GDGT cyclization proteins identify the dominant archaeal sources of tetraether lipids in the ocean

Abstract

Glycerol dibiphytanyl glycerol tetraethers (GDGTs) are distinctive archaeal membrane-spanning lipids with up to eight cyclopentane rings and/or one cyclohexane ring. The number of rings added to the GDGT core structure can vary as a function of environmental conditions, such as changes in growth temperature. This physiological response enables cyclic GDGTs preserved in sediments to be employed as proxies for reconstructing past global and regional temperatures and to provide fundamental insights into ancient climate variability. Yet, confidence in GDGT-based paleotemperature proxies is hindered by uncertainty concerning the archaeal communities contributing to GDGT pools in modern environments and ambiguity in the environmental and physiological factors that affect GDGT cyclization in extant archaea. To properly constrain these uncertainties, a comprehensive understanding of GDGT biosynthesis is required. Here, we identify 2 GDGT ring synthases, GrsA and GrsB, essential for GDGT ring formation in Sulfolobus acidocaldarius Both proteins are radical S-adenosylmethionine proteins, indicating that GDGT cyclization occurs through a free radical mechanism. In addition, we demonstrate that GrsA introduces rings specifically at the C-7 position of the core GDGT lipid, while GrsB cyclizes at the C-3 position, suggesting that cyclization patterns are differentially controlled by 2 separate enzymes and potentially influenced by distinct environmental factors. Finally, phylogenetic analyses of the Grs proteins reveal that marine Thaumarchaeota, and not Euryarchaeota, are the dominant source of cyclized GDGTs in open ocean settings, addressing a major source of uncertainty in GDGT-based paleotemperature proxy applications.

Keywords: GDGT; Sulfolobus; paleotemperature proxies; radical SAM.

Fig. 1.

GrsA (Saci_1585) and GrsB (Saci_0240) are required for GDGT ring formation. (A) Various GDGT structures produced by S. acidocaldarius. Acyclic GDGT-0 is hypothesized to be the biosynthetic precursor to cyclic GDGTs. The C-7 and C-3 positions are indicated on GDGT-8. (B) LC-MS merged extracted ion chromatograms (EICs) of acid-hydrolyzed lipid extracts of S. acidocaldarius wild type (WT), grs single and double mutants, and the complemented double deletion strain. Grey boxes indicate amplification of y axis to highlight smaller peaks. (C) LC-MS merged EICs of acid-hydrolyzed lipid extracts of M. acetivorans WT with empty plasmid pJK031A showing only a minor production of GDGT-0 and M. acetivorans WT with S. acidocaldarius grsA and grsB heterologously expressed producing GDGTs with up to 2 rings. The numbers above each chromatographic peak indicate the number of cyclopentane rings in the GDGT structure. Multiple isomers of ring-containing GDGTs and their mass spectra are shown in SI Appendix, Fig. S1.

Fig. 2.

GrsA and GrsB insert rings at distinct locations on the core GDGT structure. (A) LC-MS merged EICs of acid-hydrolyzed extracts from the complemented ΔgrsAΔgrsB deletion strains showing that GrsA can generate GDGT-1 to GDGT-4 (Top) and GrsB only produces GDGT-1 and GDGT-2 (Bottom). (B) GrsA is proposed to utilize GDGT-0 as a substrate and introduces 4 rings at the C-7 position, while GrsB prefers a substrate with rings at C-7 and introduces rings at the C-3 position. GrsB is also able to introduce rings at the C-3 position of GDGT-0, but this is thought to be an unfavorable side reaction in the complementation experiments. (C) GC-MS total ion chromatograms of biphytanes released by ether cleavage of lipid extracts from S. acidocaldarius WT, ΔgrsAΔgrsB complemented with grsA alone, grsB alone, and both genes together, showing that GrsA synthesizes rings exclusively at C-7 and GrsB at C-3. Grey boxes indicate amplification of y axis to highlight smaller peaks. bp, biphytane. Mass spectra of biphytanes are shown in SI Appendix, Fig. S4.

Fig. 3.

S. solfataricus Grs homologs are functional. (A) LC-MS merged EICs of acid-hydrolyzed lipid extracts of S. acidocaldarius ∆grsA∆grsB expressing S. solfataricus grs homologs. Expression of SSO0604 results in the production of GDGT-1 through GDGT-4, expression of SSO0477 produces GDGT-1 and GDGT-2, and expression of SSO2881 produces only GDGT-1. (B) LC-MS merged EICs of acid-hydrolyzed lipid extracts of S. acidocaldarius ∆grsB expressing the 2 S. solfataricus GrsB homologs (SSO0477 and SSO2881). In this strain, the S. solfataricus GrsB1 (SSO0477) produces GDGT-5 through GDGT-7, while GrsB2 (SSO2881) produces GDGT-5 through GDGT-8 (bold numbers). Because the S. acidocaldarius ∆grsB strain produces GDGT-1 through GDGT-4, these results suggest that S. solfataricus GrsB1 and GrsB2 prefer cyclized GDGT substrates. (C) Quantification of grsA and grsB transcripts in S. acidocaldarius and S. solfataricus. In S. acidocaldarius, grsA is expressed 9-fold higher than grsB. In S. solfataricus, grsA is expressed 18-fold higher than grsB1 and 134-fold higher than grsB2, while grsB1 is expressed 7-fold higher than grsB2.

Fig. 4.

NPSG metagenomic Grs homologs cluster only with Thaumarchaeota Grs homologs. Two clades from a rooted maximum-likelihood phylogenetic tree of Grs homologs (e value, <1e−20; protein identity, >20%) identified in cultured archaeal genomes, single-cell acquired genomes (SAGs), MAGs, or NPSG metagenomes. Grs homologs from the NPSG metagenomes are shown in red. Full phylogenetic maximum likelihood (PhyML) tree is shown in SI Appendix, Fig. S5. Black circles indicate branches that have bootstrap values greater than 0.9, and the scale bar represents one change per nucleotide site.