Paula V. Welander

All Publications

• Identification of two archaeal GDGT lipid-modifying proteins reveals diverse microbes capable of GMGT biosynthesis and modification. Proceedings of the National Academy of Sciences of the United States of America Garcia, A. A., Chadwick, G. L., Liu, X. L., Welander, P. V. 2024; 121 (26): e2318761121

  Abstract

  Archaea produce unique membrane-spanning lipids (MSLs), termed glycerol dialkyl glycerol tetraethers (GDGTs), which aid in adaptive responses to various environmental challenges. GDGTs can be modified through cyclization, cross-linking, methylation, hydroxylation, and desaturation, resulting in structurally distinct GDGT lipids. Here, we report the identification of radical SAM proteins responsible for two of these modifications-a glycerol monoalkyl glycerol tetraether (GMGT) synthase (Gms), responsible for covalently cross-linking the two hydrocarbon tails of a GDGT to produce GMGTs, and a GMGT methylase (Gmm), capable of methylating the core hydrocarbon tail. Heterologous expression of Gms proteins from various archaea in Thermococcus kodakarensis results in the production of GMGTs in two isomeric forms. Further, coexpression of Gms and Gmm produces mono- and dimethylated GMGTs and minor amounts of trimethylated GMGTs with only trace GDGT methylation. Phylogenetic analyses reveal the presence of Gms homologs in diverse archaeal genomes spanning all four archaeal superphyla and in multiple bacterial phyla with the genetic potential to synthesize fatty acid-based MSLs, demonstrating that GMGT production may be more widespread than previously appreciated. We demonstrate GMGT production in three Gms-encoding archaea, identifying an increase in GMGTs in response to elevated temperature in two Archaeoglobus species and the production of GMGTs with up to six rings in Vulcanisaeta distributa. The occurrence of such highly cyclized GMGTs has been limited to environmental samples and their detection in culture demonstrates the utility of combining genetic, bioinformatic, and lipid analyses to identify producers of distinct archaeal membrane lipids.

  View details for DOI 10.1073/pnas.2318761121

  View details for PubMedID 38885389

• Editorial: Emergence of dynamic membranes - role of terpenoids in the evolution of membrane organization and regulation FRONTIERS IN ECOLOGY AND EVOLUTION Nettersheim, B. J., Hoshino, Y., Welander, P. V. 2024; 12

  View details for DOI 10.3389/fevo.2024.1420158

  View details for Web of Science ID 001248987400001

• Tetraether archaeal lipids promote long-term survival in extreme conditions. Molecular microbiology Liman, G. L., Garcia, A. A., Fluke, K. A., Anderson, H. R., Davidson, S. C., Welander, P. V., Santangelo, T. J. 2024

  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 hyperthermophile Thermococcus kodakarensis remains 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 in T. 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.

  View details for DOI 10.1111/mmi.15240

  View details for PubMedID 38372181

• Novel sterol binding domains in bacteria. eLife Zhai, L., Bonds, A. C., Smith, C. A., Oo, H., Chou, J. C., Welander, P. V., Dassama, L. M. 2024; 12

  Abstract

  Sterol lipids are widely present in eukaryotes and play essential roles in signaling and modulating membrane fluidity. Although rare, some bacteria also produce sterols, but their function in bacteria is not known. Moreover, many more species, including pathogens and commensal microbes, acquire or modify sterols from eukaryotic hosts through poorly understood molecular mechanisms. The aerobic methanotroph Methylococcus capsulatus was the first bacterium shown to synthesize sterols, producing a mixture of C-4 methylated sterols that are distinct from those observed in eukaryotes. C-4 methylated sterols are synthesized in the cytosol and localized to the outer membrane, suggesting that a bacterial sterol transport machinery exists. Until now, the identity of such machinery remained a mystery. In this study, we identified three novel proteins that may be the first examples of transporters for bacterial sterol lipids. The proteins, which all belong to well-studied families of bacterial metabolite transporters, are predicted to reside in the inner membrane, periplasm, and outer membrane of M. capsulatus, and may work as a conduit to move modified sterols to the outer membrane. Quantitative analysis of ligand binding revealed their remarkable specificity for 4-methylsterols, and crystallographic structures coupled with docking and molecular dynamics simulations revealed the structural bases for substrate binding by two of the putative transporters. Their striking structural divergence from eukaryotic sterol transporters signals that they form a distinct sterol transport system within the bacterial domain. Finally, bioinformatics revealed the widespread presence of similar transporters in bacterial genomes, including in some pathogens that use host sterol lipids to construct their cell envelopes. The unique folds of these bacterial sterol binding proteins should now guide the discovery of other proteins that handle this essential metabolite.

  View details for DOI 10.7554/eLife.90696

  View details for PubMedID 38329015

• De novo cholesterol biosynthesis in bacteria. Nature communications Lee, A. K., Wei, J. H., Welander, P. V. 2023; 14 (1): 2904

  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 myxobacterium Enhygromyxa salina produces cholesterol and provide evidence for further downstream modifications. Through bioinformatic analysis we identify a putative cholesterol biosynthesis pathway in E. salina largely 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 cyanobacterium Calothrix sp. 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.

  View details for DOI 10.1038/s41467-023-38638-8

  View details for PubMedID 37217541

• Sterol methyltransferases in uncultured bacteria complicate eukaryotic biomarker interpretations. Nature communications Brown, M. O., Olagunju, B. O., Giner, J. L., Welander, P. V. 2023; 14 (1): 1859

  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.

  View details for DOI 10.1038/s41467-023-37552-3

  View details for PubMedID 37012227

  View details for PubMedCentralID PMC10070321

• Reply to Oren et al., "New Phylum Names Harmonize Prokaryotic Nomenclature". mBio Sharma, G., Islam, S. T., Rahi, P., Silby, M. W., Giovannelli, D., Welander, P. V., Ehling-Schulz, M., Chaudhry, V., Molloy, E., Hertweck, C., Mundra, S., Kalia, V. C., Lal, R., Singh, Y., Ruby, E., Weigel, C., Kolter, R. 2022: e0232322

  View details for DOI 10.1128/mbio.02323-22

  View details for PubMedID 36106733

• Genomic Features of the Bundle-Forming Heliobacterium Heliophilum fasciatum. Microorganisms Bender, K. S., Madigan, M. T., Williamson, K. L., Mayer, M. H., Parenteau, M. N., Jahnke, L. L., Welander, P. V., Sanguedolce, S. A., Brown, A. C., Sattley, W. M. 2022; 10 (5)

  Abstract

  Eight species of heliobacteria have had their genomes sequenced. However, only two of these genomes have been analyzed in detail, those from the thermophilic Heliomicrobium (Hmi.) modesticaldum and the alkaliphilic Heliorestis (Hrs.) convoluta. Here we present analyses of the draft genome sequence of a species of heliobacterium that grows optimally at a moderate temperature and neutral pH. The organism, Heliophilum (Hph.) fasciatum, is phylogenetically unique among cultured heliobacteria and was isolated from rice soil, a common habitat for heliobacteria. The Hph. fasciatum genome contains 3.14 Mbp-similar to that of other reported heliobacteria-but has a G+C base ratio that lies between that of Hmi. modesticaldum and Hrs. convoluta. Many of the genomic features of Hmi. modesticaldum and Hrs. convoluta, such as the absence of genes encoding autotrophic pathways, the presence of a superoperonal cluster of photosynthesis-related genes, and genes encoding endospore-specific proteins, are also characteristic of the Hph. fasciatum genome. However, despite the fact that Hph. fasciatum is diazotrophic, classical nif genes encoding the alpha and beta subunits of dinitrogenase (nifDK) present in other heliobacteria could not be identified. Instead, genes encoding several highly divergent NifDK homologs were present, at least one of which likely encodes a functional dinitrogenase and another a methylthio-alkane reductase (MarDK) for sulfur assimilation. A classical NifH (dinitrogenase reductase) homolog was also absent in Hph. fasciatum, but a related protein was identified that likely carries out this function as well as electron delivery to MarDK. The N2-fixing system of Hph. fasciatum is therefore distinct from that of other heliobacteria and may have unusual properties.

  View details for DOI 10.3390/microorganisms10050869

  View details for PubMedID 35630314

• Identification of a protein responsible for the synthesis of archaeal membrane-spanning GDGT lipids. Nature communications Zeng, Z., Chen, H., Yang, H., Chen, Y., Yang, W., Feng, X., Pei, H., Welander, P. V. 2022; 13 (1): 1545

  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.

  View details for DOI 10.1038/s41467-022-29264-x

  View details for PubMedID 35318330

• Lipid biomarkers: molecular tools for illuminating the history of microbial life. Nature reviews. Microbiology Summons, R. E., Welander, P. V., Gold, D. A. 2021

  Abstract

  Fossilized lipids preserved in sedimentary rocks offer singular insights into the Earth's palaeobiology. These 'biomarkers' encode information pertaining to the oxygenation of the atmosphere and oceans, transitions in ocean plankton, the greening of continents, mass extinctions and climate change. Historically, biomarker interpretations relied on inventories of lipids present in extant microorganisms and counterparts in natural environments. However, progress has been impeded because only a small fraction of the Earth's microorganisms can be cultured, many environmentally significant microorganisms from the past no longer exist and there are gaping holes in knowledge concerning lipid biosynthesis. The revolution in genomics and bioinformatics has provided new tools to expand our understanding of lipid biomarkers, their biosynthetic pathways and distributions in nature. In this Review, we explore how preserved organic molecules provide a unique perspective on the history of the Earth's microbial life. We discuss how advances in molecular biology have helped elucidate biomarker origins and afforded more robust interpretations of fossil lipids and how the rock record provides vital calibration points for molecular clocks. Such studies are open to further exploitation with the expansion of sequenced microbial genomes in accessible databases.

  View details for DOI 10.1038/s41579-021-00636-2

  View details for PubMedID 34635851

• Anaerobic 3-methylhopanoid production by an acidophilic photosynthetic purple bacterium. Archives of microbiology Mayer, M. H., Parenteau, M. N., Kempher, M. L., Madigan, M. T., Jahnke, L. L., Welander, P. V. 2021

  Abstract

  Bacterial lipids are well-preserved in ancient rocks and certain ones have been used as indicators of specific bacterial metabolisms or environmental conditions existing at the time of rock deposition. Here we show that an anaerobic bacterium produces 3-methylhopanoids, pentacyclic lipids previously detected only in aerobic bacteria and widely used as biomarkers for methane-oxidizing bacteria. Both Rhodopila globiformis, a phototrophic purple nonsulfur bacterium isolated from an acidic warm spring in Yellowstone, and a newly isolated Rhodopila species from a geochemically similar spring in Lassen Volcanic National Park (USA), synthesized 3-methylhopanoids and a suite of related hopanoids and contained the genes encoding the necessary biosynthetic enzymes. Our results show that 3-methylhopanoids can be produced under anoxic conditions and challenges the use of 3-methylhopanoids as biomarkers of oxic conditions in ancient rocks and as prima facie evidence that methanotrophic bacteria were active when the rocks were deposited.

  View details for DOI 10.1007/s00203-021-02561-7

  View details for PubMedID 34528111

• Enantioselective Total Synthesis of the Archaeal Lipid Parallel GDGT-0 (Isocaldarchaeol). Angewandte Chemie (International ed. in English) Falk, I. D., Gál, B. n., Bhattacharya, A. n., Wei, J. H., Welander, P. V., Boxer, S. G., Burns, N. Z. 2021

  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.

  View details for DOI 10.1002/anie.202104051

  View details for PubMedID 33930240

• Deciphering the evolutionary history of microbial cyclic triterpenoids FREE RADICAL BIOLOGY AND MEDICINE Welander, P. V. 2019; 140: 270–78

  View details for DOI 10.1016/j.freeradbiomed.2019.05.002

  View details for Web of Science ID 000485873700024

• Soil exchange rates of COS and (COO)-O-18 differ with the diversity of microbial communities and their carbonic anhydrase enzymes ISME JOURNAL Meredith, L. K., Ogee, J., Boye, K., Singer, E., Wingate, L., von Sperber, C., Sengupta, A., Whelan, M., Pang, E., Keiluweit, M., Brueggemann, N., Berry, J. A., Welander, P. V. 2019; 13 (2): 290–300

  View details for DOI 10.1038/s41396-018-0270-2

  View details for Web of Science ID 000455747900005

• GDGT cyclization proteins identify the dominant archaeal sources of tetraether lipids in the ocean. Proceedings of the National Academy of Sciences of the United States of America Zeng, Z. n., Liu, X. L., Farley, K. R., Wei, J. H., Metcalf, W. W., Summons, R. E., Welander, P. V. 2019

  Abstract

  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.

  View details for DOI 10.1073/pnas.1909306116

  View details for PubMedID 31591189

• Calditol-linked membrane lipids are required for acid tolerance in Sulfolobus acidocaldarius. Proceedings of the National Academy of Sciences of the United States of America Zeng, Z., Liu, X., Wei, J. H., Summons, R. E., Welander, P. V. 2018

  Abstract

  Archaea have many unique physiological features of which the lipid composition of their cellular membranes is the most striking. Archaeal ether-linked isoprenoidal membranes can occur as bilayers or monolayers, possess diverse polar head groups, and a multiplicity of ring structures in the isoprenoidal cores. These lipid structures are proposed to provide protection from the extreme temperature, pH, salinity, and nutrient-starved conditions that many archaea inhabit. However, many questions remain regarding the synthesis and physiological role of some of the more complex archaeal lipids. In this study, we identify a radical S-adenosylmethionine (SAM) protein in Sulfolobus acidocaldarius required for the synthesis of a unique cyclopentyl head group, known as calditol. Calditol-linked glycerol dibiphytanyl glycerol tetraethers (GDGTs) are membrane spanning lipids in which calditol is ether bonded to the glycerol backbone and whose production is restricted to a subset of thermoacidophilic archaea of the Sulfolobales order within the Crenarchaeota phylum. Several studies have focused on the enzymatic mechanism for the synthesis of the calditol moiety, but to date no protein that catalyzes this reaction has been discovered. Phylogenetic analyses of this putative calditol synthase (Cds) reveal the genetic potential for calditol-GDGT synthesis in phyla other than the Crenarchaeota, including the Korarchaeota and Marsarchaeota. In addition, we identify Cds homologs in metagenomes predominantly from acidic ecosystems. Finally, we demonstrate that deletion of calditol synthesis renders S. acidocaldarius sensitive to extremely low pH, indicating that calditol plays a critical role in protecting archaeal cells from acidic stress.

  View details for PubMedID 30518563

• C-4 sterol demethylation enzymes distinguish bacterial and eukaryotic sterol synthesis. Proceedings of the National Academy of Sciences of the United States of America Lee, A. K., Banta, A. B., Wei, J. H., Kiemle, D. J., Feng, J. n., Giner, J. L., Welander, P. V. 2018; 115 (23): 5884–89

  Abstract

  Sterols are essential eukaryotic lipids that are required for a variety of physiological roles. The diagenetic products of sterol lipids, sterane hydrocarbons, are preserved in ancient sedimentary rocks and are utilized as geological biomarkers, indicating the presence of both eukaryotes and oxic environments throughout Earth's history. However, a few bacterial species are also known to produce sterols, bringing into question the significance of bacterial sterol synthesis for our interpretation of sterane biomarkers. Recent studies suggest that bacterial sterol synthesis may be distinct from what is observed in eukaryotes. In particular, phylogenomic analyses of sterol-producing bacteria have failed to identify homologs of several key eukaryotic sterol synthesis enzymes, most notably those required for demethylation at the C-4 position. In this study, we identified two genes of previously unknown function in the aerobic methanotrophic γ-Proteobacterium Methylococcus capsulatus that encode sterol demethylase proteins (Sdm). We show that a Rieske-type oxygenase (SdmA) and an NAD(P)-dependent reductase (SdmB) are responsible for converting 4,4-dimethylsterols to 4α-methylsterols. Identification of intermediate products synthesized during heterologous expression of SdmA-SdmB along with 13C-labeling studies support a sterol C-4 demethylation mechanism distinct from that of eukaryotes. SdmA-SdmB homologs were identified in several other sterol-producing bacterial genomes but not in any eukaryotic genomes, indicating that these proteins are unrelated to the eukaryotic C-4 sterol demethylase enzymes. These findings reveal a separate pathway for sterol synthesis exclusive to bacteria and show that demethylation of sterols evolved at least twice-once in bacteria and once in eukaryotes.

  View details for PubMedID 29784781

• Microbial communities and organic biomarkers in a Proterozoic-analog sinkhole. Geobiology Hamilton, T. L., Welander, P. V., Albrecht, H. L., Fulton, J. M., Schaperdoth, I., Bird, L. R., Summons, R. E., Freeman, K. H., Macalady, J. L. 2017; 15 (6): 784-797

  Abstract

  Little Salt Spring (Sarasota County, FL, USA) is a sinkhole with groundwater vents at ~77 m depth. The entire water column experiences sulfidic (~50 μM) conditions seasonally, resulting in a system poised between oxic and sulfidic conditions. Red pinnacle mats occupy the sediment-water interface in the sunlit upper basin of the sinkhole, and yielded 16S rRNA gene clones affiliated with Cyanobacteria, Chlorobi, and sulfate-reducing clades of Deltaproteobacteria. Nine bacteriochlorophyll e homologues and isorenieratene indicate contributions from Chlorobi, and abundant chlorophyll a and pheophytin a are consistent with the presence of Cyanobacteria. The red pinnacle mat contains hopanoids, including 2-methyl structures that have been interpreted as biomarkers for Cyanobacteria. A single sequence of hpnP, the gene required for methylation of hopanoids at the C-2 position, was recovered in both DNA and cDNA libraries from the red pinnacle mat. The hpnP sequence was most closely related to cyanobacterial hpnP sequences, implying that Cyanobacteria are a source of 2-methyl hopanoids present in the mat. The mats are capable of light-dependent primary productivity as evidenced by (13) C-bicarbonate photoassimilation. We also observed (13) C-bicarbonate photoassimilation in the presence of DCMU, an inhibitor of electron transfer to Photosystem II. Our results indicate that the mats carry out light-driven primary production in the absence of oxygen production-a mechanism that may have delayed the oxygenation of the Earth's oceans and atmosphere during the Proterozoic Eon. Furthermore, our observations of the production of 2-methyl hopanoids by Cyanobacteria under conditions of low oxygen and low light are consistent with the recovery of these structures from ancient black shales as well as their paucity in modern marine environments.

  View details for DOI 10.1111/gbi.12252

  View details for PubMedID 29035021

• Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Banta, A. B., Wei, J. H., Gill, C. C., Giner, J., Welander, P. V. 2017; 114 (2): 245-250

  Abstract

  Cyclic triterpenoids are a broad class of polycyclic lipids produced by bacteria and eukaryotes. They are biologically relevant for their roles in cellular physiology, including membrane structure and function, and biochemically relevant for their exquisite enzymatic cyclization mechanism. Cyclic triterpenoids are also geobiologically significant as they are readily preserved in sediments and are used as biomarkers for ancient life throughout Earth's history. Isoarborinol is one such triterpenoid whose only known biological sources are certain angiosperms and whose diagenetic derivatives (arboranes) are often used as indicators of terrestrial input into aquatic environments. However, the occurrence of arborane biomarkers in Permian and Triassic sediments, which predates the accepted origin of angiosperms, suggests that microbial sources of these lipids may also exist. In this study, we identify two isoarborinol-like lipids, eudoraenol and adriaticol, produced by the aerobic marine heterotrophic bacterium Eudoraea adriatica Phylogenetic analysis demonstrates that the E. adriatica eudoraenol synthase is an oxidosqualene cyclase homologous to bacterial lanosterol synthases and distinct from plant triterpenoid synthases. Using an Escherichia coli heterologous sterol expression system, we demonstrate that substitution of four amino acid residues in a bacterial lanosterol synthase enabled synthesis of pentacyclic arborinols in addition to tetracyclic sterols. This variant provides valuable mechanistic insight into triterpenoid synthesis and reveals diagnostic amino acid residues to differentiate between sterol and arborinol synthases in genomic and metagenomic datasets. Our data suggest that there may be additional bacterial arborinol producers in marine and freshwater environments that could expand our understanding of these geologically informative lipids.

  View details for DOI 10.1073/pnas.1617231114

  View details for Web of Science ID 000391439300033

  View details for PubMedID 28028245

  View details for PubMedCentralID PMC5240688

• Fractionation of the methane isotopologues (CH4)-C-13, (CH3D)-C-12, and (CH3D)-C-13 during aerobic oxidation of methane by Methylococcus capsulatus (Bath) GEOCHIMICA ET COSMOCHIMICA ACTA Wang, D. T., Welander, P. V., Ono, S. 2016; 192: 186-202

  View details for DOI 10.1016/j.gca.2016.07.031

  View details for Web of Science ID 000384105700010

• Sterol Synthesis in Diverse Bacteria FRONTIERS IN MICROBIOLOGY Wei, J. H., Yin, X., Welander, P. V. 2016; 7

  Abstract

  Sterols are essential components of eukaryotic cells whose biosynthesis and function has been studied extensively. Sterols are also recognized as the diagenetic precursors of steranes preserved in sedimentary rocks where they can function as geological proxies for eukaryotic organisms and/or aerobic metabolisms and environments. However, production of these lipids is not restricted to the eukaryotic domain as a few bacterial species also synthesize sterols. Phylogenomic studies have identified genes encoding homologs of sterol biosynthesis proteins in the genomes of several additional species, indicating that sterol production may be more widespread in the bacterial domain than previously thought. Although the occurrence of sterol synthesis genes in a genome indicates the potential for sterol production, it provides neither conclusive evidence of sterol synthesis nor information about the composition and abundance of basic and modified sterols that are actually being produced. Here, we coupled bioinformatics with lipid analyses to investigate the scope of bacterial sterol production. We identified oxidosqualene cyclase (Osc), which catalyzes the initial cyclization of oxidosqualene to the basic sterol structure, in 34 bacterial genomes from five phyla (Bacteroidetes, Cyanobacteria, Planctomycetes, Proteobacteria, and Verrucomicrobia) and in 176 metagenomes. Our data indicate that bacterial sterol synthesis likely occurs in diverse organisms and environments and also provides evidence that there are as yet uncultured groups of bacterial sterol producers. Phylogenetic analysis of bacterial and eukaryotic Osc sequences confirmed a complex evolutionary history of sterol synthesis in this domain. Finally, we characterized the lipids produced by Osc-containing bacteria and found that we could generally predict the ability to synthesize sterols. However, predicting the final modified sterol based on our current knowledge of sterol synthesis was difficult. Some bacteria produced demethylated and saturated sterol products even though they lacked homologs of the eukaryotic proteins required for these modifications emphasizing that several aspects of bacterial sterol synthesis are still completely unknown.

  View details for DOI 10.3389/fmicb.2016.00990

  View details for Web of Science ID 000378390400001

  View details for PubMedID 27446030

  View details for PubMedCentralID PMC4919349

• A distinct pathway for tetrahymanol synthesis in bacteria PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Banta, A. B., Wei, J. H., Welander, P. V. 2015; 112 (44): 13478-13483

  Abstract

  Tetrahymanol is a polycyclic triterpenoid lipid first discovered in the ciliate Tetrahymena pyriformis whose potential diagenetic product, gammacerane, is often used as a biomarker for water column stratification in ancient ecosystems. Bacteria are also a potential source of tetrahymanol, but neither the distribution of this lipid in extant bacteria nor the significance of bacterial tetrahymanol synthesis for interpreting gammacerane biosignatures is known. Here we couple comparative genomics with genetic and lipid analyses to link a protein of unknown function to tetrahymanol synthesis in bacteria. This tetrahymanol synthase (Ths) is found in a variety of bacterial genomes, including aerobic methanotrophs, nitrite-oxidizers, and sulfate-reducers, and in a subset of aquatic and terrestrial metagenomes. Thus, the potential to produce tetrahymanol is more widespread in the bacterial domain than previously thought. However, Ths is not encoded in any eukaryotic genomes, nor is it homologous to eukaryotic squalene-tetrahymanol cyclase, which catalyzes the cyclization of squalene directly to tetrahymanol. Rather, heterologous expression studies suggest that bacteria couple the cyclization of squalene to a hopene molecule by squalene-hopene cyclase with a subsequent Ths-dependent ring expansion to form tetrahymanol. Thus, bacteria and eukaryotes have evolved distinct biochemical mechanisms for producing tetrahymanol.

  View details for DOI 10.1073/pnas.1511482112

  View details for Web of Science ID 000364164900041

  View details for PubMedID 26483502

• Methane Oxidation and Molecular Characterization of Methanotrophs from a Former Mercury Mine Impoundment. Microorganisms Baesman, S. M., Miller, L. G., Wei, J. H., Cho, Y., Matys, E. D., Summons, R. E., Welander, P. V., Oremland, R. S. 2015; 3 (2): 290-309

  Abstract

  The Herman Pit, once a mercury mine, is an impoundment located in an active geothermal area. Its acidic waters are permeated by hundreds of gas seeps. One seep was sampled and found to be composed of mostly CO₂ with some CH₄ present. The δ(13)CH₄ value suggested a complex origin for the methane: i.e., a thermogenic component plus a biological methanogenic portion. The relatively (12)C-enriched CO₂ suggested a reworking of the ebullitive methane by methanotrophic bacteria. Therefore, we tested bottom sediments for their ability to consume methane by conducting aerobic incubations of slurried materials. Methane was removed from the headspace of live slurries, and subsequent additions of methane resulted in faster removal rates. This activity could be transferred to an artificial, acidic medium, indicating the presence of acidophilic or acid-tolerant methanotrophs, the latter reinforced by the observation of maximum activity at pH = 4.5 with incubated slurries. A successful extraction of sterol and hopanoid lipids characteristic of methanotrophs was achieved, and their abundances greatly increased with increased sediment methane consumption. DNA extracted from methane-oxidizing enrichment cultures was amplified and sequenced for pmoA genes that aligned with methanotrophic members of the Gammaproteobacteria. An enrichment culture was established that grew in an acidic (pH 4.5) medium via methane oxidation.

  View details for DOI 10.3390/microorganisms3020290

  View details for PubMedID 27682090

  View details for PubMedCentralID PMC5023233

• Elucidation of the Burkholderia cenocepacia hopanoid biosynthesis pathway uncovers functions for conserved proteins in hopanoid-producing bacteria ENVIRONMENTAL MICROBIOLOGY Schmerk, C. L., Welander, P. V., Hamad, M. A., Bain, K. L., Bernards, M. A., Summons, R. E., Valvano, M. A. 2015; 17 (3): 735-750

  Abstract

  Hopanoids are bacterial surrogates of eukaryotic membrane sterols and among earth's most abundant natural products. Their molecular fossils remain in sediments spanning more than a billion years. However, hopanoid metabolism and function are not fully understood. Burkholderia species are environmental opportunistic pathogens that produce hopanoids and also occupy diverse ecological niches. We investigated hopanoids biosynthesis in Burkholderia cenocepacia by deletion mutagenesis and structural characterization of the hopanoids produced by the mutants. The enzymes encoded by hpnH and hpnG were essential for production of all C35 extended hopanoids, including bacteriohopanetetrol (BHT), BHT glucosamine and BHT cyclitol ether. Deletion of hpnI resulted in BHT production, while ΔhpnJ produced only BHT glucosamine. Thus, HpnI is required for BHT glucosamine production while HpnJ is responsible for its conversion to the cyclitol ether. The ΔhpnH and ΔhpnG mutants could not grow under any stress condition tested, whereas ΔhpnI, ΔhpnJ and ΔhpnK displayed wild-type growth rates when exposed to detergent, but varying levels of sensitivity to low pH and polymyxin B. This study not only elucidates the biosynthetic pathway of hopanoids in B. cenocepacia, but also uncovers a biosynthetic role for the conserved proteins HpnI, HpnJ and HpnK in other hopanoid-producing bacteria.

  View details for DOI 10.1111/1462-2920.12509

  View details for Web of Science ID 000351435600019

  View details for PubMedID 24888970

• Diverse capacity for 2-methylhopanoid production correlates with a specific ecological niche ISME JOURNAL Ricci, J. N., Coleman, M. L., Welander, P. V., Sessions, A. L., Summons, R. E., Spear, J. R., Newman, D. K. 2014; 8 (3): 675-684

  Abstract

  Molecular fossils of 2-methylhopanoids are prominent biomarkers in modern and ancient sediments that have been used as proxies for cyanobacteria and their main metabolism, oxygenic photosynthesis. However, substantial culture and genomic-based evidence now indicates that organisms other than cyanobacteria can make 2-methylhopanoids. Because few data directly address which organisms produce 2-methylhopanoids in the environment, we used metagenomic and clone library methods to determine the environmental diversity of hpnP, the gene encoding the C-2 hopanoid methylase. Here we show that hpnP copies from alphaproteobacteria and as yet uncultured organisms are found in diverse modern environments, including some modern habitats representative of those preserved in the rock record. In contrast, cyanobacterial hpnP genes are rarer and tend to be localized to specific habitats. To move beyond understanding the taxonomic distribution of environmental 2-methylhopanoid producers, we asked whether hpnP presence might track with particular variables. We found hpnP to be significantly correlated with organisms, metabolisms and environments known to support plant-microbe interactions (P-value<10(-6)); in addition, we observed diverse hpnP types in closely packed microbial communities from other environments, including stromatolites, hot springs and hypersaline microbial mats. The common features of these niches indicate that 2-methylhopanoids are enriched in sessile microbial communities inhabiting environments low in oxygen and fixed nitrogen with high osmolarity. Our results support the earlier conclusion that 2-methylhopanoids are not reliable biomarkers for cyanobacteria or any other taxonomic group, and raise the new hypothesis that, instead, they are indicators of a specific environmental niche.

  View details for DOI 10.1038/ismej.2013.191

  View details for Web of Science ID 000331879900016

  View details for PubMedID 24152713

  View details for PubMedCentralID PMC3930323

• Identification and quantification of polyfunctionalized hopanoids by high temperature gas chromatography-mass spectrometry. Organic geochemistry Sessions, A. L., Zhang, L., Welander, P. V., Doughty, D., Summons, R. E., Newman, D. K. 2013; 56: 120-130

  Abstract

  Hopanoids are triterpenoids produced mainly by bacteria, are ubiquitous in the environment, and have many important applications as biological markers. A wide variety of related hopanoid structures exists, many of which are polyfunctionalized. These modifications render the hopanoids too involatile for conventional gas chromatography (GC) separation, so require either laborious oxidative cleavage of the functional groups or specialized high temperature (HT) columns. Here we describe the systematic evaluation and optimization of a HT-GC method for the analysis of polyfunctionalized hopanoids and their methylated homologs. Total lipid extracts are derivatized with acetic anhydride and no further treatment or workup is required. We show that acid or base hydrolysis to remove di- and triacylglycerides leads to degradation of several BHP structures. DB-XLB type columns can elute hopanoids up to bacteriohopane-tetrol at 350 °C, with baseline separation of all 2-methyl/desmethyl homologs. DB-5HT type columns can additionally elute bacteriohopaneaminotriol and bacteriohopaneaminotetrol, but do not fully separate 2-methyl/desmethyl homologs. The method gave 2- to 7-fold higher recovery of hopanoids than oxidative cleavage and can provide accurate quantification of all analytes including 2-methyl hopanoids. By comparing data from mass spectra with those from a flame ionization detector, we show that the mass spectromet (MS) response factors for different hopanoids using either total ion counts or m/z 191 vary substantially. Similarly, 2-methyl ratios estimated from selected-ion data are lower than those from FID by 10-30% for most hopanoids, but higher by ca. 10% for bacteriohopanetetrol. Mass spectra for a broad suite of hopanoids, including 2-methyl homologs, from Rhodopseudomonas palustris are presented, together with the tentative assignment of several new hopanoid degradation products.

  View details for DOI 10.1016/j.orggeochem.2012.12.009

  View details for PubMedID 24496464

  View details for PubMedCentralID PMC3780965

• Molecular indicators of microbial diversity in oolitic sands of Highborn Cay, Bahamas Geobiology Edgecomb, V. P., Bernhard, J. M., Beaudoin, D., Pruss, S., Welander, P. V., Shubotz, F., Mehay, S., Gillespie, A. L., Summons, R. E. 2013; 11: 234-251
• Discovery, taxonomic distribution, and phenotypic characterization of a gene required for 3-methylhopanoid production. Proceedings of the National Academy of Sciences of the United States of America Welander, P. V., Summons, R. E. 2012; 109 (32): 12905-10

  Abstract

  Hopanoids methylated at the C-3 position are a subset of bacterial triterpenoids that are readily preserved in modern and ancient sediments and in petroleum. The production of 3-methylhopanoids by extant aerobic methanotrophs and their common occurrence in modern and fossil methane seep communities, in conjunction with carbon isotope analysis, has led to their use as biomarker proxies for aerobic methanotrophy. In addition, these lipids are also produced by aerobic acetic acid bacteria and, lacking carbon isotope analysis, are more generally used as indicators for aerobiosis in ancient ecosystems. However, recent genetic studies have brought into question our current understanding of the taxonomic diversity of methylhopanoid-producing bacteria and have highlighted that a proper interpretation of methylhopanes in the rock record requires a deeper understanding of their cellular function. In this study, we identified and deleted a gene, hpnR, required for methylation of hopanoids at the C-3 position in the obligate methanotroph Methylococcus capsulatus strain Bath. Bioinformatics analysis revealed that the taxonomic distribution of HpnR extends beyond methanotrophic and acetic acid bacteria. Phenotypic analysis of the M. capsulatus hpnR deletion mutant demonstrated a potential physiological role for 3-methylhopanoids; they appear to be required for the maintenance of intracytoplasmic membranes and cell survival in late stationary phase. Therefore, 3-methylhopanoids may prove more useful as proxies for specific environmental conditions encountered during stationary phase rather than a particular bacterial group.

  View details for DOI 10.1073/pnas.1208255109

  View details for PubMedID 22826256

  View details for PubMedCentralID PMC3420191

• Identification and characterization of Rhodopseudomonas palustris hopanoid biosynthesis mutants Geobiology Welander, P. V., Doughty, D. M., Wu, C. H., Mehay, S., Summons, R. E., Newman, D. K. 2012; 10: 163-177
• Identification of the bacteriochlorophylls, carotenoids, quinones, lipids, and hopanoids of Candidatus Chloracidobacterium thermophilum Journal of Bacteriology Garcia Costas, A. M., Tsukatani, Y., Rijpstra, W. I., Schouten, S., Welander, P. V., Summons, R. E., Bryant, D. A. 2012; 194: 1158-68
• Using Taguchi-based statistics to produce robust PCR results Bioprocessing Welander, P., Cantin, E., Lundberg, P. 2011; 10: 22-26
• Identification of a methylase required for 2-methylhopanoid production and implications for the interpretation of sedimentary hopanes. Proceedings of the National Academy of Sciences of the United States of America Welander, P. V., Coleman, M. L., Sessions, A. L., Summons, R. E., Newman, D. K. 2010; 107 (19): 8537-42

  Abstract

  The rise of atmospheric oxygen has driven environmental change and biological evolution throughout much of Earth's history and was enabled by the evolution of oxygenic photosynthesis in the cyanobacteria. Dating this metabolic innovation using inorganic proxies from sedimentary rocks has been difficult and one important approach has been to study the distributions of fossil lipids, such as steranes and 2-methylhopanes, as biomarkers for this process. 2-methylhopanes arise from degradation of 2-methylbacteriohopanepolyols (2-MeBHPs), lipids thought to be synthesized primarily by cyanobacteria. The discovery that 2-MeBHPs are produced by an anoxygenic phototroph, however, challenged both their taxonomic link with cyanobacteria and their functional link with oxygenic photosynthesis. Here, we identify a radical SAM methylase encoded by the hpnP gene that is required for methylation at the C-2 position in hopanoids. This gene is found in several, but not all, cyanobacteria and also in alpha -proteobacteria and acidobacteria. Thus, one cannot extrapolate from the presence of 2-methylhopanes alone, in modern environments or ancient sedimentary rocks, to a particular taxonomic group or metabolism. To understand the origin of this gene, we reconstructed the evolutionary history of HpnP. HpnP proteins from cyanobacteria, Methylobacterium species, and other alpha-proteobacteria form distinct phylogenetic clusters, but the branching order of these clades could not be confidently resolved. Hence,it is unclear whether HpnP, and 2-methylhopanoids, originated first in the cyanobacteria. In summary, existing evidence does not support the use of 2-methylhopanes as biomarkers for oxygenic photosynthesis.

  View details for DOI 10.1073/pnas.0912949107

  View details for PubMedID 20421508

  View details for PubMedCentralID PMC2889317

• Hopanoids play a role in membrane integrity and pH homeostasis in Rhodopseudomonas palustris TIE-1. Journal of bacteriology Welander, P. V., Hunter, R. C., Zhang, L., Sessions, A. L., Summons, R. E., Newman, D. K. 2009; 191 (19): 6145-56

  Abstract

  Sedimentary hopanes are pentacyclic triterpenoids that serve as biomarker proxies for bacteria and certain bacterial metabolisms, such as oxygenic photosynthesis and aerobic methanotrophy. Their parent molecules, the bacteriohopanepolyols (BHPs), have been hypothesized to be the bacterial equivalent of sterols. However, the actual function of BHPs in bacterial cells is poorly understood. Here, we report the physiological study of a mutant in Rhodopseudomonas palustris TIE-1 that is unable to produce any hopanoids. The deletion of the gene encoding the squalene-hopene cyclase protein (Shc), which cyclizes squalene to the basic hopene structure, resulted in a strain that no longer produced any polycyclic triterpenoids. This strain was able to grow chemoheterotrophically, photoheterotrophically, and photoautotrophically, demonstrating that hopanoids are not required for growth under normal conditions. A severe growth defect, as well as significant morphological damage, was observed when cells were grown under acidic and alkaline conditions. Although minimal changes in shc transcript expression were observed under certain conditions of pH shock, the total amount of hopanoid production was unaffected; however, the abundance of methylated hopanoids significantly increased. This suggests that hopanoids may play an indirect role in pH homeostasis, with certain hopanoid derivatives being of particular importance.

  View details for DOI 10.1128/JB.00460-09

  View details for PubMedID 19592593

  View details for PubMedCentralID PMC2747905

• The continuing puzzle of the great oxidation event Current Biology Sessions, A. L., Doughty, D. M., Welander, P. V., Summons, R. E., Newman, D. K. 2009; 19: R567-R574
• Mutagenesis of the C1 oxidation pathway in Methanosarcina barkeri: new insights into the Mtr/Mer bypass pathway Journal of Bacteriology Welander, P. V., Metcalf, W. W. 2008; 190: 1928-1936
• Tumor necrosis factor (TNF) protects resistant C57BL/6 mice against herpes simplex virus-induced encephalitis independently of signaling via TNF receptor 1 or 2 Journal of Virology Lundberg, P., Welander, P. V., Edwards, 3rd, C. K., van Rooijen, N., Cantin, E. 2007; 81: 1451-1460
• Loss of the mtr operon in Methanosarcina blocks growth on methanol, but not methanogenesis, and reveals an unknown methanogenic pathway Proceedings of the National Academy of Sciences Welander, P. V., Metcalf, W. W. 2005; 102: 10664-10669
• Herpes simplex virus type 1 DNA is immunostimulatory in vitro and in vivo Journal of Virology Lundberg, P., Welander, P., Han, X., Cantin, E. 2003; 77: 11158-11169
• A locus on mouse chromosome 6 that determines resistance to herpes simplex virus also influences reactivation, while an unlinked locus augments resistance of female mice Journal of Virology Lundberg, P., Welander, P., Openshaw, H., Nalbandian, C., Edwards, C., Moldawer, L., Cantin, E. 2003; 77: 11661-11673