Biology subjects

Thongchol, J.

Publications and source records attributed to Thongchol, J..

3 recordsLinked to original sources

bioRxiv2026

A reference genome without a virus: cDNA reconstruction reveals the provenance and function of the MS2 phage sequence

Reference genomes are often treated as faithful representations of experimentally validated viral genomes, yet the relationship between historically curated reference sequences and infectivity is rarely tested experimentally. Here, we developed a cDNA-based reconstruction platform for the canonical RNA phage MS2 and used it to compare the current NCBI reference genome (RefSeq) with closely related published isolate sequences. We found that isolate-derived sequences reproducibly yielded infectious phage, whereas the current MS2 RefSeq-derived construct did not, showing that the present reference does not represent a single experimentally validated infectious genome but instead reflects sequence curation across multiple studies. We then compared conventional and AI-enabled approaches to identify minimal changes that restore infectivity to MS2 RefSeq; a human experimentalist correctly prioritized corrective changes, whereas the genome language model Evo2 did not. We also observed that closely related corrected reference-derived constructs showed a ~4-log difference in phage output, and subsequent analysis indicated that this difference was associated with an apparent replicase frameshift in the lower-output background. This suggests that the low output construct class represents rare mutations from genomes that are one mutational step away from true function, rather than uniform function of the dominant construct population. A complementary cell-free assay provided a lower-background orthogonal readout of construct-level function, yielding ~1 x106 PFU/mL from the high-output background within 2 hours while showing no detectable recovery from the low-output background. Together, these results establish a robust platform for RNA phage reconstruction and raise the possibility that historical reference genomes, especially for RNA viruses, may not always remain faithful to experimentally validated biological function. More broadly, these findings underscore the need to verify the infectivity of reference genomes, particularly when they were assembled non-contiguously or shaped by cumulative human curation. They also highlight the importance of clearly distinguishing historically curated reference sequences from experimentally validated infectious genomes when such data are used to train or evaluate AI/ML models.

microbiology↗

bioRxiv2026

Genome-wide characterization of host factors involved in single-stranded RNA and DNA phage infection pathways

Single stranded RNA (ssRNA) and single stranded DNA (ssDNA) bacteriophages represent a key component of the global virome, yet the host genetic networks supporting their infection cycles remain poorly understood. Here, we present a comprehensive, genome-wide mapping of the genetic landscape regulating infection cycles for F pilus-dependent ssRNA and ssDNA phages in Escherichia coli. Genetic screens across ssRNA phages spanning all four genogroups of the Leviviricetes revealed a highly conserved network of host dependencies, with the notable exception of the F plasmid gene traD. While primary structural receptor components and dsbA mediated disulfide bond formation are universally required across all lineages to ensure F pilus integrity, traD exhibits a strict genogroup-specific requirement during entry, showing variable essentiality across different viral groups despite sharing an identical primary receptor. Our gene dosage screens revealed that an elevated copy number of the hslU protease or the RNA chaperone stpA restricts infection, identifying clear genetic barriers that can perturb the viral life cycle. Parallel assays with filamentous ssDNA phages produced host factor profiles consistent with published literature, while revealing additional variations in host dependency. These screens confirmed that ssDNA phages strictly rely on the host TolQRA complex for entry downstream of pilus engagement. The assays tracked prominent negative fitness signatures across homeostatic clusters, highlighting how the physiological burden of continuous, non-lytic virion extrusion strains the host envelope. Finally, this comparative approach traced the selectivity of our isolation host (E. coli HSF) to a horizontally acquired capsule architecture from Klebsiella. This surface shield excludes a large panel of double stranded DNA phages isolated on diverse E. coli strains, while allowing virions from ssDNA and ssRNA phages to engage the extended F pilus and bypass the barrier via native pilus retraction. Together, this work provides a systematic, class-wide map of single stranded phage-host interactions, bridging classical genetics with modern viral discovery while establishing a robust host platform to access uncultured viral diversity and a functional blueprint to design next generation diagnostics, protein antibiotics, and biocontrol tools to halt horizontal gene transfer.

microbiology↗

bioRxiv2026

Suppressing Transfer of Antibiotic Resistance by a Small RNA Virus

The global rise of antimicrobial resistance (AMR) demands innovative strategies to limit the spread of multidrug-resistant bacteria. Conjugative plasmids, particularly those in the incompatibility group P (IncP), play a central role in disseminating resistance genes across diverse bacterial species via their encoded Type IV secretion systems (T4SS). Here, we characterize the single-stranded RNA bacteriophage (ssRNA phage) PRR1, which selectively targets AMR ESKAPEE pathogens carrying the IncP plasmid RP4, and assess its ability to inhibit conjugation. Using cryo-electron microscopy, we first resolved the mature PRR1 virion at 3.45 [A] resolution revealing two phage maturation protein (Mat)-RNA interactions within the 3 untranslated region (UTR) - a conserved interaction (Mat-U1) and a novel interaction (Mat-V1) for ssRNA phages. To characterize the PRR1-RP4 pilus interaction, we performed alanine-scanning mutagenesis and pinpointed four critical TrbC pilin residues (S12, W13, S72, and R77) for infection. Computational modeling revealed that these residues are located near the termini of the pilin at the phage-pilus interface. Notably, native and non-infectious, UV-crosslinked PRR1 were sufficient to block RP4 transfer, indicating conjugation inhibition does not require a complete infection cycle. Finally, combining PRR1 and antibiotic treatment yielded nine unique phage-resistant mutants within T4SS-associated genes on the RP4 plasmid. Eight of these mutants nearly abolished conjugation, while the trbE frameshift mutant retained [~]30% of wild-type efficiency, which is pivotal to clarifying the relationship between phage infection and pilus function. Collectively, these results establish ssRNA phages as specific T4SS plasmid targeting agents and underscore their potential to limit horizontal gene transfer in AMR pathogens. IMPORTANCEAntimicrobial resistance (AMR) spreads rapidly through horizontal gene transfer, largely driven by conjugative plasmids. Despite their central role, few strategies exist to directly block plasmid transfer. Here, we show that the IncP plasmid-dependent ssRNA phage PRR1 can inhibit the spread of antibiotic resistance genes by targeting the RP4 T4SS pilus. Structural and mutational analyses reveal previously unrecognized RNA packaging interactions and identify four pilin residues critical for infection. Remarkably, non-infectious PRR1 particles alone are sufficient to block conjugation, offering inhibition without the selective pressure from phage replication. Almost all PRR1-resistant RP4 mutants lost or had severely reduced plasmid transfer, while the remaining mutant is critical for studying the link between T4SS function and phage infection. These results highlight ssRNA phages as precise agents for limiting AMR gene dissemination.

microbiology↗