Unusual Butane- and Pentanetriol-Based Tetraether Lipids inMethanomassiliicoccus luminyensis, a Representative of the SeventhOrder of Methanogens

Kevin W. Becker,a* Felix J. Elling,a* Marcos Y. Yoshinaga,a Andrea Söllinger,b Tim Urich,b* Kai-Uwe Hinrichsa

MARUM Center for Marine Environmental Sciences and Department of Geosciences, University of Bremen, Bremen, Germanya; Department of Ecogenomics and SystemsBiology, University of Vienna, Vienna, Austriab

ABSTRACT

A new clade of archaea has recently been proposed to constitute the seventh methanogenic order, the Methanomassiliicoccales,which is related to the Thermoplasmatales and the uncultivated archaeal clades deep-sea hydrothermal vent Euryarchaeotagroup 2 and marine group II Euryarchaeota but only distantly related to other methanogens. In this study, we investigated themembrane lipid composition of Methanomassiliicoccus luminyensis, the sole cultured representative of this seventh order. Thelipid inventory of M. luminyensis comprises a unique assemblage of novel lipids as well as lipids otherwise typical for thermo-philic, methanogenic, or halophilic archaea. For instance, glycerol sesterpanyl-phytanyl diether core lipids found mainly in halo-philic archaea were detected, and so were compounds bearing either heptose or methoxylated glycosidic head groups, neither ofwhich have been reported so far for other archaea. The absence of quinones or methanophenazines is consistent with a biochem-istry of methanogenesis different from that of the methanophenazine-containing methylotrophic methanogens. The most dis-tinctive characteristic of the membrane lipid composition of M. luminyensis, however, is the presence of tetraether lipids inwhich one glycerol backbone is replaced by either butane- or pentanetriol, i.e., lipids recently discovered in marine sediments.Butanetriol dibiphytanyl glycerol tetraether (BDGT) constitutes the most abundant core lipid type (>50% relative abundance)in M. luminyensis. We have thus identified a source for these unusual orphan lipids. The complementary analysis of diverse ma-rine sediment samples showed that BDGTs are widespread in anoxic layers, suggesting an environmental significance of Metha-nomassiliicoccales and/or related BDGT producers beyond gastrointestinal tracts.

IMPORTANCE

Cellular membranes of members of all three domains of life, Archaea, Bacteria, and Eukarya, are largely formed by lipids inwhich glycerol serves as backbone for the hydrophobic alkyl chains. Recently, however, archaeal tetraether lipids with either bu-tanetriol or pentanetriol as a backbone were identified in marine sediments and attributed to uncultured sediment-dwelling ar-chaea. Here we show that the butanetriol-based dibiphytanyl tetraethers constitute the major lipids in Methanomassiliicoccusluminyensis, currently the only isolate of the novel seventh order of methanogens. Given the absence of these lipids in a large setof archaeal isolates, these compounds may be diagnostic for the Methanomassiliicoccales and/or closely related archaea.

Methane is a potent greenhouse gas and an important inter-mediate in the global carbon cycle (1–3). Biogenic methane

is produced predominantly by archaea inhabiting diverse anoxicenvironments such as sediments, soils, wetlands, and the digestivetracts of termites and ruminants (2, 4). All cultured methanogensto date belong to the phylum Euryarchaeota, while metagenomicsequencing revealed a putative methanogenic metabolism formembers of the uncultivated Bathyarchaeota (formerly known asthe miscellaneous crenarchaeotal group [MCG]), indicating thatmethanogenesis might not be restricted to the Euryarchaeota (5).

Methanogens are classified into seven orders (Methanobacte-riales, Methanococcales, Methanomicrobiales, Methanosarcinales,Methanocellales, Methanopyrales, and Methanomassiliicoccales)that generate methane from H2/CO2, acetate, formate, or methyl-ated substrates (2, 6–8). Of these, the Methanomassiliicoccales haveonly recently been described, representing the seventh order ofmethanogens (6, 7, 9). These Euryarchaeota have been detectedbased on gene biomarker analyses in diverse environments such aslakes, soils, and marine sediments but are particularly abundant inthe digestive tracts of ruminants (10–15). A single pure culture,Methanomassiliicoccus luminyensis, as well as a few enrichment

cultures, have been obtained, all of which reduce methanol ormethylamines with H2 as the electron donor (6, 16–20).

The Methanomassiliicoccales are only distantly related to othermethanogens and form a distinct cluster within the Thermoplas-mata with the nonmethanogenic thermoacidophilic Thermoplas-matales and other related lineages, such as the deep-sea hydrother-

Received 8 March 2016 Accepted 10 May 2016

Accepted manuscript posted online 13 May 2016

Citation Becker KW, Elling FJ, Yoshinaga MY, Söllinger A, Urich T, Hinrichs K-U.2016. Unusual butane- and pentanetriol-based tetraether lipids inMethanomassiliicoccus luminyensis, a representative of the seventh order ofmethanogens. Appl Environ Microbiol 82:4505–4516. doi:10.1128/AEM.00772-16.

Editor: V. Müller, Goethe University Frankfurt am Main

Address correspondence to Kevin W. Becker, kbecker@whoi.edu.

* Present address: Kevin W. Becker, Department of Marine Chemistry andGeochemistry, Woods Hole Oceanographic Institution, Woods Hole,Massachusetts, USA; Felix J. Elling, Department of Earth and Planetary Sciences,Harvard University, Cambridge, Massachusetts, USA; Tim Urich, Institute ofMicrobiology, Ernst Moritz Arndt University Greifswald, Greifswald, Germany.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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mal vent Euryarchaeota group 2 (DHVEG-2) and the uncultivatedterrestrial miscellaneous Euryarchaeota group (TMEG), marinebenthic group D (MBG-D), and marine group II Euryarchaeota(MG-II) (Fig. 1) (7, 9). Especially the last two groups are widelydistributed in marine sediments and the surface ocean, respec-tively, but they lack cultured representatives (21, 22). Along withthe Methanomassiliicoccales, MBG-D and other benthic Euryar-chaeota are of particular interest in environmental microbiologyand geosciences, as they could be important contributors to mi-crobial biomass and activity in the sedimentary biosphere (23–25). In samples where MBG-D and MG-II dominated 16S rRNAgene libraries, glycerol dibiphytanyl glycerol tetraethers (GDGTs)have frequently been detected as major archaeal lipids, indicatingthat these archaeal groups may be able to synthesize these lipids(26–28). Moreover, intact GDGTs, e.g., GDGTs attached to gly-cosidic polar head groups, are commonly used for quantifyingarchaeal abundance in the subseafloor biosphere (24, 29–32). Un-derstanding the potential sources of GDGTs is of primary impor-tance for reliable quantification of benthic archaeal biomass usinglipid biomarkers (30, 31).

Here, we report the lipid composition of the sole isolated rep-resentative of the Methanomassiliicoccales, M. luminyensis strainB10T. The lipid analyses were facilitated by recently developedhigh-performance liquid chromatography (HPLC)–mass spec-trometry (MS) methods that allow the comprehensive, simulta-neous analysis of archaeal core and intact polar glycerol-basedmembrane lipids as well as respiratory quinones, i.e., membrane-bound electron carriers (33, 34). We show that M. luminyensisstrain B10T contains a diverse suite of unique tetraether lipids witheither butanetriol or pentanetriol replacing a glycerol backbonemoiety. Such lipids were recently found in marine and estuarinesediments but have not previously been detected in cultured ar-chaeal representatives (35–37). We further documented the dis-tribution of butanetriol-based lipids in diverse marine sediments,which suggested the widespread presence of relatives of M. lu-minyensis.

MATERIALS AND METHODSPhylogenetic analysis. High-quality 16S rRNA gene sequences of ar-chaeal groups of interest (alignment quality, �90; pintail, 100; sequencequality, �90) with a minimum length of 1,400 nucleotides (nt) were ob-tained from the SILVA RefNR SSU r123 database (38). If more than 10sequences per group were downloaded, the sequences were clustered with94.5% sequence identity using cd-hit-est of the CD-HIT suite (94) toobtain representative sequences of different genera (39). After aligning thesequences using the SINA online alignment tool (40), the alignment wasimproved by gap removal with Gblocks (95) using the least stringentparameters to avoid losing phylogenetic information (41). The alignmentwas uploaded to the Model Selection tool of the IQ-TREE web server toselect the best-suited nucleotide substitution model. A maximum-likeli-hood tree was calculated with IQ-TREE, applying the GTR model(�F�I�G4) (42). Ultrafast bootstrap (1,000 replicates) was used to verifybranch support (43). FigTree (http://tree.bio.ed.ac.uk/software/figtree/) andAdobe Illustrator (Adobe Systems Inc., San Jose, CA) were used for visu-alizing the phylogenetic tree.

Cultivation and lipid extraction. M. luminyensis was grown in ananaerobic medium based on the medium described by Lang et al. (20).Cultures (two, 20 ml), inoculated with 10% of a previous culture grownunder the same conditions, were grown in 120-ml serum flasks at 37°C for7 days under an atmosphere containing 80% H2 and 20% CO2. Cells wereharvested by centrifugation (20 min; 13,000 � g) and were subsequentlylyophilized.

Lipids from M. luminyensis were ultrasonically extracted following amodified Bligh-Dyer protocol (44) using a monophasic mixture of meth-anol, dichloromethane, and aqueous buffer (2:1:0.8, vol/vol/vol). A 50mM phosphate buffer (pH 7.4) was used for the first two extractions,while a 50 mM trichloroacetic acid buffer (pH 2) was used for two addi-tional extractions. The total lipid extracts (TLE) were dried under a streamof N2 and stored at �20°C until measurement. In addition to M. luminy-ensis, 12 marine sediment samples from a variety of depositional environ-ments (see Table 2) were analyzed and prepared as described by Liu et al.(30).

Intact polar and core lipid analysis. Intact polar and core lipids wereanalyzed by injecting TLE aliquots dissolved in methanol-dichlorometh-ane (9:1, vol/vol) on a Dionex Ultimate 3000 high-performance liquidchromatography (HPLC) system connected to a Bruker maXis ultrahigh-resolution quadrupole time of flight tandem mass spectrometer equippedwith an electrospray ion source operating in positive mode (Bruker Dal-tonik, Bremen, Germany). The mass spectrometer was set to a resolvingpower of 27,000 at m/z 1222, and every analysis was mass calibrated byloop injections of a calibration standard and correction by lock mass,leading to a mass accuracy of better than 1 to 3 ppm. Ion source and otherMS parameters were optimized by infusion of standards (acyclic GDGT

0.2

MBG A / Thaumarchaeota-

MBG D-

MG I / Thaumarchaeota-

ANME 1 / Methanomicrobia- les

MBG B / Lokiarchaeota-

DHVEG 2-

Crenarchaeota

MG II-

M IIIG-

Methanomassiliicoccales + TMEG

MCG / Bathyarchaeota

Thermoplasmatales

ANME 2 + 3 / Methanosarcinales-

?

?

?

?

?

ArchaeolUns-Archaeol

OH-ArchaeolExt-Archaeol

Acyclic GDGTCyclic GDGTCrenarchaeolOH-GDGT

GDDBDDBDGTPDGT

GTGTMe-GDGT

H-GDGT

?

M-Archaeol

Uns-GDGT

MeO-Archaeol

FIG 1 Phylogenetic tree of archaea, including methanogens and clades foundin marine sediments, and the major core lipids described for cultivated andenriched representatives. Lipid data for Methanomassiliicoccales are from thisstudy, those for other cultivated archaea are from references 26, 78, 45, 86, 87,88, and 89, and those for anaerobic methanotroph enrichments are from ref-erences 90 and 91. The maximum-likelihood tree is derived from nearly full-length 16S rRNA gene sequences. Bootstrap values (1,000 replicates) werecalculated to verify branch support (�, �95%; Œ, �80%). The scale barindicates substitutions per site. Abbreviations: MG-II, marine group II;DHVEG-2, deep-sea hydrothermal vent Euryarchaeota group 2; TMEG, ter-restrial miscellaneous Euryarchaeota group; MBG, marine benthic group; MG,marine group; ANME, anaerobic methanotroph; MCG, miscellaneous cren-archaeotal group; GDGT, glycerol dibiphytanyl glycerol tetraether; GTGT,glycerol trialkyl glycerol tetraether; GDD, glycerol dibiphytanyl diether; BDD,butanetriol dibiphytanyl diether; BDGT, butanetriol dibiphytanyl glycerol tet-raether; PDGT, pentanetriol dibiphytanyl glycerol tetraether; Uns, unsatu-rated; Ext, extended, OH, hydroxy; M, macrocyclic; MeO, methoxy; Me,methylated; H, H shaped.

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[GDGT-0], monoglycosidic [1G] GDGT-0, and diglycosidic [2G]GDGT-0) into the eluent flow from the LC system using a T-piece.

Analyte separation was achieved using reversed-phase HPLC (RP-HPLC) on an Acquity UPLC BEH C18 column (1.7 �m, 2.1 by 150 mm;Waters, Eschborn, Germany) maintained at 65°C as described by Wörmeret al. (33). The injection volumes was 10 �l, and analytes were eluted at aflow rate of 0.4 ml min�1 using linear gradients of methanol-water (85:15,vol/vol) (eluent A) to methanol-isopropanol (50:50, vol/vol) (eluent B),both with 0.04% formic acid and 0.1% NH3. The initial condition was

100% A held for 2 min, followed by a gradient to 15% B in 0.1 min and agradient to 85% B in 18 min. The column was then washed with 100% Bfor 8 min.

To determine the relative abundances of core lipids, 50% of the TLEwas hydrolyzed with 1 M HCl in methanol for 3 h at 70°C to yield corelipids (45). Additionally, biomass was hydrolyzed directly using 1 M HClin methanol for 16 h at 70°C; subsequently, lipids were ultrasonicallyextracted three times from hydrolyzed biomass using dichloromethane-methanol (DCM-MeOH) (5:1, vol/vol). The hydrolyzed TLE and the ex-

GDD-0

BDD-0

Archaeol

Extendedarchaeol

Diextendedarchaeol

Core lipids

Head groups

Phosphatidylglycerol(PG)

Monohexose (1G) n = 1Dihexose (2G) n = 2Trihexose (3G) n = 3

Methoxylated hexose(1MeOG)

Heptose(1Hp)

HO O

OH

P

OH

OO

OH

OOH

HO

HO

HO

O

O

OOH

HO

HO

CH3

O

OH

OOH

HO

HO

n

Polyprenols H

9OH H

10OH

GDGT-0

BDGT-0

PDGT-0

BDGT-1

BDGT-2

PDGT-1

PDGT-2

HOO

O OH

OH

O

O OH

OH

HOO

O

HOO

O

HOO

O

HOO

O O

O

OH

O

O

OHO

O

O

O O

O

OH

O

O

OH

O

O

O

OHO

OO

O

O

OHO

O

O

OHO

OO

[C H ]4 7HO

[C H ]4 7HO

[C H ]4 7HO

[C H ]5 9HO

[C H ]5 9HO

[C H ]5 9HO[C H ]4 7HO

FIG 2 Molecular structures of all identified intact polar and core lipids in Methanomassiliicoccus luminyensis. Lipids include glycerol diphytanyl diether(archaeol), glycerol sesterpanyl-phytanyl diether (extended archaeol), glycerol disesterpanyl diether (diextended archaeol), glycerol dibiphytanyl glycerol tetra-ether (GDGT), glycerol dibiphytanol diether (GDD), butanetriol dibiphytanyl glycerol tetraether (BDGT), butanetriol dibiphytanol diether (BDD), pentanetrioldibiphytanyl glycerol tetraether (PDGT) core lipids, and saturated and unsaturated C45 and C50 polyprenols with up to one double bond per isoprenoid unit.BDGT and PDGT core lipids with one and two cyclopentyl moieties are also shown. Intact polar lipids consist of di- and tetraether core lipids attached to a polarhead group.

Lipid Composition of Methanomassiliicoccus luminyensis

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tract obtained from hydrolyzed biomass were analyzed on the sameHPLC-MS system using normal-phase (NP) chromatography and an at-mospheric pressure chemical ionization-II ion source operated in positivemode, as described by Becker et al. (46). Briefly, hydrolyzed TLE aliquotswere dissolved in n-hexane–2-propanol (99.5:0.5, vol/vol) and injectedonto two coupled Acquity BEH amide columns (2.1 by 150 mm, 1.7-�mparticle size; Waters, Eschborn, Germany) maintained at 50°C. The injec-tion volume was 10 �l. Lipids were eluted using linear gradients of n-hexane (eluent A) to n-hexane–2-propanol (90:10, vol/vol) (eluent B) at aflow rate of 0.5 ml min�1. The initial gradient was 3% B to 5% B in 2 min,followed by increasing B to 10% in 8 min, to 20% in 10 min, to 50% in 15min, and to 100% in 10 min, followed by 6 min at 100% B to flush and 9min at 3% B to reequilibrate the columns.

Lipids were identified by retention time as well as accurate molecularmass and isotope pattern matching of proposed sum formulas in full-scanmode and tandem MS (MS2) fragment spectra. Integration of peaks wasperformed on extracted ion chromatograms using an isolation widthof �10 mDa and included the (M � H)� ions for NP-HPLC-MS andadditionally the (M � NH4)� and (M � Na)� ions for RP-HPLC-MS.Where applicable, doubly charged ions were included in the integration.

Lipid abundances were corrected for response factors of commercially

available as well as purified standards. Purified standards were obtainedfrom extracts of Archaeoglobus fulgidus as described by Elling et al. (45).The abundances of monoglycosidic (1G) glycerol dibiphytanyl glyceroltetraethers (GDGTs) and butanetriol dibiphytanyl glycerol tetraethers(BDGTs) were corrected for the response of a purified acyclic 1G-GDGTstandard, while monoheptose (1Hp) 1G-BDGT was corrected for the re-sponse of a purified acyclic 2G-GDGT standard due to the structuralsimilarity of the lipids (Fig. 1). The abundances of phosphatidylglycerol(PG), 1G-PG-BDGTs, and 1Hp-1G-PG-BDGT were corrected for the re-sponse of a commercially available 1G-PG-GDGT standard (MatreyaLLC, Pleasant Gap, PA, USA). The abundances of 1G- and 2G-archaeols(ARs) were corrected for the responses of the respective purified stan-dards, while triglycosidic (3G) ARs as well as methoxy-1G (1MeOG) 1Gand 1MeOG-2G-ARs were corrected for the response of 2G-AR. PG-ARabundances were corrected for the response of a commercial phosphati-dylethanolamine archaeol standard (Avanti Polar Lipids Inc., Alabaster,AL, USA). Due to the lack of appropriate standards, polyprenols were notcorrected for their relative response. The abundances of core GDGTs,BDGTs, pentanetriol dibiphytanyl glycerol tetraethers (PDGTs), glyceroldibiphytanol diethers (GDDs), and butanetriol dibiphytanol diethers(BDDs) were corrected for the response factors of purified GDGT-0, while

500

750

1000

1250

1500

1750

m/z

14 16 18 20 22 24

Retention time (min)

Inte

nsity

Core GDGT& BDGTCore GDD

& BDD

1G-GDGT& BDGT

1Hp-1G-PG-BDGT

PG-BDGT

1G-PG-BDGT

1Hp-1G-BDGT

PG-AR

AR

C -polyprenols50

C -polyprenols45

1G-Ext-AR

1G-diExt-AR1MeOG-1G-Ext-AR

2G-Ext-AR

1MeOG-1G-diExt-AR

2G-diExt-AR

1MeOG-2G-Ext-AR

3G-Ext-AR3G-diExt-AR

1MeOG-2G-diExt-AR

PG-AR

CoreGDGT

CoreBDGT

1G-GDGT1G-BDGT

1Hp-1G-BDGT

CoreBDD

CoreGDD

PG-BDGT

1G-PG-BDGT

1Hp-1G-PG-BDGT

AR

1MeOG-1G-Ext-AR

1MeOG-2G-Ext-AR

2G-Ext-AR

3G-Ext-AR

1MeoG-1G-diExt-AR

1MeoG-2G-diExt-AR

2G-diExt-AR

1G-diExt-AR1G-Ext-AR

3G-diExt-AR

Methanomassiliicoccus luminyensis

C -GTGT std.46

C -GTGT std.46

(a)

(b)

FIG 3 Reversed-phase HPLC-MS analyses of M. luminyensis TLE, showing an extracted ion chromatogram of all identified lipids (including the C46-GTGTinjection standard) (a) and a density map plot allowing a three-dimensional view on chromatographic separation and mass-to-charge ratio (m/z) with intensityon the z axis (from low intensities indicated by white color to intermediate intensities indicated by blue color and high intensities indicated by red color) (b). Lipidnomenclature designates combinations of core lipid types (AR, archaeol; Ext-AR, extended AR; diExt-AR, diextended AR; GDGT, glycerol dibiphytanyl glyceroltetraether; GDD, glycerol dialkanol diether; BDGT, butanetriol dibiphytanyl glycerol tetraether; BDD, butanetriol dibiphytanol diether) and head groups (PG,phosphatidylglycerol; 1G, monoglycosyl; 2G, diglycosyl; 3G, triglycosyl; PG, phosphatidyl glycerol; 1Hp-1G, monoheptose-1G; 1G-PG; 1Hp-1G-PG; 1MeOG-1G, methoxy-1G; 1MeOG-2G). For structures of lipids, see Fig. 2.

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the abundance of core AR was corrected for the response factor of therespective purified standard. The lower limit of detection for lipids was�1 pg �l�1.

RESULTSIntact polar and core lipid composition. Eighteen different intactpolar lipids (IPLs) with either di- or tetraether core structure andnine different polar head groups were detected in M. luminyensis.Head groups include mono-, di-, and trihexose, methoxy hexose,phosphatidylglycerol, monoheptose, and combinations of the dif-ferent head group types (Fig. 2 and 3). The detected IPLs compriseAR (two C20 isoprenoid side chains), GDGT-0, and extended(Ext) and diextended (diExt) AR, with the last two containingC20 –25 and C25–25 isoprenoidal chains, respectively, as core lipidstructures. Methoxy hexose- and heptose-containing lipids havebeen tentatively identified by multiple-stage mass spectrometry(Fig. 4; Table 1). Moreover, the dominant compounds were iden-tified as IPLs possessing a butanetriol dibiphytanyl glycerol tetra-ether (BDGT) core (Fig. 5; Table 1). These unusual tetraetherlipids are characterized by the replacement of one glycerol moietywith a butanetriol (36) and have not been found in any othercultured archaea to date. Free core lipids were relatively abundantand occurred as AR, GDGT, and BDGT, as well as GDD and BDD.Neither GDD nor BDD lipids were detected as IPLs (Fig. 3). Be-sides these IPLs and core lipids, we detected saturated and unsat-

urated C45 and C50 polyprenols, which contained up to one doublebond per isoprenoid unit. Methanophenazines and respiratoryquinones were not detected.

Di- and tetraether-based IPLs with glycosidic head groups ac-count for 49% of the total lipids, Total phosphate-based lipidscomprise 33%, while nonpolar free core lipids and polyprenolscontribute the remaining 18%. The most abundant single lipid inM. luminyensis is a PG-BDGT, contributing 20% to the total lipidpool (Table 1). Phosphatidylglycerol is the dominant single headgroup, representing 25% of total head groups (Fig. 6a), followedby 1G and 1Hp-1G, with 14% and 16%, respectively, while otherhead groups are equally distributed with 8 to 9%, except for 3G,which showed the lowest relative abundance (3%). The dominantcore structure in M. luminyensis in the total di- and tetraether lipidpool, including IPLs, is BDGT, accounting for more than 50%(Fig. 6b). The second most abundant core lipid is diExt-AR with30%, while all other core lipids comprise �10% of total corelipids.

Since not all lipids might be solvent extractable from cells (47,48), we acid hydrolyzed the biomass and compared the core lipiddistribution with that obtained from the TLE as well as that ob-tained from acid hydrolysis of the TLE (Fig. 6b). The relativeabundance of BDGTs was substantially higher (up to 82%) in theextracts obtained after acid hydrolysis of the TLE and direct hy-

200 400 600 800 1000 1200 1400 1600m/z

1800

Inte

nsity

1316.3336

[M+Na]⁺1692.4325

[M+NH ]₄ ⁺1687.4799

557.5993

575.6087

615.6447

633.6510

651.6579

669.6673 685.6798

703.6919721.7029

739.7211

757.7221

O

OH

OHHO

HO

HO

O

OH

OHHO

O1316

1Hp-1G-BDGT

373.3655

723.7555

[M+Na]⁺1083.8590

[M+NH₄]⁺1078.9049

Inte

nsity

O

O

OHHO

HO

CH3

O

OH

OHHO

O

OO

O

723

373

1MeOG-1G-Ext-AR

BDGT

FIG 4 MS2 spectra of ammoniated ([M � NH4]�) 1Hp-1G-BDGT (m/z 1687.5) and 1MeOG-1G-AR (m/z 1078.9). The chemical structures and the formationof major product ions are also drawn. The glycerol extension in the BDGT structure is located at either the sn-1 or sn-3 position of the glycerol. Both the 1MeOGand 1Hp head group structures have only been tentatively identified based on their exact mass in full scan and MS2 experiments, and their full characterizationrequires further structural elucidation. However, for example, for the peak at m/z 1078.9 ([M � NH4]�), we observed a dominant fragment ion associated withcore Ext-AR (61) in the MS2 spectrum, resulting from a neutral loss of 1G plus 176.1 Da plus NH3 and likely indicating a methylated dihexose head group (92).We interpreted the spectrum to represent a 1MeO-1G-Ext-AR. Similarly, we observed a loss of 2G plus CH2O plus NH3 (354.1 Da) and BDGT core lipid fragmentions (36, 37) for the peak at m/z 1687.3 ([M � NH4]�) and tentatively identified this IPL as heptose-containing lipid, 1Hp-1G-BDGT. The polar head group islocated at either the glycerol or butanetriol moiety.

Lipid Composition of Methanomassiliicoccus luminyensis

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drolysis of the biomass than in that from the TLE (Fig. 6b). Sim-ilarly, the relative abundance of GDGTs increased to almost 20%.Consequently, several lipids showed strongly reduced abundancesin the hydrolyzed extracts or were no longer detectable as in case ofthe diether lipids Ext- and diExt-AR. While AR showed a relativeabundance in the hydrolyzed TLE similar to that found by directanalysis of the TLE, its abundance was particularly low in thehydrolyzed biomass extract. As shown by Huguet et al. (48), con-centrations of GDGTs were substantially higher (one to two or-ders of magnitude) in directly hydrolyzed biomass than in thatfrom regular lipid extraction protocols for Nitrosopumilus mariti-mus biomass. Thus, although we did not generate quantitativeinformation, the BDGT and GDGT concentrations in the hydro-lyzed biomass might be so high that they overwhelm the signal ofthe diether compounds during mass spectrometry. Interestingly,in the hydrolyzed extracts, acyclic to dicyclic PDGT and mono-and bicyclic BDGTs were detected; both compound groups werenot detectable in the TLE.

Occurrence of BDGTs in the marine environment. To furtherexamine the environmental significance of the unusual BDGTs,we investigated their distribution in 12 marine sediments fromdiverse settings (Table 2). BDGTs were detected in two-thirds ofthe samples, including the Peru Margin, Hydrate Ridge, Mediter-ranean sapropels, Cascadia Margin, and Namibia Margin. In thesesamples, BDGT core lipids accounted for 0.1 and 3.5% of archaeal

core tetraethers (GDGTs plus BDGTs) (Table 2). BDGTs were notdetected in sediments from the equatorial Pacific, Namibia Mar-gin surface sediment, and deep subsurface of the Cascadia Margin.

DISCUSSIONLipid inventory of M. luminyensis compared to other archaea.The most distinctive characteristics of the membrane lipid com-position of M. luminyensis are BDGTs and PDGTs, which arepresent as IPLs and free core lipids and have not been reportedfrom other archaea. Thus, these unusual lipids may be diagnosticfor members or close relatives of the Methanomassiliicoccales.Both BDGT and PDGT lipids seem to be selectively bound in theacid-hydrolyzable fraction, as indicated by higher relative abun-dances in the hydrolyzed biomass than in the Bligh-Dyer extract(Fig. 6). Potentially, these lipids are preferentially bound toproteins in the membranes and were released by the acid treat-ment. In bacteria as well as in archaea, membrane proteins havebeen shown to selectively bind lipids, such as fatty acids, iso-prenoids, and different phospho- and glycolipids, influencingthe structural and functional integrity of proteins (see, e.g.,references 49, 50, 51, and 52).

However, the biological function and the biosynthetic pathwayof BDGTs and PDGTs remain unknown. Since the biosynthesis ofarchaeal membrane lipids typically involves dihydroxyacetonephosphate (DHAP) as an intermediate that is converted to glyc-

TABLE 1 IPLs and core lipids in the TLE of M. luminyensisa

Compoundb

m/z ([M � H]�; [M � NH4]�;[M � Na]�)c

Characteristic fragmentions in MS2

Retentiontime (min)

Relative lipidabundance (%)

GDD 1246.2965; 1263.3230; 1268.2784 669.7 23.0 0.3BDD 1260.3121; 1277.3387; 1282.2940 683.7 23.2 0.3GDGT 1302.3227; 1319.3492; 1324.3046 743.7 24.5 1.3BDGT 1316.3383; 1333.3649; 1338.3203 757.7 24.8 2.11G-GDGT 1464.3755; 1481.4020; 1486.3574 1302.3; 743.7 23.5 3.01G-BDGT 1478.3911; 1495.4177; 1500.3731 1316.3; 757.7 23.6 6.7PG-BDGT 1470.3414; 1487.3680; 1492.3234 —d 22.8 19.01G-PG-BDGT 1632.3943; 1649.4208; 1654.3762 1470.3 22.2 6.81Hp-1G-BDGT 1670.4545; 1687.4811; 1692.4365 1316.3 23.1 13.11Hp-1G-PG-BDGT 1824.4576; 1841.4842; 1846.4396 NAe 21.6 6.2AR 653.6806; 670.7072; 675.6626 373.4 19.0 0.8PG-AR 807.6837; 824.7103; 829.6657 733.6; 537.4 14.8 0.81G-Ext-AR 885.8117; 902.8382; 907.7936 373.4; 443.5; 723.8 19.3 1.11G-diExt-AR 955.8899; 972.9165; 977.8719 443.5; 793.8 21.1 0.41MeOG-1G-Ext-AR 1061.8802; 1078.9067; 1083.8621 373.4; 443.5; 723.8 19.0 5.52G-Ext-AR 1047.8645; 1064.8911; 1069.8465 373.4; 443.5; 723.8 18.4 6.11MeOG-1G-diExt-AR 1131.9584; 1148.9850; 1153.9404 443.5; 793.8 20.8 1.32G-diExt-AR 1117.9428; 1134.9693; 1139.9247 443.5; 793.8 20.3 1.71MeOG-2G-Ext-AR 1223.9330; 1240.9595; 1245.9149 373.4; 443.5; 723.8 18.6 4.93G-Ext-AR (a) 1209.9173; 1226.9439; 1231.8993 373.4; 443.5; 723.8 17.5 0.63G-Ext-AR (b) 1209.9173; 1226.9439; 1231.8993 373.4; 443.5; 723.8 18.0 1.41MeOG-2G-diExt-AR 1294.0112; 1311.0378; 1315.9932 443.5; 793.8 20.5 1.63G-diExt-AR (a) 1279.9956; 1297.0221; 1301.9775 443.5; 793.8 19.5 0.33G-diExt-AR (b) 1279.9956; 1297.0221; 1301.9775 443.5; 793.8 20.0 0.5C50:1–C50:10 polyprenols 699.6–717.8; 716.7–734.8; 721.6–739.8 Loss of H2O (�18.0 Da) 18.1–22.6 8.5C45:0–C45:9 polyprenols 631.6–649.7; 648.6–666.7; 653.6–671.7 Loss of H2O (�18.0 Da) 16.4–21.7 5.6a For interpretations of mass spectra, see the text.b Abbreviations: AR, glycerol diphytanyl diether (archaeol); Ext-AR, glycerol sesterpanyl-phytanyl diether (extended archaeol); diExt-AR, glycerol disesterpanyl diether (diextendedarchaeol); GDGT, glycerol dibiphytanyl glycerol tetraether; GDD, glycerol dibiphytanol diether; BDGT, butanetriol dibiphytanyl glycerol tetraether; BDD, butanetriol dibiphytanoldiether; 1G, monoglycosyl; 2G, diglycosyl; 3G, triglycosyl; 1Hp, monoheptose; PG, phosphatidylglycerol; 1MeOG, methoxyglycosyl.c Molecular masses of adducts in positive-ion mode RP-HPLC-MS1.d —, fragmentation not fully resolved.e NA, no MS2 data available.

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erol-1-phosphate (G1P) (53), a different biosynthetic pathwaymight be required for butane- and pentanetriol-containing lipids(36). The genomes of Methanomassiliicoccales contain genes thatare well known to be involved in archaeal ether lipid biosynthesis,including genes encoding homologues of G1P dehydrogenase,3-O-geranylgeranyl-sn-glycerlyl-1-phosphate (GGGP), and dig-eranylgeranylglyceryl phosphate (DGGGP) synthases and fourenzymes responsible for the activation of the diglyceride, the ad-dition of polar head groups to the glycerol moiety, and the finalproduction of archaeol via the subsequent reduction of the unsat-urated isoprenoid chains (9, 20). Only one gene for GGGP syn-thase, and no second homologue to this gene, could be identifiedthat might encode a hypothetical enzyme catalyzing the formationof a GG-butanetriyl-P or GG-pentanetriyl-P intermediate from

butanetriol or pentanetriol, respectively, and geranylgeranyldiphosphate (GGPP). Nevertheless, future studies on Methano-massiliicoccus BDGT and PDGT biosynthesis might help to eluci-date their unresolved biochemistry. Moreover, although the iden-tification of BDGTs and PDGTs based on HPLC-MS as well asdegradation experiments by Zhu et al. (36) seems conclusive, theexact structures of these unusual lipids, e.g., the stereochemistry ofthe butane- and pentanetriol backbone, remain to be fully re-solved (e.g., by using nuclear magnetic resonance [NMR] spec-troscopy).

M. luminyensis further possesses a unique membrane lipidcomposition of mixed di- and tetraethers with glycosidic andphosphatidylglycerol head groups, which differs distinctly fromall other methanogens and archaea. While Ext-AR as major core

2524232221 26

1300

1500

1700

m/z

Retention time (min)

Inte

nsity

core BDGTcore GDGT

core BDDcore GDD

97.1013 1228.2883

1338.3229[M+Na]⁺core BDGT

1316.3998

111.1174 1228.2845

1316.3401

1500.3767[M+Na]⁺

200 400 600 800 1000 1200 1400m/z

1G-BDGT575.6126 669.6760

739.7182

Methanomassiliicoccus luminyensis

757.7257

757.7302

721.7072

703.6963

651.6641615.6440

633.6591

1G-BDGT

557.6020

-H O₂ -H O₂ -H O₂ -H O₂ -H O₂ -H O₂ -H O₂

-C₄H₈O₂-C₃H₆O₂

OO

OO

HO

O O

O OOH

1G-GDGT

loss of G1head group

[M+H]⁺

-C₄H₈O₂

575.6126

669.6752

739.7174557.6019615.6439

721.7077

703.6963

651.6655

633.6591

595.6354

1Hp-1G-BDGT1G-PG-BDGT

PG-BDGT

1Hp-1G-PG-BDGT

Inte

nsity

(a)

(b)

[C H ]4 7HO

O O

O OOH

757557

757 557

[C H ]4 7HO

OHO

HO

OH HO

OHO

HO

OHHO

O

+

FIG 5 (a) Magnified section of a density map plot in the tetraether area, showing the major diagnostic ions of butanetriol and the corresponding solelyglycerol-containing lipids in the TLE of M. luminyensis, analyzed by RP-HPLC-MS. (b) MS2 spectra of sodiated ([M � Na]�) core BDGT (m/z 1338.3) and1G-BDGT (m/z 1500.4). The BDGT spectra match those shown by Zhu et al. (36) and Knappy et al. (37). The glycerol extension in the BDGT structure is locatedat either the sn-1 or sn-3 position of the glycerol, and the polar head group of intact BDGTs is located at either the glycerol or butanetriol moiety.

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lipid in M. luminyensis is widespread in halophilic archaea (54–56), it is present in only trace amounts in other methanogens,including Methanosarcina barkeri (57) and Methanothermobacterthermautotrophicus (58). In environmental samples, the detectionof Ext-AR has been frequently associated with methane-oxidizingarchaea (59–61). The diExt-AR that we detected in M. luminyensishas so far been reported only in halophiles (62) and as the domi-nant lipid in the thermophile Aeropyrum pernix (63) but not inmethanogens. Moreover, heptose-based membrane lipids have not

been reported in Archaea, while heptose is a common constituent ofpolysaccharides in Bacteria (see, e.g., references 64, 65, and 66).

Phosphate-bound polyprenols are widespread in all domainsof life, and they function mainly as membrane-bound (poly)sac-charide carriers involved in cell wall assembly (67–71). We foundabundant phosphate-free polyprenols in M. luminyensis, whichhave previously been detected in the thermophilic methanogen M.thermautotrophicus grown under hydrogen limitation; it was sug-gested that they may play a role in membrane stabilization (72).

0 20 40 60 80 100Relative abundance (%)

Ext-AR diExt-ARAR Me-AR

GDD-0 GDGT-0 BDD-0 BDGT-0

PDGT-0,-1,-2

TLE

Hydrolyzed TLE

Hydrolyzed biomass

1G 2G 3G

PG 1G-PG 1Hp-1G1Hp-1G-PG

1MeO-1G

1MeO-2G

0 20 40 60 80 100Relative abundance (%)

TLE

(a) Head group composition

(b) Core lipid composition

BDGT-1 BDGT-2

FIG 6 (a) Relative abundances of different head groups in the TLE of M. luminyensis. (b) Relative abundances of core lipids in the TLE, acid-hydrolyzed TLE,and acid-hydrolyzed biomass of M. luminyensis. For the TLE, free and head group-bound core lipids were considered. For chemical structures and abbreviations,see Fig. 2.

TABLE 2 Percentage of core BDGTs relative to total isoprenoidal core tetraethers in selected sediment samples

Cruise Site and corea

Sedimentdepth (m)

Total organiccarbon (wt%) % BDGTsb

M76/1 (Namibia Margin) GeoB12806-2 0.1 8.9 NDGeoB12807-2 3.1 7.4 0.21

ODP201 (Peru Margin) 1229D 4H4 30.7 4.7 0.361229A 22H1 185.9 0.47 3.5

ODP201 (equatorial Pacific) 1226B 10H3 83.8 1.1 ND1226E 20H3 320 0.28 ND

ODP204 (Hydrate Ridge) 1250D 6H5 43.5 0.96 1.11250D 12H5 100.3 1.3 0.12

IODP311 (Cascadia Margin) 1237C 10H5 79.8 0.64 ND1237C 13C6 109.8 0.56 0.21

ODP 160 (Mediterranean Sapropel) 966C 5H02 40 5.7 0.81966C 7H04 65 7.4 0.34

a Detailed information on sampling sites has been published by Liu et al. (30, 93).b Calculated as BDGTs/(BDGTs � GDGTs) � 100. ND, not detected.

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Their high relative abundance (14% of total detected lipids [Table1]) in M. luminyensis implies an important role of free polyprenolsalso in this archaeon. However, their distribution among otherarchaea has not yet been studied, and the function of phosphate-free polyprenols in archaeal cells remains elusive.

The unusual membrane lipid composition of M. luminyensis isconsistent with its phylogenetically distant relationship to otherorders of methanogens (9). Additionally, in contrast to the highdiversity of respiratory quinones in related members of the Ther-moplasmatales (34, 73, 74), no quinones were detected in M. lu-minyensis. Similarly, no methanophenazines, respiratory quinoneanalogues found in Methanosarcinales (34, 75) and Methanosaeta(76) were detected in M. luminyensis. This finding supports stud-ies by Lang et al. (20) and Söllinger et al. (15), who suggested thatthe biochemistry of methanogenesis in Methanomassiliicoccalesmay be fundamentally different from that in other, methano-phenazine- and cytochrome-containing methylotrophic archaea.

We further validated the potential of BDGTs and PDGTs asbiomarkers for M. luminyensis by analyzing 25 cultured archaea thatwe recently analyzed for their respiratory quinone compositions (34).These species cover the phyla Euryarchaeota, Crenarchaeota, andThaumarchaeota and within the Euryarchaeota several methano-gens as well as the Methanomassiliicoccales-related thermoacido-phile Thermoplasma acidophilum. We did not detect BDGTs orPDGTs in any of these archaea. This indicates a high chemotaxo-nomic potential of these lipids for Methanomassiliicoccales, al-though we cannot exclude that other, uncultured archaeal lineagesalso synthesize these lipids. Thus, BDGT biosynthesis might rep-resent another evolutionarily distinct feature of Methanomassili-icoccales similar to the unique pathways for methanogenesis and en-ergy conservation (9, 20, 77). Specific membrane lipid adaptationwithin the Thermoplasmata is supported by the fact that, for exam-ple, Aciduliprofundum boonei, belonging to DSHVE-2, contains H-shaped GDGTs (78), while they have not been reported from Ther-moplasmatales species (79) and were not detected in M. luminyensis.Analyses of the lipid inventories of other cultured representatives ofthe seventh order of methanogens are, however, required to providedetailed information about the phylogenetic patterns for the biosyn-thesis of butane- and pentanetriol-based lipids.

Implications for environmental studies. Butane- and pentan-etriol-based tetraether lipids have recently been identified in anumber of environmental settings, such as deeply buried marine(36) and shallow estuarine (35) sediments. Meador et al. (35) sug-gested a potential association of BDGTs with the miscellaneouscrenarchaeotal group (MCG) due to the positive correlation ofBDGT lipids with the relative abundance of MCG 16S rRNA se-quences in microbial communities of estuarine sediments fromthe White Oak River, NC, USA (WOR). The authors further in-terpreted the notable 13C depletion of BDGTs as evidence forBDGT producers being either autotrophs or heterotrophs feedingon 13C-depleted substrates. While being consistent with the isoto-pic lines of evidence, our results suggest that the source of BDGTsin WOR sediments might instead be members or relatives of theMethanomassiliicoccales (Fig. 1). In fact, members of the “environ-mental clade” of Methanomassiliicoccales were previously assignedto TMEG (15). Although Methanomassiliicoccales were not specif-ically described in WOR sediments, which is likely due to the lowcoverage of commonly used primers for Methanomassiliicoccales,closely related clades such as MBG-D and TMEG were abundantin these samples (35, 80). Indeed, we evaluated commonly used

primers against Methanomassiliicoccales, including the one used tosequence archaea from WOR sediments (22, 80), using TestPrime1.0 and the SILVA SSU r126 RefNR database (81), and the cover-age was only between 0.5% and 56% for zero mismatches. Accord-ingly, the positive correlation of MCG with BDGTs in the WORsediments (35) may instead result from the cooccurrence of MCGand Methanomassiliicoccales and/or uncultivated Thermoplas-mata, such as TMEG and MBG-D, which was observed by opera-tional taxonomic unit network analysis in various marine sediments(82). However, in samples where MBG-D dominate 16S rRNA clonelibraries, both BDGTs and PDGTs were not detected, while GDGTswere the most abundant lipids (26). This suggests that members ofthe MBG-D inhabiting the Pakistan margin sediments are not a ma-jor source for BDGT and PDGT lipids and that BDGT synthesis islimited to a subgroup within the Thermoplasmata.

The analysis of 12 globally distributed marine sediments fromvarious environmental settings revealed the widespread occur-rence of BDGT core lipids (Table 2), implying a large environ-mental distribution of Methanomassiliicoccales and/or relativesthat may constitute additional sources. This supports recent met-agenomic studies, which showed that besides gut and rumen en-vironments (6, 13, 14), Methanomassiliicoccales also occur ubiq-uitously in marine and terrestrial anaerobic environments (10, 15,82, 83). Similarly to the case for WOR, 16S rRNA gene sequencesof Methanomassiliicoccales were not reported for the investigatedsites, likely due to a mismatch of commonly used primers againsthitherto-undetected clades (22), while other uncultured Thermo-plasmata were detected (84). In some samples, for instance in theequatorial Pacific, BDGTs were not detected, which is probablyrelated to low total organic carbon (TOC) concentrations (29)and sulfate-reducing conditions (85) at this site. The relativeabundance of BDGTs in the 12 samples analyzed in this study(Table 2) is much lower than that in the estuarine WOR sedi-ments, where BDGTs accounted for 15% of the total archaeal corelipid pool on average (35). While these data indicate that the con-ditions in the WOR sediments select for the BDGT producers, thefactors controlling the distribution of the seventh order of meth-anogens and how they compete with hydrogenotrophic andmethylotrophic methanogens, as they require both H2 and meth-anol, remain targets for future studies. If future studies confirmthe specificity of BDGTs as biomarkers for the seventh order ofmethanogens, their detection will enrich strategies for investigat-ing these aspects in environmental samples, cultivation experi-ments, and the gastrointestinal tracts of humans and ruminantanimals (see, e.g., references 14 and 17).

ACKNOWLEDGMENTS

We thank X.-L. Liu for providing sediment TLE and T. W. Evans forassistance with sample processing.

This study was funded by the European Research Council under theEuropean Union’s Seventh Framework Programme “Ideas” Specific Pro-gramme, ERC grant agreement no. 247153 (Advanced Grant DARCLIFE;principal investigator, K.-U.H.), and by the Deutsche Forschungsge-meinschaft through the Gottfried Wilhelm Leibniz Prize awarded toK.-U.H. (Hi 616-14-1) and instrument grant Inst 144/300-1 (LC-qToFsystem). A.S. and T.U. were financially supported by a UNI:DOCSfellowship and a short-term travel grant (KWA) from the University ofVienna (Austria).

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