Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.
TL;DR: 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.
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/).
Citations
More filters
TL;DR: The Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals are described, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species.
Abstract: To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, ~150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively
9,268 citations
TL;DR: The results suggest that all three major classes of bactericidal drugs can be potentiated by targeting bacterial systems that remediate hydroxyl radical damage, including proteins involved in triggering the DNA damage response, e.g., RecA.
2,420 citations
Cites methods from "Construction of Escherichia coli K-..."
...The recA, iscS, and TCA-cycle knockouts were constructed using P1 phage transduction and were derived from an E. coli single-gene knockout library (Baba et al., 2006) (Table S4)....
[...]
TL;DR: System-wide analyses of protein and mRNA expression in individual cells with single-molecule sensitivity using a newly constructed yellow fluorescent protein fusion library for Escherichia coli found that almost all protein number distributions can be described by the gamma distribution with two fitting parameters which, at low expression levels, have clear physical interpretations as the transcription rate and protein burst size.
Abstract: Protein and messenger RNA (mRNA) copy numbers vary from cell to cell in isogenic bacterial populations. However, these molecules often exist in low copy numbers and are difficult to detect in single cells. We carried out quantitative system-wide analyses of protein and mRNA expression in individual cells with single-molecule sensitivity using a newly constructed yellow fluorescent protein fusion library for Escherichia coli. We found that almost all protein number distributions can be described by the gamma distribution with two fitting parameters which, at low expression levels, have clear physical interpretations as the transcription rate and protein burst size. At high expression levels, the distributions are dominated by extrinsic noise. We found that a single cell's protein and mRNA copy numbers for any given gene are uncorrelated.
1,970 citations
TL;DR: This work reviews strategies for natural product screening that harness the recent technical advances that have reduced technical barriers and assess the use of genomic and metabolomic approaches to augment traditional methods of studying natural products.
Abstract: Natural products have been a rich source of compounds for drug discovery. However, their use has diminished in the past two decades, in part because of technical barriers to screening natural products in high-throughput assays against molecular targets. Here, we review strategies for natural product screening that harness the recent technical advances that have reduced these barriers. We also assess the use of genomic and metabolomic approaches to augment traditional methods of studying natural products, and highlight recent examples of natural products in antimicrobial drug discovery and as inhibitors of protein-protein interactions. The growing appreciation of functional assays and phenotypic screens may further contribute to a revival of interest in natural products for drug discovery.
1,822 citations
TL;DR: The looming antibiotic-resistance crisis has penetrated the consciousness of clinicians, researchers, policymakers, politicians and the public at large as discussed by the authors, and the evolution and widespread distribution of antibiotic-resistant elements in bacterial pathogens has made diseases that were once easily treatable deadly again.
Abstract: The looming antibiotic-resistance crisis has penetrated the consciousness of clinicians, researchers, policymakers, politicians and the public at large. The evolution and widespread distribution of antibiotic-resistance elements in bacterial pathogens has made diseases that were once easily treatable deadly again. Unfortunately, accompanying the rise in global resistance is a failure in antibacterial drug discovery. Lessons from the history of antibiotic discovery and fresh understanding of antibiotic action and the cell biology of microorganisms have the potential to deliver twenty-first century medicines that are able to control infection in the resistance era.
1,481 citations
References
More filters
Book•
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
215,169 citations
Book•
01 Jan 1992
TL;DR: The most widely read reference in the water industry, Water Industry Reference as discussed by the authors, is a comprehensive reference tool for water analysis methods that covers all aspects of USEPA-approved water analysis.
Abstract: Set your standards with these standard methods. This is it: the most widely read publication in the water industry, your all-inclusive reference tool. This comprehensive reference covers all aspects of USEPA-approved water analysis methods. More than 400 methods - all detailed step-by-step; 8 vibrant, full-color pages of aquatic algae illustrations; Never-before-seen figures that will help users with toxicity testing and the identification of apparatus used in the methods; Over 300 superbly illustrated figures; A new analytical tool for a number of inorganic nonmetals; Improved coverage of data evaluation, sample preservation, and reagant water; And much more!
78,324 citations
TL;DR: A simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s), which should be widely useful, especially in genome analysis of E. coli and other bacteria.
Abstract: We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.
14,389 citations
"Construction of Escherichia coli K-..." refers background or methods in this paper
...While the project for building a transposon library was underway in Japan, a highly efficient method for direct inactivation of chromosomal genes in E. coli K-12 was reported (Datsenko and Wanner, 2000)....
[...]
...PCRverificationwith kanamycin-specific primers k1 and k2 and locus-specific primers U and D (Figure 2) was carried out as described previously (Datsenko and Wanner, 2000)....
[...]
...…other group subjected PCR products encoding ORFs to in vitro Tn5 transposition (Goryshin et al, 2000), and then recombined the mutations onto the chromosome by l Red-mediated recombination (Datsenko and Wanner, 2000), which led to the creation of insertion alleles for 1976 ORFs (Kang et al, 2004)....
[...]
...ORFs not targeted include 79 IS genes, four genes for small toxic polypeptides (ldrA, ldrB, ldrC, and ldrD), and seven genes already disrupted in BW25113 (araBAD, lacZ, and rhaBAD; Datsenko and Wanner, 2000)....
[...]
...…recA1 rph-1) was used for maintenance of the template plasmid pKD13 (GenBankt Accession number AY048744). pKD46 (GenBankt Accession number AY048746; Datsenko and Wanner, 2000) was made by PCR amplification of the Red recombinase genes from phage l and cloning into pKD16, a derivative of INT-ts…...
[...]
TL;DR: It is hypothesized that producer and consumer communities characteristic of a given river reach become established in harmony with the dynamic physical conditions of the channel.
Abstract: From headwaters to mouth, the physical variables within a river system present a continuous gradient of physical conditions. This gradient should elicit a series of responses within the constituent...
9,145 citations
TL;DR: The 4,639,221-base pair sequence of Escherichia coli K-12 is presented and reveals ubiquitous as well as narrowly distributed gene families; many families of similar genes within E. coli are also evident.
Abstract: The 4,639,221-base pair sequence of Escherichia coli K-12 is presented. Of 4288 protein-coding genes annotated, 38 percent have no attributed function. Comparison with five other sequenced microbes reveals ubiquitous as well as narrowly distributed gene families; many families of similar genes within E. coli are also evident. The largest family of paralogous proteins contains 80 ABC transporters. The genome as a whole is strikingly organized with respect to the local direction of replication; guanines, oligonucleotides possibly related to replication and recombination, and most genes are so oriented. The genome also contains insertion sequence (IS) elements, phage remnants, and many other patches of unusual composition indicating genome plasticity through horizontal transfer.
7,723 citations
"Construction of Escherichia coli K-..." refers background in this paper
...Our ORF deletions include 3912 genes annotated in both E. coli K-12 MG1655 and W3110 and 73 previously annotated genes (Supplementary Table 4)....
[...]
...The vast majority (243) were due to errors in the original 4.5- Mb E. coli K-12 MG1655 genome (an error rate of less than 1 per 13 000 nt 8 years later); 16 were due to errors in the 2.6Mb of the W3110 genome reported from 1992 to 1997....
[...]
...Consequently, MG1655 and W3110 have two and 17 extra copies of IS genes, respectively, and MG1655 has 11 andW3110 has 21 unique genes (including seven additional pseudogenes)....
[...]
...In addition to updating annotations of gene functions, start sites were changed for 682 MG1655 ORFs (Riley et al, 2006)....
[...]
...Sequence corrections resulted in major changes in the translation of 111 MG1655 open-reading frames (ORFs), mostly due to frame shifting (85), but also due to gene fissions (2), gene fusions (23), and inversion (1; Hayashi et al, 2006)....
[...]