial assay or kitGenome sequencing of Psychrobacter sp. D2Biozeron Biotechnology Co., Ltd, ChinaNCBI: JACDXZ000000000
Commercial assay or kitTranscriptome sequencing of Psychrobacter sp. D2BGI Tech Solutions Co., Ltd, ChinaNCBI: PRJNA646786
Software, algorithmHKL3000 programMinor et al., 2006Diffraction data analysis
Software, algorithmCCP4 program PhaserWinn et al., 2011Diffraction data analysis
Software, algorithmCootEmsley et al., 2010Diffraction data analysis
Software, algorithmPhenixAdams et al., 2010Diffraction data analysis
Software, algorithmPyMOLSchrödinger, LLChttp://www.pymol.org/
Software, algorithmMEGA 7Kumar et al., 2016Phylogenetic analysis

Bacterial strains, plasmids, and growth conditions

Strains and plasmids used in this study are shown in Supplementary file 1f. Isolates were cultured in the marine broth 2,216 medium or the basal medium (Supplementary file 1g) with 5 mM DMSP as the sole carbon source at 15°C–25°C. Psychrobacter sp. D2 was cultured in the marine broth 2,216 medium or the basal medium (Supplementary file 1g) supplied with different carbon sources (sodium pyruvate, acrylate or DMSP at a final concentration of 5 mM) at 15–25°C. The E. coli strains DH5α and BL21(DE3) were grown in the Lysogeny Broth (LB) medium at 37°C. Diaminopimelic acid (0.3 mM) was added to culture the E. coli WM3064 strain.

Isolation of bacterial strains from Antarctic samples

Request a detailed protocol

A total of five samples were collected from the Great Wall Station of Antarctica during the Chinese Antarctic Great Wall Station Expedition in January, 2017. Information of samples is shown in Figure 2—figure supplement 1 and Supplementary file 1a. Algae and sediments were collected using a grab sampler and stored in airtight sterile plastic bags at 4°C. Seawater samples were filtered through polycarbonate membranes with 0.22 μm pores (Millipore Co., United States). The filtered membranes were stored in sterile tubes (Corning Inc, United States) at 4°C. All samples were transferred into a 50 ml flask containing 20 ml 3% (w/v) seasalt solution (SS) and shaken at 100 rpm at 15°C for 2 hr. The suspension obtained was subsequently diluted to 10–6 with sterile SS. An aliquot (200 μl) of each dilution was spread on the basal medium (Supplementary file 1g) plates with 5 mM DMSP as the sole carbon source. The plates were then incubated at 15°C in the dark for 2–3 weeks. Colonies with different appearances were picked up and were further purified by streaking on the marine 2,216 agar plates for at least three passages. The abilities of the colonies for DMSP catabolism were verified in a liquid basal medium with DMSP (5 mM) as the sole carbon source. The isolates were stored at –80°C in the marine broth 2,216 medium containing 20 % (v/v) glycerol.

Sequence analysis of bacterial 16s rRNA genes

Request a detailed protocol

Genomic DNA of the isolates was extracted using a bacterial genomic DNA isolation kit (BioTeke Corporation, China) according to the manufacturer’s instructions. The 16 S rRNA genes of these strains were amplified using the primers 27 F/1492 R (Supplementary file 1h) and sequenced to determine their taxonomy. Pairwise similarity values for the 16 S rRNA gene of the cultivated strains were calculated through the EzBiocloud server (http://www.ezbiocloud.net/) (Yoon et al., 2017).

Bacterial growth assay with DMSP as the sole carbon source

Request a detailed protocol

Cells were grown in the marine broth 2,216 medium, harvested after incubation at 15 °C for 24 hr, and then washed three times with sterile SS. The washed cells were diluted to the same density of OD600 ≈ 2.0, and then 1 % (v/v) cells were inoculated into the basal medium with DMSP, sodium acetate, or sodium propionate (5 mM) as the sole carbon source. The bacteria were cultured in the dark at 15°C. The growth of the bacteria was measured by detecting the OD600 of the cultures at different time points using a spectrophotometer V-550 (Jasco Corporation, Japan).

Quantification of DMS by GC

Request a detailed protocol

To measure the production of DMS, cells were first cultured overnight in the marine broth 2,216 medium, and then washed three times with sterile SS. The washed cells were diluted to the same density of OD600 ≈ 0.3, then diluted 1:10 into vials (Anpel, China) containing the basal medium supplied with 5 mM DMSP as the sole carbon source. The vials were crimp sealed with rubber bungs and incubated for 2 hr at 25°C. The cultures were then assayed for DMS production on a gas chromatograph (GC-2030, Shimadzu, Japan) equipped with a flame photometric detector (Liu et al., 2018). An eight-point calibration curve of DMS standards was used (Curson et al., 2017). Abiotic controls of the basal medium amended with 5 mM DMSP were set up and incubated under the same conditions to monitor the background lysis of DMSP to DMS. Following growth of all bacteria strains in the marine broth 2,216 medium, cells were collected by centrifugation, resuspended in the lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 0.5% glycerol, pH 8.0), and lysed by sonicated. The protein content in the cells was measured by Pierce BCA Protein Assay Kit (Thermo, USA). DMS production is expressed as nmol min–1 mg protein–1.

Transcriptome sequencing of Psychrobacter sp. D2

Request a detailed protocol

Cells of strain D2 were cultured in the marine broth 2,216 medium at 180 rpm at 15°C for 24 hr. The cells were collected and washed three times with sterile SS, and then cultured in sterile SS at 180 rpm at 15°C for 24 hr. Subsequently, the cells were washed twice with sterile SS, and incubated at 4°C for 24 hr. After incubation, the cells were harvested and resuspended in sterile SS, which were used as the resting cells. The resting cells were inoculated into the basal medium with DMSP (5 mM) as the sole carbon source, and incubated at 180 rpm at 15°C. When the OD600 of the cultures reached 0.3, the cells were harvested. The resting cells and those cultured in the basal medium with sodium pyruvate (5 mM) as the sole carbon source were set up as controls. Total RNA was extracted using a RNeasy Mini Kit (QIAGEN, America) according to the manufacturer’s protocol. After validating the quality, RNA samples were sent to BGI Tech Solutions Co., Ltd (China) for transcriptome sequencing and subsequent bioinformatic analysis.

Real-time qPCR analysis

Request a detailed protocol

Cells of Psychrobacter sp. D2 were cultured in the marine broth 2,216 medium at 180 rpm at 15°C to an OD600 of 0.8. Then, cells were induced by 5 mM DMSP, and the control group without DMSP was also set up. After 20 min’s induction, total RNA was extracted using a RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Genomic DNA was removed using gDNA Eraser (TaKaRa, Japan) and cDNA was synthesized using a PrimeScript RT reagent Kit. The qPCR was performed on the Light Cycler II 480 System (Roche, Switzerland) using a SYBR Premix Ex Taq (TaKaRa, Japan). Relative expression levels of target genes were calculated using the LightCycler480 software with the ‘Advanced Relative Quantification’ method. The recA gene was used as an internal reference gene. The primers used in this study are shown in Supplementary file 1h.

Genetic manipulations of Psychrobacter sp. D2

Request a detailed protocol

Deletion of the dddX gene was performed via pK18mobsacB-Ery-based homologue recombination (Wang et al., 2015a). The upstream and downstream homologous sequences of the dddX gene were amplified with primer sets dddX-UP-F/dddX-UP-R and dddX-Down-F/dddX-Down-R, respectively. Next, the PCR fragments were inserted to the vector pK18mobsacB-Ery with HindIII/BamHI as the restriction sites to generate pK18Ery-dddX, which was transferred into E. coli WM3064. The plasmid pK18Ery-dddX was then mobilized into Psychrobacter sp. D2 by intergeneric conjugation with E. coli WM3064. To select for colonies in which the pK18Ery-dddX had integrated into the Psychrobacter sp. D2 genome by a single crossover event, cells were plated on the marine 2,216 agar plates containing erythromycin (25 μg/ml). Subsequently, the resultant mutant was cultured in the marine broth 2,216 medium and plated on the marine 2,216 agar plates containing 10% (w/v) sucrose to select for colonies in which the second recombination event occurred. Single colonies appeared on the plates were streaked on the marine 2,216 agar plates containing erythromycin (25 μg/ml), and colonies sensitive to erythromycin were further validated to be the dddX gene deletion mutants by PCR with primer pairs of dddX-1000-F/dddX-1000-R and dddX-300Up-F/dddX-700Down-R.

For complementation of the ΔdddX mutant, the dddX gene with its native promoter was amplified using the primers set dddX-pBBR1-PF/dddX-pBBR1-PR. The PCR fragment was digested with KpnI and XhoI, and then inserted into the vector pBBR1MCS to generate pBBR1MCS-dddX. This plasmid was then transformed into E. coli WM3064, and mobilized into the ΔdddX mutant by intergeneric conjugation. After mating, the cells were plated on the marine 2,216 agar plates containing kanamycin (80 μg/ml) to select for the complemented mutant. The empty vector pBBR1MCS was mobilized into the ΔdddX mutant using the same protocol. Colony PCR was used to confirm the presence of the transferred plasmid. The strains, plasmids and primers used in this study are shown in Supplementary file 1f and h.

Gene cloning, point mutation, and protein expression and purification

Request a detailed protocol

The 2247 bp full-length dddX gene was amplified from the genome of Psychrobacter sp. D2 by PCR using FastPfu DNA polymerase (TransGen Biotech, China). The amplified gene was then inserted to the NdeI/XhoI restriction sites of the pET-22b vector (Novagen, Germany) with a C-terminal His tag. All the point mutations in DddX were introduced using the PCR-based method and verified by DNA sequencing. The DddX protein and its mutants were expressed in E. coli BL21 (DE3). The cells were cultured in the LB medium with 0.1 mg/ml ampicillin at 37 °C to an OD600 of 0.8–1.0 and then induced at 18°C for 16 hr with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). After induction, cells were collected by centrifugation, resuspended in the lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 0.5% glycerol, pH 8.0), and lysed by pressure crusher. The proteins were first purified by affinity chromatography on a Ni2+-NTA column (GE healthcare, America), and then fractionated by anion exchange chromatography on a Source 15Q column (GE healthcare, America) and gel filtration on a Superdex G200 column (GE healthcare, America). The Se-derivative of DddX was overexpressed in E. coli BL21 (DE3) under 0.5 mM IPTG induction in the M9 minimal medium supplemented with selenomethionine, lysine, valine, threonine, leucine, isoleucine, and phenylalanine. The recombinant Se-derivative was purified using the aforementioned protocol for the wild-type DddX.

Enzyme assay and product identification

Request a detailed protocol

For the routine enzymatic activity assay of the DddX protein, the purified DddX protein (at a final concentration of 0.1 mM) was incubated with 1 mM DMSP, 1 mM CoA, 1 mM ATP, 2 mM MgCl2 and 100 mM Tris-HCl (pH 8.0). The reaction was performed at 37°C for 0.5 hr, and terminated by adding 10% (v/v) hydrochloric acid. The control groups had the same reaction system except that the DddX protein was not added. DMS was detected by GC as described above. Products of acryloyl-CoA and DMSP-CoA were analyzed using LC-MS. Components of the reaction system were separated on a reversed-phase SunFire C18 column (Waters, Ireland) connected to a high performance liquid chromatography (HPLC) system (Dionex, United States). The ultraviolet absorbance of samples was detected by HPLC under 260 nm. The samples were eluted with a linear gradient of 1–20% (v/v) acetonitrile in 50 mM ammonium acetate (pH 5.5) over 24 min. The HPLC system was coupled to an impact HD mass spectrometer (Bruker, Germany) for m/z determination. To determine the optimal temperature for DddX enzymatic activity, reaction mixtures containing 5 mM DMSP, 5 mM CoA, 5 mM ATP, 6 mM MgCl2, 100 mM Tris-HCl (pH 8.5), and 10 μM DddX were incubated at 5–50°C (with a 5°C interval) for 15 min. The optimum pH for DddX enzymatic activity was examined at 40°C (the optimal temperature for DddX enzymatic activity) using Britton-Robinson Buffer (Britton, 1952) with pH from 7.5 to 11.0, with a 0.5 interval. The kinetic parameters of DddX were measured by determining the production of DMS with nonlinear analysis based on the initial rates, and all the measurements were performed at the optimal pH and temperature.

The enzymatic activity of DddX toward sodium acetate or sodium propionate was measured by determining the production of acetyl-CoA or propionyl-CoA using HPLC as described above with DMSP replaced by sodium acetate or sodium propionate. To determine the effects of sodium acetate or sodium propionate on the enzymatic activity of DddX toward DMSP, sodium acetate or sodium propionate at a final concentration of 1 mM, 2 mM or 5 mM were individually added to the reaction mixture. All the measurements were performed at the optimum pH and temperature for DddX.

The enzymatic activity of 0105 (AcuI) toward acryloyl-CoA was measured by determining the production of propionate-CoA using HPLC as described above. The reaction mixture contained 2 mM DMSP, 2 mM CoA, 2 mM ATP, 10 mM MgCl2, 1 mM NADPH, 100 mM Tris-HCl (pH 8.5), 0.1 mM DddX, and 0.9 mM 0105. The reaction was performed at 40°C, pH 8.5 for 2 hr, and terminated by adding 10 % (v/v) hydrochloric acid.

Crystallization and data collection

Request a detailed protocol

The purified DddX protein was concentrated to ~8 mg/ml in 10 mM Tris–HCl (pH 8.0) and 100 mM NaCl. The DddX protein was mixed with ATP (1 mM), and the mixtures were incubated at 0°C for 1 hr. Initial crystallization trials for DddX/ATP complex were performed at 18°C using the sitting-drop vapor diffusion method. Diffraction-quality crystals of DddX/ATP complex were obtained in hanging drops containing 0.1 M lithium sulfate monohydrate, 0.1 M sodium citrate tribasic dihydrate (pH 5.5) and 20 % (w/v) polyethylene glycol (PEG) 1000 at 18°C after 2-week incubation. Crystals of the DddX Se-derivative were obtained in hanging drops containing 0.1 M HEPES (pH 7.5), 10% PEG 6000% and 5% (v/v) (+/-)–2-Methyl-2,4-pentanediol at 18°C after 2-week incubation. X-ray diffraction data were collected on the BL18U1 and BL19U1 beamlines at the Shanghai Synchrotron Radiation Facility. The initial diffraction data sets were processed using the HKL3000 program with its default settings (Minor et al., 2006).

Structure determination and refinement

Request a detailed protocol

The crystals of DddX/ATP complex belong to the C2 space group, and Se-derivative of DddX belong to the P212121 space group. The structure of DddX Se-derivative was determined by single-wavelength anomalous dispersion phasing. The crystal structure of DddX/ATP complex was determined by molecular replacement using the CCP4 program Phaser (Winn et al., 2011) with the structure of DddX Se-derivative as the search model. The refinements of these structures were performed using Coot (Emsley et al., 2010) and Phenix (Adams et al., 2010). All structure figures were processed using the program PyMOL (http://www.pymol.org/).

Circular dichroism (CD) spectroscopy

Request a detailed protocol

CD spectra for WT DddX and its mutants were carried out in a 0.1 cm-path length cell on a JASCO J-1500 Spectrometer (Japan). All proteins were adjusted to a final concentration of 0.2 mg/ml in 10 mM Tris-HCl (pH 8.0) and 100 mM NaCl. Spectra were recorded from 250 to 200 nm at a scan speed of 200 nm/min.

Molecular docking simulations

Request a detailed protocol

The structure of the DddX/ATP complex containing a pair of subunits, α and β was loaded and energy minimised in Flare (v3.0, Cresset) involving 11,248 moving heavy atoms (Chain A: 5312, Chain B: 5312, Chain G: 10 and Chain S Water: 614). The molecule minimized with 2000 iterations using a gradient of 0.657 kcal/A. The minimised structure had an RMSD 0.82 Å relative to the starting structure and a decrease in starting energy from 134999.58 kcal/mol to a final energy of 6888.60 kcal/mol. The DMSP, CoA and DMSP-CoA molecules were drawn in MarvinSketch (v19.10.0, 2019, ChemAxon for Mac) and exported as a Mol SDF format. The molecules were imported into Flare and docked into the proposed CoA/DMSP binding site using the software’s default docking parameters for intensive pose searching and scoring.

Identification of DddX homologs in bacteria and phylogenetic analysis

Request a detailed protocol

DddX (1697) of Psychrobacter sp. D2 was used as the query sequence to search for homologs in genome-sequenced bacteria in the NCBI Reference Sequence Database (RefSeq, https://www.ncbi.nlm.nih.gov/refseq/) using BLastP with a stringent setting with an e-value cut-off < –75, sequence coverage >70% and percentage identity >30%. These high stringency settings are necessary to exclude other acetyl-CoA synthetase family proteins (ACS) which are unlikely to be involved in DMSP catabolism. Multiple sequence alignment was carried out using MEGA 7 (Kumar et al., 2016) and the presence of histidine 292, tryptophan 391 and glutamate 432 was manually inspected. To confirm the activity of DddX homologs from retrieved sequences from these genome-sequenced bacteria, four sequences (Sporosarcina sp. P33; Psychrobacter sp. P11G5; Marinobacterium jannaschii; Pelagicola sp. LXJ1103) were chemically synthesized and their enzyme activity for DMSP degradation was confirmed experimentally (Figure 6—figure supplement 1). The phylogenetic tree was constructed using the neighbour-joining method with 500 bootstraps using MEGA 7 (Kumar et al., 2016). The characterized ACS ACD1 (Weiße et al., 2016) was used as the outgroup.

Data availability

The draft genome sequences of Psychrobacter sp. D2 have been deposited in the National Center for Biotechnology Information (NCBI) Genome database under accession number JACDXZ000000000. All the RNA-seq read data have been deposited in NCBI's sequence read archive (SRA) under project accession number PRJNA646786. The structure of DddX/ATP complex has been deposited in the PDB under the accession code 7CM9.

The following data sets were generated
    1. Wang X
    (2020) NCBI GenBank
    The draft genome sequence of Psychrobacter sp.D2.

References

  1. Book
    1. Britton HT
    (1952)
    Hydrogen Lons (4th edn)
    London: Chapman and Hall.
    1. Emsley P
    2. Lohkamp B
    3. Scott WG
    4. Cowtan K
    (2010) Features and development of Coot
    Acta Crystallographica Section D-Biological Crystallography 66:486–501.
    https://doi.org/10.1107/S0907444910007493
  2. Book
    1. Gasteiger E
    2. Bairoch A
    3. Appel RD
    (2005) Protein identification and analysis tools on the ExPASy server
    In: Walker JM, editors. The Proteomics Protocols Handbook. Springer Protocols Handbooks. Humana Press. pp. 571–607.
    https://doi.org/10.1385/1-59259-890-0:571
  3. Book
    1. Karsten U
    2. Kück K
    3. Vogt C
    4. Kirst GO
    (1996) Dimethylsulfoniopropionate production in phototrophic organisms and its physiological functions as a cryoprotectant
    In: Kiene RP, Visscher PT, Keller MD, Kirst GO, editors. Biological and Environmental Chemistry of DMSP and Related Sulfonium Compounds. Boston, MA: Springer. pp. 143–153.
    https://doi.org/10.1007/978-1-4613-0377-0_13

Decision letter

  1. Georg Pohnert
    Reviewing Editor; University of Jena, Germany
  2. Meredith C Schuman
    Senior Editor; University of Zurich, Switzerland
  3. Georg Pohnert
    Reviewer; University of Jena, Germany

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Following the very interesting discussion with the referees and the resulting thorough revision I am happy to see this work now ready for publication in eLife. It will add to the growing knowledge of the marine sulfur cycle and maybe even stimulate follow-up work on the soil systems you mention. All in all I thank you for your additional effort you put into the work and I am looking forward to seeing it published.

Decision letter after peer review:

Thank you for submitting your article "Novel enzyme for dimethyl sulfide-releasing in bacteria reveals a missing route in the marine sulfur cycle" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Geog Pohnert as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Meredith Schuman as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

The study isolates bacteria from diverse Antarctic samples which utilise DMSP as the sole carbon source. It initially focuses on a Gammaproteobacterium, Psychrobacter sp.D2, which the authors establish lacks a known DMSP lyase enzyme despite having DMSP lyase activity (this needs to be quantified). Through RNA-seq and bioinformatics, they identify the gene cluster responsible for this activity and identify a novel DMSP lyase somewhat related to DddD in that it involves CoA, but critically also ATP, which distinguishes it from the pack of other known Ddd enzymes. This enzyme is a ATP-dependent DMSP CoA synthase required for growth on DMSP and its transcription is upregulated by DMSP availability. The novel mechanism of this enzyme is proposed from a strong structural component to the study. The authors propose the downstream pathway for DMSP catabolism, which we find to be oversold and requiring gene mutagenesis to confirm, and to be preliminary in comparison with the authors' other findings. Finally, the study attempts to show how widespread the enzyme is in sequenced bacteria, confidently showing it to be functional in other related Gammaproteobacteria and some Firmicutes.

Essential revisions:

1. Title: “a missing route" was it really missing? We would suggest a more precise title. Would be better to say "that releases DMS" or an alternative.

2. This is a Ddd enzyme by definition and should be named as such.

Line 27- We disagree with the use of a new gene prefix when there is a strong precedent for the use of Ddd for "DMSP-dependent DMS". If this enzyme is a DMSP lyase and is in bacteria then its naming should follow protocol and be called Ddd"X"-X-being a letter not currently utilised in known systems. Deviating from this convention causes confusion and is not appropriate. Furthermore, AcoD is already assigned in some bacteria to acetaldehyde dehydrogenase II.

3. As presented, the bioinformatics-based evidence regarding the broad distribution of this enzyme (as claimed e.g. in the Abstract, line 33) does not stand up.

Currently as presented in the manuscript, especially Figure 6, we are led to believe the enzyme is more widespread than can be demonstrated based on the authors' evidence (i.e., the authors allow a very low threshold of sequence identity and claim function outside of the groups they have tested). Either more work is needed to show that claims of such a wide distribution are merited, or the authors should limit their claims to what can be substantiated by their work. Specifically, the authors cannot comment on the "functional" enzyme being widespread outside of the Gamma's and Firmicutes that were tested, let alone the importance of the role in DMSP cycling. Only three "AcoD" enzymes were ratified in this study, which are relatively closely related to each (Psychrobacter sp. D2 Sporosarcina sp. P33 and Psychrobacter sp. P11G5 that are > 77% identical to each other). As can be seen in Figure 6, these three proteins cluster together and are far removed from all the other sequences on the figure, for which we have no evidence of their function (i.e., nothing can realistically be said on Deltas, Actinos or Alphas or the MAGS). Just to be clear, these other proteins shown in clades above and below the functional "AcoDs" in Figure 6 are only ~30% identical to ratified "AcoD".

Furthermore, only strain D2 was shown to make DMS; none of the other strains were tested. Far more testing of the diverse enzymes and strains are needed to make these statements as this study only tests one strain and three of the closely related enzymes (defined on Figure 6).

Additional specific comments on this issue:

Line 280. The sentence on MAGS and the environments containing them does not stand up for reasons summarised above. All MAGS shown on Figure 6 are not similar enough to "AcoD" to be termed as functional Ddd enzymes. More work has to be done on the strains and enzymes that are more divergent to true "AcoDs" before such a statement is supported. Please delete.

Line 509-We agree with what the authors write about stringency. However, these parameters do not seem to have been utilised as stated here. Their stringency statement holds up for comparison between the D2 "AcoD" and two other tested "AcoD" enzymes and all those in the middle clade on Figure 6. But this is not the case for the proteins shown above and below this "AcoD" clade in Figure 6 which have at best around 30% identity to characterised enzymes. See below for examples. As the authors state in their methods, high-stringency methods are needed to exclude other acetyl-CoA synthetase family proteins. Thus, most of the genes shown on fig6 cannot be taken as having this Ddd activity.

"To further validate that these AcoD homologs" the authors examined the activity of two closely related enzymes from a group of nine homologs with > 65 % sequence identity (starting line 283, Figure 6). It is not surprising that these enzymes have the same activity. Homologs outside this group of nine (Figure 6) are far less related to the characterized AcoD (< 32 % seq. identity). Conservation of the phosphate-transferring His (His292) and an active site Trp (Trp391) does not seem to be strong evidence for functional conservation. The manuscript does not provide any additional evidence that these less related enzymes also degrade DMSP. Either more experimentation is necessary, or the paragraph on the "Distribution of the ATP DMSP lysis pathway in bacteria" must be revised. For example:

Psychrobacter AcoD (WP_068035783.1) is 31% identical to Bilophila sp. 4_1_30 (WP_009381183.1) in the below group of bacteria on Figure 6.

Psychrobacter AcoD (WP_068035783.1) is 29% identical to Thermomicrobium roseum (WP_041435830.1) in the above group of bacteria on Figure 6.

Line 283. This is not the case! The two sequences that were chosen to "validate" are far to close to the D2 "AcoD" than to MAGS and other potential "AcoDs" shown above and below the functional Ddd clade on Figure 6. This section design is weak and does not lend weight to the expansiveness of this family. More work on the more diverse enzymes and bacteria is needed to support the authors claims. Please delete or study the activity of the more diverse strains and their candidate "AcoDs".

Figure 6. This is a nicely presented figure that unfortunately slightly deceives the reader. The authors need to clearly show which strains they have shown to have Ddd activity (currently one as I understand it) and which enzymes they have shown to have the appropriate activity (currently three closely related enzymes as I understand it). If I am not wrong these are all confined to the middle clade of Gammas and Firmicutes. These stand clearly apart form the other strains (above and below) which have not been studied and which are only ~ 30% Identical to "AcoD" at the protein level. This is not clear on the figure and definitely misleads in the abstract and throughout the manuscript.

4. We expect to see kinetics done on the new enzyme in line with what the authors have done in other related studies on Ddd and Dmd enzymes.

This is important to place the work in context with previously identified Ddd and Dmd enzymes, many of which have been analysed by these authors in previous publications. The characterization of the AcoD activity remains entirely qualitative. The authors only provide relative activities measured at a single substrate concentration. This data does not support the following statement: "Mutations of these two residues significantly decreased the enzymatic activities of AcoD, suggesting that these residues play important roles in stabilizing the DMSP-CoA intermediate" (l.223-225).

5. The manuscript does provide unambiguous evidence for the activity of AcoD and its function during growth on DMSP. On the other hand, the description of the "ATP DMSP lysis pathway" is less clear.

Transcriptomics analysis (Figure 2C) suggest that growth on DMSP upregulate the genes 1696 (BCCT), 1697 (AcoD), 1698 and 1699. The function of the third and fourth protein remain unclear (line 253). Instead, a reductase (AcuI) encoded somewhere else on the same genome was shown to transform the acryloyl-CoA to propionate-CoA. What was the transcription profile of acuI acuH in the RNA-seq? were they induced by growth on DMSP? Is the 1696-1697-1698-1699 gene cluster conserved? What is the function of 1698 and 1699? These questions are only relevant if the authors plan to maintain the claim of having identified a new pathway. This pathway prediction component is very weak and could be supplemented by KO mutagenesis of the dddCB and acuI. Without such work this is speculation and needs to be written as such.

6. Appropriate controls, units and quantification should be used:

Line 102- Please give a normalised value for the level of DMS produced from DMSP per time and protein/cells.

Figure 2.

A. One would expect to see a growth curve of D2 on DMSP compared to acrylate, a conventional carbon source (e.g. pyruvate, glycerol or succinate) and a no carbon control. As "AcoD" is predicted to ligate CoA to DMSP it would be good to know if the strain grows on acrylate. It might be predicted to have different properties to e.g. Halomonas which does grow on acrylate. At least a no carbon and conventional carbon source should definitely be included.

B. The units for this figure are not appropriate. It would be more appropriate to show the actual amount of DMS that is produced by the strain, ideally normalised to protein, cells or absorbance and time. Detail in the figure what the control is.

C. Would like to see error bars on this figure. Also would have been sensible to colour code these to match panel D.

Figure 3.

B and C. as with Figure 2 we need to see levels of DMS normalised to cells/protein and time.

Line 374- No controls. Please include these as detailed above. No carbon, conventional carbon source, acrylate?

Quantitative data supporting Supplementary Figure 12 would be helpful. After all this route would have to explain that the bacteria can use acrylate CoA as sole carbon source (or at least alternatives would have to be discussed). Is the identified activity sufficient for this task?

Line 388- This method is/should be quantitative. It is standard practice to report DMS production normalised to time and cells/protein. Here we are only given peak area.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "A novel ATP dependent dimethylsulfoniopropionate lyase in bacteria that releases dimethyl sulfide and acryloyl-CoA" for further consideration by eLife. Your revised article has been reviewed by two peer reviewers and the evaluation has been overseen by Meredith Schuman as the Senior Editor and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Both previous referees consider the revisions to be entirely satisfactory and have no further concerns. Given that one of the earlier referees is now a co-author, we decided to include a new third expert previously not involved.

This additional referee raises a significant concern regarding the appropriateness of re-naming DddX from an Acetyl-CoA lyase to DMSP lyase, and suggests comparably simple experiments for clarification: "In light of the above, before changing the annotation of the DddX enzyme to DMSP lyase, the authors should test if DddX lost its ability to catalyse its original and primary activity, which is to ligate acetate or propionate to CoA in the presence of ATP. A substrate competition assay that tests the ability of acetate and propionate to inhibit DMSP lyase activity would also be useful. Finally, the ability of the mutation ΔDddX to inhibit growth on acetate or propionate compared with the wild type would also be important to test."

Further, the reviewer suggests additional critical discussion of the distribution of the new enzyme that could be included in a revised version.

Please address all comments of the review.

Reviewer #3:

In my opinion, the authors addressed all important concerns.

Reviewer #4:

This study will be of interest to researchers within the fields of marine microbiology and enzymology. The study identified and characterized a new DMSP lyase enzyme in an Antarctic marine bacterium. Together with the eight previously identified DMSP lyase families, this enzyme may play an important role in the ocean sulfur cycle. While an experiment to verify the substrate specificity of the enzyme is still lacking, the majority of the conclusions drawn from the study are justified, and the data analysis is thorough.

This paper describes the identification and characterization of the dimethylsulfoniopropionate (DMSP) lyase from the Antarctic marine bacterium Psychrobacter sp. D2. The authors show that using these enzymes, Psychrobacter can catabolize DMSP and use it as a sole carbon source while releasing the ubiquitous organosulfur gas dimethyl sulfide (DMS). DMS is a bioactive signalling molecule that plays a key role in the oceanic food web, it is also responsible for the formation of cloud condensation nuclei in the atmosphere and is the primary source of biogenic sulfur emitted from the ocean. The authors use a diverse set of tools from classic microbiology, molecular biology, enzymology and crystallography to identify and characterize the enzyme. The newly identified DMSP lyase belongs to the acetyl-CoA synthase family and uses CoA and ATP to transform DMSP to acryloyl-CoA and DMS. The discovery of another DMSP lyase family is surprising given that eight DMSP lyase families, belonging to four distinct superfamilies, had already been identified and are widespread in the marine environment.

Strengths:

The researchers provides unambiguous evidence that the newly identified enzyme, DddX, allows Psychrobacter sp. D2 to grow on DMSP as a sole carbon source while releasing DMS. They also present rigorous characterization of the enzyme that includes structural analysis, proposed mode of action and kinetic parameters. Finally, the researchers provide evidence that other DddX homologous genes, obtained from a few species within the alphaproteobacteria, gammaproteobacteria and firmicutes, can also catalyse this reaction. In summary, the authors support most of their claims with data, and their findings are relevant for the understanding of the role of bacteria in the oceanic sulfur cycle.

Weaknesses:

Although the paper has several strengths, further work is necessary to test the specificity of DddX. Since DddX shows strong structure and sequence similarity to acetyl-CoA synthase enzymes (ACS), and due to its low catalytic activity toward DMSP, its low kcat / KM values, and its presence in a non-functional operon and in the genomes of soil bacteria (where DMSP is not commonly present) the substrate specificity of DddX toward DMSP is still puzzling. Given that acetyl-CoA synthase enzymes have been shown to have promiscuous activity toward different short chain fatty acids including unnatural substrates (Patel et al. JBC, 1987), it is feasible that in this study the authors actually describe an acetyl-CoA synthase enzyme with a mild promiscuous activity toward DMSP. In light of the above, before changing the annotation of the DddX enzyme to DMSP lyase, the authors should test if DddX lost its ability to catalyse its original and primary activity, which is to ligate acetate or propionate to CoA in the presence of ATP. A substrate competition assay that tests the ability of acetate and propionate to inhibit DMSP lyase activity would also be useful. Finally, the ability of the mutation ΔDddX to inhibit growth on acetate or propionate compared with the wild type would also be important to test. The results from the proposed experiments would not change the main conclusion of the paper, that DddX can catalyse the formation of DMS. Yet it may have a significant impact on the interpretation of the finding.

To assess the generality of their finding, the authors should also provide further details about the diversity and abundance of the DddX enzyme in the oceans and in other environments. For example, it would be appropriate to discuss why DddX enzymes are found also in soil and other non-marine environments, where, according to current knowledge, DMSP is not commonly produced (note that DMSP is produced by many organisms including marine algae, marine bacteria and corals and can be found in abundance in the oceans, in salt marshes, sea sediments and in the roots of specialized DMSP-producing plants). Is DddX a "niche" enzyme that can operate in multiple specialized species? Or is it an enzyme that is present in many bacteria in the ocean? As it stands, additional clarification and discussion of those points is required in the text.

Comments for authors:

1. The authors show that, under the conditions tested in the lab, DddX clearly supports the growth of Psychrobacter sp. D2 on DMSP as a sole carbon source and the DddX enzyme is able to catalyze the conversion of DMSP to DMS. However, the relatively low catalytic activity of DddX, low kcat/KM values, and the appearance of DddX in non-functioning operon and in the genomes of soil bacteria, raise questions about DddX substrate specificity. Given that Acetyl-CoA synthase enzymes have been shown to have promiscuous activity toward different short chain fatty acids and even towards unnatural substrates (Smita S. Patel and David R. Walt. JBC, 1987), It is possible, and not unlikely, that the authors actually describe in their study an acetyl-CoA synthase enzyme with a mild promiscuous activity toward DMSP. In Fact from the partial alignment the authors present in Figure 4—figure supplement 3, DddX is extremely similar to other Acetyl-CoA synthase enzymes (ACS). And as mentioned by the authors, it is also very similar in structure, having all the characteristics and active sites residues of other ACS enzymes. In light of the above, I think it's important that the authors test if the enzyme lost its original activity, to ligate acetate or propionate to CoA in the presence of ATP, before changing DddX annotation from "ACS" to "DMSP lyase". A substrate competition assay that tests the ability of acetate and propionate to inhibit DMSP lyase activity would also be useful. And, testing the ability of the ΔDddX to inhibit growth on acetate or propionate and compare it with the growth of the wild-type would also be important.

If DddX lost its original function, then the authors should definitely use the name DddX and annotate their newly discovered enzyme as DMSP lyase. However, if not the author would probably want to keep the original name and maybe add a second name that includes the newly discovered function. Or at least discuss the possibility that this enzyme is bifunctional in their paper. The proposed set of experiments that we offer does not change the conclusion that DddX can catalyse the formation of DMS, but it may significantly change how we interpret the results.

Reference: Smita S Patel and David R. Walt. Substrate specificity of acetyl coenzyme A synthetase. JBC (1987) (https://doi.org/10.1016/S0021-9258(18)48214-2)

2. The authors should explain as well as discuss in the paper how DddX enzymes are found in environments such as soil and other non-marine environments, where, according to our knowledge, DMSP is not commonly produced. Note that DMSP is produced by many organisms including marine algae, marine bacteria and corals and can be found in abundance in the oceans, in salt marshes, sediments or in roots of specialized DMSP producing plants but is it also produce in other soil environments? Please provide a brief description in the text of where the bacteria with DddX in their genome can be found. Also in Figure 6, please indicate the general location of isolation near each branch as in the previous version of the figure. Please search for it for each genome in the NCBI bioSample description. For example: Sporosarcina sp. P33 (with functional DMSP lyase) is a soil bacteria – (The location of isolation is interesting) – https://www.ncbi.nlm.nih.gov/biosample/SAMN04589807

3. Please explain in more detail the general abundance of the DddX enzyme in the oceans (or other environments). Please use the "Tara oceans gene atlas" or other tools to support the high/low abundance of the DddX enzyme in the environment. Is DddX a "niche" enzyme that can operate in multiple specialized species and locations? Or is it one of the enzymes that many bacteria in the ocean possess? As it stands now it is not clear from the text/data. For example: In Line 36 – 38 the authors claims that "DddX is found in diverse marine alphaproteobacteria, gammaproteobacteria and firmicutes, suggesting that this new DMSP lyase may play an important role in DMSP/DMS cycles" – However, in Figure 6 It appears that there are not many organisms belonging to those diverse groups of bacteria. i.e. There are a very large number of bacterial species between and within alphaproteobacteria, gammaproteobacteria and firmicutes that do not have this gene. In fact from the data presented, DddX enzymes can be found sporadically in only very few groups of species which are very distinct from one another. In summary, try to be more specific in the description of the enzyme diversity and abundance throughout the paper. If DddX is only a "niche" DMSP lyase, the study is still very interesting and valid. However, one would not want to give the wrong impression about the abundance of a newly discovered enzyme. Make sure to discuss the abundance of DddX and the apparently sporadic diversity of the DddX enzyme in the discussion of the paper.

https://doi.org/10.7554/eLife.64045.sa1

Author response

Essential revisions:

1. Title: “a missing route" was it really missing? We would suggest a more precise title. Would be better to say "that releases DMS" or an alternative.

We have changed the title to "A novel ATP dependent dimethylsulfoniopropionate lyase in bacteria that releases dimethyl sulfide and acryloyl-CoA" following the reviewers’ comment.

2. This is a Ddd enzyme by definition and should be named as such.

Line 27- We disagree with the use of a new gene prefix when there is a strong precedent for the use of Ddd for "DMSP-dependent DMS". If this enzyme is a DMSP lyase and is in bacteria then its naming should follow protocol and be called Ddd"X"-X-being a letter not currently utilised in known systems. Deviating from this convention causes confusion and is not appropriate. Furthermore, AcoD is already assigned in some bacteria to acetaldehyde dehydrogenase II.

We agree with the reviewers that this novel enzyme is a Ddd enzyme. The letter “X” has not been utilized in the reported systems and thus we have changed the name of this enzyme to DddX.

3. As presented, the bioinformatics-based evidence regarding the broad distribution of this enzyme (as claimed e.g. in the Abstract, line 33) does not stand up.

Currently as presented in the manuscript, especially Figure 6, we are led to believe the enzyme is more widespread than can be demonstrated based on the authors' evidence (i.e., the authors allow a very low threshold of sequence identity and claim function outside of the groups they have tested). Either more work is needed to show that claims of such a wide distribution are merited, or the authors should limit their claims to what can be substantiated by their work. Specifically, the authors cannot comment on the "functional" enzyme being widespread outside of the Gamma's and Firmicutes that were tested, let alone the importance of the role in DMSP cycling. Only three "AcoD" enzymes were ratified in this study, which are relatively closely related to each (Psychrobacter sp. D2 Sporosarcina sp. P33 and Psychrobacter sp. P11G5 that are > 77% identical to each other). As can be seen in Figure 6, these three proteins cluster together and are far removed from all the other sequences on the figure, for which we have no evidence of their function (i.e., nothing can realistically be said on Deltas, Actinos or Alphas or the MAGS). Just to be clear, these other proteins shown in clades above and below the functional "AcoDs" in Figure 6 are only ~30% identical to ratified "AcoD".

Furthermore, only strain D2 was shown to make DMS; none of the other strains were tested. Far more testing of the diverse enzymes and strains are needed to make these statements as this study only tests one strain and three of the closely related enzymes (defined on Figure 6).

Additional specific comments on this issue:

Line 280. The sentence on MAGS and the environments containing them does not stand up for reasons summarised above. All MAGS shown on Figure 6 are not similar enough to "AcoD" to be termed as functional Ddd enzymes. More work has to be done on the strains and enzymes that are more divergent to true "AcoDs" before such a statement is supported. Please delete.

Line 509-We agree with what the authors write about stringency. However, these parameters do not seem to have been utilised as stated here. Their stringency statement holds up for comparison between the D2 "AcoD" and two other tested "AcoD" enzymes and all those in the middle clade on Figure 6. But this is not the case for the proteins shown above and below this "AcoD" clade in Figure 6 which have at best around 30% identity to characterised enzymes. See below for examples. As the authors state in their methods, high-stringency methods are needed to exclude other acetyl-CoA synthetase family proteins. Thus, most of the genes shown on fig6 cannot be taken as having this Ddd activity.

"To further validate that these AcoD homologs" the authors examined the activity of two closely related enzymes from a group of nine homologs with > 65 % sequence identity (starting line 283, Figure 6). It is not surprising that these enzymes have the same activity. Homologs outside this group of nine (Figure 6) are far less related to the characterized AcoD (< 32 % seq. identity). Conservation of the phosphate-transferring His (His292) and an active site Trp (Trp391) does not seem to be strong evidence for functional conservation. The manuscript does not provide any additional evidence that these less related enzymes also degrade DMSP. Either more experimentation is necessary, or the paragraph on the "Distribution of the ATP DMSP lysis pathway in bacteria" must be revised. For example:

Psychrobacter AcoD (WP_068035783.1) is 31% identical to Bilophila sp. 4_1_30 (WP_009381183.1) in the below group of bacteria on Figure 6.

Psychrobacter AcoD (WP_068035783.1) is 29% identical to Thermomicrobium roseum (WP_041435830.1) in the above group of bacteria on Figure 6.

Line 283. This is not the case! The two sequences that were chosen to "validate" are far to close to the D2 "AcoD" than to MAGS and other potential "AcoDs" shown above and below the functional Ddd clade on Figure 6. This section design is weak and does not lend weight to the expansiveness of this family. More work on the more diverse enzymes and bacteria is needed to support the authors claims. Please delete or study the activity of the more diverse strains and their candidate "AcoDs".

Figure 6. This is a nicely presented figure that unfortunately slightly deceives the reader. The authors need to clearly show which strains they have shown to have Ddd activity (currently one as I understand it) and which enzymes they have shown to have the appropriate activity (currently three closely related enzymes as I understand it). If I am not wrong these are all confined to the middle clade of Gammas and Firmicutes. These stand clearly apart form the other strains (above and below) which have not been studied and which are only ~ 30% Identical to "AcoD" at the protein level. This is not clear on the figure and definitely misleads in the abstract and throughout the manuscript.

Re DddX identity amino acid homology issues, we thank the reviewers for their comments and apologize for the confusion. The sequence identity cut-off was set at >30% (The 50% cut-off value was previously used to investigate the distribution of conserved ATP-grasp domain in the database in an early version of the manuscript; this error has now been corrected in this revision). After retrieving putative DddX homologues from the NCBI ref-seq database, multiple sequence alignment was manually inspected to search for the presence of conserved residues that we predict to be involved in DddX catalysis (e.g. H292, W391 and D432).

Re MAGS low homology issues, we fully agree with the reviewers on the inclusion of MAG sequences with lower identity to ratified DddX. For that reason, we have removed all mention of MAG analysis from Figure 6 and the manuscript. We thank the reviewers for pointing out these problems and feel the manuscript is tighter without their inclusion.

Re validation issues, we thank the reviewers for revealing the limitations of our initial analysis. Following the reviewers’ advice, we now include new data that validate representative DddX from the more divergent alphaproteobacterial and gammaproteobacterial DddX homologs (with only ~30% identity to Psychrobacter DddX). We chemically synthesized the dddX sequences from alphaproteobacterial Pelagicola sp. LXJ1103 and gammaproteobacterial Marinobacterium jannaschii, overexpressed them in E. coli, purified the proteins and carried out enzyme assays. Both these candidate DddX enzymes had the predicted DMSP lysis activity producing DMS and acryloyl-CoA (Figure 6—figure supplement 1). As covered above, the homology of DddX proteins to Psychrobacter DddX is now clearly shown on the new Figure 6 as is the representatives that were shown to be functional. We have also thoroughly edited the manuscript throughout to convey the real known scope of DddX. Although we could have synthesised and assayed more DddX representatives, we now feel that the spread of validation is suitable for what we want to convey.

Re strain assays, we realise that it would have been more complete assay the strains that contain the more diverse functional DddX enzymes to confirm their activity. However, in these times it was difficult to swiftly procure suitable strains. We hope that the reviewers agree with us that the functional ratification of the enzymes is sufficient for publication here.

4. We expect to see kinetics done on the new enzyme in line with what the authors have done in other related studies on Ddd and Dmd enzymes.

This is important to place the work in context with previously identified Ddd and Dmd enzymes, many of which have been analysed by these authors in previous publications. The characterization of the AcoD activity remains entirely qualitative. The authors only provide relative activities measured at a single substrate concentration. This data does not support the following statement: "Mutations of these two residues significantly decreased the enzymatic activities of AcoD, suggesting that these residues play important roles in stabilizing the DMSP-CoA intermediate" (l.223-225).

We thank the reviewers for pointing out this omission. We have now measured the enzymatic properties including the kinetic parameters of DddX from Psychrobacter sp. D2. DddX exhibited a Km value of 0.4 mM for DMSP, which is lower than DmdA and most other known DMSP lyases. The kinetic data of DddX (Figure 3—figure supplement 3), comparisons to other related enzymes (Supplementary file 1c), and the methods used are now added to the revised manuscript.

We agree with the reviewers that our current data does not support the roles of the residues Trp391 and Phe435 in stabilizing the DMSP-CoA intermediate. Because DMSP-CoA is an unstable intermediate, and it is difficult to measure the kinetic parameters of DddX mutants towards DMSP-CoA, we have now modified this sentence to "Mutations of these two residues significantly decreased the enzymatic activities of DddX, suggesting that these residues play important roles in DddX catalysis" in line 237-239 in the revised manuscript.

5. The manuscript does provide unambiguous evidence for the activity of AcoD and its function during growth on DMSP. On the other hand, the description of the "ATP DMSP lysis pathway" is less clear.

Transcriptomics analysis (Figure 2C) suggest that growth on DMSP upregulate the genes 1696 (BCCT), 1697 (AcoD), 1698 and 1699. The function of the third and fourth protein remain unclear (line 253). Instead, a reductase (AcuI) encoded somewhere else on the same genome was shown to transform the acryloyl-CoA to propionate-CoA. What was the transcription profile of acuI acuH in the RNA-seq? were they induced by growth on DMSP? Is the 1696-1697-1698-1699 gene cluster conserved? What is the function of 1698 and 1699? These questions are only relevant if the authors plan to maintain the claim of having identified a new pathway. This pathway prediction component is very weak and could be supplemented by KO mutagenesis of the dddCB and acuI. Without such work this is speculation and needs to be written as such.

We thank the reviewers for the insightful comments and agree that future experiments are required in order to firmly establish the pathway of acryloyl-CoA degradation. We have now toned down the description of the pathway and moved the related text into the Discussion section in line 315-324 in the revised manuscript, which reads:

“If Psychrobacter sp. D2 employs its AcuH homolog to convert acryloyl-CoA to 3-HP-CoA (Cao et al., 2017),then, given the high sequence identity of 1698 to DddC and 1699 to DddB, it is possible that these enzymes further catabolize 3-HP-CoA to acetyl-CoA (Alcolombri et al., 2014; Curson et al., 2011b). […] Thus, Psychrobacter sp. D2 may also employ an AcuI homolog (i.e. 0105) to convert acryloyl-CoA to propionate-CoA, which would be metabolized through the methylmalonyl-CoA pathway (Reisch et al., 2013).”

6. Appropriate controls, units and quantification should be used:

Line 102- Please give a normalised value for the level of DMS produced from DMSP per time and protein/cells.

Figure 2.

A. One would expect to see a growth curve of D2 on DMSP compared to acrylate, a conventional carbon source (e.g. pyruvate, glycerol or succinate) and a no carbon control. As "AcoD" is predicted to ligate CoA to DMSP it would be good to know if the strain grows on acrylate. It might be predicted to have different properties to e.g. Halomonas which does grow on acrylate. At least a no carbon and conventional carbon source should definitely be included.

Thank you for these important suggestions which we have now fully taken on. We have now added the quantitative data and appropriate controls in Figure 2 in the manuscript according to the reviewers’ suggestions. The growth curves of Psychrobacter sp. D2 on sodium pyruvate and acrylate, and a no carbon control are now added in Figure 2A. Psychrobacter sp. D2 could not use acrylate as a sole carbon source for growth in contrast to Halomonas, which is discussed in the discussion now in line 325-334.

B. The units for this figure are not appropriate. It would be more appropriate to show the actual amount of DMS that is produced by the strain, ideally normalised to protein, cells or absorbance and time. Detail in the figure what the control is.

We are sorry but feel it is important to show the chromatogram for DMS against a standard and a now clearly labelled media control. However, we do now report the amount of DMS that is generated normalised to protein levels both in the main text (line 106) and in the figure legend for Figure 2B. Sorry for our previous oversight.

C. Would like to see error bars on this figure. Also would have been sensible to colour code these to match panel D.

Figure 3.

B and C. as with Figure 2 we need to see levels of DMS normalised to cells/protein and time.

Line 374- No controls. Please include these as detailed above. No carbon, conventional carbon source, acrylate?

Quantitative data supporting Supplementary Figure 12 would be helpful. After all this route would have to explain that the bacteria can use acrylate CoA as sole carbon source (or at least alternatives would have to be discussed). Is the identified activity sufficient for this task?

Line 388- This method is/should be quantitative. It is standard practice to report DMS production normalised to time and cells/protein. Here we are only given peak area.

Thanks for these sensible suggestions. We have now coordinated the colour of Figure 2C and D. This is much improved for this. Re the error bars, RNA sequencing was carried out on a pool of three biological samples to produce a single library. We thus could not provide error bars here. However, the upregulation of these genes was confirmed by RT-qPCR that was done in triplicate. Thus, instead of the RNA seq data we include the RT-qPCR data in Figure 2C with error bars. We feel that this is much more appropriate. As covered above, DMS levels are reported appropriately normalised in Figure 3.

Furthermore, we acknowledge that the genes and metabolic pathway involved in acryloyl-CoA metabolism in this bacterium is unclear and requires further work. The amount of propionate-CoA normalised to protein levels is reported in the figure legend for Figure 1—figure supplement 2 (supplementary Figure 12 in the original version of manuscript). We now discuss several possibilities in line 312-324 in the revised version of manuscript.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Reviewer #4:

[…] Comments for authors:

1. The authors show that, under the conditions tested in the lab, DddX clearly supports the growth of Psychrobacter sp. D2 on DMSP as a sole carbon source and the DddX enzyme is able to catalyze the conversion of DMSP to DMS. However, the relatively low catalytic activity of DddX, low kcat/KM values, and the appearance of DddX in non-functioning operon and in the genomes of soil bacteria, raise questions about DddX substrate specificity. Given that Acetyl-CoA synthase enzymes have been shown to have promiscuous activity toward different short chain fatty acids and even towards unnatural substrates (Smita S. Patel and David R. Walt. JBC, 1987), It is possible, and not unlikely, that the authors actually describe in their study an acetyl-CoA synthase enzyme with a mild promiscuous activity toward DMSP. In Fact from the partial alignment the authors present in Figure 4—figure supplement 3, DddX is extremely similar to other Acetyl-CoA synthase enzymes (ACS). And as mentioned by the authors, it is also very similar in structure, having all the characteristics and active sites residues of other ACS enzymes. In light of the above, I think it's important that the authors test if the enzyme lost its original activity, to ligate acetate or propionate to CoA in the presence of ATP, before changing DddX annotation from "ACS" to "DMSP lyase". A substrate competition assay that tests the ability of acetate and propionate to inhibit DMSP lyase activity would also be useful. And, testing the ability of the ΔDddX to inhibit growth on acetate or propionate and compare it with the growth of the wild-type would also be important.

If DddX lost its original function, then the authors should definitely use the name DddX and annotate their newly discovered enzyme as DMSP lyase. However, if not the author would probably want to keep the original name and maybe add a second name that includes the newly discovered function. Or at least discuss the possibility that this enzyme is bifunctional in their paper. The proposed set of experiments that we offer does not change the conclusion that DddX can catalyse the formation of DMS, but it may significantly change how we interpret the results.

Reference: Smita S Patel and David R. Walt. Substrate specificity of acetyl coenzyme A synthetase. JBC (1987) (https://doi.org/10.1016/S0021-9258(18)48214-2)

Re the substrate specificity of DddX, we thank the reviewer for the insightful comments. We tested the enzymatic activities of recombinant DddX towards acetate and propionate. While DddX converted DMSP to DMS and acryloyl-CoA, it exhibited no activity towards acetate or propionate (Figure 3—figure supplement 4).

Re the substrate competition assay, we tested the enzymatic activity of DddX towards DMSP in the presence of acetate or propionate. Acetate or propionate (with a final concentration of up to 5 mM) had little effect on the enzymatic activity of DddX towards DMSP (Figure 3—figure supplement 5).

Re strain growth experiment, we tested the ability of the wild-type strain D2 and its mutants to grow with acetate or propionate as the sole carbon source. The wild-type strain D2 could use acetate or propionate as the sole carbon source, and the deletion of dddX has little effect on its growth on acetate or propionate (Figure 3—figure supplement 6), suggesting that dddX is unlikely to be involved in acetate and propionate catabolism in strain D2.

The results above also indicate that DddX does not use acetate nor propionate as a substrate, as such it does not function as an acetate-CoA ligase. We therefore propose to keep the name “DddX” in the manuscript.

2. The authors should explain as well as discuss in the paper how DddX enzymes are found in environments such as soil and other non-marine environments, where, according to our knowledge, DMSP is not commonly produced. Note that DMSP is produced by many organisms including marine algae, marine bacteria and corals and can be found in abundance in the oceans, in salt marshes, sediments or in roots of specialized DMSP producing plants but is it also produce in other soil environments? Please provide a brief description in the text of where the bacteria with DddX in their genome can be found. Also in Figure 6, please indicate the general location of isolation near each branch as in the previous version of the figure. Please search for it for each genome in the NCBI bioSample description. For example: Sporosarcina sp. P33 (with functional DMSP lyase) is a soil bacteria – (The location of isolation is interesting) – https://www.ncbi.nlm.nih.gov/biosample/SAMN04589807

We thank this reviewer for the comments and we have now added back the location/environment of isolation for the strains shown in the phylogenetic tree (see revised Figure 6). It is difficulty to speculate the source of DMSP in soils since this has not been studied extensively. However, it is likely that soil-dowelling microbes may benefit from DMSP released from plants. We added the following sentence in the discussion, which reads:

“Interestingly, DddX is found in several bacterial isolates which were isolated from soil or plant roots, suggesting that DMSP may also be produced in these ecosystems”.

3. Please explain in more detail the general abundance of the DddX enzyme in the oceans (or other environments). Please use the "Tara oceans gene atlas" or other tools to support the high/low abundance of the DddX enzyme in the environment. Is DddX a "niche" enzyme that can operate in multiple specialized species and locations? Or is it one of the enzymes that many bacteria in the ocean possess? As it stands now it is not clear from the text/data. For example: In Line 36 – 38 the authors claims that "DddX is found in diverse marine alphaproteobacteria, gammaproteobacteria and firmicutes, suggesting that this new DMSP lyase may play an important role in DMSP/DMS cycles" – However, in Figure 6 It appears that there are not many organisms belonging to those diverse groups of bacteria. i.e. There are a very large number of bacterial species between and within alphaproteobacteria, gammaproteobacteria and firmicutes that do not have this gene. In fact from the data presented, DddX enzymes can be found sporadically in only very few groups of species which are very distinct from one another. In summary, try to be more specific in the description of the enzyme diversity and abundance throughout the paper. If DddX is only a "niche" DMSP lyase, the study is still very interesting and valid. However, one would not want to give the wrong impression about the abundance of a newly discovered enzyme. Make sure to discuss the abundance of DddX and the apparently sporadic diversity of the DddX enzyme in the discussion of the paper.

We thank the reviewer for this comment. We have focused our analysis of the distribution of DddX in genome sequenced bacteria but not environmental omics datasets. As pointed out previously by other reviewers, it is necessary to test the function of those homologous sequences retrieved from environmental metagenomics datasets in order to ascertain their role in DMSP degradation. This proves challenging since those homologs returned from Tara Oceans datasets are usually short and do not always contain the full open reading frame, making it difficult for gene synthesis and overexpression in E. coli. In agreeance with the comments from other reviewers, we have focused on our analysis of DddX in genome-sequence bacteria isolates instead, and have chemically-synthesize several of these homologs to validate their role in DMSP catabolism (Figure 6). We have now modified the following sentences in the abstract, which reads:

“DddX is found in several Alphaproteobacteria, Gammaproteobacteria and Firmicutes, suggesting that this new DMSP lyase may play an overlooked role in DMSP/DMS cycles.”

We also revised the last paragraph of discussion to reflect the fact that DddX is found in several bacterial isolates.

https://doi.org/10.7554/eLife.64045.sa2

Article and author information

Author details

  1. Chun-Yang Li

    1. State Key Lab of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Qingdao, China
    2. College of Marine Life Sciences, Ocean University of China, Qingdao, China
    Contribution
    investigation, validation, writing-original-draft
    Contributed equally with
    Xiu-Juan Wang
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1151-4897
  2. Xiu-Juan Wang

    State Key Lab of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Qingdao, China
    Contribution
    investigation, writing-original-draft
    Contributed equally with
    Chun-Yang Li
    Competing interests
    No competing interests declared
  3. Xiu-Lan Chen

    1. State Key Lab of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Qingdao, China
    2. Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China
    Contribution
    supervision, validation, writing-review-and-editing
    Competing interests
    No competing interests declared
  4. Qi Sheng

    State Key Lab of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Qingdao, China
    Contribution
    investigation
    Competing interests
    No competing interests declared
  5. Shan Zhang

    State Key Lab of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Qingdao, China
    Contribution
    investigation
    Competing interests
    No competing interests declared
  6. Peng Wang

    College of Marine Life Sciences, Ocean University of China, Qingdao, China
    Contribution
    investigation
    Competing interests
    No competing interests declared
  7. Mussa Quareshy

    School of Life Sciences, University of Warwick, Coventry, United Kingdom
    Contribution
    investigation, writing-original-draft
    Competing interests
    No competing interests declared
  8. Branko Rihtman

    School of Life Sciences, University of Warwick, Coventry, United Kingdom
    Contribution
    investigation, writing-original-draft
    Competing interests
    No competing interests declared
  9. Xuan Shao

    State Key Lab of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Qingdao, China
    Contribution
    investigation
    Competing interests
    No competing interests declared
  10. Chao Gao

    State Key Lab of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Qingdao, China
    Contribution
    investigation
    Competing interests
    No competing interests declared
  11. Fuchuan Li

    National Glycoengineering Research Center and Shandong Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Qingdao, China
    Contribution
    writing-review-and-editing
    Competing interests
    No competing interests declared
  12. Shengying Li

    State Key Lab of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Qingdao, China
    Contribution
    writing-review-and-editing
    Competing interests
    No competing interests declared
  13. Weipeng Zhang

    College of Marine Life Sciences, Ocean University of China, Qingdao, China
    Contribution
    writing-review-and-editing
    Competing interests
    No competing interests declared
  14. Xiao-Hua Zhang

    College of Marine Life Sciences, Ocean University of China, Qingdao, China
    Contribution
    writing-review-and-editing
    Competing interests
    No competing interests declared
  15. Gui-Peng Yang

    Frontiers Science Center for Deep Ocean Multispheres and Earth System, Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao, China
    Contribution
    writing-review-and-editing
    Competing interests
    No competing interests declared
  16. Jonathan D Todd

    School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, United Kingdom
    Contribution
    writing-review-and-editing
    Competing interests
    No competing interests declared
  17. Yin Chen

    1. College of Marine Life Sciences, Ocean University of China, Qingdao, China
    2. School of Life Sciences, University of Warwick, Coventry, United Kingdom
    Contribution
    investigation, writing-original-draft, writing-review-and-editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0367-4276
  18. Yu-Zhong Zhang

    1. College of Marine Life Sciences, Ocean University of China, Qingdao, China
    2. Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China
    3. Marine Biotechnology Research Center, State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China
    Contribution
    conceptualization, funding-acquisition, supervision
    For correspondence
    zhangyz@sdu.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2017-1005

Funding

National Key Research and Development Program of China (2018YFC1406700)

  • Yu-Zhong Zhang

National Science Foundation of China (91851205)

  • Yu-Zhong Zhang

National Science Foundation of China (31630012)

  • Yu-Zhong Zhang

National Key Research and Development Program of China (2016YFA0601303)

  • Yu-Zhong Zhang

National Science Foundation of China (U1706207)

  • Yu-Zhong Zhang

National Science Foundation of China (42076229)

  • Chun-Yang Li

National Science Foundation of China (31870052)

  • Xiu-Lan Chen

National Science Foundation of China (31800107)

  • Peng Wang

Major Scientific and Technological Innovation Project of Shandong Province (2019JZZY010817)

  • Yu-Zhong Zhang

Program of Shandong for Taishan Scholars (tspd20181203)

  • Yu-Zhong Zhang

Natural Environment Research Council (NE/N002385)

  • Jonathan D Todd

Natural Environment Research Council (NE/P012671)

  • Jonathan D Todd

Natural Environment Research Council (NE/S001352)

  • Jonathan D Todd

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank the staffs from BL18U1 & BL19U1 beamlines of National Facility for Protein Sciences Shanghai (NFPS) and Shanghai Synchrotron Radiation Facility, for assistance during data collection. We thank Caiyun Sun and Jingyao Qu from State Key laboratory of Microbial Technology of Shandong University for their help in HPLC and LC-MS. This work was supported by the National Key Research and Development Program of China (2018YFC1406700, 2016YFA0601303), the National Science Foundation of China (grants 91851205, 31630012, U1706207, 42076229, 31870052, 31800107), Major Scientific and Technological Innovation Project (MSTIP) of Shandong Province (2019JZZY010817), the Program of Shandong for Taishan Scholars (tspd20181203) and Natural Environment Research Council Standard grants (NE/N002385, NE/P012671 and NE/S001352).

Senior Editor

  1. Meredith C Schuman, University of Zurich, Switzerland

Reviewing Editor

  1. Georg Pohnert, University of Jena, Germany

Reviewer

  1. Georg Pohnert, University of Jena, Germany

Publication history

  1. Received: October 15, 2020
  2. Accepted: May 9, 2021
  3. Accepted Manuscript published: May 10, 2021 (version 1)
  4. Version of Record published: May 28, 2021 (version 2)

Copyright

© 2021, Li et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 824
    Page views
  • 152
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Ecology
    Brendan Cornwell et al.
    Short Report Updated

    Climate change is dramatically changing ecosystem composition and productivity, leading scientists to consider the best approaches to map natural resistance and foster ecosystem resilience in the face of these changes. Here, we present results from a large-scale experimental assessment of coral bleaching resistance, a critical trait for coral population persistence as oceans warm, in 221 colonies of the coral Acropora hyacinthus across 37 reefs in Palau. We find that bleaching-resistant individuals inhabit most reefs but are found more often in warmer microhabitats. Our survey also found wide variation in symbiont concentration among colonies, and that colonies with lower symbiont load tended to be more bleaching-resistant. By contrast, our data show that low symbiont load comes at the cost of lower growth rate, a tradeoff that may operate widely among corals across environments. Corals with high bleaching resistance have been suggested as a source for habitat restoration or selective breeding in order to increase coral reef resilience to climate change. Our maps show where these resistant corals can be found, but the existence of tradeoffs with heat resistance may suggest caution in unilateral use of this one trait in restoration.

    1. Ecology
    Line K Bay, Emily J Howells
    Insight

    The ability of corals to adapt to global warming may involve trade-offs among the traits that influence their success as the foundational species of coral reefs.