DSYB catalyses the key step of dimethylsulfoniopropionate biosynthesis in many phytoplankton

Article metrics

Abstract

Dimethylsulfoniopropionate (DMSP) is a globally important organosulfur molecule and the major precursor for dimethyl sulfide. These compounds are important info-chemicals, key nutrients for marine microorganisms, and are involved in global sulfur cycling, atmospheric chemistry and cloud formation1,2,3. DMSP production was thought to be confined to eukaryotes, but heterotrophic bacteria can also produce DMSP through the pathway used by most phytoplankton4, and the DsyB enzyme catalysing the key step of this pathway in bacteria was recently identified5. However, eukaryotic phytoplankton probably produce most of Earth’s DMSP, yet no DMSP biosynthesis genes have been identified in any such organisms. Here we identify functional dsyB homologues, termed DSYB, in many phytoplankton and corals. DSYB is a methylthiohydroxybutryate methyltransferase enzyme localized in the chloroplasts and mitochondria of the haptophyte Prymnesium parvum, and stable isotope tracking experiments support these organelles as sites of DMSP synthesis. DSYB transcription levels increased with DMSP concentrations in different phytoplankton and were indicative of intracellular DMSP. Identification of the eukaryotic DSYB sequences, along with bacterial dsyB, provides the first molecular tools to predict the relative contributions of eukaryotes and prokaryotes to global DMSP production. Furthermore, evolutionary analysis suggests that eukaryotic DSYB originated in bacteria and was passed to eukaryotes early in their evolution.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Transamination pathway for DMSP biosynthesis pathway in bacteria and marine algae and phylogenetic tree of DsyB/DSYB proteins.
Fig. 2: Immunogold localization of DSYB in P. parvum CCAP946/6.
Fig. 3: Subcellular distribution of 34S in P. parvum CCAP946/6 following sulfur uptake for 48 h.

Change history

  • 30 January 2019

    In the version of this Letter originally published, the Methods incorrectly stated that all phytoplankton cultures were sampled in mid-exponential phase. The low-nitrogen cultures were sampled in early stationary phase and at the point at which Fv/Fm values decreased, to indicate that cultures were experiencing low-nitrogen conditions. All other phytoplankton cultures were sampled in exponential phase. Growth and Fv/Fm data are provided here on high- and low-nitrogen cultures (Figs 1, 2 and 3) to clarify and support this correction. The Methods also stated that cell counting was done using a Beckman Multisizer 3 Coulter Counter, but a CASY Model TT Cell Counter was used.

References

  1. 1.

    Nevitt, G. A. The neuroecology of dimethyl sulfide: a global-climate regulator turned marine infochemical. Integr. Comp. Biol. 51, 819–825 (2011).

  2. 2.

    Sievert, S. M., Kiene, R. P. & Schulz-Vogt, H. N. The sulfur cycle. Oceanography 20, 117–123 (2007).

  3. 3.

    Curson, A. R., Todd, J. D., Sullivan, M. J. & Johnston, A. W. Catabolism of dimethylsulphoniopropionate: microorganisms, enzymes and genes. Nat. Rev. Microbiol. 9, 849–859 (2011).

  4. 4.

    Summers, P. S. et al. Identification and stereospecificity of the first three enzymes of 3-dimethylsulfoniopropionate biosynthesis in a chlorophyte alga. Plant Physiol. 116, 369–378 (1998).

  5. 5.

    Curson, A. R. et al. Dimethylsulfoniopropionate biosynthesis in marine bacteria and identification of the key gene in this process. Nat. Microbiol. 2, 17009 (2017).

  6. 6.

    Caruana, A. M. N. & Malin, G. The variability in DMSP content and DMSP lyase activity in marine dinoflagellates. Prog. Oceanogr. 120, 410–424 (2014).

  7. 7.

    Lyon, B. R., Lee, P. A., Bennett, J. M., DiTullio, G. R. & Janech, M. G. Proteomic analysis of a sea-ice diatom: salinity acclimation provides new insight into the dimethylsulfoniopropionate production pathway. Plant Physiol. 157, 1926–1941 (2011).

  8. 8.

    Raina, J. B. et al. DMSP biosynthesis by an animal and its role in coral thermal stress response. Nature 502, 677–680 (2013).

  9. 9.

    Keller, M. D., Bellows, W. K. & Guillard, R. R. L. in Biogenic Sulfur in the Environment (eds Saltzman, E. S. & Cooper, W. J.) Ch. 11 (American Chemical Society, Washington DC, 1989).

  10. 10.

    Nei, M. & Rooney, A. P. Concerted and birth-and-death evolution of multigene families. Annu. Rev. Genet. 39, 121–152 (2005).

  11. 11.

    Ku, C. et al. Endosymbiotic origin and differential loss of eukaryotic genes. Nature 524, 427–432 (2015).

  12. 12.

    Baumgarten, S. et al. The genome of Aiptasia, a sea anemone model for coral symbiosis. Proc. Natl Acad. Sci. USA 112, 11893–11898 (2015).

  13. 13.

    Van Alstyne, K. L. & Puglisi, M. P. DMSP in marine macroalgae and macroinvertebrates: distribution, function, and ecological impacts. Aquat. Sci. 69, 394–402 (2007).

  14. 14.

    Spielmeyer, A. & Pohnert, G. Influence of temperature and elevated carbon dioxide on the production of dimethylsulfoniopropionate and glycine betaine by marine phytoplankton. Mar. Environ. Res. 73, 62–69 (2012).

  15. 15.

    Dickschat, J. S., Rabe, P. & Citron, C. A. The chemical biology of dimethylsulfoniopropionate. Org. Biomol. Chem. 13, 1954–1968 (2015).

  16. 16.

    Hovde, B. T. et al. Genome sequence and transcriptome analyses of Chrysochromulina tobin: metabolic tools for enhanced algal fitness in the prominent order Prymnesiales (Haptophyceae). PLoS Genet. 11, e1005469 (2015).

  17. 17.

    Jones, H. L. J., Leadbeater, B. S. C. & Green, J. C. Mixotrophy in marine species of Chrysochromulina (Prymnesiophyceae) – ingestion and digestion of a small green flagellate. J. Mar. Biol. Assoc. UK 73, 283–296 (1993).

  18. 18.

    Kettles, N. L., Kopriva, S. & Malin, G. Insights into the regulation of DMSP synthesis in the diatom Thalassiosira pseudonana through APR activity, proteomics and gene expression analyses on cells acclimating to changes in salinity, light and nitrogen. PLoS ONE 9, e94795 (2014).

  19. 19.

    Dickson, D. M. J. & Kirst, G. O. Osmotic adjustment in marine eukaryotic algae: the role of inorganic-ions, quaternary ammonium, tertiary sulfonium and carbohydrate solutes. II Prasinophytes and Haptophytes. New Phytol. 106, 657–666 (1987).

  20. 20.

    Trossat, C. et al. Salinity promotes accumulation of 3-dimethylsulfoniopropionate and its precursor S-methylmethionine in chloroplasts. Plant Physiol. 116, 165–171 (1998).

  21. 21.

    Gruber, A. et al. Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif. Plant Mol. Biol. 64, 519–530 (2007).

  22. 22.

    Raina, J. B. et al. Subcellular tracking reveals the location of dimethylsulfoniopropionate in microalgae and visualises its uptake by marine bacteria. eLife 6, e23008 (2017).

  23. 23.

    Matrai, P. A. & Keller, M. D. Total organic sulfur and dimethylsulfoniopropionate in marine phytoplankton: intracellular variations. Mar. Biol. 119, 61–68 (1994).

  24. 24.

    Stefels, J. Physiological aspects of the production and conversion of DMSP in marine algae and higher plants. J. Sea Res. 43, 183–197 (2000).

  25. 25.

    Sunda, W., Kieber, D. J., Kiene, R. P. & Huntsman, S. An antioxidant function for DMSP and DMS in marine algae. Nature 418, 317–320 (2002).

  26. 26.

    Sunagawa, S. et al. Structure and function of the global ocean microbiome. Science 348, 1261359 (2015).

  27. 27.

    Johnston, A. W. B., Green, R. T. & Todd, J. D. Enzymatic breakage of dimethylsulfoniopropionate – a signature molecule for life at sea. Curr. Opin. Chem. Biol. 31, 58–65 (2016).

  28. 28.

    Belviso, S. et al. Size distribution of dimethylsulfoniopropionate (DMSP) in areas of the tropical northeastern Atlantic Ocean and the Mediterranean Sea. Mar. Chem. 44, 55–71 (1993).

  29. 29.

    Amin, S. A. et al. Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria. Nature 522, 98–101 (2015).

  30. 30.

    Guillard, R. R. L. in Culture of Marine Invertebrate Animals (eds Smith, W. L. & Chanley, M. H.) 29–60 (Plenum Press, New York, 1975).

  31. 31.

    Berges, J. A., Franklin, D. J. & Harrison, P.J. Evolution of an artificial seawater medium: improvements in enriched seawater, artificial water over the last two decades. J. Phycol. 37, 1138–1145 (2001).

  32. 32.

    Mock, T. et al. Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus. Nature 541, 536–540 (2017).

  33. 33.

    Fixen, K. R. et al. Genome sequences of eight bacterial species found in coculture with the haptophyte Chrysochromulina tobin. Genome Announc. 4, e01162-16 (2016).

  34. 34.

    Sambrook, J., Fritsch, E. F., Maniatis, T. & Nolan, C. Molecular Cloning: A Laboratory Manual 2nd edn, Vol. 3 (Cold Spring Harbor Laboratory Press, New York, 1989).

  35. 35.

    Beringer, J. E. R factor transfer in Rhizobium leguminosarum. J. Gen. Microbiol. 84, 188–198 (1974).

  36. 36.

    Gonzalez, J. M., Whitman, W. B., Hodson, R. E. & Moran, M. A. Identifying numerically abundant culturable bacteria from complex communities: an example from a lignin enrichment culture. Appl. Environ. Microbiol. 62, 4433–4440 (1996).

  37. 37.

    Baumann, P. & Baumann, L. in The Prokaryotes: A Handbook on Habitats, Isolation and Identification of Bacteria 1st edn (eds Starr, M. P., Stolp, H., Truper, H. G., Balows, A. & Schlegel, H. G.) 1302–1331 (Springer-Verlag, Berlin, 1981).

  38. 38.

    Porter, K. G. & Feig, Y. S. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25, 943–948 (1980).

  39. 39.

    Figurski, D. H. & Helinski, D. R. Replication of an origin-containing derivative of plasmid Rk2 dependent on a plasmid function provided in trans. Proc. Natl Acad. Sci. USA 76, 1648–1652 (1979).

  40. 40.

    Downie, J. A. et al. Cloned nodulation genes of Rhizobium leguminosarum determine host range specificity. Mol. Gen. Genet. 190, 359–365 (1983).

  41. 41.

    Keen, N. T., Tamaki, S., Kobayashi, D. & Trollinger, D. Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria. Gene 70, 191–197 (1988).

  42. 42.

    Tett, A. J., Rudder, S. J., Bourdes, A., Karunakaran, R. & Poole, P. S. Regulatable vectors for environmental gene expression in Alphaproteobacteria. Appl. Environ. Microbiol. 78, 7137–7140 (2012).

  43. 43.

    Untergasser, A. et al. Primer3 – new capabilities and interfaces. Nucleic Acids Res. 40, e115 (2012).

  44. 44.

    Heid, C. A., Stevens, J., Livak, K. J. & Williams, P. M. Real time quantitative PCR. Genome Res. 6, 986–994 (1996).

  45. 45.

    Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the method. Methods 25, 402–408 (2001).

  46. 46.

    Mahmood, T. & Yang, P. C. Western blot: technique, theory, and trouble shooting. N. Am. J. Med. Sci. 4, 429–434 (2012).

  47. 47.

    Perez-Cruz, C. et al. New type of outer membrane vesicle produced by the Gram-negative bacterium Shewanella vesiculosa M7T: implications for DNA content. Appl. Environ. Microbiol. 79, 1874–1881 (2013).

  48. 48.

    Kilburn, M. R. & Clode, P. L. in Electron Microscopy: Methods and Protocols 3rd edn, Vol. 1117 (ed. Walker, J. M.) Ch. 33 (Humana Press, New York, 2014).

  49. 49.

    Schindelin, J. et al. Fiji: an open source platform for biological image analysis. Nat. Methods 9, 676–682 (2012).

  50. 50.

    Hillion, F., Kilburn, M. R., Hoppe, P., Messenger, S. & Webers, P. K. The effect of QSA on S, C, O and Siisotopic ratio measurements. Geochim. Cosmochim. Acta 72, A377 (2008).

  51. 51.

    R Development Core Team. R : A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, Vienna, 2008).

  52. 52.

    Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

  53. 53.

    Keeling, P. J. et al. The marine microbial eukaryote transcriptome sequencing project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing. PLoS Biol. 12, e1001889 (2014).

  54. 54.

    Toribio, A. L. et al. European nucleotide archive in 2016. Nucleic Acids Res. 45, 32–36 (2017).

  55. 55.

    Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal probabilistic RNA-seq quantification. Nat. Biotechnol. 34, 525–527 (2016).

  56. 56.

    Katoh, K., Misawa, K., Kuma, K. & Miyata, T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30, 3059–3066 (2002).

  57. 57.

    Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

  58. 58.

    Schwarz, G. Estimating dimension of a model. Ann. Stat. 6, 461–464 (1978).

  59. 59.

    Le, S. Q. & Gascuel, O. An improved general amino acid replacement matrix. Mol. Biol. Evol. 25, 1307–1320 (2008).

  60. 60.

    Nguyen, L. T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).

  61. 61.

    Trifinopoulos, J., Nguyen, L. T., von Haeseler, A. & Minh, B. Q. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res. 44, 232–235 (2016).

  62. 62.

    Minh, B. Q., Nguyen, M. A. T. & von Haeseler, A. Ultrafast approximation for phylogenetic bootstrap. Mol. Biol. Evol. 30, 1188–1195 (2013).

  63. 63.

    Yu, G. C., Smith, D. K., Zhu, H. C., Guan, Y. & Lam, T. T. Y. GGTREE: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods Ecol. Evol. 8, 28–36 (2017).

  64. 64.

    Kumaresan, D. et al. Aerobic proteobacterial methylotrophs in Movile Cave: genomic and metagenomic analyses. Microbiome 6, 1 (2018).

  65. 65.

    Todd, J. D. et al. Structural and regulatory genes required to make the gas dimethyl sulfide in bacteria. Science 315, 666–669 (2007).

  66. 66.

    Todd, J. D. et al. Molecular dissection of bacterial acrylate catabolism – unexpected links with dimethylsulfoniopropionate catabolism and dimethyl sulfide production. Environ. Microbiol. 12, 327–343 (2010).

  67. 67.

    Curson, A. R. J., Sullivan, M. J., Todd, J. D. & Johnston, A. W. B. Identification of genes for dimethyl sulfide production in bacteria in the gut of Atlantic Herring (Clupea harengus). ISME J. 4, 144–146 (2010).

  68. 68.

    Curson, A. R. J., Fowler, E. K., Dickens, S., Johnston, A. W. B. & Todd, J. D. Multiple DMSP lyases in the gamma-proteobacterium Oceanimonas doudoroffii. Biogeochemistry 110, 109–119 (2012).

  69. 69.

    Sun, J. et al. The abundant marine bacterium Pelagibacter simultaneously catabolizes dimethylsulfoniopropionate to the gases dimethyl sulfide and methanethiol. Nat. Microbiol. 1, 16065 (2016).

  70. 70.

    Curson, A. R., Rogers, R., Todd, J. D., Brearley, C. A. & Johnston, A. W. Molecular genetic analysis of a dimethylsulfoniopropionate lyase that liberates the climate-changing gas dimethylsulfide in several marine α-proteobacteria and Rhodobacter sphaeroides. Environ. Microbiol. 10, 757–767 (2008).

  71. 71.

    Todd, J. D., Curson, A. R. J., Dupont, C. L., Nicholson, P. & Johnston, A. W. B. The ddd P gene, encoding a novel enzyme that converts dimethylsulfoniopropionate into dimethyl sulfide, is widespread in ocean metagenomes and marine bacteria and also occurs in some Ascomycete fungi. Environ. Microbiol. 11, 1376–1385 (2009).

  72. 72.

    Todd, J. D. et al. DddQ, a novel, cupin-containing, dimethylsulfoniopropionate lyase in marine roseobacters and in uncultured marine bacteria. Environ. Microbiol. 13, 427–438 (2011).

  73. 73.

    Curson, A. R. J., Sullivan, M. J., Todd, J. D. & Johnston, A. W. B. DddY, a periplasmic dimethylsulfoniopropionate lyase found in taxonomically diverse species of Proteobacteria. ISME J. 5, 1191–1200 (2011).

  74. 74.

    Todd, J. D., Kirkwood, M., Newton-Payne, S. & Johnston, A. W. B. DddW, a third DMSP lyase in a model Roseobacter marine bacterium, Ruegeria pomeroyi DSS-3. ISME J. 6, 223–226 (2012).

  75. 75.

    Alcolombri, U. et al. Identification of the algal dimethyl sulfide-releasing enzyme: a missing link in the marine sulfur cycle. Science 348, 1466–1469 (2015).

  76. 76.

    Eddy, S. R. Accelerated profile HMM searches. PLoS Comput. Biol. 7, e1002195 (2011).

  77. 77.

    Fish, J. A. et al. FunGene: the functional gene pipeline and repository. Front. Microbiol. 4, 291 (2013).

  78. 78.

    Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2 – approximately maximum-likelihood trees for large alignments. PLoS ONE 5, e9490 (2010).

  79. 79.

    Letunic, I. & Bork, P. Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 44, 242–245 (2016).

  80. 80.

    Masella, A. P., Bartram, A. K., Truszkowski, J. M., Brown, D. G. & Neufeld, J. D. PANDAseq: paired-end assembler for illumina sequences. BMC Bioinformatics 13, 31 (2012).

Download references

Acknowledgements

Funding from the Natural Environment Research Council (NE/J01138X/1, NE/M004449/1, NE/N002385/1 and NE/P012671/1) supported work in J.D.T.’s laboratory. B.T.W. was supported by an NERC EnvEast grant (NE/L002582/1) and A.B.M. was supported by a BBSRC Norwich Research Park Biosciences Doctoral Training Partnership grant (BB/M011216/1). The NanoSIMS work was supported by an Australian Research Council Grant (DE160100636) to J.-B.R. We thank P. Wells and M. Giardina for general technical support, T. Mock for supplying Fragilariopsis cylindrus, and R. Green, J. Liu and C. Murrell for advice and discussion of results. We also acknowledge the Tara Oceans Consortium for providing metagenomic sequence data, and the facilities at the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation & Analysis, University of Western Australia, a facility funded by the University, State and Commonwealth Governments.

Author information

J.D.T. wrote the paper, designed experiments, performed experiments (gene cloning, enzyme assays, bioinformatics) and analysed data; A.R.J.C. wrote the paper, designed experiments, performed experiments (gene cloning, enzyme assays, GC to quantify DMSP/DMSHB, phytoplankton growth experiments), analysed data and prepared figures/tables; B.T.W. performed experiments (bioinformatics analysis of DsyB/DSYB in transcriptomes, metagenomes and metatranscriptomes, phylogenetic tree construction), analysed data and prepared figures/tables; B.J.P. performed experiments (gene cloning, RNA isolation, RT–qPCR experiments, protein purification, in vitro enzyme assays and western blots) and analysed data; L.P.S. performed experiments (gene cloning) and analysed data; A.B.M. performed experiments (LC-MS detection of DMSP and glycine betaine) and analysed data; P.P.L.R. performed experiments (phytoplankton growth experiments); D.K. performed experiments (bioinformatic analysis and phylogenetic tree construction); E.M. performed experiments (immunogold labelling, microscopy) and prepared figures; L.G.S. wrote the paper, performed experiments (evolutionary analysis of DsyB and DSYB sequences and phylogenetic tree construction) and prepared figures/tables; J.-B.R. wrote the paper, performed experiments (NanoSIMS, LC-MRM-MS) and prepared figures; U.K. performed experiments (LC-MRM-MS); P.L.C. and P.G. performed experiments (NanoSIMS); O.C. designed antibodies and prepared materials for microscopy; S.M. performed experiments (bioinformatic analysis); and R.A.C. supplied C. tobin CCMP291 strain. All authors reviewed the manuscript before submission.

Correspondence to Jonathan D. Todd.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–5, 7; Supplementary Tables 1,2,4,5,7,8,10 and 11; and Supplementary References

Life Sciences Reporting Summary

Supplementary Figure 6

Phylogenetic tree of environmental DsyB/DSYB protein sequences

Supplementary Table 3

DSYB proteins identified from genomes and transcriptomes

Supplementary Table 6

Metagenome information and results of DsyB and DSYB metagenomic searches

Supplementary Table 9.

GeoMICS metatranscriptome dsyB, DSYB and DMSP lyase gene transcript abundance

Supplementary Data 1

DSYB amino acid sequences identified from genomes or transcriptomes. Presented in FASTA format

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Further reading