Pseudomonas aeruginosa bacteriophage phiKMV requires type IV pili for infection, as observed from the phenotypic characterization and phage adsorption assays on a phage infection-resistant host strain mutant. A cosmid clone library of the host (P. aeruginosa PAO1) genomic DNA was generated and used to select for a clone that was able to restore phiKMV infection in the resistant mutant. This complementing cosmid also re-established type IV pili-dependent twitching motility. The correlation between bacteriophage phiKMV infectivity and type IV pili, along with its associated twitching motility, was confirmed by the resistance of a P. aeruginosa PAO1DeltapilA mutant to the phage. Subcloning of the complementing cosmid and further phage infection analysis and motility assays suggests that a common regulatory mechanism and/or interaction between the ponA and pilMNOPQ gene products are essential for bacteriophage phiKMV infectivity.