Biology subjects

Bae, H.-W.

Publications and source records attributed to Bae, H.-W..

3 recordsLinked to original sources

bioRxiv2026

Systematic mapping of insertion-tolerant regions enables capsid engineering of the RNA phage PP7

RNA phages are attractive platforms for the design of programmable bioparticles, but their development has been constrained by limited knowledge of genomic sites that can tolerate sequence insertion. Here, we combined MuA transposase-mediated in vitro insertion mutagenesis with our established reverse genetics systems to systematically identify insertion-tolerant regions (ITRs) in the RNA phages MS2 and PP7. Screening of 4,555 MS2 and 2,228 PP7 random insertion clones identified 29 and 26 nonredundant ITRs, respectively. We further analyzed and compared these ITRs in the context of RNA genome organization and virion architecture. Both phages contained ITRs within the maturation protein (MP), whereas only PP7 tolerated insertions within the coat protein (CP). On the basis of structural location and plaque-forming capacity, an ITR situated between Gly74 and Glu75 (GGC^GAG) in the PP7 CP was selected for further study. Infectious phage particles generated from cDNA clones retained the 15-bp insertion at both the RNA and protein levels. Engineered PP7 phages carrying an RGD motif inserted into the CP at this ITR displayed enhanced in vivo clearance in a Drosophila model, despite having in vitro stability comparable to that of the wild type. These findings provide the first example of CP engineering in an RNA phage and establish a framework for engineering RNA phages for biological and biotechnological applications. IMPORTANCEA major obstacle to developing RNA phages as synthetic biology platforms is the lack of design principles for genomic insertion. Here, we address this limitation by establishing a mutagenesis-and-recovery workflow that systematically identifies insertion-tolerant regions (ITRs) in the RNA phages MS2 and PP7. The resulting maps reveal distinct structural constraints in the two phages and enable rational engineering of a peptide-display site in the PP7 capsid. Using this approach, we generated an engineered infectious phage with a modified capsid, thereby providing the first demonstration of capsid engineering in an RNA phage. This study lays the groundwork for the rational design of RNA phage virions as tractable and engineerable scaffolds for future biological and biotechnological applications.

microbiology↗

bioRxiv2026

cDNA-guided functional selection uncovers selective defense systems against RNA phages

Bacteria encode diverse antiphage defense systems, yet mechanisms that target RNA phages remain comparatively underexplored. Here, we used a cDNA-based functional selection strategy to systematically identify genes that confer resistance to RNA phage infection independently of receptor variation in Pseudomonas aeruginosa. This approach uncovered previously uncharacterized antiphage defense systems, most of which are located within genomic islands, consistent with their being bona fide components of bacterial immune systems. Several systems conferred selective resistance to RNA phages and their carriage was associated with pilin variability, suggesting layered anti-phage immunity. Among these systems, Zws is the most prevalent RNA phage defense system and functions as a multidomain effector. Structural modeling and in vitro cleavage assays showed that ZwsA is an RNA endonuclease that selectively cleaves RNA phage genomes through a predicted NERD domain. Together, these findings expand the current framework of bacterial antiphage immunity and highlight the power of functional genomics to uncover cryptic components of the bacterial antiviral arsenal. IMPORTANCEBacteria harbor a broad repertoire of antiphage defense systems, but our understanding of mechanisms that target RNA phages remains limited, being heavily biased toward defenses against DNA phages. By applying a cDNA-based functional selection strategy, this study overcomes a major obstacle in defense-gene discovery and uncovers previously uncharacterized genes that represent bona fide components of the bacterial immune arsenal against RNA phages. The identification and characterization of Zowangsin (ZwsA), a NERD-domain RNA endonuclease that selectively cleaves specific signatures within RNA phage genomes, establish targeted RNA degradation as a central principle of bacterial defense against RNA phages. More broadly, this work expands the conceptual framework of bacterial antiviral immunity and illustrates the utility of functional selection for uncovering cryptic immune systems with implications for phage biology, RNA biology, and biotechnology. HIGHLIGTHSO_LIcDNA-based functional screen identifies six defense systems that restrict RNA phages C_LIO_LIThese defense systems are enriched within genomic islands of Pseudomonas aeruginosa C_LIO_LIZws, Szs, and Mws systems confer selective defense against RNA phages C_LIO_LIZwsA is a signature-selective RNA endonuclease that targets phage genomic RNA C_LI

microbiology↗

bioRxiv2024

A bacterial site 2 protease contributes to RNA phage resistance by targeting phage lysis protein

Lytic phages culminate their lifecycle by causing lysis of the infected host cell. Despite extensive research on the molecular mechanisms of phage lysis, our understanding of anti-phage resistance mechanisms during the lysis stage remains less understood. Here, we demonstrated that MucP, a site 2 protease of Pseudomonas aeruginosa (PA), mitigates the activity of the RNA phage PP7 lysis protein (LP), which contains a transmembrane (TM) helix, suggesting that MucP act as a resistance mechanism against PP7-induced lysis. We identified an LP variant (LP*) having enhanced helical propensity due to P26L and S40L mutations, which was unaffected by MucP and exhibited killing activity against PA strains that are resistant to the wild type LP, with an inverse correlation between MucP activity and LP susceptibility. A PP7 mutant with LP* exhibited MucP-escaper phenotype such as discernable plaque formation on MucP-expressing cells. These results suggest that MucP targets the RNA phage LP at the TM helix in certain strains, providing a resistance function compromising phage lysis by utilizing an existing bacterial enzyme in PA. IMPORTANCEDespite the importance of cell lysis as the last stage of phage lifecycle, the factors influencing coordinated lysis remain elusive in the context of phage-bacteria interactions. Our study identifies MucP, a membrane protease in Pseudomonas aeruginosa (PA), as a resistance factor against the RNA phage PP7 by destabilizing its lysis protein (LP), with lower helical tendency at transmembrane (TM) domain. An LP variant with higher helical tendency exhibits strong killing activity against PA isolates, revealing an inverse correlation between MucP activity and LP susceptibility. Since MucP is a conserved protease in mucoid conversion of PA, we propose that MucP offers an intrinsic or passive defense or resistance mechanism against the RNA phage, whose activities vary among the diverse PA isolates.

microbiology↗