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Abstract Acinetobacters pose a significant threat to human health, especially those with weakened immune systems. Type IV pili of acinetobacters play crucial roles in virulence and antibiotic resistance. Single-stranded RNA bacteriophages target the bacterial retractile pili, including type IV. Our study delves into the interaction betweenAcinetobacterphage AP205 and type IV pili. Using cryo-electron microscopy, we solve structures of the AP205 virion with an asymmetric dimer of maturation proteins, the nativeAcinetobactertype IV pili bearing a distinct post-translational pilin cleavage, and the pili-bound AP205 showing its maturation proteins adapted to pilin modifications, allowing each phage to bind to one or two pili. Leveraging these results, we develop a 20-kilodalton AP205-derived protein scaffold targeting type IV pili in situ, with potential for research and diagnostics. 

Abstract Leviviruses are bacteriophages with small single-stranded RNA genomes consisting of 3-4 genes, one of which (sgl) encodes a protein that induces the host to undergo autolysis and liberate progeny virions. Recent meta-transcriptomic studies have uncovered thousands of leviviral genomes, but most of these lack an annotatedsgl, mainly due to the small size, lack of sequence similarity, and embedded nature of these genes. Here, we identifysglgenes in 244 leviviral genomes and functionally characterize them inEscherichia coli. We show that leviviruses readily evolvesglgenes and sometimes have more than one per genome. Moreover, these genes share little to no similarity with each other or to previously knownsglgenes, thus representing a rich source for potential protein antibiotics. 

Although the F-specific ssRNA phage MS2 has long had paradigm status, little is known about penetration of the genomic RNA (gRNA) into the cell. The phage initially binds to the F-pilus using its maturation protein (Mat), and then the Mat-bound gRNA is released from the viral capsid and somehow crosses the bacterial envelope into the cytoplasm. To address the mechanics of this process, we fluorescently labeled the ssRNA phage MS2 to track F-pilus dynamics during infection. We discovered that ssRNA phage infection triggers the release of F-pili from host cells, and that higher multiplicity of infection (MOI) correlates with detachment of longer F-pili. We also report that entry of gRNA into the host cytoplasm requires the F-plasmid–encoded coupling protein, TraD, which is located at the cytoplasmic entrance of the F-encoded type IV secretion system (T4SS). However, TraD is not essential for pilus detachment, indicating that detachment is triggered by an early step of MS2 engagement with the F-pilus or T4SS. We propose a multistep model in which the ssRNA phage binds to the F-pilus and through pilus retraction engages with the distal end of the T4SS channel at the cell surface. Continued pilus retraction pulls the Mat-gRNA complex out of the virion into the T4SS channel, causing a torsional stress that breaks the mature F-pilus at the cell surface. We propose that phage-induced disruptions of F-pilus dynamics provides a selective advantage for infecting phages and thus may be prevalent among the phages specific for retractile pili.