Unusual Butane- and Pentanetriol-Based Tetraether Lipids in Methanomassiliicoccus luminyensis, a Representative of the Seventh Order of Methanogens

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 Euryarchaeota group 2 and marine group II Euryarchaeota but only distantly related to other methanogens. In this study, we investigated the membrane lipid composition of Methanomassiliicoccus luminyensis, the sole cultured representative of this seventh order. The lipid inventory of M. luminyensis comprises a unique assemblage of novel lipids as well as lipids otherwise typical for thermophilic, methanogenic, or halophilic archaea. For instance, glycerol sesterpanyl-phytanyl diether core lipids found mainly in halophilic archaea were detected, and so were compounds bearing either heptose or methoxylated glycosidic head groups, neither of which have been reported so far for other archaea. The absence of quinones or methanophenazines is consistent with a biochemistry of methanogenesis different from that of the methanophenazine-containing methylotrophic methanogens. The most distinctive characteristic of the membrane lipid composition of M. luminyensis, however, is the presence of tetraether lipids in which 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 marine sediment samples showed that BDGTs are widespread in anoxic layers, suggesting an environmental significance of Methanomassiliicoccales 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 in which glycerol serves as backbone for the hydrophobic alkyl chains. Recently, however, archaeal tetraether lipids with either butanetriol or pentanetriol as a backbone were identified in marine sediments and attributed to uncultured sediment-dwelling archaea. Here we show that the butanetriol-based dibiphytanyl tetraethers constitute the major lipids in Methanomassiliicoccus luminyensis, currently the only isolate of the novel seventh order of methanogens. Given the absence of these lipids in a large set of archaeal isolates, these compounds may be diagnostic for the Methanomassiliicoccales and/or closely related archaea.

FIG 1

Phylogenetic tree of archaea, including methanogens and clades found in marine sediments, and the major core lipids described for cultivated and enriched representatives. Lipid data for Methanomassiliicoccales are from this study, those for other cultivated archaea are from references , , , , , , and , and those for anaerobic methanotroph enrichments are from references and . The maximum-likelihood tree is derived from nearly full-length 16S rRNA gene sequences. Bootstrap values (1,000 replicates) were calculated to verify branch support (●, ≥95%; ○, >80%). The scale bar indicates substitutions per site. Abbreviations: MG-II, marine group II; DHVEG-2, deep-sea hydrothermal vent Euryarchaeota group 2; TMEG, terrestrial miscellaneous Euryarchaeota group; MBG, marine benthic group; MG, marine group; ANME, anaerobic methanotroph; MCG, miscellaneous crenarchaeotal group; GDGT, glycerol dibiphytanyl glycerol tetraether; GTGT, glycerol trialkyl glycerol tetraether; GDD, glycerol dibiphytanyl diether; BDD, butanetriol dibiphytanyl diether; BDGT, butanetriol dibiphytanyl glycerol tetraether; PDGT, pentanetriol dibiphytanyl glycerol tetraether; Uns, unsaturated; Ext, extended, OH, hydroxy; M, macrocyclic; MeO, methoxy; Me, methylated; H, H shaped.

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 tetraether (GDGT), glycerol dibiphytanol diether (GDD), butanetriol dibiphytanyl glycerol tetraether (BDGT), butanetriol dibiphytanol diether (BDD), pentanetriol dibiphytanyl 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 polar head group.

FIG 3

Reversed-phase HPLC-MS analyses of M. luminyensis TLE, showing an extracted ion chromatogram of all identified lipids (including the C₄₆-GTGT injection standard) (a) and a density map plot allowing a three-dimensional view on chromatographic separation and mass-to-charge ratio (m/z) with intensity on 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). Lipid nomenclature designates combinations of core lipid types (AR, archaeol; Ext-AR, extended AR; diExt-AR, diextended AR; GDGT, glycerol dibiphytanyl glycerol tetraether; 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.

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 formation of 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 1MeOG and 1Hp head group structures have only been tentatively identified based on their exact mass in full scan and MS² experiments, and their full characterization requires further structural elucidation. However, for example, for the peak at m/z 1078.9 ([M + NH₄]⁺), we observed a dominant fragment ion associated with core 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 fragment ions (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 is located at either the glycerol or butanetriol moiety.

FIG 5

(a) Magnified section of a density map plot in the tetraether area, showing the major diagnostic ions of butanetriol and the corresponding solely glycerol-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) and 1G-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 located at 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.

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.