Biology subjects

Egorov, A. A.

Publications and source records attributed to Egorov, A. A..

12 recordsLinked to original sources

bioRxiv2026

Characterization of six environmental coli-phages isolated in Astana, Kazakhstan, during the School of Molecular and Theoretical Biology

Bacteriophage (phage) collections are essential resources for studying virus-host interactions in bacterial species. Here, we report six Escherichia coli-infecting phages that expand the Lund Collection of Bacteriophages. These phages were isolated in 2025 within the framework of the School of Molecular and Theoretical Biology for high-school students, from samples collected in Lake Taldykol, Astana, Kazakhstan, using E. coli strains MG1655{Delta}RM and EV36 as hosts. The isolated phages comprise Taldykol (LuPh6), a member of the genus Kagunavirus; Aidakhar (LuPh7) of the genus Phapecoctavirus; Samruk (LuPh8) of the genus Tequintavirus; the T-odd-like phage Baiterek (LuPh9) of the genus Vequintavirus; and two T-even-like phages Tulpar (LuPh10) and Shurale (LuPh11) that belong to the Tequatrovirus genus. This expanded phage collection enhances the toolkit for investigating phage-host interactions and their molecular mechanisms and highlights the use of phage isolation as a component of high school research education. ImportancePhage collections are a key resource for studying phage biology, phage-bacteria interactions and bacterial immune systems. Here, we extend the Lund Phage Collection through the isolation and characterisation of six E. coli-infecting phages, including three novel species (LuPh6, LuPh8 and LuPh11) as well as a member of the genus Phapecoctavirus that not represented in widely used collections such as BASEL (LuPh7). This study expands the resources available for probing phage-host interactions and demonstrates an example of integrating phage research into education of high school students.

microbiology↗

bioRxiv2026

Structures and enzymatic mechanisms of DRT7/UG10 antiphage reverse transcriptases

The ongoing evolutionary arms race between bacteria and bacteriophages has driven the emergence of numerous anti-phage defense systems. Several of these defenses contain domains related to reverse transcriptases (RTs), DNA polymerases that utilize RNA as a template. Certain members of the Abi/UG RT family employ a unique enzymatic mechanism combining protein-primed DNA synthesis with template-independent polymerization. A subgroup, UG10 - also known as Defense-related RT 7 (DRT7) - is characterized by an enzymatic core comprising an RT-like domain and a primase domain, accompanied by a poorly characterized accessory element. This element is either C-terminally fused to the core or encoded by a separate open reading frame. Here, we present cryo-EM structures of protein-DNA conjugates from two UG10 enzymes, revealing a compact yet flexible domain architecture. We demonstrate that the poly(dT) product synthesized by the RT-like domain serves as a template for the primase domain to generate a complementary poly(A) strand. Combining biochemical experiments with structural modeling, we propose the conformational changes necessary for protein priming and coordination between the RT and primase domains. Finally, we show that DRT7 exhibits broad-spectrum anti-phage activity and demonstrate that the {lambda} phage DNA mimic protein Gam can trigger this defense system.

molecular biology↗

bioRxiv2025

Characterization of five environmental phages infecting Escherichia coli K-12 isolated during a phage biology training course

Phage collections are essential tools for discovering and dissecting bacterial anti-phage defense systems. Here, we report the isolation and characterization of five environmental Escherichia coli-infecting phages, obtained during the 2023 Fundamentals of Basic and Applied Phage Biology course at Lund University. The phages were isolated using a motile E. coli K-12 BW25113 strain, whose motility is conferred by an IS5 insertion upstream of the flhDC operon, the master regulator of flagellar synthesis. The isolated Escherichia phages include Lubas (LuPh1) and Lucat (LuPh2) of the genus Tequatrovirus; Lupin (LuPh3) and Lucris (LuPh4) of the genus Tequintavirus; and Kompetensportalen (LuPh5) of the genus Chivirus. Transmission electron microscopy confirmed myovirus and siphovirus morphologies consistent with these genera. As expected for phages in the flagellotropic Chivirus genus, LuPh5 failed to infect a poorly motile BW25113 strain lacking the IS5 element upstream of flhDC. By testing a panel of eight previously described anti-phage defense systems, we found that LuPh1 and LuPh2 are inhibited by the toxin-antitoxin-chaperone CmdTAC system; LuPh5 is inhibited by both the restriction-modification system EcoRI and the abortive infection reverse transcriptase AbiK; and all five phages are sensitive to the hybrid artificial CmdTA-HigC system. Collectively, our findings expand the toolkit for probing phage-host interactions and underscore the pedagogical value of incorporating phage isolation into practical training for emerging researchers.

microbiology↗

bioRxiv2024

Systematic annotation of hyper-variability hotspots in phage genomes and plasmids

Bacterial and bacteriophage genomes contain genomic regions of hyper-variability (diversity hotspots) caused by insertions of mobile genetic elements (MGEs), non-homologous recombination events and non-horizontal hypermutation. Accessory genes encoded in the diversity hotspots are involved in anti-MGE defence and counter-defence, virulence and antimicrobial resistance (AMR), thus playing key roles in interactions amongst phages, MGEs, bacteria and eukaryotic hosts. To date the majority of research has been focused on either individual hotspots or on relatively limited sets of hotspots in a small set of genomes, typically from a single species. A global understanding of hotspot diversity and dynamics still lacking. To address this gap, we developed iLund4u, an algorithm for the systematic annotation of hotspots across millions of sequences. Using a proteome composition approach, iLund4u detects proteome communities, annotates accessory proteins and identifies hotspots. By analysing 873K phage genomes and 696K plasmid sequences we identified 13.7K hotspots and 171K diverse protein families encoded there as cargo. Furthermore, iLund4u allows for protein search and proteome annotation functions versus a precomputed iLund4u database. In the protein search mode iLund4u identifies all hotspots that encode homologues of a query protein. In the proteome annotation mode iLund4u annotates hotspots by searching for communities of similar proteomes. Detailed documentation, user guide and the source code are available at the iLund4u home page: art-egorov.github.io/ilund4u.

microbiology↗

bioRxiv2024

LoVis4u: Locus Visualisation tool for comparative genomics

SummaryComparative genomic analysis often involves visualisation of alignments of genomic loci. While several software tools are available for this task, ranging from Python and R libraries to standalone graphical user interfaces, there is lack of a tool that offers fast, automated usage and the production of publication-ready vector images. Here we present LoVis4u, a command-line tool and Python API designed for highly customizable and fast visualisation of multiple genomic loci. LoVis4u generates vector images in PDF format based on annotation data from GenBank or GFF files. It is capable of visualising entire genomes of bacteriophages as well as plasmids and user-defined regions of longer prokaryotic genomes. Additionally, LoVis4u offers optional data processing steps to identify and highlight accessory and core genes in input sequences. Availability and ImplementationLoVis4u is implemented in Python3 and runs on Linux and MacOS. The command-line interface covers most practical use cases, while the provided Python API allows usage within a Python program, integration into external tools, and additional customisation. Source code is available at the GitHub page: github.com/art-egorov/lovis4u. Detailed documentation that includes an example-driven guide is available from the software home page: art-egorov.github.io/lovis4u.

bioinformatics↗

bioRxiv2024

The ABCF ATPase New1 resolves translation termination defects associated with specific tRNAArg and tRNALys> isoacceptors in the P site

The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts.

molecular biology↗

bioRxiv2024

Mechanism of phage sensing and abortion by toxin-antitoxin-chaperone systems

Toxin-antitoxins (TAs) are prokaryotic two-gene systems comprised of a toxin neutralised by an antitoxin. Toxin-antitoxin-chaperone (TAC) systems additionally include a SecB-like chaperone that stabilises the antitoxin by recognising its chaperone addiction (ChAD) element. TACs have been shown to mediate antiphage defence, but the mechanisms of viral sensing and restriction are unexplored. We identify and characterise two Escherichia coli antiphage TAC systems containing HigBA and CmdTA TA units, HigBAC and CmdTAC. The HigBAC is triggered through recognition of the gpV major tail protein of phage {lambda}. Both the ChAD and gpV are recognised by the HigC chaperone through analogous aromatic molecular patterns, explaining the mechanism of activation. We show that the CmdT ADP-ribosyltransferase toxin modifies mRNA to shut down protein synthesis. We establish the modularity of TACs by creating a hybrid broad-spectrum antiphage system combining the CmdTA TA warhead with the HigC chaperone phage sensor. HighlightsE. coli HigBAC and CmdTAC are translation-targeting phage immunity TAC systems HigC chaperone recognises phage {lambda} major tail protein to trigger HigBAC toxicity CmdT ADP-ribosyltransferase toxin abrogates translation through modification of mRNA HigC combined with CmdTA yields hybrid broad-spectrum antiphage defence system

microbiology↗

bioRxiv2024

A Comprehensive Resource for Exploring Antiphage Defense: DefenseFinder Webservice, Wiki and Databases.

In recent years, a vast number of novel antiphage defense mechanisms were uncovered. To facilitate the exploration of mechanistic, ecological, and evolutionary aspects related to antiphage defense systems, we released DefenseFinder in 2021 (Tesson et al., 2022). DefenseFinder is a bioinformatic program designed for the systematic identification of known antiphage defense mechanisms. The initial release of DefenseFinder v1.0.0 included 60 systems. Over the past three years, the number of antiphage systems incorporated into DefenseFinder has grown to 152. The increasing number of known systems makes it a challenge to enter the field and makes the interpretation of detections of antiphage systems difficult. Moreover, the rapid development of sequence-based predictions of structures offers novel possibilities of analysis and should be easily available. To overcome these challenges, we present a hub of resources on defense systems, including: 1) an updated version of DefenseFinder with a web-service search function, 2) a community-curated repository of knowledge on the systems, and 3) precomputed databases, which include annotations done on RefSeq genomes and structure predictions generated by AlphaFold. These pages can be freely accessed for users as a starting point on their journey to better understand a given system. We anticipate that these resources will foster the use of bioinformatics in the study of antiphage systems and will serve the community of researchers who study antiphage systems. This resource is available at: https://defensefinder.mdmlab.fr.

genomics↗

bioRxiv2023

Human tissues exhibit diverse composition of translation machinery

While protein synthesis is vital for the majority of cell types of the human body, diversely differentiated cells require specific translation regulation. This suggests specialization of translation machinery across tissues and organs. Using transcriptomic data from GTEx, FANTOM, and Gene Atlas we systematically explored the abundance of transcripts encoding translation factors and aminoacyl-tRNA synthetases (ARSases) in human tissues. We revised a few known and identified several novel translation-related genes exhibiting strict tissue-specific expression. The proteins they encode include eEF1A1, eEF1A2, PABPC1L, PABPC3, eIF1B, eIF4E1B, eIF4ENIF1, and eIF5AL1. Furthermore, our analysis revealed a pervasive tissue-specific relative abundance of translation machinery components (e.g. PABP and eRF3 paralogs, eIF2B subunits, eIF5MPs, and some ARSases), suggesting presumptive variance in the composition of translation initiation, elongation, and termination complexes. These conclusions were largely confirmed by the analysis of proteomic data. Finally, we paid attention to sexual dimorphism in the repertoire of translation factors encoded in sex chromosomes (eIF1A, eIF2{gamma}, and DDX3), and identified testis and brain as organs with the most diverged expression of translation-associated genes.

molecular biology↗

bioRxiv2022

Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria: VmlR2, Ard1 and CplR

Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge.

microbiology↗

bioRxiv2022

uORF4u: a tool for annotation of conserved upstream open reading frames

SummaryUpstream open reading frames (uORFs, encoding so-called leader peptides) can regulate translation and transcription of downstream main ORFs (mORFs) in prokaryotes and eukaryotes. However, annotation of novel functional uORFs is challenging due their short size of usually less than 100 codons. While transcription- and translation-level next generation sequencing (NGS) methods can be used for genome-wide uORF identification, this data is not available for the vast majority of species with sequenced genomes. At the same time, the exponentially increasing amount of genome assemblies gives us the opportunity to take advantage of evolutionary conservation in our predictions of ORFs. Here we present a tool for conserved uORF annotation in 5' upstream sequences of a user-defined protein of interest or a set of protein homologues. It can also be used to find small ORFs within a set of nucleotide sequences. The output includes publication-quality figures with multiple sequence alignments, sequence logos and locus annotation of the predicted uORFs in graphical vector format. Availability and ImplementationuORF4u is written in Python3 and runs on Linux and MacOS. The command-line interface covers most practical use cases, while the provided Python API allows usage within a Python program and additional customisation. Source code is available from the GitHub page: https://github.com/art-egorov/uorf4u. Detailed documentation that includes an example-driven guide available at the software home page: https://art-egorov.github.io/uorf4u.

bioinformatics↗

bioRxiv2021

A standard knockout procedure alters expression of adjacent loci at the translational level

The S. cerevisiae gene deletion collection is widely used for functional gene annotation and genetic interaction analyses. However, the standard G418-resistance cassette used to produce knockout mutants delivers strong regulatory elements into the target genetic loci. To date, its side effects on the expression of neighboring genes have never been systematically assessed. Here, using ribosome profiling data, RT-qPCR, and reporter expression, we investigated perturbations induced by the KanMX module. Our analysis revealed significant alterations in the transcription efficiency of neighboring genes and, more importantly, severe impairment of their mRNA translation, leading to changes in protein abundance. In the "head-to-head" orientation of the neighbor and the deleted gene, knockout often led to a shift of the transcription start site of the neighboring gene, introducing new uAUG codon(s) into the expanded 5 untranslated region (5 UTR). In the "tail-to-tail" arrangement, knockout led to activation of alternative polyadenylation signals in the neighboring gene, thus altering its 3 UTR. These events may explain the so-called neighboring gene effect (NGE), i.e. false genetic interactions of the deleted genes. We estimate that in as much as [~]1/5 of knockout strains the expression of neighboring genes may be substantially (>2-fold) deregulated at the level of translation.

molecular biology↗