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Role of nitrogen and carbon transport, regulation, and metabolism genes for Saccharomyces cerevisiae survival in vivo.

ABSTRACT: Saccharomyces cerevisiae is both an emerging opportunistic pathogen and a close relative of pathogenic Candida species. To better understand the ecology of fungal infection, we investigated the importance of pathways involved in uptake, metabolism, and biosynthesis of nitrogen and carbon compounds for survival of a clinical S. cerevisiae strain in a murine host. Potential nitrogen sources in vivo include ammonium, urea, and amino acids, while potential carbon sources include glucose, lactate, pyruvate, and fatty acids. Using mutants unable to either transport or utilize these compounds, we demonstrated that no individual nitrogen source was essential, while glucose was the most significant primary carbon source for yeast survival in vivo. Hydrolysis of the storage carbohydrate glycogen made a slight contribution for in vivo survival compared with a substantial requirement for trehalose hydrolysis. The ability to sense and respond to low glucose concentrations was also important for survival. In contrast, there was little or no requirement in vivo in this assay for any of the nitrogen-sensing pathways, nitrogen catabolite repression, the ammonium- or amino acid-sensing pathways, or general control. By using auxotrophic mutants, we found that some nitrogenous compounds (polyamines, methionine, and lysine) can be acquired from the host, while others (threonine, aromatic amino acids, isoleucine, and valine) must be synthesized by the pathogen. Our studies provide insights into the yeast-host environment interaction and identify potential antifungal drug targets.

SUBMITTER: Kingsbury JM

PROVIDER: S-EPMC1459679 | biostudies-literature | 2006 May

REPOSITORIES: biostudies-literature

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Role of nitrogen and carbon transport, regulation, and metabolism genes for Saccharomyces cerevisiae survival in vivo.

Kingsbury Joanne M JM Goldstein Alan L AL McCusker John H JH

Eukaryotic cell 20060501 5

Saccharomyces cerevisiae is both an emerging opportunistic pathogen and a close relative of pathogenic Candida species. To better understand the ecology of fungal infection, we investigated the importance of pathways involved in uptake, metabolism, and biosynthesis of nitrogen and carbon compounds for survival of a clinical S. cerevisiae strain in a murine host. Potential nitrogen sources in vivo include ammonium, urea, and amino acids, while potential carbon sources include glucose, lactate, py ...[more]

PMID: 16682459

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Project description:Ammonium is the most common N source for yeast fermentations. Although its transport and assimilation mechanisms are well documented, there have been only a few attempts to measure the in vivo intracellular concentration of ammonium and assess its impact on gene expression. Using an isotope dilution mass spectrometry (IDMS)-based method, we were able to measure the intracellular ammonium concentration in N-limited aerobic chemostat cultivations using three different N sources (ammonium, urea, and glutamate) at the same growth rate (0.05 h-1). The experimental results suggest that, at this growth rate, a similar concentration of intracellular (IC) ammonium, about 3.6 mmol NH4+/literIC, is required to supply the reactions in the central N metabolism, independent of the N source. Based on the experimental results and different assumptions, the vacuolar and cytosolic ammonium concentrations were estimated. Furthermore, we identified a futile cycle caused by NH3 leakage into the extracellular space, which can cost up to 30% of the ATP production of the cell under N-limited conditions, and a futile redox cycle between Gdh1 and Gdh2 reactions. Finally, using shotgun proteomics with protein expression determined relative to a labeled reference, differences between the various environmental conditions were identified and correlated with previously identified N compound-sensing mechanisms.IMPORTANCE In our work, we studied central N metabolism using quantitative approaches. First, intracellular ammonium was measured under different N sources. The results suggest that Saccharomyces cerevisiae cells maintain a constant NH4+ concentration (around 3 mmol NH4+/literIC), independent of the applied nitrogen source. We hypothesize that this amount of intracellular ammonium is required to obtain sufficient thermodynamic driving force. Furthermore, our calculations based on thermodynamic analysis of the transport mechanisms of ammonium suggest that ammonium is not equally distributed, indicating a high degree of compartmentalization in the vacuole. Additionally, metabolomic analysis results were used to calculate the thermodynamic driving forces in the central N metabolism reactions, revealing that the main reactions in the central N metabolism are far from equilibrium. Using proteomics approaches, we were able to identify major changes, not only in N metabolism, but also in C metabolism and regulation.

Project description:BackgroundConsiderable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose.ResultsGenes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators HAP4, CAT8 and SIP1-2 and 4. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor RGT1 and its target genes HXT2-3, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells.ConclusionThe results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant S. cerevisiae strains. Phosphorylation of different isoforms of glycolytic enzymes suggests that regulation of glycolysis also occurred at a post-translational level, supporting prior findings.

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Role of nitrogen and carbon transport, regulation, and metabolism genes for Saccharomyces cerevisiae survival in vivo.

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Role of nitrogen and carbon transport, regulation, and metabolism genes for Saccharomyces cerevisiae survival in vivo.

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