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.
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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.
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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.).
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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.
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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.
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.
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.
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.
Supplementary information
Supplementary Information
Supplementary Methods and Tables 1–6.
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.
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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
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DOI: https://doi.org/10.1038/s41589-021-00948-7
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