Characterization of the bacteriophage PhiKMV DNA ligase.

Gene 17 product (gp17) of the Pseudomonas aeruginosa-infecting bacteriophage phiKMV shows in silico similarity to T7 DNA ligase. In a semi-quantitative activity assay, it is shown that gp17 is a functional, ATP-dependent DNA ligase, in spite of some structural differences related to DNA-binding properties). Enzymatic activity of His6-based purified expression product was optimised (4 degrees C at 24h for sticky end double-stranded DNA fragments) and estimated at 0.5 Weiss U/microg.

Homotypic interactions among bacteriophage phiKMV early proteins.

Little is known about the bacteriophage proteins expressed immediately after infection of the host cell. Most of these early proteins are probably involved in bacteriophage-host interactions redirecting the bacterial metabolism to phage production. Interaction analysis of the first 16 phiKMV gene products (gp) identified homotypic interactions of gp7, gp9 and gp15. Two related yeast two-hybrid procedures, a matrix and a minilibrary approach, were applied to detect protein-protein interactions. A two-step selection procedure enabled drastic reduction of the background. Interactions were confirmed by drop tests. Multimerization of gp15 is consistent with its putative function as a DNA helicase involved in DNA replication. Homotypic interaction of gp7 and gp9 suggests they function as dimers or multimers. The absence of heterotypic interactions among early phiKMV proteins hints at their functional independence from other early phage proteins and their involvement in phage-host interactions that are important for creating optimal conditions for phage propagation. Besides, these results demonstrate the compatibility of phiKMV early gene products with the yeast two-hybrid system. Therefore, they are promising candidates to screen for interactions with host proteins.