Project description:We conducted whole genome sequencing on eight evolved E. coli strains (S1–S8) and the parental wild-type (WT) strain to identify mutations arising from ofloxacin treatments. These strains (S1-S8), generated through fluoroquinolone-mediated adaptive laboratory evolution (ALE), exhibited varying levels of tolerance and resistance. The ALE experiment involved intermittent antibiotic treatments of eight independent cultures over 22 days. The untreated WT strain served as a baseline to pinpoint mutations in the evolved strains.
Project description:Using a synthetic biosensor to couple production of a specific metabolite with cell growth, we spontaneously evolved cells under the selective condition toward the acquisition of genotypes that optimally reallocated cellular resources. Using 3-hydroxypropionic acid (3-HP) production from glycerol in Escherichia coli as a model system, we determined that spontaneous mutations in the conserved regions of proteins involved in global transcriptional regulation altered the expression of several genes associated with central carbon metabolism. Our study provides a new perspective on adaptive laboratory evolution (ALE) using synthetic biosensors, thereby supporting future efforts in metabolic pathway optimization.
Project description:Fit phenotypes are achieved through optimal transcriptomic allocation. Here, we performed a high-resolution, multi-scale study of the transcriptomic tradeoff between two key fitness phenotypes, stress response (fear) and growth (greed), in Escherichia coli. We introduced 12 RNA polymerase (RNAP) mutations commonly acquired during adaptive laboratory evolution (ALE) and found that single mutations could result in large shifts in this fear vs. greed tradeoff likely through destabilizing the rpoB-rpoC interface. RpoS and GAD regulons drive the fear response while ribosomal proteins and the ppGpp regulon underlie greed. Growth rate selection pressure during ALE results in endpoint strains that universally have RNAP mutations, with synergistic mutations reflective of particular conditions. A phylogenetic analysis found the tradeoff in numerous bacteria species and one archaea species. The results suggest that the tradeoff represents a general principle of transcriptome allocation in bacteria where small genetic changes can result in large phenotypic adaptations to growth conditions.
Project description:We obtained pfkAB-deleted E. coli K-12 MG1655 strain that can thrive on glucose minimal medium with adaptive laboratory evolution (pfk_ALE-1 strain). Functional analysis of the mutations detected in the pfk_ALE-1 strain was conducted to elucidate the molecular mechanisms underlying the effects of these mutations. We performed transcriptome analysis with RNA-seq to investigate the transcriptomic effects of mutations involved in the glycolytic pathway and global transcriptional regulation. Transcriptomic analysis revealed the expression levels of 4,497 genes on the chromosome of MG1655 and ALE-1 strains.
Project description:Chloroplast RNA processing requires a large number of nuclear-encoded RNA binding proteins (RBPs) that are imported post-translationally into the organelle. Most of these RBPs are highly specific for one or few target RNAs. By contrast, members of the chloroplast ribonucleoprotein family (cpRNPs) have a wider RNA target range. We here present a quantitative analysis of RNA targets of the cpRNP CP31A using digestion-optimized RNA co-immunoprecipitation with deep sequencing (DO-RIP-seq). This identifies the mRNAs coding for subunits of the chloroplast NDH complex as main targets for CP31A. We demonstrate using whole-genome gene expression analysis and targeted RNA gel blot hybridization that the ndh mRNAs are all down-regulated in cp31a mutants. This diminishes the activity of the NDH complex. Our findings demonstrate how a chloroplast RNA binding protein can combine functionally related RNAs into one post-transcriptional operon.
Project description:Using a synthetic biosensor to couple production of a specific metabolite with cell growth, we spontaneously evolved cells under the selective condition toward the acquisition of genotypes that optimally reallocated cellular resources. Using 3-hydroxypropionic acid (3-HP) production from glycerol in Escherichia coli as a model system, we determined that spontaneous mutations in the conserved regions of proteins involved in global transcriptional regulation altered the expression of several genes associated with central carbon metabolism. Our study provides a new perspective on adaptive laboratory evolution (ALE) using synthetic biosensors, thereby supporting future efforts in metabolic pathway optimization.