Biology subjects

Isaev, A.

Publications and source records attributed to Isaev, A..

7 recordsLinked to original sources

bioRxiv2025

A census of anti-CRISPR proteins reveals AcrIE9 as an inhibitor of Escherichia coli K12 Type IE CRISPR-Cas system

CRISPR-Cas adaptive immunity systems provide defense against mobile genetic elements and are often countered by diverse anti-CRISPR (Acr) proteins. The Type IE CRISPR-Cas of Escherichia coli K12 has been a model for structural and functional studies and is a part of the species core genome. However, this system is transcriptionally silent, which has fueled questions about its true biological function. To clarify the role of this system in defense, we carried out a census of Acr proteins found in Enterobacterales and identified AcrIE9 as a potent inhibitor of the E. coli K12 Type IE CRISPR-Cas system. While sharing little sequence identity, AcrIE9 proteins from Pseudomonas and Escherichia both interact with the Cas7 subunit of the Cascade complex, thus preventing its binding to DNA. We further show that AcrIE9 is genetically linked to AcrIE10, forming the most widespread anti-CRISPR cluster in Enterobacterales, and this module often co-occurs with a novel HTH-like protein with unusual architecture.

microbiology↗

bioRxiv2025

Taxonomically different symbiotic communities of sympatric Arctic sponge species show functional similarity with specialization at species level

Marine sponges harbor diverse communities of associated organisms, including eukaryotes, viruses, and bacteria. Sponge associated microbiomes contribute to the health of the host organisms by defending them against invading bacteria and providing them with essential metabolites. Here we describe microbiomes of three sympatric species of cold-water marine sponges - Halichondria panicea, Halichondria sitiens, and Isodictya palmata - sampled over a period of six years at the White Sea. We identified the sponges as low microbial abundance species and detected stably associated bacteria that represent new taxa of sponge symbionts within Alpha- and Gammaproteobacteria. The sponges carried unique sets of unrelated species of symbiotic bacteria illustrating varying complexity of microbiomes. On a community level, sponge associated microbiomes shared common symbiotic features; they encoded multiple eukaryotic-like proteins, biosynthetic pathways, and transporters of amino acids and vitamins essential for sponges. On a species level, however, different classes of eukaryotic-like proteins and pathways were distributed between dominant and minor symbionts indicating specialization within microbiomes. Particularly, taurine and sulfoacetate metabolism pathways were associated exclusively with dominant symbionts in all three sponge species. Our study demonstrates strong functional convergence and co-evolution of microbiomes of sympatric cold-water sponge species with a distribution of functions between community members. Additionally, we observed dramatic shifts in compositions of sponge microbiomes coinciding with abnormally high water temperatures during the 2018 season, highlighting the vulnerability of cold-water ecosystems to global warming.

microbiology↗

bioRxiv2025

Specificity and Mechanism of tRNA cleavage by the AriB Toprim nuclease of the PARIS bacterial immune system

Transfer RNA molecules have been recently recognized as widespread targets of bacterial immune systems. Translation inhibition through tRNA cleavage or modification inhibits phage propagation, thereby protecting the bacterial population. To counteract this, some viruses encode their own tRNA molecules, allowing infection to take place. The AriB effector of the PARIS defence system is a Toprim nuclease previously shown to target the E. coli tRNALys(UUU), but not a tRNALys(UUU) variant encoded by bacteriophage T5. We demonstrate here that the T5-tRNALys(UUU) is required but not sufficient to bypass PARIS immunity. Combining tRNA-sequencing, genetics, phage infection and in vitro biochemical data, we reveal that the E. coli tRNAThr(UGU), is another prime target of AriB and tRNAAsn(GUU) represents a secondary, yet biologically relevant, target of the PARIS effector. Activated AriB protein cleaves these targets in vitro, and the cleavage reaction is not dependent on the presence of specific tRNA modifications. We show that the overexpression of phage T5 tRNALys(UUU), tRNAThr(UGU) and tRNAAsn(GUU) variants is sufficient to inhibit PARIS anti-viral defence. Finally, we propose a model for tRNA recognition by the AriB dimer and provide molecular details of its nuclease activity and specificity.

microbiology↗

bioRxiv2024

Molecular basis of foreign DNA recognition by BREX anti-phage immunity system

Anti-phage systems of the BREX (BacteRiophage EXclusion) superfamily rely on epigenetic DNA methylation to discriminate between the host and invading DNA, but their mechanism of protection remains enigmatic. We demonstrate that in Type I BREX systems, both defense and methylation are based on site-specific DNA recognition by the BrxX (PglX) methyltransferase and require the S-adenosyl methionine cofactor. We present a 2.2-[A] cryoEM structure of Escherichia coli BrxX bound to target dsDNA, which reveals the molecular details of DNA recognition by BREX and paves the way for rational engineering of BREX specificity. We show that BrxX alone does not support methylation, and BREX activity requires an assembly of a supramolecular BrxBCXZ immune complex. Finally, we present a cryoEM structure of BrxX bound to a phage-encoded inhibitor Ocr that sequesters an inactive dimeric form of BrxX. Together, these results allow us to propose a model of BREX-mediated DNA sensing and anti-phage defense.

molecular biology↗

bioRxiv2024

Viral proteins activate PARIS-mediated tRNA degradation and viral tRNAs rescue infection

Viruses compete with each other for limited cellular resources, and some viruses deliver defense mechanisms that protect the host from competing genetic parasites. PARIS is a defense system, often encoded in viral genomes, that is composed of a 53 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB). Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-EM to determine the structure of this complex which explains how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving the host tRNALys. Phage T5 subverts PARIS immunity through expression of a tRNALys variant that prevents PARIS-mediated cleavage, and thereby restores viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids.

biochemistry↗

bioRxiv2023

Benchmarking DNA Isolation Methods for Marine Metagenomics Studies

Metagenomics is a powerful tool to study marine microbial communities. However, obtaining high-quality environmental DNA suitable for downstream sequencing applications is a challenging task. The quality and quantity of isolated DNA heavily depend on the choice of purification procedure and the type of sample. Selection of an appropriate DNA isolation method for a new type of material often entails a lengthy trial and error process. Further, each DNA purification approach introduces biases and thus affects the composition of the studied community. To account for these problems and biases, we systematically investigated efficiency of DNA purification from three types of samples (water, sea sediment, and digestive tract of a model invertebrate Magallana gigas) with eight commercially available microbial DNA isolation kits. For each kit-sample combination we measured the quantity of purified DNA, extent of DNA fragmentation, the presence of PCR-inhibiting contaminants, admixture of eukaryotic DNA, alpha-diversity, and reproducibility of the resulting community composition based on 16S rRNA amplicons sequencing. Additionally, we determined a "kitome", e.g., a set of contaminating taxa inherent for each type of purification kit used. The resulting matrix of evaluated parameters allows one to select the best DNA purification procedure for a given type of sample.

bioinformatics↗

bioRxiv2023

Phage T3 overcomes the BREX defence through SAM cleavage and inhibition of SAM synthesis

Bacteriophage T3 encodes a SAMase that through cleavage of S-adenosyl-methionine (SAM) circumvents the SAM-dependent Type I Restriction-Modification defence of the host bacterium Escherichia coli. Here, we show that the SAMase also allows T3 to evade BREX defence. SAM degradation weakly affects BREX methylation of host DNA, but completely inhibits the defensive function of BREX, suggesting that SAM is required as a co-factor for BREX-mediated exclusion of phage DNA. The anti-BREX activity of the T3 SAMase is mediated by two independent mechanisms: enzymatic degradation of SAM and downregulation of SAM synthesis through direct inhibition of the host SAM synthase MetK. We determined a 2.8 [A] cryo-EM structure of the eight-subunit T3 SAMase-MetK complex. Structure guided mutagenesis of the SAMase-MetK interface revealed that the interaction with MetK stabilizes the T3 SAMase in vivo, thus further stimulating its anti-BREX activity. This work provides insights in the versatility and intricacy of bacteriophage counter-defence mechanisms and highlights the role of SAM as an important co-factor of diverse phage-defence systems.

microbiology↗