Project description:We carried out adaptive laboratory evolution of an E. coli strain lacking four genes (adhE, pta, ldhA, frdA) involved in acetyl-CoA consumption, allowing the efficient utilization of acetate as its sole carbon and energy source. The transcriptomes according to the medium status (M9 aceate, M9 glucose) of the evolved strain (SBA01) and its parent strain (DSM01) were compared using RNA-seq.
Project description:Transcription profiling of wild type E. coli MG1655, intestine-adapted E. coli MG1655star, and E. coli MG1655 flhD mutant grown on glucose, mannose, and mucus. We previously isolated a spontaneous mutant of E. coli K-12, strain MG1655, following passage through the streptomycin-treated mouse intestine, which has colonization traits superior to the wild-type parent strain (Leatham, et. al., 2005, Infect Immun 73:8039-49) The intestine-adapted strain (E. coli MG1655star) grew faster on several different carbon sources compared to the wild-type and was non-motile due to deletion of the flhD gene. To further characterize E. coli MG1655star, we used several high-throughput genomic approaches. Whole-genome pyrosequencing did not reveal any changes on its genome, aside from the deletion at the flhDC locus, that could explain the colonization advantage of E. coli MG1655star. Microarray analysis revealed modest, yet significant induction of catabolic gene systems across the genome in both E. coli MG1655star and the isogenic flhD mutant. Catabolome analysis with Biolog GN2 Microplates revealed an enhanced ability of both E. coli MG1655star and the isogenic flhD mutant to oxidize a wide variety of carbon sources. The results show that intestine-adapted E. coli MG1655star is more fit than the wild-type for intestinal colonization because loss of FlhD results in elevated expression of genes involved in carbon and energy metabolism, leading to more efficient carbon source utilization, which results in a higher population size in the intestine. Hence mutations that enhance metabolic efficiency confer a colonization advantage.
Project description:It is now possible to discover the physiological function of LysR-family transcription factors (LLTF) in E. coli using ChIP-Exo (in vivo DNA-binding), growth phenotype, conserved gene clustering, and transcriptional analysis (RNA-seq deletion mutants). The LysR-family regulator phenotype microarray has been detected for YbdO, YbeF, YcaN, YiaU, and YgfI deletion mutants and for the isogenic E. coli BW25113 strain in minimal medium at carbon/nitrogen starvation or L-threonine supplement conditions. The LLTF YbdO and YgfI regulation is important for bacterial growth adaptation to low pH with citrate supplementation or glycerol as the carbon source, respectively. A systems analysis approach allows for the identification of the YneJ (putrescine utilization), YgfI, and YbdO transcription factor regulated genes in Escherichia coli. YgfI regulates DhaKLM, the phosphotransferase system involved in glycerol and dihydroxyacetone utilization. YbdO is a repressor for ybdMN and is likely involved in citrate lyase regulation. YneJ, re-named PtrR, directly controls the expression of the succinate-semialdehyde dehydrogenase Sad (YneI) and is important for bacterial growth in the presence of L-glutamate as a nitrogen source. The sad promoter is repressed by PtrR. PtrR is also a repressor of the fnrS gene, encoding a small regulatory RNA involved in the regulation of the sodB gene, as shown by RNA-seq data. A 15-bp palindromic PtrR-binding site was identified in the upstream regions of the sad / ptrR and fnrS genes and confirmed by ChIP-Exo and fluorescent polarization assays. The PtrR-dependent regulation of fnrS likely leads to a regulatory cascade induced by this small RNA.
Project description:Mapping the occupancy of ArcA throughout the genome of Escherchia coli MG1655 K-12 using an affinity purified antibody under anaerobic and aerobic growth conditions. As a control, we also performed ChIP-chip onArcA in a ∆arcA mutant strain of Escherchia coli MG1655 K-12. Described in the manuscript The response regulator ArcA uses a diverse binding site architechture to globally regulate carbon oxidation in E. coli
Project description:Transcription profiling of wild type E. coli MG1655, intestine-adapted E. coli MG1655star, and E. coli MG1655 flhD mutant grown on glucose, mannose, and mucus. We previously isolated a spontaneous mutant of E. coli K-12, strain MG1655, following passage through the streptomycin-treated mouse intestine, which has colonization traits superior to the wild-type parent strain (Leatham, et. al., 2005, Infect Immun 73:8039-49) The intestine-adapted strain (E. coli MG1655star) grew faster on several different carbon sources compared to the wild-type and was non-motile due to deletion of the flhD gene. To further characterize E. coli MG1655star, we used several high-throughput genomic approaches. Whole-genome pyrosequencing did not reveal any changes on its genome, aside from the deletion at the flhDC locus, that could explain the colonization advantage of E. coli MG1655star. Microarray analysis revealed modest, yet significant induction of catabolic gene systems across the genome in both E. coli MG1655star and the isogenic flhD mutant. Catabolome analysis with Biolog GN2 Microplates revealed an enhanced ability of both E. coli MG1655star and the isogenic flhD mutant to oxidize a wide variety of carbon sources. The results show that intestine-adapted E. coli MG1655star is more fit than the wild-type for intestinal colonization because loss of FlhD results in elevated expression of genes involved in carbon and energy metabolism, leading to more efficient carbon source utilization, which results in a higher population size in the intestine. Hence mutations that enhance metabolic efficiency confer a colonization advantage. Three strains were profiled: E. coli MG1655 wildtype, E. coli flhD, and an intestine adapted strain, MG1655star, derived from the wildtype and isolated from feces after 15 days in the streptomycin treated mouse intestine, which proved to be a better colonizer than the wildtype, were grown on MOPS minimal medium containing 0.2% glucose or mannose, or mucus (10 mg/ml) and RNA was extracted from logarithmic phase cultures, and also from mucus grown cells in late log phase.
Project description:Escherichia coli, the common inhabitant of the mammalian intestine, exhibits considerable intraspecies genomic variation, which has been suggested to reflect adaptation to different ecological niches. Also, regulatory trade-offs, e.g., between catabolic versatility and stress protection, are thought to result in significant physiological differences between strains. For these reasons, the relevance of experimental observations made for “domesticated” E. coli strains with regard to the behaviour of this species in its natural environments is often questioned and frequently doubts are raised on the status of E. coli as a defined species. We therefore investigated the variability of important eco-physiological functions such as carbon substrate uptake and breakdown capabilities as well as stress defence mechanisms in the genomes of commensal and pathogenic E. coli strains. Furthermore, eco-physiological properties of environmental strains were compared to standard laboratory strain K-12 MG1655. Catabolic, stress protection, and carbon- and energy source transport operons showed a very low intraspecies variability in 57 commensal and pathogenic E. coli. Environmental isolates adapted to glucose-limited growth in a similar way as E. coli MG1655, namely by increasing their catabolic flexibility and by inducing high affinity substrate uptake systems. Our results indicate that the major eco-physiological properties are highly conserved in the natural population of E. coli. This questions the proposed dominant role of horizontal gene transfer for niche adaptation. Keywords: comparative genomic hybridisation
Project description:Escherichia coli exhibits diauxic growth in sugar mixtures due to CRP-mediated catabolite repression and inducer exclusion related to phosphotransferase system enzyme activity. Replacement of the native crp gene with a catabolite repression mutant (referred to as crp*) alleviates diauxic effects in E. coli and enables co-utilization of glucose and other sugars. While previous studies have examined the effects of expressing CRP* mutants on the expression of specific catabolic genes, little is known about the global transcriptional effects of CRP* expression. In this study, we compare the transcriptome of E. coli W3110 (expressing wild-type CRP) to that of mutant strain PC05 (expressing CRP*) in the presence and absence of glucose.
Project description:E. coli isolates from different CF patients demonstrate increased growth rate when grown with glycerol, a major component of fecal fat, as the sole carbon source compared to E. coli from healthy controls. CF and control E. coli isolates have differential gene expression when grown in minimal media with glycerol as the sole carbon source. While CF isolates display a growth promoting transcriptional profile, control isolates engage stress and stationary phase programs, which likely results in slower growth rates.
Project description:The purpose of this study is to determine whether the presence of pathogenic Escherichia coli in colon is associated with psychiatric disorders.