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/).

Analysis of cellular components at multiple levels of biological information can provide valuable functional insights. We performed multiple high-throughput measurements to study the response of Escherichia coli cells to genetic and environmental perturbations. Analysis of metabolic enzyme gene disruptants revealed unexpectedly small changes in messenger RNA and proteins for most disruptants. Overall, metabolite levels were also stable, reflecting the rerouting of fluxes in the metabolic network. In contrast, E. coli actively regulated enzyme levels to maintain a stable metabolic state in response to changes in growth rate. E. coli thus seems to use complementary strategies that result in a metabolic network robust against perturbations.

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Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple-knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose-7-phosphate to erythrose-4-phosphate and dihydroxyacetone phosphate were observed in transaldolase-deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose-7-phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP-dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks.

Tetracycline-inhibited ribosome profiling (TetRP) provides a powerful new experimental tool for comprehensive genome-wide identification of translation initiation sites in bacteria. We validated TetRP by confirming the translation start sites of protein-coding genes in accordance with the 2006 version of Escherichia coli K-12 annotation record (GenBank U00096.2) and found ∼150 new start sites within 60 nucleotides of the annotated site. This analysis revealed 72 per cent of the genes whose initiation site annotations were changed from the 2006 GenBank record to the newer 2014 annotation record (GenBank U00096.3), indicating a high sensitivity. Also, results from reporter fusion and proteomics of N-terminally enriched peptides showed high specificity of the TetRP results. In addition, we discovered over 300 translation start sites within non-coding, intergenic regions of the genome, using a threshold that retains ∼2,000 known coding genes. While some appear to correspond to pseudogenes, others may encode small peptides or have previously unforeseen roles. In summary, we showed that ribosome profiling upon translation inhibition by tetracycline offers a simple, reliable and comprehensive experimental tool for precise annotation of translation start sites of expressed genes in bacteria.

BackgroundThere is a significant difference between synonymous codon usage in many organisms, and it is known that codons used more frequently generally showed efficient decoding rate. At the gene level, however, there are conflicting reports on the existence of a correlation between codon adaptation and translation efficiency, even in the same organism.ResultsTo resolve this issue, we cultured Escherichia coli under conditions designed to maintain constant levels of mRNA and protein and subjected the cells to ribosome profiling (RP) and mRNA-seq analyses. We showed that the RP results correlated more closely with protein levels generated under similar culture conditions than with the mRNA abundance from the mRNA-seq. Our result indicated that RP/mRNA ratio could be used as a measure of translation efficiency at gene level. On the other hand, the RP data showed that codon-specific ribosome density at the decoding site negatively correlated with codon usage, consistent with the hypothesis that preferred codons display lower ribosome densities due to their faster decoding rate. However, highly codon-adapted genes showed higher ribosome densities at the gene level, indicating that the efficiency of translation initiation, rather than higher elongation efficiency of preferred codons, exerted a greater effect on ribosome density and thus translation efficiency.ConclusionsThese findings indicate that evolutionary pressure on highly expressed genes influenced both codon bias and translation initiation efficiency and therefore explains contradictory findings that codon usage bias correlates with translation efficiency of native genes, but not with the artificially created gene pool, which was not subjected to evolution pressure.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1115) contains supplementary material, which is available to authorized users.

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 gamma-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.

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