Biology subjects

Pozhydaieva, N.

Publications and source records attributed to Pozhydaieva, N..

5 recordsLinked to original sources

bioRxiv2026

DNA-intercalating antiphage molecules trigger abortive infection through mutual destruction and synergize with bacterial immunity

Bacteria deploy diverse antiphage defense systems, including small bioactive molecules providing protection at the multicellular level. DNA-intercalating anthracyclines, such as daunorubicin, exhibit broad antiphage activity, but the underlying mechanism has remained elusive. Here, we systematically screened the Escherichia coli BASEL phage collection to elucidate the mode of action of DNA-intercalating antiphage molecules. We identified taxonomically distinct clusters of susceptible viral groups and show that, in T5-like phages (Markadamsvirinae), daunorubicin blocks infection after first-step transfer (FST). In the presence of daunorubicin, continued expression of pre-early genes leads to abortive infection via mutual destruction, where both phage and host succumb. Analogous abortive-infection phenotypes occur across taxonomically diverse phages exposed to chemically distinct DNA-intercalating molecules. Notably, we show that daunorubicin synergizes with downstream nucleic acid-targeting defenses underscoring context-dependent outcomes. Together, these findings reveal how chemical defense contributes to the multilayered antiviral immunity and highlight the intricate interplay between mechanistic inhibition and infection outcome.

microbiology↗

bioRxiv2025

Strain-level diversity of giant viruses infecting chlorarachniophyte algae in the subtropical North Pacific

Giant DNA viruses are ubiquitous among unicellular eukaryotes and occur in marine, freshwater, and terrestrial environments. Despite intense metagenomic data mining, their strain-level diversity remains largely unexplored. Here we introduce a model system comprising four isolates of a giant virus called ChlorV, which infects marine microalgae of the class Chlorarachniophyceae (Rhizaria) from station ALOHA, Hawaii. The ChlorV genomes are 469 kbp to 493 kbp long and encode approximately 400 proteins, at least 106 of which are present n purified virions. Although the four viral genomes are highly syntenic, they differ by several insertions and deletions that often encode methyltransferases. Interestingly, we found that some of these methyltransferase genes correlated with specific DNA methylation patterns in the ChlorV strain Our study describes the first giant viruses infecting the eukaryotic supergroup Rhizaria and demonstrates how viral strain-level variation in gene content and epigenetic features may affect eco-evolutionary processes in marine microalgae.

microbiology↗

bioRxiv2024

T4 phage RNA is NAD-capped and alters the NAD-capepitranscriptome of Escherichia coli during infection through a phage-encoded decapping enzyme

Nicotinamide adenine dinucleotide (NAD+) serves as a cap-like structure on cellular RNAs (NAD-RNAs) across all domains of life, including Escherichia coli. Beyond its role in metabolism, NAD+ also functions as a regulatory molecule in phage defense mechanisms. However, NAD-RNAs have not yet been identified during bacteriophage infections, and the mechanisms governing their synthesis and degradation in this context remain unknown. To address this gap, we used T4 phage infection of E. coli as a defined and well-characterized model system to study NAD-RNAs at the virus-host interface. Here, we report the first identification and characterization of NAD-RNAs during phage infection. Using time-resolved NAD captureSeq, we identified NAD-capped host and T4 phage transcripts and observed that the set of enriched NAD-capped RNAs varies across different infection phases. Importantly, NAD captureSeq identifies NAD-capped transcripts based on enrichment and does not directly report the fraction of molecules that are NAD-capped. Consequently, temporal changes in enrichment may reflect altered NAD-capping, altered transcript abundance, or a combination of both. We provide evidence that NAD-RNAs are generated by the host RNA polymerase by initiating transcription with NAD+ at canonical transcription start sites. By quantifying intracellular NAD+ and bulk NAD-capped RNA during infection, we observe parallel decreases in both parameters over the course of infection. Furthermore, we characterize NudE.1, a T4 phage-encoded Nudix hydrolase previously shown to have in vitro NAD-RNA decapping activity. Together, our work presents the first time-resolved analysis of an RNA modification in a host-phage system, defining the landscape, dynamics, and turnover of NAD-capped RNAs during infection and providing a framework for future studies addressing their regulatory functions in phage biology.

microbiology↗

bioRxiv2024

Temporal epigenome modulation enables efficient bacteriophage engineering and functional analysis of phage DNA modifications

Lytic bacteriophages hold substantial promise in medical and biotechnological applications. CRISPR-Cas systems offer a way to explore these mechanisms via site-specific phage mutagenesis. However, phages can resist Cas-mediated cleavage through extensive DNA modifications like cytosine glycosylation, hindering mutagenesis efficiency. Our study utilizes the eukaryotic enzyme NgTET to temporarily reduce phage DNA modifications, facilitating Cas nuclease cleavage and enhancing mutagenesis efficiency. This approach enables precise DNA targeting and seamless point mutation integration, exemplified by deactivating specific ADP-ribosyltransferases crucial for phage infection. Furthermore, by temporally removing DNA modifications, we elucidated the effects of these modifications on T4 phage infections without necessitating gene deletions. Our results present a strategy enabling the investigation of phage epigenome functions and streamlining the engineering of phages with cytosine DNA modifications. The described temporal modulation of the phage epigenome is valuable for synthetic biology and fundamental research to comprehend phage infection mechanisms through the generation of mutants.

microbiology↗

bioRxiv2022

Identification of NAD-RNAs and ADPR-RNA decapping in the archaeal model organisms Sulfolobus acidocaldarius and Haloferax volcanii

NAD is a coenzyme central to metabolism that was also found to serve as a 5-terminal cap of bacterial and eukaryotic RNA species. The presence and functionality of NAD-capped RNAs (NAD-RNAs) in the archaeal domain remain to be characterized in detail. Here, by combining LC-MS and NAD captureSeq methodology, we quantified the total levels of NAD-RNAs and determined the identity of NAD-RNAs in the two model archaea, Sulfolobus acidocaldarius and Haloferax volcanii. A complementary differential RNA-Seq (dRNA-Seq) analysis revealed that NAD transcription start sites (NAD-TSS) correlate with well-defined promoter regions and often overlap with primary transcription start sites (pTSS). The population of NAD-RNAs in the two archaeal organisms shows clear differences, with S. acidocaldarius possessing more capped small non-coding RNAs (sncRNAs) and leader sequences. The NAD-cap did not prevent 5[->]3 exonucleolytic activity by the RNase Saci-aCPSF2. To investigate enzymes that facilitate the removal of the NAD-cap, four Nudix proteins of S. acidocaldarius were screened. None of the recombinant proteins showed NAD decapping activity. Instead, the Nudix protein Saci_NudT5 showed activity after incubating NAD-RNAs at elevated temperatures. Hyperthermophilic environments promote the thermal degradation of NAD into the toxic product ADPR. Incorporating NAD into RNAs and the regulation of ADPR-RNA decapping by Saci_NudT5 is proposed to provide additional layers of maintaining stable NAD levels in archaeal cells. ImportanceThis study reports the first characterization of 5-terminally modified RNA molecules in Archaea and establishes that NAD-RNA modifications, previously only identified in the other two domains of life, are also prevalent in the archaeal model organisms Sulfolobus acidocaldarius and Haloferax volcanii. We screened for NUDIX hydrolases that could remove the NAD-RNA cap and showed that none of these enzymes removed NAD modifications, but we discovered an enzyme that hydrolyzes ADPR-RNA. We propose that these activities influence the stabilization of NAD and its thermal degradation to potentially toxic ADPR products at elevated growth temperatures.

molecular biology↗