Comparative Evaluation of Carbohydrate Metabolism in Perch (Perca fluviatilis L.) from Water Bodies with Different Contents of Humic Acids

Characteristic features of energy metabolism, related to the adaptation of fish to adverse environmental conditions, were detected in perch (Perca fluviatilis L.) inhabiting the lake with a high humus content, compared to perch from the control lake. We studied a complex of enzymes involved in carbohydrate metabolism and assessed the relationship between metabolic pathways using correlation analysis. The intensity of oxidative metabolism was found to increase in the gills. Activation of energy metabolism in the liver was characterized by an increased consumption of carbohydrates. In addition, the role of the liver-specific lactate dehydrogenase (LDH) isoform D₄ also increased. The activity of enzymes involved in carbohydrate metabolism decreased most strongly in white skeletal muscles, whereas the role of the pentose-phosphate pathway (along with glycolysis) in the production of muscle lactate increased. The adaptation of fish to a high-humus aquatic environment was accompanied by a slight decrease in their fatness.     

Opine Dehydrogenases in Marine Invertebrates

It is well known today that opine production anaerobic pathways are analogs to the classical glycolytic pathway (lactate production pathway). These pathways, catalyzed by a group of enzymes called opine dehydrogenases (OpDHs), ensure continuous flux of glycolysis and a constant supply of ATP by maintaining the NADH/NAD⁺ ratio during exercise and hypoxia, thus regulating the cytosolic redox balance in glycolysis under anoxia. OpDHs are distributed in a wide range of marine invertebrate phyla, including sponges (Porifera). Phylogenetic analyses supported with enzymatic assays strongly indicate that sponge OpDHs constitute an enzyme class unrelated to other OpDHs. Therefore, OpDHs in marine invertebrates are divided into two groups, a mollusk/annelid type and a sponge type, which belongs to the OCD/mu-crystallin family.     

Further studies on the phylogenetic distribution of pyruvate oxidoreductase activities

• 1.1. The phylogenetic distribution of lactate, octopine, alanopine and strombine dehydrogenase activities (respectively, LDH, ODH, ADH and SDH) was examined in over 60 species from seven phyla and from three continents.
• 2.2. The results confirm and extend previously published data. Consistencies of distribution are observed at the levels of phyla, class, order and family.
• 3.3. Major observations include prominent SDH in the Porifera; LDH only in the Polyplacophera, Nudibranchia and Myidae (Mollusca) and nereid worms (Polychaeta); ODH and SDH in the marine pulmonate Melampus bidentatus (Basommatophora); high ADH to SDH ratios in marine gastropods; high ODH in active molluscs; and apparent SDH in the barnacle Lepas anatifera.
• 4.4. The results are discussed in relation to theories of opine pathway evolution and the newly discovered tauropine and β-alanopine opine dehydrogenases.

     

Agrobacterium tumefaciens TR-DNA encodes a pathway for agropine biosynthesis

Summary A pathway for biosynthesis of the crown-gall opine, agropine is proposed and three potential new precursors characterised. The location of genes involved in the three steps of this pathway was determined by site directed insertions and deletions in the T_R region of the octopine Ti plasmid, pTiB6Tra^c. The proposed biosynthetic pathway for agropine which involves three T-DNA genes is in contrast to the biosynthesis of octopine and nopaline where single T-DNA genes are involved.     

Arginine catabolism: a new function of both octopine and nopaline Ti-plasmids of Agrobacterium.

Summary The oncogenic plasmids of Agrobacterium, the Ti-plasmids, carry genes that enable their bacterial host to catabolize opines. Opines are unusual amino acid derivatives that are only produced in crown gall tumours incited by oncogenic strains of Agrobacterium. The 2 opines, octopine and nopaline, are degraded by Agrobacterium strains carrying the octopine or the nopoline Ti-plasmid, respectively, to arginine and pyruvic acid, and to arginine and -ketoglutaric acid. In this paper it is shown that the Ti-plasmids carry gene(s) involved in the utilisation of arginine as a carbon source. Strains harbouring wild type octopine or nopaline Ti-plasmids in the chromosomal context of strain C58C1 do not grow on arginine as a carbon source. However, they are able to grow on arginine provided that they are induced, or constitutive for opine catabolism. The features of ornithine utilisation are identical. The gene(s) involved in arginine and ornithine utilization in C58C1 (pTi-oct) or C58C1 (pTi-nop) are under the control of the regulator gene that controls octopine or nopaline catabolism. A tentative pathway of octopine utilization is proposed, in which at least two steps are Ti-plasmid coded, and probably belong to the same operon: 1-scission of octopine into arginine and pyruvic acid 2-transformation of an arginine derivative (GSA?) to glutamic acid.Arginine utilization as a carbon source is therefore a new function of the Ti-plasmid. As this function is not inducible by arginine but by opines, it provides a method for selecting regulatory mutants of opine catabolism in the genetic background of strain C58.     

Structure and characterization of the crown gall opines heliopine, vitopine and ridéopine

The crown gall opines heliopine from tumors induced by octopine type Agrobacterium tumefaciens strains A6, A136(pTiB6-806), E9, A652 and 1590-1 and vitopine from tumor induced by grapevine strains S4 and T2 are identical to synthetic N2-(1'R-carboxyethyl)-L-glutamine. Tumors produced by strains S4 and T2 do not contain octopine or lysopine, but they do contain heliopine and the new opine ridéopine identified as N-(4'-aminobutyl)-D-glutamic acid. Grapevine strains S4 and T2 grow normally on tumor heliopine or synthetic heliopine and on tumor and synthetic ridéopine as well as on ridéopine lactam as sole carbon source. While octopine strains A6 and A136(pTiB6-806) do not grow on heliopine, mutant colonies do appear after a few weeks. Heliopine catabolism by octopine strains is not induced by octopine.     

Specificity of Octopine Uptake by Rhizobium and Pseudomonas Strains

The octopine-utilizing strain Agrobacterium tumefaciens B6S3 and three nonagrobacteria which had the capacity to utilize this opine were compared for octopine uptake. The characteristics of uptake by Rhizobium meliloti A3 and strain B6S3 were similar. In both bacteria, uptake activity was inducible by octopine and by the related opine octopinic acid, and competition assays showed that these two opine substrates were accepted by the same uptake system with an equivalent affinity. Cells of Pseudomonas putida 203 accumulated octopine against a concentration gradient, and this activity was induced specifically by octopine. While strain 203 did not utilize octopinic acid, a spontaneous mutant with a combined capacity for octopine and octopinic acid utilization was obtained. Both opines induced octopine uptake by this mutant, but octopinic acid was not a substrate for the induced system. Thus, the Pseudomonas uptake system exhibited a different specificity for octopine than the corresponding Agrobacterium system. The nonfluorescent pseudomonad GU187j, which utilized the three related opines octopine, octopinic acid, and nopaline, was constitutive for octopine uptake. Strain GU187j possessed a system which accepted these three opines, but not arginine or ornithine, with a similar affinity.     

Meiotic transmission of T-DNA genes inArabidopsis thaliana plants and their expression after 5-azacytidine treatment

Arabidopsis thaliana tumors were induced by octopine strain ofAgrobacterium tumefaciens B6S3 and its derivatives with modified T-DNA. Flowering shoots appeared sponta-neously onin vitro cultivated tumors and set seeds. R₁ and R₂ progeny of octopine synthesizing plants segregated in opine synthesis activities 3:1 and 15:1. Octopine synthase activity showed absolute linkage with agropine synthesis in most lines. In R₃ and R₄ progenies, the fraction of octopine synthase and agropine synthesis positive plants was lower than expected, but Mendelian segregation was restored if plants were cultivated on medium with 5-azacytidine. The most probable mechanism of disapearance of opine synthesis is cytosine methylation. The effect of 5-azacytidine lasted for at least next two generations.     

Parameters controlling opine formation during muscular activity and environmental hypoxia

Summary In order to elucidate the regulatory parameters which determine multiple opine formation in marine invertebrates, anaerobiosis was induced in 25 species from several phyla by stimulating the animals to vigorous muscular activity or by subjecting them to environmental hypoxia. The quantity of glycolytic end products and the corresponding amino acids were measured. In a second set of experiments the amounts of substrates and products of the opine dehydrogenase reactions in the isolated introvert retractor muscle (IRM) ofSipunculus nudus were determined in both situations.During environmental hypoxia opines accumulated according to the contents of the corresponding amino acids. Mass action ratios (MAR) of the opine dehydrogenase reactions in the isolated IRM were in the range of control values (octopine dehydrogenase 1.9·10¹¹ mol^–2·l², strombine dehydrogenase 2.2·10¹⁰ mol^–2·l²). During muscular activity those opines accumulated preferentially which corresponded to the highest opine dehydrogenase activities. In the isolated IRM only octopine accumulated during contractile activity; the MAR of the octopine dehydrogenase reaction was near the control value while the MAR of the strombine dehydrogenase reaction deviated by a factor of 9.The results indicate that during environmental hypoxia the opine dehydrogenases present in a tissue catalyze near equilibrium and the relative amount of opines accumulated is dictated by the concentration of the corresponding amino acids. During muscular activity only those opine dehydrogenases catalyze near equilibrium which are present in sufficiently high activities to keep pace with an increased glycolytic flux. Therefore, different opines may accumulate in the same animal during muscular activity and during environmental hypoxia.     

Anaerobiosis and acid-base status in marine invertebrates: a theoretical analysis of proton generation by anaerobic metabolism

Summary In animals, various organic acids are accumulated during hypoxia or anoxia as products of anaerobic energy metabolism. The diversity of such acids is largest in marine invertebrates where succinate, propionate, acetate, lactate, alanine, octopine, strombine, and alanopine, are produced mainly from glycogen and aspartate. The effect of these substances on the acid-base status was assessed by a theoretical analysis of the respective metabolic pathways. This resulted in a general rule which was applied to evaluate the proton balance of the reactions in energy metabolism: net changes in the number of carboxyl groups or changes in the degree of dissociation of other groups (e.g. phosphate or ammonia) determine the net amount of H⁺ ions released or bound by the substrates and the metabolic end products.For marine invertebrates the results of the analysis can be summarized as follows: In glycogenolysis one mol of protons per mol of end products is released during cytosolic glycolysis, independent of the type of metabolic end product (lactate, octopine, alanopine, strombine, or alanine). The same applies for mitochondrial production of propionate and acetate, whereas formation of succinate results in dissociation of two mol H⁺ per mol. Fermentation of aspartate, however, diminishes the amount of protons which is produced in the succinate-propionate pathway. Net metabolisation of Mg ATP^2– yields extra protons, whereas the cleavage of phosphagens (e.g. creatine phosphate, arginine phosphate) consumes protons.Additionally the break-down of energy-rich phosphates to inorganic phosphate has to be taken into account because of the shift of the intracellular buffer curve caused by changes of the respective effective pK values.     

Determination of octopine by pre-column fluorescence derivatization using benzoin

A sensitive fluorimetric method for the determination of octopine, a member of opine family, is presented. The method is based on the formation of a fluorescent derivative of octopine with benzoin and the separation by high performance liquid chromatography using a reversed-phase column (Kaseisorb LC ODS-300) within 20 min. The octopine derivative is completely separated from other guanidino compounds including arginine which is generally very high in marine invertebrates. This method gives higher sensitivity, 5 pmol minimum detection, and better reproducibility than the electrophoresis method and colorimetric method.     

Isolation of Agrobacterium Ti-plasmid regulatory mutants

Summary Crown gall tumors incited by Agrobacterium tumefaciens synthesize basic amino acid derivatives called opines. Opine production in tumours and opine catabolism by A. tumefaciens are coded by Ti-plasmids which confer oncogenicity on this bacterium. Catabolism of opines is inducible, and a method for isolation of regulatory mutants is described. From octopine-type bacteria, by plating on non-inducing substrates (noroctopine, noroctopine acid, D-histopine) we have isolated regulatory mutants of three types: constitutive, partially constitutive, and fully inducible by the analogue. From nopaline-type bacteria, by plating on octopine (a non inducing substrate) we have isolated analogous regulatory mutants.Synthetic opines, in which the amino acid moiety has been replaced by toxic arginine analogues, are toxic for these regulatory mutants. We isolated mutants resistant to such synthetic opines, and found that some had lost the capacity to utilize octopine. A survey of a large number of such mutants revealed that all of them still incited octopine synthetizing tumors.Mutants constitutive for octopine catabolism are in some instances also constitutive for Ti-plasmid transfer. A simple method for screening regulatory mutants for constitutive Ti-plasmid transfer is described.This work has been supported in part by grants from the Centre National de la Recherche Scientifique (contrats ATP 2814 and 3363).     

Endogenous carbohydrate esterases of Clostridium thermocellum are identified and disrupted for enhanced isobutyl acetate production from cellulose

Medium-chain esters are versatile chemicals with broad applications as flavors, fragrances, solvents, and potential drop-in biofuels. Currently, these esters are largely produced by the conventional chemical process that uses harsh operating conditions and requires high energy input. Alternatively, the microbial conversion route has recently emerged as a promising platform for sustainable and renewable ester production. The ester biosynthesis pathways can utilize either lipases or alcohol acyltransferase (AAT), but the AAT-dependent pathway is more thermodynamically favorable in an aqueous fermentation environment. Even though a cellulolytic thermophile Clostridium thermocellum harboring an AAT-dependent pathway has recently been engineered for direct conversion of lignocellulosic biomass into esters, the production is not efficient. One potential bottleneck is the ester degradation caused by the endogenous carbohydrate esterases (CEs) whose functional roles are poorly understood. The challenge is to identify and disrupt CEs that can alleviate ester degradation while not negatively affecting the efficient and robust capability of C. thermocellum for lignocellulosic biomass deconstruction. In this study, by using bioinformatics, comparative genomics, and enzymatic analysis to screen a library of CEs, we identified and disrupted the two most critical CEs, Clo1313_0613 and Clo1313_0693, that significantly contribute to isobutyl acetate degradation in C. thermocellum. We demonstrated that an engineered esterase-deficient C. thermocellum strain not only reduced ester hydrolysis but also improved isobutyl acetate production while maintaining effective cellulose assimilation.     

Uncovering the metabolic response of abalone (<Emphasis Type="Italic">Haliotis midae)</Emphasis> to environmental hypoxia through metabolomics

Introduction

Oxygen is essential for metabolic processes and in the absence thereof alternative metabolic pathways are required for energy production, as seen in marine invertebrates like abalone. Even though hypoxia has been responsible for significant losses to the aquaculture industry, the overall metabolic adaptations of abalone in response to environmental hypoxia are as yet, not fully elucidated.

Objective

To use a multiplatform metabolomics approach to characterize the metabolic changes associated with energy production in abalone (Haliotis midae) when exposed to environmental hypoxia.

Methods

Metabolomics analysis of abalone adductor and foot muscle, left and right gill, hemolymph, and epipodial tissue samples were conducted using a multiplatform approach, which included untargeted NMR spectroscopy, untargeted and targeted LC–MS spectrometry, and untargeted and semi-targeted GC-MS spectrometric analyses.

Results

Increased levels of anaerobic end-products specific to marine animals were found which include alanopine, strombine, tauropine and octopine. These were accompanied by elevated lactate, succinate and arginine, of which the latter is a product of phosphoarginine breakdown in abalone. Primarily amino acid metabolism was affected, with carbohydrate and lipid metabolism assisting with anaerobic energy production to a lesser extent. Different tissues showed varied metabolic responses to hypoxia, with the largest metabolic changes in the adductor muscle.

Conclusions

From this investigation, it becomes evident that abalone have well-developed (yet understudied) metabolic mechanisms for surviving hypoxic periods. Furthermore, metabolomics serves as a powerful tool for investigating the altered metabolic processes in abalone.

     

Octopine- and Nopaline-Inducible Proteins in Agrobacterium tumefaciens Are Also Induced by Arginine

Octopine induced the synthesis of 83, 76, 62, 58, 44, 42, 31, and 22 kDa proteins in Agrobacterium tumefaciens strains harboring the tumor-inducing (Ti) plasmids pTiA6 and pTiAch5. Nopaline induced the synthesis of 83, 76, 62, 58, 56, 44, 42, 31, and 22 kDa proteins in A. tumefaciens strains harboring the Ti plasmids pTiC58 and pTiT37. The molecular masses of proteins induced by octopine and nopaline were very similar. In accordance with the ‘opine concept’, octopine and nopaline were found to induce protein synthesis only in strains harboring the respective Ti plasmids. Arginine, a common catabolic product of octopine and nopaline, induced the synthesis of most of the proteins induced by the two opines. Our results show that only the initial step(s) of octopine and nopaline catabolism are induced by specific opines in the respective strains. The subsequent steps are likely to be regulated by arginine in both strains. Received: 5 January 1996 / Accepted: 21 February 1996     

Evaluation of Physical and Functional Protein-Protein Interaction Prediction Methods for Detecting Biological Pathways

Background

Cellular activities are governed by the physical and the functional interactions among several proteins involved in various biological pathways. With the availability of sequenced genomes and high-throughput experimental data one can identify genome-wide protein-protein interactions using various computational techniques. Comparative assessments of these techniques in predicting protein interactions have been frequently reported in the literature but not their ability to elucidate a particular biological pathway.

Methods

Towards the goal of understanding the prediction capabilities of interactions among the specific biological pathway proteins, we report the analyses of 14 biological pathways of Escherichia coli catalogued in KEGG database using five protein-protein functional linkage prediction methods. These methods are phylogenetic profiling, gene neighborhood, co-presence of orthologous genes in the same gene clusters, a mirrortree variant, and expression similarity.

Conclusions

Our results reveal that the prediction of metabolic pathway protein interactions continues to be a challenging task for all methods which possibly reflect flexible/independent evolutionary histories of these proteins. These methods have predicted functional associations of proteins involved in amino acids, nucleotide, glycans and vitamins & co-factors pathways slightly better than the random performance on carbohydrate, lipid and energy metabolism. We also make similar observations for interactions involved among the environmental information processing proteins. On the contrary, genetic information processing or specialized processes such as motility related protein-protein linkages that occur in the subset of organisms are predicted with comparable accuracy. Metabolic pathways are best predicted by using neighborhood of orthologous genes whereas phyletic pattern is good enough to reconstruct central dogma pathway protein interactions. We have also shown that the effective use of a particular prediction method depends on the pathway under investigation. In case one is not focused on specific pathway, gene expression similarity method is the best option.     

Differential modulation of glucose,lactate, and pyruvate oxidation by insulin and dichloroacetate in the rat heart

Despite the fact that lactate and pyruvate are potential substrates for energy production in vivo, our understanding of the control and regulation of carbohydrate metabolism is based principally on studies where glucose is the only available carbohydrate. Therefore, the purpose of this study was to determine the contributions of lactate, pyruvate, and glucose to energy production in the isolated, perfused rat heart over a range of insulin concentrations and after activation of pyruvate dehydrogenase with dichloroacetate (DCA). Hearts were perfused with physiological concentrations of [1-13C]glucose, [U-13C]lactate, [2-13C]pyruvate, and unlabeled palmitate for 45 min. Hearts were freeze clamped, and 13C NMR glutamate isotopomer analysis was performed on tissue extracts. Glucose, lactate, and pyruvate all contributed significantly to myocardial energy production; however, in the absence of insulin, glucose contributed only 25-30% of total pyruvate oxidation. Even under conditions where carbohydrates represented >95% of substrate entering the tricarboxylic acid (TCA) cycle, we found that glucose contributed at most 50-60% of total carbohydrate oxidation. Despite being present at only 0.1 mM, pyruvate contributed between approximately 10% and 30% of total acetyl-CoA entry into the TCA cycle. We also found that insulin and DCA not only increased glucose oxidation but also exogenous pyruvate oxidation; however, lactate oxidation was not increased. The differential effects of insulin and DCA on pyruvate and lactate oxidation provide further evidence for compartmentation of cardiac carbohydrate metabolism. These results may have important implications for understanding the mechanisms underlying the beneficial effects of increasing cardiac carbohydrate metabolism.     

Studies on the phylogenetic distribution of pyruvate oxidoreductases

Lactate dehydrogenase is present in all phyla examined. Alanopine and strombine dehydrogenase activities are present in all major phyla except the Arthropoda, Echinodermata and Chordata. Octopine dehydrogenase is also absent from the latter phyla and virtually absent from the Annelida. The opine dehydrogenase activities are generally absent from non-marine species. Relationships can be observed between malate dehydrogenase and glutamic-oxaloacetic transaminase and between the latter and glutamic-pyruvic transaminase in certain phyla. The results support the suggestion that the opine pathways are used in tissues to generate high (relatively) rates of energy production.     

Detection of opines by colorimetric assay

A colorimetric procedure for confirming the presence of arginine-derived opines (nopaline and octopine) in plant tissue extracts is described. Those materials are widely used as markers of plant cell transformation and tumorigenesis mediated by the tumor-inducing plasmids of Agrobacterium tumefaciens. Nopaline and octopine are generally detected, following resolution by paper electrophoresis, by observation of the uv-fluorescent products formed upon reaction with phenanthrenequinone. We found that a further heat treatment step, compatible with paper electrophoresis, results in rapid production of a red-purple pigment. Our colorimetric assay is sensitive to 1.25-micrograms quantities of opine and eliminates problems of background fluorescence encountered with crude plant extract in the usual assay.