The genome sequences of thousands of bacteriophages have been determined and functions for many of the encoded genes have been assigned based on homology to characterized sequences. However, functions have not been assigned to more than two-thirds of the identified phage genes as they have no recognizable sequence features. Recent genome-wide overexpression screens have begun to identify bacteriophage genes that encode proteins that reduce or inhibit bacterial growth. This study describes the construction of a plasmid-based overexpression library of 76 genes encoded by Cluster K1 mycobacteriophage Amelie, which is genetically similar to cluster K phages Waterfoul and Hammy recently described in similar screens and closely related to phages that infect clinically important mycobacteria. 26 out of the 76 genes evaluated in our screen, encompassing 34% of the genome, reduced growth of the host Mycobacterium smegmatis to various degrees. More than one-third of these 26 toxic genes have no known function, and 10 of the 26 genes almost completely abolished host growth upon overexpression. Notably, while several of the toxic genes identified in Amelie shared homologs with other Cluster K phages recently screened, this study uncovered seven previously unknown gene families that exhibit cytotoxic properties, thereby broadening the repertoire of known phage-encoded growth inhibitors. This work, carried out under the HHMI-supported SEA-GENES project (Science Education Alliance Gene-function Exploration by a Network of Emerging Scientists), underscores the importance of comprehensive overexpression screens in elucidating genome-wide patterns of phage gene function and novel interactions between phages and their hosts.
Keywords: Mycobacterium smegmatis; cytotoxicity; genome-wide screen; mycobacteriophage.
© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.