Identification of a protein responsible for the synthesis of archaeal membrane-spanning GDGT lipids

Abstract

Glycerol dibiphytanyl glycerol tetraethers (GDGTs) are archaeal monolayer membrane lipids that can provide a competitive advantage in extreme environments. Here, we identify a radical SAM protein, tetraether synthase (Tes), that participates in the synthesis of GDGTs. Attempts to generate a tes-deleted mutant in Sulfolobus acidocaldarius were unsuccessful, suggesting that the gene is essential in this organism. Heterologous expression of tes homologues leads to production of GDGT and structurally related lipids in the methanogen Methanococcus maripaludis (which otherwise does not synthesize GDGTs and lacks a tes homolog, but produces a putative GDGT precursor, archaeol). Tes homologues are encoded in the genomes of many archaea, as well as in some bacteria, in which they might be involved in the synthesis of bacterial branched glycerol dialkyl glycerol tetraethers.

Fig. 1. Tes (Maeo_0574 or MA_1486) is sufficient for GDGT synthesis.

LC-MS extracted ion chromatograms of acid hydrolyzed lipid extracts of M. maripaludis wild type (WT) with empty plasmid pMEV4 or ma_1114 heterologous expression showing only production of archaeol, and M. maripaludis WT with maeo_0574 or ma_1486 heterologous expression producing GDGT. Mass spectra are shown in Supplementary Fig. 1. Source data are provided as a Source Data file.

Fig. 2. GDGT biosynthesis pathway, intermediate, and side product.

a LC-MS extracted ion chromatograms of acid hydrolyzed extracts from heterologous expression strains showing that Tes (maeo_0574) can generate not only GDGT, but also the potential biosynthetic intermediate GTGT, and the side product macrocyclic archaeol. b Proposed GDGT biosynthesis pathway based on the Tes heterologous expression products. The chromatograms of all Tes homologs expression strains are shown in Supplementary Fig. 2, and mass spectra are shown in Supplementary Fig. 1. Notice that the double bonds on archaeol may be required for the condensation reaction (see discussion). Source data are provided as a Source Data file.

Fig. 3. Quantification of lipids formation during Tes expression in M. maripaludis.

The abundance of core GDGT-related lipids (ng/mg dry cell mass) from heterologous expression of Tes genes maeo_0574, ma_1486, or ma_1114, showing maeo_0574 is more efficient to synthesize GDGT and related lipids than the other homologs. The abundance of core lipids was calculated relative to the peak area of the synthetic C46 GTGT internal standard (5). Results are the mean ± standard error from three biological replicates. Source data are provided as a Source Data file.

Fig. 4. Tes homologs are found in diverse archaeal and bacterial phyla.

These data were generated through BLASTP searches of Tes homologs (e-value < 1e⁻⁵⁰, identity > 20%) in the NCBI database and searching published studies demonstrating GDGT production from pure cultures^,,,,,–. At least one strain or one genome in the phylum, class or order containing the Tes homolog and GDGTs (at least GDGT-0) is marked with a black circle. Otherwise, archaea or bacteria without any Tes homolog or GDGTs are marked with white circles. Uncultured archaeal strains or with cultured strains not yet tested for GDGT production, are marked with gray circles. Only one putative Tes homolog (e-value = 6e⁻⁵³, identity = 28%) was identified from 231 MG-II MAGs (*). Phylogenetic lineages of archaea and bacteria are modified from references^,,. Proteobacteria and acidobacteria are divided into class level and order level, respectively. Terrabacteria, FCB (Fibrobacteres-Chlorobi-Bacteroidetes), PVC (Planctomycetes-Verrucomicrobia-Chlamydiae) and CPR (Candidate Phyla Radiation) are bacterial superphyla^, and only several phyla are shown in each group. Bacterial phyla that do not belong to these groups are assigned “Other” in the figure. Details and references are listed in Supplementary Table 2.