Project description:One longstanding question in microbiology is how microbes buffer perturbations in energy metabolism. In this study, we systematically analyzed the impact of different levels of ATP demand in Escherichia coli under various conditions (aerobic and anaerobic, with and without cell growth). One key finding is that, under all conditions tested, the glucose uptake increases with rising ATP demand, but only to a critical level beyond which it drops markedly, even below wild-type levels. Focusing on anaerobic growth and using metabolomics and proteomics data in combination with a kinetic model, we show that this biphasic behavior is induced by the dual dependency of the phosphofructokinase on ATP (substrate) and ADP (allosteric activator). This mechanism buffers increased ATP demands by a higher glycolytic flux but, as shown herein, it collapses under very low ATP concentrations. Model analysis also revealed two major rate-controlling steps in the glycolysis under high ATP demand, which could be confirmed experimentally. Our results provide new insights on fundamental mechanisms of bacterial energy metabolism and guide the rational engineering of highly productive cell factories.
Project description:Comparative analysis of changes in gene and protein expression and fatty acid profiles between Escherichia coli K-12 MG1655 ΔfadD ΔaraBAD expressing an acyl-acyl carrier protein thioesterase from Umbellularia californica (BTE) or a non-functional mutant thioesterase (BTE-H204A) to determine the functional basis for losses in cell viability, membrane integrity, or other stresses and metabolic perturbations that may be present. New hypotheses obtained from the study will assist in metabolic engineering efforts of improved strains exhibiting higher fatty acid yields and productivities.
Project description:Bacterial transcription factors (TFs) regulate gene expression to adapt to changing environments; when combined, the TF’s regulatory actions comprise transcriptional regulatory networks (TRNs). The chromatin immunoprecipitation (ChIP) assay is the major contemporary method for mapping in vivo protein-DNA interactions in the genome. It enables the genome-wide study of transcription factor binding sites (TFBSs) and gene regulation. Here, we present the genome-wide binding for major TFs in E. coli K-12 MG1655.
Project description:Escherichia coli DH1 cultures with treated with 6% 1,4 Butanediol for 1 hour and compared with untreated cultures The data from this experiment was used to identify a candidate for further study as described in Szmidt et al 2013 Utilizing a highly responsive gene, yhjX, in E. coli based production of 1,4-Butanediol submitted to Chemical Engineering Science
Project description:Cellular metabolism controls gene expression through allosteric interactions between metabolites and transcription factors. Methods to detect these regulatory interactions are mostly based on in vitro binding assays, but there are no methods to identify them at a genome-scale in vivo. Here we show that dynamic transcriptome and metabolome data identify metabolites that are potential effectors of transcription factors in E. coli. By switching the culture conditions between starvation and growth for 20 hours, we induced strong metabolite concentration changes and accompanying gene expression changes, which were measured by LC-MS/MS and RNA sequencing. From the transcriptome data we calculated the activity of 209 transcriptional regulators with Network Component Analysis, and then tested which metabolites correlated with these activities. This approach captured, for instance, the in vivo Hill-kinetics of CRP regulation by cyclic-AMP, a canonical example of allosteric transcription factor regulation in E. coli. By testing correlations between all pairs of transcription factors and metabolites, we predicted putative effectors of 65 transcription factors, and validated five of them in vitro. These results show that the combination of transcriptomics and metabolomics can generate hypotheses about metabolism-transcription interactions that are relevant in vivo and drive transitions between physiological states.
Project description:The only target locus of transcription factor BglJ known to date is the bgl operon, and activation of bgl by BglJ requires RcsB. Transcription factor LeuO is involved in stress responses and known as antagonist of H-NS. To identifiy novel targets of BglJ, we overexpressed BglJ in Escherichia coli K12 and measured differential gene expression by performing DNA microarray analysis. Moreover, to analyze whether all targets of BglJ require RcsB, we overexpressed BglJ in an rcsB deletion background. In addition, we overexpressed LeuO to identifiy targets of LeuO.
Project description:Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its co-factor NusG is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global co-localization of the nucleoid-associated protein H-NS with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes.
Project description:Metabolism controls gene expression through allosteric interactions between metabolites and transcription factors. These interactions are usually measured with in vitro assays, but there are no methods to identify them at a genome-scale in vivo. Here we show that dynamic transcriptome and metabolome data identify metabolites that control transcription factors in E. coli. By switching an E. coli culture between starvation and growth, we induce strong metabolite concentration changes and gene expression changes. Using Network Component Analysis we calculate the activities of 209 transcriptional regulators and correlate them with metabolites. This approach captures, for instance, the in vivo kinetics of CRP regulation by cyclic-AMP. By testing correlations between all pairs of transcription factors and metabolites, we predict putative effectors of 71 transcription factors, and validate five interactions in vitro. These results show that combining transcriptomics and metabolomics generates hypotheses about metabolism-transcription interactions that drive transitions between physiological states.