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  1. Abstract

    Eukaryotes produce highly modified sterols, including cholesterol, essential to eukaryotic physiology. Although few bacterial species are known to produce sterols, de novo production of cholesterol or other complex sterols in bacteria has not been reported. Here, we show that the marine myxobacteriumEnhygromyxa salinaproduces cholesterol and provide evidence for further downstream modifications. Through bioinformatic analysis we identify a putative cholesterol biosynthesis pathway inE. salinalargely homologous to the eukaryotic pathway. However, experimental evidence indicates that complete demethylation at C-4 occurs through unique bacterial proteins, distinguishing bacterial and eukaryotic cholesterol biosynthesis. Additionally, proteins from the cyanobacteriumCalothrixsp. NIES-4105 are also capable of fully demethylating sterols at the C-4 position, suggesting complex sterol biosynthesis may be found in other bacterial phyla. Our results reveal an unappreciated complexity in bacterial sterol production that rivals eukaryotes and highlight the complicated evolutionary relationship between sterol biosynthesis in the bacterial and eukaryotic domains.

     
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  2. Abstract

    Sterane molecular fossils are broadly interpreted as eukaryotic biomarkers, although diverse bacteria also produce sterols. Steranes with side-chain methylations can act as more specific biomarkers if their sterol precursors are limited to particular eukaryotes and are absent in bacteria. One such sterane, 24-isopropylcholestane, has been attributed to demosponges and potentially represents the earliest evidence for animals on Earth, but enzymes that methylate sterols to give the 24-isopropyl side-chain remain undiscovered. Here, we show that sterol methyltransferases from both sponges and yet-uncultured bacteria function in vitro and identify three methyltransferases from symbiotic bacteria each capable of sequential methylations resulting in the 24-isopropyl sterol side-chain. We demonstrate that bacteria have the genomic capacity to synthesize side-chain alkylated sterols, and that bacterial symbionts may contribute to 24-isopropyl sterol biosynthesis in demosponges. Together, our results suggest bacteria should not be dismissed as potential contributing sources of side-chain alkylated sterane biomarkers in the rock record.

     
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  3. Abstract Glycerol dibiphytanyl glycerol tetraethers (GDGTs) are archaeal monolayer membrane lipids that can provide a competitive advantage in extreme environments. Here, we identify a radical SAM protein, tetraether synthase (Tes), that participates in the synthesis of GDGTs. Attempts to generate a tes-deleted mutant in Sulfolobus acidocaldarius were unsuccessful, suggesting that the gene is essential in this organism. Heterologous expression of tes homologues leads to production of GDGT and structurally related lipids in the methanogen Methanococcus maripaludis (which otherwise does not synthesize GDGTs and lacks a tes homolog, but produces a putative GDGT precursor, archaeol). Tes homologues are encoded in the genomes of many archaea, as well as in some bacteria, in which they might be involved in the synthesis of bacterial branched glycerol dialkyl glycerol tetraethers. 
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  4. Abstract

    The sole unifying feature of the incredibly diverse Archaea is their isoprenoid‐based ether‐linked lipid membranes. Unique lipid membrane composition, including an abundance of membrane‐spanning tetraether lipids, impart resistance to extreme conditions. Many questions remain, however, regarding the synthesis and modification of tetraether lipids and how dynamic changes to archaeal lipid membrane composition support hyperthermophily. Tetraether membranes, termed glycerol dibiphytanyl glycerol tetraethers (GDGTs), are generated by tetraether synthase (Tes) by joining the tails of two bilayer lipids known as archaeol. GDGTs are often further specialized through the addition of cyclopentane rings by GDGT ring synthase (Grs). A positive correlation between relative GDGT abundance and entry into stationary phase growth has been observed, but the physiological impact of inhibiting GDGT synthesis has not previously been reported. Here, we demonstrate that the model hyperthermophileThermococcus kodakarensisremains viable when Tes (TK2145) or Grs (TK0167) are deleted, permitting phenotypic and lipid analyses at different temperatures. The absence of cyclopentane rings in GDGTs does not impact growth inT. kodakarensis, but an overabundance of rings due to ectopic Grs expression is highly fitness negative at supra‐optimal temperatures. In contrast, deletion of Tes resulted in the loss of all GDGTs, cyclization of archaeol, and loss of viability upon transition to the stationary phase in this model archaea. These results demonstrate the critical roles of highly specialized, dynamic, isoprenoid‐based lipid membranes for archaeal survival at high temperatures.

     
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  5. Glycerol dibiphytanyl glycerol tetraethers (GDGTs) are distinctive archaeal membrane-spanning lipids with up to eight cyclopentane rings and/or one cyclohexane ring. The number of rings added to the GDGT core structure can vary as a function of environmental conditions, such as changes in growth temperature. This physiological response enables cyclic GDGTs preserved in sediments to be employed as proxies for reconstructing past global and regional temperatures and to provide fundamental insights into ancient climate variability. Yet, confidence in GDGT-based paleotemperature proxies is hindered by uncertainty concerning the archaeal communities contributing to GDGT pools in modern environments and ambiguity in the environmental and physiological factors that affect GDGT cyclization in extant archaea. To properly constrain these uncertainties, a comprehensive understanding of GDGT biosynthesis is required. Here, we identify 2 GDGT ring synthases, GrsA and GrsB, essential for GDGT ring formation in Sulfolobus acidocaldarius . Both proteins are radical S-adenosylmethionine proteins, indicating that GDGT cyclization occurs through a free radical mechanism. In addition, we demonstrate that GrsA introduces rings specifically at the C-7 position of the core GDGT lipid, while GrsB cyclizes at the C-3 position, suggesting that cyclization patterns are differentially controlled by 2 separate enzymes and potentially influenced by distinct environmental factors. Finally, phylogenetic analyses of the Grs proteins reveal that marine Thaumarchaeota, and not Euryarchaeota, are the dominant source of cyclized GDGTs in open ocean settings, addressing a major source of uncertainty in GDGT-based paleotemperature proxy applications. 
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  6. Abstract

    Archaeal glycerol dibiphytanyl glycerol tetraethers (GDGT) are some of the most unusual membrane lipids identified in nature. These amphiphiles are the major constituents of the membranes of numerous Archaea, some of which are extremophilic organisms. Due to their unique structures, there has been significant interest in studying both the biophysical properties and the biosynthesis of these molecules. However, these studies have thus far been hampered by limited access to chemically pure samples. Herein, we report a concise and stereoselective synthesis of the archaeal tetraether lipid parallel GDGT‐0 and the synthesis and self‐assembly of derivatives bearing different polar groups.

     
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  7. Abstract

    Archaeal glycerol dibiphytanyl glycerol tetraethers (GDGT) are some of the most unusual membrane lipids identified in nature. These amphiphiles are the major constituents of the membranes of numerous Archaea, some of which are extremophilic organisms. Due to their unique structures, there has been significant interest in studying both the biophysical properties and the biosynthesis of these molecules. However, these studies have thus far been hampered by limited access to chemically pure samples. Herein, we report a concise and stereoselective synthesis of the archaeal tetraether lipid parallel GDGT‐0 and the synthesis and self‐assembly of derivatives bearing different polar groups.

     
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  8. Abstract

    Differentiating the contributions of photosynthesis and respiration to the global carbon cycle is critical for improving predictive climate models. Carbonic anhydrase (CA) activity in leaves is responsible for the largest biosphere-atmosphere trace gas fluxes of carbonyl sulfide (COS) and the oxygen-18 isotopologue of carbon dioxide (CO18O) that both reflect gross photosynthetic rates. However, CA activity also occurs in soils and will be a source of uncertainty in the use of COS and CO18O as carbon cycle tracers until process-based constraints are improved. In this study, we measured COS and CO18O exchange rates and estimated the corresponding CA activity in soils from a range of biomes and land use types. Soil CA activity was not uniform for COS and CO2, and patterns of divergence were related to microbial community composition and CA gene expression patterns. In some cases, the same microbial taxa and CA classes catalyzed both COS and CO2 reactions in soil, but in other cases the specificity towards the two substrates differed markedly. CA activity for COS was related to fungal taxa and β-D-CA expression, whereas CA activity for CO2 was related to algal and bacterial taxa and α-CA expression. This study integrates gas exchange measurements, enzyme activity models, and characterization of soil taxonomic and genetic diversity to build connections between CA activity and the soil microbiome. Importantly, our results identify kinetic parameters to represent soil CA activity during application of COS and CO18O as carbon cycle tracers.

     
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