The network structure and the metabolic fluxes in central carbon metabolism were characterized in aerobically grown cells of Saccharomyces cerevisiae. The cells were grown under both high and low glucose concentrations, i.e., either in a chemostat at steady state with a specific growth rate of 0.1 h ؊1 or in a batch culture with a specific growth rate of 0.37 h ؊1 . Experiments were carried out using [1-13 C]glucose as the limiting substrate, and the resulting summed fractional labelings of intracellular metabolites were measured by gas chromatography coupled to mass spectrometry. The data were used as inputs to a flux estimation routine that involved appropriate mathematical modelling of the central carbon metabolism of S. cerevisiae. The results showed that the analysis is very robust, and it was possible to quantify the fluxes in the central carbon metabolism under both growth conditions. In the batch culture, 16.2 of every 100 molecules of glucose consumed by the cells entered the pentose-phosphate pathway, whereas the same relative flux was 44.2 per 100 molecules in the chemostat. The tricarboxylic acid cycle does not operate as a cycle in batch-growing cells, in contrast to the chemostat condition. Quantitative evidence was also found for threonine aldolase and malic enzyme activities, in accordance with published data. Disruption of the MIG1 gene did not cause changes in the metabolic network structure or in the flux pattern.Since the whole genome of Saccharomyces cerevisiae has been sequenced (15), several kinds of analyses have been applied in order to assign function to orphan genes. In many of these analyses, the aim has been to determine how the different genes (both the ones with known function and those open reading frames that have no assigned function) interact with each other, enabling the cells to take up nutrients, grow, divide, regulate their metabolism, release products to the environment, and respond to different stimuli (31).The different approaches that have been used for this purpose can be classified as transcriptome, proteome, and metabolome analyses, depending on the type of compounds measured, i.e., transcripts, proteins, or metabolites (31). Besides these approaches, other types of phenotypic investigations have been performed, such as growth on synthetic or rich media, growth on different carbon sources, determination of the ability to consume oxygen, and measurement of enzyme activities in cell extracts. In recent work, Entian and coworkers (9) showed that the type and accuracy of the methods used are very important for the identification of phenotypes in singledeletion mutants.A very prominent phenotypic investigation method that has been employed for the analysis of cells in different environments, as well as for the analysis of different mutants, is the quantification of metabolic fluxes in cells grown under balanced conditions, such as in a steady-state chemostat or during the exponential phase of a batch culture. This quantification can be carried out by metabolite balancing ...

A detailed characterization of the central metabolic network of Saccharomyces cerevisiae CEN.PK 113-7D was carried out during cometabolism of different mixtures of glucose and acetate, using aerobic C-limited chemostats in which one of these two substrates was labeled with 13 C. To confirm the role of malic enzyme, an isogenic strain with the corresponding gene deleted was grown under the same conditions. The labeling patterns of proteinogenic amino acids were analyzed and used to estimate metabolic fluxes and/or make inferences about the in vivo activities of enzymes of the central carbon metabolism and amino acid biosynthesis. Malic enzyme flux increased linearly with increasing acetate fraction. During growth on a very-high-acetate fraction, the activity of malic enzyme satisfied the biosynthetic needs of pyruvate in the mitochondria, while in the cytosol pyruvate was supplied via pyruvate kinase. In several cases enzyme activities were unexpectedly detected, e.g., the glyoxylate shunt for a very-low-acetate fraction, phosphoenolpyruvate carboxykinase for an acetate fraction of 0.46 C-mol of acetate/C-mol of substrate, and glucose catabolism to CO 2 via the tricarboxylic acid cycle for a very-highacetate fraction. Cytoplasmic alanine aminotransferase activity was detected, and evidence was found that ␣-isopropylmalate synthase has two active forms in vivo, one mitochondrial and the other a short cytoplasmic form.

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Modern medicine is founded on the discovery of penicillin and subsequent small molecules that inhibit bacterial peptidoglycan (PG) and cell wall synthesis. However, the discovery of new chemically and mechanistically distinct classes of PG inhibitors has become exceedingly rare, prompting speculation that intracellular enzymes involved in PG precursor synthesis are not 'druggable' targets. Here, we describe a β-lactam potentiation screen to identify small molecules that augment the activity of β-lactams against methicillin-resistant Staphylococcus aureus (MRSA) and mechanistically characterize a compound resulting from this screen, which we have named murgocil. We provide extensive genetic, biochemical, and structural modeling data demonstrating both in vitro and in whole cells that murgocil specifically inhibits the intracellular membrane-associated glycosyltransferase, MurG, which synthesizes the lipid II PG substrate that penicillin binding proteins (PBPs) polymerize and cross-link into the cell wall. Further, we demonstrate that the chemical synergy and cidality achieved between murgocil and the β-lactam imipenem is mediated through MurG dependent localization of PBP2 to the division septum. Collectively, these data validate our approach to rationally identify new target-specific bioactive β-lactam potentiation agents and demonstrate that murgocil now serves as a highly selective and potent chemical probe to assist our understanding of PG biosynthesis and cell wall biogenesis across Staphylococcal species.

S. cerevisiae responds to the presence of amino acids in the environment through the membrane-bound complex SPS, by altering transcription of several genes. Global transcription analysis shows that 46 genes are induced by L-citrulline. Under the given conditions there appears to be only one pathway for induction with L-citrulline, and this pathway is completely dependent on the SPS component, Ssy1p, and either of the transcription factors, Stp1p and Stp2p. Besides the effects on amino acid permease genes, an ssy1 and an stp1 stp2 mutant exhibit a number of other transcriptional phenotypes, such as increased expression of genes subject to nitrogen catabolite repression and genes involved in stress response. A group of genes involved in the upper part of the glycolysis, including those encoding hexose transporters Hxt4p, Hxt5p, Hxt6p, Hxt7p, hexokinase Hxk1p, glyceraldehyde 3-phosphate dehydrogenase Tdh1p and glucokinase (Glk1p), shows increased transcription levels in either or both of the mutants. Also, most of the structural genes involved in trehalose and glycogen synthesis and a few genes in the glyoxylate cycle and the pentose phosphate pathway are derepressed in the ssy1 and stp1 stp2 strains.