Functional and Phenotypic Analysis of Kimona gp55 and gp56
College
College of Arts and Sciences
Mentor Information
Richard Pollenz
Description
Bacteriophages encode many proteins with limited functional annotation, making it difficult to predict how individual gene products influence bacterial hosts. Phenotypic assays that directly test expressed proteins provide a more reliable way to assess biological function. Here, I evaluated the effects of Kimona phage proteins gp55 and gp56 following inducible expression in Mycobacterium smegmatis. Based on conserved sequence features, I hypothesized that gp55 functions as a DNA primase but lacks the metal-binding motif, whereas gp56 lacks the catalytic domains required for primase activity, and that neither protein would exhibit cytotoxic effects. Gene 55 encodes gp55, a 219-amino-acid protein containing core catalytic domains characteristic of DNA primases but lacking the N-terminal zinc-ribbon domain typically involved in DNA binding. Gene 56 encodes gp56, a 134-amino-acid protein that lacks these catalytic regions and instead contains only a partial zinc-binding motif consistent with an accessory primase domain. Together, these proteins are hypothesized to function as a complete primase system. Both are smaller than known cytotoxic primase-associated cytotoxic proteins, such as the Waterfowl phage gp65 primase/polymerase, a 719-amino-acid multi-domain enzyme whose catalytic architecture likely underlies its toxicity when expressed in M. smegmatis. To assess phenotypic effects, gp55 and gp56 were induced using the pExTra system activated by anhydrotetracycline (aTc) and evaluated using a plate-based cytotoxicity dilution assay on 7H9 medium. Neither protein causes detectable growth inhibition across the dilution series, thus both may be needed to show toxicity. This supports that gp55 and gp56 are consistent with other genetic screens of primases lacking cytotoxicity.
Functional and Phenotypic Analysis of Kimona gp55 and gp56
Bacteriophages encode many proteins with limited functional annotation, making it difficult to predict how individual gene products influence bacterial hosts. Phenotypic assays that directly test expressed proteins provide a more reliable way to assess biological function. Here, I evaluated the effects of Kimona phage proteins gp55 and gp56 following inducible expression in Mycobacterium smegmatis. Based on conserved sequence features, I hypothesized that gp55 functions as a DNA primase but lacks the metal-binding motif, whereas gp56 lacks the catalytic domains required for primase activity, and that neither protein would exhibit cytotoxic effects. Gene 55 encodes gp55, a 219-amino-acid protein containing core catalytic domains characteristic of DNA primases but lacking the N-terminal zinc-ribbon domain typically involved in DNA binding. Gene 56 encodes gp56, a 134-amino-acid protein that lacks these catalytic regions and instead contains only a partial zinc-binding motif consistent with an accessory primase domain. Together, these proteins are hypothesized to function as a complete primase system. Both are smaller than known cytotoxic primase-associated cytotoxic proteins, such as the Waterfowl phage gp65 primase/polymerase, a 719-amino-acid multi-domain enzyme whose catalytic architecture likely underlies its toxicity when expressed in M. smegmatis. To assess phenotypic effects, gp55 and gp56 were induced using the pExTra system activated by anhydrotetracycline (aTc) and evaluated using a plate-based cytotoxicity dilution assay on 7H9 medium. Neither protein causes detectable growth inhibition across the dilution series, thus both may be needed to show toxicity. This supports that gp55 and gp56 are consistent with other genetic screens of primases lacking cytotoxicity.
