Biosynthesis of GMGT lipids by a radical SAM enzyme associated with anaerobic archaea and oxygen-deficient environments

Domain: earth_science
Published: 2024-06-19
Editorial outcome: accept

Abstract
Archaeapossesscharacteristicmembrane-spanninglipidsthatarethoughttocontributetotheadaptationtoextremeenvironments.However,thebiosynthesisoftheselipidsispoorlyunderstood.Here,weidentifyaradicalS-adenosyl-L-methionine(SAM)enzymethatsynthesizesglycerolmonoalkylglyceroltetraethers(GMGTs).Theenzyme,whichwenameGMGTsynthase(Gms),catalyzestheformationofaC(sp3)–C(sp3)linkagebetweenthetwoisoprenoidchainsofglyceroldialkylglyceroltetraethers(GDGTs).ThisconclusionissupportedbyheterologousexpressionofgenegmsfromaGMGT-producingspeciesinamethanogen,aswellasdemonstrationofinvitroactivityusingpurifiedGmsenzyme.Additionally,weshowthatgenesencodingputativeGmshomologsarepresentinobligateanaerobicarchaeaandinmetagenomesobtainedfromoxygen-deficientenvironments,andappeartobeabsentinmetagenomesfromoxicsettings.

Introduction
Archaeal GDGTs (glycerol dialkyl glycerol tetraethers) membrane-spanning lipids provide structural advantages that help archaea adapt to extreme environments1 and serve as crucial as lipid biomarkers in the reconstruction of paleoclimate changes2. Recent advancementsinunravelingGDGTsbiosyntheticpathwayshavelaidastrongbiologicalfoundationfortheeffectiveutilizationofGDGTsproxies. The key enzymes responsible for GDGTs biosynthesis, particularly the processes of C(sp3)-C(sp3) cross-linking3,4 and cyclization5, belong to the radical S-adenosyl-L-methionine (SAM) superfamily.TheseenzymesharboraradicalSAM[4Fe–4S]clusterthatutilizesSAMtogenerateahighlyreactive5′-deoxyadenosineradical(5′-dAdo•)viathereductivecleavageofSAM.The5′-dAdo•theninitiatesdiverseradicalchemistriesthrough either hydrogen atom (H-atom) abstraction or substrate adenosylation6. InthebiosynthesisofGDGTs,theorchestratedutilizationofsuchahigh-energyradicalproveshighlyadvantageousforformingachallengingC(sp3)–C(sp3)linkage.

GMGTs (glycerol monoalkyl glycerol tetraethers) constitute an importantsubsetofGDGTsderivatives,distinguishedbyanadditionalC–Clinkagebetweenthetwoisoprenoidchains7.Theseuniquelipids havebeenidentifiedinmultiplethermophilicarchaeaandarethoughttocontributetoincreasedmembranerigidityathightemperature8. This is consistent with the observed positive correlation between GMGTs abundance and temperature in global peat environments9. However, findings from culture experiments exhibited a conflicting trend, wherein the abundanceof GMGTs decreased astemperature increasedinthearchaeonPyrococcusfuriosus10.ThisresultsuggestsGMGTsformationwasnotsolelyinresponsetoenvironmentalheatstress.Nevertheless,thelackofknowledgeregardingtheirbiosynthesis,biochemicalmechanisms,andcomprehensivebiologicalsourceshashinderedourunderstandingoftheirphysiologicalsignificance.Inthisstudy,throughtheintegrationofinvivogeneexpressionandinvitroenzymaticactivityassays,wehaveidentifiedthekeygeneresponsibleforGMGTsbiosynthesis.Moreover,thisgeneisexclusivelypresentinanaerobic archaealgenomes and metagenomesobtained from oxygen-deficient environments, suggesting the potential associationofGMGTsproducerswithoxygen-deficienthabitats.

Results
DiscoveryandidentificationofGMGTssynthaseinvivoandinvitro

ThebiosynthesisofGMGTslikelyoccursviadirectgenerationofaC–CbridgebetweentwoisoprenoidchainsonGDGTs,i.e.,formingacovalentbondbetweentwomethylgroups11.InspiredbytheradicalSAM(RS)enzymeTes3anditshomologMJ06194thatactivateinertsp3-hybridizedcarboncenters,wesearchedthegenomeforcandidateradicalSAMenzymes.IthasbeenobservedthatThermococcusguaymasensisproducesGMGTs12whileitsclosesisterspeciesThermococcuskodakarensisdoesnot13.Similarly,inthecaseofPyrococcusfuriosusandPyrococcusyayanosii,theformerproducesGMGTs10,whereasthelatterdoesnot13. By comparing the homology of all radical SAM enzymes (pfam04055) between species with or without GMGTs formation, we successfully found a promising candidate protein (WP_062369926.1 from T. guaymasensis, or AAL80771.1 from P. furiosus),whichispresentinGMGTs-formingspeciesbutabsentinspecieslackingGMGTsformation(SupplementaryTable1).TheseproteinscontainingacanonicalCX3CX2Cmotifwhichprovidesthreecysteine(Cys)residuestoligateaRS[4Fe–4S]clusterforperformingradicalSAMchemistry(SupplementaryFig.1).We thoughtit highly likelythatthisproteinisresponsibleforformingthebridgingcovalentbondwithinisoprenoidalGDGTsthatleadstotheformationofGMGTs.

To test our hypothesis, we conducted in vivo studies using an engineered methanogen Methanococcus maripaludis asthe hostfor heterologousexpression.Thisparticularmethanogenharborsthetesgene3andcanproduceGDGT-0asthesubstrateforGMGTssynthase(Fig. 1a). We also cloned and expressed the gene homolog of okinawensiswhichproducesGMGTs14,knownasMETOK_RS04425,toimprove the expression efficiency of exogenous protein in the methanogenhost(Fig.1bleft).Thecloneswereculturedat37°Cfor7daysandthenharvestedforlipidanalysiswithreverse-phaseultra-highperformanceliquidchromatographymassspectrometry(UPLC-MS/MS). The results clearly showed the production of a lipid compoundwithm/z=1300.3042,twoDaltonslessthanthem/zofGDGT-0(1302.3259).Inaddition,thefragmentationpatternfromasingleisoprenoidchainintandemMS/MSofGDGT-0isabsentinthepatternoftheproduct(SupplementaryFig.2).Thisisconsistentwithpublishedspectra7andsupportstheformationofaC–Cbondbetweenthetwoisoprenoidchains.ThisanalysisconfirmstheinvivoformationofGMGT-0,indicatingthattheradicalSAMenzymeencodedbyMETOK_RS04425isresponsibleforthebiosynthesisofGMGTs(Fig.1a).WehavethusnamedthisenzymeGMGTssynthase(Gms).

To confirm the function of Gms, we purified Gms enzyme and performedinvitrostudies.RecombinantGmsfromM.okinawensiswasheterologouslyexpressedinEscherichiacoliBL21(DE3)andpurifiedanaerobicallyusingStrep-Tactinaffinitychromatography.AmutantstrainofSulfolobusacidocaldarius,whichproducesGDGT-0asitsdominantmembranelipid5,wasemployedforisolatingthesubstrateGDGT-0usingpreparativeLC-MS.AninvitroactivityassaywasthenperformedbyincubatingGmsanaerobicallywith30equiv.ofreductantdithionite(DTH),30equiv.ofSAM,and10equiv.ofGDGT-0at37°Cfor72h.ThepresenceofGMGT-0wasconfirmed,withayieldofapproximately4.8%(Fig. 1b right, Supplementary Figs. 3, 4). These resultsunequivocallydemonstratethatGmscatalyzestheformationofaC–ClinkagebetweentwoisoprenoidchainsonGDGT-0toproduceGMGT-0(Fig.1a).

To further elucidate the details of GMGT-0 biosynthesis, we employed electron paramagnetic resonance (EPR) spectroscopy to characterizetheFe–Scluster(s)inpurifiedGms.EPRspectroscopyisatechniquethatprobesparamagneticspecies,e.g.,organicradicals,metalionswithspecificoxidationstates,ormulti-nuclearclustersincertainredoxstates15.AsshowninFig.2,theEPRspectrumoftheas-purifiedGmsreducedbyDTHrevealsacomplexFe–Sclustersignal.SubsequentadditionofSAMresultsinanimmediatespectralchange,suggestingSAMbindstotheradicalSAM[4Fe–4S]+clusterandsignificantlyaffectstheelectronicstructureofthecluster.Additionally,weobservedmorethanoneFe–Sclustersignalinthespectra.Inordertodeconvolutethesignal,weemployedAlphaFold216topredictthestructureofGms.ThemodelshowsthatGmscontainsfourdomains,includinganN-terminaldomaincomprisedofsixhelices,aconservedradicalSAMdomain(RS-domain),abridgingregionwithtwohelices,aβ-harpinmotif,andaC-terminalSPASMdomain(Fig.2aandSupplementaryFig.1).ByaligningwiththeatomicstructureofSPASM-domaincontainingradicalSAMenzymeSuiB(PDB:5V1T)17, three [4Fe–4S] clusters (RS, AuxI and AuxII clusters) were displayedinourGmsmodel,consistentwiththecomplexFe–SclustersignalobservedintheEPRspectra(Fig.2bandSupplementaryFig.1).Withtheaidofthisstructuralmodel,wepreparedcorrespondingGmsmutantsbyknockingouttwo[4Fe–4S]clusters,leavingonlyoneclusterineach(Fig.2e,f).Thegtensorsforthethreeclusterswereclearlydeterminedviaspectralsimulation,i.e.,[2.051,1.884,1.817]fortheSAM-boundradicalSAMcluster,[2.045,1.930,1.885]fortheAuxIcluster,and[2.036,1.924,1.871]fortheAuxIIcluster(Fig.2f).Thegtensorsarecharacteristicoftwoauxiliary[4Fe–4S]clusters,buttheg-tensorforSAM-boundradicalSAMclusterisdistinctfromthetypicaltensorwithg ~2.0016,18. In addition, we did not observe a SAM response for both AuxI and AuxII clusters (Supplementary Fig. 5), suggestingthatSAMdoesnotbindtotheauxiliaryclustersasitdoesfortheradicalSAMcluster.ThisfurthercorroboratesthestructuralmodelshowingthetwoauxiliaryclustersfullyligatedbyfourCysresidues.

TheradicalSAMdomainofGmsencompassesthree-quartersofthetriosephosphateisomerase(TIM)barrelfoldandfeaturesasite-differentiated[4Fe–4S]cluster,whichissharedamongallRSenzymes(Fig.2b).Gmsexhibitsasubstrate-bindingpocketsimilartoTes,butthispocketissignificantlyreducedinsizeduetotheinsertionofaβ-hairpinwithinthebridgingregion,renderingitincapableofaccommodatingaGDGT-0substrateadequately(SupplementaryFig.6).Theinteractionbetweentheβ-hairpinandthepocketprimarilyinvolvestwohydrogenbondsbetweenF326andN351,aswellasS334andE346(Fig. 2c). On the opposite side of the β-hairpin, there are multiple hydrophobic amino acids. Therefore, it is hypothesized thatduringGDGT-0substratebinding,thishairpinflipsoutwards,providingsufficientspaceforsubstratebinding(SupplementaryFig.7).Additionally,thehydrophobiccavityfromtheN-terminaldomainofGms,whichfacethesubstrate-bindingpocket(Fig.2dandSupplementaryFig.7),mayalsobeinvolvedinsubstratebinding(Fig. 2d and SupplementaryFig.7).

We then compared the structural models of four RS enzymes, includingGms,Tes,GrsAandSuiB(PDB:5V1T)(SupplementaryFig.8and SupplementaryDatasets 1–3). While Tes primarily catalyzes the synthesisofGDGT-04,GrsA,likeGms,utilizesGDGT-0asthesubstrate,butcatalyzestheformationofacyclopentaneringattheC-7positionofGDGT-05.Ontheotherhand,SuiBisapeptide-modifyingSPASM-domain-containingRSenzymethatinstallsaLys–Trpcross-linkinitspeptidesubstrate17.ThesefourproteinshavedifferentdomaincompositionsbutpossesssimilarRSdomains(SupplementaryFig.8).ItisworthnotingthatGmsandTesbothhaveanα-domainoverlayingabovetheRS-domainasa‘cap’,buttheyareonoppositesides(SupplementaryFig.8a,b).This‘cap’andtheRS-domaininTesprovidetwohydrophobicpocketsforsubstratebinding.ThisobservationsuggeststhatthesubstrateinGmsmayhaveadifferentorientationforreachingtheRS catalytic center (Supplementary Figs. 7, 8). Although GrsA sharesthesamesubstrateasGms,itsstructuralcharacteristicsdivergenotablyfromGms(SupplementaryFig.8d).Firstly,GrsAlacksthe‘cap’abovetheRSdomain,whichformsanunclosedpocketnearitsN-domainandRS-domain. ThisstructuralmodelsuggeststhatGrsAdoesnotneedtheassistanceofthe‘cap’abovetheRS-domaintoefficientlybindthesubstrate.Secondly,GrsAhastwoelongatedhydrophobichelicesinitsC-terminus.GiventhatthesubstrateGDGT-0localizestothecellmembrane,itisconceivablethattheβ-hairpinofGmsandtheC-terminalhelixofGrsAmayactasanchorstothecellmembrane,facilitatingpotentialsubstrateinteractionswiththeseproteins.Thestructural similarities between SuiB and Gms are pronounced, buttheirdisparatepocketsresultindistinctsubstrateaffinitiesandbindingspecificities(SupplementaryFig.8a,c).WhilethiscomparisonbetweenGms,Tes,SuiB,andGrsAoffersinsightsintosubstratebindingmechanisms,furtherexperimentalvalidationisneeded.

ThebiologicalsourcesofGMGTs

ToidentifythebiologicalsourcesofGMGTs,weperformedaBLASTPsearchagainsttheNon-redundantproteinsequences(nr)databaseinNCBIrestrictedtoarchaeagenomesforGmsproteinhomologs.Weinitiallyobtained649GmshomologsfromtheBLASTPsearch(e-value<1e−50,identity>30%,sequencelength>420aminoacids),followedbysequenceclusteringata90%sequenceidentityusingCD-HITtool19,resultingin441homologs.Subsequently,these441homologsunderwentfilteringwithsequencesimilaritynetworks(SSNs)20toidentifyisofunctionalgroupsofradicalSAMenzymes,leadingtotheidentificationof413Gmshomologsbasedonanalignmentscoreof90.These 413 homologs are classified within the mega-1-1-24 subgroup andcanbedistinguishedfromotheridentifiedradicalSAMenzymes,suchasTes3,4andGrs5involvedinGDGTsbiosynthesis(SupplementaryFig.9),andAhbC/AhbD21,22involvedintheanaerobicbiosynthesisofhemebinmethanogensandsulfate-reducingbacteria(SupplementaryFig.10).Phylogeneticanalysisindicatesthese413Gmshomologsaredistributedwidelyinallthreearchaealsuperphyla(Asgard,Tack,andDPANN)andEuryarchaeota(Fig.3aandSupplementaryFig.11).Significantly,itisintriguingtonotethatallGmshomologsareexclusivelypresentinobligateanaerobicarchaealgenomes(Fig.3a).

Lipid analysis of culturable archaea from previous studies was further applied to explore the potential link between GMGTs production (Gms) and anaerobic respiration. Some obligate anaerobic archaea,includingMethanothermobacterthermautotrophicus,Palaeococcushelgesonii,Pyrococcusfuriosus,Thermococcusacidaminovorans,Aciduliprofundumboonei,andIgnisphaeraaggregans,containGmshomologsandwerereportedpreviouslytoproduceGMGTs8,10,23,24.Incontrast,aerobicarchaea,suchasmarinethaumarchaea,oneofthemostabundantarchaeainmarinewatercolumns,donothaveaGmshomologanddonotproduceGMGTs25.Moreover,eventhefacultativeanaerobicarchaea,suchasMetallosphaerasedulaandS.acidocaldarius,lacktheGmshomologanddonotmakeGMGTslipids26,27.Thus,theseobservationssuggestthebiosynthesisofGMGTsisexclusivetoobligateanaerobicarchaea.

gmsgenepresentinoxygen-deficientenvironments

ThedistinctbiologicalsourceofGMGTsthatwehaveidentifiedsuggeststhatGMGTsproducersshouldbeabundantinoxygen-deficientecosystemsandenvironments.Wenextinvestigatedthepresenceofgmsgenehomologsinthemetagenomesextractedfromoxicandoxygen-deficientenvironmentswithtBLASTnsearches(e-value<1e−20,identity>60%,sequencelength>80bp),whichindicatesthemicrobialcommunitieshavethegeneticcapacitytosynthesizeGMGTs.The relative abundances of gms were calculated as RPKM (reads per kilobase of sequence per million reads) values. We defined oxygen-deficientenvironmentsasO2<25μM,aconcentrationlowenoughtoinduceanaerobicmetabolism28.

Oceanmetagenomesequences,inconjunctionwithoxygenlevelinformation, were obtained from the NCBI SRA (Sequence Read Archive)database.ThesesequenceswereextractedfromthePacificOcean,theBlackSea,theBalticSea,andtheSouthChinaSeabluehole,encompassingvariousoceanicenvironments,includingtheoceansurface,suboxicwater(oxygenminimumzone,OMZ),andanoxicmarinesediment/water.Thesemetagenomesequencesweresubsequentlyanalyzedforthepresenceofgms.Consistentwiththecultureresults,gmswasexclusivelydetectedinsuboxic/anoxic(oxygen-deficient)environments,andabsentunderoxicconditions(Fig.3b and SupplementaryFig.12a).However,gmswasnotfoundineveryoxygen-deficientsample.Forexample,gmswasnotfoundincertainsamplesfromtheBalticSeasuboxicwatercolumnandBlackSeasediments(Fig. 3b). This implies that fluctuations in GMGTs producers within oxygen-deficient environments could be influenced by additional environmentalfactorssuchasspecificredoxconditions.

Inadditiontotheocean,weobservedacomparabledistributionpatternofgmsinfreshwaterlakes(Fig.3bandSupplementaryFig.12b).Wespecificallychoserepresentativelakeswithavailablemetagenomedataandoxygenlevelmeasurementsforouranalysis.TheselakesincludedLake227inCanada,KivuLakeinRwanda,andLakeTanganyikainEastAfrica.Unliketheopenocean,thebottomwatersofmostlakesareanoxicoreveneuxinicduetorestrictedcirculation29.Intheselakeenvironments,gmshomologswerefoundexclusivelyinoxygen-deficientwaters.TheanalysisofmetagenomesfrombothoceansandlakessuggeststhatGMGTsproducersarewidespreadinavarietyofoxygen-deficientenvironmentsbutabsentinoxicenvironments.

Methods
Microbialstrains,media,andgrowthconditions
Microbial strains used in this study are shown in Supplementary Table2.
M. maripaludis with pMEV4 plasmid was cultured under an anaerobic conditionwith puromycin(2.5μg/mL) inMcFmedium at37°C, and the solid medium was prepared by adding 1.5% agar45. LysogenybrothwasusedtocultureE.coliDH10BandBL21(DE3)ΔiscR(withkanamycinresistance)at37°C.
S. acidocaldarius ΔgrsAΔgrsB mutant was grown aerobically at 70°C,200rpminbasalBrockmediumsupplementedwith0.1%(wt/vol) NZ-amine, 0.2% (wt/vol) sucrose, 10μg/mL (wt/vol) uracil, and adjusted to pH 3 with sulfuric acid46. To cultivate on plates, Brock mediumwassupplementedwithafinalconcentrationof0.6%–0.8%(w/v)gelrite,10mMMgCl2and3mMCaCl2.

Plasmidconstruction
PlasmidsandprimersusedinthisstudyareshowninSupplementaryTables3,4.
PrimersweresynthesizedbyTsingkeCompany(Beijing,China).
PCR was performed using Phusion high-fidelity DNA Polymerase (Vazyme).TheFastPurePlasmidMiniKit(Vazyme)wasusedfortheisolationofE.coliplasmidDNA.DNAfragmentswerepurifiedbytheFastPureGelDNAExtractionMiniKit(Vazyme)forcloning.TheClonExpressOneStepCloningKit(Vazyme)wasusedfortheconstructionoftheplasmidsviastepwiseGibsonassembly.DNAsequenceswereconfirmedbysequencingatTsingke(Beijing,China).Intheinvivostudies,toconstructtheplasmidsfortheexpressioninM.maripaludis,theMETOK_RS04425genewassynthesizedbyGENEWIZCompanyandclonedintotheAfeIsiteofpMEV4-maeo_0574.
TheplasmidusedintheinvitrostudiesisaC-terminalstrep-tagIIcontainingpET-21b(+)vectorwithacodon-optimizedMETOK_RS04425geneforE.coli expression system. To construct the METOK_RS04425 mutant plasmids, the ΔAuxIΔAuxII, ΔRSΔAuxII and ΔRSΔAuxI mutants weregeneratedbyoverlapextensionPCRwiththecorrespondinginnerprimers(SupplementaryTable4)viathestepwiseGibsonassembly.

Transformationofplasmidsintohostcells
Intheinvivostudies,theconstructedplasmidsweretransformedintoM.maripaludisbypolyethyleneglycol(PEG)-mediatedtransformation,screenedonpuromycin(2.5μg/mL)inMcFagarmedium,andverifiedbyPCRamplificationwithprimersP1F/1R47.Intheinvitrostudies,theplasmidwastransformedtotheelectrocompetentcellsofE. coli DH10B or BL21 (DE3) ΔiscR by electroporation using a Micro-Pulser Electroporator (BioRad). The transformants were selected on agar plateswith100μg/mLampicillinand40μg/mLkanamycin.

Lipidextraction,purificationandanalysis
M.maripaludistransformants(100mL)wereculturedanaerobicallyat37°C and harvested in the stationary phase. S. acidocaldarius ΔgrsAΔgrsBmutantwasculturedaerobicallyat70°Cwithshakingat150rpm, and harvested in the stationary phase. Cultures were collected by centrifugation at 10,000×g for 10min, and pellets were storedat−80°Cbeforeextraction.
The pellets were acid hydrolyzed with 7mL of 10% (vol/vol) hydrochloric acid (HCl) and methanol (MeOH) in a 40mL covered glassbottleovernightat70°C.10mLofdichloromethane(DCM)and10mLofpurewaterwereaddedtotheacid-treatedmixture,andweretransferredtoa50mLTeflontube.Thesampleswerecentrifugedat2800×gfor10minforseparatingtheaqueousphasefromtheorganicphase.Theorganicphasewasthentransferredtothecollectionglassbottle.ThelipidswereextractedthreetimesbyDCM.Priortofurtheranalysis, the organic phase was combined and filtered through a 0.22μmPTFEfilter,thendriedwithnitrogengas3.
The GDGT-0 was purified from the lipids extracted from the ΔgrsAΔgrsBmutantofS.acidocaldariususingthepreparativeLC-MS.
GDGT-0purificationwasperformedonanAgilent1260InfinitIIHPLC-LC/MSD XT system with a fraction collector. Compound separation was achieved with a C18 column (150mm×10mm, ACE) at 45°C. Lipidswereelutedat45°Candforthefirst2minwith100%Aand0%B,whereA=MeOHandB=IPA,followedbythefollowinggradient:90/10A/Bfrom2minto5min,76/24A/Bfrom5minto15min,35/65A/Bfrom 15min to20min,10/90A/Bfrom20minto 32min and finally 100%Afrom32mintotheend.ThepurifiedGDGT-0wasusedasthesubstrateforinvitroGmsactivityassay.
Lipidswereanalyzedbyreversed-phaseliquidchromatography-high-resolution mass spectrometry (RP-LC-HRMS). Waters ACQUITY ClassIultra-highperformanceliquidchromatography(UPLC)isusedincombinationwithaSYNAPTG2-SiquadrupoleTimeofFlight(qTOF)high-resolution mass spectrometer and an electrospray ionization (ESI) source in positive mode. C18-AR UPLC column (2.1×150mm, 2μm;ACE)maintainedat55°C.Thetotalrunningtimewas40min,andthe sample was stored at 7°C during the running. The flow rate is 0.3ml/min. The mass spectrawere collected by FAST-DDA, MS m/z 100–2000 and MS2 m/z 50–2000. Using the collision-induced dissociation(CID)method,thefirst5ionswiththehighestintensityandthe total ion chromatographic (TIC) threshold > 20000 were fragmentedtoobtaintheproductionspectrum.Real-timedynamicmassexclusioncanobtaintheMS2spectrumoflowintensityions48.

Gmsexpressionandpurification
Theplasmidsofstrep-taggedGmsanditsvariantsweretransformedintothecompetentcellsofE.coliBL21(DE3)ΔiscRwithkanamycinresistance. The culture of each recombinant was grown in Luria-Bertani broth with 100mg/L ampicillin, 40mg/L kanamycin, 2mM ammoniumferriccitrate,0.5%(w/v)glucose,and100mM3-(N-morpholino) propanesulfonic acid (MOPS, pH 8.0) at 28°C to an OD600 ≈0.4.ThentheculturesweretransferredintotheCoyanaerobic chambers, and supplemented with 5mM Cysteine and 10mM fumarate.Isopropylβ-D-1-Dthiogalactopyranoside(IPTG)wasaddedtoafinalconcentrateof0.25mMtoinduceproteinexpression49.Thecultures werestirred for 22h, and the cells wereharvested by centrifugationat7800×gfor30minat4°C.Thenthecellswerefrozeninliquidnitrogenandstoredat−80°C.
ThecellswerelysedusingaLysisbuffer(100mMHEPES,150mMKCl,pH8.0)containingBugbusterdetergentsolution(EMDMillipore),benzonanse (EMD Millipore), rLysozyme (EMD Millipore), and one EDTAfreeproteaseinhibitorcocktailtablet(Roche).Thecelldebriswascentrifugedat29,000×gat4°Cfor30min,andthesupernatantwasappliedtoa50mLcolumnvolumeofstrep-tactionresin.Thecolumnwaswashedwith100mLbufferW(50mMHEPES,150mMKCl,pH8.0)toremovenonspecificbindingproteins.Theproteinwasthenelutedusing80mLbufferWcontaining3mMdesthiobiotin,andthedarkbrownproteinfractionwascollected.Theas-elutedprotein,withaconcentrationof~300μM,wasaliquoted,flashfrozenandstoredinliquidnitrogen.

EPRspectroscopyandanalysis
Gms reaction mixtures were prepared anaerobically. 100μL of as-elutedGms(~300μM)orGmsvariants(~300μM)inbufferWsolutionwasincubatedwith40equiv.ofsodiumdithionite(DTH)or/and40equiv.ofSAM.SampleswerefrozenandstoredinliquidnitrogenforEPRcharacterization.
X-band (9.35GHz) CW EPR spectra were recorded on a Bruker EleXsysE500spectrometerequippedwithasuper-highQresonator(ER4122SHQE)intheDepartmentofChemistryattheSouthernUniversityofScienceandTechnology(SUSTech).CryogenictemperatureswereobtainedandcontrolledbyusinganESR900liquidheliumcryostatinconjunctionwithatemperaturecontroller(OxfordInstrumentsMercuryiTC)andagasflowcontroller.CWEPRspectrawere recorded at 10 K using the following parameters: microwave power = 0.02mW;conversiontime=40ms;modulationamplitude=0.05mT;modulationfrequency=100kHz.SimulationsoftheCWspectraandthefollowingpulseEPRspectrawereperformedusingEasySpin5.2.35toolbox50,51 within the Matlab 2014a software suite (The Mathworks Inc.,Natick,MA).

Structureprediction
The Gms, Tes, and GrsA structures were predicted in AlphaFold216. BecausethestructureofGrsAdoesnotexistintheAlphaFold2proteinstructuredatabase(https://alphafold.ebi.ac.uk),toensureparameterconsistency,wepredictedallthreeproteinsusingastandaloneplatform, which was set up on a local computer running the CentOS operatingsystem,andacceleratedbyoneNVIDIAGeForceRTX2080Ti GPU with the parameters set as MAX_TEMPLATE_HITS=20, RELAX_MAX_ITERATIONS=0, RELAX_ENERGY_TOLERANCE=2.39, RELAX_STIFFNESS=10.0,RELAX_MAX_OUTER_ITERATIONS=20. Five models were predicted and ranked based on the pLDDT values, representingdifferentlevelsofconfidence.Finally,fivemodelswithsimilar interaction modes weregenerated, and rank_0wasused for further analysis. Compared to AF-F8AME3-F1-model_v4 and AF-Q4JAU6-F1-model_v4 in the AlphaFold database, the RMSD (root-mean-squaredeviation)valuesofGmsandTesare0.178and0.220,respectively,indicatingaproperstructureprediction.ThepredictedmodelofTesshowedasimilarstructuretothepublishedhomolog,GDGT-MAS (PDB: 7TOM, RMSD=0.992). Since GDGT-MAS had a C-terminalmissinginthestructure,wehereusedthepredictedmodelofTesforcomparison.

Gmsactivityassay
ThepurifiedGDGT-0wasresuspendedin20μLDMSOand100μLofbufferW,byincubatingthesolutionatroomtemperatureforhours.The reaction solution contained 25μM Gms, 30 equiv. of DTH, 30 equiv.ofSAMand10equiv.ofGDGT-0in1.5mLbufferWat37°Cfor72hr.Analiquotofthereactionsolutionwasextractedby10%(vol/vol)hydrochloric acid (HCl) in methanol (MeOH) for lipid analysis. The activityassayswerecarriedoutintriplicate.

Bioinformaticanalyzes
BLASTP searches of Gms homologs were conducted in the Non-redundant protein sequences (nr) database in NCBI with METOK_RS04425 amino acid sequence from M. okinawensis as the searchquery.Defaultsettingswereusedforallsearches.Thesearches wererestrictedtoAsgard(taxid:1935183),TACK(taxid:1783275),Euryarchaeota (taxid:28890), DPANN (taxid:1783276), respectively, to identifytheGmshomologs(e-value<1e−50,identity>30%,length>420aa). Redundant sequences were removed by clustering at a 90% sequence identity using CD-HIT version 4.6.819. SSNs analysis was permformed by RadicalSAM.org20 with an alignment scoreof 90 to identify radical SAM protein isofunctional groups. Twenty archaeal GrsAsequenceswereusedastheoutgroup.Proteinsequenceswerealigned via MUSCLE version 3.8.3152 with the outgroup included. MaximumlikelihoodtreeswerebuiltbyRAxMLversion8.2.1253usingtheCATmodelofrateheterogeneitywiththeLGsubstitutionmatrixand1000bootstrapiterations.interactiveTreeOfLife(iTOL)wasusedfortreevisualizationandannotation(http://itol.embl.de/)54.
To search gms gene homologs in metagenomes, the program NCBI-BLAST2.2.27+wasusedforTBLASTN.Defaultsettingswereusedforallsearches.ThegmshomologswereidentifiedbyusingTBLASTNsearches (e-value<1e−20, identity >60%, length >80bp) of the SRA (SequencesReadArchive)databaseonNCBIwiththeMETOK_RS04425DNAsequencefromofM.okinawensisasthesearchquery.TheGenBank metagenomes accessions for searches in Fig. 3 are listed in SourceData.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Review Li et al., Nature Comm.\nThis manuscript deals with various aspects (biosynthesis, enzymology, physiology, biogeochemistry and molecular paleontology) of so-called isoprenoid GMGTs, archaeal membrane lipids that are produced by “cross-linking” of the much more common GDGTs. These GMGTs have been reported before in archaea but the exact chemical structure and biosynthetic pathway by which GDGTs are produced and potential biomarker value remains elusive. The novel aspect of this manuscript is that it for the first time (although a similar manuscript from the group where the last and corresponding author performed a post-doc from 2015-2019 has been posted on a preprint server; https://www.biorxiv.org/ content/10.1101/ 2023.10.20.563219v1.abstract) identifies the gene encoding the enzyme responsible for the “crosslinking”. This allowed to identify the presence of this gene in archaeal (meta)genomes, revealing that its occurrence is limited to anaerobic archaea, which is suggested to result in a biomarker potential of isoprenoid GMGTs.\nAlthough I feel the microbiological work is very interesting and well executed, there are several major issues that prevent me to recommend this manuscript for publication in Nature Communications.\n1) It starts with the nomenclature: the authors call the archaeal membrane lipid of interest H- GDGT-0, which is chemically incorrect. When the two biphytanyl chains are cross-linked the GDGT becomes a glycerol MONOalkyl glycerol tetraether (GMGT); this is NOT a GDGT and this should be reflected in its name and, consequently, in the name of the enzyme and gene encoding the enzyme (the H in Hbs does not make sense). If you look to the “general” structure of GMGT-0, the introduction of the bridge actually results in a structure that looks like an eight (8; two macrocycles connected) not an H-structure (that only holds when you break the four ether linkages). I guess the origin of the use of H in the name is the publication of Morii et al. Biochim Biophys Acta 1390, 339-345 (1998) but there the authors specifically refer to the H-shaped core of the membrane lipid.\n2) I find it peculiar that the authors have done a lot of experiments to establish the enzyme producing a biochemical product that has not been fully characterized chemically. Lutnaes et al. (2007) elucidated the structure of a series of octaterpene tetracarboxylic acids with 4–6 cyclopentane moieties in petroleum by two-dimensional NMR studies. This established the position of the bridge, i.e. between two mid-chain methyl groups of each biphytanyl chain (although this still leaves various isomers possible). These tetracarboxylic acids were interpreted to be diagenetic products of isoGMGTs derived from hyperthermophilic archaea. Hence, direct structural identification of isoGMGTs in cultures of archaea is still lacking; the assignment of the position of the C-C bridge in their presumed diagenetic products by Lutnaes et al. (2006, 2007) is currently the best available indirect evidence. The authors do not provide any other chemical evidence other than showing a mass spectrum revealing a molecular ion that is two daltons lower than that of GDGT-0 (actually the spectra they show in Fig. 1C have been published previously and there is no good reason to show them again) and fail to acknowledge this weakness. In fact, a substantial part of the manuscript (lines 121-169) deals with details of GMGT-0 biosynthesis with predictions of the active sites of the enzyme (see Extended Data Fig. 6, where specifically two methyl groups (in red) of GDGT-0 are indicated to enter the active site) without knowing with certainty what the structure of the product of the enzyme reaction is. I find this unacceptable: the authors should isolate GMGT-0 from one of their cultures and prove the chemical structure they propose by 2D-NMR or related techniques. In the abstract they claim “This marks the second known example of direct linkage of two sp3 carbons by archaea”, but in fact we are not sure of the structure of the product produced and thus the sites of crosslinking, so this is highly speculative. The authors have isolated the enzyme and performed an in vitro study, which is very nice, but they should have isolated the product produced by this enzyme as well.\n3) The finding that the enzyme responsible for the production of GMGT-0 is restricted to anaerobic archaea is interesting but it remains unclear how they have established this since many of the investigated species seem not to be available in culture (species names in Extended Data Fig. 7 are unreadable; many seem to belong to groups that contain no cultured relative). Also, it remains entirely unclear what the role of temperature is in the formation of GMGT-0. This membrane lipid has mostly been reported to occur in (hyper)thermophilic archaea and the “bridging” has been proposed as an adaptation to temperature to rigidify the membrane. This is not discussed at all. Furthermore, the authors do not provide a plausible explanation why “bridging” of GDGTs would be an advantage for an archaeon in an anaerobic environment. They hypothesize that it could be an adaptation to oxidative stress but fail to present data to support that. Furthermore, this would be in contradiction with their proposal that GMGT-0 is a “novel biomarker for anoxia”.\n4) The quest for the “hbs gene” in the environment generates questions. It remains unclear why different tBLASTn criteria (lines 194) were used as compared to those (lines 172-173) in the search in archaeal genomes. Especially the use of a sequence length of only >80 bp (equivalent to >27 aa?) seems risky. But I am not an expert in this field, so I might be wrong.\n5) The environmental dataset on GMGT-0 presented are not convincing. Firstly, the relative abundance of GMGT-0 (it is actually not clear if this is the only GMGT taken into account or if this includes GMGTs with cyclopentane moieties) is extremely low. In the SPM of the Baltic Sea and Black Sea it is never >0.2% of the total GDGTs (I guess they mean total isoprenoid GDGTs). This is extremely low and, in fact, generates analytical challenges to assess their relative amount. The profile of the Black Sea also shows the presence in oxic waters and a decrease in the euxinic waters. Actually, the Black Sea is 2200 m deep and the sampling stops at 110 m, so this sample set cannot reveal what is going on in this euxinic basin since only 5% of the depth range is covered. The SPM profile of the Baltic Sea contains only four datapoints and the two in the sub- oxic zone shows the highest relative abundance, whilst there is only one sample in the oxic zone obtained from the surface where the abundance of archaeal GDGTs is typically below detection limit. Hence, both SPM profiles do not contribute to the story. The data from modern wetlands shows indeed a higher relative abundance (ca. 4%) of GMGT-0 in anoxic soil but this dataset has been published in 2018 by Naafs et al., so this is telling nothing new (“we suggest that H-GDGTs are preferentially produced at depth, in the anoxic, water-saturated catotelm by anaerobic archaea and bacteria. This is consistent with the observation that all reported organisms that produce H-isoGDGTs are strict anaerobes”; quote from Naafs et al.). The data presented also only provide the relative abundance of GMGT-0 whereas absolute concentrations (normalized on TOC) would be required; changes in the absolute abundance of GDGTs now also affect the relative abundance of GMGT-0. In conclusion, the environmental datasets do not contribute anything convincing or new and should be left out.\n6) The whole story is framed as the data would provide a novel biomarker “for studying ocean anoxic events dating back hundreds of millions of years in Earth’s history” (see also the start of the introduction). I don’t see the advantage of using GMGT-0 in the study of OAEs. We already have many biomarkers to indicate that the water column has been anoxic (e.g. dinorhopane, lycopene etc.) and some of these even allow us to assess that the lower part of photic zone was euxinic (e.g., isorenieratene derivatives; Sinninghe Damste and Koster, 1998; Kuypers et al., 2002). What is GMGT-0 offering in addition, assuming we are convinced it can only originate from anaerobic archaea? The authors already acknowledge themselves that they can’t discriminate between archaea residing in the water column or in the sediment (that ultimately will become anoxic upon burial). So, what is going to reveal this in addition to what all the biomarker data on OAEs that have been obtained in the last 4-5 decades? I don’t see the point and it is also not provided in the manuscript. If we look to the data of OAE2 (Demerara Rise), we again see that GMGT-0 (perhaps including GMGTs with cyclopentane moieties; no information provided) is again present in very low relative abundance (ca. 0.5% on average). This strongly contrasts the statement in the abstract “remarkably abundant in ancient sediments during past periods of oxygen deficiency, such as OAE- 2”. A slight increase is seen during OAE-2 but at this site bottom waters were probably also anoxic before and after the OAE, Otherwise, it would have been difficult to explain TOC levels of 10% throughout this section. This is also concluded on the basis of more established biomarkers in a recent paper (Abraham et al., 2023), which is co-authored by one of the co-authors of this manuscript. So, it remains totally unclear what the GMGT-0 profile is indicating.\n7) The scientific contribution of some of the authors also raises questions. The author contribution mentions “X.L., H.Y., J.K., G.T.C., A.K.W., B.D.A.N., L.D. and F.W. provided environmental samples and performed lipid analysis” but as far as I can see there is only data of three co-authors: JK and AKW (environmental GDGT and GMGT data Black Sea and Baltic Sea; Fig. 4a) and BDAN (GDGT and GMGT data Modern Wetland (Fig. 4b) and OAE2 Demerara Rise (Fig. 4c). The GDGT data for these sites have previously been published in Wittenborn et al (2023), Naafs et al. (2018), and Abraham et al. (2023). The relative quantitation of GMGT-0 in these cases only required the integration of one additional peak in their dataset (in case of Naafs et al., 2018 this was already reported). So, it remains unclear why there is such an extensive experimental description of GDGT/GMGT analysis in this manuscript; the authors could have just refereed to these paper. The contribution of the other five authors remains a mystery. There is a reference to a paper of Connock et al. (ref 45) in the legend of Fig. 4 but this paper refers to a site in the South Atlantic and not in the proto North Atlantic Ocean where Demerara Rise is located.\nI would strongly suggest focusing on the strong point of the paper (the biochemical characterization of the enzyme and apparent limitation to anaerobic archaea) and leave the rest out.\nMore detailed comments:\nLine 1 Title doesn’t read well. Furthermore, the authors do not provide any mechanism why GMGT- 0 is only produced in anaerobic archaea. The title should reflect what has really been demonstrated (e.g. “Anaerobic archaea produce GMGT-0 by a novel biphytanyl crosslinking enzyme”).\nLine 30 “Often overlooked subset” Why?\nLine 31 Clumsy writing: it is the enzyme that is primarily responsible. This sentence can be written in a more clear way.\nLines 41-44 Very speculative and not supported by the data provided.\nLines 47-70 The introduction has no focus and is poorly written (e.g. no connection between paragraph #1 and #2).\nLine 55 If you want to refer to a paper that applies GDGT for SST reconstruction during OAEs an original paper needs to be quoted. Ref 4 does not make sense in this respect.\nLine 66 “extra cross link”? How do you mean? GDGDT have no cross-link\nLine 69 “synthetic mechanisms”. Unclear what this means.\nI have only annotated page 1-2. This paper has 20 authors, some are based on highly ranked universities where English is the mother tongue. I can hardly believe that all of them have thoroughly gone through the text and made scientific and editorial comments. It is not the task of a referee to do this job.\nJaap Sinninghe Damsté\nFebruary 5, 2024", "reviewer_2": "This manuscript is about the identification of the enzyme responsible of converting the membrane- spanning tetra ether lipid GDGT-0 into the bridged derivative H-GDGT-0. Through heterologous expression and in vivo lipid analysis, as well as protein purification and in vitro enzyme assays, the authors convincely show that this C-C bonding enzyme belongs to the radical SAM class of enzymes, elucidating a further enzymatic step in archaeal lipid biosynthesis.\ncomments\nFig. 1b, the in vitro activity of the purified enzyme seems very low. Likely the enzym readily inactivates when exposed to oxygen; or cofactor incorporation might be very poor. The authors should consider the optimised conditions for radical SAM protein expression as described in ref 6. This is important as now it is not entirely clear if the further analysis (EPR) was done on a fully functional enzyme.\nOtherwise, in this in vitro assay, it could be an issue of substrate exposure to the enzyme. I noticed the assays do not contain detergent which would be necessary to keep the GDGT-0 monodispersed.\nThe authors propose a putative mechanism using the alfafold2 model and insights on other radical SAM proteins. However, what I entirely miss in the discussion is how this enzyme can act on a substrate that supposingly is membrane spanning. How does the binding site every get access to the relevant part of the substrate, that in the membrane spanning state will be in the middle of the membrane! A transparent discussion is needed.", "reviewer_3": "Glycerol dialkyl glycerol teraethers (GDGTs) are a special class of archaeal ether lipids that are membrane-spanning and can have several modifications/variations like crosslinks between the isoprenoid chains (H-GDGTs) or rings within the isoprenoid chains. Only in the last few years, the discovery of the enzymes that catalyze the reactions at the isoprenoid chains began (Tes, GrsA, GrsB), they are all radical SAM (RS) enzymes. The presented manuscript is a continuation of the elucidation of these interesting enzymes, as it describes for the first time the RS enzyme that introduces the crosslinks in H-GDGTs, why the authors call it “H-HDGT bridge synthase” (Hbs).\nThe authors study the Hbs activity in vitro and in vivo. Furthermore, they explore the occurrence of Hbs homologues in fully sequenced species and in metagenomic data. They also provide new own data on the occurrence of H-GDGTs in oxygen-deficient and oxic water samples. From all those data, they deduce that H-GDGT producing species are obligate anaerobic, and therefore only occur in (almost) oxygen-free environments. They suggest that H-GDGTs might be suitable as a new biomarker for studying oxygen deficiency in the geological past.\nThe study is, in most parts, properly done, and the results are properly presented. However, I have one main concern. Minor issues are listed below.\nMain concern:\nThe authors claim that Hbs homologues only occur in obligate anaerobic species (l. 171 ff), which is stressed as one of the major conclusions from this study. For this, they do BLAST searches within archaeal genomes with defined search parameters (e-value limit, min. identity, …). The authors do, however, not prove that these parameters are suited to distinguish between “true” Hbs and other related RS enzymes like for example Tes that also occur in aerobic species. The authors must somehow show that the hits they get are homogenous in their sequence properties, but different from Tes, Grs, or other, not yet characterized RS enzymes. This could, for example, be done by using the sequence similarity network (SSN) technique, or at least by showing adequate multiple sequence alignments or phylogenetic trees with all these RS enzymes (not only Grs as an outgroup as in ext. data Fig. 7). The same applies for the search in the metagenomes. For clarity, the authors should not only show the hits among metagenomic data in source data (is this really what is shown according to l. 606 in “Archaeal domain-Hbs-homologs”?) but also all the hits from the BLASTP search.\nIn general (see l.130ff, and also 1st paragraph of discussion), the whole story would benefit from a more detailed description of the structural properties of Hbs in comparison with Tes (which also has auxiliary Fe-S clusters), GrsA/B (which have no auxiliary Fe-S clusters) and SuiB. This could be done as extensions to Fig. 1 and ext. data Fig. 1, the EPR data then could be a separate figure.\nOther issues, in order of appearance:\nCarefully read again the manuscript for good language and typos, especially ll. 530-545 are full of them.\nll. 78-81: The result of this comparison should be better presented\nl. 82f: a gene cannot contain a motif defined by amino acids, only a protein can contain such a motif\nl. 121 ff: As EPR is a rarely seen method, it might be helpful for readers not so familiar with it to very briefly resume at the beginning of the paragraph what can be measured with this method.\next. data Fig. 1: At the time of the first reference to this figure in the text, nobody knows yet that WP_62369926.1 is Hbs, so both names should be used in the figure legend. It must also be introduced in the legend that the structure is a model, as also this is only introduced later in the text.\nFig. 2: The language of the legend should be improved (e.g.:”a, Four domains shown in Hbs”).\nl. 172/173: a) BLASTP can only search in protein databases, but not in “archaeal genomes” = DNA sequences; b) Is it really the case that the search was limited to archaea? This does not become clear from the according methods section. What about hits in bacteria? This might be interesting for some people because Tes homologues also occur in bacteria.\nl. 174: the authors state that they got 649 hits. In methods, they state that they cluster sequences with 90% similarity, in ext. data Fig. 7 they mention 177 unique homologues from 910 genomes. How do those numbers fit together, especially the 910 genomes?\next. data Fig. 7: “Based on 177 unique archaea Hbs homologues and 19 archaea Grs homologues from 910 archaea genomes in JGI IMG 691 database as outgroups […]”: the sentence as a whole seems strange: are the 177 unique homologues from the 910 genomes, or the 19 Grs sequences, or both together? Why more genomes than sequences?\next. data Fig. 7: I cannot see any blue circles as mentioned in the legend.\next. data Fig. 7: It would be appropriate to list also the Grs sequences in “source data”. With respect to my main concern: Would Tes as an outgroup also work?\nl. 212: ext. data Fig. 8b? 8c does not exist.\nFig. 3b, c: What is the y-axis of these diagrams? Water/Sediment depth like in Fig. 4?\nl. 249ff: Shouldn’t equation 1 better already appear two paragraphs above (ll. 236-241)? Or move it to methods?\nl. 308f: “The presence of H-GDGTs primarily depends on shift in its associated microbial communities in the water column or sediment, which are highly sensitive and responsive to the rise of anoxic condition.”: I do not completely understand the meaning of this sentence. Better: … depends on the distribution of the species that produce the H-GDGTs?\nl. 312 f.: “[…] the H-GDGTs proxy has the advantage to yield a more precise and regional representation of ocean oxygen deficiency supported by its well-established biological basis.” I also do not understand this completely. What is meant with “its well-established biological basis”?\nl. 539 and elsewhere: “centrifugation at 6,000 rpm for 30 min at 4 \"”: it makes only sense to give the rpm if you also mention the rotor. Alternatively, give the g value.\nl. 593: is really clustered at 90% sequence similarity (using a scoring matrix) or rather at 90% sequence identity?\nExt. data Table 1: separating horizontal lines between the M. maripaludis, E. coli and S. acidocaldarius strains might improve clarity. Typo “Escherichia coil”."}, "author_response": "Point-by-Point Response\nReviewer #1 (Remarks to the Author):\nReview Li et al., Nature Comm.\nThis manuscript deals with various aspects (biosynthesis, enzymology, physiology, biogeochemistry and molecular paleontology) of so-called isoprenoid GMGTs, archaeal membrane lipids that are produced by “cross-linking” of the much more common GDGTs. These GMGTs have been reported before in archaea but the exact chemical structure and biosynthetic pathway by which GDGTs are produced and potential biomarker value remains elusive. The novel aspect of this manuscript is that it for the first time (although a similar manuscript from the group where the last and corresponding author performed a post-doc from 2015-2019 has been posted on a preprint server; https://www.biorxiv.org/ content/10.1101/ 2023.10.20.563219v1.abstract) identifies the gene encoding the enzyme responsible for the “crosslinking”. This allowed to identify the presence of this gene in archaeal (meta)genomes, revealing that its occurrence is limited to anaerobic archaea, which is suggested to result in a biomarker potential of isoprenoid GMGTs.\nAlthough I feel the microbiological work is very interesting and well executed, there are several major issues that prevent me to recommend this manuscript for publication in Nature Communications.\nWe thank Dr. Sinninghe Damsté for the insight review and valuable comments. In response to his suggestions, we have decided to remove the environmental GMGT lipid data and instead focus on the rest aspects of our study.\n1) It starts with the nomenclature: the authors call the archaeal membrane lipid of interest H- GDGT-0, which is chemically incorrect. When the two biphytanyl chains are cross-linked the GDGT becomes a glycerol MONOalkyl glycerol tetraether (GMGT); this is NOT a GDGT and this should be reflected in its name and, consequently, in the name of the enzyme and gene encoding the enzyme (the H in Hbs does not make sense). If you look to the “general” structure of GMGT-0, the introduction of the bridge actually results in a structure that looks like an eight (8; two macrocycles connected) not an H-structure (that only holds when you break the four ether linkages). I guess the origin of the use of H in the name is the publication of Morii et al. Biochim Biophys Acta 1390, 339-345 (1998) but there the authors specifically refer to the H-shaped core of the membrane lipid.\nWe included both \"H-GDGT\" and \"GMGT\" terminologies in our manuscript. The reason we used \"H-GDGT\" as the primary term is its accessibility for general audiences and its association with GDGT molecules as a derivative. This study showing GDGT is the substrate for H-GDGT through cross linking of isoprenoid chains in GDGT. Initially, we considered \"H- GDGT,\" while less chemically precise than \"GMGT,\" to hold value for general audiences due to its straightforward recognition and its direct link to GDGT molecules.\nWe understood the reviewer's concerns and have made the necessary adjustments. Thus, we have used \"GMGT\" as the primary term, and renamed the enzyme as \"GMGT synthase (Gms)\" in manuscript.\n2) I find it peculiar that the authors have done a lot of experiments to establish the enzyme producing a biochemical product that has not been fully characterized chemically. Lutnaes et al. (2007) elucidated the structure of a series of octaterpene tetracarboxylic acids with 4–6 cyclopentane moieties in petroleum by two-dimensional NMR studies. This established the position of the bridge, i.e. between two mid-chain methyl groups of each biphytanyl chain (although this still leaves various isomers possible). These tetracarboxylic acids were interpreted to be diagenetic products of isoGMGTs derived from hyperthermophilic archaea. Hence, direct structural identification of isoGMGTs in cultures of archaea is still lacking; the assignment of the position of the C-C bridge in their presumed diagenetic products by Lutnaes et al. (2006, 2007) is currently the best available indirect evidence. The authors do not provide any other chemical evidence other than showing a mass spectrum revealing a molecular ion that is two daltons lower than that of GDGT-0 (actually the spectra they show in Fig. 1C have been published previously and there is no good reason to show them again) and fail to acknowledge this weakness. In fact, a substantial part of the manuscript (lines 121-169) deals with details of GMGT-0 biosynthesis with predictions of the active sites of the enzyme (see Extended Data Fig. 6, where specifically two methyl groups (in red) of GDGT-0 are indicated to enter the active site) without knowing with certainty what the structure of the product of the enzyme reaction is. I find this unacceptable: the authors should isolate GMGT-0 from one of their cultures and prove the chemical structure they propose by 2D-NMR or related techniques. In the abstract they claim “This marks the second known example of direct linkage of two sp3 carbons by archaea”, but in fact we are not sure of the structure of the product produced and thus the sites of crosslinking, so this is highly speculative. The authors have isolated the enzyme and performed an in vitro study, which is very nice, but they should have isolated the product produced by this enzyme as well.\nPurification of milligrams of lipid substrates GDGT-0 and the product GMGT-0 is a challenging and time-consuming task, which are not commercially available. Despite our numerous efforts to purify these compounds, we have encountered difficulties in obtaining a sufficient amount for NMR analysis. Additionally, the NMR data presented by Lutnaes et al. (2006, 2007) already provided clear evidence for the position of cross-linkage. Thus, we believed that the NMR characterization of GMGT required additional study that is beyond the scope of this manuscript.\nThe Fig. 1c has been removed to supplementary as requested. The spectra in Fig. 1c were used to show the identification of GMGT compound in our samples, rather than to declaim the novelty of these spectra.\nReferences\n1. Lutnaes, Bjart Frode, et al. Archaeal C 80 isoprenoid tetraacids responsible for naphthenate deposition in crude oil processing. Organic & biomolecular chemistry 4.4 (2006): 616-620.\n2. Lutnaes, Bjart F., et al. Structure elucidation of C 80, C 81 and C 82 isoprenoid tetraacids responsible for naphthenate deposition in crude oil production. Organic & Biomolecular Chemistry 5.12 (2007): 1873-1877.\n3) The finding that the enzyme responsible for the production of GMGT-0 is restricted to anaerobic archaea is interesting but it remains unclear how they have established this since many of the investigated species seem not to be available in culture (species names in Extended Data Fig. 7 are unreadable; many seem to belong to groups that contain no cultured relative). Also, it remains entirely unclear what the role of temperature is in the formation of GMGT-0. This membrane lipid has mostly been reported to occur in (hyper)thermophilic archaea and the “bridging” has been proposed as an adaptation to temperature to rigidify the membrane. This is not discussed at all. Furthermore, the authors do not provide a plausible explanation why “bridging” of GDGTs would be an advantage for an archaeon in an anaerobic environment. They hypothesize that it could be an adaptation to oxidative stress but fail to present data to support that. Furthermore, this would be in contradiction with their proposal that GMGT-0 is a “novel biomarker for anoxia”.\nA new phylogenetic tree figure (Supplementary Fig. 9) has been added, with improved figure resolution.\nGMGTs analysis from peatlands suggested a positive correlation between the relative abundance of GMGTs and temperature (Naafs et al., 2018). In contrast, culture experiments with the thermophilic archaeon indicated a negative correlation between GMGT abundance and temperature (Tourte et al., 2022), suggesting that GMGT formation may not primarily be a response to heat stress, but rather to other environmental factors. Furthermore, our homolog search for Gms revealed the genetic capability of many mesophilic anaerobic archaea, such as Bathyarchaea, to produce GMGTs, despite not experiencing heat stress. Hence, in this study, we do not consider temperature as the primary factor influencing GMGT formation; instead, we propose that anoxic conditions and oxidative stress play more significant roles. These statements have been incorporated into the Introduction section (line 50 to 64).\nWe found that GMGTs were produced by anaerobic archaea, which mainly live in anoxic environments. We also observed that the abundance GMGTs lipids were more enriched in suboxic zone than anoxic zone in modern sea (previous Fig. 4A). Based on these observations, we hypothesized that trace of oxygen in suboxic environments, such as oxic-anoxic fluctuation environments, maybe stimulate GMGTs producers to produce more GMGTs to tolerate oxidative stress. Therefore, this hypothesis aligns with our proposal that GMGTs serve as a potential proxy for oxygen deficiency, without contradiction.\nReferences\n1. Naafs, B. D. A., et al. \"Archaeal and bacterial H-GDGTs are abundant in peat and their relative abundance is positively correlated with temperature.\" Geochimica et Cosmochimica Acta 227 (2018): 156-170.\n2. Tourte, Maxime, et al. \"Membrane adaptation in the hyperthermophilic archaeon relies upon a novel strategy involving glycerol monoalkyl glycerol tetraether lipids.\" Environmental Microbiology 24.4 (2022): 2029-2046.\n4) The quest for the “hbs gene” in the environment generates questions. It remains unclear why different tBLASTn criteria (lines 194) were used as compared to those (lines 172-173) in the search in archaeal genomes. Especially the use of a sequence length of only >80 bp (equivalent to >27 aa?) seems risky. But I am not an expert in this field, so I might be wrong.\nTwo different methods were used for the search of Gms protein/gene homologs in archaeal genomes and environmental metagenomes, respectively.\nFor the search of protein homologs in archaeal genomes, the full length of the Gms protein amino acid sequence was used as a query to conduct a BLASTP search in NCBI archaeal genomes. (e-value < 1e-50, identity > 30%, sequence length > 420 amino acids).\nHowever, to quantify the abundance of gene homologs in environmental metagenomes, a different search method was used. This is because the raw data of metagenomes in the SRA (Sequence Read Archive) database consist of unassembled short reads (~300bp). Thus, a tBLASTn search was conducted to quantify the abundance of gene homologs in environment. The tBLASTn search parameters used in this study (e-value < 1e-20, identity > 60%, DNA sequence length > 80 bp) have been successfully applied by other studies (Saunders et al. 2019; Lee et al., 2016; Ogilvie et al., 2013.).\nReferences:\n1. Saunders, Jaclyn K., et al. Complete arsenic-based respiratory cycle in the marine microbial communities of pelagic oxygen-deficient zones. Proceeding of the National Academy of Sciences of the United States of America 14;116(2019):9925-9930.\n2. Lee, Jin-Woo, et al. Metagenomic analysis reveals the contribution of anaerobic methanotroph-1b in the oxidation of methane at the Ulleung Basin, East Sea of Korea. Journal of Microbiology 54(2016):814-822.\n3. Ogilvie, Lesley A., et al. Genome signature-based dissection of human gut metagenomes to extract subliminal viral sequences. Nature Communications 4(2013):2420.\n5) The environmental dataset on GMGT-0 presented are not convincing. Firstly, the relative abundance of GMGT-0 (it is actually not clear if this is the only GMGT taken into account or if this includes GMGTs with cyclopentane moieties) is extremely low. In the SPM of the Baltic Sea and Black Sea it is never >0.2% of the total GDGTs (I guess they mean total isoprenoid GDGTs). This is extremely low and, in fact, generates analytical challenges to assess their relative amount. The profile of the Black Sea also shows the presence in oxic waters and a decrease in the euxinic waters. Actually, the Black Sea is 2200 m deep and the sampling stops at 110 m, so this sample set cannot reveal what is going on in this euxinic basin since only 5% of the depth range is covered. The SPM profile of the Baltic Sea contains only four datapoints and the two in the sub-oxic zone shows the highest relative abundance, whilst there is only one sample in the oxic zone obtained from the surface where the abundance of archaeal GDGTs is typically below detection limit. Hence, both SPM profiles do not contribute to the story. The data from modern wetlands shows indeed a higher relative abundance (ca. 4%) of GMGT-0 in anoxic soil but this dataset has been published in 2018 by Naafs et al., so this is telling nothing new (“we suggest that H-GDGTs are preferentially produced at depth, in the anoxic, water-saturated catotelm by anaerobic archaea and bacteria. This is consistent with the observation that all reported organisms that produce H-isoGDGTs are strict anaerobes”; quote from Naafs et al.). The data presented also only provide the relative abundance of GMGT-0 whereas absolute concentrations (normalized on TOC) would be required; changes in the absolute abundance of GDGTs now also affect the relative abundance of GMGT-0. In conclusion, the environmental datasets do not contribute anything convincing or new and should be left out.\nWe understand reviewer's concerns about the complexity of developing a new biomarker, and agree to remove the GMGT lipid data, focusing on biology aspects.\nOur initial proposal for developing GMGT as an anoxia proxy follows a logical sequence:\n1) Discovery of GMGT Synthase: the identification of GMGT synthase enabled a comprehensive investigation of its biological sources by examining all archaeal genomes in the database, rather than relying on a few reported culture species.\n2) Gene Present in Anoxic Environments: the presence of the GMGT synthase gene was exclusively detected in metagenomes from anoxic environments, suggesting a potential association between GMGT and anoxic conditions.\n3) Enrichment of GMGT Lipids in Anoxic Environments: lipid analysis revealed an enrichment of GMGT lipids in anoxic environments, providing evidence of its potential as an anoxia biomarker.\nThis sequential approach demonstrates a systematic progression from gene discovery to environmental distribution, resulting in the recognition of GMGT as a promising indicator of anoxic conditions.\nFollowing stage 2), our hypothesis suggested that GMGT lipids might exhibit a similar distribution pattern in anaerobic environments. To test this hypothesis, we had conducted an extensive search for published GMGT lipid data and reached out to collaborators who might possess relevant samples. Despite months of effort, it turns out that the quality of existing environmental GMGT lipid data did not meet the criteria set by reviewer for establishing GMGT lipid as an anoxia biomarker and proxy.\nOur strategy to develop a new biomarker begins with the discovery of a novel lipid biosynthesis gene. This approach contrasts with the traditional procedure, which typically starts by correlating lipid profiles with specific environmental factors, and then explores their biological basis afterward. While our approach stands on a robust biological foundation from the outset, its weakness lies in the potential for low abundance of the target lipid biomarker in certain sites, leading to limitations in broader applicability. Nonetheless, by establishing the biomarker on a solid biological footing from the start, we aim to promote its reliability and effectiveness through extensive examination of environmental samples in follow-up studies.\nIn summary, we maintain confidence in the association between the distribution of GMGT lipids and environmental anoxic conditions, supported by their origins from anaerobic archaea and the distribution pattern of their gene in anoxic environments. However, we also realize the complexity of lipid distribution in nature, particularly cross geological time spans. Thus, we have agreed with the reviewer to remove current environmental GMGT lipid data from this study, recognizing the potential challenges and uncertainties associated with interpreting such data.\n7) The scientific contribution of some of the authors also raises questions. The author contribution mentions “X.L., H.Y., J.K., G.T.C., A.K.W., B.D.A.N., L.D. and F.W. provided environmental samples and performed lipid analysis” but as far as I can see there is only data of three co-authors: JK and AKW (environmental GDGT and GMGT data Black Sea and Baltic Sea; Fig. 4a) and BDAN (GDGT and GMGT data Modern Wetland (Fig. 4b) and OAE2 Demerara Rise (Fig. 4c). The GDGT data for these sites have previously been published in Wittenborn et al (2023), Naafs et al. (2018), and Abraham et al. (2023). The relative quantitation of GMGT-0 in these cases only required the integration of one additional peak in their dataset (in case of Naafs et al., 2018 this was already reported). So, it remains unclear why there is such an extensive experimental description of GDGT/GMGT analysis in this manuscript; the authors could have just refereed to these papers. The contribution of the other five authors remains a mystery. There is a reference to a paper of Connock et al. (ref 45) in the legend of Fig. 4 but this paper refers to a site in the South Atlantic and not in the proto North Atlantic Ocean where Demerara Rise is located.\nThe author list has been changed to accompany with the removal of environmental GMGT lipid data.\nI would strongly suggest focusing on the strong point of the paper (the biochemical characterization of the enzyme and apparent limitation to anaerobic archaea) and leave the rest out.\nAs explain above, we have removed all the environmental GMGT lipid data, and focused on the rest aspects.\nMore detailed comments:\nLine 1 Title doesn’t read well. Furthermore, the authors do not provide any mechanism why GMGT- 0 is only produced in anaerobic archaea. The title should reflect what has really been demonstrated (e.g. “Anaerobic archaea produce GMGT-0 by a novel biphytanyl crosslinking enzyme”).\nThe title has been changed to “Biosynthesis of GMGTs by a radical SAM enzyme associated with anaerobic archaea and environmental oxygen deficiency”.\nLine 30 “Often overlooked subset” Why?\nWe mean the physiology and geological significance of GMGT are not clear, and thus researchers pay less attention to GMGT compared to cyclic GDGTs. We have removed these words to prevent confusion.\nLine 31 Clumsy writing: it is the enzyme that is primarily responsible. This sentence can be written in a more clear way.\nEdits made as suggested.\nLines 41-44 Very speculative and not supported by the data provided.\nThese statements have been removed.\nLines 47-70 The introduction has no focus and is poorly written (e.g. no connection between paragraph #1 and #2).\nThe induction was rewritten, the paragraph 1# (oxygen deficiency content) has been removed.\nLine 55 If you want to refer to a paper that applies GDGT for SST reconstruction during OAEs an original paper needs to be quoted. Ref 4 does not make sense in this respect.\nOAEs content has been removed, and the introduction has been rewritten, and Schouten et al., OG, 2013 review paper has been cited.\nLine 77 “extra cross link”? How do you mean? GDGDT have no cross-link\nWe tend to express GMGT has an extra cross link on isoprenoid than GDGT.\nThe sentence has been revised to “distinguished by an additional C-C linkage between the two isoprenoid chains”.\nLine 79 “synthetic mechanisms”. Unclear what this means.\nIt has been revised to “biochemical mechanisms”\nI have only annotated page 1-2. This paper has 20 authors, some are based on highly ranked universities where English is the mother tongue. I can hardly believe that all of them have thoroughly gone through the text and made scientific and editorial comments. It is not the task of a referee to do this job.\nWe have done careful proofreading and corrected language errors.\nJaap Sinninghe Damsté\nFebruary 5, 2024\nAbraham, M. A. F., Naafs, B. D. A., Lauretano, V., Sgouridis, F., & Pancost, R. D. (2023). Warming drove the Expansion of Marine Anoxia in the Equatorial Atlantic during the Cenomanian Leading up to Oceanic Anoxic Event 2. Clim. Past, 19, 2569–2580.\nKuypers, M. M., Pancost, R. D., Nijenhuis, I. A., & Sinninghe Damsté, J. S. (2002). Enhanced productivity led to increased organic carbon burial in the euxinic North Atlantic basin during the late Cenomanian oceanic anoxic event. Paleoceanography, 17(4), 3-1.\nLutnaes, B. F., Krane, J., Smith, B. E., & Rowland, S. J. (2007). Structure elucidation of C 80, C 81 and C 82 isoprenoid tetraacids responsible for naphthenate deposition in crude oil production. Organic & Biomolecular Chemistry, 5(12), 1873-1877.\nNaafs, B. D. A., McCormick, D., Inglis, G. N., & Pancost, R. D. (2018). Archaeal and bacterial H- GDGTs are abundant in peat and their relative abundance is positively correlated with temperature. Geochimica et Cosmochimica Acta, 227, 156-170.\nSinninghe Damsté, J. S., & Köster, J. (1998). A euxinic southern North Atlantic Ocean during the Cenomanian/Turonian oceanic anoxic event. Earth and Planetary Science Letters, 158(3-4), 165-173.\nWittenborn, A. K., Bauersachs, T., Hassenrück, C., Käding, K., Wäge-Recchioni, J., Jürgens, K., ... & Kaiser, J. (2023). Nitrosopumilus as main source of isoprenoid glycerol dialkyl glycerol tetraether lipids in the central Baltic Sea. Frontiers in Microbiology, 14.\nReviewer #2 (Remarks to the Author):\nThis manuscript is about the identification of the enzyme responsible of converting the membrane-spanning tetra ether lipid GDGT-0 into the bridged derivative H-GDGT-0. Through heterologous expression and in vivo lipid analysis, as well as protein purification and in vitro enzyme assays, the authors convincely show that this C-C bonding enzyme belongs to the radical SAM class of enzymes, elucidating a further enzymatic step in archaeal lipid biosynthesis.\ncomments\nFig. 1b, the in vitro activity of the purified enzyme seems very low. Likely the enzyme readily inactivates when exposed to oxygen; or cofactor incorporation might be very poor. The authors should consider the optimised conditions for radical SAM protein expression as described in ref 6. This is important as now it is not entirely clear if the further analysis (EPR) was done on a fully functional enzyme.\nOtherwise, in this in vitro assay, it could be an issue of substrate exposure to the enzyme. I noticed the assays do not contain detergent which would be necessary to keep the GDGT-0 monodispersed.\nWe performed the in vitro activity assay under anaerobic conditions. And actually, both expression and purification procedures were carried out in the anaerobic chamber to avoid any damage due to oxygen. We have specifically emphasized this point now.\nThe EPR spectrum clearly shows signals from all three FeS clusters, suggesting that the enzyme we purified is fully active.\nFor the in vitro activity assay, we first dissolved GDGT-0 in DMSO, and then slowly added it to the enzyme in a buffer solution. For complete incubation of GDGT-0 within the enzyme, we monitored the mixture to make sure it reached a homogeneous state before adding SAM to initiate the reaction. Although the in vitro activity is lower than in vivo activity, this could be due to the slow-release process of the product.\nThe authors propose a putative mechanism using the alfafold2 model and insights on other radical SAM proteins. However, what I entirely miss in the discussion is how this enzyme can act on a substrate that supposingly is membrane spanning. How does the binding site every get access to the relevant part of the substrate, that in the membrane spanning state will be in the middle of the membrane! A transparent discussion is needed.\nBy comparing the structure of Gms to Tes, SuiB, and GrsA, we found some structural differences in Gms and speculated that the β-hairpin might be involved in anchoring the protein to the membrane. A detailed discussion has been added to the revised manuscript (see new Supplementary Fig. 8, and line: 149-170).\nReviewer #3 (Remarks to the Author):\nGlycerol dialkyl glycerol teraethers (GDGTs) are a special class of archaeal ether lipids that are membrane-spanning and can have several modifications/variations like crosslinks between the isoprenoid chains (H-GDGTs) or rings within the isoprenoid chains. Only in the last few years, the discovery of the enzymes that catalyze the reactions at the isoprenoid chains began (Tes, GrsA, GrsB), they are all radical SAM (RS) enzymes. The presented manuscript is a continuation of the elucidation of these interesting enzymes, as it describes for the first time the RS enzyme that introduces the crosslinks in H-GDGTs, why the authors call it “H-HDGT bridge synthase” (Hbs).\nGiven Reviewer 1 raised similar concern about the terminology, we have used “GMGT”, instead of “H-GDGT”, as the primary term, and renamed the enzyme as “GMGT synthase (Gms)”.\nThe authors study the Hbs activity in vitro and in vivo. Furthermore, they explore the occurrence of Hbs homologues in fully sequenced species and in metagenomic data. They also provide new own data on the occurrence of H-GDGTs in oxygen-deficient and oxic water samples. From all those data, they deduce that H-GDGT producing species are obligate anaerobic, and therefore only occur in (almost) oxygen-free environments. They suggest that H-GDGTs might be suitable as a new biomarker for studying oxygen deficiency in the geological past.\nThe study is, in most parts, properly done, and the results are properly presented. However, I have one main concern. Minor issues are listed below.\nMain concern:\nThe authors claim that Hbs homologues only occur in obligate anaerobic species (l. 171 ff), which is stressed as one of the major conclusions from this study. For this, they do BLAST searches within archaeal genomes with defined search parameters (e-value limit, min. identity, …). The authors do, however, not prove that these parameters are suited to distinguish between “true” Hbs and other related RS enzymes like for example Tes that also occur in aerobic species. The authors must somehow show that the hits they get are homogenous in their sequence properties, but different from Tes, Grs, or other, not yet characterized RS enzymes. This could, for example, be done by using the sequence similarity network (SSN) technique, or at least by showing adequate multiple sequence alignments or phylogenetic trees with all these RS enzymes (not only Grs as an outgroup as in ext. data Fig. 7). The same applies for the search in the metagenomes. For clarity, the authors should not only show the hits among metagenomic data in source data (is this really what is shown according to l. 606 in “Archaeal domain-Hbs-homologs”?) but also all the hits from the BLASTP search.\nFollowing these suggestions, we have used the SSN method to distinguish Gms from Grs and Tes. The SSN analysis results clearly indicated that Gms form distinct from Grs or Tes enzymes. Additionally, sequence comparison revealed minimal homology between Gms and Grs or Tes. As a result, it is unlikely that Gms homology searches would include any Grs or Tes enzymes. For detailed information, please refer to the new supplementary Fig. 10 and included below.\nSupplementary Fig. 10 | The homology analysis of Gms, Tes, and Grs sequences.\nIn our search for similarity with other radical SAM proteins, we found that Gms sequence shares similarity with AhbC and AhbD proteins (32-39% identities). These two proteins are known to be involved in the anaerobic biosynthesis of heme b in methanogens (anaerobic archaea) and sulfate-reducing bacteria (anaerobic bacteria). However, Gms protein possesses an additional ~100 amino acid sequence in the N-terminal region compared to AhbC and AhbD. As a result, Gms can be distinguished from AhbC and AhbD by setting a length cutoff of 420aa in the search parameters. Furthermore, in the SSN method, using an alignment score of 90 is sufficient to differentiate Gms from AhbC and AhbD. For detailed information, please refer to the new supplementary Fig. 11 and included below.\nSupplementary Fig. 11 | The homology analysis of Gms and AhbC/AhbD.\nIn source data, “Archaeal domain-Gms-homologs” is the hits of BLASTP from archaeal genome database, and the “Lake-Gms (TPKM)” and “Lake-Gms (TPKM)” are the hits of metagenomes search with TBLASTN.\nIn general (see l.130ff, and also 1st paragraph of discussion), the whole story would benefit from a more detailed description of the structural properties of Hbs in comparison with Tes (which also has auxiliary Fe-S clusters), GrsA/B (which have no auxiliary Fe-S clusters) and SuiB. This could be done as extensions to Fig. 1 and ext. data Fig. 1, the EPR data then could be a separate figure.\nWe have incorporated a more detailed description of the structural comparison with Tes, SuiB, and GrsA, along with the addition of a new supplementary Fig. 8.\nFor the suggestion of separation of protein structure and EPR data into two figures, we believe that displaying the EPR data together with the structure model in one figure would make it easier for readers to understand the signals of each cluster. EPR is a unique spectroscopic technique used for characterizing paramagnetic FeS clusters, and presenting the data alongside the structure model would provide a better context for its interpretation. Therefore, we would like to proceed with presenting the EPR data with the structure model in one figure.\nOther issues, in order of appearance:\nCarefully read again the manuscript for good language and typos, especially ll. 530-545 are full of them.\nErrors have been corrected.\nll. 78-81: The result of this comparison should be better presented\nWe have added a new supplementary table 4 to list this comparison.\nl. 82f: a gene cannot contain a motif defined by amino acids, only a protein can contain such a motif\nEdits made as suggested.\nl. 121 ff: As EPR is a rarely seen method, it might be helpful for readers not so familiar with it to very briefly resume at the beginning of the paragraph what can be measured with this method.\nWe have added a brief introduction to EPR before presenting the EPR results. “EPR spectroscopy is a technique that only probes paramagnetic species, e.g., organic radical, metal ions with specific oxidation states, or multi-nuclear cluster in certain redox states.”\next. data Fig. 1: At the time of the first reference to this figure in the text, nobody knows yet that WP_62369926.1 is Hbs, so both names should be used in the figure legend. It must also be introduced in the legend that the structure is a model, as also this is only introduced later in the text.\nEdits made as suggested.\nFig. 2: The language of the legend should be improved (e.g.:”a, Four domains shown in Hbs”).\nThe legend language has been revised.\nl. 172/173: a) BLASTP can only search in protein databases, but not in “archaeal genomes” = DNA sequences; b) Is it really the case that the search was limited to archaea? This does not become clear from the according methods section. What about hits in bacteria? This might be interesting for some people because Tes homologues also occur in bacteria.\nIndeed, we conducted a search for Gms protein homologs in the NCBI \"non-redundant protein sequence\" database. The NCBI BLASTP website enables searches within specific organism genomes, as illustrated in the figure below. For this study, we restricted the search to archaeal genomes, resulting in Gms homologs exclusively from archaea. We have revised the method of this part to avoid confusion.\nWhile we did find Gms homologs are present in some bacterial genomes. However, bacteria do not synthesize archaeal-type GDGT or GMGT. Therefore, these bacterial Gms homologs likely have different biological function with different substrate. Studying the function of these bacterial Gms homologs falls outside the scope of this study.\nl. 174: the authors state that they got 649 hits. In methods, they state that they cluster sequences with 90% similarity, in ext. data Fig. 7 they mention 177 unique homologues from 910 genomes. How do those numbers fit together, especially the 910 genomes?\nThe 177 Gms homolog hits used for the construction of phylogenetic tree (previous ext. data Fig. 7) were obtained from JGI IMG database through BLASTP searches conducted on 910 archaeal genomes. However, the 649 hits used for Fig. 3A were acquired from NCBI database. It's not unexpected that conducting BLASTP searches for Gms in two different databases yielded different numbers of hits (see below figure).\nTo be consistent, we have constructed a new phylogenetic tree with Gms homologs from NCBI database BLASTP search (see new Supplementary Fig. 9).\next. data Fig. 7: “Based on 177 unique archaea Hbs homologues and 19 archaea Grs homologues from 910 archaea genomes in JGI IMG 691 database as outgroups […]”: the sentence as a whole seems strange: are the 177 unique homologues from the 910 genomes, or the 19 Grs sequences, or both together? Why more genomes than sequences?\nThe legend of supplementary Fig. 9 has been revised to clarify confusion (Suplementary file line: 85-91). Because only a subset of archaeal genomes contains the Gms gene or protein, resulting in fewer total Gms hits than the total number of archaeal genomes.\next. data Fig. 7: I cannot see any blue circles as mentioned in the legend.\nA new Gms phylogenetic tree has been constructed as described above with improved figure resolution (see new Supplementary Fig. 9).\next. data Fig. 7: It would be appropriate to list also the Grs sequences in “source data”. With respect to my main concern: Would Tes as an outgroup also work?\nIn our response to your above main concern, we have compared the similarity between Gms with other RS proteins including Tes and Grs. Here, we constructed a Gms phylogenetic tree using Tes sequences as outgroup (below figure). These results showed both Grs and Tes sequences work fine as an outgroup in Gms phylogenetic tree construction.\nl. 212: ext. data Fig. 8b? 8c does not exist.\nThanks for pointing this out, and we have corrected that.\nFig. 3b, c: What is the y-axis of these diagrams? Water/Sediment depth like in Fig. 4?\nYes, and we have added instruction in figure legend: “The y-axis represents the approximate depth of water and sediment.” (line 252-253)\nl. 249ff: Shouldn’t equation 1 better already appear two paragraphs above (ll. 236-241)? Or move it to methods?\nWe have removed these environmental lipid data in response to the Reviewer 1's request.\nl. 308f: “The presence of H-GDGTs primarily depends on shift in its associated microbial communities in the water column or sediment, which are highly sensitive and responsive to the rise of anoxic condition.”: I do not completely understand the meaning of this sentence. Better: … depends on the distribution of the species that produce the H-GDGTs?\nThanks for the suggestion, but this paragraph discussing environmental lipid data has been removed in response to the Reviewer 1's request.\nl. 312 f.: “[…] the H-GDGTs proxy has the advantage to yield a more precise and regional representation of ocean oxygen deficiency supported by its well-established biological basis.” I also do not understand this completely. What is meant with “its well-established biological basis”?\nLike above, this paragraph has been removed.\nl. 539 and elsewhere: “centrifugation at 6,000 rpm for 30 min at 4 \"”: it makes only sense to give the rpm if you also mention the rotor. Alternatively, give the g value.\nThe speed has been converted to g value in manuscript.\nl. 593: is really clustered at 90% sequence similarity (using a scoring matrix) or rather at 90% sequence identity?\nWe have revised that to “90% sequence identity”.\nExt. data Table 1: separating horizontal lines between the M. maripaludis, E. coli and S. acidocaldarius strains might improve clarity. Typo “Escherichia coil”.\nEdits made as suggested."}]

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