Butanetriol and pentanetriol dialkyl glycerol tetraethers (BDGTs and PDGTs)
are membrane lipids, recently discovered in sedimentary environments and in
the methanogenic archaeon
Unique membrane lipids formed a key argument in the postulation of the
existence of Archaea as a third and independent domain of life, as distantly
related to Bacteria as to Eukarya, when Woese et al. (1990) proposed their
revised tree of life. Membrane lipids form an envelope that separates cells
from their environment and protects their interior components. Specific
chemical properties define the fluidity and permeability of the membrane
barrier, regulating what can enter the internal cell compartment. On the one
hand, membrane lipids from members of all domains of life share some common
characteristics, such as their amphiphilic nature. That is, they all possess
apolar alkyl chains and polar headgroups held together by a glycerol moiety
(Lombard et al., 2012). On the other hand, membrane lipids of Archaea
fundamentally differ from those of Bacteria and Eukarya in that they contain
(bi)phytanyl chains constituted from the condensation of several isoprenoid
units and ether linkages to the
The recent identification of butanetriol and pentanetriol dialkyl glycerol
tetraethers (BDGTs and PDGTs, Knappy et al., 2014; Zhu et al., 2014), in
which one glycerol is substituted by a butanetriol or pentanetriol,
challenges this assumption. Tandem mass spectrometry complemented with gas
chromatography (GC) detection of butanetriol after hydrolysis (Zhu et al.,
2014) demonstrated the presence of a 1,2,3 butanetriol backbone in BDGTs but
did not specify its configuration within the lipid molecule, notably its
linkages with the biphytanyl side chains. Subsequently, BDGTs and PDGTs were
observed in diverse samples, both as intact polar lipids (IPLs, lipids with
polar headgroups) and core lipids (CLs, lipids without polar headgroups),
from recent organic-rich estuarine sediments (Meador et al., 2015) to old
Jurassic marine shales (Knappy et al., 2014) and deep subsurface sediments
(Becker et al., 2016; Zhu et al., 2014). Furthermore, Becker et al. (2016)
identified BDGTs as prominent membrane lipids in
In the present study, the exact structure of the core BDGT molecule was
elucidated through high-field one- and two-dimensional nuclear magnetic resonance
(NMR) analysis of BDGT purified from a pure culture of
Marine sediment samples were collected with a combination of multi-corer and
gravity coring at eight different sites (Fig. 1) in the Mediterranean and
Black seas during two expeditions: RV
Sampling sites in the Black Sea and Mediterranean Sea. The map was generated with GMT software (Wessel et al., 2013).
Lipid extraction of sediment samples was performed according to a modified
Bligh and Dyer method (Sturt et al., 2004). Samples (ca. 50–60 g wet
weight) were lyophilized and ultrasonically extracted four times with a
mixture of dichloromethane (DCM)
Detection and quantification of intact polar lipids (IPLs) were carried out
on a maXis plus ultra-high-resolution quadrupole time-of-flight mass
spectrometer (Q-ToF-MS; Bruker), coupled to an Ultimate 3000RS
ultra-high-pressure liquid chromatography (UHPLC) instrument (Dionex). The
IPLs were chromatographically separated by an ACE3 C18 column (
Six of the forty-eight marine sediment TLEs from the Rhone Delta, the eastern Mediterranean Sea and the Black Sea (sample nos. 5, 8, 20, 22, 34, 38 of the data set; detailed information available in Table S1) were selected to investigate the natural stable isotopic composition of the BDGT-derived biphytanes. Only IPL-BDGTs were analyzed, as they are more likely to derive from living organisms. Before isotopic analysis via gas chromatography coupled to isotope ratio mass spectrometry (GC-IRMS), IPL-BDGTs were purified with two steps of preparative HPLC and then cleaved into biphytanes (bp), as detailed below.
The TLE samples were first separated into IPL and CL fractions by preparative
HPLC (Agilent 1200 series) with a modified version of the protocol reported
by Meador et al. (2015). TLE separation was performed on an LiChrospher Diol
column (
The CL-BDGTs were further separated from CL-GDGTs according to Zhu et
al. (2014). The hydrolyzed IPL fraction was injected into an Agilent 1200
normal-phase HPLC system equipped with a PerfectSil CN-3 column (
Ether cleavage with
In order to gain information on the C sources of BDGTs, the stable carbon
isotope analysis of total organic carbon (TOC), dissolved inorganic carbon
(DIC) and methane (
Direct acid hydrolysis of the freeze-dried biomass pellet was performed
according to Becker et al. (2016) using 1 M HCl in MeOH for 16 h at 70
BDGT-0 (860
Principal component analysis (PCA) was performed with the R software using FactoMineR and vegan packages. PCA requires all variables to follow a normal distribution; thus all data were reduced and centered before analysis.
Analysis of high-resolution
Continued.
Detailed structure of
However, a number of new
Geochemical parameters of the 48 analyzed samples were previously described
in Schmidt et al. (2017). Notably, TOC values ranged between 0.08 % and
4.37 % (Table S1). The highest TOC content was measured in the sapropel
layers of the eastern Mediterranean Basin (GeoB15103), while the basin sites,
i.e., eastern Mediterranean Basin (excluding the sapropel layers), Cap de
Creus Canyon and Ligurian–Provençal Basin (GeoB15103, GeoB17302,
GeoB17304), exhibited the lowest TOC contents. From the 48 samples, ranging
in depth from surface to 635 cm and ages from the modern to
Intact polar lipid concentration against total lipid concentration (log scales) for each archaeal lipid type discussed in this study (PDGTs in orange, BDGTs in red and GDGTs in blue). The dashed line represents the 1 : 1 line.
In order to evaluate the variability in BDGT and PDGT distribution within the
data set, a PCA was performed with the major environmental variables and
indices of BDGT and PDGT relative abundances. In addition to the fractional
abundance for each BDGT and PDGT pool [
Principal component analysis biplot showing relationships between major geochemical parameters (grey arrows), indices illustrating BDGT and PDGT distribution (red arrows), and 45 sediment samples (filled circles) from the Mediterranean and Black seas where BDGTs and PDGTs were detected.
Six samples were selected for analysis of the
Stable carbon isotopic composition (
In the Black Sea and Rhone Delta (GeoB15105 and GeoB17306), BDGT-derived
biphytanes had similarly low
A predominant methanotrophic origin for BDGTs is unlikely, as
Multivariate analysis of the marine sediment sample set (
Overall, our data infer that BDGT (and PDGT) producers may comprise an
autotrophic, potentially methanogenic, community as well as a heterotrophic,
likely not methanogenic, community. The methanogenic origin of BDGTs (and
PDGTs) is in agreement with their prominence in
Zhu et al. (2014) demonstrated the 1,2,3 butanetriol structure of the BDGT backbone via gas chromatography MS, following ether cleavage from its biphytanyl chains. In the present study, 2-D NMR analysis confirmed the presence of a butanetriol backbone and unequivocally determined its configuration in the tetraether molecule (Fig. 2). Additional methylations have been previously observed on the isoprenoid chains or as methoxylation on the glycerol in different lipid classes (e.g., Elling et al., 2017; Knappy et al., 2015). However, BDGTs (and PDGTs) stand out as unique archaeal membrane lipids that contain a non-glycerol moiety. This raises two fundamental questions: (i) how are these lipids biosynthesized? (ii) Why do microorganisms produce them?
For every domain of life, it is known that dihydroxyacetone phosphate (DHAP),
an intermediate in glycolysis, serves as a precursor of the glycerol moiety
in membrane lipid backbones (Koga and Morii, 2007). At the early stage of
membrane lipid biosynthesis, DHAP is converted by stereospecific glycerol
dehydrogenase enzymes into either a glycerol-3-phosphate (G-3-P) or a
glycerol-1-phosphate (G-1-P) in Bacteria and Archaea, respectively. The
existence of BDGTs (and PDGTs) implies that different precursors must be
involved at the very first steps of lipid biosynthesis. Knappy et al. (2014)
suggested, for example, the involvement of putative butanetriol or
pentanetriol phosphate. However, in the genomes of Methanomassiliicoccales,
only one gene for 3-O-geranylgeranyl-sn-glyceryl-1-phosphate (GGGP) synthase
was identified (Becker et al., 2016), and no second homologue that might
encode a hypothetical enzyme catalyzing the formation of butanetriol- or
pentanetriol-based intermediates. Alternatively, BDGTs and PDGTs might be
regular GDGTs that underwent additional methylation at the final stages of
their biosynthesis. Welander et al. (2010) showed that an
S-adenosylmethionine (SAM) enzyme catalyzing a radical reaction was
responsible for the methylation of certain bacterial hopanoids at the C2
position. We found 13 genes annotated as belonging to the radical SAM
superfamily in the permanent draft genome of
The ubiquitous presence of BDGTs and PDGTs in the environment and their
active biosynthesis by certain organisms, notably
The unique structure of BDGTs, here unambiguously elucidated by NMR
experiments, further increases the diversity of membrane lipids observed in
Archaea. BDGTs and PDGTs were detected in a large set of marine sediment
samples from diverse geochemical, depth and age conditions, highlighting
their widespread presence in marine sediments. Within the data set, major
differences are also observed in the BDGT and PDGT headgroup distribution
patterns and
Data will be made available in PANGAEA under
Samples are stored at MARUM – Center for Marine Environmental Sciences, University of Bremen, Germany. Sample aliquots may be requested from Kai-Uwe Hinrichs.
The supplement related to this article is available online at:
SC, KUH, JSL and VBH designed the study; SC, TBM, KWB and JS performed laboratory work and lipid quantification; LM and QZZ performed the isotope analysis; MPC performed the NMR-based structural elucidation; VBH selected sites and led sample collection and curation; SC performed the statistical analysis, interpreted the results and wrote the paper with significant input from TBM and KUH; all co-authors commented on the manuscript.
The authors declare that they have no conflict of interest.
The authors acknowledge the participants and the crew members of the two
DARCSEAS cruises: RV
This research has been supported by the European Research Council (DARCLIFE (grant no. 247153)) and the Deutsche Forschungsgemeinschaft (grant no. HI 616-14-1). Sarah Coffinet was financially supported by the Institutional Strategy of the University of Bremen, funded by the German Excellence Initiative.The article processing charges for this open-access publication were covered by the University of Bremen.
This paper was edited by Marcel van der Meer and reviewed by Darci Rush and one anonymous referee.