Aerial warfare: An inducible production of volatile bioactive metabolites in a novel species of Scytinostroma sp.

Antimicrobial volatile organic compounds (VOCs) may provide fungi an advantage over other competing microorganisms. As these defensive metabolites are often produced in response to microbial competitors, they are easily overlooked in axenic cultures. We used media supplemented with spent medium from Candida albicans to induce the expression of a broad-spectrum antimicrobial response in a previously uncharacterised white-rot fungus, Scytinostroma sp. Crude extractions of Scytinostroma sp. metabolites were found to be cytotoxic to fibroblast cells and antimicrobial to filamentous fungi, yeasts and Gram-positive bacteria. Volatile antimicrobial activity was observed for Scytinostroma sp. cultures and metabolite extracts using antimicrobial assays in bi-compartmentalised plates. Culture headspace analysis using solid-phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) revealed a pronounced shift in Scytinostroma sp. VOCs when cultured on media supplemented with C. albicans spent medium. We observed a significant increase in the levels of 45 identified VOCs, including 7 metabolites with reported antimicrobial activity. Using preparative HPLC combined with GC-MS, we determined that isovelleral is likely to be the main broad-spectrum antimicrobial metabolite produced by Scytinostroma sp. Isovelleral is a sesquiterpene dialdehyde with both antibiotic and antifeedant properties, previously detected in fruit bodies of other Basidiomycetes.

Epipyrone A, a Broad-Spectrum Antifungal Compound Produced by Epicoccum nigrum ICMP 19927.

We have isolated a filamentous fungus that actively secretes a pigmented exudate when growing on agar plates. The fungus was identified as being a strain of Epicoccum nigrum. The fungal exudate presented strong antifungal activity against both yeasts and filamentous fungi, and inhibited the germination of fungal spores. The chemical characterization of the exudate showed that the pigmented molecule presenting antifungal activity is the disalt of epipyrone A-a water-soluble polyene metabolite with a molecular mass of 612.29 and maximal UV-Vis absorbance at 428 nm. This antifungal compound showed excellent stability to different temperatures and neutral to alkaline pH.

Juice Index: an integrated Sauvignon blanc grape and wine metabolomics database shows mainly seasonal differences.

INTRODUCTION: Although Sauvignon Blanc (SB) grapes are cultivated widely throughout New Zealand, wines from the Marlborough region are most famous for their typical varietal combination of tropical and vegetal aromas. These wines differ in composition from season to season as well as among locations within the region, which makes the continual production of good quality wines challenging. Here, we developed a unique database of New Zealand SB grape juices and wines to develop tools to help winemakers to make blending decisions and assist in the development of new wine styles. METHODS: About 400 juices were collected from different regions in New Zealand over three harvest seasons (2011-2013), which were then fermented under controlled conditions using a commercial yeast strain Saccharomyces cerevisiae EC1118. Comprehensive metabolite profiling of these juices and wines by gas chromatography-mass spectrometry (GC-MS) was combined with their detailed oenological parameters and associated meteorological data. RESULTS: These combined metabolomics data clearly demonstrate that seasonal variation is more prominent than regional difference in both SB grape juices and wines, despite almost universal use of vineyard irrigation to mitigate seasonal rainfall and evapotranspiration differences, Additionally, we identified a group of juice metabolites that play central roles behind these variations, which may represent chemical signatures for juice and wine quality assessment. CONCLUSION: This database is the first of its kind in the world to be available for the wider scientific community and offers potential as a predictive tool for wine quality and innovation when combined with mathematical modelling.

The fate of linoleic acid on Saccharomyces cerevisiae metabolism under aerobic and anaerobic conditions.

INTRODUCTION: Saccharomyces cerevisiae has been widely used for fermenting food and beverages for over thousands years. Its metabolism together with the substrate composition play an important role in determining the characteristics of the final fermented products. We previously showed that the polyunsaturated fatty acid, linoleic acid, which is present in the grape juice at trace levels, significantly affected the development of aroma compounds of the wines. However, the effect of linoleic acid on the overall cell metabolism of S. cerevisiae is still not clear. Therefore, we aimed to unlock the metabolic response of S. cerevisiae to linoleic acid using metabolomics and isotope labelling experiments. METHODS: We cultured the cells on a minimal mineral medium supplementing them with linoleic acid isomers and 13C-linoleic acid. Both intracellular and extracellular metabolite profiles were determined using gas chromatography coupled to mass spectrometry (GC-MS) to investigate which S. cerevisiae pathways were affected by linoleic acid supplementation. RESULTS: The utilisation of linoleic acid by S. cerevisiae had a significant impact on the primary carbon metabolism increasing the glucose consumption and the ethanol production under anaerobic condition. The energetic state of the cell was, therefore, affected and the glycolytic pathway, the TCA cycle and the amino acid production were up-regulated. We also observed that linoleic acid was transported into the cell and converted into other fatty acids affecting their profile even under anaerobic condition. CONCLUSION: Our data clearly shows that linoleic acid supplementation in growth medium increased glucose consumption and ethanol production by S. cerevisiae under anaerobic condition. We also suggest that S. cerevisiae might be able to perform an alternative anaerobic pathway to ß-oxidation, which has not been reported yet.

Metabolite secretion in microorganisms: the theory of metabolic overflow put to the test.

INTRODUCTION: Microbial cells secrete many metabolites during growth, including important intermediates of the central carbon metabolism. This has not been taken into account by researchers when modeling microbial metabolism for metabolic engineering and systems biology studies. MATERIALS AND METHODS: The uptake of metabolites by microorganisms is well studied, but our knowledge of how and why they secrete different intracellular compounds is poor. The secretion of metabolites by microbial cells has traditionally been regarded as a consequence of intracellular metabolic overflow. CONCLUSIONS: Here, we provide evidence based on time-series metabolomics data that microbial cells eliminate some metabolites in response to environmental cues, independent of metabolic overflow. Moreover, we review the different mechanisms of metabolite secretion and explore how this knowledge can benefit metabolic modeling and engineering.

Modulation of Nitrous Oxide (N2O) Accumulation by Primary Metabolites in Denitrifying Cultures Adapting to Changes in Environmental C and N.

Metabolomics provides insights into the actual physiology of cells rather than their mere "potential", as provided by genomic and transcriptomic analysis. We investigate the modulation of nitrous oxide (N2O) accumulation by intracellular metabolites in denitrifying bacteria using metabolomics and genome-based metabolic network modeling. Profiles of metabolites and their rates of production/consumption were obtained for denitrifying batch cultures under four conditions: initial COD:N ratios of 11:1 and 4:1 with and without nitrite spiking (28 mg-N L-1). Only the nitrite-spiked cultures accumulated N2O. The NO2- spiked cultures with an initial COD:N = 11:1 accumulated 3.3 ± 0.57% of the total nitrogen added as N2O and large pools of tricarboxylic acid cycle intermediates and amino acids. In comparison, the NO2- spiked cultures with COD:N = 4:1 showed significantly higher (p = 0.028) N2O accumulation (8.5.3 ± 0.9% of the total nitrogen added), which was linked to the depletion of C11-C20 fatty acids. Metabolic modeling analysis shows that at COD:N of 4:1 the denitrifying cells slowly generate electron equivalents as FADH2 through ß-oxidation of saturated fatty acids, while COD:N of 11:1 do it through the TCA cycle. When combined with NO2- shock, this prolonged the duration over which insufficient electron equivalents were available to completely reduce NOx to N2, resulting in increased N2O accumulation. Results extend the understanding of how organic carbon and nitrite loads modulate N2O accumulation in denitrification, which may contribute to further design strategies to control greenhouse gas emissions from agricultural soils or wastewater treatment systems.

The effect of linoleic acid on the Sauvignon blanc fermentation by different wine yeast strains.

The level of linoleic acid in the Sauvignon blanc (SB) grape juice affects the development of different aroma compounds during fermentation by Saccharomyces cerevisiae EC1118, including key varietal thiols such as 3-mercaptohexanol (3MH) and 3-mercaptohexyl acetate (3MHA). However, it is still unknown if linoleic acid would affect in a similar way other commonly used S. cerevisiae wine strains. Here we investigated the effect of grape juice linoleic acid on the development of aroma compounds and other metabolites of SB wines using different wine yeast strains: EC1118, AWRI796 and VIN13. Linoleic acid clearly affected the levels of acetylated aroma compounds, several amino acids, and antioxidant molecules, independent of yeast strain, but the production of 3MH was affected by linoleic acid in a strain-specific manner. Moreover, the supplementation of deuterium-labelled 3MH also affected the production of varietal thiols in a strain-specific way. Linoleic acid reduced the acetylation process probably by inhibiting an acetyltransferase, an effect that was independent of the yeast strain. However, regulation of the 3MH biosynthesis is strain-specific, which suggests a mindful consideration not only towards the wine yeast but also to the linoleic acid concentration in the grape juice in order to obtain the desired wine aroma characteristics.

Assessment of nitric oxide (NO) redox reactions contribution to nitrous oxide (N2 O) formation during nitrification using a multispecies metabolic network model.

Over the coming decades nitrous oxide (N2O) is expected to become a dominant greenhouse gas and atmospheric ozone depleting substance. In wastewater treatment systems, N2O is majorly produced by nitrifying microbes through biochemical reduction of nitrite (NO2(-)) and nitric oxide (NO). However it is unknown if the amount of N2O formed is affected by alternative NO redox reactions catalyzed by oxidative nitrite oxidoreductase (NirK), cytochromes (i.e., P460 [CytP460] and 554 [Cyt554 ]) and flavohemoglobins (Hmp) in ammonia- and nitrite-oxidizing bacteria (AOB and NOB, respectively). In this study, a mathematical model is developed to assess how N2O formation is affected by such alternative nitrogen redox transformations. The developed multispecies metabolic network model captures the nitrogen respiratory pathways inferred from genomes of eight AOB and NOB species. The performance of model variants, obtained as different combinations of active NO redox reactions, was assessed against nine experimental datasets for nitrifying cultures producing N2O at different concentration of electron donor and acceptor. Model predicted metabolic fluxes show that only variants that included NO oxidation to NO2(-) by CytP460 and Hmp in AOB gave statistically similar estimates to observed production rates of N2O, NO, NO2(-) and nitrate (NO3(-)), together with fractions of AOB and NOB species in biomass. Simulations showed that NO oxidation to NO2(-) decreased N2O formation by 60% without changing culture's NO2(-) production rate. Model variants including NO reduction to N2O by Cyt554 and cNor in NOB did not improve the accuracy of experimental datasets estimates, suggesting null N2O production by NOB during nitrification. Finally, the analysis shows that in nitrifying cultures transitioning from dissolved oxygen levels above 3.8 ± 0.38 to <1.5 ± 0.8 mg/L, NOB cells can oxidize the NO produced by AOB through reactions catalyzed by oxidative NirK.

Use of metabolomics for the identification and validation of clinical biomarkers for preterm birth: Preterm SAMBA.

BACKGROUND: Spontaneous preterm birth is a complex syndrome with multiple pathways interactions determining its occurrence, including genetic, immunological, physiologic, biochemical and environmental factors. Despite great worldwide efforts in preterm birth prevention, there are no recent effective therapeutic strategies able to decrease spontaneous preterm birth rates or their consequent neonatal morbidity/mortality. The Preterm SAMBA study will associate metabolomics technologies to identify clinical and metabolite predictors for preterm birth. These innovative and unbiased techniques might be a strategic key to advance spontaneous preterm birth prediction. METHODS/DESIGN: Preterm SAMBA study consists of a discovery phase to identify biophysical and untargeted metabolomics from blood and hair samples associated with preterm birth, plus a validation phase to evaluate the performance of the predictive modelling. The first phase, a case-control study, will randomly select 100 women who had a spontaneous preterm birth (before 37 weeks) and 100 women who had term birth in the Cork Ireland and Auckland New Zealand cohorts within the SCOPE study, an international consortium aimed to identify potential metabolomic predictors using biophysical data and blood samples collected at 20 weeks of gestation. The validation phase will recruit 1150 Brazilian pregnant women from five participant centres and will collect blood and hair samples at 20 weeks of gestation to evaluate the performance of the algorithm model (sensitivity, specificity, predictive values and likelihood ratios) in predicting spontaneous preterm birth (before 34 weeks, with a secondary analysis of delivery before 37 weeks). DISCUSSION: The Preterm SAMBA study intends to step forward on preterm birth prediction using metabolomics techniques, and accurate protocols for sample collection among multi-ethnic populations. The use of metabolomics in medical science research is innovative and promises to provide solutions for disorders with multiple complex underlying determinants such as spontaneous preterm birth.

Chemicals eluting from disposable plastic syringes and syringe filters alter neurite growth, axogenesis and the microtubule cytoskeleton in cultured hippocampal neurons.

Cultures of dissociated hippocampal neurons are often used to study neuronal cell biology. We report that the development of these neurons is strongly affected by chemicals leaching from commonly used disposable medical-grade syringes and syringe filters. Contamination of culture medium by bioactive substance(s) from syringes and filters occurred with multiple manufacturing lots and filter types under normal use conditions and resulted in changes to neurite growth, axon formation and the neuronal microtubule cytoskeleton. The effects on neuronal morphology were concentration dependent and significant effects were detected even after substantial dilution of the contaminated medium. Gas chromatography-mass spectrometry analyses revealed many chemicals eluting from the syringes and filters. Three of these chemicals (stearic acid, palmitic acid and 1,2-ethanediol monoacetate) were tested but showed no effects on neurite growth. Similar changes in neuronal morphology were seen with high concentrations of bisphenol A and dibutyl phthalate, two hormonally active plasticisers. Although no such compounds were detected by gas chromatography­mass spectrometry, unknown plasticisers in leachates may affect neurites. This is the first study to show that leachates from laboratory consumables can alter the growth of cultured hippocampal neurons. We highlight important considerations to ensure leachate contamination does not compromise cell biology experiments.

Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy.

BACKGROUND: Quenching in cold buffered methanol at -40 °C has long been the preferred method for sub-second inactivation of cell metabolism during metabolic fingerprinting. However, methanol is known to cause intracellular metabolite leakage of microbial cells, making the distinction between intra- and extracellular metabolites in microbial systems challenging. In this paper, we tested three quenching protocols proposed for microbial cultures: fast filtration, cold buffered methanol and cold glycerol saline. RESULTS: Our results clearly showed that cold glycerol saline quenching resulted in the best recovery of intracellular metabolites in Lactobacillus paracasei subsp. paracasei (L. paracasei). Membrane integrity assayed by propidium iodide revealed that approximately 100 % [Corrected] of the L. paracasei cell membranes were damaged by contact with the cold buffered methanol solution, whilst cold glycerol saline quenching led to minimal cell damage. Due to the nature of the L. paracasei culture, fast filtration took several minutes, which is far from ideal for metabolites with high intracellular turnover rates. CONCLUSION: The implementation of a reliable, reproducible quenching method is essential within the metabolomics community. Cold glycerol saline prevented leakage of intracellular metabolites, and, thus, allowed more accurate determinations of intracellular metabolite levels.

Metabolite Profile of Cervicovaginal Fluids from Early Pregnancy Is Not Predictive of Spontaneous Preterm Birth.

In our study, we used a mass spectrometry-based metabolomic approach to search for biomarkers that may act as early indicators of spontaneous preterm birth (sPTB). Samples were selected as a nested case-control study from the Screening for Pregnancy Endpoints (SCOPE) biobank in Auckland, New Zealand. Cervicovaginal swabs were collected at 20 weeks from women who were originally assessed as being at low risk of sPTB. Samples were analysed using gas chromatography-mass spectrometry (GC-MS). Despite the low amount of biomass (16-23 mg), 112 compounds were detected. Statistical analysis showed no significant correlations with sPTB. Comparison of reported infection and plasma inflammatory markers from early pregnancy showed two inflammatory markers were correlated with reported infection, but no correlation with any compounds in the metabolite profile was observed. We hypothesise that the lack of biomarkers of sPTB in the cervicovaginal fluid metabolome is simply because it lacks such markers in early pregnancy. We propose alternative biofluids be investigated for markers of sPTB. Our results lead us to call for greater scrutiny of previously published metabolomic data relating to biomarkers of sPTB in cervicovaginal fluids, as the use of small, high risk, or late pregnancy cohorts may identify metabolite biomarkers that are irrelevant for predicting risk in normal populations.

Global metabolic response of Enterococcus faecalis to oxygen.

Oxygen and oxidative stress have become relevant components in clarifying the mechanism that weakens bacterial cells in parallel to the mode of action of bactericidal antibiotics. Given the importance of oxidative stress in the overall defense mechanism of bacteria and their apparent role in the antimicrobial mode of action, it is important to understand how bacteria respond to this stress at a metabolic level. The aim of this study was to determine the impact of oxygen on the metabolism of the facultative anaerobe Enterococcus faecalis using continuous culture, metabolomics, and (13)C enrichment of metabolic intermediates. When E. faecalis was rapidly transitioned from anaerobic to aerobic growth, cellular metabolism was directed toward intracellular glutathione production and glycolysis was upregulated 2-fold, which increased the supply of critical metabolite precursors (e.g., glycine and glutamate) for sulfur metabolism and glutathione biosynthesis as well as reducing power for cellular respiration in the presence of hemin. The ultimate metabolic response of E. faecalis to an aerobic environment was the upregulation of fatty acid metabolism and benzoate degradation, which was linked to important changes in the bacterial membrane composition as evidenced by changes in membrane fatty acid composition and the reduction of membrane-associated demethylmenaquinone. These key metabolic pathways associated with the response of E. faecalis to oxygen may represent potential new targets to increase the susceptibility of this bacterium to bactericidal drugs.

Nitrogen and carbon assimilation by Saccharomyces cerevisiae during Sauvignon blanc juice fermentation.

To investigate the assimilation and production of juice metabolites by Saccharomyces cerevisiae during winemaking, we compared the metabolite profiles of 63 Sauvignon blanc (SB) grape juices collected over five harvesting seasons from different locations of New Zealand before and after fermentation by the commercial wine yeast strain EC1118 at 15 °C. Metabolite profiles were obtained using gas chromatography-mass spectrometry and nuclear magnetic resonance and the oenological parameters were determined by Fourier transform infrared spectroscopy. Our results revealed that the amino acids threonine and serine were the most consumed organic nitrogen sources, while proline and gamma-aminobutyric acid were the least consumed amino acids during SB juice fermentation. Saccharomyces cerevisiae metabolised some uncommon nitrogen sources (e.g. norleucine, norvaline and pyroglutamic acid) and several organic acids, including some fatty acids, most likely after fermenting the main juice sugars (glucose, fructose and mannose). However, consumption showed large variation between juices and in some cases between seasons. Our study clearly shows that preferred nitrogen and carbon sources were consumed by S. cerevisiae EC1118 independent of the juice fine composition, whilst the consumption of other nutrient sources mainly depended on the concentration of other juice metabolites, which explains the uniqueness of each barrel of wine.

Constitutive homologous expression of phosphoglucomutase and transaldolase increases the metabolic flux of Fusarium oxysporum.

BACKGROUND: Fusarium oxysporum is among the few filamentous fungi that have been reported of being able to directly ferment biomass to ethanol in a consolidated bioprocess. Understanding its metabolic pathways and their limitations can provide some insights on the genetic modifications required to enhance its growth and subsequent fermentation capability. In this study, we investigated the hypothesis reported previously that phosphoglucomutase and transaldolase are metabolic bottlenecks in the glycolysis and pentose phosphate pathway of the F. oxysporum metabolism. RESULTS: Both enzymes were homologously overexpressed in F. oxysporum F3 using the gpdA promoter of Aspergillus nidulans for constitutive expression. Transformants were screened for their phosphoglucomutase and transaldolase genes expression levels with northern blot. The selected transformant exhibited high mRNA levels for both genes, as well as higher specific activities of the corresponding enzymes, compared to the wild type. It also displayed more than 20 and 15% higher specific growth rate upon aerobic growth on glucose and xylose, respectively, as carbon sources and 30% higher biomass to xylose yield. The determination of the relative intracellular amino and non-amino organic acid concentrations at the end of growth on glucose revealed higher abundance of most determined metabolites between 1.5- and 3-times in the recombinant strain compared to the wild type. Lower abundance of the determined metabolites of the Krebs cycle and an 68-fold more glutamate were observed at the end of the cultivation, when xylose was used as carbon source. CONCLUSIONS: Homologous overexpression of phosphoglucomutase and transaldolase in F. oxysporum was shown to enhance the growth characteristics of the strain in both xylose and glucose in aerobic conditions. The intracellular metabolites profile indicated how the changes in the metabolome could have resulted in the observed growth characteristics.

Toxin-antitoxin systems of Mycobacterium smegmatis are essential for cell survival.

The role of chromosomal toxin-antitoxin (TA) modules in bacterial physiology remains enigmatic despite their abundance in the genomes of many bacteria. Mycobacterium smegmatis contains three putative TA systems, VapBC, MazEF, and Phd/Doc, and previous work from our group has shown VapBC to be a bona fide TA system. In this study, we show that MazEF and Phd/Doc are also TA systems that are constitutively expressed, transcribed as leaderless transcripts, and subject to autoregulation, and expression of the toxin component leads to growth inhibition that can be rescued by the cognate antitoxin. No phenotype was identified for deletions of the individual TA systems, but a triple deletion strain (ΔvapBC, mazEF, phd/doc), designated ΔTA(triple), exhibited a survival defect in complex growth medium demonstrating an essential role for these TA modules in mycobacterial survival. Transcriptomic analysis revealed no significant differences in gene expression between wild type and the ΔTA(triple) mutant under these conditions suggesting that the growth defect was not at a transcriptional level. Metabolomic analysis demonstrated that in response to starvation in complex medium, both the wild type and ΔTA(triple) mutant consumed a wide range of amino acids from the external milieu. Analysis of intracellular metabolites revealed a significant difference in the levels of branched-chain amino acids between the wild type and ΔTA(triple) mutant, which are proposed to play essential roles in monitoring the nutritional supply and physiological state of the cell and linking catabolic with anabolic reactions. Disruption of this balance in the ΔTA(triple) mutant may explain the survival defect in complex growth medium.

Direct liquid transmission of sound has little impact on fermentation performance in Saccharomyces cerevisiae.

Sound is a physical stimulus that has the potential to affect various growth parameters of microorganisms. However, the effects of audible sound on microbes reported in the literature are inconsistent. Most published studies involve transmitting sound from external speakers through air toward liquid cultures of the microorganisms. However, the density differential between air and liquid culture could greatly alter the sound characteristics to which the microorganisms are exposed. In this study we apply white noise sound in a highly controlled experimental system that we previously established for transmitting sound underwater directly into liquid cultures to examine the effects of two key sound parameters, frequency and intensity, on the fermentation performance of a commercial Saccharomyces cerevisiae ale yeast growing in a maltose minimal medium. We performed these experiments in an anechoic chamber to minimise extraneous sound, and find little consistent effect of either sound frequency or intensity on the growth rate, maltose consumption, or ethanol production of this yeast strain. These results, while in contrast to those reported in most published studies, are consistent with our previous study showing that direct underwater exposure to white noise sound has little impact on S. cerevisiae volatile production and sugar utilization in beer medium. Thus, our results suggest the possibility that reported microorganism responses to sound may be an artefact associated with applying sound to cultures externally via transmission through air.

Metabolic Regulation of Copper Toxicity during Marine Mussel Embryogenesis.

The development of new tools for assessing the health of cultured shellfish larvae is crucial for aquaculture industries to develop and refine hatchery methodologies. We established a large-volume ecotoxicology/health stressor trial, exposing mussel (Perna canaliculus) embryos to copper in the presence of ethylenediaminetetraacetic acid (EDTA). GC/MS-based metabolomics was applied to identify potential biomarkers for monitoring embryonic/larval health and to characterise mechanisms of metal toxicity. Cellular viability, developmental abnormalities, larval behaviour, mortality, and a targeted analysis of proteins involved in the regulation of reactive oxygen species were simultaneously evaluated to provide a complementary framework for interpretative purposes and authenticate the metabolomics data. Trace metal analysis and speciation modelling verified EDTA as an effective copper chelator. Toxicity thresholds for P. canaliculus were low, with 10% developmental abnormalities in D-stage larvae being recorded upon exposure to 1.10 µg·L-1 bioavailable copper for 66 h. Sublethal levels of bioavailable copper (0.04 and 1.10 µg·L-1) caused coordinated fluctuations in metabolite profiles, which were dependent on development stage, treatment level, and exposure duration. Larvae appeared to successfully employ various mechanisms involving the biosynthesis of antioxidants and a restructuring of energy-related metabolism to alleviate the toxic effects of copper on cells and developing tissues. These results suggest that regulation of trace metal-induced toxicity is tightly linked with metabolism during the early ontogenic development of marine mussels. Lethal-level bioavailable copper (50.3 µg·L-1) caused severe metabolic dysregulation after 3 h of exposure, which worsened with time, substantially delayed embryonic development, induced critical oxidative damage, initiated the apoptotic pathway, and resulted in cell/organism death shortly after 18 h of exposure. Metabolite profiling is a useful approach to (1) assess the health status of marine invertebrate embryos and larvae, (2) detect early warning biomarkers for trace metal contamination, and (3) identify novel regulatory mechanisms of copper-induced toxicity.

The metabolic response of Candida albicans to farnesol under hyphae-inducing conditions.

Farnesol is a quorum-sensing molecule (QSM) produced, and sensed, by the polymorphic fungus, Candida albicans. This cell-to-cell communication molecule is known to suppress the hyphal formation of C. albicans at high cell density. Despite many studies investigating the signalling mechanisms by which QSMs influence the morphogenesis of C. albicans, the downstream metabolic effect of these signalling pathways in response to farnesol-mediated morphogenesis remains obscure. Here, we have used metabolomics to investigate the metabolic response of C. albicans upon exposure to farnesol under hyphae-inducing conditions. We have found a general up-regulation of central carbon metabolic pathways when hyphal formation was suppressed by farnesol evidenced by a considerably larger number of central carbon metabolic intermediates detected under this condition at an overall lower intracellular level. By combining the metabolic profiles from farnesol-exposed cells with previous metabolomics data for C. albicans undergoing morphogenesis, we have identified several metabolic pathways that are likely to be associated with the morphogenetic process of C. albicans, as well as metabolic pathways such as those involved in lipid metabolism that appeared to be specifically affected by farnesol. Therefore, our results provide important new insights into the metabolic role of farnesol in C. albicans metabolism.

Linking high-resolution metabolic flux phenotypes and transcriptional regulation in yeast modulated by the global regulator Gcn4p.

Genome sequencing dramatically increased our ability to understand cellular response to perturbation. Integrating system-wide measurements such as gene expression with networks of protein-protein interactions and transcription factor binding revealed critical insights into cellular behavior. However, the potential of systems biology approaches is limited by difficulties in integrating metabolic measurements across the functional levels of the cell despite their being most closely linked to cellular phenotype. To address this limitation, we developed a model-based approach to correlate mRNA and metabolic flux data that combines information from both interaction network models and flux determination models. We started by quantifying 5,764 mRNAs, 54 metabolites, and 83 experimental (13)C-based reaction fluxes in continuous cultures of yeast under stress in the absence or presence of global regulator Gcn4p. Although mRNA expression alone did not directly predict metabolic response, this correlation improved through incorporating a network-based model of amino acid biosynthesis (from r = 0.07 to 0.80 for mRNA-flux agreement). The model provides evidence of general biological principles: rewiring of metabolic flux (i.e., use of different reaction pathways) by transcriptional regulation and metabolite interaction density (i.e., level of pairwise metabolite-protein interactions) as a key biosynthetic control determinant. Furthermore, this model predicted flux rewiring in studies of follow-on transcriptional regulators that were experimentally validated with additional (13)C-based flux measurements. As a first step in linking metabolic control and genetic regulatory networks, this model underscores the importance of integrating diverse data types in large-scale cellular models. We anticipate that an integrated approach focusing on metabolic measurements will facilitate construction of more realistic models of cellular regulation for understanding diseases and constructing strains for industrial applications.