Tom M Conrad

Nara Institute of Science and Technology, Ikuma, Nara, Japan

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Publications (10)75.82 Total impact

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    ABSTRACT: Genetic interaction networks are especially useful for functional assignment of genes and gaining new insights into the systems-level organization of the cell. While studying interactions of nonessential genes can be relatively straight-forward via use of deletion mutants, different approaches must be used to reveal interactions of essential genes due to their indispensability. One method shown to be useful for revealing interactions of essential genes requires tagging the query protein. However, this approach can be complicated by mutational effects of potential hypomorphic alleles. Here, we describe a pilot study for a new scheme of systematically studying the interactions of essential genes. Our method uses a low-copy, F-based, complementing plasmid, pFE604T, from which the essential gene is conditionally expressed. The essential gene is expressed at lower levels, producing a moderate growth defect in a query host. Secondary mutations are introduced into the query host by conjugation and the resultant exconjugants are scored for growth by imaging them over time. We report results from studying five essential query genes: dnaN, ftsW, trmD, yrfF and yjgP, showing (on average) interactions with nearly 80 nonessential genes. This system should prove useful for genome-wide analyses of other essential genes in E. coli K-12.
    Preview · Article · Aug 2013 · Genes & Genetic Systems
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    ABSTRACT: The initial genome-scale reconstruction of the metabolic network of Escherichia coli K-12 MG1655 was assembled in 2000. It has been updated and periodically released since then based on new and curated genomic and biochemical knowledge. An update has now been built, named iJO1366, which accounts for 1366 genes, 2251 metabolic reactions, and 1136 unique metabolites. iJO1366 was (1) updated in part using a new experimental screen of 1075 gene knockout strains, illuminating cases where alternative pathways and isozymes are yet to be discovered, (2) compared with its predecessor and to experimental data sets to confirm that it continues to make accurate phenotypic predictions of growth on different substrates and for gene knockout strains, and (3) mapped to the genomes of all available sequenced E. coli strains, including pathogens, leading to the identification of hundreds of unannotated genes in these organisms. Like its predecessors, the iJO1366 reconstruction is expected to be widely deployed for studying the systems biology of E. coli and for metabolic engineering applications.
    Preview · Article · Oct 2011 · Molecular Systems Biology
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    Tom M Conrad · Nathan E Lewis · Bernhard Ø Palsson
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    ABSTRACT: Laboratory evolution studies provide fundamental biological insight through direct observation of the evolution process. They not only enable testing of evolutionary theory and principles, but also have applications to metabolic engineering and human health. Genome-scale tools are revolutionizing studies of laboratory evolution by providing complete determination of the genetic basis of adaptation and the changes in the organism's gene expression state. Here, we review studies centered on four central themes of laboratory evolution studies: (1) the genetic basis of adaptation; (2) the importance of mutations to genes that encode regulatory hubs; (3) the view of adaptive evolution as an optimization process; and (4) the dynamics with which laboratory populations evolve.
    Full-text · Article · Jul 2011 · Molecular Systems Biology
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    ABSTRACT: Herein we measure the effect of four adaptive non-synonymous mutations to the glycerol kinase (glpK) gene on catalytic function and regulation, to identify changes that correlate to increased fitness in glycerol media. The mutations significantly reduce affinity for the allosteric inhibitor fructose-1,6-bisphosphate (FBP) and formation of the tetramer, which are structurally related, in a manner that correlates inversely with imparted fitness during growth on glycerol, which strongly suggests that these enzymatic parameters drive growth improvement. Counterintuitively, the glpK mutations also increase glycerol-induced auto-catabolite repression that reduces glpK transcription in a manner that correlates to fitness. This suggests that increased specific GlpK activity is attenuated by negative feedback on glpK expression via catabolite repression, possibly to prevent methylglyoxal toxicity. We additionally report that glpK mutations were fixed in 47 of 50 independent glycerol-adapted lineages. By far the most frequently mutated locus (nucleotide 218) was mutated in 20 lineages, strongly suggesting this position has an elevated mutation rate. This study demonstrates that fitness correlations can be used to interrogate adaptive processes at the protein level and to identify the regulatory constraints underlying selection and improved growth.
    Preview · Article · May 2011 · Journal of Biological Chemistry
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    Hojung Nam · Tom M Conrad · Nathan E Lewis
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    ABSTRACT: Evolution results from molecular-level changes in an organism, thereby producing novel phenotypes and, eventually novel species. However, changes in a single gene can lead to significant changes in biomolecular networks through the gain and loss of many molecular interactions. Thus, significant insights into microbial evolution have been gained through the analysis and comparison of reconstructed metabolic networks. However, challenges remain from reconstruction incompleteness and the inability to experiment with evolution on the timescale necessary for new species to arise. Despite these challenges, experimental laboratory evolution of microbes has provided some insights into the cellular objectives underlying evolution, under the constraints of nutrient availability and the use of mechanisms that protect cells from extreme conditions.
    Full-text · Article · Apr 2011 · Current Opinion in Biotechnology
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    Dataset: Table S3
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    ABSTRACT: Primers used for Sanger sequencing to confirm mutations reported by Nimblegen and Illumina sequencing technologies. The rpoS, udhA, pntA, and pntB genes were sequenced in their entirety in all nine evolved strains using Sanger sequencing, not just regions immediately surrounding reported mutations. Primers were designed for this purpose by dividing the four genes into regions of approximately 800–900 base pairs with an overlap between regions of approximately 100 base pairs. (0.06 MB DOC)
    Preview · Dataset · Nov 2010
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    Dataset: Table S2
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    ABSTRACT: Primers used to introduce mutations detected after adaptive evolution back into the starting unevolved Δpgi strain according to the method of Tischer et al [46]. (0.04 MB DOC)
    Preview · Dataset · Nov 2010
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    Dataset: Table S1
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    ABSTRACT: Indirect assessment of RpoS activity utilizing the peroxidase assay [19]. Colonies with functional RpoS exhibit vigorous bubbling when they come into contact with hydrogen peroxide via a mechanism based on rpoS control of katE expression. We defined vigorous bubbling (v) as bubble formation occurring within five seconds after contact with hydrogen peroxide; medium bubbling (m) as bubble formation occurring between five and ten seconds after contact; and slight bubbling (s) as bubble formation occurring ten seconds after contact. This assay was performed 24 and 48 hours after colonies had been inoculated onto LB plates. Abbreviations: repl - replicate. (0.06 MB DOC)
    Preview · Dataset · Nov 2010
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    ABSTRACT: Bacterial survival requires adaptation to different environmental perturbations such as exposure to antibiotics, changes in temperature or oxygen levels, DNA damage, and alternative nutrient sources. During adaptation, bacteria often develop beneficial mutations that confer increased fitness in the new environment. Adaptation to the loss of a major non-essential gene product that cripples growth, however, has not been studied at the whole-genome level. We investigated the ability of Escherichia coli K-12 MG1655 to overcome the loss of phosphoglucose isomerase (pgi) by adaptively evolving ten replicates of E. coli lacking pgi for 50 days in glucose M9 minimal medium and by characterizing endpoint clones through whole-genome re-sequencing and phenotype profiling. We found that 1) the growth rates for all ten endpoint clones increased approximately 3-fold over the 50-day period; 2) two to five mutations arose during adaptation, most frequently in the NADH/NADPH transhydrogenases udhA and pntAB and in the stress-associated sigma factor rpoS; and 3) despite similar growth rates, at least three distinct endpoint phenotypes developed as defined by different rates of acetate and formate secretion. These results demonstrate that E. coli can adapt to the loss of a major metabolic gene product with only a handful of mutations and that adaptation can result in multiple, alternative phenotypes.
    Full-text · Article · Nov 2010 · PLoS Genetics
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    ABSTRACT: Specific small deletions within the rpoC gene encoding the β'-subunit of RNA polymerase (RNAP) are found repeatedly after adaptation of Escherichia coli K-12 MG1655 to growth in minimal media. Here we present a multiscale analysis of these mutations. At the physiological level, the mutants grow 60% faster than the parent strain and convert the carbon source 15-35% more efficiently to biomass, but grow about 30% slower than the parent strain in rich medium. At the molecular level, the kinetic parameters of the mutated RNAP were found to be altered, resulting in a 4- to 30-fold decrease in open complex longevity at an rRNA promoter and a ∼10-fold decrease in transcriptional pausing, with consequent increase in transcript elongation rate. At a genome-scale, systems biology level, gene expression changes between the parent strain and adapted RNAP mutants reveal large-scale systematic transcriptional changes that influence specific cellular processes, including strong down-regulation of motility, acid resistance, fimbria, and curlin genes. RNAP genome-binding maps reveal redistribution of RNAP that may facilitate relief of a metabolic bottleneck to growth. These findings suggest that reprogramming the kinetic parameters of RNAP through specific mutations allows regulatory adaptation for optimal growth in new environments.
    Full-text · Article · Nov 2010 · Proceedings of the National Academy of Sciences
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    ABSTRACT: After hundreds of generations of adaptive evolution at exponential growth, Escherichia coli grows as predicted using flux balance analysis (FBA) on genome-scale metabolic models (GEMs). However, it is not known whether the predicted pathway usage in FBA solutions is consistent with gene and protein expression in the wild-type and evolved strains. Here, we report that >98% of active reactions from FBA optimal growth solutions are supported by transcriptomic and proteomic data. Moreover, when E. coli adapts to growth rate selective pressure, the evolved strains upregulate genes within the optimal growth predictions, and downregulate genes outside of the optimal growth solutions. In addition, bottlenecks from dosage limitations of computationally predicted essential genes are overcome in the evolved strains. We also identify regulatory processes that may contribute to the development of the optimal growth phenotype in the evolved strains, such as the downregulation of known regulons and stringent response suppression. Thus, differential gene and protein expression from wild-type and adaptively evolved strains supports observed growth phenotype changes, and is consistent with GEM-computed optimal growth states. Molecular Systems Biology 6: 390; published online 27 July 2010; doi:10.1038/msb.2010.47 Subject Categories: functional genomics; simulation and data analysis
    Full-text · Article · Jul 2010 · Molecular Systems Biology
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    ABSTRACT: Genome-scale metabolic reconstructions under the Constraint Based Reconstruction and Analysis (COBRA) framework are valuable tools for analyzing the metabolic capabilities of organisms and interpreting experimental data. As the number of such reconstructions and analysis methods increases, there is a greater need for data uniformity and ease of distribution and use. We describe BiGG, a knowledgebase of Biochemically, Genetically and Genomically structured genome-scale metabolic network reconstructions. BiGG integrates several published genome-scale metabolic networks into one resource with standard nomenclature which allows components to be compared across different organisms. BiGG can be used to browse model content, visualize metabolic pathway maps, and export SBML files of the models for further analysis by external software packages. Users may follow links from BiGG to several external databases to obtain additional information on genes, proteins, reactions, metabolites and citations of interest. BiGG addresses a need in the systems biology community to have access to high quality curated metabolic models and reconstructions. It is freely available for academic use at http://bigg.ucsd.edu.
    Preview · Article · Apr 2010 · BMC Bioinformatics
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    ABSTRACT: Table 2 - Orphan reactions. 58 E. coli iJR904 orphan (non-gene associated) reactions are categorized by current status. 'Found in 1260' shows whether the reactions are found in reconstruction iAF1260. 'Y*' indicates that the reaction is found in iAF1260 in modified form (usually because of the addition of the periplasm compartment). Notes: spont. -spontaneous, lump - lumped reaction, virtual - virtual reaction, P - H. pylori, S - S. aureus, Y - S. cerevisiae, H - H. sapiens,
    Preview · Dataset · Apr 2010
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    ABSTRACT: Table 1 - Contents of BiGG. BiGG currently contains 7 reconstructions including two versions of E. coli. There are a total of 7234 unique reactions and exchanges in the database. Exchange reactions carry metabolites from the extracellular 'compartment' across the system boundary and are not technically part of the metabolic reconstruction. Translocation reactions carry a metabolite between compartments (possibly performing other transformations). Reactions can be gene associated or not. Every reconstruction contains the 'cytosol' and 'extracellular' compartment. Human and yeast contain 'endoplasmic reticulum', 'mitochondria', 'peroxisome', 'nucleus'. The 'periplasm' in iAF1260, vacuole in yeast, and lysosome in human are unique to these reconstructions.
    Preview · Dataset · Apr 2010
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    ABSTRACT: Mutations reported for LactA, LactB, LactC, LactD, and LactE strains using Nimblegen CGS arrays.
    Preview · Dataset · Oct 2009
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    ABSTRACT: Regulons enriched for differential expression in LactA, LactB, LactC, LactD, and LactE strains.
    Preview · Dataset · Oct 2009
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    ABSTRACT: False negative rate of our mutation detection algorithm using a reference sequence genome with 'mutations' inserted at known locations.
    Preview · Dataset · Oct 2009
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    ABSTRACT: The presence and absence of mutations at time points or in colonies were used for determination of mutation trajectory and population mutation penetration.
    Preview · Dataset · Oct 2009
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    ABSTRACT: Mutations reported for all strains using Solexa sequencing.
    Preview · Dataset · Oct 2009
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    ABSTRACT: Primers used in this study.
    Preview · Dataset · Oct 2009