Comparative genomics and structure-informed analysis identify a putative tandem receptor-binding module in Pseudomonas phage UF_RH1

Abstract Bacteriophage receptor-binding proteins (RBPs) determine host recognition and host range but are frequently missed by conventional annotation because they evolve rapidly and often lack recognizable sequence domains. Pseudomonas phage UF_RH1 is a lytic Septimatrevirus with a 42,567 bp double-stranded DNA genome encoding 57 predicted open reading frames, several of which remain annotated as hypothetical proteins. Here, we reanalyzed the UF_RH1 distal-tail region using comparative synteny, phage RBP specific prediction tools, structure-informed domain analysis, oligomeric-symmetry prediction, and independent physicochemical calculations. Two adjacent proteins, WCF59042.1 (317 aa) and WCF59043.1 (320 aa), emerged as the strongest candidates for functional reannotation. WCF59042.1 was classified by DepoScope as a tail-spike protein with probability 1.00 and contains a high confidence C terminal tail-spike-associated segment spanning residues 210 to 317. WCF59043.1 was independently classified as a tail-spike candidate by RBPdetect2 (probability 0.51) and showed a strong Seq2Symm prediction for C3 homo-trimeric symmetry (0.990). Comparative genomic analysis places these proteins at positions corresponding to DLP2 gp22 and gp23, two similarly sized hypothetical virion proteins immediately downstream of the tape measure gene in a conserved distal-tail morphogenesis cassette. Because the closely related phages DLP1 and DLP2 use type IV pili for adsorption, we propose a testable model in which WCF59042.1 functions as a proximal connector/adaptor and WCF59043.1 contributes a distal trimeric receptor binding element. The model is computational and does not establish direct binding to PilA or assembled pili. Together, the convergent sequence, structural, oligomeric, and syntenic evidence supports a previously unrecognized tandem distal tail receptor-binding module in UF_RH1 and provides experimentally tractable targets for defining Septimatrevirus adsorption and host range.