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

The spread of resistant bacteria, leading to untreatable infections, is a major public health threat but the pace of antibiotic discovery to combat these pathogens has slowed down. Most antibiotics were originally isolated by screening soil-derived actinomycetes during the golden era of antibiotic discovery in the 1940s to 1960s. However, diminishing returns from this discovery platform led to its collapse, and efforts to create a new platform based on target-focused screening of large libraries of synthetic compounds failed, in part owing to the lack of penetration of such compounds through the bacterial envelope. This article considers strategies to re-establish viable platforms for antibiotic discovery. These include investigating untapped natural product sources such as uncultured bacteria, establishing rules of compound penetration to enable the development of synthetic antibiotics, developing species-specific antibiotics and identifying prodrugs that have the potential to eradicate dormant persisters, which are often responsible for hard-to-treat infections.

Platforms for antibiotic discovery

Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species This notion has been supported by many groups but has been challenged recently Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments We first used a novel intracellular H2O2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H2O2 We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality

https://www.pnas.org/content/pnas/111/20/E2100.full.pdf

Antibiotics induce redox-related physiological alterations as part of their lethality

Phages are the most abundant entity in the biosphere and outnumber bacteria by a factor of 10. Phage DNA may also constitute 20% of bacterial genomes; however, its role is ill defined. Here, we explore the impact of cryptic prophages on cell physiology by precisely deleting all nine prophage elements (166 kbp) using Escherichia coli. We find that cryptic prophages contribute significantly to resistance to sub-lethal concentrations of quinolone and β-lactam antibiotics primarily through proteins that inhibit cell division (for example, KilR of rac and DicB of Qin). Moreover, the prophages are beneficial for withstanding osmotic, oxidative and acid stresses, for increasing growth, and for influencing biofilm formation. Prophage CPS-53 proteins YfdK, YfdO and YfdS enhanced resistance to oxidative stress, prophages e14, CPS-53 and CP4-57 increased resistance to acid, and e14 and rac proteins increased early biofilm formation. Therefore, cryptic prophages provide multiple benefits to the host for surviving adverse environmental conditions.

/pdf/cryptic-prophages-help-bacteria-cope-with-adverse-3aj6137gpr.pdf

Cryptic prophages help bacteria cope with adverse environments

Here we describe a straightforward, efficient, and reliable way to clone an insert of choice into a plasmid of choice without restriction endonucleases or T4 DNA ligase Chimeric primers containing

Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids

Based on the genomic sequence data of Escherichia coli K-12 strain, we have constructed a complete set of cloned individual genes encoding Histidine-tagged proteins with or without GFP fused for functional genomic analysis. Each clone encodes a protein of predicted ORF attached by Histidines and seven spacer amino acids at the N-terminal end, and five spacer amino acids and GFP at the C-terminal end. SfiI restriction sites are generated at both the N- and C-terminal boundaries of ORF upon cloning, which enables easy transfer of ORF to other vector systems by cutting with SfiI. Expression of cloned ORF is under the control of an IPTG-inducible promoter, which is strictly repressed by lacI(q) repressor gene product. The set of cloned ORFs described here should provide unique resources for systematic functional genomic approaches including (i) construction of DNA microarray, (ii) production and purification of proteins, (iii) analysis of protein localization by monitoring GFP fluorescence and (iv) analysis of protein-protein interaction.

Complete set of ORF clones of Escherichia coli ASKA library (A Complete Set of E. coli K-12 ORF Archive): Unique Resources for Biological Research

Remineralization of organic matter in deep-sea sediments is important in oceanic biogeochemical cycles, and bacteria play a major role in this process. Shewanella violacea DSS12 is a psychrophilic and piezophilic γ-proteobacterium that was isolated from the surface layer of deep sea sediment at a depth of 5110 m. Here, we report the complete genome sequence of S. violacea and comparative analysis with the genome of S. oneidensis MR-1, isolated from sediments of a freshwater lake. Unlike S. oneidensis, this deep-sea Shewanella possesses very few terminal reductases for anaerobic respiration and no c-type cytochromes or outer membrane proteins involved in respiratory Fe(III) reduction, which is characteristic of most Shewanella species. Instead, the S. violacea genome contains more terminal oxidases for aerobic respiration and a much greater number of putative secreted proteases and polysaccharases, in particular, for hydrolysis of collagen, cellulose and chitin, than are encoded in S. oneidensis. Transporters and assimilatory reductases for nitrate and nitrite, and nitric oxide-detoxifying mechanisms (flavohemoglobin and flavorubredoxin) are found in S. violacea. Comparative analysis of the S. violacea genome revealed the respiratory adaptation of this bacterium to aerobiosis, leading to predominantly aerobic oxidation of organic matter in surface sediments, as well as its ability to efficiently use diverse organic matter and to assimilate inorganic nitrogen as a survival strategy in the nutrient-poor deep-sea floor.

Complete genome sequence and comparative analysis of Shewanella violacea, a psychrophilic and piezophilic bacterium from deep sea floor sediments

It is an objection of the present invention to provide a fluorescence reading apparatus in view of the influence of fluorescence derived from a fluorescence substance that is not involved with an interaction between a probe substance and a target substance. The above-mentioned problem is solved by a fluorescence reading apparatus 12 comprising an incidence angle adjusting means 10 for adjusting an incidence angle when light from a light source 7 is incident on a substrate 2; and a controller 11 that controls an amount of the incidence angle adjusted by the incidence angle adjusting means 10 and also comprises a means for receiving information on the incidence angle and information on the fluorescence intensities or fluorescence images at a plurality of incidence angles and obtaining the penetration depths of the evanescent fields with respect to the plurality of incidence angles from the information on the incidence angles; and a means for obtaining information on the fluorescence intensities in the obtained plurality of penetration depths.

Apparatus and method for reading fluorescence

[PROBLEMS] To provide a fluorescence reader such as a microarray reader structured in consideration of the influence originated from a fluorescent substance not concerned in the interaction between the probe material and a target substance. [MEANS FOR SOLVING PROBLEMS] A fluorescence reader (12) comprises an angle-of-incidence adjusting means (10) for adjusting the angle of incidence when light from a light source (7) is incident on a substrate (2) and a controller (11) having means for controlling the angle of incidence adjusted by the angle-of-incidence adjusting means (10), receiving information on the angle of incidence and informationon the intensities of fluorescence at a plurality of angles of incidence or information on a fluorescence image, and determining the penetration depth of the evanescent field from the information on the angle of incidence, and means for acquiring information on the fluorescence intensities at the determined penetration depths.

Eiji Inamoto

Papers

Complete set of ORF clones of Escherichia coli ASKA library (A Complete Set of E. coli K-12 ORF Archive): Unique Resources for Biological Research

Complete genome sequence and comparative analysis of Shewanella violacea, a psychrophilic and piezophilic bacterium from deep sea floor sediments

Apparatus and method for reading fluorescence

Fluorescence reader and fluorescence reading method