Changes In Substrate Availability Research Articles

Alterations in rat adipose tissue transcriptome and proteome in response to prolonged fasting.

Various pathophysiological situations of negative energy balance involve the intense depletion of the body's energy reserves. White adipose tissue is a central place to store energy and a major endocrine organ. As a model of choice to better understand how the white adipose tissue dynamically responds to changes in substrate availability, we used the prolonged fasting paradigm, which is characterized by successive periods of stimulated (phase 2) and then reduced (phase 3) lipid mobilization/utilization. Using omics analyses, we report a regulatory transcriptional program in rat epididymal (EPI) adipose tissue favoring lipolysis during phase 2 and repressing it during phase 3. Changes in gene expression levels of lipases, lipid droplet-associated factors, and the proteins involved in cAMP-dependent and cAMP-independent regulation of lipolysis are highlighted. The mRNA and circulating levels of adipose-secreted factors were consistent with the repression of insulin signaling during prolonged fasting. Other molecular responses are discussed, including the regulation of leptin and adiponectin levels, the specific changes reflecting an increased fibrinolysis and a possible protein catabolism-related energy saving mechanism in late fasting. Finally, some differences between internal and subcutaneous (SC) adipose tissues are also reported. These data provide a comprehensive molecular basis of adipose tissue responses when facing a major energetic challenge.

Quantitative Flow Cytometry to Understand Population Heterogeneity in Response to Changes in Substrate Availability in Escherichia coli and Saccharomyces cerevisiae Chemostats.

Microbial cells in bioprocesses are usually described with averaged parameters. But in fact, single cells within populations vary greatly in characteristics such as stress resistance, especially in response to carbon source gradients. Our aim was to introduce tools to quantify population heterogeneity in bioprocesses using a combination of reporter strains, flow cytometry, and easily comprehensible parameters. We calculated mean, mode, peak width, and coefficient of variance to describe distribution characteristics and temporal shifts in fluorescence intensity. The skewness and the slope of cumulative distribution function plots illustrated differences in distribution shape. These parameters are person-independent and precise. We demonstrated this by quantifying growth-related population heterogeneity of Saccharomyces cerevisiae and Escherichia coli reporter strains in steady-state of aerobic glucose-limited chemostat cultures at different dilution rates and in response to glucose pulses. Generally, slow-growing cells showed stronger responses to glucose excess than fast-growing cells. Cell robustness, measured as membrane integrity after exposure to freeze-thaw treatment, of fast-growing cells was strongly affected in subpopulations of low membrane robustness. Glucose pulses protected subpopulations of fast-growing but not slower-growing yeast cells against membrane damage. Our parameters could successfully describe population heterogeneity, thereby revealing physiological characteristics that might have been overlooked during traditional averaged analysis.

Coenzyme A–mediated degradation of pyruvate dehydrogenase kinase 4 promotes cardiac metabolic flexibility after high-fat feeding in mice

Cardiac energy is produced primarily by oxidation of fatty acids and glucose, with the relative contributions of each nutrient being sensitive to changes in substrate availability and energetic demand. A major contributor to cardiac metabolic flexibility is pyruvate dehydrogenase (PDH), which converts glucose-derived pyruvate to acetyl-CoA within the mitochondria. PDH is inhibited by phosphorylation dependent on the competing activities of pyruvate dehydrogenase kinases (PDK1-4) and phosphatases (PDP1-2). A single high-fat meal increases cardiac PDK4 content and subsequently inhibits PDH activity, reducing pyruvate utilization when abundant fatty acids are available. In this study, we demonstrate that diet-induced increases in PDK4 are reversible and characterize a novel pathway that regulates PDK4 degradation in response to the cardiac metabolic environment. We found that PDK4 degradation is promoted by CoA (CoASH), the levels of which declined in mice fed a high-fat diet and normalized following transition to a control diet. We conclude that CoASH functions as a metabolic sensor linking the rate of PDK4 degradation to fatty acid availability in the heart. However, prolonged high-fat feeding followed by return to a low-fat diet resulted in persistent in vitro sensitivity of PDH to fatty acid-induced inhibition despite reductions in PDK4 content. Moreover, increases in the levels of proteins responsible for β-oxidation and rates of palmitate oxidation by isolated cardiac mitochondria following long-term consumption of high dietary fat persisted after transition to the control diet. We propose that these changes prime PDH for inhibition upon reintroduction of fatty acids.

Kruppel-like factor 15 is required for the cardiac adaptive response to fasting.

Cardiac metabolism is highly adaptive in response to changes in substrate availability, as occur during fasting. This metabolic flexibility is essential to the maintenance of contractile function and is under the control of a group of select transcriptional regulators, notably the nuclear receptor family of factors member PPARα. However, the diversity of physiologic and pathologic states through which the heart must sustain function suggests the possible existence of additional transcriptional regulators that play a role in matching cardiac metabolism to energetic demand. Here we show that cardiac KLF15 is required for the normal cardiac response to fasting. Specifically, we find that cardiac function is impaired upon fasting in systemic and cardiac specific Klf15-null mice. Further, cardiac specific Klf15-null mice display a fasting-dependent accumulation of long chain acylcarnitine species along with a decrease in expression of the carnitine translocase Slc25a20. Treatment with a diet high in short chain fatty acids relieves the KLF15-dependent long chain acylcarnitine accumulation and impaired cardiac function in response to fasting. Our observations establish KLF15 as a critical mediator of the cardiac adaptive response to fasting through its regulation of myocardial lipid utilization.

Effects of glucose availability in Lactobacillus sakei; metabolic change and regulation of the proteome and transcriptome.

Effects of glucose availability were investigated in Lactobacillus sakei strains 23K and LS25 cultivated in anaerobic, glucose-limited chemostats set at high (D = 0.357 h-1) and low (D = 0.045 h-1) dilution rates. We observed for both strains a shift from homolactic towards more mixed acid fermentation when comparing high to low growth rates. However, this change was more pronounced for LS25 than for 23K, where dominating products were lactate>formate>acetate≥ethanol at both conditions. A multivariate approach was used for analyzing proteome and transcriptome data from the bacterial cultures, where the predictive power of the omics data was used for identifying features that can explain the differences in the end-product profiles. We show that the different degree of response to the same energy restriction revealed interesting strain specific regulation. An elevated formate production level during slow growth, more for LS25 than for 23K, was clearly reflected in correlating pyruvate formate lyase expression. With stronger effect for LS25, differential expression of the Rex transcriptional regulator and NADH oxidase, a target of Rex, indicated that maintainance of the cell redox balance, in terms of the NADH/NAD+ ratio, may be a key process during the metabolic change. The results provide a better understanding of different strategies that cells may deploy in response to changes in substrate availability.

Brain metabolism in health, aging, andneurodegeneration.

Brain cells normally respond adaptively to bioenergetic challenges resulting from ongoing activity in neuronal circuits, and from environmental energetic stressors such as food deprivation and physical exertion. At the cellular level, such adaptive responses include the "strengthening" of existing synapses, the formation of new synapses, and the production of new neurons from stem cells. At the molecular level, bioenergetic challenges result in the activation of transcription factors that induce the expression of proteins that bolster the resistance of neurons to the kinds of metabolic, oxidative, excitotoxic, and proteotoxic stresses involved in the pathogenesis of brain disorders including stroke, and Alzheimer's and Parkinson's diseases. Emerging findings suggest that lifestyles that include intermittent bioenergetic challenges, most notably exercise and dietary energy restriction, can increase the likelihood that the brain will function optimally and in the absence of disease throughout life. Here, we provide an overview of cellular and molecular mechanisms that regulate brain energy metabolism, how such mechanisms are altered during aging and in neurodegenerative disorders, and the potential applications to brain health and disease of interventions that engage pathways involved in neuronal adaptations to metabolic stress.

Changes in substrate availability drive carbon cycle response to chronic warming

As earth's climate continues to warm, it is important to understand how the capacity of terrestrial ecosystems to retain carbon (C) will be affected. We combined measurements of microbial activity with the concentration, quality, and physical accessibility of soil carbon to microorganisms to evaluate the mechanisms by which more than two decades of experimental warming has altered the carbon cycle in a Northeast US temperate deciduous forest. We found that concentrations of soil organic matter were reduced in both the organic and mineral soil horizons. The molecular composition of the carbon was altered in the mineral soil with significant reductions in the relative abundance of polysaccharides and lignin, and an increase in lipids. Mineral-associated organic matter was preferentially depleted by warming in the top 3 cm of mineral soil. We found that potential extracellular enzyme activity per gram of soil at a common temperature was generally unaffected by warming treatment. However, by measuring potential extracellular enzyme activities between 4 and 30 °C, we found that activity per unit microbial biomass at in-situ temperatures was increased by warming. This was associated with a tendency for microbial biomass to decrease with warming. These results indicate that chronic warming has reduced soil organic matter concentrations, selecting for a smaller but more active microbial community increasingly dependent on mineral-associated organic matter.

Microbial community response to changes in substrate availability and habitat conditions in a reciprocal subsoil transfer experiment

While habitat conditions influencing the abundance of microorganisms in topsoil are well known, these dynamics have been largely unexplored in deeper soil horizons. We investigated the effects of different substrate availabilities and environmental conditions on microbial community composition and carbon flow into specific groups of microorganisms in subsoils using a reciprocal soil transfer experiment within an acid and sandy Dystric Cambisol from a ∼100-year old European beech (Fagus sylvatica L.) forest in Lower Saxony, Germany. Containers filled with subsoil from 10 to 20 cm (SUB20) and 110 to 120 cm (SUB120) soil depths and with additions of different amounts of 13C labelled cellulose (1% and 5% of the respective organic carbon content of both soil layers) were exposed either in their home field environment or transferred reciprocally between SUB20 and SUB120 horizons for periods of one, four and twelve months. During the exposure of twelve months, 13C accumulated up to 15 percent in total microbial biomass and up to 25 percent in fungal PLFAs. Similar microbial 13C incorporation rates in SUB20 samples located at either 20 or 120 cm depth indicated comparable microclimatic conditions in both soil environments with no depth-dependent effects on the decomposer communities. While low nitrogen availability (when primary C-limitation was alleviated) and water content limited bacterial growth and activity at both depths, fungal abundance and activity were less affected due to their ability to efficiently exploit resources in surrounding soil by hyphal growth and higher drought resistance. Consequently, bacterial PLFAs (phospholipid fatty acids) incorporated less 13C than fungi. The relatively high, from 1% to 5% cellulose addition linearly increased, 13C incorporation rates in SUB120 samples at 120 cm depth clearly showed the potential of efficient carbon turnover in deeper soil layers. Spatial separation between subsoil microorganisms and their substrates may therefore be an important factor influencing carbon accumulation in subsoil.

Spatial and temporal dynamics of nitrogen fixing, nitrifying and denitrifying microbes in an unfertilized grassland soil

The microbial groups of nitrogen fixers, ammonia oxidizers, and denitrifiers largely drive the inorganic nitrogen cycle in temperate terrestrial ecosystems. Their spatial and temporal dynamics, however, vary depending on the studied scale. The present study aimed to fill a knowledge gap by providing an explicit picture of spatial and temporal dynamics of a subset of these soil microorganisms at the plot scale. We selected an unfertilized perennial grassland, where nitrogen cycling is considered to be efficient and tightly coupled to plant growth. At six times over one growing season 60 soil samples were taken from a 10 m × 10 m area and abundances of marker genes for total archaea and bacteria (16S rRNA), nitrogen fixing bacteria (nifH), ammonia oxidizing archaea (amoA AOA) and bacteria (amoA AOB), and denitrifying bacteria (nirS, nirK and nosZ) were determined by qPCR. Potential nitrification activity (PNA) and denitrifying enzyme activity (DEA) were determined. Seasonal changes in abundance patterns of marker genes were detected, and were associated with changes in substrate availability associated with plant growth stages. Potential nitrification and denitrification enzyme activities were strongly spatially structured at the studied scale, corresponding to periods of rapid plant growth, June and October, and their spatial distributions were similar, providing visual evidence of highly localized spatial and temporal conditions at this scale. Temporal variability in the N-cycling communities versus the stability of their respective potential activities provided evidence of both short-lived temporal niche partitioning and a degree of microbial functional redundancy. Our results indicate that in an unfertilized grassland, at the meter scale, abundances of microbial N-cycling organisms can exhibit transient changes, while nitrogen cycling processes remain stable.

The Role of Microbial Community Composition in Controlling Soil Respiration Responses to Temperature.

Rising global temperatures may increase the rates of soil organic matter decomposition by heterotrophic microorganisms, potentially accelerating climate change further by releasing additional carbon dioxide (CO2) to the atmosphere. However, the possibility that microbial community responses to prolonged warming may modify the temperature sensitivity of soil respiration creates large uncertainty in the strength of this positive feedback. Both compensatory responses (decreasing temperature sensitivity of soil respiration in the long-term) and enhancing responses (increasing temperature sensitivity) have been reported, but the mechanisms underlying these responses are poorly understood. In this study, microbial biomass, community structure and the activities of dehydrogenase and β-glucosidase enzymes were determined for 18 soils that had previously demonstrated either no response or varying magnitude of enhancing or compensatory responses of temperature sensitivity of heterotrophic microbial respiration to prolonged cooling. The soil cooling approach, in contrast to warming experiments, discriminates between microbial community responses and the consequences of substrate depletion, by minimising changes in substrate availability. The initial microbial community composition, determined by molecular analysis of soils showing contrasting respiration responses to cooling, provided evidence that the magnitude of enhancing responses was partly related to microbial community composition. There was also evidence that higher relative abundance of saprophytic Basidiomycota may explain the compensatory response observed in one soil, but neither microbial biomass nor enzymatic capacity were significantly affected by cooling. Our findings emphasise the key importance of soil microbial community responses for feedbacks to global change, but also highlight important areas where our understanding remains limited.

Oviposition Substrate of the Mountain Fly Drosophila nigrosparsa (Diptera: Drosophilidae)

The survival of insect larvae often depends on the mother’s choice of oviposition substrate, and thus, this choice is an essential part of an insect species’ ecology. Especially species with narrow substrate preferences may suffer from changes in substrate availability triggered by, for example, climate change. Recent climate warming is affecting species directly (e.g., physiology) but also indirectly (e.g., biological interactions) leading to mismatching phenologies and distributions. However, the preferred oviposition substrate is still unknown for many drosophilid species, especially for those at higher elevations. In this study, we investigated the oviposition-substrate preference of the montane-alpine fly Drosophila nigrosparsa in rearing and multiple-choice experiments using natural substrates in the laboratory. Insect emergence from field-collected substrates was tested. More than 650 insects were reared from natural substrates, among them 152 drosophilids but no individual of D. nigrosparsa. In the multiple-choice experiments, D. nigrosparsa preferred ovipositing on mushrooms (> 93% of eggs); additionally, a few eggs were laid on berries but none on other substrates such as cow faeces, rotten plant material, and soil. The flies laid 24 times more eggs per day when mushrooms were included in the substrates than when they were excluded. We infer that D. nigrosparsa is a mushroom breeder with some variation in oviposition choice. The flies favoured some mushrooms over others, but they were not specialised on a single fungal taxon. Although it is unclear if and how climate change will affect D. nigrosparsa, our results indicate that this species will not be threatened by oviposition-substrate limitations in the near future because of the broad altitudinal distribution of the mushrooms considered here, even if the flies will have to shift upwards to withstand increasing temperatures.

Limits to soil carbon stability; Deep, ancient soil carbon decomposition stimulated by new labile organic inputs

Carbon (C) buried deep in soil (below 1 m) is often hundreds to thousands of years old, though the stability and sensitivity of this deep C to environmental change are not well understood. We examined the C dynamics in three soil horizons and their responses to changes in substrate availability in a coarse-textured sandy spodosol (0.0–0.1, 1.0–1.3, and 2.7–3.0 m deep). Substrate additions were intended to mimic an increase in root exudates and available inorganic nitrogen (N) that would follow an increase of belowground biomass at depth, as previously found in a long-term CO2 enrichment experiment at this site. We incubated these soils for 60 days with glucose, alanine, and leaf litter, crossed with an inorganic N amendment equivalent to three times ambient concentrations. The organic substrates were isotopically labeled (13C), allowing us to determine the source of mineralized C and assess the priming effect. Enzyme activity increased as much as 13 times in the two deeper horizons (1.0–1.3, and 2.7–3.0 m) after the addition of the organic substrates, even though the deepest horizon had microbial biomass and microbial phospholipid fatty acids below the level of detection before the experiment. The deepest horizon (2.7–3.0 m) yielded the largest priming response under alanine, indicating that microorganisms in these soil horizons can become active in response to input of organic substrates. Inorganic N amendments significantly decreased the priming effect, suggesting that decomposition may not be N limited. However, alanine (organic N) yielded the highest priming effect at every soil depth, indicating the importance of differentiating effect of organic and inorganic N on decomposition. Distinct priming effects with depth suggest that portions of the soil profile can respond differently to organic inputs. Our findings indicate that the deep soil C pools might be more vulnerable to environmental or anthropogenic change than previously thought, potentially influencing net CO2 exchange estimates between the land and the atmosphere.

Succession of soil microbial communities and enzyme activities in artificial soils

Soil microorganisms are frequently attached to mineral surfaces or organo-mineral complexes, yet little is known about the microbial colonization of different soil minerals. The use of artificial soils that differ only in their mineral composition (illite, montmorillonite, ferrihydrite, boehmite) and the presence of charcoal, but not in soil texture and organic composition, offered a unique opportunity to study composition, function and succession of soil microorganisms colonizing newly exposed organo-mineral surfaces. Artificial soils were incubated with a microbial inoculum from an arable topsoil at constant temperature (20°C) and moisture conditions for up to 18 months. The succession of enzyme activities involved in C-, N- and P-cycling gave clear evidence that nutrient limitation drove microbial community structure during the incubation independent of mineral composition. Discriminant analyses of principal components of PLFAs showed that microbial community structure changed over a period of 18 months toward similar communities for all artificial soils at the end of incubation. This was supported by a shift in the soil microbial community from dominance of specific phyla like Betaproteobacteria, which is often referred to as copiotrophic organisms, during the first 6 months of the incubation, toward systems with a higher dominance of e.g. Acidobacteria, which are suggested to follow the oligotrophic life-strategy. The effect of mineral surface properties on enzyme activities was pronounced during the first 6 months of incubation. Microbial colonization and succession on mineral surfaces was likely affected by mineral properties such as surface charge and, at the end of incubation, availability of beneficial nutrients. Charcoal affected the microbial community only during the first 6 months of incubation with slightly increased colonization by bacteria which are often described as oligotrophic organisms. In contrast, illite and montmorillonite probably provided nutrient rich environments with montmorillonite supplying more exchangeable cations. The artificial soils experiment clearly showed that changes in substrate availability as well as mineral properties are important drivers for the development of microbial communities.

Are variations in heterotrophic soil respiration related to changes in substrate availability and microbial biomass carbon in the subtropical forests?

Soil temperature and moisture are widely-recognized controlling factors on heterotrophic soil respiration (Rh), although they often explain only a portion of Rh variability. How other soil physicochemical and microbial properties may contribute to Rh variability has been less studied. We conducted field measurements on Rh half-monthly and associated soil properties monthly for two years in four subtropical forests of southern China to assess influences of carbon availability and microbial properties on Rh. Rh in coniferous forest was significantly lower than that in the other three broadleaf species-dominated forests and exhibited obvious seasonal variations in the four forests (P < 0.05). Temperature was the primary factor influencing the seasonal variability of Rh while moisture was not in these humid subtropical forests. The quantity and decomposability of dissolved organic carbon (DOC) were significantly important to Rh variations, but the effect of DOC content on Rh was confounded with temperature, as revealed by partial mantel test. Microbial biomass carbon (MBC) was significantly related to Rh variations across forests during the warm season (P = 0.043). Our results suggest that DOC and MBC may be important when predicting Rh under some conditions, and highlight the complexity by mutual effects of them with environmental factors on Rh variations.

Temperature dependences of growth rates and carrying capacities of marine bacteria depart from metabolic theoretical predictions.

Using the metabolic theory of ecology (MTE) framework, we evaluated over a whole annual cycle the monthly responses to temperature of the growth rates (μ) and carrying capacities (K) of heterotrophic bacterioplankton at a temperate coastal site. We used experimental incubations spanning 6ºC with bacterial physiological groups identified by flow cytometry according to membrane integrity (live), nucleic acid content (HNA and LNA) and respiratory activity (CTC+). The temperature dependence of μ at the exponential phase of growth was summarized by the activation energy (E), which was variable (-0.52 to 0.72 eV) but followed a seasonal pattern, only reaching the hypothesized value for aerobic heterotrophs of 0.65 eV during the spring bloom for the most active bacterial groups (live, HNA, CTC+). K (i.e. maximum experimental abundance) peaked at 4 × 10(6) cells mL(-1) and generally covaried with μ but, contrary to MTE predictions, it did not decrease consistently with temperature. In the case of live cells, the responses of μ and K to temperature were positively correlated and related to seasonal changes in substrate availability, indicating that the responses of bacteria to warming are far from homogeneous and poorly explained by MTE at our site.

Changes in Macrofungal Communities Following Forest Conversion into Tree Plantations in Southern Brazil

AbstractConversion of diverse native forests to tree monocultures remains an ongoing, worldwide threat to biodiversity. Although the effects of forest conversion have been studied in a wide range of taxonomic groups, the effects on macrofungal communities remain poorly understood. We sampled macrofungal fruiting bodies in the National Forest of São Francisco de Paula in Southern Brazil over 12 months in four different forest habitats: native Araucaria angustifolia forest, A. angustifolia plantation, Pinus taeda or P. elliottii plantation, and Eucalyptus saligna plantation. The distribution of macrofungal species in different functional groups varied among habitats: the macrofungal species composition of the A. angustifolia plantation was more similar to that of the native forest, while the exotic Pinus or Eucalyptus plantations were less similar to the native forest. The conversion of native forest to exotic tree plantations reduced the number of macrofungal decomposer species, probably due to changes in substrate availability and quality. We conclude that fungal diversity and ecosystem functionality require the preservation of native, mature forests and suggest a shift of Brazilian forestry guidelines to encourage the plantations of native species instead of exotics.

Shifts in the microbial community structure explain the response of soil respiration to land-use change but not to climate warming

Soil stores more carbon (C) than plants and atmosphere combined and it is vulnerable to increased microbial respiration under projected global changes including land-use change and future climate scenarios (mainly elevated temperature). Land-use change is known to have a direct impact on soil organic C and soil respiration (Rs) but the mechanisms that drive these changes remain debatable. Similarly, recent studies and simulation models predict that Rs will respond positively to projected climate warming. However, there are significant uncertainties in the magnitude and mechanisms of this feedback response of Rs to global change. To identify the mechanisms of Rs response to land-use change and climate warming, we first investigated Rs from different land use types. Soil respiration was estimated seasonally from four different Scottish land uses: moorland, birch woodland, grassland and pine forest (n = 24). Our results demonstrated that despite a dramatic loss of total C and nitrogen (N) in the soils under birch trees, the Rs in the birch woodland was similar to that of the moorland and pine forest, with Rs in the grassland being significantly higher. The microbial community structure, estimated by Multiplex Terminal-Restriction Fragment Length Polymorphism (MT-RFLP) and 454 pyrosequencing, was significantly different under each land use type. A strong correlation of Rs with soil properties (pH, inorganic N, C:N ratio and moisture) and with microbial community structure was identified.To test the impact of elevated temperature on Rs and to identify potential microbial mechanisms, we performed laboratory incubation studies. Soils from different land uses were incubated at 7 °C (mean annual temperature (MAT) in Scotland) and 10 °C (MAT + 3 °C) with and without the presence of a labile (13C-glucose) and recalcitrant (13C-lignin) form of C to identify the active groups of microbes and to determine the role of substrate availability on feedback response. The warming treatment induced an increase in Rs rates in all soils. The magnitude of the Rs response to warming was modulated by the land use types, and the Rs was more prominent in soils with high C contents. The addition of glucose substantially increased both total and rate of Rs compared to no substrate- and lignin-amended soils, providing evidence of labile C depletion as a mechanism for the thermal response of Rs. The warming treatment did not impact the composition of the active or total microbial community as revealed by phospholipid fatty acid-stable isotope probing (PLFA-SIP), MT-RFLP and 454 pyrosequencing. Our results showed that the microbial metabolic activity was higher under warming treatment suggesting that a positive feedback of Rs to increased temperature is mediated by changes in substrate availability and microbial metabolic rates.

Methenyl-Dephosphotetrahydromethanopterin Is a Regulatory Signal for Acclimation to Changes in Substrate Availability in Methylobacterium extorquens AM1.

During an environmental perturbation, the survival of a cell and its response to the perturbation depend on both the robustness and functionality of the metabolic network. The regulatory mechanisms that allow the facultative methylotrophic bacterium Methylobacterium extorquens AM1 to effect the metabolic transition from succinate to methanol growth are not well understood. Methenyl-dephosphotetrahydromethanopterin (methenyl-dH4MPT), an early intermediate during methanol metabolism, transiently accumulated 7- to 11-fold after addition of methanol to a succinate-limited culture. This accumulation partially inhibited the activity of the methylene-H4MPT dehydrogenase, MtdA, restricting carbon flux to the assimilation cycles. A strain overexpressing the gene (mch) encoding the enzyme that consumes methenyl-dH4MPT did not accumulate methenyl-dH4MPT and had a growth rate that was 2.7-fold lower than that of the wild type. This growth defect demonstrates the physiological relevance of this enzymatic regulatory mechanism during the acclimation period. Changes in metabolites and enzymatic activities were analyzed in the strain overexpressing mch. Under these conditions, the activity of the enzyme coupling formaldehyde with dH4MPT (Fae) remained constant, with concomitant formaldehyde accumulation. Release of methenyl-dH4MPT regulation did not affect the induction of the serine cycle enzyme activities immediately after methanol addition, but after 1 h, the activity of these enzymes decreased, likely due to the toxicity of formaldehyde accumulation. Our results support the hypothesis that in a changing environment, the transient accumulation of methenyl-dH4MPT and inhibition of MtdA activity are strategies that permit flexibility and acclimation of the metabolic network while preventing the accumulation of the toxic compound formaldehyde. The identification and characterization of regulatory mechanisms for methylotrophy are in the early stages. We report a nontranscriptional regulatory mechanism that was found to operate as an immediate response for acclimation during changes in substrate availability. Methenyl-dH4MPT, an early intermediate during methanol oxidation, reversibly inhibits the methylene-H4MPT dehydrogenase, MtdA, when Methylobacterium extorquens is challenged to switch from succinate to methanol growth. Bypassing this regulatory mechanism causes formaldehyde to accumulate. Fae, the enzyme catalyzing the conversion of formaldehyde to methylene-dH4MPT, was also identified as another potential regulatory target using this strategy. The results herein further our understanding of the complex regulatory network in methylotrophy and will allow us to improve metabolic engineering strategies of methylotrophs for the production of value-added products.

A mitochondrial CO2-adenylyl cyclase-cAMP signalosome controls yeast normoxic cytochrome c oxidase activity.

Mitochondria, the major source of cellular energy in the form of ATP, respond to changes in substrate availability and bioenergetic demands by employing rapid, short-term, metabolic adaptation mechanisms, such as phosphorylation-dependent protein regulation. In mammalian cells, an intramitochondrial CO2-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway regulates aerobic energy production. One target of this pathway involves phosphorylation of cytochrome c oxidase (COX) subunit 4-isoform 1 (COX4i1), which modulates COX allosteric regulation by ATP. However, the role of the CO2-sAC-cAMP-PKA signalosome in regulating COX activity and mitochondrial metabolism and its evolutionary conservation remain to be fully established. We show that in Saccharomyces cerevisiae, normoxic COX activity measured in the presence of ATP is 55% lower than in the presence of ADP. Moreover, the adenylyl cyclase Cyr1 activity is present in mitochondria, and it contributes to the ATP-mediated regulation of COX through the normoxic subunit Cox5a, homologue of human COX4i1, in a bicarbonate-sensitive manner. Furthermore, we have identified 2 phosphorylation targets in Cox5a (T65 and S43) that modulate its allosteric regulation by ATP. These residues are not conserved in the Cox5b-containing hypoxic enzyme, which is not regulated by ATP. We conclude that across evolution, a CO2-sAC-cAMP-PKA axis regulates normoxic COX activity.

Fast "Feast/Famine" Cycles for Studying Microbial Physiology Under Dynamic Conditions: A Case Study with Saccharomyces cerevisiae.

Microorganisms are constantly exposed to rapidly changing conditions, under natural as well as industrial production scale environments, especially due to large-scale substrate mixing limitations. In this work, we present an experimental approach based on a dynamic feast/famine regime (400 s) that leads to repetitive cycles with moderate changes in substrate availability in an aerobic glucose cultivation of Saccharomyces cerevisiae. After a few cycles, the feast/famine produced a stable and repetitive pattern with a reproducible metabolic response in time, thus providing a robust platform for studying the microorganism’s physiology under dynamic conditions. We found that the biomass yield was slightly reduced (−5%) under the feast/famine regime, while the averaged substrate and oxygen consumption as well as the carbon dioxide production rates were comparable. The dynamic response of the intracellular metabolites showed specific differences in comparison to other dynamic experiments (especially stimulus-response experiments, SRE). Remarkably, the frequently reported ATP paradox observed in single pulse experiments was not present during the repetitive perturbations applied here. We found that intracellular dynamic accumulations led to an uncoupling of the substrate uptake rate (up to 9-fold change at 20 s.) Moreover, the dynamic profiles of the intracellular metabolites obtained with the feast/famine suggest the presence of regulatory mechanisms that resulted in a delayed response. With the feast famine setup many cellular states can be measured at high frequency given the feature of reproducible cycles. The feast/famine regime is thus a versatile platform for systems biology approaches, which can help us to identify and investigate metabolite regulations under realistic conditions (e.g., large-scale bioreactors or natural environments).