Microbial sulfate reduction has governed Earth's biogeochemical sulfur cycle for at least 2.5 billion years. However, the enzymatic mechanisms behind this pathway are incompletely understood, ...particulary for the reduction of sulfite—a key intermediate in the pathway. This critical reaction is performed by DsrAB, a widespread enzyme also involved in other dissimilatory sulfur metabolisms. Using in vitro assays with an archaeal DsrAB, supported with genetic experiments in a bacterial system, we show that the product of sulfite reduction by DsrAB is a protein-based trisulfide, in which a sulfite-derived sulfur is bridging two conserved cysteines of DsrC. Physiological studies also reveal that sulfate reduction rates are determined by cellular levels of DsrC. Dissimilatory sulfate reduction couples the four-electron reduction of the DsrC trisulfide to energy conservation.
Biological nitrogen fixation is catalyzed by the enzyme nitrogenase. Two forms of this metalloenzyme, the vanadium (V)- and iron (Fe)-only nitrogenases, were recently found to reduce small amounts of ...carbon dioxide (CO
) into the potent greenhouse gas methane (CH
). Here, we report carbon (
C/
C) and hydrogen (
H/
H) stable isotopic compositions and fractionations of methane generated by V- and Fe-only nitrogenases in the metabolically versatile nitrogen fixer
The stable carbon isotope fractionation imparted by both forms of alternative nitrogenase are within the range observed for hydrogenotrophic methanogenesis (
α
= 1.051 ± 0.002 for V-nitrogenase and 1.055 ± 0.001 for Fe-only nitrogenase; values are means ± standard errors). In contrast, the hydrogen isotope fractionations (
α
= 2.071 ± 0.014 for V-nitrogenase and 2.078 ± 0.018 for Fe-only nitrogenase) are the largest of any known biogenic or geogenic pathway. The large
α
shows that the reaction pathway nitrogenases use to form methane strongly discriminates against
H, and that
α
distinguishes nitrogenase-derived methane from all other known biotic and abiotic sources. These findings on nitrogenase-derived methane will help constrain carbon and nitrogen flows in microbial communities and the role of the alternative nitrogenases in global biogeochemical cycles.
All forms of life require nitrogen for growth. Many different kinds of microbes living in diverse environments make inert nitrogen gas from the atmosphere bioavailable using a special enzyme, nitrogenase. Nitrogenase has a wide substrate range, and, in addition to producing bioavailable nitrogen, some forms of nitrogenase also produce small amounts of the greenhouse gas methane. This is different from other microbes that produce methane to generate energy. Until now, there was no good way to determine when microbes with nitrogenases are making methane in nature. Here, we present an isotopic fingerprint that allows scientists to distinguish methane from microbes making it for energy versus those making it as a by-product of nitrogen acquisition. With this new fingerprint, it will be possible to improve our understanding of the relationship between methane production and nitrogen acquisition in nature.
Many Archaea produce membrane‐spanning lipids that enable life in extreme environments. These isoprenoid glycerol dibiphytanyl glycerol tetraethers (GDGTs) may contain up to eight cyclopentyl and one ...cyclohexyl ring, where higher degrees of cyclization are associated with more acidic, hotter or energy‐limited conditions. Recently, the genes encoding GDGT ring synthases, grsAB, were identified in two Sulfolobaceae; however, the distribution and abundance of grs homologs across environments inhabited by these and related organisms remain a mystery. To address this, we examined the distribution of grs homologs in relation to environmental temperature and pH, from thermal springs across Earth, where sequences derive from metagenomes, metatranscriptomes, single‐cell and cultivar genomes. The abundance of grs homologs shows a strong negative correlation to pH, but a weak positive correlation to temperature. Archaeal genomes and metagenome‐assembled genomes (MAGs) that carry two or more grs copies are more abundant in low pH springs. We also find grs in 12 archaeal classes, with the most representatives in Thermoproteia, followed by MAGs of the uncultured Korarchaeia, Bathyarchaeia and Hadarchaeia, while several Nitrososphaeria encodes >3 copies. Our findings highlight the key role of grs‐catalysed lipid cyclization in archaeal diversification across hot and acidic environments.
Summary
Adaptation of lipid membrane composition is an important component of archaeal homeostatic response. Historically, the number of cyclopentyl and cyclohexyl rings in the glycerol dibiphytanyl ...glycerol tetraether (GDGT) Archaeal lipids has been linked to variation in environmental temperature. However, recent work with GDGT‐making archaea highlight the roles of other factors, such as pH or energy availability, in influencing the degree of GDGT cyclization. To better understand the role of multiple variables in a consistent experimental framework and organism, we cultivated the model Crenarchaeon Sulfolobus acidocaldarius DSM639 at different combinations of temperature, pH, oxygen flux, or agitation speed. We quantified responses in growth rate, biomass yield, and core lipid compositions, specifically the degree of core GDGT cyclization. The degree of GDGT cyclization correlated with growth rate under most conditions. The results suggest the degree of cyclization in archaeal lipids records a universal response to energy availability at the cellular level, both in thermoacidophiles, and in other recent findings in the mesoneutrophilic Thaumarchaea. Although we isolated the effects of key individual parameters, there remains a need for multi‐factor experiments (e.g., pH + temperature + redox) in order to more robustly establish a framework to better understand homeostatic membrane responses.
Archaea adjust the number of cyclopentane rings in their glycerol dibiphytanyl glycerol tetraether (GDGT) membrane lipids as a homeostatic response to environmental stressors such as temperature, pH, ...and energy availability shifts. However, archaeal expression patterns that correspond with changes in GDGT composition are less understood. Here we characterize the acid and cold stress responses of the thermoacidophilic crenarchaeon
Saccharolobus islandicus
REY15A using growth rates, core GDGT lipid profiles, transcriptomics and proteomics. We show that both stressors result in impaired growth, lower average GDGT cyclization, and differences in gene and protein expression. Transcription data revealed differential expression of the GDGT ring synthase
grsB
in response to both acid stress and cold stress. Although the GDGT ring synthase encoded by
grsB
forms highly cyclized GDGTs with ≥5 ring moieties,
S. islandicus grsB
upregulation under acidic pH conditions did not correspond with increased abundances of highly cyclized GDGTs. Our observations highlight the inability to predict GDGT changes from transcription data alone. Broader analysis of transcriptomic data revealed that
S. islandicus
differentially expresses many of the same transcripts in response to both acid and cold stress. These included upregulation of several biosynthetic pathways and downregulation of oxidative phosphorylation and motility. Transcript responses specific to either of the two stressors tested here included upregulation of genes related to proton pumping and molecular turnover in acid stress conditions and upregulation of transposases in cold stress conditions. Overall, our study provides a comprehensive understanding of the GDGT modifications and differential expression characteristic of the acid stress and cold stress responses in
S. islandicus
.
Summary
Microorganisms regulate the composition of their membranes in response to environmental cues. Many Archaea maintain the fluidity and permeability of their membranes by adjusting the number of ...cyclic moieties within the cores of their glycerol dibiphytanyl glycerol tetraether (GDGT) lipids. Cyclized GDGTs increase membrane packing and stability, which has been shown to help cells survive shifts in temperature and pH. However, the extent of this cyclization also varies with growth phase and electron acceptor or donor limitation. These observations indicate a relationship between energy metabolism and membrane composition. Here we show that the average degree of GDGT cyclization increases with doubling time in continuous cultures of the thermoacidophile Sulfolobus acidocaldarius (DSM 639). This is consistent with the behavior of a mesoneutrophile, Nitrosopumilus maritimus SCM1. Together, these results demonstrate that archaeal GDGT distributions can shift in response to electron donor flux and energy availability, independent of pH or temperature. Paleoenvironmental reconstructions based on GDGTs thus capture the energy available to microbes, which encompasses fluctuations in temperature and pH, as well as electron donor and acceptor availability. The ability of Archaea to adjust membrane composition and packing may be an important strategy that enables survival during episodes of energy stress.
Methane generated by microorganisms is most often depleted in the doubly substituted isotopologue 12 CH 2 D 2 relative to the stochastic reference distribution. To constrain the controls on depleted ...∆ 12 CH 2 D 2 values, we experimentally isolated the root cause with microorganisms that produce methane from methylphosphonate via the C-P lyase pathway. This mechanism of methane production preserves the three hydrogens from methylphosphonate and adds one hydrogen from water. When maintaining the same methylphosphonate source, but varying the D/H composition of growth medium water, we observed significant shifts in methane ∆ 12 CH 2 D 2 values, but little to no change in ∆ 13 CH 3 D values. We reproduced these observations with a model that considers only the combinatorial isotope effect. The variation in ∆ 12 CH 2 D 2 values of product methane resulted from the differences in D/H between reactants water and methylphosphonate. This work validates the hypothesis that combinatorial effects can strongly influence methane ∆ 12 CH 2 D 2 values, and must be considered for low temperature, abiotic or biotic systems where methane hydrogen is derived from multiple reservoirs.
The degree of cyclization, or ring index (RI), in archaeal glycerol dibiphytanyl glycerol tetraether (GDGT) lipids was long thought to reflect homeoviscous adaptation to temperature. However, more ...recent experiments show that other factors (e.g., pH, growth phase, and energy flux) can also affect membrane composition. The main objective of this study was to investigate the effect of carbon and energy metabolism on membrane cyclization. To do so, we cultivated
sp. DS80, a metabolically flexible and thermoacidophilic archaeon, on different electron donor, acceptor, and carbon source combinations (S
/Fe
/CO
, H
/Fe
/CO
, H
/S
/CO
, or H
/S
/glucose). We show that differences in energy and carbon metabolism can result in over a full unit of change in RI in the thermoacidophile
sp. DS80. The patterns in RI correlated with the normalized electron transfer rate between the electron donor and acceptor and did not always align with thermodynamic predictions of energy yield. In light of this, we discuss other factors that may affect the kinetics of cellular energy metabolism: electron transfer chain (ETC) efficiency, location of ETC reaction components (cytoplasmic
extracellular), and the physical state of electron donors and acceptors (gas
solid). Furthermore, the assimilation of a more reduced form of carbon during heterotrophy appears to decrease the demand for reducing equivalents during lipid biosynthesis, resulting in lower RI. Together, these results point to the fundamental role of the cellular energy state in dictating GDGT cyclization, with those cells experiencing greater energy limitation synthesizing more cyclized GDGTs.IMPORTANCESome archaea make unique membrane-spanning lipids with different numbers of five- or six-membered rings in the core structure, which modulate membrane fluidity and permeability. Changes in membrane core lipid composition reflect the fundamental adaptation strategies of archaea in response to stress, but multiple environmental and physiological factors may affect the needs for membrane fluidity and permeability. In this study, we tested how
sp. DS80 changed its core lipid composition when grown with different electron donor/acceptor pairs. We show that changes in energy and carbon metabolisms significantly affected the relative abundance of rings in the core lipids of DS80. These observations highlight the need to better constrain metabolic parameters, in addition to environmental factors, which may influence changes in membrane physiology in Archaea. Such consideration would be particularly important for studying archaeal lipids from habitats that experience frequent environmental fluctuations and/or where metabolically diverse archaea thrive.
The precise interpretation of environmental sulfur isotope records requires a quantitative understanding of the biochemical controls on sulfur isotope fractionation by the principle ...isotope-fractionating process within the S cycle, microbial sulfate reduction (MSR). Here we provide the only direct observation of the major ((34)S/(32)S) and minor ((33)S/(32)S, (36)S/(32)S) sulfur isotope fractionations imparted by a central enzyme in the energy metabolism of sulfate reducers, dissimilatory sulfite reductase (DsrAB). Results from in vitro sulfite reduction experiments allow us to calculate the in vitro DsrAB isotope effect in (34)S/(32)S (hereafter, Formula: see text) to be 15.3 ± 2‰, 2σ. The accompanying minor isotope effect in (33)S, described as Formula: see text, is calculated to be 0.5150 ± 0.0012, 2σ. These observations facilitate a rigorous evaluation of the isotopic fractionation associated with the dissimilatory MSR pathway, as well as of the environmental variables that govern the overall magnitude of fractionation by natural communities of sulfate reducers. The isotope effect induced by DsrAB upon sulfite reduction is a factor of 0.3-0.6 times prior indirect estimates, which have ranged from 25 to 53‰ in (34)εDsrAB. The minor isotope fractionation observed from DsrAB is consistent with a kinetic or equilibrium effect. Our in vitro constraints on the magnitude of Formula: see text is similar to the median value of experimental observations compiled from all known published work, where (34)ε r-p = 16.1‰ (r-p indicates reactant vs. product, n = 648). This value closely matches those of MSR operating at high sulfate reduction rates in both laboratory chemostat experiments (Formula: see text 17.3 ± 1.5‰, 2σ) and in modern marine sediments (Formula: see text 17.3 ± 3.8‰). Targeting the direct isotopic consequences of a specific enzymatic processes is a fundamental step toward a biochemical foundation for reinterpreting the biogeochemical and geobiological sulfur isotope records in modern and ancient environments.
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