ABSTRACT: GNPS melHKO Mm
untargeted metabolomics
(glycerol/propionate/cholesterol/oleic acid/pyruvate as sole carbon source)
RP C18/HILIC/HIPLEX column
Positive/Negative phase
Project description:A proteome and acetylome study under different culture conditions was carried out by quantitative label-free mass spectrometry analysis. E. coli K12 BW25113 strain was grown employing TB7 complex medium or M9 minimal medium, supplemented with glucose 20 mM or glycerol 40 mM as carbon source. Therefore, 4 culture conditions (TB7-glucose, TB7-glycerol, MM9-glucose, and MM9-glycerol) were used. Samples were taken in exponential and in stationary growth phase, resulting in 8 experimental samples: TB7-glucose in exponential phase, TB7-glucose in stationary phase, TB7-glycerol in exponential phase, TB7-glycerol in stationary phase, MM9-glucose in exponential phase, MM9-glucose in stationary phase, MM9-glycerol in exponential phase, and MM9-glycerol in stationary phase, with 4 biological replicates from each. This work emphasises the importance of the culture conditions choice, as they determine both, location of acetylation, and acetylation level, which may have an impact on regulation of the central metabolism.
Project description:Rsf1p is a putative transcription factor required for efficient growth using glycerol as sole carbon source but not for growth on the alternative respiratory carbon source ethanol. We use microarrays to determine the differences in the transcriptional program between the Δrsf1 mutant and the wild type during respiratory growth on glycerol as well as the transition to growth on glycerol as sole carbon source. Keywords: Mutant analysis during timecourse following switching carbon source from dextrose to glycerol
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:Rsf1p is a putative transcription factor required for efficient growth using glycerol as sole carbon source but not for growth on the alternative respiratory carbon source ethanol. We use microarrays to determine the differences in the transcriptional program between the delta-rsf1 mutant and the wild type during respiratory growth on glycerol as well as the transition to growth on glycerol as sole carbon source. Experiment Overall Design: delta-rsf1or the isogenic parent strain were grown to early log (A600=0.6) in YPD and then washed twice in prewarmed YPG (30 C) and returned to the air shaker in YPG for 15, 30 or 60 minutes. "Limit" conditions were provided by harvesting cells grown in YPD to early log phase without shifting to YPG and by harvesting cells grown in YPG to early log phase. Since the delta-rsf1 mutant and its isogenic parent strain grow equally well on the respiratory carbon source ethanol, cells were also harvested after being grown in ethanol to early log phase.
Project description:The purpose of this study was to examine how Mtb integrates acidic pH and available carbon sources as environmental cues to regulate its metabolism and growth rate. RNA-seq transcriptional profiling of M. tuberculosis growing at acidic or neutral pH, in pyruvate or glycerol, was examined. These studies identified carbon source-dependent and -independent pH-dependent adaptations. Mtb strain CDC1551 was grown in standing T-75 flasks in 40 mL of medium seeded an initial OD of 0.1. We examined medium in four conditions pH 7.0 10 mM glycerol, pH 5.7 10 mM glycerol, pH 7.0 10 mM pyruvate, pH 5.7 10 mM pyruvate. Following 3 days of incubation at 37C, RNA was isolated from the bacterial cultures and used for RNA-seq.
Project description:Propionate is an abundant carboxylic acid in nature. Microorganisms metabolize propionate aerobically via the 2-methylcitrate pathway. This pathway depends on a series of three reactions in the citric acid cycle that leads to the conversion of succinate to oxaloacetate. Interestingly, the gamma-proteobacterium Escherichia coli can use propionate as a carbon and electron source under oxic but not under anoxic conditions. The typical downregulation of the citric acid cycle under anoxic conditions is only partially responsible for the inability to use propionate under anoxic conditions since an arcA mutant shows very limited growth on propionate. RT-PCR and transcriptomic analysis revealed a post-transcriptional regulation of the prp-genecluster encoding the necessary enzymes for propionate metabolism. The polycistronic mRNA was hydrolyzed in the 3`-5` direction under anoxic conditions. This regulatory strategy is highly constructive because the last gene of the operon encodes the first enzyme of the propionate metabolism. Further analysis revealed that RNase R catalyzes the hydrolysis of the prp transcripts. Consequently, an rnr-deletion strain could metabolize propionate under anoxic conditions. To the best of our knowledge, this is the first study describing the influence of RNase R on the anaerobic metabolism of E. coli.
Project description:Diatoms are prominent marine microalgae, interesting not only from an ecological point of view, but also for their possible use for biotechnology applications. They can be cultivated in phototrophic conditions, using sunlight as the only energy source. Some diatoms, however, can also grow in mixotrophic mode, where both light and external reduced carbon contribute to biomass accumulation. In this study, we investigated the consequences of mixotrophy on the growth and metabolism of the pennate diatom Phaeodactylum tricornutum, using glycerol as a source of reduced carbon. Transcriptomic, metabolomic and physiological data indicate that glycerol affects the central-carbon, carbon-storage and lipid metabolism of the diatom. In particular, glycerol addition mimics some typical responses of nitrogen limitation on lipid metabolism at the level of TAG accumulation and fatty acid composition. However, this compound does not diminish photosynthetic activity and cell growth, at variance with nutrient limitation, revealing essential aspects of the metabolic flexibility of these microalgae and suggesting possible biotechnological applications of mixotrophy.
Project description:We report the application of a high-throughput technique, RNA-seq, to study the transcriptomic pattern of P. putida KT2440 cultures using myristic acid as the sole carbon source at the exponential phase of growth compared with cultures grown with glucose as the sole carbon source. Thus, we can explore the expression of genes involved in fatty acid degradation.
Project description:PrfA activity was studied in L. monocytogenes strain EGD and in an isogenic prfA deletion mutant (EGDΔprfA) carrying multiple copies of the wild-type prfA or the mutant prfA* gene (strains EGDΔprfApPrfA and EGDΔprfApPrfA*) after growth in brain heart infusion (BHI), Luria-Bertani broth (LB) or a defined minimal medium (MM) supplemented either with one of the three PTS-carbohydrates, glucose, mannose and cellobiose, or the non-PTS carbon source glycerol. Low PrfA activity was observed in the wild-type EGD strain in BHI and LB with either of these carbon sources, while PrfA activity was high in minimal medium in presence of glycerol but significantly reduced in presence of cellobiose. The strains expressing the prfA and prfA* gene under the prfA promoters, P1 and P2, produced equally large amounts of PrfA protein and high PrfA activity was observed in strain EGDΔprfApPrfA* under all growth conditions. In contrast, high PrfA activity in strain EGDΔprfApPrfA was only observed when this strain was cultured in BHI but not in LB or MM (in presence of either carbon source). A ptsH mutant (lacking a functional HPr) was able to grow in BHI suggesting that growth of L. monocytogenes in this culture medium is supported by carbon sources whose uptake and metabolism are independent of the PTS pathway. However, this mutant was unable to grow in LB and MM regardless which of the four carbon sources was added, suggesting that uptake of the used carbohydrates and the catabolism of glycerol depend fully on the functional common PTS pathway. Furthermore, the growth rates of L. monocytogenes are strongly reduced in presence of large amounts of PrfA protein when growing MM but less in LB and only slightly in BHI. The expression profiles of the genes encoding PTS permeases were determined in the three strains under various growth conditions. The data suggest that PrfA activity correlates with the expression level and the phosphorylation state of specific PTS permeases. This SuperSeries is composed of the SubSeries listed below.
Project description:RNA-Seq results accompanying submission of a manuscript: "Cholesterol-dependent transcriptome remodeling reveals new insight into the contribution of cholesterol to Mycobacterium tuberculosis pathogenesis" describing the role of cholesterol and vitamin B12 in shaping the transcriptome of the Mycobacterium tuberculosis H37Rv and M. tuberculosis ∆prpR - propionate regulator (PrpR) mutant. Next generation sequencing results are provided in three independent biological replicates for each strain growing in three different media - minimal medium with glycerol or cholesterol as the sole carbon source and standard 7H9/10% OADC medium. The influence of vitamin B12 on M. tuberculosis transcriptome was analysed on 7H9/10% OADC medium supplemented with B12. The study allowed us to re-establish the list of genes potentially involved in cholesterol metabolism. We further proposed a novel regulatory function of vitamin B12 and PrpR, a propionate regulator, in coordinated cholesterol breakdown metabolite dissipation and virulent phenotype induction. Finally, we demonstrated that a key role of cholesterol in Mtb metabolism is not only providing carbon and energy but also inducing a transcriptome remodeling program that helps in developing tolerance to the unfavorable host cell environment.