RNA polymerase mutants found through adaptive evolution reprogram Escherichia coli for optimal growth in minimal media

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.kinetics | stringent response | transcription

Global metabolic network reorganization by adaptive mutations allows fast growth of Escherichia coli on glycerol

“…1c). Consistent with previous results 11 , biomass yield was increased by the rpoC mutation (expressed as decreased glycerol consumption for equal biomass increase), although it grew faster than both WT and GLPK (Fig. 1a).…”

“…Together, these results suggest that the rpoC mutation improves growth through major reorganization of the metabolic network that reflects increased anabolic needs to cope with rapid growth. Moreover, since the growth rate enhancing effects of the rpoC mutation are observable only on minimal media 11 , these global changes can also be interpreted as depicting a response optimizing the synthesis of most biomass precursors, which might explain the generally lower levels of precursor metabolites.…”

“…Most of the acquired mutations affecting growth phenotype can be grouped into those affecting condition-specific function (for example, glycerol kinase or glpK) or global transcription patterns (for example, RNA polymerase b 0 subunit encoded by rpoC). Although the biochemical characteristics of some of the naturally occurring mutations in glycerol kinase and RNA polymerase have been studied 10,11 , the underlying molecular and network-level consequences and mechanisms promoting cell growth in vivo have not been uncovered. While adaptive evolution and identification of mutations by resequencing has been made easy and cheap, the elucidation of network-level mechanisms remains a major bottleneck in the study of adaptive evolution 4,12 .…”

“…However, the genotype-phenotype relationship is multilayered and hierarchical and the measured genotypes and phenotypes are the opposite ends of this hierarchy. The in vitro assessment of the effects of the mutated gene products relative to the wild type (WT) can help determine how molecular properties change 10,11 but studies at the level of individual gene-product properties give limited insights into the mechanisms that change the phenotype. Network-level changes resulting from the introduction of causal mutations can now be characterized through the generation and integrative analysis of polyomic data sets.…”

Laboratory evolution of Geobacter sulfurreducens for enhanced growth on lactate via a single-base-pair substitution in a transcriptional regulator

“…The availability of technology to rapidly identify mutations in adapted strains, and then evaluate the contributions of those mutations to adaptation with genetic manipulation, has greatly accelerated the understanding of the mechanisms for adaptation to new substrates (Brockhurst et al, 2010). Genomic changes promoting adaptation to new substrates have included mutations that enhance the kinetics of enzymes acting on the substrate (Fong et al, 2005a, b;Herring et al, 2006;Conrad et al, 2009;Lee and Palsson, 2010) and mutations in promoter regions and global regulatory elements (Treves et al, 1998;Herring et al, 2006;Pelosi et al, 2006); as well as RNA polymerases (Herring et al, 2006;Conrad et al, 2010). Surprisingly, there have been fewer reports of adaptation to the use of a new substrate via mutations in known transcriptional regulators (Philippe et al, 2007;Barrick et al, 2009) despite the fact that changes in transcriptional regulators are thought to have key roles in the evolution of new microbial species (Dekel and Alon, 2005;Babu and Aravind, 2006;Babu et al, 2007), a concept further supported with the observation that transcriptional regulatory networks evolve and change faster than other collaborative biological networks such as protein interaction networks and metabolic pathway networks (Shou et al, 2011).…”