Emmanuele Severi (@emmseveri.bsky.social)

Cross-family and phage-specific gene requirements for Klebsiella infection revealed by scalable RB-TnSeq genetic screens Bacteriophages (phages) are being cataloged at unprecedented rates and are recognized as key players in nutrient and energy cycling across ecosystems. Yet, the biological determinants of phage-host specificity and infection success remain poorly understood. Here, we used a randomly barcoded, genome-wide, loss-of-function transposon mutant library (RB-TnSeq) of Klebsiella sp. M5al, a plant-associated, nitrogen-fixing rhizobacterium, to identify bacterial genetic determinants necessary for the infection of 25 double-stranded DNA phages spanning five viral families. This approach identified 42 bacterial genes associated with phage infection, encompassing genes involved in receptor biosynthesis, regulatory pathways, electron transport, and unknown functions. Disruption of genes involved in receptor biosynthesis, such as glycosyltransferases involved in lipopolysaccharide (LPS) production, conferred cross-resistance to half of the phages, while intracellular gene disruptions had phage-specific effects. Taxonomically, we found significant differences in RB-TnSeq profiles across phage families and genera. While bacterial gene requirements are generally clustered by phage genus, we also observed notable divergences within genera. These differences were associated with variation in receptor usage, likely driven by divergence in tail fiber or other host recognition proteins. In some cases, differential reliance on bacterial intracellular factors suggested that even closely related phages may depend on distinct infection strategies, potentially involving phage-specific genes of unknown function. Our findings reveal key genetic dependencies shaping phage-host interactions and offer a framework for predicting infection outcomes in natural and applied settings. ### Competing Interest Statement The authors have declared no competing interest. Department of Energy and Environment, https://ror.org/05d5hbz44, #DE-SC0020173, #DE-SC0023307 https://www.biorxiv.org/content/10.64898/2026.03.14.711032v1