title: Revealing bacterial targets of growth inhibitors encoded by bacteriophage T7.
abstract: Today's arsenal of antibiotics is ineffective against some emerging strains of antibiotic-resistant pathogens. Novel inhibitors of bacterial growth therefore need to be found. The target of such bacterial-growth inhibitors must be identified, and one way to achieve this is by locating mutations that suppress their inhibitory effect. Here, we identified five growth inhibitors encoded by T7 bacteriophage. High-throughput sequencing of genomic DNA of resistant bacterial mutants evolving against three of these inhibitors revealed unique mutations in three specific genes. We found that a nonessential host gene, ppiB, is required for growth inhibition by one bacteriophage inhibitor and another nonessential gene, pcnB, is required for growth inhibition by a different inhibitor. Notably, we found a previously unidentified growth inhibitor, gene product (Gp) 0.6, that interacts with the essential cytoskeleton protein MreB and inhibits its function. We further identified mutations in two distinct regions in the mreB gene that overcome this inhibition. Bacterial two-hybrid assay and accumulation of Gp0.6 only in MreB-expressing bacteria confirmed interaction of MreB and Gp0.6. Expression of Gp0.6 resulted in lemon-shaped bacteria followed by cell lysis, as previously reported for MreB inhibitors. The described approach may be extended for the identification of new growth inhibitors and their targets across bacterial species and in higher organisms.
authors.affiliation: Department of Clinical Microbiology and Immunology, Sackler School of Medicine, and.
authors.forename: Shahar
authors.initials: S
authors.lastname: Molshanski-Mor
authors.affiliation: Department of Clinical Microbiology and Immunology, Sackler School of Medicine, and.
authors.forename: Ido
authors.initials: I
authors.lastname: Yosef
authors.affiliation: Department of Clinical Microbiology and Immunology, Sackler School of Medicine, and.
authors.forename: Ruth
authors.initials: R
authors.lastname: Kiro
authors.affiliation: Department of Clinical Microbiology and Immunology, Sackler School of Medicine, and.
authors.forename: Rotem
authors.initials: R
authors.lastname: Edgar
authors.affiliation: Department of Clinical Microbiology and Immunology, Sackler School of Medicine, and.
authors.forename: Miriam
authors.initials: M
authors.lastname: Manor
authors.affiliation: Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Michael
authors.initials: M
authors.lastname: Gershovits
authors.affiliation: Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Mia
authors.initials: M
authors.lastname: Laserson
authors.affiliation: Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Tal
authors.initials: T
authors.lastname: Pupko
authors.affiliation: Department of Clinical Microbiology and Immunology, Sackler School of Medicine, and ehudq@post.tau.ac.il.
authors.forename: Udi
authors.identifier: http://orcid.org/0000-0003-2169-5270
authors.initials: U
authors.lastname: Qimron
mesh_terms: D017123:Bacteriophage T7 / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure / Q000821:virology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D016679:Genome, Viral / Q000502:physiology*; D011062:Polynucleotide Adenylyltransferase / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D029968:Escherichia coli Proteins; D014764:Viral Proteins; C078473:MreB protein, E coli; D011062:Polynucleotide Adenylyltransferase; C063643:pcnB protein, E coli
keywords: bacterial cytoskeleton; bacteriophage biology; high-throughput DNA sequencing; host takeover; target identification