Biology subjects

Plewka, J.

Publications and source records attributed to Plewka, J..

11 recordsLinked to original sources

bioRxiv2025

Crystallographic characterisation and development of bi-substrate inhibitors of coronavirus nsp14 methyltransferase

SARS-CoV-2 non-structural protein 14 (nsp14) is essential for viral mRNA cap guanine-N7 methylation and represents a promising but underexplored antiviral target. Herein we describe a structure-guided campaign based on a hit from a focussed SAM mimetic library. Systematic SAR exploration guided by six X-ray co-crystal structures in complex with SARS-CoV-2 led to compound 26, a bi-substrate inhibitor that bridges the SAM and RNA cap binding sites. Compound 26 achieved nanomolar potency against nsp14 from SARS-CoV-2 (IC50 = 53 nM), SARS-CoV-1, and two alphacoronaviruses, with excellent selectivity over human RNMT and flaviviral MTase. In general, the compounds demonstrated favourable metabolic stability, passive permeability, and no HepG2 cytotoxicity. However, cellular antiviral activity was limited, revealing disconnects between enzyme inhibition and phenotypic response. These findings provide a structural framework for optimizing bi-substrate methyltransferase inhibitors against coronaviruses with a view for pan-coronaviral activity.

biochemistry↗

bioRxiv2025

Microbial Diversity and Community Shifts in a Petroleum Reservoir under Production: Effects of Water Breakthrough and Anthropogenic Alterations

Subsurface petroleum reservoirs host indigenous microorganisms that survive extreme conditions and long-term isolation. Microbial activity in these environments can contribute to adverse effects such as oil biodegradation and reservoir souring. Unlike the broader deep biosphere, oil reservoirs are frequently subjected to anthropogenic disturbances, particularly during production processes like water injection, which introduces external microbes and electron acceptors. In this study, we investigated microbial diversity, community structure, and the impact of water breakthrough in the Edvard Grieg oil reservoir offshore Norway using 16S rRNA gene and metagenomic sequencing. We found clear regional heterogeneity in community composition, with low diversity dominated by thermophilic, anaerobic, and halotolerant taxa. The southern region (wells A13, A17, A18, and A19) exhibited lower diversity, while well A07 showed a distinct microbial signature. The dominant genera included the strictly anaerobic bacterium Thermoanaerobacter and the hyperthermophilic archaeon Thermococcus. Water breakthrough triggered shifts in community structure, not due to widespread replacement by injected microbes, but via the increase of sulfate-reducing bacteria. Metagenomic data supported these observations and suggested rapid microbial transport between injection water and the water separator. These findings support the use of microbial signatures as cost-effective tools for monitoring oil reservoir processes and integrity.

microbiology↗

bioRxiv2025

Microbial Worlds Apart: Distinct Communities in Crude Oil and Production Waters

Genomic analyses of microbial community composition are used to improve oil reservoir engineering and monitor reservoir dynamics. Given the challenges of extracting nucleic acids from oil, production water samples are often used as proxies from which to infer microbial community information from oil reservoirs. We employed 16S rRNA gene amplicon and metagenomic sequencing on samples of crude oil and production water from four North Sea oil fields. Taxonomic profiling revealed differences in microbial compositions and functions between production water and crude oil. Production water was more homogeneous, less diverse, harboured taxa associated with conditions non-native to the reservoir (e.g., seawater), and exhibited evidence of contact with atmospheric oxygen, most likely from passing through the water separators. Conversely, crude oil samples harboured microbial taxa typically associated with oil reservoirs. Despite long-term production and, in some cases, re-injection of production waters, the putative native microbial communities were still present in the oil. These findings demonstrate that crude oil samples are much more representative of oil reservoir microbiomes than their production water proxies.

microbiology↗

bioRxiv2023

Metaproteogenomics resolution of a high-CO2 aquifer community suggests an active symbiotic lifestyle of groundwater Gracilibacteria

BackgroundBacteria of the Candidate Phyla Radiation (CPR), constituting about 25% of the bacterial biodiversity, are characterized by small cell size and patchy genomes without complete key metabolic pathways, suggesting a symbiotic lifestyle. Gracilibacteria (BD1-5), which are part of the CPR branch, possess alternate coded genomes and have not yet been cultivated. However, besides genomic evidence, little is known about the lifestyle of Gracilibacteria, their temporal dynamics, and activity in natural ecosystems, particularly in groundwater, where they were initially been genomically resolved. Therefore, we here aimed to investigate Gracilibacteria activity in situ and to discern expressed genes involved in their lifestyle, using the metaproteogenome of Gracilibacteria as a function of time in the cold-water geyser Wallender Born in the Volcanic Eifel region in Germany. ResultsWe coupled genome-resolved metagenomics and metaproteomics to investigate a cold-water geyser microbial community enriched in Gracilibacteria across a 12-day time-series. Groundwater was collected and sequentially filtered to fraction CPR and other bacteria. Based on 670 Gbps of metagenomic data, 1129 different ribosomal protein S3 marker genes and 751 high-quality genomes (123 population genomes after dereplication), we identified dominant bacteria belonging to Galionellales and Gracilibacteria along with keystone microbes, which were low in genomic abundance but substantially contributing to proteomic abundance. Seven high-quality Gracilibacteria genomes showed typical limitations, such as limited amino acid or nucleotide synthesis, in their central metabolism but no co-occurrence with potential hosts. The genomes of these Gracilibacteria encoded for a high number of proteins related to a symbiotic or even predatory lifestyle, e.g., type IV and type II secretion system subunits and features related to cell-cell interactions and cell motility, which were also detected on protein level. ConclusionsCoupling metagenomics to metaproteomics enabled us to identify microbial keystone taxa in a high-CO2 aquifer, and to reveal microbial dynamics of Gracilibacteria. We posit that Gracilibacteria might be successful microbial predators in this ecosystem, potentially aiding in population control of this highly perturbed microbial geyser community from the deep biosphere.

ecology↗

bioRxiv2023

Differential expression of core metabolic functions in Candidatus Altiarchaeum inhabiting distinct subsurface ecosystems

Candidatus Altiarchaea are widespread across aquatic subsurface ecosystems and possess a highly conserved core genome, yet adaptations of this core genome to different biotic and abiotic factors based on gene expression remain unknown. Here, we investigated the metatranscriptome of two Ca. Altiarchaeum populations that thrive in two substantially different subsurface ecosystems. In Crystal Geyser, a high-CO2 groundwater system in the USA, Ca. Altiarchaeum crystalense co-occurs with the symbiont Ca. Huberiarchaeum crystalense, while in the Muehlbacher sulfidic spring in Germany, an artesian spring high in sulfide concentration, Ca. A. hamiconexum is heavily infected with viruses. We here mapped metatranscriptome reads against their genomes to analyze the in situ expression profile of their core genomes. Out of 537 shared gene clusters, 331 were functionally annotated and 130 differed significantly in expression between the two sites. Main differences were related to genes involved in cell defense like CRISPR-Cas, virus defense, replication, and transcription as well as energy and carbon metabolism. Our results demonstrate that altiarchaeal populations in the subsurface are likely adapted to their environment while influenced by other biological entities that tamper with their core metabolism. We consequently posit that viruses and symbiotic interactions can be major energy sinks for organisms in the deep biosphere. (Originality-Significance StatementOrganisms of the uncultivated phylum Ca. Altiarchaeota are globally widespread and fulfill essential roles in carbon cycling, e.g., carbon fixation in the continental subsurface. Here, we show that the transcriptional activity of organisms in the continental subsurface differ significantly depending on the geological and microbial setting of the ecosystem explaining many of the previously observed physiological traits of this organism group.)

microbiology↗

bioRxiv2022

Heads in the clouds: marine viruses disperse bidirectionally along the natural water cycle

Marine viruses have thoroughly been studied in seawater, yet their dispersal from neuston ecosystems at the air-sea interface towards the atmosphere remains a knowledge gap. Here, we show that 6.2 % of the studied virus population were shared between air-sea interface ecosystems and rainwater. Virus enrichment in the 1-mm thin surface microlayer and sea foams happened selectively, and variant analysis proved virus transfer to aerosols and rain. Viruses detected in rain and aerosols showed a significantly higher percent G/C base content compared to marine viruses, and a genetically distinct rain virome supports that those viruses could inhabit higher air masses. CRISPR spacer matches of marine prokaryotes to "foreign" viruses from rainwater prove regular virus-host encounters at the air-sea interface. Our findings on aerosolization and long-range atmospheric dispersal implicate virus-mediated carbon turnover in remote areas, viral dispersal mechanisms relevant to human health, and involvement of viruses in atmospheric processes like ice-nucleation.

microbiology↗

bioRxiv2022

MuDoGeR: Multi-Domain Genome Recovery from metagenomes made easy

Several computational frameworks and workflows that recover genomes from prokaryotes, eukaryotes, and viruses from metagenomes exist. However, it is difficult for scientists with little bioinformatics experience to evaluate quality, annotate genes, dereplicate, assign taxonomy and calculate relative abundance and coverage of genomes belonging to different domains. MuDoGeR is a user-friendly tool accessible for non-bioinformaticians that make it easy to recover genomes of prokaryotes, eukaryotes, and viruses from metagenomes, either alone or in combination. We tested MuDoGer using 24 individual-isolated genomes and 574 metagenomes, demonstrating the applicability for a few samples and high throughput. MuDoGeR is open-source software available at https://github.com/mdsufz/MuDoGeR.

bioinformatics↗

bioRxiv2022

Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity.

During the RNA replication, coronaviruses require proofreading to maintain the integrity of their large genomes. Nsp14 associates with viral polymerase complex to excise the mismatched nucleotides. Aside from the exonuclease activity, nsp14 methyltransferase domain mediates cap methylation, facilitating translation initiation and protecting viral RNA from recognition by the innate immune sensors. The nsp14 exonuclease activity is modulated by a protein co-factor nsp10. While the nsp10/nsp14 complex structure is available, the mechanistic basis for nsp10 mediated modulation remains unclear in the absence of nsp14 structure. Here we provide a crystal structure of nsp14 in an apo-form. Comparative analysis of the apo- and nsp10 bound structures explain the modulatory role of the co-factor protein. Further, the structure presented in this study rationalizes the recently proposed idea of nsp14/nsp10/nsp16 ternary complex.

microbiology↗

bioRxiv2022

Despite the odds: formation of the SARS-CoV-2 methylation complex.

Coronaviruses protect their single-stranded RNA genome with a methylated cap during replication. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16 where nsp10 acts as a co-factor to both. Aditionally, nsp14 carries an exonuclease domain, which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins should be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex. This interaction is expected to encourage formation of mature capped viral mRNA, modulating the nsp14s exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.

microbiology↗

bioRxiv2020

Competition NMR for Detection of Hit/Lead Inhibitors of Protein-Protein Interactions

Screening for small-molecule fragments that can lead to potent inhibitors of protein-protein interactions (PPIs) is often a laborious step as the fragments cannot dissociate the targeted PPI due to their low M-mM affinities. Here, we describe an NMR competition assay - called w-AIDA-NMR (weak-Antagonist Induced Dissociation Assay-NMR) - that is sensitive to weak M-mM ligand-protein interactions and which can be used in initial fragment screening campaigns. By introducing point mutations in the complexs protein that is not targeted by the inhibitor, we lower the effective affinity of the complex allowing for short fragments to dissociate the complex. We illustrate the method with the compounds that block the Mdm2/X-p53 and PD-1/PD-L1 oncogenic interactions. Targeting the PD-/PD-L1 PPI has profoundly advanced the treatment of different types of cancers.

biophysics↗

bioRxiv2019

CA-170 a potent small-molecule PD-L1 inhibitor or not?

CA-170 is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins - important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between CA-170 and PD-L1. To strengthen our reasoning, we performed control experiments on AUNP-12 - a 29-mer peptide, which is a precursor of CA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: BMS-1166 and peptide p57.

cancer biology↗