A genome-wide overexpression screen reveals Mycobacterium smegmatis growth inhibitors encoded by mycobacteriophage Hammy
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Published:2023-11-02
Issue:12
Volume:13
Page:
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ISSN:2160-1836
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Container-title:G3: Genes, Genomes, Genetics
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language:en
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Short-container-title:
Author:
Amaya Isabel1ORCID, Edwards Kaylia1ORCID, Wise Bethany M1ORCID, Bhattacharyya Ankita2, Pablo Clint H D2, Mushrush Ember3, Coats Amber N2, Dao Sara2, Dittmar Grace2, Gore Taylor2, Jarva Taiya M2, Kenkebashvili Giorgi2, Rathan-Kumar Sudiksha2, Reyes Gabriella M2, Watts Garrett L2, Watts Victoria Kalene2, Dubrow Deena4, Lewis Gabrielle4, Stone Benjamin H4, Xue Bingjie4, Cresawn Steven G4, Mavrodi Dmitri2, Sivanathan Viknesh1ORCID, Heller Danielle1ORCID
Affiliation:
1. Center for the Advancement of Science Leadership and Culture, Howard Hughes Medical Institute , Chevy Chase, MD 20185 , USA 2. School of Biological, Environmental, and Earth Sciences, University of Southern Mississippi , Hattiesburg, MS 39406 , USA 3. Department of Biology, University of Maryland Baltimore County , Baltimore, MD 21250 , USA 4. Department of Biology, James Madison University , Harrisonburg, VA 22807 , USA
Abstract
Abstract
During infection, bacteriophages produce diverse gene products to overcome bacterial antiphage defenses, to outcompete other phages, and to take over cellular processes. Even in the best-studied model phages, the roles of most phage-encoded gene products are unknown, and the phage population represents a largely untapped reservoir of novel gene functions. Considering the sheer size of this population, experimental screening methods are needed to sort through the enormous collection of available sequences and identify gene products that can modulate bacterial behavior for downstream functional characterization. Here, we describe the construction of a plasmid-based overexpression library of 94 genes encoded by Hammy, a Cluster K mycobacteriophage closely related to those infecting clinically important mycobacteria. The arrayed library was systematically screened in a plate-based cytotoxicity assay, identifying a diverse set of 24 gene products (representing ∼25% of the Hammy genome) capable of inhibiting growth of the host bacterium Mycobacterium smegmatis. Half of these are related to growth inhibitors previously identified in related phage Waterfoul, supporting their functional conservation; the other genes represent novel additions to the list of known antimycobacterial growth inhibitors. This work, conducted as part of the HHMI-supported Science Education Alliance Gene-function Exploration by a Network of Emerging Scientists (SEA-GENES) project, highlights the value of parallel, comprehensive overexpression screens in exploring genome-wide patterns of phage gene function and novel interactions between phages and their hosts.
Funder
HHMI-supported Science Education Alliance GENES Gene function Exploration Network of Emerging Scientists
Publisher
Oxford University Press (OUP)
Subject
Genetics (clinical),Genetics,Molecular Biology
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