Biology subjects

Buck, M.

Publications and source records attributed to Buck, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Genome size variation between pelagic and benthic communities across prokaryotic taxonomy and environmental gradients in the Baltic Sea

While theories and models have appeared to explain genome size as a result of evolutionary processes, little work has shown that genome sizes carry ecological signatures. Our work delves into the ecological implications of microbial genome size variation in benthic and pelagic habitats across environmental gradients of the brackish Baltic Sea. While depth is significantly associated with genome size in benthic and pelagic brackish metagenomes, salinity is only correlated to genome size in benthic metagenomes. Overall, we confirm that prokaryotic genome sizes in Baltic sediments (3.47 Mbp) are significantly bigger than in the water column (2.96 Mbp). While benthic genomes have a higher number of functions than pelagic genomes, the smallest genomes coded for a higher number of module steps per Mbp for most of the functions irrespective of their environment. Some examples of this functions are amino acid metabolism and central carbohydrate metabolism. However, we observed that nitrogen metabolism was almost absent in pelagic genomes and was mostly present in benthic genomes. Finally, we also show that Bacteria inhabiting Baltic sediments and water column not only differ in taxonomy, but also in their metabolic potential, such as the Wood-Ljungdahl pathway or the presence of different hydrogenases. Our work shows how microbial genome size is linked to abiotic factors in the environment, metabolic potential and taxonomic identity of Bacteria and Archaea within aquatic ecosystems.

microbiology↗

bioRxiv2022

Development of novel cytoprotective small compounds inhibiting mitochondria-dependent apoptosis

We identified cyto-protective small molecules (CSMs) by a cell-based high-throughput screening of Bax inhibitors. Through a medicinal chemistry program, M109S was developed, which is orally bioactive and penetrates the blood-brain/retina barriers. M109S protected retinal cells in the mouse models of Stargardt disease and macular degeneration. M109S directly interacted with Bax and inhibited the conformational change and mitochondrial translocation of Bax. M109S inhibited ABT-737-induced apoptosis both in Bax-only and Bak-only MEFs. M109S also inhibited apoptosis induced by staurosporine (mouse embryonic fibroblasts), etoposide (Neuro2a cells), and obatoclax (ARPE19 cells). M109S is a novel small molecule protecting cells from mitochondria-dependent apoptosis both in vitro and in vivo. M109S has the potential to become a new research tool for studying cell death mechanisms and to develop therapeutics targeting mitochondria-dependent cell death pathway. (128words)

cell biology↗

bioRxiv2022

A metagenomic view on fungal diversity in freshwater lakes

Fungi are essential components in a wide range of ecosystems and while major efforts have been spent on disentangling the diversity and functional roles of fungi in terrestrial environments, our knowledge about aquatic fungi is lagging. To address this knowledge gap, we explored metagenomes from 25 lakes from the arctic and boreal zone and one tropical reservoir with the aim of describing the community structure of fungi and fungi-like organisms (Oomycota). A second objective was to identify possible environmental factors influencing the composition of the fungal communities. Our results show that the main fungal phyla and orders are the same across all the lakes despite the differences in geographic location and prevailing climate. Still, there was differential distribution of more highly resolved taxa across the lakes that accordingly featured distinct communities, possibly caused by differential availability of carbon substrates in the lakes. A more detailed classification of sequences related to the pathogenic Oomycota genus Phytophthora clearly demonstrated that while technologies now exist for sequencing entire microbial communities in great detail, we are still severely limited by insufficient coverage of eukaryotic sequences and genomes in public databases.

ecology↗

bioRxiv2022

Lola-I is a developmentally regulated promoter pioneer factor

While enhancers are often regulated at the level of accessibility by pioneer factors, promoters tend to be constitutively accessible and poised for activation by paused Pol II -- thus are often not considered as sites of developmental regulation. Here we show that the accessibility of promoters and the acquisition of paused Pol II can be subject to developmental regulation by pioneer factors. We show that Lola-I, a Drosophila zinc finger transcription factor, is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene expression. Lola-I mediates this function by binding to the edges of the promoter nucleosomes, which leads to their depletion, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers, providing further evidence that promoters and enhancers display unexpectedly similar characteristics.

genomics↗

bioRxiv2022

Exploring intra-species diversity through non-redundant pangenome assemblies

At the genome level, microorganisms are highly adaptable both in terms of allele and gene composition. Such heritable traits emerge in response to different environmental niches and can have a profound influence on microbial community dynamics. As a consequence of this, any individual genome or clonal population will contain merely a fraction of the total genetic diversity of any operationally defined "species", with the collective from that group presenting the broader genomic diversity known as the pangenome. Pangenomes are valuable concepts for studying evolution and adaptation in microorganisms, as they partition genomes into core regions (present in all the genomes, and responsible for housekeeping and species-level niche adaptation) and accessory regions (present only in some genomes, and responsible for ecotype divergence). Here we present SuperPang, an algorithm capable of producing pangenome assemblies from a set of input genomes of varying quality, including metagenome-assembled genomes or MAGs. SuperPang runs in linear time and its results are complete, non-redundant, preserve gene ordering and contain both coding and non-coding regions. Our approach provides a modular view of the pangenome, identifying operons and genomic islands, and allowing to track their prevalence in different populations. We illustrate our approach by analyzing the intra-species diversity of Polynucleobacter, a clade of ubiquitous freshwater microorganisms characterized by their streamlined genomes and their ecological versatility. We show how SuperPang facilitates the simultaneous analysis of allelic and gene content variation under different environmental pressures, allowing us to study the drivers of microbial diversification at unprecedented resolution.

bioinformatics↗

bioRxiv2022

Taxonomic and functional diversity of aquatic heterotrophs is sustained by dissolved organic matter chemodiversity

Dissolved organic matter (DOM) is ubiquitous in aquatic ecosystems and fundamental for planetary processes and ecosystem functioning. While the link between microbial community composition and heterotrophic utilization of DOM has been recognized, the full diversity of organic compounds, their bioavailability, degradability and specific influences on the diversity and function of heterotrophs are still not clear. Here we experimentally investigate heterotrophic bacteria in thirty-three freshwater model communities. We identified 34 different heterotrophs growing in ambient lake DOM with taxonomic affiliations matching abundant freshwater bacterioplankton. We further describe 25 different heterotrophs growing in the phycosphere of M. Aeruginosa and 6 heterotrophs growing on the DOM produced by M. Aeruginosa with taxonomic affiliation in accord to phycosphere heterotrophs. In our experiment we observed that heterotrophs that live in the phycosphere remove more dissolved organic carbon than abundant freshwater heterotrophs. Moreover, phycosphere heterotrophs have bigger genomes than abundant lake bacteria. Altogether of the 4224 chemical features that were resolved by LC-MS, only 1229 were seen in all three treatments. None of the common/shared compounds were removed across all the model communities, suggesting contrasting niches of the studied taxa. Altogether our study highlights how each model community, with its unique taxonomic assemblages and organotroph functioning is upkept by the chemodiversity of DOM.

ecology↗

bioRxiv2022

A consensus protocol for the recovery of mercury methylation genes from metagenomes

Mercury methylation genes (hgcAB) mediate the formation of the toxic methylmercury and have been identified from diverse environments, including freshwater and marine ecosystems, Arctic permafrost, forest and paddy soils, coal-ash amended sediments, chlor-alkali plants discharges and geothermal springs. Here we present the first attempt at a standardized protocol for the detection, identification and quantification of hgc genes from metagenomes. Our Hg-MATE (Hg-cycling Microorganisms in Aquatic and Terrestrial Ecosystems) database, a catalogue of hgc genes, provides the most accurate information to date on the taxonomic identity and functional/metabolic attributes of microorganisms responsible for Hg methylation in the environment. Furthermore, we introduce "marky-coco", a ready-to-use bioinformatic pipeline based on de novo single-metagenome assembly, for easy and accurate characterization of hgc genes from environmental samples. We compared the recovery of hgc genes from environmental metagenomes using the marky-coco pipeline with an approach based on co-assembly of multiple metagenomes. Our data show similar efficiency in both approaches for most environments except those with high diversity (i.e., paddy soils) for which a co-assembly approach was preferred. Finally, we discuss the definition of true hgc genes and methods to normalize hgc gene counts from metagenomes.

microbiology↗

bioRxiv2022

Abundance and expression of hgcAB genes and mercury availability jointly explain methylmercury formation in stratified brackish waters

Neurotoxic methylmercury (MeHg) is formed by microbial methylation of inorganic divalent Hg (HgII) and constitutes severe environmental and human health risks. The methylation is enabled by hgcA and hgcB genes, but it is not known if the associated molecular-level processes are rate-limiting or enable accurate prediction of MeHg formation in nature. In this study, we investigated the relationships between hgcA genes and MeHg across redox stratified water columns in the brackish Baltic Sea. We found that the abundance of hgcA genes and transcripts combined with the concentration of dissolved HgII-sulfide species were strong predictors of both HgII methylation rate and MeHg concentration, implying their roles as principal joint drivers of MeHg formation in these systems. In establishing relationships between hgcA genes and MeHg, we advance the fundamental understanding of mechanistic principles governing MeHg formation in nature and enable refined predictions of MeHg levels in coastal seas in response to the accelerating spread of oxygen deficient zones.

ecology↗

bioRxiv2021

16S rRNA gene sequences of Candidatus Methylumidiphilus (Methylococcales), a putative methanotrophic genus in lakes and ponds

A putative novel methanotrophic genus, Candidatus Methylumidiphilus (Methylococcales), was recently shown to be ubiquitous and one of the most abundant methanotrophic genera in water columns of oxygen-stratified lakes and ponds of boreal and subarctic area. However, it has probably escaped detection in many previous studies using 16S rRNA gene amplicon sequencing due to insufficient database coverage, which is because Ca. Methylumidiphilus lacks cultured representatives and previously analysed metagenome assembled genomes (MAGs) affiliated with it do not contain 16S rRNA genes. Therefore, we screened MAGs affiliated with the genus for their 16S rRNA gene sequences in a recently published lake and pond MAG dataset. Among 66 MAGs classified as Ca. Methylumidiphilus (with completeness over 40% and contamination less than 5%) originating from lakes in Finland, Sweden and Switzerland as well as from ponds in Canada, we could find 5 MAGs each containing one 1532 bp long sequence spanning the V1-V9 regions of the 16S rRNA gene. After removal of sequence redundancy, this resulted in two unique 16S rRNA gene sequences. These sequences represented two different putative species, i.e. Ca. Methylumidiphilus alinenensis (Genbank accession: OK236221) as well as another so far unnamed species of Ca. Methylumidiphilus (Genbank accession: OK236220). We suggest that including these two sequences in reference databases will enhance 16S rRNA gene - based detection of members of this genus from environmental samples.

ecology↗

bioRxiv2021

Predicting Transmembrane (TM) Domain Dimer Structures using Martini 3

Determination of the structure and dynamics of transmembrane (TM) regions of single-transmembrane receptors is key to understanding their mechanism of signal transduction across the plasma membrane. Although many studies have been performed on isolated soluble extra- and intracellular receptor domains in aqueous solutions, limited knowledge exists on the lipid embedded TM domain. In this study, we examine the assembly of configurations of receptor TM region dimers using the Martini 3 force field for coarse-grain (CG) molecular dynamics simulations. This recently published version of Martini has new bead types and sizes, which allows more accurate predictions of molecular interactions compared to the previous versions. At first glance our results with Martini 3 simulations show only a reasonable agreement with ab initio predictions using PREDDIMER (for TM domains only), AlphaFold2 Multimer and with available NMR derived structures for TM helix dimers. Specifically, 6 of 11 CG TM structures are similar to the NMR structures (within < 3.5 [A] mainchain RMSD) compared to 10 of 11 and 9 of 11 using PREDDIMER and Alphafold, respectively (7 structures of the latter are within 1.5 [A]) Surprisingly, AlphaFold2 predictions are more comparable with NMR structures when the database of 2001 (mainly composed of soluble proteins) instead of 2020 PDB structures are used. While there are some differences in the conditions used, the CG simulations primarily reveal that alternate configurations of the TM dimers that are sampled, which readily interconvert with a predominant population. The implications of these findings for our understanding of the signalling mechanism of TM receptors are discussed, including opportunities for the development of new pharmaceuticals, some of which are peptide based.

biophysics↗

bioRxiv2021

mOTUpan: a robust Bayesian approach to leverage metagenome assembled genomes for core-genome estimation

Recent advances in sequencing and bioinformatics have expanded the tree of life by providing genomes for uncultured environmentally relevant clades, either through metagenome assembled genomes (MAGs) or single-cell assembled genomes (SAGs). While this expanded diversity can provide novel insights about microbial population structure, most tools available for core-genome estimation are overly sensitive to genome completeness. Consequently a major portion of the huge phylogenetic diversity uncovered by environmental genomics approaches remain excluded from such analyses. We present mOTUpan, a novel iterative Bayesian method for computing the core-genome for sets of genomes of highly diverse completeness range. The likelihood for each gene-cluster to belong to core- or accessory-genome is estimated by computing the probability of its presence/absence pattern in the target set of genomes. The core-genome prediction is computationally efficient and can be scaled up to thousands of genomes. It has shown comparable estimates as state-of-the-art tools Roary and PPanGGoLiN for high-quality genomes and outperforms them at lower completeness thresholds. mOTUpan wraps a bootstrapping procedure to estimate the quality of a specific core-genome prediction, as the accuracy of each run will depend on the specific completeness distribution and the number of genomes in the dataset under scrutiny. mOTUpan is implemented in the mOTUlizer software package, and available at github.com/moritzbuck/mOTUlizer, under GPL 3.0 license.

bioinformatics↗

bioRxiv2021

Cell Surface Multimeric Assemblies Regulate Canonical and Noncanonical EphA2 Receptor Tyrosine Kinase Signaling

The EphA2 receptor tyrosine kinase mediates ligand-induced canonical signaling associated with tumor suppression and ligand-independent noncanonical signaling implicated in tumor progression. Using time-resolved fluorescence spectroscopy in live cells, we find that unliganded EphA2 receptors pre-assemble into multimers, which is mediated by two symmetric and one asymmetric interfaces in the ectodomain. Upon ligand binding, EphA2 receptors are further assemble into large clusters that also requires the three interfaces. Functionally, disrupting either the symmetric or asymmetric contacts individually blocks the autorecycling of the EphA2 apo receptor. However, only symmetric contact disruption promotes noncanonical signaling and inhibits ligand-induced catalytic activation and endocytosis, which are associated with increased cell migration in vitro and reduced survival in a syngeneic murine glioblastoma model. Our results reveal the pivotal role of EphA2 assembly in dictating canonical vs. noncanonical signaling, and identify the precise molecular interfaces that mediate the formation of the EphA2 signaling clusters.

biophysics↗

bioRxiv2021

Conformational clamping by a membrane ligand activates the EphA2 receptor

The EphA2 receptor is a promising drug target for cancer treatment, since EphA2 activation can inhibit metastasis and tumor progression. It has been recently described that the TYPE7 peptide activates EphA2 using a novel mechanism that involves binding to the single transmembrane domain of the receptor. TYPE7 is a conditional transmembrane (TM) ligand, which only inserts into membranes at neutral pH in the presence of the TM region of EphA2. However, how membrane interactions can activate EphA2 is not known. We systematically altered the sequence of TYPE7 to identify the binding motif used to activate EphA2. With the resulting six peptides, we performed biophysical and cell migration assays that identified a new potent peptide variant. We also performed a mutational screen that determined the helical interface that mediates dimerization of the TM domain of EphA2 in cells. These results, together with molecular dynamic simulations, allowed to elucidate the molecular mechanism that TYPE7 uses to activate EphA2, where the membrane peptide acts as a molecular clamp that wraps around the TM dimer of the receptor. We propose that this binding mode stabilizes the active conformation of EphA2. Our data, additionally, provide clues into the properties that TM ligands need to have in order to achieve activation of membrane receptors.

biophysics↗

bioRxiv2021

Towards the NMR solution Structure and the Dynamics of the C-terminal Region of APOL1 and its G1, G2 Variants with a Membrane Mimetic

Secreted apolipoprotein L1 (APOL1) is well known as an innate immune factor, protecting against African trypanosomiasis. The intracellular form has multiple functions, including regulating autophagy, intracellular vesicle trafficking, and ion channel activity. The APOL1 protein (G0) has two common variants (denoted G1 and G2) in the C-terminal region and are associated with a high risk of chronic kidney disease (CKD) and progression to end-stage kidney disease. Our previous studies using molecular modeling suggested that APOL1 G1 and G2 stabilize an autoinhibited state of the C-terminus, leading to impaired intracellular interactions with SNARE proteins. To characterize the structural consequence of kidney disease-associated APOL1 variants further, we assigned the C-terminal region proteins using 1H, 13C, 15N multidimensional nuclear magnetic resonance (NMR) spectra in solution in the presence of membrane mimetic dodecylphosphocholine micelles. We then derived models for the three-dimensional structure of APOL1-G0, and -G1 and -G2 variant C-terminal regions using the chemical shifts of the main chain nuclei followed by NMR relaxation measurements. The data suggest that changes in the three-dimensional structure of APOL1 C-terminal region induced by kidney disease-associated variants, not least the alteration of key sidechains and their interactions, could disrupt membrane association and the yet to be characterized protein-protein interactions including its binding partners, such as SNARE proteins. Such interactions could underlie the intracellular mechanisms that mediate the pathogenesis of CKD. In the future, one may try to reverse such structural and dynamics changes in the protein by designing agents that may bind and then mitigate APOL1 variant-associated CKD.

biophysics↗

bioRxiv2021

Comprehensive analysis of chemical and biological problems associated with browning agents used in aquatic studies

Inland waters receive and process large amounts of colored organic matter from the terrestrial surroundings. These inputs dramatically affect the chemical, physical, and biological properties of water bodies, as well as their roles as global carbon sinks and sources. To understand the complex changes associated with allochthonous inputs, experiments are needed. However, manipulative studies, especially at ecosystem scales, require large amounts of dissolved organic matter with optical and chemical properties resembling indigenous organic matter. Here we compared the chemical and biological impacts of two leonardite products (HuminFeed (HF) and SuperHume (SH)) and a freshly derived reverse osmosis concentrate of organic matter (RO) in a set of comprehensive mesocosm- and laboratory-scale experiments and analyses. The chemical properties of RO concentrate and the leonardite products were very different with leonardite products being low and RO being high in carboxylic functional groups. Light had a strong impact on the properties of leonardite products, including loss of color and increased particle formation. Furthermore, HF had drastic impacts on bacteria as light stimulated bacterial production and modified community composition, while dark conditions appeared to inhibit bacterial processes. While none of the browning agents inhibited the growth of the tested phytoplankton, Gonyostomum semen, leonardite products had detrimental effects on zooplankton abundance and Daphnia reproduction. We conclude that the effects of browning agents extracted from leonardite are in sharp contrast to those originating from terrestrially-derived DOM. Hence, they should be used with great caution in experimental studies on the consequences of terrestrial carbon for aquatic systems.

ecology↗

bioRxiv2021

Resolving the Interactions Between Class 3 Semaphorin Receptors in Live Cells

The plexin/neuropilin/semaphorin family of proteins is involved with tissue patterning in the developing embryo. These proteins play roles in cell migration and adhesion, but are also important in disease, including cancer angiogenesis and metastasis. While some structures of the soluble domains of these proteins have been determined, the conformations of full-length receptor complexes are just beginning to be studied, especially within the context of the cell plasma membrane. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) allows direct insight to the formation of protein-protein interactions in the membrane of live cells. Here we investigated the homodimerization of neuropilin-1, Plexin A2, Plexin A4, and Plexin D1. Consistent with previous studies, we found that neuropilin-1, Plexin A2 and Plexin A4 are dimers in the absence of exogenous ligand. Plexin D1, on the other hand, was monomeric under similar conditions, which had not been previously reported. We also found that Plexin A2 and A4 assemble into a heteromeric complex. Stimulation with Semaphorin 3A or Semaphorin 3C ligand neither disrupts nor enhances the dimerization of the receptors when they are expressed alone, suggesting that activation involves a conformational change rather than a shift in the monomer-dimer equilibrium. However, upon stimulation with Semaphorin 3C, Plexin D1 and neuropilin-1 form a heteromeric complex, while Semaphorin 3A does not induce a stable complex with these receptors. This analysis of interactions by PIE-FCCS provides a complementary approach to the existing structural and biochemical data that will aid in the development of new therapeutic strategies to target these receptors during disease.

biophysics↗

bioRxiv2021

A genomic perspective on genome size distribution across Earth's microbiomes reveals a tendency to gene loss

Our view of genome size in Archaea and Bacteria has remained skewed as the data used to paint its picture has been dominated by genomes of microorganisms that can be cultivated under laboratory settings. However, the continuous effort to catalog the genetic make-up of Earths microbiomes specifically propelled by recent extensive work on uncultivated microorganisms, provides a unique opportunity to revise our perspective on genome size distribution. Capitalizing on a recently released extensive catalog of tens of thousands of metagenome-assembled genomes, we provide a comprehensive overview of genome size distributions. We observe that the known phylogenetic diversity of environmental microorganisms possesses significantly smaller genomes than the collection of laboratory isolated microorganisms. Aquatic microorganisms average 3.1 Mb, host-associated microbial genomes average 3.0 Mb, terrestrial microorganism average 3.7 Mb and isolated microorganisms average 4.3 Mb. While the environment where the microorganisms live can certainly be linked to genome size, in some cases, evolutionary phylogenetic history can be a stronger predictor. Moreover, ecological strategies such as auxotrophies have a direct impact on genome size. To better understand the ecological drivers of genome size, we expand on the known and the overlooked factors that influence genome size in different environments, phylogenetic groups and trophic strategies.

microbiology↗

bioRxiv2021

Neuropilin-1 Assists SARS-CoV-2 Infection by Stimulating the Separation of Spike Protein Domains S1 and S2

The cell surface receptor Neuropilin-1 (Nrp1) was recently identified as a host factor for SARS-CoV-2 entry. As the Spike protein of SARS-CoV-2 is cleaved into the S1 and the S2 domain by furin protease, Nrp1 binds to the newly created C-terminal RRAR amino acid sequence of the S1 domain. In this study, we model the association of a Nrp1 (a2-b1-b2) protein with the Spike protein computationally and analyze the topological constraints in the SARS-CoV-2 Spike protein for binding with Nrp1 and ACE2. Importantly, we study the exit mechanism of S2 from the S1 domain with the assistance of ACE2 as well as Nrp1 by molecular dynamics pulling simulations. In the presence of Nrp1, by binding the S1 more strongly to the host membrane, there is a high probability of S2 being pulled out, rather than S1 being stretched. Thus, Nrp1 binding could stimulate the exit of S2 from the S1 domain, which will likely increase virus infectivity as the liberated S2 domain mediates the fusion of virus and host membranes. Understanding of such a Nrp1-assisted viral infection opens the gate for the generation of protein-protein inhibitors, such as antibodies, which could attenuate the infection mechanism and protect certain cells in a future combination therapy.

biophysics↗