Glyoxylate metabolism is a key feature of the metabolic degradation of 1,4-dioxane by Pseudonocardia dioxanivorans strain CB1190.
ABSTRACT: The groundwater contaminant 1,4-dioxane (dioxane) is transformed by several monooxygenase-expressing microorganisms, but only a few of these, including Pseudonocardia dioxanivorans strain CB1190, can metabolize the compound as a sole carbon and energy source. However, nothing is yet known about the genetic basis of dioxane metabolism. In this study, we used a microarray to study differential expression of genes in strain CB1190 grown on dioxane, glycolate (a previously identified intermediate of dioxane degradation), or pyruvate. Of eight multicomponent monooxygenase gene clusters carried by the strain CB1190 genome, only the monooxygenase gene cluster located on plasmid pPSED02 was upregulated with dioxane relative to pyruvate. Plasmid-borne genes for putative aldehyde dehydrogenases, an aldehyde reductase, and an alcohol oxidoreductase were also induced during growth with dioxane. With both dioxane and glycolate, a chromosomal gene cluster encoding a putative glycolate oxidase was upregulated, as were chromosomal genes related to glyoxylate metabolism through the glyoxylate carboligase pathway. Glyoxylate carboligase activity in cell extracts from cells pregrown with dioxane and in Rhodococcus jostii strain RHA1 cells expressing the putative strain CB1190 glyoxylate carboligase gene further demonstrated the role of glyoxylate metabolism in the degradation of dioxane. Finally, we used (13)C-labeled dioxane amino acid isotopomer analysis to provide additional evidence that metabolites of dioxane enter central metabolism as three-carbon compounds, likely as phosphoglycerate. The routing of dioxane metabolites via the glyoxylate carboligase pathway helps to explain how dioxane is metabolized as a sole carbon and energy source for strain CB1190.
Project description:Bacterial multicomponent monooxygenase gene targets in Pseudonocardia dioxanivorans CB1190 were evaluated for their use as biomarkers to identify the potential for 1,4-dioxane biodegradation in pure cultures and environmental samples. Our studies using laboratory pure cultures and industrial activated sludge samples suggest that the presence of genes associated with dioxane monooxygenase, propane monooxygenase, alcohol dehydrogenase, and aldehyde dehydrogenase are promising indicators of 1,4-dioxane biotransformation; however, gene abundance was insufficient to predict actual biodegradation. A time course gene expression analysis of dioxane and propane monooxygenases in Pseudonocardia dioxanivorans CB1190 and mixed communities in wastewater samples revealed important associations with the rates of 1,4-dioxane removal. In addition, transcripts of alcohol dehydrogenase and aldehyde dehydrogenase genes were upregulated during biodegradation, although only the aldehyde dehydrogenase was significantly correlated with 1,4-dioxane concentrations. Expression of the propane monooxygenase demonstrated a time-dependent relationship with 1,4-dioxane biodegradation in P. dioxanivorans CB1190, with increased expression occurring after over 50% of the 1,4-dioxane had been removed. While the fraction of P. dioxanivorans CB1190-like bacteria among the total bacterial population significantly increased with decrease in 1,4-dioxane concentrations in wastewater treatment samples undergoing active biodegradation, the abundance and expression of monooxygenase-based biomarkers were better predictors of 1,4-dioxane degradation than taxonomic 16S rRNA genes. This study illustrates that specific bacterial monooxygenase and dehydrogenase gene targets together can serve as effective biomarkers for 1,4-dioxane biodegradation in the environment.
Project description:Analysis of gene expression in Pseudonocardia dioxanivorans strain CB1190 during growth with dioxane, glycolate or pyruvate. Three treatments, based on carbon source used for growth: pyruvate, dioxane and glycolate. Triplicate microarrays (biological replicates) were prepared for each treatment. Pyruvate was used as the reference treatment for subsequent analysis. Gene expression changes were compared pair-wise: dioxane vs. pyruvate, and glycolate vs. pyruvate
Project description:The bacterium Pseudonocardia dioxanivorans CB1190 grows on the cyclic ethers 1,4-dioxane (dioxane) and tetrahydrofuran (THF) as sole carbon and energy sources. Prior transcriptional studies indicated that an annotated THF monooxygenase (THF MO) gene cluster, thmADBC, located on a plasmid in CB1190 is upregulated during growth on dioxane. In this work, transcriptional analysis demonstrates that upregulation of thmADBC occurs during growth on the dioxane metabolite β-hydroxyethoxyacetic acid (HEAA) and on THF. Comparison of the transcriptomes of CB1190 grown on THF and succinate (an intermediate of THF degradation) permitted the identification of other genes involved in THF metabolism. Dioxane and THF oxidation activity of the THF MO was verified in Rhodococcus jostii RHA1 cells heterologously expressing the CB1190 thmADBC gene cluster. Interestingly, these thmADBC expression clones accumulated HEAA as a dead-end product of dioxane transformation, indicating that despite its genes being transcriptionally upregulated during growth on HEAA, the THF MO enzyme is not responsible for degradation of HEAA in CB1190. Similar activities were also observed in RHA1 cells heterologously expressing the thmADBC gene cluster from Pseudonocardia tetrahydrofuranoxydans K1.
Project description:Analysis of gene expression in Pseudonocardia dioxanivorans strain CB1190 during growth on THF (tetradhydrofuran) or succinate, and re-analysis of GSE33197 published expression data. Two new treatments, based on carbon/energy source used for growth: THF and succinate. Three previously published treatments (pyruvate, dioxane, and glycolate) from GEO Series GSE33197 were also reanalyzed. Triplicate microarrays (biological replicates) were prepared for each treatment. Gene expression changes were compared directly between the new treatments (THF vs. succinate) and to previously published data during growth on pyruvate (GEO accession numbers GSM821862-GSM821864) in order to yield THF vs. pyruvate and succinate vs. pyruvate contrasts. Included as part of the re-analysis of GSE33197 were data from dioxane and glycolate treatments (GSM821865-GSM821870). The complete dataset representing: (1) the THF- and succinate-treated CB1190 Samples and (2) the pyruvate-, dioxane- and glycolate-treated CB1190 Samples from Series GSE33197 (re-processed using RMA), is linked below as a supplementary file (GSE48814_complete_data.txt).
Project description:Pseudonocardia dioxanivorans CB1190 is the first bacterium reported to be capable of growth on the environmental contaminant 1,4-dioxane and the first member of the genus Pseudonocardia for which there is an annotated genome sequence. Preliminary analysis of the genome (chromosome and three plasmids) indicates that strain CB1190 possesses several multicomponent monooxygenases that could be involved in the aerobic degradation of 1,4-dioxane and other environmental contaminants.
Project description:Carbon dioxide (CO2) is very important for photosynthesis of green plants. CO2 concentration in the atmosphere is relatively stable, but it drops sharply after sunrise due to the tightness of the greenhouse and the absorption of CO2 by vegetable crops. Vegetables in greenhouses are chronically CO2 starved. To investigate the feasibility of using genetic engineering to improve the photosynthesis and yield of greenhouse cucumber in a low CO2 environment, five genes encoding glyoxylate carboligase (GCL), tartronic semialdehyde reductase (TSR), and glycolate dehydrogenase (GlcDH) in the glycolate catabolic pathway of Escherichia coli were partially or completely introduced into cucumber chloroplast. Both partial pathway by introducing GlcDH and full pathway expressing lines exhibited higher photosynthetic efficiency and biomass yield than wild-type (WT) controls in low CO2 environments. Expression of partial pathway by introducing GlcDH increased net photosynthesis by 14.9% and biomass yield by 44.9%, whereas the expression of the full pathway increased seed yield by 33.4% and biomass yield by 59.0%. Photosynthesis, fluorescence parameters, and enzymatic measurements confirmed that the introduction of glycolate catabolic pathway increased the activity of photosynthetic carbon assimilation-related enzymes and reduced the activity of photorespiration-related enzymes in cucumber, thereby promoting the operation of Calvin cycle and resulting in higher net photosynthetic rate even in low CO2 environments. This increase shows an improvement in the efficiency of the operation of the photosynthetic loop. However, the utilization of cucumber of low concentration CO2 was not alleviated. This study demonstrated the feasibility of introducing the pathway of exogenous glycolate catabolic pathway to improve the photosynthetic and bio-yield of cucumber in a low CO2 environment. These findings are of great significance for high photosynthetic efficiency breeding of greenhouse cucumber.
Project description:Background:Glycolate is an important ?-hydroxy carboxylic acid widely used in industrial and consumer applications. The production of glycolate from glucose in Escherichia coli is generally carried out by glycolysis and glyoxylate shunt pathways, followed by reduction to glycolate. Glycolate accumulation was significantly affected by nitrogen sources and isocitrate dehydrogenase (ICDH), which influenced carbon flux distribution between the tricarboxylic acid (TCA) cycle and the glyoxylate shunt, however, the mechanism was unclear. Results:Herein, we used RNA-Seq to explore the effects of nitrogen sources and ICDH knockout on glycolate production. The Mgly534 strain and the Mgly624 strain (with the ICDH deletion in Mgly534), displaying different phenotypes on organic nitrogen sources, were also adopted for the exploration. Though the growth of Mgly534 was improved on organic nitrogen sources, glycolate production decreased and acetate accumulated, while Mgly624 achieved a balance between cell growth and glycolate production, reaching 0.81?g glycolate/OD (2.6-fold higher than Mgly534). To further study Mgly624, the significant changed genes related to N-regulation, oxidative stress response and iron transport were analyzed. Glutamate and serine were found to increase the biomass and productivity respectively. Meanwhile, overexpressing the arginine transport gene argT accelerated the cell growth rate and increased the biomass. Further, the presence of Fe2+ also speeded up the cells growth and compensated for the lack of reducing equivalents. Conclusion:Our studies identified that ICDH knockout strain was more suitable for glycolate production. RNA-Seq provided a better understanding of the ICDH knockout on cellular physiology and glycolate production.
Project description:Mycobacterium dioxanotrophicus PH-06 can degrade 1,4-dioxane (dioxane), which is a groundwater contaminant of emerging concern. In order to find the genes involved in dioxane degradation. RNA sequencing was first used to investigate gene expression levels of PH-06 during growth on two different carbon sources (dioxane and glucose). The sequencing shows that a monooxygenase gene cluster was upregulated when treated with dioxane relative to glucose. Overall design: 2 samples, no replicate
Project description:Failure to detoxify the intermediary metabolite glyoxylate in human hepatocytes underlies the metabolic pathology of two potentially lethal hereditary calcium oxalate kidney stone diseases, PH (primary hyperoxaluria) types 1 and 2. In order to define more clearly the roles of enzymes involved in the metabolism of glyoxylate, we have established singly, doubly and triply transformed CHO (Chinese-hamster ovary) cell lines, expressing all combinations of normal human AGT (alanine:glyoxylate aminotransferase; the enzyme deficient in PH1), GR/HPR (glyoxylate/hydroxypyruvate reductase; the enzyme deficient in PH2), and GO (glycolate oxidase). We have embarked on the preliminary metabolic analysis of these transformants by studying the indirect toxicity of glycolate as a simple measure of the net intracellular production of glyoxylate. Our results show that glycolate is toxic only to those cells expressing GO and that this toxicity is diminished when AGT and/or GR/HPR are expressed in addition to GO. This finding indicates that we have been able to reconstruct the glycolate-->glyoxylate, glyoxylate-->glycine, and glyoxylate-->glycolate metabolic pathways, catalysed by GO, AGT, and GR/HPR respectively, in cells that do not normally express them. These results are compatible with the findings in PH1 and PH2, in which AGT and GR/HPR deficiencies lead to increased oxalate synthesis, due to the failure to detoxify its immediate precursor glyoxylate. These CHO cell transformants have a potential use as a cell-based bioassay for screening small molecules that stabilize AGT or GR/HPR and might have use in the treatment of PH1 or PH2.
Project description:<h4>Background</h4>The photorespiratory nitrogen cycle in C? plants involves an extensive diversion of carbon and nitrogen away from the direct pathways of assimilation. The liberated ammonia is re-assimilated, but up to 25% of the carbon may be released into the atmosphere as CO?. Because of the loss of CO? and high energy costs, there has been considerable interest in attempts to decrease the flux through the cycle in C? plants. Transgenic tobacco plants were generated that contained the genes gcl and hyi from E. coli encoding glyoxylate carboligase (EC 184.108.40.206) and hydroxypyruvate isomerase (EC 220.127.116.11) respectively, targeted to the peroxisomes. It was presumed that the two enzymes could work together and compete with the aminotransferases that convert glyoxylate to glycine, thus avoiding ammonia production in the photorespiratory nitrogen cycle.<h4>Results</h4>When grown in ambient air, but not in elevated CO?, the transgenic tobacco lines had a distinctive phenotype of necrotic lesions on the leaves. Three of the six lines chosen for a detailed study contained single copies of the gcl gene, two contained single copies of both the gcl and hyi genes and one line contained multiple copies of both gcl and hyi genes. The gcl protein was detected in the five transgenic lines containing single copies of the gcl gene but hyi protein was not detected in any of the transgenic lines. The content of soluble amino acids including glycine and serine, was generally increased in the transgenic lines growing in air, when compared to the wild type. The content of soluble sugars, glucose, fructose and sucrose in the shoot was decreased in transgenic lines growing in air, consistent with decreased carbon assimilation.<h4>Conclusions</h4>Tobacco plants have been generated that produce bacterial glyoxylate carboligase but not hydroxypyruvate isomerase. The transgenic plants exhibit a stress response when exposed to air, suggesting that some glyoxylate is diverted away from conversion to glycine in a deleterious short-circuit of the photorespiratory nitrogen cycle. This diversion in metabolism gave rise to increased concentrations of amino acids, in particular glutamine and asparagine in the leaves and a decrease of soluble sugars.