Calditol-linked membrane lipids are required for acid tolerance in Sulfolobus acidocaldarius Zhirui Zenga,1, Xiao-Lei Liub,1, Jeremy H. Weia, Roger E. Summonsc, and Paula V. Welandera,2 aDepartment of Earth System Science, Stanford University, Stanford, CA 94305; bDepartment of Geology and Geophysics, University of Oklahoma, Norman, OK 73019; and cDepartment of Earth, Atmospheric and Planetary Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139 Edited by Katherine H. Freeman, Pennsylvania State University, University Park, PA, and approved November 7, 2018 (received for review August 14, 2018) Archaea have many unique physiological features of which the lipid and sea-surface temperatures in the Mesozoic and early Cenozoic, composition of their cellular membranes is the most striking. and the geologic history of archaea in ancient sediments (13, 14). Archaeal ether-linked isoprenoidal membranes can occur as bilayers However, deciphering the evolutionary implications of archaeal or monolayers, possess diverse polar head groups, and a multiplicity lipid biosynthesis, as well as being able to properly interpret ar- of ring structures in the isoprenoidal cores. These lipid structures chaeal lipid biomarkers, requires a full understanding of the are proposed to provide protection from the extreme temperature, biosynthetic pathways and the physiological roles of these various pH, salinity, and nutrient-starved conditions that many archaea structures in extant archaea. inhabit. However, many questions remain regarding the synthesis Studies of archaeal membrane lipid synthesis have revealed and physiological role of some of the more complex archaeal lipids. distinct proteins and biochemical reactions, but several open In this study, we identify a radical S-adenosylmethionine (SAM) questions remain (4, 15). In particular, the production of a unique protein in Sulfolobus acidocaldarius required for the synthesis of five-membered ring, calditol, that is ether-linked directly to the a unique cyclopentyl head group, known as calditol. Calditol- glycerol backbone of GDGTs is both an enzymatic and physio- linked glycerol dibiphytanyl glycerol tetraethers (GDGTs) are mem- logical mystery (Fig. 1) (16). Calditol-linked GDGTs were first brane spanning lipids in which calditol is ether bonded to the glyc- discovered in Sulfolobus acidocaldarius, where they comprise up to erol backbone and whose production is restricted to a subset of 90% of the polar lipid fraction (17). Calditol–GDGTs have been thermoacidophilic archaea of the Sulfolobales order within the detected subsequently in two other species of the Sulfolobales Crenarchaeota phylum. Several studies have focused on the enzy- order within the Crenarchaeota phylum, Acidianus and Metal- EARTH, ATMOSPHERIC, matic mechanism for the synthesis of the calditol moiety, but to losphaera (18, 19). The enzyme(s) required for calditol synthesis has AND PLANETARY SCIENCES date no protein that catalyzes this reaction has been discovered. not been identified, although several structural and biosynthetic Phylogenetic analyses of this putative calditol synthase (Cds) reveal studies have validated the cyclic calditol structure (20–23) and the genetic potential for calditol–GDGT synthesis in phyla other demonstrated that calditol is derived from glucose (16, 24, 25). than the Crenarchaeota, including the Korarchaeota and Marsarch- The physiological significance of calditol-linked membranes is aeota. In addition, we identify Cds homologs in metagenomes pre- dominantly from acidic ecosystems. Finally, we demonstrate that also unknown. All calditol-producing archaea isolated thus far – MICROBIOLOGY S. acidocaldarius are thermoacidophiles with a pH range of 0.4 6 and a temper- deletion of calditol synthesis renders sensitive to – extremely low pH, indicating that calditol plays a critical role in ature range of 44 °C 85 °C (18, 19, 26). The ability of these protecting archaeal cells from acidic stress. calditol-producing archaea to grow in such environmental calditol | glycerol dibiphytanyl glycerol tetraethers | GDGT | radical SAM | Significance Sulfolobus It is thought that the distinct ether lipid membranes of archaea ne of the defining features that distinguishes archaea from allow them to thrive in environmental extremes. However, it O both eukaryotes and bacteria is the structure of their has been difficult to demonstrate this physiological role di- membrane lipids (1, 2). Bacterial and eukaryotic membranes are rectly. Here, we identify a protein required for the biosynthesis composed of phospholipid bilayers made of fatty acid chains ester of a unique archaeal lipid head group, calditol, whose pro- linked to glycerol-3-phosphate. Archaeal membranes have a duction was considered to be restricted to a subset of archaeal similar overall amphiphilic structure but are composed of iso- thermoacidophiles. We show that deletion of this protein in Sulfolobus acidocaldarius prenoidal chains ether bonded to glycerol-1-phosphate (3, 4). The prevents production of calditol- majority of archaea can also generate membrane spanning mono- linked membrane lipids and, in turn, inhibits cell growth at extremely low pH. Our findings also suggest that archaea more layers known as glycerol dibiphytanyl glycerol tetraethers (GDGTs), S which can contain cyclopentyl or cyclohexyl rings (SI Appendix, broadly, like bacteria, employ radical -adenosylmethionine proteins to modify membrane lipids in ways that confer pro- Fig. S1) (3, 4). In addition, archaea possess a diverse array of tective effects when environmental conditions, such as pH, polar head groups, including phosphoesters of ethanolamine, serine, fluctuate significantly. and myo-inositol, or glycosyl groups, such as glucose or galactose linked directly to the glycerol backbone (5, 6). Author contributions: Z.Z., X.-L.L., R.E.S., and P.V.W. designed research; Z.Z., X.-L.L., and P. These unique archaeal membrane lipids are significant from V.W. performed research; X.-L.L. and J.H.W. contributed new reagents/analytic tools; Z.Z., several perspectives. From an evolutionary standpoint, the “lipid X.-L.L., J.H.W., R.E.S., and P.V.W. analyzed data; and Z.Z., X.-L.L., J.H.W., R.E.S., and P.V.W. divide” between archaeal membranes and those of bacteria and wrote the paper. eukaryotes raises interesting questions regarding the evolution of The authors declare no conflict of interest. membrane lipid biosynthesis and what the membrane composition This article is a PNAS Direct Submission. of the last universal common ancestor may have been (7–9). Geo- Published under the PNAS license. chemically, archaeal lipids detectedinbothmodernenvironments 1Z.Z. and X.-L.L. contributed equally to this work. and ancient sediments can function as taxonomic indicators and/or 2To whom correspondence should be addressed. Email: [email protected]. paleotemperature proxies (10–12). Archaeal biomarker studies have This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. provided insight into a variety of ecological topics including the role 1073/pnas.1814048115/-/DCSupplemental. of archaea in marine biogeochemistry, predictions of atmospheric www.pnas.org/cgi/doi/10.1073/pnas.1814048115 PNAS Latest Articles | 1of6 Downloaded by guest on September 30, 2021 A calditol-linked lipids provide a protective role under environmen- HO tally relevant conditions. O HO O O Results HO OH HO O hexose HO OH acid HO S. acidocaldarius [CORE] O Identification of a Calditol Synthesis Protein in . HO OH hydrolysis hexose O P O OH [CORE] Previous studies have proposed that a cyclase-like enzyme syn- O- OH HO OH thesized the formation of calditol through the C-4 oxidation of phospho-myo-inositol core GDGT 0-6 glucose in an “inositol-like” mechanism, followed by ether- ? ification to the core GDGT lipid (16, 24, 25). However, a second HO mechanism has been put forth that involves an unusual ring O contraction of a glucose molecule linked to the GDGT core (27). HO O OH HO O OH OH This mechanism is similar to the proposed formation of the HO OH HO OH acid HO [CORE] O cyclitol ether side chain of bacteriohopanepolyols (BHPs). hexose HO OH O hydrolysis O P O OH Synthesis of this BHP side chain requires a radical SAM protein calditol HO OH [CORE] O- hpnJ HO OH OH encoded by the gene (27, 28). Radical SAM enzymes phospho-myo-inositol calditol-GDGT 0-6 catalyze a diverse set of difficult reactions through the gener- ation of a 5-deoxyadenosyl radical (dAdo·) intermediate (29). B wild type Δsaci_0343 Therefore, we hypothesized that a radical SAM protein may 6 6 x10 core x10 core also be required to synthesize the calditol carbocycle observed 6.0 calditol GDGT 4 6.0 calditol GDGT 4 Sulfolobus GDGT 4 GDGT 4 in membrane lipids. We searched the S. acidocaldarius genome for all genes that 3.0 3.0 encode proteins with a radical SAM motif [protein family (Pfam) identifier pfam04055] and identified 18 candidates (SI Appendix, 0.0 0.0 Table S1). We used the Basic Local Alignment Search Tool (BLASTP) (30) to determine if any of these radical SAM proteins − Δcds Δcds + p1561_cds had homologs (e value < 1e 50; >30% identity) in other calditol- x106 x106 calditol producing archaea (18, 19) but were absent from non–calditol- 8.0 core 4.0 GDGT 4 core – e GDGT 4 GDGT 4 producing species (31 33). We utilized this stringent value be- Relative intensity cause radical SAM proteins are primarily identified by the short 4.0 2.0 amino acid sequence motif CxxxCxxC. As a result, less-stringent BLAST analyses of these proteins may pull out other radical SAM 0.0 0.0 proteins that are not involved in calditol synthesis. Using this 35 40 45 50 55 60 35 40 45 50 55 60 cutoff, 3 of the 18 candidate proteins, Saci_0343, Saci_0344, and Elution time (min) Saci_1489, were found only in the calditol-producing archaea, and we attempted to delete all three genes separately in S. acid- Fig. 1. The radical SAM protein encoded by cds is required for calditol syn- ocaldarius. We were able to construct markerless deletion mutants thesis. (A) Example of the polar head groups identified in archaeal mem- of saci_0343 and saci_1489 but not saci_0344.