Quantitative analysis of the bioenergetics of Mycobacterium tuberculosis along with Glyoxylate cycle as a drug target under inhibition of enzymes using Petri net.

The bacteria Mycobacterium tuberculosis is responsible for the infectious disease Tuberculosis. Targeting the tubercule bacteria is an important challenge in developing the antimycobacterials. The glyoxylate cycle is considered as a potential target for the development of anti-tuberculosis agents, due to its absence in the humans. Humans only possess tricarboxylic acid cycle, while this cycle gets connected to glyoxylate cycle in microbes. Glyoxylate cycle is essential to the Mycobacterium for its growth and survival. Due to this reason, it is considered as a potential therapeutic target for the development of anti-tuberculosis agents. Here, we explore the effect on the behavior of the tricarboxylic acid cycle, glyoxylate cycle and their integrated pathway with the bioenergetics of the Mycobacterium, under the inhibition of key glyoxylate cycle enzymes using Continuous Petri net. Continuous Petri net is a special Petri net used to perform the quantitative analysis of the networks. We first study the tricarboxylic acid cycle and glyoxylate cycle of the tubercule bacteria by simulating its Continuous Petri net model under different scenarios. Both the cycles are then integrated with the bioenergetics of the bacteria and the integrated pathway is again simulated under different conditions. The simulation graphs show the metabolic consequences of inhibiting the key glyoxylate cycle enzymes and adding the uncouplers on the individual as well as integrated pathway. The uncouplers that inhibit the synthesis of adenosine triphosphate, play an important role as anti-mycobacterials. The simulation study done here validates the proposed Continuous Petri net model as compared with the experimental outcomes and also explains the consequences of the enzyme inhibition on the biochemical reactions involved in the metabolic pathways of the mycobacterium.

[Mechanism of Cordyceps militaris against non-small cell lung cancer: based on serum metabolomics].

This study investigated the potential mechanism of Cordyceps militaris(CM) against non-small cell lung cancer(NSCLC) based on serum untargeted metabolomics. Specifically, Balb/c nude mice were used to generate the human lung cancer A549 xenograft mouse model. The tumor volume, tumor weight, and tumor inhibition rate in mice in the model, cisplatin, Cordyceps(low-, medium-, and high-dose), and CM(low-, medium-, and high-dose) groups were compared to evaluate the influence of CM on lung cancer. Gas chromatography-mass spectrometry(GC-MS) was used for the analysis of mouse serum, SIMCA 13.0 for the compa-rison of metabolic profiles, and MetaboAnalyst 5.0 for the analysis of metabolic pathways. According to the pharmacodynamic data, the tumor volume and tumor weight of mice in high-dose CM group and cisplatin group decreased as compared with those in the model group(P<0.05 or P<0.01). The results of serum metabolomics showed that the metabolic profiles of the model group were significantly different from those of the high-dose CM group, and the content of endogenous metabolites was adjusted to different degrees. A total of 42 differential metabolites and 7 differential metabolic pathways were identified. In conclusion, CM could significantly inhibit the tumor growth of lung cancer xenograft mice. The mechanism is the likelihood that it influences the aminoacyl-tRNA biosynthesis, the metabolism of D-glutamine and D-glutamate, metabolism of alanine, aspartate, and glutamate, metabolism of glyoxylate and dicarboxylic acid, biosynthesis of phenylalanine, tyrosine, and tryptophan, arginine biosynthesis as well as nitrogen metabolism. This study elucidated the underlying mechanism of CM against NSCLC from the point of metabolites. The results would lay a foundation for the anticancer research and clinical application of CM.

Effect of synbiotic yogurt fortified with monk fruit extract on hepatic lipid biomarkers and metabolism in rats with type 2 diabetes.

Monk fruit extract (MFE) is widely used as a sweetener in foods. In this study, the effects of the consumption of MFE-sweetened synbiotic yogurt on the lipid biomarkers and metabolism in the livers of type 2 diabetic rats were evaluated. The results revealed that the MFE-sweetened symbiotic yogurt affected the phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerol, lysophosphatidic acids, lysophosphatidylcholines, lysophosphatidylethanolamines, lysophosphatidylglycerols, lysophosphatidylinositols, lysophosphatidylserines, and fatty acid-hydroxy fatty acids biomarkers in the livers of type 2 diabetic rats. In addition, the consumption of the MFE-sweetened synbiotic yogurt significantly altered 12 hepatic metabolites, which are involved in phenylalanine metabolism, sphingolipid metabolism, bile secretion, and glyoxylate and dicarboxylate metabolism in the liver. Furthermore, a multiomics (metabolomic and transcriptomic) association study revealed that there was a significant correlation between the MFE-sweetened synbiotic yogurt and the metabolites and genes involved in fatty acid biosynthesis, bile secretion, and glyoxylate and dicarboxylate metabolism. The findings of this study will provide new insights on exploring the function of sweeteners for improving type 2 diabetes mellitus liver lipid biomarkers.

Glyoxylate reductase/hydroxypyruvate reductase regulates the free d-aspartate level in mammalian cells.

Multiple d-amino acids are present in mammalian cells, and these compounds have distinctive physiological functions. Among the free d-amino acids identified in mammals, d-aspartate plays critical roles in the neuroendocrine and endocrine systems, as well as in the central nervous system. Mammalian cells have the molecular apparatus necessary to take up, degrade, synthesize, and release d-aspartate. In particular, d-aspartate is degraded by d-aspartate oxidase (DDO), a peroxisome-localized enzyme that catalyzes the oxidative deamination of d-aspartate to generate oxaloacetate, hydrogen peroxide, and ammonia. However, little is known about the molecular mechanisms underlying d-aspartate homeostasis in cells. In this study, we established a cell line that overexpresses cytoplasm-localized DDO; this cell line cannot survive in the presence of high concentrations of d-aspartate, presumably because high levels of toxic hydrogen peroxide are produced by metabolism of abundant d-aspartate by DDO in the cytoplasm, where hydrogen peroxide cannot be removed due to the absence of catalase. Next, we transfected these cells with a complementary DNA library derived from the human brain and screened for clones that affected d-aspartate metabolism and improved cell survival, even when the cells were challenged with high concentrations of d-aspartate. The screen identified a clone of glyoxylate reductase/hydroxypyruvate reductase (GRHPR). Moreover, the GRHPR metabolites glyoxylate and hydroxypyruvate inhibited the enzymatic activity of DDO. Furthermore, we evaluated the effects of GRHPR and peroxisome-localized DDO on d- and l-aspartate levels in cultured mammalian cells. Our findings show that GRHPR contributes to the homeostasis of these amino acids in mammalian cells.

BtsT/ BtsS is involved in glyoxylate transport in E. coli and its mutations facilitated glyoxylate utilization.

Glyoxylate is an important chemical and is also an intermediate involved in metabolic pathways of living microorganisms. However, it cannot be rapidly utilized by many microbes. We observed a very long lag phase (up to 120 h) when E. coli is growing in a mineral medium supplemented with 50 mM glyoxylate. To better understand this strange growth pattern on glyoxylate and accelerate glyoxylate utilization, a random genomic library of E. coli was transformed into E. coli BW25113, and mutants that showed significantly shortened lag phase on glyoxylate were obtained. Interestingly, mutations in BtsT/BtsS, a two component system that is involved in pyruvate transport, were found to be a common feature in all mutants retrieved. We further demonstrated, through reverse engineering, that the mutations in BtsT/BtsS can indeed increase glyoxylate uptake. Growth experiments with different concentration of glyoxylate also showed the higher the concentration of glyoxylate, the shorter the lag phase. These new findings thus increased our understanding on microbial utilization of glyoxylate.

Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt.

BACKGROUND: Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium. RESULTS: We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium. CONCLUSIONS: Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.

Lipid Composition and Associated Gene Expression Patterns during Pollen Germination and Pollen Tube Growth in Olive (Olea europaea L.).

Pollen lipids are essential for sexual reproduction, but our current knowledge regarding lipid dynamics in growing pollen tubes is still very scarce. Here, we report unique lipid composition and associated gene expression patterns during olive pollen germination. Up to 376 genes involved in the biosynthesis of all lipid classes, except suberin, cutin and lipopolysaccharides, are expressed in olive pollen. The fatty acid profile of olive pollen is markedly different compared with other plant organs. Triacylglycerol (TAG), containing mostly C12-C16 saturated fatty acids, constitutes the bulk of olive pollen lipids. These compounds are partially mobilized, and the released fatty acids enter the ß-oxidation pathway to yield acetyl-CoA, which is converted into sugars through the glyoxylate cycle during the course of pollen germination. Our data suggest that fatty acids are synthesized de novo and incorporated into glycerolipids by the 'eukaryotic pathway' in elongating pollen tubes. Phosphatidic acid is synthesized de novo in the endomembrane system during pollen germination and seems to have a central role in pollen tube lipid metabolism. The coordinated action of fatty acid desaturases FAD2-3 and FAD3B might explain the increase in linoleic and alpha-linolenic acids observed in germinating pollen. Continuous synthesis of TAG by the action of diacylglycerol acyltransferase 1 (DGAT1) enzyme, but not phosphoplipid:diacylglycerol acyltransferase (PDAT), also seems plausible. All these data allow for a better understanding of lipid metabolism during the olive reproductive process, which can impact, in the future, on the increase in olive fruit yield and, therefore, olive oil production.

[Primary hyperoxaluria: case report and therapeutic perspectives].

Primary hyperoxaluria (PH) is a rare genetic disorder with autosomal recessive transmission, characterized by high endogenous production and markedly excessive urinary excretion of oxalate (Ox). It causes the accumulation of calcium oxide crystals in organs and tissues including bones, heart, arteries, skin and kidneys, where it may cause oxalo-calcic nephrolithiasis, nephrocalcinosis and chronic renal failure. Some forms are secondary to enteric diseases, drugs or dietetic substances, while three primitive forms, caused by various enzymatic defects, are currently known: PH1, PH2 and PH3. An early diagnosis, with the aid of biochemical and genetic investigations, helps prevent complications and establish a therapeutic strategy that often includes liver and liver-kidney transplantation, improving the prognosis of these patients. In this work we describe the clinical case of a patient with PH1 undergoing extracorporeal hemodialysis treatment and we report the latest research results that could change the life of patients with PH.

Gains and losses of metabolic function inferred from a phylotranscriptomic analysis of algae.

Hidden Markov models representing 167 protein sequence families were used to infer the presence or absence of homologs within the transcriptomes of 183 algal species/strains. Statistical analyses of the distribution of HMM hits across major clades of algae, or at branch points on the phylogenetic tree of 98 chlorophytes, confirmed and extended known cases of metabolic loss and gain, most notably the loss of the mevalonate pathway for terpenoid synthesis in green algae but not, as we show here, in the streptophyte algae. Evidence for novel events was found as well, most remarkably in the recurrent and coordinated gain or loss of enzymes for the glyoxylate shunt. We find, as well, a curious pattern of retention (or re-gain) of HMG-CoA synthase in chlorophytes that have otherwise lost the mevalonate pathway, suggesting a novel, co-opted function for this enzyme in select lineages. Finally, we find striking, phylogenetically linked distributions of coding sequences for three pathways that synthesize the major membrane lipid phosphatidylcholine, and a complementary phylogenetic distribution pattern for the non-phospholipid DGTS (diacyl-glyceryl-trimethylhomoserine). Mass spectrometric analysis of lipids from 25 species was used to validate the inference of DGTS synthesis from sequence data.

Metatranscriptomic exploration of microbial functioning in clouds.

Clouds constitute the uppermost layer of the biosphere. They host diverse communities whose functioning remains obscure, although biological activity potentially participates to atmospheric chemical and physical processes. In order to gain information on the metabolic functioning of microbial communities in clouds, we conducted coordinated metagenomics/metatranscriptomics profiling of cloud water microbial communities. Samples were collected from a high altitude atmospheric station in France and examined for biological content after untargeted amplification of nucleic acids. Living microorganisms, essentially bacteria, maintained transcriptional and translational activities and expressed many known complementary physiological responses intended to fight oxidants, osmotic variations and cold. These included activities of oxidant detoxification and regulation, synthesis of osmoprotectants/cryoprotectants, modifications of membranes, iron uptake. Consistently these energy-demanding processes were fueled by central metabolic routes involved in oxidative stress response and redox homeostasis management, such as pentose phosphate and glyoxylate pathways. Elevated binding and transmembrane ion transports demonstrated important interactions between cells and their cloud droplet chemical environments. In addition, polysaccharides, potentially beneficial for survival like exopolysaccharides, biosurfactants and adhesins, were synthesized. Our results support a biological influence on cloud physical and chemical processes, acting notably on the oxidant capacity, iron speciation and availability, amino-acids distribution and carbon and nitrogen fates.

Precise tuning of the glyoxylate cycle in Escherichia coli for efficient tyrosine production from acetate.

BACKGROUND: Acetate is one of promising feedstocks owing to its cheap price and great abundance. Considering that tyrosine production is gradually shifting to microbial production method, its production from acetate can be attempted to further improve the economic feasibility of its production. RESULTS: Here, we engineered a previously reported strain, SCK1, for efficient production of tyrosine from acetate. Initially, the acetate uptake and gluconeogenic pathway were amplified to maximize the flux toward tyrosine. As flux distribution between glyoxylate and TCA cycles is critical for efficient precursor supplementation, the activity of the glyoxylate cycle was precisely controlled by expression of isocitrate lyase gene under different-strength promoters. Consequently, the engineered strain with optimal flux distribution produced 0.70 g/L tyrosine with 20% of the theoretical maximum yield which are 1.6-fold and 1.9-fold increased values of the parental strain. CONCLUSIONS: Tyrosine production from acetate requires precise tuning of the glyoxylate cycle and we obtained substantial improvements in production titer and yield by synthetic promoters and 5' untranslated regions (UTRs). This is the first demonstration of tyrosine production from acetate. Our strategies would be widely applicable to the production of various chemicals from acetate in future.

Extracellular Acidic pH Inhibits Acetate Consumption by Decreasing Gene Transcription of the Tricarboxylic Acid Cycle and the Glyoxylate Shunt.

Escherichia coli produces acetate during aerobic growth on various carbon sources. After consuming the carbon substrate, E. coli can further grow on the acetate. This phenomenon is known as the acetate switch, where cells transition from producing acetate to consuming it. In this study, we investigated how pH governs the acetate switch. When E. coli was grown on a glucose-supplemented medium initially buffered to pH 7, the cells produced and then consumed the acetate. However, when the initial pH was dropped to 6, the cells still produced acetate but were only able to consume it when little (<10 mM) acetate was produced. When significant acetate was produced in acidic medium, which occurs when the growth medium contains magnesium, amino acids, and sugar, the cells were unable to consume the acetate. To determine the mechanism, we characterized a set of metabolic mutants and found that those defective in the tricarboxylic acid (TCA) cycle or glyoxylate shunt exhibited reduced rates of acetate consumption. We further found that the expression of the genes in these pathways was reduced during growth in acidic medium. The expression of the genes involved in the AckA-Pta pathway, which provides the principal route for both acetate production and consumption, was also inhibited in acidic medium but only after glucose was depleted, which correlates with the acetate consumption phase. On the basis of these results, we conclude that growth in acidic environments inhibits the expression of the acetate catabolism genes, which in turn prevents acetate consumption.IMPORTANCE Many microorganisms produce fermentation products during aerobic growth on sugars. One of the best-known examples is the production of acetate by Escherichia coli during aerobic growth on sugars. In E. coli, acetate production is reversible: once the cells consume the available sugar, they can consume the acetate previously produced during aerobic fermentation. We found that pH affects the reversibility of acetate production. When the cells produce significant acetate during growth in acidic environments, they are unable to consume it. Unconsumed acetate may accumulate in the cell and inhibit the expression of pathways required for acetate catabolism. These findings demonstrate how acetate alters cell metabolism; they also may be useful for the design of aerobic fermentation processes.

Lack of glyoxylate shunt dysregulates iron homeostasis in Pseudomonas aeruginosa.

The aceA and glcB genes, encoding isocitrate lyase (ICL) and malate synthase, respectively, are not in an operon in many bacteria, including Pseudomonas aeruginosa, unlike in Escherichia coli. Here, we show that expression of aceA in P. aeruginosa is specifically upregulated under H2O2-induced oxidative stress and under iron-limiting conditions. In contrast, the addition of exogenous redox active compounds or antibiotics increases the expression of glcB. The transcriptional start sites of aceA under iron-limiting conditions and in the presence of iron were found to be identical by 5' RACE. Interestingly, the enzymatic activities of ICL and isocitrate dehydrogenase had opposite responses under different iron conditions, suggesting that the glyoxylate shunt (GS) might be important under iron-limiting conditions. Remarkably, the intracellular iron concentration was lower while the iron demand was higher in the GS-activated cells growing on acetate compared to cells growing on glucose. Absence of GS dysregulated iron homeostasis led to changes in the cellular iron pool, with higher intracellular chelatable iron levels. In addition, GS mutants were found to have higher cytochrome c oxidase activity on iron-supplemented agar plates of minimal media, which promoted the growth of the GS mutants. However, deletion of the GS genes resulted in higher sensitivity to a high concentration of H2O2, presumably due to iron-mediated killing. In conclusion, the GS system appears to be tightly linked to iron homeostasis in the promotion of P. aeruginosa survival under oxidative stress.

Investigation of the Antifatigue Effects of Korean Ginseng on Professional Athletes by Gas Chromatography-Time-of-Flight-Mass Spectrometry-Based Metabolomics.

Ginseng is usually used for alleviating fatigue. The purpose of this paper was to evaluate the regulatory effect of Korean ginseng on the metabolic pattern in professional athletes, and, further, to explore the underlying mechanism of the antifatigue effect of Korean ginseng. GC-time-of-flight-MS was used to profile serum samples from professional athletes before training and after 15 and 30 day training, and professional athletes administered with Korean ginseng in the meanwhile. Biochemical parameters of all athletes were also analyzed. For the athlete control group, strength-endurance training resulted in an elevation of creatine kinase (CK) and blood urea nitrogen (BUN), and a reduction in blood hemoglobin, and a dynamic trajectory of the metabolomic profile which were related to fatigue. Korean ginseng treatment not only lead to a marked reduction in CK and blood urea nitrogen (BUN) in serum, but also showed regulatory effects on the serum metabolic profile and restored scores plots close to normal, suggesting that the change in metabolic profiling could reflect the antifatigue effect of Korean ginseng. Furthermore, perturbed levels of 11 endogenous metabolites were regulated by Korean ginseng significantly, which might be primarily involved in lipid metabolism, energy balance, and chemical signaling. These findings suggest that metabolomics is a potential tool for the evaluation of the antifatigue effect of Korean ginseng and for the elucidation of its pharmacological mechanism.

A reverse glyoxylate shunt to build a non-native route from C4 to C2 in Escherichia coli.

Most central metabolic pathways such as glycolysis, fatty acid synthesis, and the TCA cycle have complementary pathways that run in the reverse direction to allow flexible storage and utilization of resources. However, the glyoxylate shunt, which allows for the synthesis of four-carbon TCA cycle intermediates from acetyl-CoA, has not been found to be reversible to date. As a result, glucose can only be converted to acetyl-CoA via the decarboxylation of the three-carbon molecule pyruvate in heterotrophs. A reverse glyoxylate shunt (rGS) could be extended into a pathway that converts C4 carboxylates into two molecules of acetyl-CoA without loss of CO2. Here, as a proof of concept, we engineered in Escherichia coli such a pathway to convert malate and succinate to oxaloacetate and two molecules of acetyl-CoA. We introduced ATP-coupled heterologous enzymes at the thermodynamically unfavorable steps to drive the pathway in the desired direction. This synthetic pathway in essence reverses the glyoxylate shunt at the expense of ATP. When integrated with central metabolism, this pathway has the potential to increase the carbon yield of acetate and biofuels from many carbon sources in heterotrophic microorganisms, and could be the basis of novel carbon fixation cycles.

Malate and fumarate extend lifespan in Caenorhabditis elegans.

Malate, the tricarboxylic acid (TCA) cycle metabolite, increased lifespan and thermotolerance in the nematode C. elegans. Malate can be synthesized from fumarate by the enzyme fumarase and further oxidized to oxaloacetate by malate dehydrogenase with the accompanying reduction of NAD. Addition of fumarate also extended lifespan, but succinate addition did not, although all three intermediates activated nuclear translocation of the cytoprotective DAF-16/FOXO transcription factor and protected from paraquat-induced oxidative stress. The glyoxylate shunt, an anabolic pathway linked to lifespan extension in C. elegans, reversibly converts isocitrate and acetyl-CoA to succinate, malate, and CoA. The increased longevity provided by malate addition did not occur in fumarase (fum-1), glyoxylate shunt (gei-7), succinate dehydrogenase flavoprotein (sdha-2), or soluble fumarate reductase F48E8.3 RNAi knockdown worms. Therefore, to increase lifespan, malate must be first converted to fumarate, then fumarate must be reduced to succinate by soluble fumarate reductase and the mitochondrial electron transport chain complex II. Reduction of fumarate to succinate is coupled with the oxidation of FADH2 to FAD. Lifespan extension induced by malate depended upon the longevity regulators DAF-16 and SIR-2.1. Malate supplementation did not extend the lifespan of long-lived eat-2 mutant worms, a model of dietary restriction. Malate and fumarate addition increased oxygen consumption, but decreased ATP levels and mitochondrial membrane potential suggesting a mild uncoupling of oxidative phosphorylation. Malate also increased NADPH, NAD, and the NAD/NADH ratio. Fumarate reduction, glyoxylate shunt activity, and mild mitochondrial uncoupling likely contribute to the lifespan extension induced by malate and fumarate by increasing the amount of oxidized NAD and FAD cofactors.

Non-traditional antibacterial screening approaches for the identification of novel inhibitors of the glyoxylate shunt in gram-negative pathogens.

Antibacterial compounds that affect bacterial viability have traditionally been identified, confirmed, and characterized in standard laboratory media. The historical success of identifying new antibiotics via this route has justifiably established a traditional means of screening for new antimicrobials. The emergence of multi-drug-resistant (MDR) bacterial pathogens has expedited the need for new antibiotics, though many in the industry have questioned the source(s) of these new compounds. As many pharmaceutical companies' chemical libraries have been exhaustively screened via the traditional route, we have concluded that all compounds with any antibacterial potential have been identified. While new compound libraries and platforms are being pursued, it also seems prudent to screen the libraries we currently have in hand using alternative screening approaches. One strategy involves screening under conditions that better reflect the environment pathogens experience during an infection, and identifying in vivo essential targets and pathways that are dispensable for growth in standard laboratory media in vitro. Here we describe a novel screening strategy for identifying compounds that inhibit the glyoxylate shunt in Pseudomonas aeruginosa, a pathway that is required for bacterial survival in the pulmonary environment. We demonstrate that these compounds, which were not previously identified using traditional screening approaches, have broad-spectrum antibacterial activity when they are tested under in vivo-relevant conditions. We also show that these compounds have potent activity on both enzymes that comprise the glyoxylate shunt, a feature that was supported by computational homology modeling. By dual-targeting both enzymes in this pathway, we would expect to see a reduced propensity for resistance development to these compounds. Taken together, these data suggest that understanding the in vivo environment that bacterial pathogens must tolerate, and adjusting the antibacterial screening paradigm to reflect those conditions, could identify novel antibiotics for the treatment of serious MDR pathogens.

Comparative transcriptomic profiling of two tomato lines with different ascorbate content in the fruit.

In recent years, interest in tomato breeding for enhanced antioxidant content has increased as medical research has pointed to human health benefits from antioxidant dietary intake. Ascorbate is one of the major antioxidants present in tomato, and little is known about mechanisms governing ascorbate pool size in this fruit. In order to provide further insights into genetic mechanisms controlling ascorbate biosynthesis and accumulation in tomato, we investigated the fruit transcriptome profile of the Solanum pennellii introgression line 10-1 that exhibits a lower fruit ascorbate level than its cultivated parental genotype. Our results showed that this reduced ascorbate level is associated with an increased antioxidant demand arising from an accelerated oxidative metabolism mainly involving mitochondria, peroxisomes, and cytoplasm. Candidate genes for controlling ascorbate level in tomato fruit were identified, highlighting the role of glycolysis, glyoxylate metabolism, and purine breakdown in modulating the ascorbate pool size.

Adaptation of Rhizobium leguminosarum to pea, alfalfa and sugar beet rhizospheres investigated by comparative transcriptomics.

BACKGROUND: The rhizosphere is the microbe-rich zone around plant roots and is a key determinant of the biosphere's productivity. Comparative transcriptomics was used to investigate general and plant-specific adaptations during rhizosphere colonization. Rhizobium leguminosarum biovar viciae was grown in the rhizospheres of pea (its legume nodulation host), alfalfa (a non-host legume) and sugar beet (non-legume). Gene expression data were compared to metabolic and transportome maps to understand adaptation to the rhizosphere. RESULTS: Carbon metabolism was dominated by organic acids, with a strong bias towards aromatic amino acids, C1 and C2 compounds. This was confirmed by induction of the glyoxylate cycle required for C2 metabolism and gluconeogenesis in all rhizospheres. Gluconeogenesis is repressed in R. leguminosarum by sugars, suggesting that although numerous sugar and putative complex carbohydrate transport systems are induced in the rhizosphere, they are less important carbon sources than organic acids. A common core of rhizosphere-induced genes was identified, of which 66% are of unknown function. Many genes were induced in the rhizosphere of the legumes, but not sugar beet, and several were plant specific. The plasmid pRL8 can be considered pea rhizosphere specific, enabling adaptation of R. leguminosarum to its host. Mutation of many of the up-regulated genes reduced competitiveness for pea rhizosphere colonization, while two genes specifically up-regulated in the pea rhizosphere reduced colonization of the pea but not alfalfa rhizosphere. CONCLUSIONS: Comparative transcriptome analysis has enabled differentiation between factors conserved across plants for rhizosphere colonization as well as identification of exquisite specific adaptation to host plants.

Increased riboflavin production from activated bleaching earth by a mutant strain of Ashbya gossypii.

The production of riboflavin from vegetable oil was increased using a mutant strain of Ashbya gossypii. This mutant was generated by treating the wild-type strain with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Riboflavin production was 10-fold higher in the mutant compared to the wild-type strain. The specific intracellular catalase activity after 3 d of culture was 6-fold higher in the mutant than in the wild-type strain. For the mutant, riboflavin production in the presence of 40 mM hydrogen peroxide was 16% less than that in the absence of hydrogen peroxide, whereas it was 56% less for the wild-type strain. The isocitrate lyase (ICL) activity of the mutant was 0.26 mU/mg of protein during the active riboflavin production phase, which was 2.6-fold higher than the wild-type strain. These data indicate that the mutant utilizes the carbon flux from the TCA cycle to the glyoxylate cycle more efficiently than the wild-type strain, resulting in enhanced riboflavin production. This novel mutant has the potential to be of use for industrial-scale riboflavin production from waste-activated bleaching earth (ABE), thereby transforming a useless material into a valuable bioproduct.