E. coli intracellular metabolism across various growth conditions
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ABSTRACT: FIA-TOF measurements of intracellular metabolites in E. coli cells grown across a range of 40 diverse conditions. See associated publication for details.
Project description:FIA-TOF measurements of intracellular metabolites in E. coli cells grown across a range of 40 diverse conditions. See associated publication for details.
Project description:Mycobacterium tuberculosis (Mtb) infection of macrophages reprograms cellular lipid metabolism to promote the formation of cytosolic lipid droplets. While it is clearly known that intracellular Mtb utilize host derived fatty acids and cholesterol to fuel a majority of its biosynthetic demands, the role of macrophage lipid metabolism on the bacteria’s ability to access the intracellular lipid pool remains controversial. We have utilized a CRISPR genetic knockdown approach to, systematically and comprehensively, characterize the role of macrophage fatty acid metabolism on the intracellular growth of Mtb. Our analyzes demonstrate that knockdown of lipid import, sequestration and metabolism genes collectively impair the intracellular growth of Mtb in macrophages. We further demonstrate that modulating fatty acids homeostasis in macrophages impair Mtb replication by enhancing production of pro-inflammatory cytokines, restricting the bacteria access to nutrients and increasing oxidative stress in a manner which is surprisingly divergent. We also demonstrate that impaired macrophage lipid droplet biogenesis is restrictive to intracellular Mtb growth. However, increased induction of lipid droplet formation by downstream blockade of fatty acid oxidation does not rescue the impaired Mtb growth phenotypes. Our work provides a characterization of macrophage fatty acid homeostasis and how its modulation impacts Mtb intracellular growth.
Project description:This project makes in depth measurements of protein and rna gene expression of E. coli under various environmental conditions including glycerol, lactate, gluconate, NaCl and MgSO4.
Project description:Salmonella invasion of non-phagocytic cells is a dynamic process that requires the translocation of effector proteins into host cells by a type III secretion system (T3SS1) encoded on Salmonella pathogenicity island 1 (SPI1). The signals involved in T3SS1 induction in vivo are not known, but SPI1-induced invasive Salmonella can be prepared by growth in synthetic media. Here we compared two different methods of preparing SPI1-induced bacteria by using a combination of transcriptome analysis, a single-cell reporter assay and intracellular gene expression analysis to reveal clear differences between these two bacterial populations that are reflected in their ability to interact with cultured epithelial cells. Invasion efficiency and intracellular replication were greater for bacteria grown with aeration to late-log phase compared to those grown without aeration to stationary phase. No significant differences in SPI1 regulon expression were revealed by transcriptome analysis. By contrast, surface appendage and stress response genes were considerably changed. Single cell analyses of gene expression revealed variation in the frequency of SPI1-induced bacteria and a correlation between SPI1-induction and the presence of flagella. The expression of virulence genes following invasion of cultured epithelial cells was also found to be predetermined by pre-invasion growth conditions. Key words: epithelial, flagella, invasion, motility, Salmonella-containing vacuole, treatment of shaking versus treatment of static growth conditions for St-SL1344
Project description:Salmonella invasion of non-phagocytic cells is a dynamic process that requires the translocation of effector proteins into host cells by a type III secretion system (T3SS1) encoded on Salmonella pathogenicity island 1 (SPI1). The signals involved in T3SS1 induction in vivo are not known, but SPI1-induced invasive Salmonella can be prepared by growth in synthetic media. Here we compared two different methods of preparing SPI1-induced bacteria by using a combination of transcriptome analysis, a single-cell reporter assay and intracellular gene expression analysis to reveal clear differences between these two bacterial populations that are reflected in their ability to interact with cultured epithelial cells. Invasion efficiency and intracellular replication were greater for bacteria grown with aeration to late-log phase compared to those grown without aeration to stationary phase. No significant differences in SPI1 regulon expression were revealed by transcriptome analysis. By contrast, surface appendage and stress response genes were considerably changed. Single cell analyses of gene expression revealed variation in the frequency of SPI1-induced bacteria and a correlation between SPI1-induction and the presence of flagella. The expression of virulence genes following invasion of cultured epithelial cells was also found to be predetermined by pre-invasion growth conditions. Key words: epithelial, flagella, invasion, motility, Salmonella-containing vacuole,
Project description:The aim of the project is to identify transcriptome-wide binding sites for the global RNA-binding protein ProQ in Salmonella during intracellular-like conditions.
Project description:A genome reduced E. coli strain MDS42ΔgalK::Ptet-gfp-kan were applied for the comparative transcriptome analysis. Genome-wide transcriptional changes under high osmotic prresure, high temperature condition and starvation were evaluated.
Project description:Accurate measurements of cellular protein concentrations are invaluable to quantitative studies of gene expression and physiology in living cells. Here, we developed a versatile mass spectrometric workflow based on data-independent acquisition proteomics (DIA/SWATH) together with a novel protein inference algorithm (xTop). We used this workflow to accurately quantify absolute protein abundances in E. coli for >2000 proteins over >60 growth conditions, including nutrient limitations, non-metabolic stresses and non-planktonic states. The resulting high-quality dataset of protein mass fractions allowed us to characterize proteome responses from a coarse (groups of related proteins) to a fine (individual) protein level. Hereby, a plethora of novel biological findings could be elucidated, including the generic upregulation of low-abundant proteins under various metabolic limitations, the non-specificity of catabolic enzymes upregulated under carbon limitation, the lack of large-scale proteome reallocation under stress compared to nutrient limitations, as well as surprising strain-dependent effects important for biofilm formation. These results present valuable resources for the systems biology community and can be used for future multi-omics studies of gene regulation and metabolic control in E. coli.