Biology subjects

Villanueva, L.

Publications and source records attributed to Villanueva, L..

14 recordsLinked to original sources

bioRxiv2026

Distinct taxonomic groups of sponge symbionts have the biosynthetic capacity for the production of ether lipids

The marine sponge holobiont, composed of the sponge host and its microbial symbionts, is a known source of abundant and diverse ether lipids (ELs). Apart from their structural role in the cytoplasmic membrane of archaea and some bacteria, ELs have often been linked to signaling functions and defense against pathogens. Despite the relevance of ELs, their biosynthesis, as well as the identity of their producers, remain elusive. Here, we report the analysis of potential ether lipid producing genes and gene clusters, detected in marine sponge metagenomes as well as public sponge genomes. We show that the sponge holobiont has the capacity to synthesize ELs via several pathways, and suggest the ability of the sponge holobiont to synthesize ELs under different O2 levels. Finally, targeted lipidome analysis confirmed that ELs are present in the lipid profiles of all of the studied sponge holobiont samples, and indicates that the biosynthesis of the plasmalogens detected is likely restricted to the sponge host itself, based on the detected hydrocarbon chain lengths. This work provides a basis for the challenging quest to decipher intricate EL biosynthesis in marine sponges and their associated microbes.

microbiology↗

bioRxiv2026

Bridging the lipid divide: archaeal ESCRT-III binds phosphoinositol and polarises the cytokinetic membrane

All cells remodel their membranes to divide. The highly conserved ESCRT-III system forms contractile polymers which, through direct interactions with membrane lipids, remodel membranes across the tree of life. In exploring how ESCRT-III divides the chemically and structurally unique archaeal membrane, we reveal that the homologue CdvB1 is required for the establishment of a distinct membrane domain within the division bridge of Sulfolobus acidocaldarius, associated with an accumulation of membrane-spanning inositol phosphate lipids. We show that CdvB1 associates with phosphoinositides in vitro and that this interaction aids cytokinesis in vivo. Together, we suggest that although eukaryotes inherited their membrane lipids from bacteria during eukaryogenesis, key features of the ESCRT-III:membrane interface that allow these polymers to bind, organise, and remodel eukaryotic membranes, may originate in archaea.

cell biology↗

bioRxiv2026

Towards understanding the bacterial biosynthesis of branched GDGTs: Identification of iso-diabolic acid-based tetraester and mixed ether/ester, membrane-spanning lipid intermediates in members of the Bacillota

Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are bacterial membrane-spanning lipids (MSLs) resembling archaeal membrane lipids, as they form monolayers and are linked to glycerol backbones via ether bonds. Ubiquitous in soils, sediments, and aquatic environments, their distributions are widely applied as paleoclimate proxies for reconstructing past temperature and pH. Despite this, understanding of their biological origins and functional role in cells remain incomplete. While some Acidobacteria are known producers of brGDGTs, genomic evidence and environmental surveys indicate additional bacterial contributors. In this study, we report the first detection of potential brGDGT biosynthetic intermediates in Bacillota. Ultra-high pressure liquid chromatography high resolution multi-stage mass spectrometry (UHPLC-HRMSn) revealed membrane-spanning diglycerol lipids which contained iso-diabolic acid (13,16-dimethyl octacosanedioic acid)-derived alkyl chains. These diglycerol lipids displayed diverse structures, including tetraesters, mixed ester/ether combinations, and vinyl ether bonds. Additionally, open membrane spanning lipids analogous to brGTGTs were also identified. Notably, all brGDGT and brGTGT analogues were detected with a phosphatidylglycerol head group. Experiments showed that the two Bacillota strains, which produce these brGDGT biosynthetic intermediates, responded differently to changes in temperature and oxygen availability, suggesting that environmental regulation of brGDGT-related lipids is taxonomically dependent. Based on these findings, we propose a biosynthetic pathway for brGDGT formation and highlight the physiological implications for interpreting brGDGT-based paleoclimate proxies. This work expands the known diversity of bacterial sources of brGDGTs and provides new insights into the ecological and evolutionary significance of these lipids. IMPORTANCEBranched GDGTs (brGDGTs) are bacterial membrane spanning lipids which form a monolayer, linked through ether bonds to the glycerol backbone, characteristics more commonly found in archaeal membrane lipids. They are commonly used in paleoclimate proxies to assess past temperature and pH but their predictive power is hampered by the lack of information regarding their biological producers. Branched GDGTs have been detected in just a few species of the Acidobacteria but there are strong indications that other bacterial phyla also contribute to the pool of brGDGTs in the environment. Here, we report for the first time the production of potential brGDGT intermediates in Bacillota species. This study demonstrates that brGDGTs likely occur much more widespread in the bacterial domain than previously thought and opens a new chapter both in the understanding of the function of these membrane lipids and their use in paleoclimatology.

microbiology↗

bioRxiv2025

Proton stress adaptation in acidophilic sulfate-reducing bacteria: insights from Acididesulfobacillus acetoxydans for acid mine drainage bioremediation

Acid mine drainage (AMD) waters are a global environmental threat due to their extremely low pH (<3) and high metal loads. Acidophilic sulfate-reducing bacteria (aSRB) can mitigate AMD by reducing sulfate to sulfide, a proton-consuming process that also precipitates metals as metal sulfides. Although sulfate reduction has been observed in AMD waters, most characterized aSRB are only moderately acidophilic and originate from protected microniches. Here, we examined the pH tolerance and proton stress adaptation of the complete organic acid-oxidizing aSRB Acididesulfobacillus acetoxydans. Continuous chemostat cultivations were operated across a pH gradient, achieving steady states from pH 5.0 (optimum) down to 2.9, with microcosms showing metabolic activity even at pH 2.5 which is typical AMD-acidity. Transcriptomic profiles remained remarkably stable across conditions, except for upregulation of the K+-transporting ATPase (kdpABC) at lower pH, suggesting an increased reliance on the chemiosmotic gradient to impede proton influx. Lipid analysis revealed increased core lipid saturation, mid-chain methylation and a shift in priming precursors from leucine to valine at low pH, indicating reduced membrane permeability and more energy-efficient biosynthetic pathways. These adaptations impede proton entry demonstrating adaptation of aSRB to AMD-like acidity and removes the critical pH bottleneck for AMD bioremediation and metal recovery. SynopsisThis study shows acidophilic sulfate-reducing bacteria can adapt to AMD-like acidity while retaining metabolic activity, underscoring their potential for AMD bioremediation and biotechnology.

microbiology↗

bioRxiv2025

Near equilibrium unbinding of streptavidin/biotin using single molecule acoustic force spectroscopy.

The dissociation of the streptavidin-biotin (SA-b) bond has been widely characterized using bulk and single molecule force spectroscopy (SMFS) techniques. However, the dissociation rates (koff) from SMFS ([~]10-1 s-1) typically do not align with those from bulk approaches ([~]10-6 - 10-5 s-1), likely because SMFS measurements are conducted far from equilibrium. Near equilibrium SMFS requires high throughput measurements to obtain large enough statistics, and high stability over long time measurements at ultraslow loading force rates, impractical in most SMFS techniques. Here, we developed in-situ force calibration strategies for acoustic force spectroscopy (AFS) to probe the unbinding forces of SA-b in the near equilibrium regime, from 10 pN/s down to 10- 3 pN/s. The resulting koff was in excellent agreement with that from bulk measurements on the very same system. Combined with our previous SA-b data, we covered 15 orders of magnitude in loading rate, setting the ground for SMFS over the widest dynamic range, essential to fully describe the energy landscape of biomolecular processes.

biophysics↗

bioRxiv2024

Unraveling an unknown diversity of archaeal and bacterial tetraether membrane lipid producers in a euxinic marine system

Bacterial membrane lipids have been traditionally defined as fatty acids (FAs) bilayers linked through ester bonds, while those of Archaea as ether-linked isoprenoids forming bilayers or monolayers of membrane spanning lipids (MSLs) known as isoprenoidal glycerol dialkyl glycerol tetraethers (isoGDGTs). This paradigm has been challenged with the discovery of branched GDGTs (brGDGTs), membrane spanning ether-bound branched alkyl FAs, that are of bacterial origin but whose specific producers in the environment are often unknown. The limited number of available microbial cultures restricts the knowledge of the biological sources of membrane lipids, which in turn limits their potential applicability as biomarkers. To address this limitation, we detected membrane lipids in the Black Sea using high resolution accurate mass/mass spectrometry and inferred their potential producers by targeting lipid biosynthetic pathways encoded on the metagenome, in metagenome-assembled genomes and unbinned scaffolds. We also detected brGDGTs and overly branched GDGTs in the suboxic and euxinic waters, which are potentially attributed, to members of the Planctomycetota, Cloacimonadota, Desulfobacterota, Chloroflexota, Actinobacteria and Myxococcota--all anaerobic microorganisms. These results open a new chapter in the use of specific brGDGTs as biomarkers of anoxic conditions in marine settings and of the role of these membrane lipids in microbial adaptation.

microbiology↗

bioRxiv2024

Emergence and evolution of heterocyte glycolipid biosynthesis enabled specialized nitrogen fixation in cyanobacteria

Paleontological and phylogenomic observations have shed light on the evolution of cyanobacteria. Nevertheless, the emergence of heterocytes, specialized cells for nitrogen fixation, remains unclear. Heterocytes are surrounded by heterocyte glycolipids (HGs), which contribute to protection of the nitrogenase enzyme from oxygen. Here, by comprehensive HG identification and screening of HG biosynthesis genes throughout cyanobacteria, we identify HG analogs produced by specific and distantly related non-heterocytous cyanobacteria. These structurally less complex molecules probably acted as precursors of HGs, suggesting that HGs arose after a genomic reorganization and expansion of ancestral biosynthetic machinery, enabling the rise of cyanobacterial heterocytes in an increasingly oxygenated atmosphere. Subsequently, HG chemical structure evolved convergently in response to environmental pressures. Our results open a new chapter in the potential use of diagenetic products of HGs and HG analogs as fossils for reconstructing the evolution of multicellularity and division of labor in cyanobacteria.

microbiology↗

bioRxiv2024

Coupled metalipidomics-metagenomics reveal structurally diverse sphingolipids produced by a wide variety of marine bacteria

Microbial lipids, used as taxonomic markers and physiological indicators, have mainly been studied through cultivation. However, this approach is limited due to the scarcity of cultures of environmental microbes, thereby restricting insights into the diversity of lipids and their ecological roles. Addressing this limitation, here we apply for the first time metalipidomics combined with metagenomics in the Black Sea, classifying and tentatively identifying 1,623 lipid-like species across 18 lipid classes. We discovered over 200 novel, abundant, and structurally diverse sphingolipids in euxinic waters, including unique 1-deoxysphingolipids with long-chain fatty acids and sulfur-containing groups. Genomic analysis revealed that members of 38 bacterial phyla in the Black Sea can synthesize sphingolipids, representing a fourfold increase from previously known capabilities and accounting for up to 25% of the microbial community. These sphingolipids appear to be involved in oxidative stress response and cell wall remodeling. Our findings underscore the effectiveness of multi-omics approaches in exploring microbial chemical ecology.

microbiology↗

bioRxiv2023

Organic matter degradation in the deep, sulfidic waters of the Black Sea: Insights into the ecophysiology of novel anaerobic bacteria

Our knowledge about the physiology of deep sea (>1,000 m) microorganisms involved in organic matter (OM) degradation is still scare due to the lack of available isolates, especially from sulfidic environments. In this study, we successfully cultivated and characterized the physiology of a wide range of novel piezotolerant anaerobic bacteria affiliated with the phyla Fusobacteriota, Bacillota, Spirochaetota, Bacteroidota, Cloacimonadota, Planctomycetota, Mycoplasmatota and Chloroflexota involved in OM degradation in deep sulfidic waters of the Black Sea. The novel taxa are specialized in degrading specific types of OM and cover a wide range of physiological categories, including primary degraders, fermenters, and terminal oxidizers. This is the first report which demonstrates this for such a diverse physiological group from any sulfidic marine habitat. Collectively, this study provides a step forward in our understanding of the microbes thriving in the extreme conditions of the deep sulfidic waters of the Black Sea.

microbiology↗

bioRxiv2022

Combining DNA scaffolds and acoustic force spectroscopy to characterize individual protein bonds

Single-molecule data are of great significance in biology, chemistry, and medicine. However, experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are needed. Acoustic force spectroscopy (AFS) is an emerging manipulation technique which generates acoustic waves to apply force in parallel on a large population of microbeads tethered to a surface. We have exploited this configuration on a recently developed modular Junctured-DNA (J-DNA) scaffold designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measured its unbinding kinetics under force at the single-bond level. Special effort was made in analyzing the data in order to identify potential pitfalls. We established a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy for measuring the force dependent rupture of a single domain antibody with its antigen. We get a good agreement with standard measurement at zero force. Our technique offers single molecule precision for multiplexed measurements of interactions of biotechnological and medical interest.

biophysics↗

bioRxiv2022

Disentangling the lipid divide: Identification of key enzymes for the biosynthesis of unusual Membrane-spanning and Ether lipids in Bacteria

Bacterial membranes are composed of fatty acids (FAs) ester-linked to glycerol-3-phosphate, while archaea possess membranes made of isoprenoid chains ether-linked to glycerol-1-phosphate. Many archaeal species organize their membrane as a monolayer of membrane-spanning lipids (MSLs). Exceptions to this lipid divide are the production by some bacterial species of (ether-bound) MSLs, formed by tail-tail condensation of fatty acids resulting in the formation of (iso) diabolic acids (DAs), which are the likely precursors of paleoclimatological relevant branched glycerol dialkyl glycerol tetraether molecules. However, the enzymes responsible for their production are unknown. Here, we report the discovery of bacterial enzymes responsible for the condensation reaction of fatty acids and for ether bond formation, and confirm that the building blocks of iso-DA are branched iso-FAs. Phylogenomic analyses of the key biosynthetic genes reveal a much wider diversity of potential MSL (ether)-producing bacteria than previously thought, with significant implications for our understanding of the evolution of lipid membranes.

microbiology↗

bioRxiv2021

Unraveling the critical growth factors for stable cultivation of (nano-sized) Micrarchaeota

Micrarchaeota are members of the archaeal DPANN superphylum. These so far poorly characterized archaea have been found to have reduced genomes and likely depend on interactions with host organisms for growth and survival. Here we report on the enrichment of the first stable co-culture of a member of the Micrarchaeota together with its host, as well as the isolation of the latter. Electron microscopic analysis suggest that growth is dependent on the physical interaction of the two organisms within a biofilm. The interaction seems to be ensured by the necessity to grow in form of a biofilm. Furthermore, transcriptomic analyses indicate a shift towards biofilm formation of the host as a result of co-cultivation. Finally, genomic, metabolomic, extracellular polymeric substance (EPSs) and lipid content analyses reveal that the Micrarchaeon symbiont relies on the acquisition of metabolites from its host and thereby provide first insights into the basis of symbiont-host interactions.

microbiology↗

bioRxiv2019

Cascabel: a flexible, scalable and easy-to-use amplicon sequence data analysis pipeline

Marker gene sequencing of the rRNA operon (16S, 18S, ITS) or cytochrome c oxidase I (CO1) is a popular means to assess microbial communities of the environment, microbiomes associated with plants and animals, as well as communities of multicellular organisms via environmental DNA sequencing. Since this technique is based on sequencing a single gene rather than the entire genome, the number of reads needed per sample is lower than that required for metagenome sequencing, making marker gene sequencing affordable to nearly any laboratory. Despite the relative ease and cost-efficiency of data generation, analyzing the resulting sequence data requires computational skills that may go beyond the standard repertoire of a current molecular biologist/ecologist. We have developed Cascabel, a flexible and easy-to-use amplicon sequence data analysis pipeline, which uses Snakemake and a combination of existing and newly developed solutions for its computational steps. Cascabel takes the raw data as input and delivers a table of operational taxonomic units (OTUs) and a representative sequence tree. Our pipeline allows customizing the analyses by offering several choices for most of the steps, for example different OTU generating methods. The pipeline can make use of multiple computing nodes and scales from personal computers to computing servers. The analyses and results are fully reproducible and documented in an HTML and optional pdf report. Cascabel is freely available at Github: https://github.com/AlejandroAb/CASCABEL and licensed under GNU GPLv3.

bioinformatics↗

bioRxiv2018

Bridging the divide: bacteria synthesizing archaeal membrane lipids

Archaea synthesize membranes of isoprenoid lipids that are ether-linked to glycerol, while Bacteria/Eukarya produce membranes consisting of ester-bound fatty acids. This dichotomy in membrane lipid composition or lipid divide is believed to have arisen after the Last Universal Common Ancestor (LUCA). A leading hypothesis is that LUCA possessed a mixed heterochiral archaeal/bacterial membrane, however no natural microbial representatives supporting this scenario have been shown to exist today. Here, we demonstrate that bacteria of the Fibrobacteres-Chlorobi-Bacteroidetes (FCB) group superphylum and related candidate phyla encode a complete pathway for archaeal membrane lipid biosynthesis in addition to the bacterial fatty acid membrane pathway. Key genes were expressed in the environment and their recombinant expression in E. coli resulted in the formation of a mixed archaeal/bacterial membrane. Our results support the existence of mixed membranes in natural environments and their stability over large evolutionary timescales, thereby bridging a once-thought fundamental divide in biology.

evolutionary biology↗