Age dependency of the metabolic conversion of polyamines into amino acids in IMR-90 human embryonic lung diploid fibroblasts

When radioactive polyamines (putrescine or spermidine) were incubated with mammalian cells in tissue culture, the radioactivity was incorporated into cellular proteins via two different metabolic pathways; one is metabolic labeling of an 18,000-dalton protein via hypusine formation, and the other is general protein synthesis employing radioactive amino acids derived from biodegradation of polyamines via GABA shunt and Krebs cycle. Aminoguanidine, a potent inhibitor of diamine oxidase, blocked the metabolic conversion of polyamines to amino acids but had no effect on the metabolic labeling of the 18,000-dalton protein. We have investigated these two polyamine-associated biochemical events in IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL). We found that (1) the metabolic labeling of the 18,000-dalton protein was about two-fold greater in young cells (PDL = 22) than that in old cells (PDL = 48), and (2) the metabolic labeling of other cellular proteins, employing amino acids derived from putrescine via polyamine catabolic pathway, was more than six-fold greater in the old cells (PDL = 48) than in the young cells (PDL = 22). Since the rate of protein synthesis was about 1.4-fold higher in the young cells as compared to the old cells, our data indicated that the activity of catabolic conversion of putrescine (or spermidine) to amino acids in old IMR-90 cells was about eight-fold greater than that in young cells. This remarkable increase of polyamine catabolism and the slight decrease of metabolic labeling of the 18,000-dalton protein were also observed in cell strains derived from patients with premature aging disease.     

Catabolite-repression-like phenomenon in Rhizobium meliloti.

We report a phenomenon similar to catabolite repression in Rhizobium meliloti. Succinate, which allows the highest observed rate of growth of R. meliloti, caused an immediate reduction of beta-galactosidase activity when added to cells growing in lactose. A Lac- mutant was unaltered in nodulation and nitrogen fixation capacities, but a pleiotropic mutant deficient in several catabolic properties was unable to produce effective nitrogen-fixing nodules.     

Early steps of metabolism evolution inferred by cladistic analysis of amino acid catabolic pathways

Among abiotic molecules available in primitive environments, free amino acids are good candidates as the first source of energy and molecules for early protocells. Amino acid catabolic pathways are likely to be one of the very first metabolic pathways of life. Among them, which ones were the first to emerge? A cladistic analysis of catabolic pathways of the sixteen aliphatic amino acids and two portions of the Krebs cycle is performed using four criteria of homology. The cladogram shows that the earliest pathways to emerge are not portions of the Krebs cycle but catabolisms of aspartate, asparagine, glutamate, glutamine, proline, arginine. Earliest enzymatic catabolic functions were deaminations and transaminations. Later on appeared enzymatic decarboxylations. The consensus tree allows to propose four time spans for catabolism development and corroborates the views of Cordón in 1990 about the evolution of catabolism.     

Functional and evolutionary relatedness of genes for exopolysaccharide synthesis in Rhizobium meliloti and Rhizobium sp. strain NGR234.

Rhizobium meliloti SU47 and Rhizobium sp. strain NGR234 produce distinct exopolysaccharides that have some similarities in structure. R. meliloti has a narrow host range, whereas Rhizobium strain NGR234 has a very broad host range. In cross-species complementation and hybridization experiments, we found that several of the genes required for the production of the two polysaccharides were functionally interchangeable and similar in evolutionary origin. NGR234 exoC and exoY corresponded to R. meliloti exoB and exoF, respectively. NGR234 exoD was found to be an operon that included genes equivalent to exoM, exoA, and exoL in R. meliloti. Complementation of R. meliloti exoP, -N, and -G by NGR234 R'3222 indicated that additional equivalent genes remain to be found on the R-prime. We were not able to complement NGR234 exoB with R. meliloti DNA. In addition to functional and evolutionary equivalence of individual genes, the general organization of the exo regions was similar between the two species. It is likely that the same ancestral genes were used in the evolution of both exopolysaccharide biosynthetic pathways and probably of pathways in other species as well.     

Experimental identification and quantification of glucose metabolism in seven bacterial species

The structurally conserved and ubiquitous pathways of central carbon metabolism provide building blocks and cofactors for the biosynthesis of cellular macromolecules. The relative uses of pathways and reactions, however, vary widely among species and depend upon conditions, and some are not used at all. Here we identify the network topology of glucose metabolism and its in vivo operation by quantification of intracellular carbon fluxes from 13C tracer experiments. Specifically, we investigated Agrobacterium tumefaciens, two pseudomonads, Sinorhizobium meliloti, Rhodobacter sphaeroides, Zymomonas mobilis, and Paracoccus versutus, which grow on glucose as the sole carbon source, represent fundamentally different metabolic lifestyles (aerobic, anaerobic, photoheterotrophic, and chemoheterotrophic), and are phylogenetically distinct (firmicutes, gamma-proteobacteria, and alpha-proteobacteria). Compared to those of the model bacteria Escherichia coli and Bacillus subtilis, metabolisms of the investigated species differed significantly in several respects: (i) the Entner-Doudoroff pathway was the almost exclusive catabolic route; (ii) the pentose phosphate pathway exhibited exclusively biosynthetic functions, in many cases also requiring flux through the nonoxidative branch; (iii) all aerobes exhibited fully respiratory metabolism without significant overflow metabolism; and (iv) all aerobes used the pyruvate bypass of the malate dehydrogenase reaction to a significant extent. Exclusively, Pseudomonas fluorescens converted most glucose extracellularly to gluconate and 2-ketogluconate. Overall, the results suggest that metabolic data from model species with extensive industrial and laboratory history are not representative of microbial metabolism, at least not quantitatively.     

Control of fatty acid and glycerol release in adipose tissue lipolysis

Adipose tissue lipolysis is the catabolic process leading to the breakdown of triglycerides stored in fat cells and the release of fatty acids and glycerol. Recent work has revealed that lipolysis is not a simple metabolic pathway stimulated by catecholamines and inhibited by insulin. New discoveries on the regulation of lipolysis by endocrine and paracrine factors and on the proteins involved in triglyceride hydrolysis have led to a reappraisal of the complexity of the various signal transduction pathways. The steps involved in the dysregulation of lipolysis observed in obesity have partly been identified.     

Identification of bacterial periplasmic glycine betaine-binding protein after electrophoresis and affinity labeling

Antibodies were elicited in rabbits against periplasmic proteins obtained by cold osmotic shock from the Gram-negative eubacterium Rhizobium meliloti. When analyzed by crossed immunoelectrophoresis (CIE), the periplasmic proteins gave rise to 20 distinct immunoprecipitates corresponding to the same number of bands in polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions and in SDS-PAGE. The periplasmic glycine betaine-binding protein (GB-BP) was identified by autoradiography after affinity labeling with [14C]glycine betaine in PAGE and in CIE gels. The binding proved to be quite specific to glycine betaine, since the GB-BP was not labeled by choline (a metabolic precursor of glycine betaine in Escherichia coli and Rhizobium meliloti) and 15 distinct L-amino acids, including L-proline which, like glycine betaine is also an osmoprotectant. Affinity labeling of the GB-BP with [14C]glycine betaine after protein separation by PAGE or CIE is a simple and sensitive technique permitting the GB-BP to the unambiguously detected and identified in samples of complex protein mixtures containing down to 2 micrograms of GB-BP in PAGE and only 0.2 micrograms in CIE.     

Sinorhizobium meliloti metabolism in the root nodule: a proteomic perspective

The proteome of the model symbiotic bacterium, Sinorhizobium meliloti was examined to determine the enzymatic reactions and cell processes that occur when S. meliloti occupies the root nodules of Medicago truncatula and Melilotus alba. The proteomes of the nodule bacteria were compared to that of S. meliloti grown under laboratory cultured conditions as an additional control. All the detectable protein spots on the two-dimensional (2-D) gels between pH 4-7 were analyzed. In total, the identity of proteins in 1545 spots from 2-D gels was determined using peptide mass fingerprinting. There were clear differences in the proteome of nodule bacteria and cultured bacteria and putative nodule-specific and nodule suppressed proteins were identified. The data were analyzed using metabolic pathway prediction programs and used to review the biochemical and genetic studies that had been done previously on S. meliloti over several decades. There was a broad congruency between the proteomic and biochemical data when the overall pathways of central carbon and nitrogen metabolism were considered. A selective suite of ABC-type transporters was present in nodule bacteria that were biased towards the transport of amino acids and inorganic ions (P and Fe) suggesting that a highly specialized nutrient exchange was occurring between the nodule bacteria and the host. Proteins prominent in nodule bacteria were those involved in the pathways for vitamin synthesis and stress-related processes (chaperoning, heat shock, detoxification of reactive oxygen species, regulation of stress and osmo-regulation). Some of these proteins were found only in nodule bacteria. These results show the extent of the shift in metabolism that occurs when S. meliloti invades legume plants and establishes a nitrogen fixing symbiosis.     

Phosphoketolase Pathway for Xylose Catabolism in Clostridium acetobutylicum Revealed by 13C Metabolic Flux Analysis

Solvent-producing clostridia are capable of utilizing pentose sugars, including xylose and arabinose; however, little is known about how pentose sugars are catabolized through the metabolic pathways in clostridia. In this study, we identified the xylose catabolic pathways and quantified their fluxes in Clostridium acetobutylicum based on [1-(13)C]xylose labeling experiments. The phosphoketolase pathway was found to be active, which contributed up to 40% of the xylose catabolic flux in C. acetobutylicum. The split ratio of the phosphoketolase pathway to the pentose phosphate pathway was markedly increased when the xylose concentration in the culture medium was increased from 10 to 20 g liter(-1). To our knowledge, this is the first time that the in vivo activity of the phosphoketolase pathway in clostridia has been revealed. A phosphoketolase from C. acetobutylicum was purified and characterized, and its activity with xylulose-5-P was verified. The phosphoketolase was overexpressed in C. acetobutylicum, which resulted in slightly increased xylose consumption rates during the exponential growth phase and a high level of acetate accumulation.     

Microbial degradation of components of waste water from phenol-producing industry

Processes of aerobic biodegradation of components of phenol production sewage (phenol, acetophenone, dimethylphenylcarbinol, cumene hydroperoxide, alpha-methylstyrene, benzoate, and p-hydroxybenzoate) by bacterial strains obtained from the collection of Saratov Institute of Biocatalysis were studied. The metabolic reactions were shown to be oxidative and have a common catabolic sequence (cumene hydroperoxide-dimethylphenyl-carbinol alpha-methylstyrene-acetophenone-phenyl acetate-phenol-pyrocatechol-aromatic ring breakage). Benzoate and p-hydroxybenzoate were degraded through the formation of pyrocatechol and protocatechuate, respectively. Metabolic pathways were similar in model mixtures of components and sewage samples.     

Metabolism of apoB lipoproteins of intestinal and hepatic origin during constant feeding of small amounts of fat

We aimed to identify mechanisms by which apolipoprotein B-48 (apoB-48) could have an atherogenic role by simultaneously studying the metabolism of postprandial apoB-48 and apoB-100 lipoproteins. The kinetics of apoB-48 and apoB-100, each in four density subfractions of VLDL and intermediate density lipoprotein (IDL), were studied by stable isotope labeling in a constantly fed state with half-hourly administration of almond oil in five postmenopausal women. A non-steady-state, multicompartmental model was used. Despite a much lower production rate, VLDL and IDL apoB-48 shared a similar secretion pattern with apoB-100: both were directly secreted into all fractions with similar percentage mass distributions. Fractional catabolic rates (FCRs) of apoB-48 and apoB-100 were similar in VLDL and IDL. We identified a fast turnover compartment of light VLDL that had a residence time of <30 min for apoB-48 and apoB-100. Finally, a high secretion rate of apoB-48 was associated with a slow FCR of VLDL and IDL apoB-100. In conclusion, the intestine secretes a spectrum of apoB lipoproteins, similar to what the liver secretes, albeit with a much lower secretion rate. Once in plasma, intestinal and hepatic triglyceride-rich lipoproteins have similar rates of clearance and participate interactively in similar metabolic pathways, with high apoB-48 production inhibiting the clearance of apoB-100.     

A non-nodulating alfalfa mutant displays neither root hair curling nor early cell division in response to Rhizobium meliloti.

The early events in the alfalfa-Rhizobium meliloti symbiosis include deformation of epidermal root hairs and the approximately concurrent stimulation of cell dedifferentiation and cell division in the root inner cortex. These early steps have been studied previously by analysis of R. meliloti mutants. Bacterial strains mutated in nodABC, for example, fail to stimulate either root hair curling or cell division events in the plant host, whereas exopolysaccharide (exo) mutants of R. meliloti stimulate host cell division but the resulting nodules are uninfected. As a further approach to understanding early symbiotic interactions, we have investigated the phenotype of a non-nodulating alfalfa mutant, MnNC-1008 (NN) (referred to as MN-1008). Nodulating and non-nodulating plants were inoculated with wild-type R. meliloti and scored for root hair curling and cell divisions. MN-1008 was found to be defective in both responses. Mutant plants inoculated with Exo- bacteria also showed no cell division response. Therefore, the genetic function mutated in MN-1008 is required for both root hair curling and cell division, as is true for the R. meliloti nodABC genes. These observations support the model that the distinct cellular processes of root hair curling and cell division are triggered by related mechanisms or components, or are causally linked.     

The early ontogeny of digestive and metabolic enzyme activities in two commercial strains of arctic charr (Salvelinus alpinus L.)

The extent to which growth performance is linked to digestive or energetic capacities in the early life stages of a salmonid species was investigated. We compared two strains of Arctic charr known to have different growth potentials during their early development (Fraser and Yukon gold). Trypsin, lipase, and amylase activities of whole alevins were measured at regular intervals from hatching through 65 days of development. To assess catabolic ability, we also measured five enzymes representing the following metabolic pathways: amino acid oxidation (amino aspartate transferase), fatty acid oxidation (beta-hydroxy acyl CoA-dehydrogenase), tricarboxylic acid cycle (citrate synthase), glycolysis (pyruvate kinase), and anaerobic glycolysis (lactate dehydrogenase). The measurement of these enzyme activities in individual fish allowed a clear evaluation of digestive capacity in relation to energetic demand. We also compared triploid and diploid individuals within the Yukon gold strain. For the whole experimental period, diploid Yukon gold fish exhibited the highest growth rate (1.08+/-0.18% length/day) followed by triploid Yukon gold fish (1.00+/-0.28% length/day) and finally Fraser strain fish (0.84+/-0.28% length/day). When differences in enzyme activities were observed, the Fraser strain showed higher enzyme activities at a given length than the Yukon gold strain (diploid and triploid). Higher growth performance appears to be linked to lower metabolic capacity. Our results suggest that fish may have to reach an important increase in the ratio of digestive to catabolic enzyme activities or a leveling off of metabolic enzyme activities before the onset of large increases in mass.     

Enzyme-specific profiles for genome annotation: PRIAM

The advent of fully sequenced genomes opens the ground for the reconstruction of metabolic pathways on the basis of the identification of enzyme-coding genes. Here we describe PRIAM, a method for automated enzyme detection in a fully sequenced genome, based on the classification of enzymes in the ENZYME database. PRIAM relies on sets of position-specific scoring matrices ('profiles') automatically tailored for each ENZYME entry. Automatically generated logical rules define which of these profiles is required in order to infer the presence of the corresponding enzyme in an organism. As an example, PRIAM was applied to identify potential metabolic pathways from the complete genome of the nitrogen-fixing bacterium Sinorhizobium meliloti. The results of this automated method were compared with the original genome annotation and visualised on KEGG graphs in order to facilitate the interpretation of metabolic pathways and to highlight potentially missing enzymes.     

Sequence analysis of the 181-kb accessory plasmid pSmeSM11b, isolated from a dominant Sinorhizobium meliloti strain identified during a long-term field release experiment

The 181 251 bp accessory plasmid pSmeSM11b of Sinorhizobium meliloti strain SM11, belonging to a dominant indigenous S. meliloti subpopulation identified during a long-term field release experiment, was sequenced. This plasmid has 166 coding sequences (CDSs), 42% of which encode proteins with homology to proteins of known function. Plasmid pSmeSM11b is a member of the repABC replicon family and contains a large gene region coding for a conjugation system similar to that of other self-transmissible plasmids in Rhizobium and Agrobacterium. Another pSmeSM11b gene region, possibly involved in sugar metabolism and polysaccharide catabolism, resembled a region of S. meliloti 1021 megaplasmid pSymB and in the genome of Sinorhizobium medicae WSM419. Another module of plasmid pSmeSM11b encodes proteins similar to those of the nitrogen-fixing actinomycete Frankia CcI3, and which are likely to be involved in the synthesis of a secondary metabolite. Several ORFs of pSmeSM11b were predicted to play a role in nonribosomal peptide synthesis. Plasmid pSmeSM11b has many mobile genetic elements, which contribute to the mosaic composition of the plasmid.     

The Medicago truncatula CRE1 cytokinin receptor regulates lateral root development and early symbiotic interaction with Sinorhizobium meliloti

Legumes develop different types of lateral organs from their primary root, lateral roots and nodules, the latter depending on a symbiotic interaction with Sinorhizobium meliloti. Phytohormones have been shown to function in the control of these organogeneses. However, related signaling pathways have not been identified in legumes. We cloned and characterized the expression of Medicago truncatula genes encoding members of cytokinin signaling pathways. RNA interference of the cytokinin receptor homolog Cytokinin Response1 (Mt CRE1) led to cytokinin-insensitive roots, which showed an increased number of lateral roots and a strong reduction in nodulation. Both the progression of S. meliloti infection and nodule primordia formation were affected. We also identified two cytokinin signaling response regulator genes, Mt RR1 and Mt RR4, which are induced early during the symbiotic interaction. Induction of these genes by S. meliloti infection is altered in mutants affected in the Nod factor signaling pathway; conversely, cytokinin regulation of the early nodulin Nodule Inception1 (Mt NIN) depends on Mt CRE1. Hence, cytokinin signaling mediated by a single receptor, Mt CRE1, leads to an opposite control of symbiotic nodule and lateral root organogenesis. Mt NIN, Mt RR1, and Mt RR4 define a common pathway activated during early S. meliloti interaction, allowing crosstalk between plant cytokinins and bacterial Nod factors signals.     

13C-NMR, 1H-NMR and gas-chromatography mass-spectrometry studies of the biosynthesis of 13C-enriched L-lysine by Brevibacterium flavum

The basic metabolic pathways of lysine biosynthesis in Brevibacterium flavum, a strain which excretes excessive amounts of L-lysine, have been followed by using two 13C-labeled precursors. 13C- and 1H-NMR spectroscopies in conjunction with gas chromatography mass spectrometry (GC-MS) have revealed the various metabolic pathways leading to L-[13C]lysine. Discrete metabolic pathways give rise to distinct labeling patterns. L-Lysine resulting from [1-13C]glucose fermentation is relatively specifically labeled: L-[3,5-13C]lysine is the main product. Experimental and theoretical approaches based on the 13C-enrichment values of intracellular glutamate, a major intermediate metabolite, allowed us to assess the relative contribution of the major metabolic pathways forming lysine. The labeling pattern of glutamate reflects the isotope distribution in 2-oxoglutarate. When [2-13C]acetate is used as the sole carbon source in the culture, the energy-producing steps of the Krebs cycle are essential. The higher activity of the Krebs cycle, when endogenous carbohydrates are exhausted from the culture, is indicated by the increased 13C enrichment in C-1 of lysine and reveal a high content of isotopomers of four, five and six 13C atoms in the lysine molecule, pointing out that the four-carbon intermediates of the cycle are being derived from the glyoxylate shunt pathway. Such a phenomenon does not occur in glucose fermentation. GC-MS analyses of 13C enrichments and isotopomer distributions in metabolites and end products are in good agreement with the predicted contribution of each metabolic pathway. This new methodological approach of combined NMR and GC-MS has been demonstrated to be applicable to various other metabolic studies.