Polyvalent phage GSP004 recognizes O-antigen polysaccharide receptors in Salmonella and Escherichia coli through tail fiber protein ORF208

ABSTRACT The majority of phages are capable of lysing only specific bacterial strains within a single species, and polyvalent phages with cross-genus lytic capability are relatively rare. In this study, we isolated a polyvalent phage, GSP004, from swine farm wastewater, which exhibited cross-genus lytic activity against multiple Salmonella serovars and Escherichia coli ( E. coli ) O157:H7. Morphological classification assigns GSP004 to the family Myoviridae within the order Caudovirales , while phylogenetic analysis of its genome identifies it as a member of the genus Kuttervirus in the family Ackermannviridae . The periodate/proteinase K assays confirmed bacterial surface polysaccharides as the host receptor targeted by GSP004. Combined with lipopolysaccharide (LPS) competitive adsorption assays, gene knockout strain spot/adsorption/dynamic lysis assays, and phage nucleic acid release experiments, we demonstrated that the O-antigen of LPS was the sole receptor for GSP004 to infect Salmonella and E. coli O157:H7, respectively. Subsequent characterization via protein competition adsorption, antibody blocking, and fluorescent labeling experiments identified the tail protein ORF208 as the receptor-binding protein (RBP) recognizing LPS O-antigens. Further studies revealed that phage GSP004 employs its tail protein ORF208 to recognize and bind to the LPS O-antigen of Salmonella and E. coli through distinct molecular mechanisms, thereby mediating cross-genus infection. This finding provides critical molecular insights into the interaction between polyvalent phages and their bacterial hosts. IMPORTANCE Elucidating the molecular mechanism of cross-genus host recognition in polyvalent phages will provide a critical theoretical foundation for the rational design of broad-host-range phages. However, research on the cross-genus host recognition mechanisms of polyvalent phages remains scarce. Here, we isolated a polyvalent phage GSP004, which serves as an exemplary model for investigating the interaction mechanisms between such polyvalent phages and their bacterial hosts. Our study elucidates the molecular basis underlying the capability of GSP004 to simultaneously infect Salmonella and E. coli O157:H7 across genera. This study provides crucial molecular evidence for understanding the evolutionary strategies by which phages expand their cross-genus host range and establishes a theoretical foundation for the rational design of broad-host-range phage therapeutics.