title: Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis.
abstract: A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria.
authors.affiliation: Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Byoung-Mo
authors.initials: BM
authors.lastname: Koo
authors.affiliation: European Molecular Biology Laboratory, Genome Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: George
authors.initials: G
authors.lastname: Kritikos
authors.affiliation: Department of Chemistry, Boston University, Boston, MA 02215, USA.
authors.forename: Jeremiah D
authors.initials: JD
authors.lastname: Farelli
authors.affiliation: Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Horia
authors.initials: H
authors.lastname: Todor
authors.affiliation: Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Tong
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Harvey
authors.initials: H
authors.lastname: Kimsey
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Ilan
authors.initials: I
authors.lastname: Wapinski
authors.affiliation: European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK.
authors.forename: Marco
authors.initials: M
authors.lastname: Galardini
authors.affiliation: Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Angelo
authors.initials: A
authors.lastname: Cabal
authors.affiliation: Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Peters
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Anna-Barbara
authors.initials: AB
authors.lastname: Hachmann
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: David Z
authors.initials: DZ
authors.lastname: Rudner
authors.affiliation: Department of Chemistry, Boston University, Boston, MA 02215, USA.
authors.forename: Karen N
authors.initials: KN
authors.lastname: Allen
authors.affiliation: European Molecular Biology Laboratory, Genome Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany. Electronic address: typas@embl.de.
authors.forename: Athanasios
authors.initials: A
authors.lastname: Typas
authors.affiliation: Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute of Quantitative Biology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: cgrossucsf@gmail.com.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Gross
mesh_terms: D000596:Amino Acids; D001412:Bacillus subtilis / Q000235:genetics*; D017353:Gene Deletion; D015723:Gene Library; D015698:Genomic Library; D023281:Genomics; D057166:High-Throughput Screening Assays / Q000379:methods*; D017384:Sequence Deletion / Q000235:genetics; D013171:Spores, Bacterial / Q000235:genetics
chemical_list: D000596:Amino Acids
keywords: Bacillus subtilis; Genome-wide screening; auxotrophic gene; competence; essential gene; growth; high-throughput methods for transformation and double-mutant analysis; ordered gene deletion mutant library; sporulation; ysaA