Tetraether archaeal lipids promote long‐term survival in extreme conditions

The sole unifying feature of the incredibly diverse Archaea is their isoprenoid‐based ether‐linked lipid membranes. Unique lipid membrane composition, including an abundance of membrane‐spanning tetraether lipids, impart resistance to extreme conditions. Many questions remain, however, regarding the synthesis and modification of tetraether lipids and how dynamic changes to archaeal lipid membrane composition support hyperthermophily. Tetraether membranes, termed glycerol dibiphytanyl glycerol tetraethers (GDGTs), are generated by tetraether synthase (Tes) by joining the tails of two bilayer lipids known as archaeol. GDGTs are often further specialized through the addition of cyclopentane rings by GDGT ring synthase (Grs). A positive correlation between relative GDGT abundance and entry into stationary phase growth has been observed, but the physiological impact of inhibiting GDGT synthesis has not previously been reported. Here, we demonstrate that the model hyperthermophile Thermococcus kodakarensis remains viable when Tes (TK2145) or Grs (TK0167) are deleted, permitting phenotypic and lipid analyses at different temperatures. The absence of cyclopentane rings in GDGTs does not impact growth in T. kodakarensis, but an overabundance of rings due to ectopic Grs expression is highly fitness negative at supra‐optimal temperatures. In contrast, deletion of Tes resulted in the loss of all GDGTs, cyclization of archaeol, and loss of viability upon transition to the stationary phase in this model archaea. These results demonstrate the critical roles of highly specialized, dynamic, isoprenoid‐based lipid membranes for archaeal survival at high temperatures.

fication of tetraether lipids and how dynamic changes to archaeal lipid membrane composition support hyperthermophily.Tetraether membranes, termed glycerol dibiphytanyl glycerol tetraethers (GDGTs), are generated by tetraether synthase (Tes) by joining the tails of two bilayer lipids known as archaeol.GDGTs are often further specialized through the addition of cyclopentane rings by GDGT ring synthase (Grs).
A positive correlation between relative GDGT abundance and entry into stationary phase growth has been observed, but the physiological impact of inhibiting GDGT synthesis has not previously been reported.Here, we demonstrate that the model hyperthermophile Thermococcus kodakarensis remains viable when Tes (TK2145) or Grs (TK0167) are deleted, permitting phenotypic and lipid analyses at different temperatures.The absence of cyclopentane rings in GDGTs does not impact growth in T.
kodakarensis, but an overabundance of rings due to ectopic Grs expression is highly fitness negative at supra-optimal temperatures.In contrast, deletion of Tes resulted in the loss of all GDGTs, cyclization of archaeol, and loss of viability upon transition to the stationary phase in this model archaea.These results demonstrate the critical roles of highly specialized, dynamic, isoprenoid-based lipid membranes for archaeal survival at high temperatures.

| INTRODUC TI ON
The diversity of marine and terrestrial environments containing an abundance of microbial life continues to expand and often beguiles the limits of life itself.Archaea often dominate and thrive in the extremes of temperature, salinity, pressure, and pH (Merino et al., 2019;Teske et al., 2021).The unique, domain-specific, isoprenoid-based lipid membranes generated by all Archaea (Koga, 2014) are thought to assist but cannot completely resolve rationales for survival in the extremes.Beyond compositional and structural differences, the dynamic response of archaeal lipid membranes to changes in growth phase, growth rate, and temperature supports adaptive responses to rapid and often dramatic stimuli in highly volatile environments (Elling et al., 2014;Hurley et al., 2016;Qin et al., 2015;Yang et al., 2023;Zhou et al., 2020).
The formation of GDGT from archaeol and subsequent cyclization are thought to primarily modify cell membrane fluidity and permeability, aiding archaeal organisms to adapt to a wide range of environmental parameters (Valentine, 2007;Van de Vossenberg et al., 1998;Zhou et al., 2020).The dynamic changes of archaeal lipid composition in response to environmental variation, F I G U R E 1 Tetraether lipid biosynthetic pathway in T. kodakarensis.The C20 isoprenoid chains of archaeol are covalently linked to generate GDGT (also termed GDGT-0), through the activity of TK2145 and Tk-Tes, with GTGT and macrocyclic archaeol (MA) as intermediate or side products, respectively.Production of cyclized-tetraether lipids (GDGT-1, 2, 3, 4) is catalyzed by TK0167, Tk-Grs.Archaeol-1 and archaeol-2 are detected only in strains ectopically expressing Tk-Grs or those with disruption of native Tk-Tes activities.
combined with the geologically relevant stability of archaeal lipids, also provide a reliable method to infer ancient sea surface temperatures, informing paleoclimate studies (Schouten et al., 2002;Wang et al., 2017;Zhang et al., 2016).Further, the unique properties of archaeal lipid membranes, often termed archaeosomes, offer promise for therapeutic approaches, including nano-drug delivery protocols and vaccine delivery as an alternative to the more conventional liposome-based systems (Jia et al., 2022;Lilia Romero & Jose Morilla, 2023;Santhosh & Genova, 2022).
Controlled and reliable production of specialized archaeosomes with novel compositions and novel properties is also of major biotechnological interest.However, while GDGT properties have been readily investigated in artificial systems, biological platforms wherein GDGT and cyclized GDGT synthesis can be controlled and regulated are lacking.
GDGT synthesis is achieved through the action of the radical Sadenosylmethionine (SAM) superfamily protein GDGT-macrocyclic archaeol synthase (GDGT-MAS) (Lloyd et al., 2022), also known as tetraether synthase (Tes) (Zeng et al., 2022).The formation of cyclopentane rings in GDGT-0 (no rings) at the C-7 and C-3 positions is catalyzed by another radical SAM superfamily protein, GDGT ring synthase (Grs), first characterized in Sulfolobus acidocaldarius (Zeng et al., 2019).While biochemical studies have determined the enzymes responsible for the production of GDGT-0 and its cyclized derivatives, biological manipulations have been lacking due to the presumed essentiality of Tes (Zeng et al., 2022).Thermococcus kodakarensis is a highly versatile, genetically tractable, hyperthermophilic, anaerobic archaeon that naturally synthesizes GDGT-0 and, to a much lesser extent, its ring-bearing derivatives.The relative abundance of GDGT lipids in T. kodakarensis under optimal growth conditions has been determined in previous studies and found to vary between ~25% and 80% based on the extraction and analysis methods used.T. kodakarensis undergoes substantial diether-totetraether lipid composition changes in response to temperature and growth phase (Matsuno et al., 2009;Tourte et al., 2020).GDGT synthesis, which involves Tes activities, contributes to hyperthermophilic physiology.In this study, we generated Tes and Grs deletion mutants, demonstrating that neither tetraether membrane formation nor membrane cyclization were essential in T. kodakarensis.Physiological characterization of these mutants and of strains ectopically expressing Tes and Grs demonstrated the importance of tetraether lipids for thermophily and fitness in the extremes for T. kodakarensis while also revealing unexpected cyclization responses.

| Microbial growth and media conditions
The constructed strains of T. kodakarensis were anaerobically cultured as previously described in artificial sea water (ASW) media supplemented with 5 g/L tryptone, 5 g/L yeast extract, 5 g/L pyruvate, 2 g/L elemental sulfur (S°), and a KOD1-vitamin mixture with or without 1 mM agmatine (Liman et al., 2022).Culture growth at 85°C or 95°C was monitored using optical density measurements at 600 nm.
The growth rates of a minimum of three independent biological replicates were monitored and plotted for each strain.Biomass harvested for lipid extractions and analyses was either grown at 85°C or 95°C, harvested 24 h post-inoculum (late-stationary phase), and stored frozen (at −80°C or on dry ice) until sample processing for lipid extraction.

| T. kodakarensis strain constructions
Strains used in this study are listed in Table 1.All T. kodakarensis deletion strains were constructed via homologous recombination, resulting in markerless deletions on the genome as previously described (Liman et al., 2022).Deletion strain genotypes were confirmed through whole genome sequencing (WGS) at >100x coverage using Oxford Nanopore MinION sequencing and visualized using the Integrative Genomics Viewer.Complemented strains were constructed as previously described (Santangelo et al., 2010;Scott et al., 2021).Briefly, strains carrying the expression vectors were selected based on agmatine autotrophy and cultured without agmatine supplementation.Retention of expression plasmids was confirmed via PCR using primers flanking the insertion site of the gene of interest.

| Lipid extraction and analyses
Frozen biomass samples were freeze-dried, resuspended in methanol (MeOH), and transferred to glass tubes where the solvent was evaporated under a N 2 stream.Samples were acid hydrolyzed in 2 mL of 1 M HCl in MeOH for 3 h at 90°C before being neutralized by the addition of 1 mL of 2 M KOH in MeOH and diluted with 5 mL of deionized water.Samples were extracted three times with 5 mL of dichloromethane (DCM), which was pooled and evaporated under a N 2 stream.Samples were then resuspended in 1 mL of 9:1 MeOH:DCM and filtered through 0.45μm polytetrafluoroethylene filters.
Core lipids were analyzed on an Agilent 1260 Infinity II series high-performance liquid chromatography (HPLC) instrument coupled to an Agilent G6125B single-quadrupole mass spectrometer with the electrospray ionization (ESI) interface in positive mode.
ESI-MS conditions were as follows: drying gas temperature 300°C, drying gas flow rate 8.0 L/min, nebulizer pressure 35 psi, capillary voltage 3500 V, and fragmentor voltage 175 V in scanning mode with a range of m/z = 600-1400.
Core lipids were separated with reverse phase chromatography on a Kinetex 1.7 μm XB-C18 100 Å LC column (150 × 2.1 mm) by a method modified from Rattray and Smittenberg (Rattray & Smittenberg, 2020) with mobile phase A: MeOH with 0.04% formic acid and 0.03% NH 3 , and mobile phase B: isopropanol with 0.04% formic acid and 0.03% NH 3 .An initial mobile phase of 60A:40B was held for 1 minute and then linearly ramped to 50A:50B over 19 min.This composition was held for 15 minutes and then linearly ramped back to 60A:40B over 5 min, which was then held for 10 min to allow for re-equilibration.A large injection volume of 25 μL was used to detect all compounds of interest.
Compounds were identified by the mass of the protonated parent ion ([M + H]+) coupled with a comparison of elution times to laboratory standards or to those found in previous literature (Rattray & Smittenberg, 2020).The nonresponse factor-corrected relative intensities of compounds were calculated using the manually integrated peak areas of the [M + H] + ions only.
Diether lipids were analyzed on an Agilent 7890B Series GC instrument coupled to an Agilent 5977A Series MSD in EI mode at 70 eV, scanning over a range of m/z = 50-900.Lipids were separated on two Agilent DB-17HT columns (30 m × 0.25 mm × 0.15 μm film thickness) connected in series using helium as the carrier gas with a constant flow rate of 1.1 mL/min.GC conditions were as follows: 60°C to 200°C at 10 degrees/min, then 200°C to 300°C at 4 degrees/min, and finally held at 300°C for 60 min.

| Culture of Halorubrum lacusprofundi and base hydrolysis of biomass
Liquid cultures (75 mL) of H. lacusprofundi DSM 5036 were grown on DSMZ Medium 372 at 10°C, shaking for five months.H. lacusprofundi biomass was base hydrolyzed in 2 mL of 1 M KOH in MeOH for 3 h at 70°C.Reactions were neutralized with 1 mL of 2 M HCl in MeOH.The core lipids were then extracted and analyzed as described for T. kodakarensis.

| Lipid sample hydrogenation
The lipid extract was resuspended in 2 mL of 1:1 MeOH:ethyl acetate (EtOAc).Argon was bubbled through the solution for 10 min before platinum (IV) oxide (~15 mg, 66 μmol) was added.The reaction mixture was placed in a Parr pressure vessel, which was then pressurized to 60 psi with H2, and the reaction mixture was continuously stirred with magnetic stir bars.After 16 h, the pressure was released, and argon was bubbled through the reaction for 10 min.The reaction was then filtered through celite with additional portions of EtOAc and then concentrated under vacuum.

| TK2145 and TK0167 encode the nonessential tetraether synthase (Tes) and GDGT-ring synthase (Grs), respectively
Analyses of the lipidome of T. kodakarensis strains revealed the presence of membrane-spanning glycerol dibiphytanyl glycerol tetraethers (GDGTs) and a small percentage of cyclized GDGTs, thus predicting the presence of an active Tes and Grs (Figure 2c,d; Figure S2a).Based on homology with recently identified Tes enzymes (Zeng et al., 2019(Zeng et al., , 2022)), TK2145 was predicted to encode the sole Tes homolog (1e −170 e-value, 45% identity) in T. kodakarensis (Tk-Tes); however, no biochemical or genetic evidence supporting TK2145 as Tk-Tes was previously reported.An unbiased mutagenesis of T. kodakarensis (Orita et al., 2019) revealed that inactivation of TK2145 resulted in reduced hyperthermotolerance in T. kodakarensis, but lipid analyses were not performed to confirm loss of GDGT production.The co-purification of the product of TK2145 with TK2140, a DNA ligase (Li et al., 2010), is the only other previous, and still unexplained, information on the role of the product of TK2145 in vivo.We also searched the T. kodakarensis genome for a potential Grs homolog and identified TK0167 as a strong candidate (8e −72 e-value, 32% identity to GrsA).Beyond basic transcriptomics (Jäger et al., 2014) or microarray data (Čuboňová et al., 2012) demonstrating expression of TK0167, no previous information on the role(s) of TK0167 has been reported.
To establish the potential roles of TK2145 and TK0167 in lipid production and maturation in T. kodakarensis, we individually targeted each locus for deletion with established genetic techniques (Liman et al., 2022) (Figure 2a,b).While the entire open reading frame of TK2145 was deleted, a portion of TK0167 was intentionally not targeted for genomic deletion due to the presence of a small RNA identified in our previous transcriptomic data that overlaps with TK0167 (Jäger et al., 2014).Deletion of TK2145 from the parental strain TS559 was successful (generating strain AL016; Δtes), as was the desired partial deletion of TK0167 (generating strain AL010; Δgrs) (Table 1).The exact endpoints of each markerless genomic deletion were confirmed first by PCR amplification of genomic regions TA B L E 1 T. kodakarensis strains used in this study.et al., 2019), suggesting that tetraether membranes may be more critical in some archaeal clades than others.

| Lack of GDGTs impacts late stationary phase survival
The biological importance of tetraether lipids has not previously been investigated in vivo due to the presumed essentiality of Tes (Zeng et al., 2022).Given the small percentage (~0.1%) of tetraethers that were cyclized in the parental strain, it was not surprising that deletion of Tk-Grs (strain AL010) did not result in a significant defect in growth rate or final cell densities at 85°C (optimal) or 95°C (supra-optimal, but tolerable) (Figure 2e,f).In contrast, eliminating Tk-Tes (strain AL016) resulted in substantial impacts on survival upon transition from the late-exponential phase to the stationary phase (Figure 2e,f).Aside from the slightly elongated lag phase, the rate of Δtes growth largely matched that of the parental strain TS559 at both optimal and supra-optimal growth temperatures.Whereas TS559 showed only minor reductions in optical density upon reaching stationary phase, suggesting minor impacts on viability, the optical density of Δtes (AL016) drops significantly upon entry into stationary phase (Figure 2e,f).
These data imply that most (~50%-75%) of the culture perishes in Tes but also confirm the stationary phase phenotype of cells lacking Tk-Tes can be rescued through complementation.

| Aberrant production of cyclized tetraether lipids dramatically impairs hyperthermophilic growth
As less than 1% of GDGTs were cyclized in the parental strain TS559, we predicted that ectopic complementation of Tk-Grs might result in an overabundance of cyclic tetraethers, permitting evaluation of the impact of increased lipid rings on microbial fitness (Figure 4; Figure S2a,g).We introduced an autonomously replicating plasmid (Santangelo et al., 2010) directing Grs expression from a strong promoter (Scott et al., 2021) into the parental strain TS559 and the Δgrs strain (Figure 4; Figure S4); strains of TS559 and Δgrs retaining the same plasmid (pTS543) lacking the grs expression cassette were also generated as controls (Table 1).
As predicted, introduction of the Tk-Grs complementation vector (pTS543-TK0167) into the Δgrs strain not only restored the production of cyclized GDGTs but also dramatically increased cyclization.The Ring Long-term survival of T. kodakarensis demands Tes (TK2145) catalyzed tetraether lipid production but not Grs (TK0167)catalyzed cyclization.(a) The genomic locus of TK0167, predicted to encode Tk-Grs (blue), shown with flanking genes (green) in the parental strain (TS559) (Top) and in the TK0167 partial deletion strain (Δgrs; AL010) (Bottom).(b) The genomic locus of TK2145, predicted to encode Tk-Tes (blue), shown with flanking genes (green) in the parental strain (TS559) (Top) and in the TK2145 deletion strain (Δtes; AL016) (Bottom).
(c) LC-MS extracted ion chromatograms of one replicate of the acid hydrolyzed lipid extracts from the parent strain TS559, the Δtes strain, and the Δtes strain complemented with TK2145.GDGT production was lost upon deletion of Tk-Tes and restored with ectopic expression of TK2145.(d) LC-MS extracted ion chromatograms of one replicate of the acid hydrolyzed lipid extracts from the parent strain TS559, the Δgrs strain, and the Δgrs strain complemented with TK0167.Deletion of Tk-Grs resulted in the loss of cyclized GDGTs and cyclization was restored with ectopic expression of TK0167.(e, f) Exponential growth rates of T. kodakarensis at 85°C (e) and 95°C (f) are not significantly impacted by deletion of TK2145 (Tk-Tes; strain AL016) or TK0167 (Tk-Grs; AL010); however, deletion of Tk-Tes (TK2145) dramatically impacts survival upon entry into the stationary phase.
Index (RI) is defined as the weighted average of ring numbers in GDGT compounds (Zhang et al., 2016).We observed a massive increase in the RI from just 0.002 and 0.004 in TS559 at 85°C and 95°C, respectively, to 0.470 and 0.740 in the Δgrs strains complemented with ectopic Grs expression at 85°C and 95°C, respectively (Figure 4d; Figure S5, Table 2).Tk-Grs ectopic expression in the parental strain TS559 also dramatically shifted the lipid composition compared to the parental strain (Figure S4c,d), increasing the average RI to 0.413 and 0.588 at 85°C and 95°C, respectively (Figure S5).Thus, ectopic expression of Tk-Grs can result in strains where ~19%-28% of the tetraether lipids are cyclized, providing a route to the large-scale production of cyclized GDGTs in T. kodakarensis (Figures 4d; Figure S4d).Additionally, when Tk-Grs is ectopically expressed in Δgrs at 60°C, an even greater average RI of 1.67 is observed, and ~ 53% of the tetraether lipids are cyclized, indicating that GDGT cyclization has a complex relationship with temperature (Figure S5, S7a).
The increased relative abundance of cyclized GDGTs in the parental and Δgrs strains ectopically expressing Tk-Grs is not benign and results in substantial defects at increased growth temperatures (Figures 4a,b; Figure S4a,b).While increased Grs activities throughout the growth cycle are tolerated without significant impact at the optimal growth temperature of 85°C, ectopic expression of Tk-Grs at 95°C dramatically slows growth, although final cell densities matching parental strains are eventually achieved (Figure 4a,andb, Figure S4a,b).Thus, while an increase in the proportion of GDGTs that are cyclized from natural levels of ~0.1% to ~19% is well tolerated at 85°C, near identical increases from ~0.3% to ~28% at 95°C result in dramatic fitness consequences (Figure 4b; Figure S4b).It will be of interest to determine how increases in cyclized GDGTs change key parameters of membrane biology (e.g., stability, fluidity, and permeability) in T. kodakarensis that result in impaired growth rates at supra-optimal temperatures.

| Disruption of native Tk-Tes or Tk-Grs activity results in unexpected lipid composition changes
As expected, the complete loss of GDGTs in the Δtes strain results in a membrane composed of only diether lipids.However, unexpectedly,   2), supported by mass spectra and hydrogenation experiments described later in the text.
In the absence of its presumed natural GDGT substrates, Tk-Grs uses archaeol as a substrate, resulting in the Δtes strain converting ~7% of its diether lipids to archaeol-1 and archaeol-2 at both 85°C and 95°C (Figure 3c and Table 2).The significance of newly cyclized archaeols is emphasized by the fact that the cyclized diethers in Δtes are ~70-fold higher (~7% of core diethers) than the abundance of all cyclized tetraethers (~0.1% of core tetraethers) in the parental strain TS559 (Figure 3c,d).
Further, our analyses reveal that two regioisomers of archaeol-1 are possible; these are isomers that differ by which tail the ring is placed in-either the C3 glycerol-bonded tail or the C2 glycerolbonded tail (Figure S9).The EI mass spectra of both TMS-archaeol-1a and TMS-archaeol-1b reveal that both isomers are actually a mixture The restoration of Tk-Tes activities in the Δtes complementation strain also resulted in unexpected changes to the lipidome.
As expected, restoring Tk-Tes expression and activities via ectopic expression in the Δtes strain restored production of GDGT-0 (Figure 3d).Surprisingly, ectopic expression of Tk-Tes also resulted in an increased abundance of GDGT-1, 2, 3, and 4 with an average ring index of 0.358 at 85°C.This was similar to that of strains ectopically expressing Tk-Grs at 85°C (Δgrs + pTS543-grs RI = 0.470 and TS559 + pTS543-grs RI = 0.413), although this increased cyclization was much less at 95°C with an RI of just 0.080 (Figure S5).An increase in the average RI was also observed for the parent strain (TS559) overexpressing Tk-Tes.However, the RI increase is much lower at both 85°C (RI = 0.016) and 95°C (RI = 0.013) than in the complementation strain, but still an increase as compared to 0.002 and 0.004 in the parental strain at 85°C and 95°C, respectively (Figure S5).These findings indicate that the activities of Grs, which may be temperature regulated, are impacted by the ectopic expression of Tes.
TA B L E 2 Core lipid analysis of all T. kodakarensis strains.Like all archaea, T. kodakarensis generates an isoprenoid-based lipid membrane that responds to environmental and growth phase changes.Instead of a conventional bilayer, the membranes of many archaea are dominated by membrane-spanning tetraether lipids termed GDGTs that are synthesized by the activities of a tetraether lipid synthase (Tes) (Lloyd et al., 2022;Zeng et al., 2022).
GDGTs can be modified through the formation of ring structures within the dibiphytanyl tails by the activity of GDGT-ring synthase (Grs) (Zeng et al., 2019).Ring-containing lipids are typically only minor constituents of the total lipidome of the Thermococcales (Tourte et al., 2020) but can be the dominant membrane lipids in other Archaea, including Thaumarchaeota (Pitcher et al., 2009), Sulfolobales (Jensen et al., 2015), and Thermoplasmatales (Uda et al., 2004) species.Cyclized GDGTS are thought to impart unique properties to membranes that support survival in the extremes (Matsuno et al., 2009;Meador et al., 2014 In this study, we established that T. kodakarensis strains lacking the tetraether synthase Tes (TK2145) are viable despite the complete lack of tetraether lipids.The hyperthermophilic growth of T.
kodakarensis is thus not dependent on tetraether lipids, although the lack of tetraether lipid synthesis leads to a dramatic loss of viability upon entry into the stationary phase.In the absence of natural GDGT substrates in the T. kodakarensis Δtes deletion strain, the GDGT cyclase Grs instead modifies ~7% of archaeol to monoor di-ring-containing compounds (archaeol-1 and archaeol-2;  throughout all growth phases does provide a route to abundant (~53%, 19%, and 28% of tetraether lipids at 60°C, 85°C, and 95°C, respectively) ringed-GDGT production, but these high levels of derivatized GDGTs led to significant growth defects at 95°C.
Furthermore, the effects of temperature on cyclization appear to be complex, as increased levels of cyclized GDGTs are observed at both supra-optimal (95°C) and sub-optimal (60°C) temperatures.While increases in cyclized GDGTs in response to increased temperature have been well documented in many archaea, other physiological factors known to influence GDGT cyclization, such as growth rate, also change with temperature and thus may have confounding effects on cyclization levels.Alternatively, the turnover of cyclized GDGTs could be reduced at suboptimal growth temperatures, or Tk-Grs may simply function more efficiently under such conditions.Taken together, our findings show the importance of tetraether lipid synthesis in the long-term survival of T. kodakarensis.The regulation of lipid composition appears to be critical for both archaeal hyperthermophily and growth phase transitions.It is also likely that combinatorial changes to environmental variables (e.g., temperature, salinity, pressure, and pH) will also direct changes to the lipidome that will reveal other critical roles for tetraether lipids and their cyclized derivatives.
Our findings also demonstrate the production of new lipid types, specifically archaeol-1 and archaeol-2.While macrocyclic archaeol containing one or two rings has been observed in environmental lipid extracts (Stadnitskaia et al., 2003), the presence of rings in archaeol and cyclized diether lipids in culture has not been previously observed.Tk-Grs appears to be promiscuous, allowing the production of cyclized diethers when ectopically expressed throughout the growth phases, especially at 60°C, where they comprise half of the diether lipids, interestingly equal to the proportion of GDGTs cyclized at this temperature.This suggests that Tk-Grs can work equally well on both diether and tetraether lipids under certain conditions.
The mass spectra of the two archaeol-1 isomers provide further biochemical insights into Tk-Grs function.Fragmentation patterns demonstrate that Tk-Grs can form the first ring in either lipid tail but that it prefers to do so on the tail furthest from the headgroup.
Additionally, the presence of multiple chromatographically resolved isomers of archaeol-1 and -2 has interesting implications for our understanding of the stereochemistry of the ring moieties in archaeal lipids (Figure S11a,b, S12a-e).While the observed isomers of archaeol-1 and -2 could in theory be the result of different ring shapes (e.g., cyclohexane) or cyclization at different positions along the phytanyl tail (e.g., at C-3), no biochemical precedence exists for such multifaceted activity of Grs.Further, we (1) do not observe LC chromatographic resolution of the isomers that would suggest varying ring shapes (Liu et al., 2016), (2) do not detect the presence of archaeol with more than 2 rings (possible if C3 and C7 cyclization occurs), and (3) do not detect fragments in the mass spectra of the archaeol-2 isomers that would indicate that the two rings can be found together on one tail.Thus, we hypothesize that the different isomers of archaeol-1 and -2 are diastereomers of one another, differing in their stereochemistry around the cyclopentane ring (cis vs. trans configuration) (Figure S11a,b, S12a-e).Previous studies have determined that the stereochemistry of the cyclopentane rings in GDGTs from S. acidocaldarius (Montenegro et al., 2003), crenarchaeol from Thaumarchaeota (Holzheimer et al., 2021), and environmental GDGT-derived compounds (Lutnaes et al., 2006) appears to be exclusively trans, particularly with a C7(S)-C10(S) configuration.However, one notable exception is the potential presence of a cis-configured cyclopentane ring in the enigmatic crenarchaeol isomer (Sinninghe Damsté et al., 2018).Thus, it is unclear if the sug- Figure S2c,d) demonstrating that Tes activities are encoded at the TK2145 locus and that no other Tes activities exist within T. kodakarensis.The viability of the Tk-Tes mutant contrasts with the presumed essentiality of the S. acidocaldarius Tes (Zeng the stationary phase due to the lack of tetraether lipids and provide in vivo evidence that tetraether lipids are necessary to promote viability in stationary phase T. kodakarensis cultures.Given the known volatile nature and ever-changing nutrient availability of hydrothermal vents, the fitness of T. kodakarensis strains lacking tetraether lipids pales in comparison to tetraether-containing strains in natural environments.The rescued production of core tetraether lipids in the Tes mutant via ectopic Tk-Tes activities restored the decline in population density that was observed in the Δtes strain upon entry to the stationary phase.Ectopic Tk-Tes expression completely restored growth phenotypes at both 85°C and 95°C (Figure3a,b).Our findings thus not only genetically confirm the function of TK2145 as a the Δtes strain is capable of cyclizing these diether lipids, producing putative rings containing derivatives of archaeol, here termed archaeol-1 and archaeol-2 with one and two rings, respectively (Figures1, 3c; FigureS6a-d, Table FigureS6e,f).Growth of the Δgrs complementation strain (Δgrs + pTS543-grs) at 60°C resulted in the robust production of archaeol-1 and -2, together accounting for ~51% of the total core diether lipids, while archaeol-1 and -2 were completely absent in of the two regioisomers, as demonstrated by the co-occurrence of the m/z = 307, 412 fragment ion pair and the m/z = 309, 410 fragment ion pair in the same mass spectrum, each pair resulting from the C2 to C3 glycerol bond cleavage of a different regioisomer (Figures S9, S14, S15).However, the m/z = 307, 412 fragment ion pair is dominant over the m/z = 309, 410 pair, indicating that the ring is more commonly found in the C3 glycerol-bonded tail (tail furthest from the headgroup) and suggesting that Tk-Grs prefers to cyclize at this location first (Figure S9, S14, S15).

Figure 1 )
Figure1) that may assist in preserving lipid membrane integrity and function in the absence of tetraether lipids.Deletion of Grs (TK0167) eliminates the production of the small (<1%) percentage of cyclic GDGTs found in natural membranes and is generally not phenotypic.While loss of ring-containing GDGTs is fitness-neutral under laboratory conditions, aberrant levels of ring-containing GDGTs are not well tolerated at supra-optimal growth temperatures upon overexpression of Grs in vivo.Overproduction of Grs gested additional stereoisomers of archaeol-1 and archaeol-2 are merely artifacts of the "unnatural" cyclization of archaeol or if they are indicative of the stereochemistry of the rings in the GDGTs of T. kodakarensis as well.Given the demands for biological routes for large-scale production of derivatized GDGTs(Jia et al., 2022;Lilia Romero & Jose Morilla, 2023;Santhosh & Genova, 2022), strains that express wildtype and potential-variant forms of Grs hold substantial promise.Rescued recovery of GDGT synthesis via ectopicTk-Tes expression in genomically deleted Tes strains also reproducibly increases the total amount of cyclic GDGTs observed in comparison to the overexpression of Tk-Tes in the parental strain.Based on previous T. kodakarensis transcriptomic data, Tk-Tes (encoded by TK2145) is co-expressed with TK2146, which is annotated as a hypothetical regulatory protein.Perhaps the co-expression of Tes and TK2146 in the parental strain but not in the Δtes strain played a role in the regulation of cyclopentane ring production in T. kodakarensis.These findings ultimately warrant more investigation into the main regulatory pathway that controls the production of cyclic GDGTs.In conclusion, the ability to manipulate lipidome composition in T. kodakarensis offers a powerful mechanism to study the impacts of tetraether lipid biosynthesis on archaeal physiology and survival in the extremes.Investigations into the interplay between GDGT biosynthesis, modification, and cellular viability in different environments will allow a better understanding of the roles of these tetraether lipids in the evolution of archaeal organisms.Although the properties of these new lipids still require further investigation, the expansion and control of lipid diversity in T. kodakarensis can be utilized for biotechnology applications in relation to drug deliveries and vaccines.