Identification of novel inhibitors of Mycobacterium smegmatis growth through genome-wide overexpression of Cluster P3 mycobacteriophage Xavia genes.
TL;DR
Functional screening is extended into a lineage of actinobacteriophages that has not previously been characterized, and several proteins with no known function are identified that warrant further mechanistic analysis.
Abstract
Bacteriophages encode numerous genes with no known function, many of which can affect essential cellular processes when expressed in the bacterial host. For mycobacteriophages, genome-wide overexpression in Mycobacterium smegmatis can be used to identify proteins that impair growth. To evaluate the cytotoxic potential of the Cluster P3 phage Xavia, we constructed a plasmid library containing 71 predicted Xavia genes under the anhydrotetracycline inducible promoter pTet and screened this library in a plate-based cytotoxicity assay to measure impacts on M. smegmatis growth. Two genes prevented transformants recovery, consistent with toxicity under basal promoter leakiness, and inducible expression of 18 additional genes impaired growth. These inhibitory proteins include structural components; factors involved in DNA metabolism, lysogeny, and lysis; and several proteins with no known function. These results extend functional screening into a lineage of actinobacteriophages that has not previously been characterized, and identify new proteins that warrant further mechanistic analysis.