[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
Molecular Systems Biology 10/2011; 7(1):535. DOI:10.1038/msb.2011.65 · 10.87 Impact Factor
[Show abstract][Hide abstract] 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.
Molecular Systems Biology 07/2011; 7(1):509. DOI:10.1038/msb.2011.42 · 10.87 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
Current Opinion in Biotechnology 04/2011; 22(4):595-600. DOI:10.1016/j.copbio.2011.03.006 · 7.12 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
Proceedings of the National Academy of Sciences 11/2010; 107(47):20500-5. DOI:10.1073/pnas.0911253107 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: When prokaryotes are maintained at early- to mid-log phase growth through serial passaging for hundreds of generations, the strains improve fitness and evolve a higher growth rate (Lenski and Travisano, 1994; Ibarra et al, 2002). This increased growth rate is the result of the appearance of a few causal mutations (Herring et al, 2006; Conrad et al, 2009). In Escherichia coli, these altered growth phenotypes are consistent with predictions from genome-scale models of metabolism (GEMs) (Ibarra et al, 2002; Fong and Palsson, 2004). However, it is still not known (1) whether absolute gene and protein expression levels and expression changes are consistent with optimal growth predictions from in silico GEMs or (2) whether measured expression changes can be linked to physiological changes that are based on known mechanisms or pathways. In this study, we begin to address these questions using constraint-based modeling of E. coli K-12 metabolism (Feist and Palsson, 2008) to analyze omic data that document the expression changes in E. coli under adaptive evolution in three different growth conditions.
Mapping high-throughput data to a network can be useful for interpretation. However, it does not account for upstream and downstream effects of gene and protein expression changes. The analysis of data in the context of GEMs can suggest if predicted activity is consistent with the data. For this work, we used a variant of flux balance analysis (FBA), called Parsimonious enzyme usage FBA (pFBA) (Figure 1), to classify all genes according to whether they are used in the optimal growth solutions. Results from these models were compared with the data to assess whether the data were consistent with genes and proteins within the predicted optimal solutions, and whether the expression changes were consistent with measured physiology. Through this analysis, we find that the data provide a high coverage of genes that contribute to the optimal growth solutions (Figure 1B). In fact, the union of the proteomic and transcriptomic data for non-essential genes provides support for 97.7% of all non-essential gene-associated reactions within the optimal growth predictions. Thus, the spectrum of expressed genes and proteins is consistent with the pathway utilization that is predicted for these optimal growth phenotypes.
Laboratory-evolved strains attain a higher growth rate. This higher growth rate is usually associated with an increased substrate uptake rate (Ibarra et al, 2002; Fong et al, 2005) and in some cases more efficient metabolism (Ibarra et al, 2002). Both of these properties are also witnessed in the strains studied here. It has been reported that in most cases, evolved strain growth phenotype is consistent with GEM predictions (Ibarra et al, 2002; Teusink et al, 2009). Here, we evaluate whether the laboratory-evolved strains adjust the gene and protein expression levels in accordance with pathway usage in the optimal growth predictions. Essential and non-essential genes and proteins within the optimal growth solutions are significantly upregulated (Figure 1B). This suggests that these proteins may be acting as bottlenecks that are relieved through the adaptive process, thereby allowing for a higher substrate uptake rate and growth rate. However, genes and proteins associated with reactions that cannot carry a flux in the given growth conditions are downregulated in the evolved strains (Figure 1B). Furthermore, there is downregulation of genes associated with less efficient pathways (Figure 5C). Thus, the omic data support the emergence of the predicted optimal growth states, consistent with the increased substrate uptake upstream and the increased biomass production downstream of these internal pathways.
Regulatory mechanisms, both known and unknown, are responsible for the changes seen here. Across all data sets, several metabolic regulons are significantly downregulated. However, no known regulons were enriched among upregulated genes or proteins for all but one data set. Aside from just regulating the metabolic pathways directly, these mechanisms lead to additional physiological changes. For example, in the minimal media growth conditions used here, the stringent response normally represses growth while upregulating amino-acid biosynthetic processes. However, evolved strain gene expression shows a suppression of the stringent response, as evolved strain gene expression shows either no expression change or changes opposite to the normal stringent response.
The implications of this work are as follows: (1) genome-scale gene and protein expression data are consistent with FBA computed optimal growth states, and evolved strains reinforce these optimal states; (2) genome-scale models will have an important function bridging the gap between genotype and phenotype; and (3) the development of additional genome-scale models of other growth-related processes such as transcription and translation (Thiele et al, 2009) will have an important function in elucidating the mechanisms that contribute the most to altered phenotypes (Lewis et al, 2009a). In addition, reconstruction of the transcriptional regulation network will aid in identifying the control of expression changes seen in the other systems.
Molecular Systems Biology 07/2010; 6(1):390. DOI:10.1038/msb.2010.47 · 10.87 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Short-term laboratory evolution of bacteria followed by genomic sequencing provides insight into the mechanism of adaptive evolution, such as the number of mutations needed for adaptation, genotype-phenotype relationships, and the reproducibility of adaptive outcomes.
In the present study, we describe the genome sequencing of 11 endpoints of Escherichia coli that underwent 60-day laboratory adaptive evolution under growth rate selection pressure in lactate minimal media. Two to eight mutations were identified per endpoint. Generally, each endpoint acquired mutations to different genes. The most notable exception was an 82 base-pair deletion in the rph-pyrE operon that appeared in 7 of the 11 adapted strains. This mutation conferred an approximately 15% increase to the growth rate when experimentally introduced to the wild-type background and resulted in an approximately 30% increase to growth rate when introduced to a background already harboring two adaptive mutations. Additionally, most endpoints had a mutation in a regulatory gene (crp or relA, for example) or the RNA polymerase.
The 82 base-pair deletion found in the rph-pyrE operon of many endpoints may function to relieve a pyrimidine biosynthesis defect present in MG1655. In contrast, a variety of regulators acquire mutations in the different endpoints, suggesting flexibility in overcoming regulatory challenges in the adaptation.