Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Convergent evolution of bacterial ceramide synthesis

Nature Chemical Biology volume 18, pages 305–312 (2022)Cite this article

Subjects

Abstract

The bacterial domain produces numerous types of sphingolipids with various physiological functions. In the human microbiome, commensal and pathogenic bacteria use these lipids to modulate the host inflammatory system. Despite their growing importance, their biosynthetic pathway remains undefined since several key eukaryotic ceramide synthesis enzymes have no bacterial homolog. Here we used genomic and biochemical approaches to identify six proteins comprising the complete pathway for bacterial ceramide synthesis. Bioinformatic analyses revealed the widespread potential for bacterial ceramide synthesis leading to our discovery of a Gram-positive species that produces ceramides. Biochemical evidence demonstrated that the bacterial pathway operates in a different order from that in eukaryotes. Furthermore, phylogenetic analyses support the hypothesis that the bacterial and eukaryotic ceramide pathways evolved independently.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: CCNA_01220 is a functional serine palmitoyltransferase (Spt).
Fig. 2: A genetic screen identified ceramide synthesis enzymes.
Fig. 3: Bioinformatic analysis identifies a wide range of potential ceramide-producing bacteria.
Fig. 4: Phylogenetic analysis indicates convergent evolution of ceramide synthesis.

Similar content being viewed by others

Data availability

The raw data for Fig. 1c and Extended Data Figs. 1c,d, 5c–e and 7a are provided as Microsoft Excel files in the Supplementary Information (Source Data 1–4). The data for the bioinformatic analyses was obtained from the following publicly available NCBI resources: NCBI Prokaryotic Representative Genomes: https://ftp.ncbi.nlm.nih.gov/genomes/GENOME_REPORTS/prok_representative_genomes.txt. Accession numbers for the proteins used for BLAST analyses are as follows. Bacterial Spt homologs: C. crescentus YP_002516593.1; P. gingivalis BAG34240; M. xanthus ABF87747; B. stolpii BAF73753. Bacterial bCerS homologs: C. crescentus YP_002516585.1; P. gingivalis BAG32893; M. xanthus ABF92629; B. stolpii WP_102243213. Bacterial CerR homologs: C. crescentus YP_002516595.1; P. gingivalis BAG34405; M. xanthus ABF87537; B. stolpii WP_102243212. Eukaryotic CerS homologs: Human P27544.1; A. thaliana NP_001184985; S. cerevisiae AAA21579.1. Eukaryotic Spt homologs: Human NP_006406.1; A. thaliana NP_190447.1; S. cerevisiae CAA56805.1. Eukaryotic KDSR homologs: Human NP_002026.1; A. thaliana NP_187257; S. cerevisiae P38342. Eukaryotic Gcn5 homologs: Human AAC39769.1 and S. cerevisiae NP_011768.1. Source data are provided with this paper.

References

  1. Harrison, P. J., Dunn, T. M. & Campopiano, D. J. Sphingolipid biosynthesis in man and microbes. Nat. Prod. Rep. 35, 921–954 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Brown, E. M. et al. Bacteroides-derived sphingolipids are critical for maintaining intestinal homeostasis and symbiosis. Cell Host Microbe 25, 668–680 e667 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Moye, Z. D., Valiuskyte, K., Dewhirst, F. E., Nichols, F. C. & Davey, M. E. Synthesis of sphingolipids impacts survival of Porphyromonas gingivalis and the presentation of surface polysaccharides. Front Microbiol. 7, 1919 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Stankeviciute, G., Guan, Z., Goldfine, H. & Klein, E. A. Caulobacter crescentus adapts to phosphate starvation by synthesizing anionic glycoglycerolipids and a novel glycosphingolipid. mBio 10, e00107–e00119 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ahrendt, T., Wolff, H. & Bode, H. B. Neutral and phospholipids of the Myxococcus xanthus lipodome during fruiting body formation and germination. Appl. Environ. Microbiol. 81, 6538–6547 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kaneshiro, E. S., Hunt, S. M. & Watanabe, Y. Bacteriovorax stolpii proliferation and predation without sphingophosphonolipids. Biochem. Biophys. Res. Commun. 367, 21–25 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Hannun, Y. A. & Obeid, L. M. Sphingolipids and their metabolism in physiology and disease. Nat. Rev. Mol. Cell Biol. 19, 175–191 (2018).

    Article  CAS  PubMed  Google Scholar 

  8. Merrill, A. H. Jr. Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chem. Rev. 111, 6387–6422 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ikushiro, H., Hayashi, H. & Kagamiyama, H. A water-soluble homodimeric serine palmitoyltransferase from Sphingomonas paucimobilis EY2395T strain. Purification, characterization, cloning, and overproduction. J. Biol. Chem. 276, 18249–18256 (2001).

    Article  CAS  PubMed  Google Scholar 

  10. Yard, B. A. et al. The structure of serine palmitoyltransferase; gateway to sphingolipid biosynthesis. J. Mol. Biol. 370, 870–886 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Geiger, O., Gonzalez-Silva, N., Lopez-Lara, I. M. & Sohlenkamp, C. Amino acid-containing membrane lipids in bacteria. Prog. Lipid Res. 49, 46–60 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Olea-Ozuna, R. J. et al. Five structural genes required for ceramide synthesis in Caulobacter and for bacterial survival. Environ. Microbiol. 23, 143–159 (2020).

  13. Wadsworth, J. M. et al. The chemical basis of serine palmitoyltransferase inhibition by myriocin. J. Am. Chem. Soc. 135, 14276–14285 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Harrison, P. J. et al. Use of isotopically labeled substrates reveals kinetic differences between human and bacterial serine palmitoyltransferase. J. Lipid Res. 60, 953–962 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Li, S., Xie, T., Liu, P., Wang, L. & Gong, X. Structural insights into the assembly and substrate selectivity of human SPT-ORMDL3 complex. Nat. Struct. Mol. Biol. 28, 249–257 (2021).

    Article  CAS  PubMed  Google Scholar 

  16. Raman, M. C. et al. The external aldimine form of serine palmitoyltransferase: structural, kinetic, and spectroscopic analysis of the wild-type enzyme and HSAN1 mutant mimics. J. Biol. Chem. 284, 17328–17339 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Raman, M. C., Johnson, K. A., Clarke, D. J., Naismith, J. H. & Campopiano, D. J. The serine palmitoyltransferase from Sphingomonas wittichii RW1: an interesting link to an unusual acyl carrier protein. Biopolymers 93, 811–822 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Ren, J. et al. Quantification of 3-ketodihydrosphingosine using HPLC-ESI-MS/MS to study SPT activity in yeast Saccharomyces cerevisiae. J. Lipid Res. 59, 162–170 (2018).

    Article  CAS  PubMed  Google Scholar 

  19. Zheng, W. et al. Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy. Biochim. Biophys. Acta 1758, 1864–1884 (2006).

    Article  CAS  PubMed  Google Scholar 

  20. Tidhar, R. et al. Eleven residues determine the acyl chain specificity of ceramide synthases. J. Biol. Chem. 293, 9912–9921 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chow, T. C. & Schmidt, J. M. Fatty acid composition of Caulobacter crescentus. Microbiology 83, 369–373 (1974).

    Google Scholar 

  22. Okino, N. et al. The reverse activity of human acid ceramidase. J. Biol. Chem. 278, 29948–29953 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Chen, M., Markham, J. E., Dietrich, C. R., Jaworski, J. G. & Cahoon, E. B. Sphingolipid long-chain base hydroxylation is important for growth and regulation of sphingolipid content and composition in Arabidopsis. Plant Cell 20, 1862–1878 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Omae, F. et al. DES2 protein is responsible for phytoceramide biosynthesis in the mouse small intestine. Biochem. J 379, 687–695 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Price, M. N. et al. Mutant phenotypes for thousands of bacterial genes of unknown function. Nature 557, 503–509 (2018).

    Article  CAS  PubMed  Google Scholar 

  26. Kawahara, K., Moll, H., Knirel, Y. A., Seydel, U. & Zähringer, U. Structural analysis of two glycosphingolipids from the lipopolysaccharide-lacking bacterium Sphingomonas capsulata. Eur. J. Biochem. 267, 1837–1846 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Feng, Y. & Cronan, J. E. Escherichia coli unsaturated fatty acid synthesis: complex transcription of the fabA gene and in vivo identification of the essential reaction catalyzed by FabB. J. Biol. Chem. 284, 29526–29535 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Christen, B. et al. The essential genome of a bacterium.Mol. Syst. Biol. 7, https://doi.org/10.1038/msb.2011.58 (2011).

  29. Stankeviciute, G. et al. Differential modes of crosslinking establish spatially distinct regions of peptidoglycan in Caulobacter crescentus. Mol. Microbiol. 111, 995–1008 (2019).

    Article  CAS  PubMed  Google Scholar 

  30. Rocha, F. G. et al. Porphyromonas gingivalis sphingolipid synthesis limits the host inflammatory response. J. Dent. Res. 99, 568–576 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Mun, J. et al. Structural confirmation of the dihydrosphinganine and fatty acid constituents of the dental pathogen Porphyromonas gingivalis. Org. Biomol. Chem. 5, 3826–3833 (2007).

    Article  CAS  PubMed  Google Scholar 

  32. Nguyen, M. & Vedantam, G. Mobile genetic elements in the genus Bacteroides, and their mechanism(s) of dissemination. Mob. Genet. Elements 1, 187–196 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Kavanagh, K. L., Jornvall, H., Persson, B. & Oppermann, U. Medium- and short-chain dehydrogenase/reductase gene and protein families: the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes. Cell. Mol. Life Sci. 65, 3895–3906 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Boyden, L. M. et al. Mutations in KDSR cause recessive progressive symmetric erythrokeratoderma. Am. J. Hum. Genet. 100, 978–984 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kageyama-Yahara, N. & Riezman, H. Transmembrane topology of ceramide synthase in yeast. Biochem. J 398, 585–593 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Spassieva, S. et al. Necessary role for the Lag1p motif in (dihydro)ceramide synthase activity. J. Biol. Chem. 281, 33931–33938 (2006).

    Article  CAS  PubMed  Google Scholar 

  37. Liebisch, G. et al. Update on LIPID MAPS classification, nomenclature and shorthand notation for MS-derived lipid structures. J. Lipid Res. 61, 1539–1555 (2020).

  38. Yao, L. et al. A selective gut bacterial bile salt hydrolase alters host metabolism. eLife 7, e37182 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Lee, M. T., Le, H. H. & Johnson, E. L. Dietary sphinganine is selectively assimilated by members of the mammalian gut microbiome. J. Lipid Res. 62, 100034 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Tudzynski, B. Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology. Appl. Microbiol. Biotechnol. 66, 597–611 (2005).

    Article  CAS  PubMed  Google Scholar 

  41. Huang, R., O’Donnell, A. J., Barboline, J. J. & Barkman, T. J. Convergent evolution of caffeine in plants by co-option of exapted ancestral enzymes. Proc. Natl Acad. Sci. USA 113, 10613–10618 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Dick, R. et al. Comparative analysis of benzoxazinoid biosynthesis in monocots and dicots: independent recruitment of stabilization and activation functions. Plant Cell 24, 915–928 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kawasaki, S. et al. The cell envelope structure of the lipopolysaccharide-lacking gram-negative bacterium Sphingomonas paucimobilis. J. Bacteriol. 176, 284–290 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Poindexter, J. S. Selection for nonbuoyant morphological mutants of Caulobacter crescentus. J. Bacteriol. 135, 1141–1145 (1978).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Ferrieres, L. et al. Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery. J. Bacteriol. 192, 6418–6427 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Martinez-Garcia, E., Calles, B., Arevalo-Rodriguez, M. & de Lorenzo, V. pBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes. BMC Microbiol. 11, 38 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bligh, E. G. & Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917 (1959).

    Article  CAS  PubMed  Google Scholar 

  48. Guan, Z., Katzianer, D., Zhu, J. & Goldfine, H. Clostridium difficile contains plasmalogen species of phospholipids and glycolipids. Biochim. Biophys. Acta 1842, 1353–1359 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Edgar, R. C. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinf. 5, 113 (2004).

    Article  Google Scholar 

  50. Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Miller, M. A., Pfeiffer, W. & Schwartz, T. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In Proc. 2010 Gateway Computing Environments Workshop (GCE), 1–8 (2010).

  52. Chamberlain, S. A. & Szocs, E. taxize: taxonomic search and retrieval in R. F1000Res. 2, 191 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Yu, G. Using ggtree to visualize data on tree-like structures. in Curr. Protoc. Bioinformatics 69, e96 (IEEE, 2020).

  54. Paradis, E. & Schliep, K. ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics 35, 526–528 (2019).

    Article  CAS  PubMed  Google Scholar 

  55. Wang, L. G. et al. Treeio: an R Package for phylogenetic tree input and output with richly annotated and associated data. Mol. Biol. Evol. 37, 599–603 (2020).

    Article  CAS  PubMed  Google Scholar 

  56. Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer-Verlag, 2016).

    Book  Google Scholar 

  57. Madeira, F. et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 47, W636–W641 (2019).

  58. Lowther, J. et al. Role of a conserved arginine residue during catalysis in serine palmitoyltransferase. FEBS Lett. 585, 1729–1734 (2011).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank C. Cugini (Rutgers University) for providing P. gingivalis genomic DNA for cloning. We also thank A. Geneva (Rutgers University-Camden) for helpful discussions. Funding was provided by National Science Foundation grant nos. MCB-1553004 and MCB-2031948 (E.A.K.), National Institutes of Health grant nos. GM069338 and R01AI148366 (Z.G.) and Biotechnology and Biological Sciences Research Council grant nos. BB/M010996/1 and BB/T016841/1 (D.J.C.).

Author information

Authors and Affiliations

  1. Center for Computational and Integrative Biology, Rutgers University-Camden, Camden, NJ, USA

    Gabriele Stankeviciute, Joshua D. Chamberlain, Aimiyah Coleman, Rachel D’Emilia, Larina Fu, Hung Nguyen & Eric A. Klein

  2. Rutgers Center for Lipid Research, Rutgers University, New Brunswick, NJ, USA

    Gabriele Stankeviciute & Eric A. Klein

  3. East Chem School of Chemistry, University of Edinburgh, Edinburgh, UK

    Peijun Tang, Ben Ashley, Eric C. Mohan & Dominic J. Campopiano

  4. Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Matthew E. B. Hansen

  5. Department of Biochemistry, Duke University Medical Center, Durham, NC, USA

    Ziqiang Guan

  6. Biology Department, Rutgers University-Camden, Camden, NJ, USA

    Eric A. Klein

Authors
  1. Gabriele Stankeviciute
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peijun Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ben Ashley
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Joshua D. Chamberlain
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Matthew E. B. Hansen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Aimiyah Coleman
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rachel D’Emilia
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Larina Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Eric C. Mohan
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Hung Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Ziqiang Guan
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Dominic J. Campopiano
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Eric A. Klein
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.S. made the mutant and complementation strains for characterizing the synthetic pathway and performed the lipid extractions. P.T., B.A. and E.C.M. purified and characterized the recombinant proteins. J.D.C. cloned and analyzed the P. buccae complementation strains. M.E.B.H. and E.A.K. performed the phylogenetic and bioinformatic analyses. E.A.K. acquired the microscopy images. A.C., R.D., L.F. and H.N. performed the transposon screen to isolate ceramide-deficient mutants. Z.G. performed the lipid mass spectrometry analyses. G.S., P.T., B.A., Z.G., D.J.C. and E.A.K. designed the experiments, interpreted the data and wrote the paper.

Corresponding authors

Correspondence to Ziqiang Guan, Dominic J. Campopiano or Eric A. Klein.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Chemical Biology thanks the anonymous reviewers for their contribution to the peer review of this work.

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

Extended data

Extended Data Fig. 1 C. crescentus Spt can use a variety of substrates.

(a) Recombinant CCNA_01220 (Spt) was purified as described in the methods. TP is total protein extract. Recombinant protein was independently purified at least three times with similar purities as judged by Coomassie staining. (b) The identity of the purified Spt protein was confirmed by LC/ESI/MS. The peaks correspond to the relative abundances of the multiple-charged species of the protein analyte; the charge is indicated in parenthesis. (c-d) Kinetic analyses of CCNA_01220 determined the Km values for C16:1-CoA (0.045 ± 0.004 mM. Panel c) and L-serine (11.97 ± 2.18 mM. Panel d) (n = 3; data are presented as mean + /- SD). (e) Positive- and negative-ion mode MS/MS analysis of ceramides from wild-type C. crescentus (see Fig. 2a) confirm that the desaturation and additional hydroxyl group are located on the acyl chain moiety. (f-g) Serine auxotrophic ΔserA cells were grown in HIGG media without serine, and lipids were isolated for MS/MS analysis. (f) Negative ion ESI/MS shows the [M + Cl]− ion of 1-deoxyceramide emerging at 2 to 3 min. The incorporation of alanine rather than serine is designated with a red-dashed oval. (g) Positive ion MS/MS analysis of the parent ion confirmed the identity of the 1-deoxyceramide.

Source data

Extended Data Fig. 2 A transposon screen for ceramide-deficient mutants yielded multiples hits in the spt genomic locus.

(a) The schematic illustrates the tandem positive/negative selection screen used to identify ceramide synthesis genes. Transposon mutants were initially screened for growth on polymyxin B. Resistant clones were then assayed for increased sensitivity to bacteriophage ϕCr30. The effects on ceramide production were assessed by LC/MS. (b) The sites of transposon insertions are indicated with a red triangle. Gene annotations are based on the results presented in Figs. 1–3 and Extended Data Fig. 3-5. The exact coordinates of the insertions are provided in Supplementary Table 1.

Extended Data Fig. 3 Acyl carrier protein and ACP-synthetase are required for ceramide synthesis in C. crescentus.

(a-b) ACP (ccna_01221) and ACP-synthetase (ccna_01223) deletion strains and the corresponding complementation strains were grown overnight in PYE with 0.3% (w/v) xylose. Negative ion ESI/MS shows the [M + Cl]− ions of the lipids emerging at 2 to 3 min. Ceramide species are labelled with a red dot. (c) A total ion chromatogram (TIC) and (d) the corresponding extracted ion chromatogram (EIC) of lipids from C. crescentus grown in PYE media show the major species present in the lipid extract.

Extended Data Fig. 4 MS/MS confirms the production of oxidized ceramide in the absence of CerR.

The oxCer lipid produced in the ΔcerR strain (Fig. 2a, middle panel) was subjected to MS/MS analysis. The fragment ions confirm the presence of two oxidized C = O bonds.

Extended Data Fig. 5 Purification of C. crescentus bCerS.

(a) bCerS was purified as described in the Materials and Methods. SDS–PAGE analysis confirmed protein purity following S200 size-exclusion chromatography. The indicated fractions were pooled for MS characterization. Recombinant protein was independently purified at least three times with similar purities as judged by Coomassie staining. (b) The identity of the purified bCerS protein was confirmed by LC/ESI/MS. The protein molecular weight was calculated using the relative abundances of the multiple-charged species of the protein analyte (bottom); the charges are indicated in parenthesis. (c) Kinetic analyses of CCNA_01212 in the absence (left) and presence (right) of 3-KDS determined the Km,app value for C16:0-CoA (21.3 ± 4.1 µM) (n = 3, data are presented as mean + /- SD). (d) To determine the substrate specificity of C. crescentus bCerS, we incubated the recombinant enzyme with 40 µM 3-KDS and an equimolar mixture of fatty acid-CoA (C8-C24) with a total final concentration of 50 µM. The reaction proceeded for 1 hr and the reaction products were analyzed by ESI/MS. The integrated ion counts were normalized to C24 (n = 3, data are presented as mean + /- SD). (e) To determine the acyl-chain saturation preference of C. crescentus bCerS, we incubated the recombinant enzyme with 40 µM 3-KDS and an equimolar mixture of C16:0-CoA and C16:1-CoA, with a total final concentration of 50 µM. The reaction proceeded for 1 hr and the reaction products were analyzed by NPLC-ESI/MS in the negative ion mode to determine the ratio of ceramide products produced (C16:0 product, [M + Cl]− at m/z 572.481; C16:1 product, [M + Cl]− at m/z 570.466) (n = 3, data are presented as mean + /- SD). (f) Recombinant bCerS was incubated with 40 µM sphinganine and 50 µM C16:0-CoA for 1 hr and the reaction product was analyzed by ESI/MS.

Source data

Extended Data Fig. 6 Phylogeny of bacterial ceramide synthesis enzymes.

Unrooted phylogenetic trees were created for the individual Spt, CerR, and bCerS proteins from the organisms identified in Fig. 3d (left). A zoomed in view of the branches of the trees containing Actinobacteria show the close relationship between these Gram-positive organisms and Deltaproteobacteria (right). For each of the individual enzymes, the closest homologues for the Actinobacteria are found in Deltaproteobacteria. Bootstrap percentages are indicated by the filled circles at each node.

Extended Data Fig. 7 Ceramide synthesis orthologues.

(a) Using the genomic coordinates acquired during the phylogenetic analysis of bacterial ceramide genes, the distances between spt, bcerS, and cerR were calculated. The histogram columns are colored by taxonomic class. The numbers above the columns are the average distance ± the standard error of the mean. (b) MS/MS analysis of ceramides from S. aurantiacus confirm that the desaturation occurs on the long-chain base. (c) MS/MS analysis of ceramides produced by bCerS homologues from P. buccae (see Fig. 3f) show that HMPREF0649_00885 preferred fully saturated substrates, while HMPREF0649_00886 used a desaturated palmitoyl-CoA substrate.

Source data

Extended Data Fig. 8 Alignment of Spt across taxonomic domains suggests a common evolutionary ancestor.

Protein alignment of several eukaryotic and bacterial Spt enzymes was performed using Clustal Omega57. Previously characterized active site residues are indicated by (*)58.

Extended Data Fig. 9 Alignment of reductase enzymes suggests independent evolution of CerR and KDSR.

(a-b) Alignments of bacterial CerR proteins (a) or C. crescentus CerR and human KDSR (b) were done using Clustal Omega57. Green-highlighted residues are active site amino acids in KDSR34. The YxxxK motif is the reductase active site and the TGxxxGxG motif is the NAD binding site. Note that the bacterial NAD binding site, TGxxGFxG, is different from the eukaryotic site.

Extended Data Fig. 10 Alignment of ceramide synthase enzymes suggests independent evolution of bCerS and CerS.

(a) bCerS homologues were aligned using Clustal Omega57. (b) The conserved Lag1P domains from eukaryotic CerS proteins35,36 were aligned with Clustal Omega57. Active-site residues are highlighted in yellow. The Lag1P domain is absent from bCerS.

Supplementary information

Supplementary Information

Supplementary Methods and Tables 1–6.

Reporting Summary

Supplementary Data 1

TBLASTN hits and query parameters used to identify bacterial species with genes encoding Spt, bCerS and CerR.

Supplementary Data 2

Bacterial species with multiple bCerS homologs.

Source data

Source Data Fig. 1

Source data for Fig. 1c.

Source Data Extended Data Fig. 1

Source data for Extended Data Fig. 1c,d.

Source Data Extended Data Fig. 5

Source data for Extended Data Fig. 5c–e.

Source Data Extended Data Fig. 7

Source data for Extended Data Fig. 7a.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Stankeviciute, G., Tang, P., Ashley, B. et al. Convergent evolution of bacterial ceramide synthesis. Nat Chem Biol 18, 305–312 (2022). https://doi.org/10.1038/s41589-021-00948-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-021-00948-7

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology