Microcalorimetric investigations of the metabolism of yeasts

Summary Microcalorimetric experiments on growth ofSaccharomyces under oxygen and nitrogen pressure between 0 and 10 kp/cm² are described. Within this range there are no alterations of the metabolism by the pressureper se but increased cell volume and a pronounced number of cells are observed. With nitrogen the enthalpy change amounts to a value of 130 cal/g glucose invariable with pressure. For oxygen a maximum heat evolution of 650 cal/g glucose is found in stirred cultures at the minimum pressure of almost 0 kp/cm². With rising O₂ pressure one observes a strong repression of heat flux which drops to a minimum value at 2 kp/cm². This repression is overcome by substrate concentrations less than 2 mg/ml. In unstirred cultures exposed to oxygen pressure the growth is determined by the geometrical and temporal distribution of cells and oxygen in the vessels. The calorimetric data are discussed in view of the mean volume and the dry weight of the cells.Herrn Prof. Dr. W. Stein zum 60. Geburtstag gewidmet.     

Growth of the yeast Saccharomyces cerevisiae on a non-fermentable substrate: control of energetic yield by the amount of mitochondria

The purpose of this study was to investigate the long-term control of ATP synthesis during the course of Saccharomyces cerevisiae batch grown on lactate, a purely respiratory substrate. For this, we used a respirometric and on-line calorimetric approach to analyse the energetic balances and the control of energetic metabolism during growth. Enthalpic growth yields assessed by enthalpy balance (taking account of substrate consumption, by-product accumulation, biomass formation and heat dissipation) remained constant during the entire exponential growth. Moreover, at the same time, a parallel decrease in basal respiratory rate and enthalpy flux occurred. It is shown that the decrease in respiration corresponds to a decrease in the amount of mitochondria per cell but not to a change of steady state of oxidative phosphorylation. Taking into account the part of energy used for maintenance, it can be concluded that mitochondria by themselves are the major heat dissipative system in a fully aerobic metabolism, and that the decrease in the amount of mitochondria when growth rate decreases leads to an enthalpic growth yield constant.     

Dissecting the energy metabolism in Mycoplasma pneumoniae through genome‐scale metabolic modeling

Mycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint‐based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time‐dependent changes, albeit using a static model. By performing an in silico knock‐out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms.     

Investigation of the anaerobic metabolism of <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> by means of a flux model

A flux model of the anaerobic metabolism of Rhodobacter capsulatus related to hydrogen production has been constructed. The performance of this model has been assessed by comparing the computed metabolic fluxes with experimental values obtained by several research groups who worked on various strains of R. capsulatus and utilized different growth setups. We have investigated the photoheterotrophic metabolism of R. capsulatus on acetate and have shown that in this mode the bacterium can produce hydrogen or biopolymers. Analysis of the flux model revealed several mutants that can evolve hydrogen with a higher rate than the wild type.     

Flux balance analysis of Chlorella sp. FC2 IITG under photoautotrophic and heterotrophic growth conditions

Quantification of carbon flux distribution in the metabolic network of microalgae remains important to understand the complex interplay between energy metabolism, carbon fixation, and assimilation pathways. This is even more relevant with respect to cyclic metabolism of microalgae under light–dark cycle. In the present study, flux balance analysis (FBA) was carried out for an indigenous isolate Chlorella sp. FC2 IITG under photoautotrophic and heterotrophic growth conditions. A shift in intracellular flux distribution was predicted during transition from nutrient sufficient phase to nutrient starvation phase of growth. Further, dynamic flux analysis (dFBA) was carried out to capture light–dark metabolism over discretized pseudo steady state time intervals. Our key findings include the following: (i) unlike heterotrophic condition, oxidative pentose phosphate (PP) pathway, and Krebs cycle were relatively inactive under photoautotrophic growth; (ii) in both growth conditions, while transhydrogenation reaction was highly active, glyoxalate shunt was found to be nonoperative; (iii) flux distribution during transition period was marked with up regulation of carbon flux toward nongrowth associated (NGA) maintenance energy, oxidative phosphorylation, and photophosphorylation; (iv) redirection of carbon flux from polysaccharide and neutral lipid resulted in up regulation of Krebs cycle flux in the dark phase; (v) elevated glycolytic and acetyl-CoA flux were coupled with induction of neutral lipid during light cycle of the growth; (vi) significantly active photophosphorylation in the light phase was able to satisfy cellular energy requirement without need of oxidative PP pathway; and (vi) unlike static FBA, dFBA predicted an unaltered NGA maintenance energy of 1.5 mmol g^?1 DCW h^?1.     

A high standard metabolic rate constrains juvenile growth

The allocation of energy to various components of an individual's energy budget is often viewed as a competitive process. As such, a tradeoff may exist between production (growth) and maintenance metabolism. One view of a potential tradeoff, termed “the principle of allocation”, suggests that individuals with lower maintenance metabolic expenditures may have higher growth rates. To determine whether such a tradeoff exists, I analyzed the relationship between growth rate and maintenance metabolism of 225 juvenile snapping turtles housed in the laboratory. I measured growth from hatching to 6 months of age, and then measured oxygen consumption and calculated standard metabolic rate. Mean growth rate was 0.19 g d⁻ and mean standard metabolic rate (SMR) was 1.41 kJ d⁻. Maintenance metabolism and growth were negatively correlated after both were adjusted for body mass. The results support the “principle of allocation” theory: individuals with higher standard metabolic rates tended to have low growth rates.     

Misconceptions About the Relation Between Plant Growth and Respiration

Abstract: The relation between plant growth rate and respiration rate is readily derived from the overall chemical reaction for aerobic metabolism. The derived relation can be used to show that separation of respiration into growth (g) and maintenance (m) components is not a useful concept. g and m cannot be unambiguously measured or defined in terms of biochemical processes. Moreover, because growth yield calculations from biochemical pathway analysis, from biomass molecular composition, from biomass heat of combustion, and from biomass elemental composition have not included all of the energy costs for biosynthesis, they are not accurate measures of the carbon cost for plant growth. Improper definitions of growth-respiration relations are impeding the use of physiological properties for prediction of plant growth as a function of environmental variables.     

Metabolic flux analysis of Shewanella spp. reveals evolutionary robustness in central carbon metabolism

Shewanella spp. are a group of facultative anaerobic bacteria widely distributed in marine and freshwater environments. In this study, we profiled the central metabolic fluxes of eight recently sequenced Shewanella species grown under the same condition in minimal medium with [3‐¹³C] lactate. Although the tested Shewanella species had slightly different growth rates (0.23–0.29 h^?1) and produced different amounts of acetate and pyruvate during early exponential growth (pseudo‐steady state), the relative intracellular metabolic flux distributions were remarkably similar. This result indicates that Shewanella species share similar regulation in regard to central carbon metabolic fluxes under steady growth conditions: the maintenance of metabolic robustness is not only evident in a single species under genetic perturbations (Fischer and Sauer, 2005; Nat Genet 37(6):636–640), but also observed through evolutionary related microbial species. This remarkable conservation of relative flux profiles through phylogenetic differences prompts us to introduce the concept of metabotype as an alternative scheme to classify microbial fluxomics. On the other hand, Shewanella spp. display flexibility in the relative flux profiles when switching their metabolism from consuming lactate to consuming pyruvate and acetate. Biotechnol. Bioeng. 2009;102: 1161–1169. © 2008 Wiley Periodicals, Inc.     

Metabolic modeling of Chlamydomonas reinhardtii: energy requirements for photoautotrophic growth and maintenance

In this study, a metabolic network describing the primary metabolism of Chlamydomonas reinhardtii was constructed. By performing chemostat experiments at different growth rates, energy parameters for maintenance and biomass formation were determined. The chemostats were run at low irradiances resulting in a high biomass yield on light of 1.25 g  mol⁻¹. The ATP requirement for biomass formation from biopolymers (K _x ) was determined to be 109 mmol g⁻¹ (18.9 mol mol⁻¹) and the maintenance requirement (m _ATP) was determined to be 2.85 mmol g⁻¹ h⁻¹. With these energy requirements included in the metabolic network, the network accurately describes the primary metabolism of C. reinhardtii and can be used for modeling of C. reinhardtii growth and metabolism. Simulations confirmed that cultivating microalgae at low growth rates is unfavorable because of the high maintenance requirements which result in low biomass yields. At high light supply rates, biomass yields will decrease due to light saturation effects. Thus, to optimize biomass yield on light energy in photobioreactors, an optimum between low and high light supply rates should be found. These simulations show that metabolic flux analysis can be used as a tool to gain insight into the metabolism of algae and ultimately can be used for the maximization of algal biomass and product yield.     

Ploidy distribution in the explant tissue and the calluses induced during the initial stage of internode segment culture of Asparagus officinalis L.

Summary To pursue the causal factors of ploidy variation in tissue culture of asparagus (Asparagus officinalis L.), ploidy status of stem explants and the calluses induced from these explants were examined using flow cytometry (FCM). The frequency of higher ploidy (>8C) cells in the stem explants increased with increasing distance from the shoot apex. Calluses derived from sections far from the apex also showed higher ploidy distributions, and also higher ploidy (16C) in general than was observed in the explant tissue. Detailed studies using FCM and charge-coupled device (CCD)-equipped fluorescence microscopy showed that 16C cells appeared in the explant tissues within 1 wk of culture, possibly induced by the plant growth regulators (PGRs), especially 1-naphthaleneacetic acid (NAA), in the culture medium.     

Butterfly seed predation: effects of landscape characteristics,plant ploidy level and population structure

Polyploidization has been suggested as one of the most common mechanisms for plant diversification. It is often associated with changes in several morphological, phenological and ecological plant traits, and therefore has the potential to alter insect–plant interactions. Nevertheless, studies evaluating the effect of plant polyploidy on interspecific interactions are still few. We investigated pre-dispersal seed predation by the butterfly Anthocharis cardamines in 195 populations of two ploidy levels of the herb Cardamine pratensis (tetraploid ssp. pratensis, 2n = 30 vs. octoploid ssp. paludosa, 2n = 56–64). We asked if differences in incidence and intensity of predation among populations were related to landscape characteristics, plant ploidy level and population structure. The incidence of the seed predator increased with increasing plant population size and decreasing distance to nearest population occupied by A. cardamines. The intensity of predation decreased with increasing plant population size and was not affected by isolation. Probability of attack decreased with increasing shading, and intensity of predation was higher in grazed than in non-grazed habitats. The attack intensity increased with increasing mean flower number of plant population, but was not affected by flowering phenology. Individuals in tetraploid populations suffered on average from higher levels of seed predation, had higher mean flower number, were less shaded and occurred more often in grazed habitats than octoploid populations. When accounting for differences in habitat preferences between ploidy levels there was no longer a difference in intensity of predation, suggesting that the observed differences in attack rates among populations of the two ploidy levels are mediated by the habitat. Overall, our results suggest that polyploidization is associated with differentiation in habitat preferences and phenotypic traits leading to differences in interspecific interaction among plant populations. This, in turn, may facilitate further divergence of ploidy levels.     

荒漠河岸胡杨林生态系统能量分配及蒸散发

生态输水工程使得黑河下游地区荒漠河岸林的面积得到了恢复,为进一步巩固生态恢复的成果,非常有必要深入研究胡杨林生态系统的水热交换过程及其对环境因子和人类活动的响应机理。基于涡度相关技术,对2013-2016年荒漠河岸胡杨林生态系统的水热交换过程进行了研究,得出的主要结论如下：（1）生长季生态系统的能量消耗以潜热通量为主,波文比为0.21;非生长季则以感热通量为主,波文比为3.61;春季和秋季的灌水过程,使得胡杨生长前期和后期的潜热通量值较高,直接影响着胡杨林生态系统的能量分配。（2）灌水的时间改变了潜热通量和蒸散发的季节变化过程,而灌水的量决定着春季和秋季潜热通量和蒸散量的年际差异。（3）2014-2016年,生态系统的年蒸散量和生长季蒸散量平均分别为1092 mm和932 mm。荒漠河岸胡杨林生态系统的蒸散量主要受系统水分状况（灌水量）及植物生长状况的影响。     

SENSITIVITY TO PHOSPHORUS LIMITATION INCREASES WITH PLOIDY LEVEL IN A NEW ZEALAND SNAIL

Evolutionary and ecological factors that explain natural variation in ploidy level remain poorly understood. One intriguing possibility is that nutrient costs associated with higher per‐cell nucleic acid content could differentially influence the fitness of different ploidy levels. Here, we test this hypothesis by determining whether access to phosphorus (P), a main component of nucleic acids, differentially affects growth rate in asexual freshwater snails (Potamopyrgus antipodarum) that differ in ploidy. As expected if larger genomes generate higher dietary P requirements, tetraploid P. antipodarum experienced a more than twofold greater reduction in growth rate in low‐P versus high‐P conditions relative to triploids. Mirroring these results, tetraploid P. antipodarum also had a significant reduction in body P content under low P relative to high P, whereas triploid body P content was unaffected. Taken together, these results set the stage for the possibility that P availability could influence the distribution and relative frequency of P. antipodarum of different ploidy levels. These findings could be applicable to many other animal taxa featuring ploidy‐level variation, which includes many mixed sexual/asexual taxa.     

A cell suspension ofLinum flavum (L.) in phosphate limited continuous culture

A cell suspension ofLinum flavum was grown in phosphate limited continuous culture at two different growth rates. Energy metabolism (respiration), coniferin and lignin production and overall biomass composition were analysed, in order to establish the relations between growth, maintenance and secondary metabolism. The ATP turnover rate was higher in the faster growing cultures, corresponding with a higher energy requirement. The coniferin production was not directly correlated with the growth rate, indicating the possibility of high production at high growth rates. Steady states grown under identical conditions showed different characteristics that may have evolved during pre-culture time.     

Response to competition of bulbous geophyte Allium oleraceum differing in ploidy level

Experimental studies that explore the possible causes of ploidy distributions and niche differentiation are rare. Increased competitive ability may be advantageous for survival in dense vegetation and may strongly affect local and regional abundances of cytotypes and potentially contribute to invasion success. We compared survival, growth and reproduction of plants originating from bulbils of three cytotypes (2n = 4x, 5x, 6x) of Allium oleraceum growing with and without a competitor (Arrhenatherum elatius). There was a strong negative effect of competition but no effect of ploidy or ploidy × competition on survivorship, height and total dry mass of A. oleraceum, i.e. no support for different competitive abilities of the ploidy levels. However, slightly different responses of populations to competition treatments within all cytotypes suggest differentiation within cytotypes. Under competition, plant survivorship was low, surviving plants were small, had low dry mass and produced neither sexual nor asexual propagules. Without competition, plant survivorship was high, and cytotypes differed in three traits after 2 year's growth: dry mass of flowers, number of flowers and ratio of the dry mass of sexual to asexual propagules all decreased with increasing ploidy level. We additionally tested tetra‐ and pentaploids as to whether plants originating from different types of propagule (bulbils, seeds) differ in survivorship, growth and reproduction when growing with and without a competitor. Plants originating from bulbils had higher survivorship, were more robust, flowered earlier and produced more propagules when compared to plants originating from seeds and grown without competition. Under competition, differences in performance between plants originating from seeds and bulbils mostly disappeared, with higher survivorship only for plants originating from bulbils.     

Myriocin-induced adaptive laboratory evolution of an industrial strain of Saccharomyces cerevisiae reveals its potential to remodel lipid composition and heat tolerance

The modification of lipid composition allows cells to adjust membrane biophysical properties in response to changes in environmental temperature. Here, we use adaptive laboratory evolution (ALE) in the presence of myriocin, a sphingolipid (SLs) biosynthesis inhibitor, to remodel the lipid profile of an industrial yeast strain (LH) of Saccharomyces cerevisiae. The approach enabled to obtain a heterogeneous population (LHev) of myriocin-tolerant evolved clones characterized by its growth capacity at high temperature. Myriocin exposure also caused tolerance to soraphen A, an inhibitor of the acetyl-CoA carboxylase Acc1, the rate-limiting enzyme in fatty acid de novo production, supporting a change in lipid metabolism during ALE. In line with this, characterization of two randomly selected clones, LH03 and LH09, showed the presence of lipids with increased saturation degree and reduced acyl length. In addition, the clone LH03, which displays the greater improvement in fitness at 40°C, exhibited higher SL content as compared with the parental strain. Analysis of the LH03 and LH09 genomes revealed a loss of chromosomes affecting genes that have a role in fatty acid synthesis and elongation. The link between ploidy level and growth at high temperature was further supported by the analysis of a fully isogenic set of yeast strains with ploidy between 1N and 4N which showed that the loss of genome content provides heat tolerance. Consistent with this, a thermotolerant evolved population (LH40°) generated from the parental LH strain by heat-driven ALE exhibited a reduction in the chromosome copy number. Thus, our results identify myriocin-driven evolution as a powerful approach to investigate the mechanisms of acquired thermotolerance and to generate improved strains.     

Myocyte ploidy in heart chambers of birds with different locomotor activity

The ploidy levels of atrio- and ventriculocytes were determined by means of cytofluorimetry in 31 species of birds. The obtained data were collated with postnatal growth rate, heart mass index, and relative masses of heart chambers. The difference between mean ploidy of cardiomyocytes in the left and right atrium is small (7.9+/-0.6%) and comparable to the difference in the masses of these chambers (10.5+/-0.8%). The difference between mean ploidy of atrio- and ventriculocytes is most pronounced for the left and right parts of heart (23.9+/-1.4% and 24.0+/-1.3%, respectively) and corresponds to considerable differences in the average masses of atria and ventricles (4.5-fold and 2.1-fold, respectively). The mean cardiomyocyte ploidy levels in the left and right ventricles differ only slightly, as in the case of atria (by 8.1+/-0.5%), whereas the average mass of the left ventricle is greater by 237+/-16%. This discord can be explained by peculiarities of the growth, which is nonproportionally faster in the left ventricle during the last stage of proliferative heart growth as compared to other chambers. The cardiomyocyte ploidy is higher in birds with a relatively small heart and lower ability to flight. Birds with a high locomotor activity in the adult state have an athletic heart (mass index >1%); they are fast growing, altricial species with a low heart workload in the early postnatal ontogenesis. Birds with a low locomotor activity at the adult state are precocial; they grow slowly and have a high locomotor activity from the first minutes of life. Thus, notwithstanding the fact that a greater elevation of cardiomyocyte ploidy level is acquired under a higher functional load (ventricles vs. atria, left vs. right part of the heart), it is associated with a lower functional potential of the organ at the adult state. The level of somatic polyploidy can be considered an indicator of developmental tensions arising due to a high workload during the growth of a given organ and deficiency of resources invested into this growth. J. Exp. Zool. 293:427-441, 2002.     

Metabolism associated with raised metabolic flux to sugar nucleotide precursors of exopolysaccharides in Lactobacillus delbrueckii subsp. bulgaricus

Exopolysaccharide (EPS) metabolism was studied in a galactose-negative strain of Lactobacillus delbrueckii subsp. bulgaricus, using two different approaches. Firstly, using both the parent strain and a chemically induced mutant with higher yield and specific productivity of EPS than the parent, comparative information was obtained relating to enzyme activities and metabolite levels associated with EPS formation when grown on lactose. Under continuous culture conditions (D=0.10 h⁻¹), the higher metabolic flux towards EPS formation in the mutant strain relative to the parent appeared to be mediated by raised levels of UDP-glucose pyrophosphorylase (UGP). Marginally raised UDP-galactose 4-epimerase (UGE) activity in the mutant strain suggested that this enzyme could also play a role in EPS overproduction. The second approach involved investigating the effect of growth rate on sugar nucleotide metabolism in the parent, as it is known that EPS production is growth-associated in this strain. UGE activity in the parent strain appeared to increase when the growth rate was elevated from 0.05 to 0.10 h⁻¹, and further to 0.35 h⁻¹, conditions that can be associated with higher levels of metabolic flux to EPS formation. Concurrent with these increments, intracellular ATP levels in the cell were raised. In both investigations glucose-6-phosphate accumulated pointing to a constriction at this branch-point, and a limitation in the flow of carbon towards fructose-6-phosphate or glucose-1-phosphate. The changes in metabolism associated with enhanced flux to EPS provide guidance as to how the yield of Lactobacillus delbrueckii subsp. bulgaricus EPS can be improved.