Whole-genome resequencing of Escherichia coli K-12 MG1655 undergoing short-term laboratory evolution in lactate minimal media reveals flexible selection of adaptive mutations
TL;DR: 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, suggesting flexibility in overcoming regulatory challenges in the adaptation.
Abstract: Background: 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. Results: 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. Conclusions: 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.
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TL;DR: Evidence for parallel and collateral evolution has been found in many taxa, and an emerging hypothesis is that they result from the fact that mutations in some genetic targets minimize pleiotropic effects while simultaneously maximizing adaptation.
Abstract: This Review distinguishes between three distinct routes by which similar genetic changes contribute to convergent evolution and discusses examples from diverse taxa. Convergent genetic evolution might result from the fact that some mutations both minimize pleiotropic effects and maximize adaptation.
651 citations
TL;DR: In this article, the authors reported that >98% of active reactions from FBA optimal growth solutions are supported by transcriptomic and proteomic data, and when E. coli adapts to growth rate selective pressure, the evolved strains upregulated genes within the optimal growth predictions, and downregulated genes outside of the optimal solutions.
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.
636 citations
Cites background or methods from "Whole-genome resequencing of Escher..."
...For example, whole-genome resequencing of adapted strains showed that only a small number of mutations arise after hundreds of generations (Herring et al, 2006; Conrad et al, 2009)....
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...Therefore, it seems that there are unknown regulatory mechanisms at work, potentially because of mutations found in transcriptional regulators in the evolved strains (Herring et al, 2006; Conrad et al, 2009; Charusanti et al, submitted for publication)....
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...The genetic and physiological characteristics of the adaptation have been described (Herring et al, 2006; Barrick et al, 2009; Conrad et al, 2009; Charusanti et al, submitted for publication)....
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...When prokaryotes are grown at low- to mid-log phase for hundreds of generations through periodic serial passaging, they acquire an increased growth rate (Lenski and Travisano, 1994; Ibarra et al, 2002; Fong et al, 2003; Barrick et al, 2009; Conrad et al, 2009; Teusink et al, 2009)....
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...…this work, each strain studied had ample time for more drastic changes in gene and protein expression, as a result of the mutations in metabolic enzymes and global regulators attained in the adaptive time course (Herring et al, 2006; Conrad et al, 2009; Charusanti et al, submitted for publication)....
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TL;DR: Fundamental issues associated with its formulation and use are reviewed and use to compute optimal growth states are reviewed.
565 citations
Cites background from "Whole-genome resequencing of Escher..."
...Evolved strains can then be re-sequenced to find all mutations generated, thus illuminating the underlying genetic and molecular biological basis for optimal growth phenotypes [31,32]....
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TL;DR: Categorization of the available tools for network inference according to the strategies that they use to deal with the problem of underdetermination allows an insight into why a certain tool is more appropriate for the specific research question or data set at hand.
Abstract: Network inference, which is the reconstruction of biological networks from high-throughput data, can provide valuable information about the regulation of gene expression in cells. However, it is an underdetermined problem, as the number of interactions that can be inferred exceeds the number of independent measurements. Different state-of-the-art tools for network inference use specific assumptions and simplifications to deal with underdetermination, and these influence the inferences. The outcome of network inference therefore varies between tools and can be highly complementary. Here we categorize the available tools according to the strategies that they use to deal with the problem of underdetermination. Such categorization allows an insight into why a certain tool is more appropriate for the specific research question or data set at hand.
520 citations
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TL;DR: A scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments with 96% coverage at 99.96% accuracy in one run of the machine is described.
Abstract: The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.
8,434 citations
"Whole-genome resequencing of Escher..." refers background in this paper
...Pulse field gel electrophoresis [13] or sequencing using longer read lengths, such as 454 [14], or paired reads can provide information on these mutation events....
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TL;DR: In this analysis, mRNA folding and associated rates of translation initiation play a predominant role in shaping expression levels of individual genes, whereas codon bias influences global translation efficiency and cellular fitness.
Abstract: Synonymous mutations do not alter the encoded protein, but they can influence gene expression. To investigate how, we engineered a synthetic library of 154 genes that varied randomly at synonymous sites, but all encoded the same green fluorescent protein (GFP). When expressed in Escherichia coli, GFP protein levels varied 250-fold across the library. GFP messenger RNA (mRNA) levels, mRNA degradation patterns, and bacterial growth rates also varied, but codon bias did not correlate with gene expression. Rather, the stability of mRNA folding near the ribosomal binding site explained more than half the variation in protein levels. In our analysis, mRNA folding and associated rates of translation initiation play a predominant role in shaping expression levels of individual genes, whereas codon bias influences global translation efficiency and cellular fitness.
1,378 citations
TL;DR: In this article, the authors examined the dynamics of evolutionary adaptation, the genetic bases of adaptation, tradeoffs and the environmental specificity of adaptation and the origin and evolutionary consequences of mutators.
Abstract: Microorganisms have been mutating and evolving on Earth for billions of years. Now, a field of research has developed around the idea of using microorganisms to study evolution in action. Controlled and replicated experiments are using viruses, bacteria and yeast to investigate how their genomes and phenotypic properties evolve over hundreds and even thousands of generations. Here, we examine the dynamics of evolutionary adaptation, the genetic bases of adaptation, tradeoffs and the environmental specificity of adaptation, the origin and evolutionary consequences of mutators, and the process of drift decay in very small populations.
1,359 citations
TL;DR: Physicians need to know whether laboratories in their area routinely test for E. coli O157 in stool specimens, and whether treatment with antimicrobial agents remains controversial: some studies suggest that treatment may precipitate haemolytic uraemic syndrome, and others suggest no effect or even a protective effect.
1,035 citations
TL;DR: The high conservation of small RNAs among closely related bacterial species, as well as analysis of transcripts detected by high-density oligonucleotide probe arrays, are used to predict the presence of novel small RNA genes in the intergenic regions of the Escherichia coli genome.
Abstract: A burgeoning list of small RNAs with a variety of regulatory functions has been identified in both prokaryotic and eukaryotic cells. However, it remains difficult to identify small RNAs by sequence inspection. We used the high conservation of small RNAs among closely related bacterial species, as well as analysis of transcripts detected by high-density oligonucleotide probe arrays, to predict the presence of novel small RNA genes in the intergenic regions of the Escherichia coli genome. The existence of 23 distinct new RNA species was confirmed by Northern analysis. Of these, six are predicted to encode short ORFs, whereas 17 are likely to be novel functional small RNAs. We discovered that many of these small RNAs interact with the RNA-binding protein Hfq, pointing to a global role of the Hfq protein in facilitating small RNA function. The approaches used here should allow identification of small RNAs in other organisms.
710 citations
"Whole-genome resequencing of Escher..." refers background in this paper
...A direct relationship also exists between the hfq and ryhA genes; ryhA codes for a small RNA that interacts with hfq and may provide regulation [20]....
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