Identification of an R1-type pyocin previously misannotated as a prophage in Pseudomonas aeruginosa ATCC 27853

While Pseudomonas aeruginosa ATCC 27853 is a widely used reference strain with previously characterized prophage regions, our use of one of the latest prophage prediction tools, PHASTEST, helped reveal a critical misclassification in its genome. Using this tool, we initially identified six prophage regions, with four classified as intact; however, in-depth analysis demonstrated that one of these predicted intact prophages was, in fact, a functional pyocin-encoding region. Specifically, the region spanning 679,586-698,056 bp, initially annotated as an intact prophage, was definitively re-identified as a region harbouring an R1-type pyocin. The most recent literature regarding prophages in P. aeruginosa ATCC 27853 classifies the region spanning 683,173-696,044 bp as a prophage. This region falls entirely within the genomic region we describe and reclassify, further emphasizing the importance of the reclassification performed in this study. The identified R1-type pyocin was induced using mitomycin C, processed via tangential flow filtration, and its bactericidal activity was confirmed against a clinical P. aeruginosa isolate via spot-test killing assays and absorbance-based assays. Transmission electron microscopy revealed R-type pyocin particles averaging 133 nm in length. This misidentification of a pyocin as a prophage critically underscores the inherent limitations of current bioinformatic tools in accurately distinguishing between these distinct phage-derived elements, thereby highlighting the urgent need for more refined annotation methodologies. Accurate identification of such elements is essential, as they may influence experimental outcomes and provide new insights into bacterial defence mechanisms.