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Cell Syst. 2017 Mar 22;4(3):291-305.e7. doi: 10.1016/j.cels.2016.12.013. Epub 2017 Feb 8.

Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis.

Author information

1
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA.
2
European Molecular Biology Laboratory, Genome Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
3
Department of Chemistry, Boston University, Boston, MA 02215, USA.
4
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
5
Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
6
European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK.
7
European Molecular Biology Laboratory, Genome Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany. Electronic address: typas@embl.de.
8
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.

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.

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

PMID:
28189581
PMCID:
PMC5400513
DOI:
10.1016/j.cels.2016.12.013
[Indexed for MEDLINE]
Free PMC Article

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