Carbon allocation in wild-type and Glc+ Rhodobacter sphaeroides under photoheterotrophic conditions.

The photosynthetic bacterium Rhodobacter sphaeroides is capable of producing H2 via nitrogenase when grown photoheterotrophically in the absence of N2. By using 14C-labeled malate, it was found that greater than 95% of this substrate was catabolized completely to CO2 during H2 production. About 60% of this catabolism was associated with H2 biosynthesis, while almost 40% provided reductant for other cellular purposes. Thus, only a small fraction of malate provided carbon skeletons. The addition of ammonium, which inhibited nitrogenase activity, increased substrate conversion into carbon skeletons threefold. Catabolism of malate occurred primarily via the tricarboxylic acid cycle, but gluconeogenesis was also observed. The wild-type organism grew poorly on glucose, accumulated gluconate and 2-keto-3-deoxygluconate, and did not produce H2. More than 50% of metabolized glucose appeared in carbon skeletons or in storage compounds. A glucose-utilizing mutant was five times more effective in utilizing this substrate. This mutant produced H2 from glucose, using 74% of metabolized substrate for this purpose. Glucose converted to storage products or to other carbon skeletons was reduced to 8%. Fixation of CO2 competed directly with H2 production for reducing equivalents and ATP. Refixation of CO2 released from these substrates under H2-producing conditions was, at most, 10 to 12%. Addition of ammonium increased refixation of respired CO2 to 83%. Patterns of carbon flow of fixation products were associated with the particular strains and culture conditions.     

Carbon allocation in wild-type and Glc+ Rhodobacter sphaeroides under photoheterotrophic conditions

The photosynthetic bacterium Rhodobacter sphaeroides is capable of producing H2 via nitrogenase when grown photoheterotrophically in the absence of N2. By using 14C-labeled malate, it was found that greater than 95% of this substrate was catabolized completely to CO2 during H2 production. About 60% of this catabolism was associated with H2 biosynthesis, while almost 40% provided reductant for other cellular purposes. Thus, only a small fraction of malate provided carbon skeletons. The addition of ammonium, which inhibited nitrogenase activity, increased substrate conversion into carbon skeletons threefold. Catabolism of malate occurred primarily via the tricarboxylic acid cycle, but gluconeogenesis was also observed. The wild-type organism grew poorly on glucose, accumulated gluconate and 2-keto-3-deoxygluconate, and did not produce H2. More than 50% of metabolized glucose appeared in carbon skeletons or in storage compounds. A glucose-utilizing mutant was five times more effective in utilizing this substrate. This mutant produced H2 from glucose, using 74% of metabolized substrate for this purpose. Glucose converted to storage products or to other carbon skeletons was reduced to 8%. Fixation of CO2 competed directly with H2 production for reducing equivalents and ATP. Refixation of CO2 released from these substrates under H2-producing conditions was, at most, 10 to 12%. Addition of ammonium increased refixation of respired CO2 to 83%. Patterns of carbon flow of fixation products were associated with the particular strains and culture conditions.     

Root respiration and associated costs in deciduous and evergreen species of Quercus

Root construction and maintenance costs were estimated in four evergreen and three deciduous Quercus species that are typical in the landscape of southern Spain. The cost quantification was based on analysis of the growth–­respiration ratio. Values observed for both construction cost (ranging from 1·17 to 1·29 g glucose g^?1 dry weight) and maintenance cost (ranging from 6·22 to 11·71 mg glucose g^?1 dry weight d^?1) were generally lower than those reported in other studies. The results showed non‐significant differences between deciduous and evergreen species. The lack of significant differences between species appeared to be due to the homogeneity of growth conditions. Hydroponic culture, with unrestricted nutrient and water supply, would lead to low tissue carbon content and low respiration rates, leading to the low costs observed. Furthermore, the fact that root organs are clearly importers of organic molecules inevitably entails some underestimation of the respiration associated with growth and, to a lesser extent, with maintenance respiration. This leads in turn to underestimation of the corresponding construction and maintenance costs. All this raises doubts as to the suitability of this method for studying root systems.     

Origins and metabolism of formate in higher plants

Formate, a simple one-carbon compound, is readily metabolized in plant tissues. In greening potato tubers, similar to some procaryotes, formate is directly synthesized via a ferredoxin-dependent fixation of CO₂, serving as the main precursor for carbon skeletons in biosynthetic pathways. In other plant species and tissues, formate appears as a side-product of photorespiration and of fermentation pathways, but possibly also as a product of direct CO₂ reduction in chloroplasts. Formate metabolism is closely related to serine synthesis and to all subsequent reactions originating from serine. Formate may have a role in biosynthesis of numerous compounds, in energetic metabolism and in signal transduction pathways related to stress response. This review summarizes the current state of formate research, physiological/biochemical and molecular aspects.     

Comparison of Four Methods Calculating the Seasonal Pattern of Plant Growth Efficiency of a Kiwifruit Berry

Four methods of determining the substrate requirements for synthesisof a kiwifruit [Actinidia deliciosa (A. Chev.) C. F. Liang etA. R. Ferguson var. deliciosa cv. Hayward] berry were comparedusing data derived from common kiwifruit berry samples collectedfrom anthesis to fruit maturity. The four methods were basedon fruit proximal analysis, elemental analysis, heats of combustion,or tissue carbon content. All methods gave similar patternsof seasonal costs and values of final cost to the plant (mean1.21 g glucose g^–1 season^–1) but there was lessagreement for growth respiration (mean 0.147 g glucose g^–1season^–1). This is the first time that a continuous recordof growth cost over the course of development has been presented,and the trends in seasonal cost reflect the uptake into andsynthesis of the different biochemical constituents in the fruit.The differences between the results of each method reflect theunderlying assumptions used in their development. It appearsfrom this work that the method of McDermitt and Loomis (1981),utilizing elemental analyses, is most preferred. Actinidia deliciosa (A. Chev.) C. F. Liang et A. R. Ferguson var deliciosa cv Haywood, kiwifruit, true growth yield, plant growth efficiency, production value, glucose value, bioenergetic cost     

Metabolic capabilities of Escherichia coli: I. synthesis of biosynthetic precursors and cofactors

Metabolism of living cells converts substrates into metabolic energy, redox potential and metabolic end products that are essential to maintain cellular function. The flux distribution among the various biochemical pathways is determined by the kinetic properties of enzymes which are subject to strict regulatory control. Simulation of metabolic behavior therefore requires the complete knowledge of biochemical pathways, enzyme kinetics as well as their regulation. Unfortunately, complete kinetic and regulatory information is not available for microbial cells, thus preventing accurate dynamic simulation of their metabolic behavior. However, it is possible to define wider limits on metabolic behavior based solely on flux balances of biochemical pathways. We present here comprehensive information about the catabolic pathways of the bacterium Escherichia coli. Using this biochemical database, we formulate a stoichiometric model of the bacterial network of fueling reactions. After logical structural reduction, the network consists of 53 metabolic fluxes and 30 metabolites. The solution space of this under-determined system of equations presents the bounds of metabolic flux distribution that the bacterial cell can achieve. We use specific objective functions and linear optimization to investigate the capability of E. coli catabolism to maximally produce the 12 biosynthetic precursors and three key cofactors within this solution space. For the three cofactors, the maximum yields are calculated to be 18.67 ATP, 11.6 NADH and 11 NADPH per glucose molecule, respectively. The yields of NADH and NADPH are less than 12 owing to the energy costs of importing glucose. These constraints are made explicit by the interpretation of shadow prices. The optimal yields of the 12 biosynthetic precursors are computed. Four of the 12 precursors (3-phosphoglycerate, phosphoenolpyruvate, pyruvate and oxaloacetate) can be made by E. coli with complete carbon conversion. Conversely, none of the sugar monophosphates can be made with 100% carbon conversion and analysis of the shadow prices reveals that this conversion is constrained by the energy cost of importing glucose. Three of the 12 precursors (acetyl-coA, α-ketoglutarate, and succinyl-coA) cannot be made with full carbon conversion owing to stoichiometric constraints; there is no route to these compounds without carrying out a decarboxylation reaction. Metabolite flux balances and linear optimization have thus been used to determine the catabolic capabilities of E. coli .     

Expanded thermodynamic model for microbial true yield prediction

Thermodynamic methods to predict true yield and stoichiometry of bacterial reactions have been widely used in biotechnology and environmental engineering. However, yield predictions are often inaccurate for certain simple organic compounds. This work evaluates an existing method and identifies the cause of prediction errors for compounds with low degree of reductance of carbon. For these compounds, carbon, not energy or reducing equivalents, constrains growth. Existing thermodynamically-based models do not account for the potential of carbon-limited growth. The improved method described here consists of four balances: carbon balance, nitrogen balance, electron balance, and energy balance. Two efficiency terms, K1 and K2 are defined and estimated from a priori analysis. The results show that K1 and K2 are nearly the same in value so that only one coefficient, K = 0.41 is used in the modified model. Comparisons with observed yields show that use of the new model and parameters results in significantly improved yield estimation based on inclusion of the carbon balance. The average estimation error is less than 6% for the data set presented.     

Carbon and nitrogen metabolism in ectomycorrhizal fungi and ectomycorrhizas

The literature concerning the metabolism of carbon and nitrogen compounds in ectomycorrhizal associations of trees is reviewed. The absorption and translocation of mineral ions by the mycelia require an energy source and a reductant which are both supplied by respiratory catabolism of carbohydrates produced by the host plant. Photosynthates are also required to generate the carbon skeletons for amino acid and carbohydrate syntheses during the growth of the mycelia. Competition for photosynthates occurs between the fungal cells and the various vegetative sinks in the host tree. The nature of carbon compounds involved in these processes, their routes of metabolism, the mechanisms of control and the partitioning of metabolites between the various sites of utilization are only poorly understood. Both ascomycetous and basidiomycetous ectomycorrhizal fungi synthesize and some, if not all, accumulate mannitol, trehalose and triglycerides. The fungal strains employ the Embden--Meyerhof pathway of glucose catabolism and the key enzymes of the pentose phosphate pathway (6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, transaldolase and transketolase). Anaplerotic CO2 fixation, via pyruvate carboxylase and/or phosphoenolpyruvate carboxykinase, provides high pools of amino acids. This process could be important in the recapture and assimilation of respired CO2 in the rhizosphere. The ectomycorrhizas are thought to contain the Embden--Meyerhof pathway, the pentose phosphate pathway and the tricarboxylic acid cycle, which provide the carbon skeletons for the assimilation of ammonia into amino acids. The main route of assimilation of ammonia appears to be through the glutamine synthetase-glutamate synthase cycle in the ectomycorrhizas. Glutamate dehydrogenase plays a minor role in this process. Glutamate dehydrogenase and glutamine synthetase are present in free-living ectomycorrhizal fungi and they participate in the assimilation of ammonia and the synthesis of amino acids through the glutamate dehydrogenase/glutamine synthetase sequence. In both in vitro cultures of fungi and ectomycorrhizas, the assimilated nitrogen accumulates in glutamine. Glutamine, but also ammonia, are thought to be exported from the fungal tissues to the host cells. Studies on the metabolism of ectomycorrhizas and ectomycorrhizal fungi have focused on the metabolic pathways and compounds which accumulate in the symbiotic tissues. Studies on regulation of the overall process, and the control of enzyme activity in particular, are still fragmentary.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure and function of polyketide biosynthetic enzymes: various strategies for production of structurally diverse polyketides

Polyketides constitute a large family of natural products that display various biological activities. Polyketides exhibit a high degree of structural diversity, although they are synthesized from simple acyl building blocks. Recent biochemical and structural studies provide a better understanding of the biosynthetic logic of polyketide diversity. This review highlights the biosynthetic mechanisms of structurally unique polyketides, β-amino acid-containing macrolactams, enterocin, and phenolic lipids. Functional and structural studies of macrolactam biosynthetic enzymes have revealed the unique biosynthetic machinery used for selective incorporation of a rare β-amino acid starter unit into the polyketide skeleton. Biochemical and structural studies of cyclization enzymes involved in the biosynthesis of enterocin and phenolic lipids provide mechanistic insights into how these enzymes diversify the carbon skeletons of their products.     

Occurrence and diversity of lipids in modern coral skeletons

Coral skeletons are composite acellular structures, in which organic macromolecules are intimately associated with mineral phases. Previous studies focussed on proteins and sugars of the soluble organic matrices extracted from the skeletons. Here we report the occurrence of diverse lipids which were extracted from the aragonitic skeletons of seven modern coral species. Using thin layer chromatography, we show that these lipids differ in quantity and composition between the species. Higher proportions of sterols and sterol esters in skeleton extracts as compared to a much higher abundance of waxes and triglycerides in previously studied extracts from scleractinian soft tissues suggest a specific, although not yet determined, role in biomineralisation. The occurrence of intraskeletal lipids along with other organic components should also be taken into account when using coral skeletons as bone allografts, as well as in fossilisation processes.     

High mineral contents explain the low construction cost of leaves, stems and fruits of tomato plants

The construction cost of plant tissues is used in crop models to convert the products of photosynthesis into biomass. As for other greenhouse crops, tomato tissues are specific in that they have a high mineral content. The consequences of this accumulation of minerals on the construction cost of the tissues and the possible interactions with the physiological age of the organs and with the CO2 concentration in the atmosphere was examined. For that purpose, three methods of estimating the construction cost were used and compared. Large quantities of minerals accumulated in the tissues of tomato plants (ranging from 0.05 in fruits to 0.26 g g^-1 DM in leaves). The subsequent dilution of the organic matter explained why the estimated construction cost of the dry matter (organic matter + minerals) was fairly low in comparison to that of other crops species. The construction cost was higher in fruits than in vegetative organs, partly because of a lower mineral content. It decreased by 7-12% from top to bottom of the canopy, following the increase in the physiological age of the tissues. This ontogenic drift was partly explained by the accumulation of minerals in the older organs. In the conditions of CO2 enrichment of a commercial greenhouse, no effect of CO2 concentration on the mineral content and on the construction cost of tissues was observed. Such a variability of the construction cost of tomato plant tissues due to the accumulation of minerals or to the ontogeny questions the use of standard values in crop models.Key words: Lycopersicon esculentum Mill., construction cost, heat of combustion, elemental composition, mineral content.      

Proximate biochemical composition and caloric content calculated from elemental CHN analysis: a stoichiometric concept

Summary Carbohydrate, lipid, and protein compositions are stoichiometrically related to organic CHN (carbon, hydrogen, nitrogen) contents. Elemental CHN analyses of total biomass and ash, therefore, provide a basis for the calculation of proximate biochemical composition and bomb caloric value. The classical nitrogen to protein conversion factor (6.25) should be replaced by 5.8±0.13. A linear relation exists between the mass fraction of non-protein carbon and the carbohydrate and lipid content. Residual water in dry organic matter can be estimated with the additional information derived from hydrogen measurements.The stoichiometric CHN method and direct biochemical analysis agreed within 10% of ash-free dry biomass (for muscle, liver and fat tissue of silver carp; gut contents composed of detritus and algae; commercial fish food). The detrital material, however, had to be corrected for non-protein nitrogen.A linear relationship between bomb caloric value and organic carbon fractions was derived on the basis of thermodynamic and stoichiometric principles, in agreement with experimental data published for bacteria, algae, protozoa and invertebrates. The highly automatic stoichiometric CHN method for the separation of nutrient contents in biomass extends existing ecophysiological concepts for the construction of balanced carbon and nitrogen, as well as biochemical and energy budgets. Offprint requests to: Institut für Zoologie, Abt. Zoophysiologie, Universität Innsbruck, Peter-Mayr-Str. 1A, A-6020 Innsbruck, Austria     

Mapping the genetic and tissular diversity of 64 phenolic compounds in Citrus species using a UPLC–MS approach

Background and Aims Phenolic compounds contribute to food quality and have potential health benefits. Consequently, they are an important target of selection for Citrus species. Numerous studies on this subject have revealed new molecules, potential biosynthetic pathways and linkage between species. Although polyphenol profiles are correlated with gene expression, which is responsive to developmental and environmental cues, these factors are not monitored in most studies. A better understanding of the biosynthetic pathway and its regulation requires more information about environmental conditions, tissue specificity and connections between competing sub-pathways. This study proposes a rapid method, from sampling to analysis, that allows the quantitation of multiclass phenolic compounds across contrasting tissues and cultivars.Methods Leaves and fruits of 11 cultivated citrus of commercial interest were collected from adult trees grown in an experimental orchard. Sixty-four phenolic compounds were simultaneously quantified by ultra-high-performance liquid chromatography coupled with mass spectrometry.Key Results Combining data from vegetative tissues with data from fruit tissues improved cultivar classification based on polyphenols. The analysis of metabolite distribution highlighted the massive accumulation of specific phenolic compounds in leaves and the external part of the fruit pericarp, which reflects their involvement in plant defence. The overview of the biosynthetic pathway obtained confirmed some regulatory steps, for example those catalysed by rhamnosyltransferases. The results suggest that three other steps are responsible for the different metabolite profiles in ‘Clementine’ and ‘Star Ruby’ grapefruit.Conclusions The method described provides a high-throughput method to study the distribution of phenolic compounds across contrasting tissues and cultivars in Citrus, and offers the opportunity to investigate their regulation and physiological roles. The method was validated in four different tissues and allowed the identification and quantitation of 64 phenolic compounds in 20 min, which represents an improvement over existing methods of analysing multiclass polyphenols.     

Soluble organic matrices of aragonitic skeletons of Merulinidae (Cnidaria, Anthozoa)

Our interpretation of the overall taxonomy and evolution of the Scleractinia, the most important reef builders in tropical areas, has long depended exclusively on morphology of the calcareous skeletons. The reported series of physical and biochemical characterizations of skeletons and the mineralizing matrices extracted from the skeletons allow, for the first time, the level of biochemical diversity among corallites of the same family to be estimated. Similarities and differences observed in the micro- and nanostructures of the skeletons reflect those of the soluble organic matrices. Sulphur is mainly associated with sulphated acidic sugars. The role of sulphated sugars on the biomineralization processes is still underestimated. The resulting data suggest that environmental conditions may act on the mineralization process through the detailed compositions of the mineralizing matrices.     

Cadmium induces acidosis in maize root cells

* Cadmium (Cd) stress increases cell metabolic demand for sulfur, reducing equivalents, and carbon skeletons, to sustain phytochelatin biosynthesis for Cd detoxification. In this condition the induction of potentially acidifying anaplerotic metabolism in root tissues may be expected. For these reasons the effects of Cd accumulation on anaplerotic metabolism, glycolysis, and cell pH control mechanisms were investigated in maize (Zea mays) roots. * The study compared root apical segments, excised from plants grown for 24 h in a nutrient solution supplemented, or not, with 10 microM CdCl(2), using physiological, biochemical and (31)P-nuclear magnetic resonance (NMR) approaches. * Cadmium exposure resulted in a significant decrease in both cytosolic and vacuolar pH of root cells and in a concomitant increase in the carbon fluxes through anaplerotic metabolism leading to malate biosynthesis, as suggested by changes in dark CO2 fixation, metabolite levels and enzyme activities along glycolysis, and mitochondrial alternative respiration capacity. This scenario was accompanied by a decrease in the net H(+) efflux from the roots, probably related to changes in plasma membrane permeability. * It is concluded that anaplerotic metabolism triggered by Cd detoxification processes might lead to an imbalance in H(+) production and consumption, and then to cell acidosis.     

Lactose biosynthesis: A sensitive radiochemical assay

A method is presented for the determination of lactose biosynthesis from labeled glucose, galactose, or other precursors based upon the addition of samples of the reaction mixture (after removal of the tissue or biosynthetic enzymes) to each of two strains of Escherichia coli. While both strains can metabolize glucose and galactose, only one is able to hydrolyze lactose. The sugars are converted by the bacteria largely to cell material and carbon dioxide. The difference between the residual, nonvolatile, soluble radioactivity in the medium from the two bacterial cultures represents the lactose unused by the strain unable to hydrolyze it.     

Carbon allocation to volatiles and other reproductive components in male Ficus carica (Moraceae)

Volatile compounds are often mediators of plant-pollinator interactions. Their emission is presumed to be costly, but this cost has seldom been quantified. Figs of Ficus carica (a dioecious species) release volatile compounds when receptive, thus attracting the agaonid wasp Blastophaga psenes. In dioecious fig species, wasps lay eggs inside male figs and pollinate female ones. For a male tree, we estimated carbon allocation to reproduction using the annual growth module (AGM) as the unit of measurement. Over the growing season, leaf and fig carbon exchange and construction costs were measured, as well as carbon investment in stamens, provisioning pollinators, and biosynthesis and release of volatile compounds. Representativity of the tree studied was evaluated by measuring some of these parameters on seven other male fig trees. The results show that 7.6-16.4% of the carbon assimilated by leaves and figs was invested in reproduction. Of the carbon invested in reproduction, pollinator attraction and feeding represented only 0.08-0.12% and 1.84-2.33%, respectively, while pollinator sheltering (fig construction and respiration) represented 97.6-98.0%. In this strict and coevolved plant-pollinator association, the main male reproductive investment was thus in the structures sheltering the associated pollinators.     

The effect of elevated CO2 on the chemical composition and construction costs of leaves of 27 C3 species

We determined the proximate chemical composition as well as the construction costs of leaves of 27 species, grown at ambient and at a twice-ambient partial pressure of atmospheric CO₂. These species comprised wild and agricultural herbaceous plants as well as tree seedlings. Both average responses across species and the range in response were considered. Expressed on a total dry weight basis, the main change in chemical composition due to CO₂ was the accumulation of total non-structural carbohydrates (TNC). To a lesser extent, decreases were found for organic N compounds and minerals. Hardly any change was observed for total structural carbohydrates (cellulose plus hemicellulose), lignin and lipids. When expressed on a TNC-free basis, decreases in organic N compounds and minerals were still present. On this basis, there was also an increase in the concentration of soluble phenolics. In terms of glucose required for biosynthesis, the increase in costs for one chemical compound – TNC – was balanced by a decrease in the costs for organic N compounds. Therefore, the construction costs, the total amount of glucose required to produce 1 g of leaf, were rather similar for the two CO₂ treatments; on average a small decrease of 3% was found. This decrease was attributable to a decrease of up to 30% in the growth respiration coefficient, the total CO₂ respired [mainly for N AD(P)H and ATP] in the process of constructing 1 g of biomass. The main reasons for this reduction were the decrease in organic N compounds and the increase in TNC.     

Chemolithoorganotrophic growth of Nitrosomonas europaea on fructose

The nitrifying bacterium Nitrosomonas europaea can obtain all its carbon for growth from CO(2) and all its energy and reductant for growth from the oxidation of NH(3) and is considered an obligate chemolithoautotroph. Previous studies have shown that N. europaea can utilize limited amounts of certain organic compounds, including amino acids, pyruvate, and acetate, although no organic compound has been reported to support the growth of N. europaea. The recently completed genomic sequence of N. europaea revealed a potential permease for fructose. With this in mind, we tested if N. europaea could utilize fructose and other compounds as carbon sources to support growth. Cultures were incubated in the presence of fructose or other organic compounds in sealed bottles purged of CO(2). In these cultures, addition of either fructose or pyruvate as the sole carbon source resulted in a two- to threefold increase in optical density and protein content in 3 to 4 days. Studies with [(14)C]fructose showed that >90% of the carbon incorporated by the cells during growth was derived from fructose. Cultures containing mannose, glucose, glycerol, mannitol, citrate, or acetate showed little or no growth. N. europaea was not able to grow with fructose as an energy source, although the presence of fructose did provide an energy benefit to the cells. These results show that N. europaea can be grown in CO(2)-free medium by using fructose and pyruvate as carbon sources and may now be considered a facultative chemolithoorganotroph.     

Inhibitory effect of organic carbon on CO₂ fixing by non-photosynthetic microbial community isolated from the ocean

The inhibitory effect of organic carbon on CO₂ fixation (CF) by the non-photosynthetic microbial community (NPMC) and its mechanism were studied. The results showed that different concentrations of glucose inhibited CF to some extent. However, when these microorganisms pre-cultured with glucose were re-cultured without organic carbon, their CF efficiency differed significantly from the control based on the glucose concentration in the pre-culture. ATP as bioenergy and NADH as reductant had no obvious inhibitory effect on CF; conversely, they improved CF efficiency to some extent, especially when both were present simultaneously. These results implied that not all organic materials inhibited CF by NPMC, and only those that acted as good carbon sources, such as glucose, inhibited CF. Moreover, some metabolites generated during the catabolism of glucose by heterotrophic metabolism of NPMC might inhibit CF, while other cumulated materials present in the cell interior, such as ATP and NADH, might improve CF.