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      Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection

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          Abstract

          We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants—the ‘Keio collection'—provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase ( http://ecoli.aist-nara.ac.jp/).

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          Experimental Determination and System Level Analysis of Essential Genes in <i>Escherichia coli</i> MG1655

          S. Y. Gerdes, M. D. Scholle, J. W. Campbell … (2003)
          Journal of Bacteriology, 185(19), 5673-5684 Defining the gene products that play an essential role in an organism's functional repertoire is vital to understanding the system level organization of living cells. We used a genetic footprinting technique for a genome-wide assessment of genes required for robust aerobic growth of in rich media. We identified 620 genes as essential and 3,126 genes as dispensable for growth under these conditions. Functional context analysis of these data allows individual functional assignments to be refined. Evolutionary context analysis demonstrates a significant tendency of essential genes to be preserved throughout the bacterial kingdom. Projection of these data over metabolic subsystems reveals topologic modules with essential and evolutionarily preserved enzymes with reduced capacity for error tolerance.
          Article 0 comments Cited 212 times – based on 0 reviews     Review now
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            Escherichia coli K-12: a cooperatively developed annotation snapshot—2005

            Monica Riley, Takashi Abe, Martha Arnaud … (2006)
            The goal of this group project has been to coordinate and bring up-to-date information on all genes of Escherichia coli K-12. Annotation of the genome of an organism entails identification of genes, the boundaries of genes in terms of precise start and end sites, and description of the gene products. Known and predicted functions were assigned to each gene product on the basis of experimental evidence or sequence analysis. Since both kinds of evidence are constantly expanding, no annotation is complete at any moment in time. This is a snapshot analysis based on the most recent genome sequences of two E.coli K-12 bacteria. An accurate and up-to-date description of E.coli K-12 genes is of particular importance to the scientific community because experimentally determined properties of its gene products provide fundamental information for annotation of innumerable genes of other organisms. Availability of the complete genome sequence of two K-12 strains allows comparison of their genotypes and mutant status of alleles.
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              Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome.

              Jun Kato, Tomotake Ote, Masayuki Hashimoto … (2004)
              The minimization of a genome is necessary to identify experimentally the minimal gene set that contains only those genes that are essential and sufficient to sustain a functioning cell. Recent developments in genetic techniques have made it possible to generate bacteria with a markedly reduced genome. We developed a simple system for formation of markerless chromosomal deletions, and constructed and characterized a series of large-scale chromosomal deletion mutants of Escherichia coli that lack between 2.4 and 29.7% of the parental chromosome. Combining deletion mutations changes cell length and width, and the mutant cells with larger deletions were even longer and wider than the parental cells. The nucleoid organization of the mutants is also changed: the nucleoids occur as multiple small nucleoids and are localized peripherally near the envelope. Inhibition of translation causes them to condense into one or two packed nucleoids, suggesting that the coupling of transcription and translation of membrane proteins peripherally localizes chromosomes. Because these phenotypes are similar to those of spherical cells, those may be a consequence of the morphological change. Based on the nucleoid localization observed with these mutants, we discuss the cellular nucleoid dynamics.
              Article 0 comments Cited 109 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): Mol Syst Biol
                Title: Molecular Systems Biology
                ISSN (Electronic): 1744-4292
                Publication date Collection: 2006
                Publication date (Electronic): 21 February 2006
                Volume: 2
                Page: 2006.0008
                Affiliations
                [1 ] Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan
                [2 ] Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan
                [3 ] CREST, JST (Japan Science and Technology), Kawaguchi, Saitama, Japan
                [4 ] Department of Biological Sciences, Purdue University, West Lafayette, IN, USA
                Author notes
                [a ] Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-2054, USA. Tel.: +1 765 494 8034; Fax: +1 765 494 0876; E-mail: blwanner@ 123456purdue.edu
                [b ] Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Tel.: +81 743 72 5660; Fax: +81 743 72 5669; E-mail: hmori@ 123456gtc.naist.jp
                Article
                Publisher Item ID: msb4100050
                DOI: 10.1038/msb4100050
                PMC ID: 1681482
                PubMed ID: 16738554
                SO-VID: 30656964-d9f5-4fbd-899e-8a69f89c9f23
                Copyright © Copyright © 2006, EMBO and Nature Publishing Group
                History
                Date received : 28 September 2005
                Date accepted : 7 December 2005
                Page count
                Pages: 1
                Categories
                Subject: Article

                ScienceOpen disciplines: Quantitative & Systems biology
                Keywords: systems biology,resources,bacterial functional genomics,essential gene,e. coli/gene,knockout mutants
                Data availability:
                ScienceOpen disciplines: Quantitative & Systems biology
                Keywords: systems biology, resources, bacterial functional genomics, essential gene, e. coli/gene, knockout mutants

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