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

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.

[1]  Peer Bork,et al.  Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees , 2016, Nucleic Acids Res..

[2]  Susumu Goto,et al.  Data, information, knowledge and principle: back to metabolism in KEGG , 2013, Nucleic Acids Res..

[3]  R. B. Johnston,et al.  Controlled lysis of bacterial cells utilizing mutants with defective synthesis of D-alanine. , 1988, Canadian journal of microbiology.

[4]  J. Hoch,et al.  Multisensory activation of the phosphorelay initiating sporulation in Bacillus subtilis: identification and sequence of the protein kinase of the alternate pathway , 1993, Molecular microbiology.

[5]  Oscar P Kuipers,et al.  Controlling competence in Bacillus subtilis: shared use of regulators. , 2003, Microbiology.

[6]  Shane T. Jensen,et al.  The sigmaE regulon and the identification of additional sporulation genes in Bacillus subtilis. , 2003, Journal of molecular biology.

[7]  Sean R. Collins,et al.  A strategy for extracting and analyzing large-scale quantitative epistatic interaction data , 2006, Genome Biology.

[8]  Vincent Schächter,et al.  A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1 , 2008, Molecular systems biology.

[9]  A. Grossman,et al.  Identification and characterization of the immunity repressor (ImmR) that controls the mobile genetic element ICEBs1 of Bacillus subtilis , 2007, Molecular microbiology.

[10]  E. O’Shea,et al.  A serine sensor for multicellularity in a bacterium , 2013, eLife.

[11]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[12]  C. Gross,et al.  High-throughput bacterial functional genomics in the sequencing era. , 2015, Current opinion in microbiology.

[13]  Xin Yan,et al.  Cre/lox System and PCR-Based Genome Engineering in Bacillus subtilis , 2008, Applied and Environmental Microbiology.

[14]  N. Krogan,et al.  A Chemical-Genomic Screen of Neglected Antibiotics Reveals Illicit Transport of Kasugamycin and Blasticidin S , 2016, PLoS genetics.

[15]  S. Ehrlich,et al.  Essential Bacillus subtilis genes , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Nevan J Krogan,et al.  High-throughput genetic interaction mapping in the fission yeast Schizosaccharomyces pombe , 2007, Nature Methods.

[17]  E. Nester,et al.  An enzyme common to histidine and aromatic amino acid biosynthesis in Bacillus subtilis , 1976, Journal of bacteriology.

[18]  Roy R Chaudhuri,et al.  Comprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH) , 2009, BMC Genomics.

[19]  S. Aymerich,et al.  CcpN (YqzB), a novel regulator for CcpA‐independent catabolite repression of Bacillus subtilis gluconeogenic genes , 2005, Molecular microbiology.

[20]  Liran Carmel,et al.  Genome-wide Analysis of Substrate Specificities of the Escherichia coli Haloacid Dehalogenase-like Phosphatase Family* , 2006, Journal of Biological Chemistry.

[21]  Raphael H. Michna,et al.  SubtiWiki–a database for the model organism Bacillus subtilis that links pathway, interaction and expression information , 2013, Nucleic Acids Res..

[22]  P. Bork,et al.  Enterotypes of the human gut microbiome , 2011, Nature.

[23]  Davide Heller,et al.  eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences , 2015, Nucleic Acids Res..

[24]  Peter D. Karp,et al.  The MetaCyc Database of metabolic pathways and enzymes and the BioCyc collection of Pathway/Genome Databases , 2007, Nucleic Acids Res..

[25]  P. Youngman,et al.  Structure and function of the Bacillus SpoIIE protein and its localization to sites of sporulation septum assembly , 1996, Molecular microbiology.

[26]  D. Haslam,et al.  The molecular basis of Clostridium difficile disease and host response , 2015, Current opinion in gastroenterology.

[27]  Philip M. Kim,et al.  Quantitative Genome-Wide Genetic Interaction Screens Reveal Global Epistatic Relationships of Protein Complexes in Escherichia coli , 2014, PLoS genetics.

[28]  M. Malecki,et al.  Bacterial adaptation to cold. , 2013, Microbiology.

[29]  Roland R. Regoes,et al.  Granulocytes Impose a Tight Bottleneck upon the Gut Luminal Pathogen Population during Salmonella Typhimurium Colitis , 2014, PLoS pathogens.

[30]  Sierd Bron,et al.  Bacillus subtilis and its closest relatives: from genes to cells , 2001 .

[31]  Michael Y. Galperin,et al.  Genomic determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation-specific genes , 2012, Environmental microbiology.

[32]  Kelly M. Wetmore,et al.  Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons , 2015, mBio.

[33]  D. Rudner,et al.  High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis , 2016, PLoS biology.

[34]  A. Driks.  Bacillus subtilis Spore Coat , 1999, Microbiology and Molecular Biology Reviews.

[35]  H. Sokol,et al.  Faecalibacterium prausnitzii and human intestinal health. , 2013, Current opinion in microbiology.

[36]  Stefan Engelen,et al.  MicroScope—an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data , 2012, Nucleic Acids Res..

[37]  B. Faircloth,et al.  Primer3—new capabilities and interfaces , 2012, Nucleic acids research.

[38]  Wen J. Li,et al.  Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation , 2015, Nucleic Acids Res..

[39]  Hua Huang,et al.  Panoramic view of a superfamily of phosphatases through substrate profiling , 2015, Proceedings of the National Academy of Sciences.

[40]  F. Guo,et al.  Essentiality drives the orientation bias of bacterial genes in a continuous manner , 2015, Scientific Reports.

[41]  M. Son,et al.  Preparing DNA libraries for multiplexed paired-end deep sequencing for Illumina GA sequencers. , 2011, Current protocols in microbiology.

[42]  María Martín,et al.  UniProt: A hub for protein information , 2015 .

[43]  The Uniprot Consortium,et al.  UniProt: a hub for protein information , 2014, Nucleic Acids Res..

[44]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.

[45]  Newton Medeiros Vidal,et al.  Uncovering major genomic features of essential genes in Bacteria and a methanogenic Archaea , 2015, The FEBS journal.

[46]  Oscar P. Kuipers,et al.  SporeWeb: an interactive journey through the complete sporulation cycle of Bacillus subtilis , 2013, Nucleic Acids Res..

[47]  Nitin Kumar,et al.  Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation , 2016, Nature.

[48]  C. Hill,et al.  Isoprenoid biosynthesis in bacterial pathogens. , 2012, Microbiology.

[49]  Renato J. Alves,et al.  A Genomic Signature and the Identification of New Sporulation Genes , 2013, Journal of bacteriology.

[50]  Michael Y. Galperin,et al.  Divergence and Convergence in Enzyme Evolution , 2011, The Journal of Biological Chemistry.

[51]  Bastien Chevreux,et al.  The Origins of 168, W23, and Other Bacillus subtilis Legacy Strains , 2008, Journal of bacteriology.

[52]  C. Gross,et al.  MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis , 2015, Proceedings of the National Academy of Sciences.

[53]  A. Grossman,et al.  Modulation of the ComA-Dependent Quorum Response in Bacillus subtilis by Multiple Rap Proteins and Phr Peptides , 2006, Journal of bacteriology.

[54]  Sean R. Collins,et al.  A tool-kit for high-throughput, quantitative analyses of genetic interactions in E. coli , 2008, Nature Methods.

[55]  Y. Fujita,et al.  Identification of additional TnrA‐regulated genes of Bacillus subtilis associated with a TnrA box , 2003, Molecular microbiology.

[56]  Ashkan Golshani,et al.  Conditional Epistatic Interaction Maps Reveal Global Functional Rewiring of Genome Integrity Pathways in Escherichia coli. , 2016, Cell reports.

[57]  Masaru Tomita,et al.  Update on the Keio collection of Escherichia coli single-gene deletion mutants , 2009, Molecular systems biology.

[58]  P. Bork,et al.  Accurate and universal delineation of prokaryotic species , 2013, Nature Methods.

[59]  J. Errington,et al.  Interlinked Sister Chromosomes Arise in the Absence of Condensin during Fast Replication in B. subtilis , 2014, Current Biology.

[60]  Ana Rita Brochado,et al.  High-throughput approaches to understanding gene function and mapping network architecture in bacteria. , 2013, Current opinion in microbiology.

[61]  Jessica M. Silvaggi,et al.  Unmasking Novel Sporulation Genes in Bacillus subtilis , 2004, Journal of bacteriology.

[62]  M. J. Jedrzejas.  The Structure and Function of Novel Proteins of Bacillus anthracis and Other Spore-Forming Bacteria: Development of Novel Prophylactic and Therapeutic Agents , 2002, Critical reviews in biochemistry and molecular biology.

[63]  M. McClelland,et al.  Defined Single-Gene and Multi-Gene Deletion Mutant Collections in Salmonella enterica sv Typhimurium , 2014, PloS one.

[64]  Marina Santiago,et al.  A new platform for ultra-high density Staphylococcus aureus transposon libraries , 2015, BMC Genomics.

[65]  D. Popham,et al.  Homologues of the Bacillus subtilis SpoVB Protein Are Involved in Cell Wall Metabolism , 2009, Journal of bacteriology.

[66]  A. Driks.  Adam Driks Spore Coat Bacillus subtilis , 1999 .

[67]  Gregory A. Buck,et al.  Genome-wide essential gene identification in Streptococcus sanguinis , 2011, Scientific reports.

[68]  Pablo Tamayo,et al.  Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[69]  S. Ben-Yehuda,et al.  A Checkpoint Protein That Scans the Chromosome for Damage at the Start of Sporulation in Bacillus subtilis , 2006, Cell.

[70]  J. Dworkin,et al.  Recent progress in Bacillus subtilis sporulation. , 2012, FEMS microbiology reviews.

[71]  A. Emili,et al.  Global Functional Atlas of Escherichia coli Encompassing Previously Uncharacterized Proteins , 2009, PLoS biology.

[72]  Yan Zhang,et al.  Replication–transcription conflicts in bacteria , 2012, Nature Reviews Microbiology.

[73]  D. Rice,et al.  Crystal structure of S. aureus YlaN, an essential leucine rich protein involved in the control of cell shape , 2007, Proteins.

[74]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[75]  E. Brown,et al.  Metabolic suppression identifies new antibacterial inhibitors under nutrient limitation , 2013, Nature chemical biology.

[76]  Jörg Stülke,et al.  Essential genes in Bacillus subtilis: a re-evaluation after ten years. , 2013, Molecular bioSystems.

[77]  N. Krogan,et al.  Phenotypic Landscape of a Bacterial Cell , 2011, Cell.

[78]  J. Errington,et al.  Complex polar machinery required for proper chromosome segregation in vegetative and sporulating cells of Bacillus subtilis , 2016, Molecular microbiology.

[79]  J. Kato,et al.  Construction of consecutive deletions of the Escherichia coli chromosome , 2007, Molecular systems biology.