Biology subjects

Welander, P. V.

Publications and source records attributed to Welander, P. V..

12 recordsLinked to original sources

bioRxiv2026

A lipid compendium of a metabolically compromised bacterium provides insights into lipid acquisition, biosynthesis, and metabolism

The Lyme disease agent Borrelia burgdorferi belongs to a class of metabolically compromised bacteria that cannot survive without host-derived lipids. Survival of the agent in tick and vertebrate hosts requires substantial nutrient acquisition and potential cell envelope remodeling. While prior studies identified cholesterol, cholesterol glycolipids, and phosphatidylcholines as membrane lipids in B. burgdorferi, the identity of many other membrane lipids, their origin, and their physiological relevance remain unknown. Here, we used a suite of untargeted and targeted high-resolution mass spectrometry methods to reveal a complex lipid profile of the pathogen and to identify the origin of its lipids. The analysis detected more than 500 lipids in B. burgdorferi, the majority of which are sourced from the environment. However, the bacterium selectively accumulates certain lipids while excluding others, suggesting discriminatory uptake. These include cholesteryl esters and triglycerides that are organized in foci within the pathogen. Intriguingly, the pathogen also synthesizes predominantly eukaryotic lipids such as the lysosomal bis(monoacylglycerol)phosphate and the plant glycolipid sulfoquinovosyl diacylglycerol (SQDG). The biosynthesis of the latter is carried out by enzymes that exhibit structural homology to plant oxidoreductases and galactosyltransferases, yet their closest orthologs are found in bacteria. This hints that the capability of SQDG synthesis is more widespread in spirochaetes and other bacteria. Together, the comprehensive lipid profiling we report here uncovers novel aspects of the physiology of the metabolically challenged B. burgdorferi and highlights lipid acquisition and synthesis pathways as potentially critical for pathogen survival.

biochemistry↗

bioRxiv2026

Characterization of aliA and aliB deletion mutants reveals a dominant role of AliA in Haloferax volcanii lipoprotein lipidation

Protein lipidation is a widespread strategy for anchoring proteins to cellular membranes across all domains of life, yet the mechanisms underlying this process in archaea remain poorly understood. Recently, the first archaeal enzymes involved in lipobox-containing protein (lipoprotein) biogenesis, AliA and AliB, were identified and characterized in the model archaeon Haloferax volcanii. Although these paralogs share significant sequence similarity, distinct deletion phenotypes suggest differences in their substrate specificity and function. Here, we employed large-scale Triton X-114 fractionation followed by quantitative proteomics and lipid-specific mass spectrometry to systematically analyze AliA- and AliB-dependent lipoprotein lipidation. Deletion of aliA affected substantially more lipoproteins in Hfx. volcanii than deletion of aliB, markedly diminishing their TX-114 enrichment--indicating reduced hydrophobicity--and abolishing thioether-linked archaeol modification. This establishes AliA as the primary enzyme responsible for archaeal lipoprotein lipidation. In contrast, deletion of aliB affected only a small subset of lipoproteins and did not significantly reduce thioether-linked archaeol levels. In addition to defining distinct and non-redundant roles for AliA and AliB, this study provides the first large-scale experimental validation of predicted archaeal lipoproteins and identifies candidate components of the archaeal lipoprotein biogenesis pathway, substantially advancing mechanistic understanding and enabling improved lipoprotein prediction in this previously underexplored field.

molecular biology↗

bioRxiv2026

A sterol reductase responsible for the unusual 8(14)-unsaturation in bacterial sterol production and degradation

Sterols are a class of lipids that play a crucial role in human health through their essential physiological roles and as a point of interaction between commensal and pathogenic bacteria. The biosynthesis and modification of these lipids is a well-characterized process in many eukaryotes and increasingly in bacteria. However, the proteins responsible for formation of the unusual 8(14)-unsaturation found in the sterols produced by aerobic methanotrophs, dinoflagellates, nematodes, and marine sponges, remains unknown. Here, we utilize a heterologous expression system to identify a bacterial 8,14-sterol reductase (8,14-Bsr) responsible for generating the 8(14)-unsaturation in the aerobic methanotroph Methylococcus capsulatus. This enzyme modifies the direct product of C-14 demethylation, reducing one double bond in the nuclear core structure and isomerizing the other to produce an 8(14)-sterol. We subsequently tested the requirement of putative active site residues for catalysis through site directed mutagenesis, identifying residues likely involved in interacting with the sterol substrate and directly catalyzing this reaction. Bioinformatic analysis of the distribution of 8,14-Bsr reveals it is unique to the bacterial domain, found primarily in the Methylococcaceae family, the Mycobacteriales order, and yet uncultured members of the Myxococcota phylum. Further phylogenetic analysis of 8,14-Bsr suggests it shares an evolutionary history with the C-14 demethylase in these organisms and that these two enzymes were likely inherited together. These results provide insight into novel sterol biochemistry, further delimiting sterol biosynthesis in the bacterial domain from eukaryotes and illustrating the importance of molecular characterization to identify bacterial proteins that interact with sterols. ImportanceMany of the eukaryotic proteins required for the biosynthesis of sterols, such as cholesterol, have been characterized. However, the pathways governing analogous processes in the bacterial domain are less characterized. Here, we identify an 8,14-sterol reductase in aerobic methanotrophs. This enzyme carries out a unique biochemical reaction, saturating and isomerizing double bonds in the nuclear core structure to produce an 8(14)-sterol. This reductase is restricted to the bacterial domain, further separating the evolution of bacterial sterol production from eukaryotes. Additionally, we find this reductase is prevalent in members of the sterol degrading order Mycobacteriales, highlighting a potential role for this protein in the remodeling of host sterol production by these pathogens.

microbiology↗

bioRxiv2025

Biochemical characterization of bacterial methyltransferases reveals necessary residues for sterol side-chain propylation

The capacity to methylate sterol side-chains via sterol methyltransferases (SMTs) was thought to be widespread in fungi, algae, and plants but limited in animals and nonexistent in bacteria. We have previously demonstrated that yet-uncultured bacteria have the genomic capacity to produce side-chain methylated sterols de novo. Further, we identified three bacterial SMTs capable of producing 24-isopropyl sterols and showed that each of these SMTs was biochemically sufficient for all three of the side-chain methylation steps necessary for 24-isopropyl sterol synthesis. To date, no eukaryotic SMT has been identified that is capable of sequentially adding three methyl groups to generate this propyl structure. To better understand this unique biochemical feature of bacterial SMTs and to potentially identify key amino acid residues involved in sequential methylations on the sterol side-chain, we performed site-directed mutagenesis of propylating bacterial SMTs. Through these analyses, we identified a glycine residue that is necessary but not sufficient for side-chain propylation. This residue is located outside known SMT substrate-binding domains, but inside the active site of an SMT protein model docked with 24-methylenecholesterol. We also show that phenylalanine residues in sterol-binding Region I increase the production of 24-isopropyl sterols, and that 25 residues are conserved among methylating, ethylating, and propylating SMTs. Together, the presence of these residues may allow us to predict if an organism has the genomic capacity to produce C-24 propylated sterols directly from sequencing data, and to generate hypotheses about the environments in which bacterial sterol side-chain propylation may be occurring through analysis of metagenomes. Given the high preservation potential of side-chain alkylated sterols and their use as molecular fossils indicative of ancient life deep in time, a better understanding of the distribution of these unique lipids in modern life and present-day ecosystems will allow for more robust interpretations of side-chain alkylated steranes in the rock record.

microbiology↗

bioRxiv2025

Identification of radical SAM enzymes responsible for the methylation and desaturation of archaeal lipids and an AttH hydratase mediating hydroxy-GDGT biosynthesis

Lipid biosynthesis in archaea is highly dependent upon radical (S)-adenosyl methionine (rSAM) enzymes. The formation of their membrane-spanning lipids, known as glycerol dibiphytanyl glycerol tetraethers (GDGTs), is catalyzed by the rSAM tetraether synthase and the subsequent cyclization of their lipid tails is performed by the B12-binding rSAM (B12-rSAM) GDGT ring synthase. Further, GDGT tails are cross-linked by the rSAM GMGT synthase and then methylated by the B12-rSAM GMGT methylase. Here, utilizing three archaeal model organisms - Sulfolobus acidocaldarius, Thermococcus kodakarensis, and Methanosarcina acetivorans - we expand this repertoire of rSAM enzymes further, identifying two B12-rSAM GDGT methylases and a novel B12-rSAM GDGT desaturase. We also extend beyond the rSAM enzyme superfamily, identifying an AttH-like hydratase mediating hydroxy-GDGT (OH-GDGT) biosynthesis. Specifically, we identify two GDGT methylases in S. acidocaldarius and Thermococcus aggregans which possess different substrate specificities. While GDGT methylation is generally low in archaea, we observe high levels of lipid methylation in response to penicillin G and hexanoic acid amphiphile exposure, suggesting a role for lipid methylation in response to membrane-destabilizing chemical agents. Utilizing heterologous expression in M. acetivorans and T. kodakarensis, we uncover a novel B12-rSAM enzyme from Candidatus Bathyarchaeota B1_G15 with unusual tetraether desaturase (Ted) activity that seemingly reverses previous saturation by geranylgeranyl reductase, anaerobically forming double bonds in the inert hydrocarbon tails of a GDGT. Finally, we show that a non-rSAM AttH-like hydratase, genomically associated with Ted, is a hydroxy-GDGT synthase (Hgs), putatively hydrating the double bond(s) introduced by Ted to form OH-GDGTs. These results highlight the value of exploring the rSAM enzyme landscape of archaea - revealing diverse and novel rSAM enzyme activities amongst homologous proteins that invoke new paradigms such as reversibility in the archaeal lipid biosynthesis pathway.

microbiology↗

bioRxiv2025

Archaeal lineages related to eukaryotes encode functional diterpenoid cyclases

The first eukaryotic cell originated through the union of an archaeon and a bacterium. Phylogenetic evidence suggests that the archaeal partner was a member of phylum Asgardarchaeota, likely within the Heimdallarchaeia clade. Although little is known about the molecular basis of eukaryogenesis, insight can be gained from studying the unique biochemistry of modern Asgardarchaeota. Here, we investigated the potential for Asgards to produce cyclic terpenoids. Phylogenomics coupled with structural prediction revealed that organisms from the clades Hodarchaeales and Kariarchaeaceae encode for diterpenoid cyclases. We tested the functionality of these enzymes in vitro, showing that they cyclize geranylgeranyl pyrophosphate to form bicyclic halimadienyl pyrophosphate. Halimadienyl nucleosides have previously been shown to mediate intracellular persistence of Mycobacterium tuberculosis in host endosomes, and may function similarly in Asgardarchaeota. The characterization of Asgard diterpenoid cyclases provides insight into the biochemistry of an elusive group of microbes that are pivotal in the evolution of complex cellular life.

microbiology↗

bioRxiv2024

A geranylgeranyl reductase homolog required for cholesterol production in Myxococcota

Myxococcota is a phylum of sterol producing bacteria. They exhibit a clade depth for sterol biosynthesis unparalleled in the bacterial domain and produce sterols of a biosynthetic complexity that rivals eukaryotes. Additionally, the sterol biosynthesis pathways found in this phylum have been proposed as a potential source for sterol biosynthesis in the last eukaryotic common ancestor, lending an evolutionary importance to our understanding of this pathway in Myxococcota. However, sterol production has only been characterized in a few species and outstanding questions about the evolutionary history of this pathway remain. Here, we identify two myxobacteria, Minicystis rosea and Sandaracinus amylolyticus, capable of cholesterol biosynthesis. These two myxobacteria possess a cholesterol biosynthesis pathway that differs in both the ordering and enzymes involved in biosynthesis compared to Enhygromyxa salina, a myxobacterium previously demonstrated to produce cholesterol, as well as the canonical pathways found in eukaryotes. We characterize an alternative bacterial reductase responsible for performing C-24 reduction, further delineating bacterial cholesterol production from eukaryotes. Finally, we examine the distribution and phylogenetic relationships of sterol biosynthesis proteins across both cultured and uncultured Myxococcota species, providing evidence for multiple acquisition events and instances of both horizontal and vertical transfer at the family level. Altogether, this work further demonstrates the capacity of myxobacteria to synthesize eukaryotic sterols but with an underlying diversity in the biochemical reactions which govern sterol synthesis, suggesting a complex evolutionary history and refining our understanding of how myxobacterial cholesterol production relates to their eukaryotic counterparts. Significance StatementSterols are essential and ubiquitous lipids in eukaryotes, but their significance in bacteria is less understood. Sterol production in Myxococcota, a phylum of developmentally complex predatory bacteria, has provided insight into novel sterol biochemistry and prompted discussion regarding the evolution of this pathway within both the eukaryotic and bacterial domains. Here, we characterize cholesterol biosynthesis in two myxobacteria, providing evidence for distinct pathway organization and identifying a unique protein responsible for C-24 reduction. We couple these results to phylogenomic analysis of sterol biosynthesis within Myxococcota revealing a complicated evolutionary history marked by vertical and horizontal transfer, suggesting a mosaic acquisition of this pathway in Myxococcota and highlighting the complex role myxobacteria may have had in sterol transfer to eukaryotes.

microbiology↗

bioRxiv2024

The evolutionary history and modern diversity of triterpenoid cyclases

Cyclic terpenoids are a class of lipid compounds containing immense structural and functional diversity, with many cyclic triterpenoids acting as regulators of the physical properties and spatial organization of lipid membranes. Cyclic terpenoids are also readily preserved as terpane fossils, such as steranes and hopanes, forming a rich record of the evolution of life on Earth. Formation of the multiple ring structure of all cyclic terpenoids is catalyzed by terpenoid cyclase enzymes, among which are whole clades of proteins - many from environmental metagenomes and uncultured organisms - whose substrates and products are completely unknown. We investigate the function of these divergent cyclases through biochemical assays, and the evolutionary processes that produced them by testing and applying a variety of evolutionary models. We find deep divergence between the diterpenoid cyclases and triterpenoid cyclases, with other clades branching between the two, rooting the triterpenoid cyclase subtree, between squalene-hopene cyclases and sterol cyclases. Through a simple test of evolutionary rate shifts, we find an elevated evolutionary rate in the enzyme active site on the squalene-hopene cyclase stem, potentially indicative of positive selection. Finally, by testing the activity of divergent cyclases for a variety of substrates, we find a group of early-branching sterol cyclases from bacteria that synthesize arborinols, two of which produce the molecular precursor to a Permian "orphan biomarker." Together, our data present an evolutionary framework for triterpenoid cyclases that can inform both the biochemical potential of these proteins and their products occurrence in the geological record.

evolutionary biology↗

bioRxiv2023

Discovery of two archaeal GDGT lipid modifying enzymes reveals diverse microbes capable of H-GDGT biosynthesis and modification

Archaea produce unique membrane-spanning lipids, termed glycerol dialkyl glycerol tetraethers (GDGTs), which are thought to aid in adaptive responses to various environmental challenges. GDGTs can be modified in a variety of ways, including cyclization, bridging or cross-linking, methylation, hydroxylation, and desaturation, to give rise to a plethora of structurally distinct GDGT lipids with different properties. Here we report the discovery of a pair of radical SAM enzymes responsible for two of these modifications - an H-GDGT bridge synthase (Hbs), responsible for cross-linking the two hydrocarbon tails of a GDGT to produce H-GDGTs and an H-GDGT methylase (Hgm), responsible for the subsequent methylation of H-GDGTs. Heterologous expression of Hbs proteins from various archaea in Thermococcus kodakarensis results in the production of H-GDGTs in two isomeric forms. Further, co-expression of Hbs and Hgm results in the production of mono- and di-methylated H-GDGTs and minor amounts of tri-methylated H- GDGTs while expression of Hgm alone results in minor production of mono- and di- methylated GDGTs. Phylogenetic analyses reveal the presence of Hbs homologs in diverse archaeal genomes spanning all four archaeal superphyla. We also find Hbs homologs in bacterial genomes that have the genetic potential to synthesize fatty acid- based membrane-spanning lipids such as brGDGTs. We subsequently demonstrate H- GDGT production in three Hbs-encoding archaea, identifying an increase in H-GDGTs in response to elevated temperature in members of the genus Archaeoglobus and observing the production of highly cyclized H-GDGTs with up to 6 rings in the Thermoproteales archaeon Vulcanisaeta distributa. Such highly cyclized H-GDGTs are the precursors of ARN acids, a class of tetraprotic naphthenic acids that cause destructive mineral deposition during crude oil processing. Co-occurrence of the H-GDGT synthase with the previously identified GDGT ring synthases in archaeal genomes allowed identification of multiple archaeal phyla with the genetic potential to produce highly cyclized H-GDGTs, with particularly interesting candidates in the class Thermoplasmata from oil rich environments.

microbiology↗

bioRxiv2022

De novo cholesterol biosynthesis in the bacterial domain

Sterols are versatile lipids primarily associated with eukaryotes. While bacteria also produce sterols and studies of bacterial biosynthesis proteins have revealed novel biosynthetic pathways and a potential evolutionary role in the origin of sterol biosynthesis, no bacterium has been shown to synthesize highly modified eukaryotic sterols, such as cholesterol. This has led to the notion that bacteria only produce biosynthetically simple sterols and has lessened the consideration of bacterial production in discussions of sterol biosynthesis. In this study, we demonstrate two phylogenetically distinct bacteria, Enhygromyxa salina and Calothrix sp. NIES-4105, are capable of de novo cholesterol production. We also identified 25-hydroxycholesterol released as a product of acid hydrolysis in extracts from both bacteria, suggesting cholesterol exists as a conjugated molecule in these organisms. We coupled our lipid extractions to bioinformatic analyses and heterologous expression experiments to identify genetic pathways driving cholesterol production in each bacterium. E. salina shares much of its cholesterol biosynthesis pathway with the canonical eukaryotic pathway, except for C-4 demethylation where we identified a unique variation on the bacterial C-4 demethylation pathway. Calothrix lacks homologs for several steps in cholesterol biosynthesis, suggesting this bacterium may harbor a novel mechanism for completing cholesterol biosynthesis. Altogether, these results demonstrate the complexity underpinning bacterial sterol biosynthesis and raise further questions about the functional and regulatory roles of sterols in bacteria.

microbiology↗

bioRxiv2022

Structural insights into bacterial sterol transport

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.

biochemistry↗

bioRxiv2022

Sterol side chain alkylation by sponge and bacterial methyltransferases

Sterol lipids are required by most eukaryotes and are readily preserved as sterane molecular fossils. These geologic steranes are broadly interpreted as biomarkers for ancient eukaryotes1,2 although diverse bacteria also produce sterols3. Steranes with side-chain methylations can act as more specific biomarkers4 if their sterol precursors are limited to particular extant eukaryotes and are absent in bacteria. An abundance of one such sterane, 24-isopropylcholestane, in late Neoproterozoic rocks has been attributed to marine demosponges and potentially represents the earliest evidence for animals on Earth5. However, debates over this interpretation6-14 continue given the potential for alternative sources of 24-isopropylcholestane and the lack of experimental evidence demonstrating the function of enzymes that methylate sterols to give the 24-isopropyl side-chain. Here we show that sterol methyltransferases from both sponges and bacteria are functional and identify three bacterial methyltransferases each capable of sequential methylations resulting in the 24-isopropyl sterol side-chain. We identified two of these propylating enzymes in a demosponge metagenome suggesting bacterial symbionts contribute to 24-isopropyl sterol biosynthesis in demosponges. Our results demonstrate yet-uncultured bacteria have the genomic capacity to synthesize side-chain alkylated sterols and should therefore be considered when interpreting side-chain alkylated sterane biomarkers in the rock record.

microbiology↗