Journal articles: 'Methylcitrate cycle'

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Savvi, Suzana, Digby F. Warner, Bavesh D. Kana, John D. McKinney, Valerie Mizrahi, and Stephanie S. Dawes. "Functional Characterization of a Vitamin B12-Dependent Methylmalonyl Pathway in Mycobacterium tuberculosis: Implications for Propionate Metabolism during Growth on Fatty Acids." Journal of Bacteriology 190, no. 11 (March 28, 2008): 3886–95. http://dx.doi.org/10.1128/jb.01767-07.

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ABSTRACT Mycobacterium tuberculosis is predicted to subsist on alternative carbon sources during persistence within the human host. Catabolism of odd- and branched-chain fatty acids, branched-chain amino acids, and cholesterol generates propionyl-coenzyme A (CoA) as a terminal, three-carbon (C3) product. Propionate constitutes a key precursor in lipid biosynthesis but is toxic if accumulated, potentially implicating its metabolism in M. tuberculosis pathogenesis. In addition to the well-characterized methylcitrate cycle, the M. tuberculosis genome contains a complete methylmalonyl pathway, including a mutAB-encoded methylmalonyl-CoA mutase (MCM) that requires a vitamin B12-derived cofactor for activity. Here, we demonstrate the ability of M. tuberculosis to utilize propionate as the sole carbon source in the absence of a functional methylcitrate cycle, provided that vitamin B12 is supplied exogenously. We show that this ability is dependent on mutAB and, furthermore, that an active methylmalonyl pathway allows the bypass of the glyoxylate cycle during growth on propionate in vitro. Importantly, although the glyoxylate and methylcitrate cycles supported robust growth of M. tuberculosis on the C17 fatty acid heptadecanoate, growth on valerate (C5) was significantly enhanced through vitamin B12 supplementation. Moreover, both wild-type and methylcitrate cycle mutant strains grew on B12-supplemented valerate in the presence of 3-nitropropionate, an inhibitor of the glyoxylate cycle enzyme isocitrate lyase, indicating an anaplerotic role for the methylmalonyl pathway. The demonstrated functionality of MCM reinforces the potential relevance of vitamin B12 to mycobacterial pathogenesis and suggests that vitamin B12 availability in vivo might resolve the paradoxical dispensability of the methylcitrate cycle for the growth and persistence of M. tuberculosis in mice.

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Horswill, Alexander R., and Jorge C. Escalante-Semerena. "Salmonella typhimurium LT2 Catabolizes Propionate via the 2-Methylcitric Acid Cycle." Journal of Bacteriology 181, no. 18 (September 15, 1999): 5615–23. http://dx.doi.org/10.1128/jb.181.18.5615-5623.1999.

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ABSTRACT We previously identified the prpBCDE operon, which encodes catabolic functions required for propionate catabolism inSalmonella typhimurium. Results from13C-labeling experiments have identified the route of propionate breakdown and determined the biochemical role of each Prp enzyme in this pathway. The identification of catabolites accumulating in wild-type and mutant strains was consistent with propionate breakdown through the 2-methylcitric acid cycle. Our experiments demonstrate that the α-carbon of propionate is oxidized to yield pyruvate. The reactions are catalyzed by propionyl coenzyme A (propionyl-CoA) synthetase (PrpE), 2-methylcitrate synthase (PrpC), 2-methylcitrate dehydratase (probably PrpD), 2-methylisocitrate hydratase (probably PrpD), and 2-methylisocitrate lyase (PrpB). In support of this conclusion, the PrpC enzyme was purified to homogeneity and shown to have 2-methylcitrate synthase activity in vitro.1H nuclear magnetic resonance spectroscopy and negative-ion electrospray ionization mass spectrometry identified 2-methylcitrate as the product of the PrpC reaction. Although PrpC could use acetyl-CoA as a substrate to synthesize citrate, kinetic analysis demonstrated that propionyl-CoA is the preferred substrate.

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Domin, Nicole, Duncan Wilson, and Matthias Brock. "Methylcitrate cycle activation during adaptation of Fusarium solani and Fusarium verticillioides to propionyl-CoA-generating carbon sources." Microbiology 155, no. 12 (December 1, 2009): 3903–12. http://dx.doi.org/10.1099/mic.0.031781-0.

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Propionyl-CoA is an inhibitor of both primary and secondary metabolism in Aspergillus species and a functional methylcitrate cycle is essential for the efficient removal of this potentially toxic metabolite. Although the genomes of most sequenced fungal species appear to contain genes coding for enzymes of the methylcitrate cycle, experimental confirmation of pathway activity in filamentous fungi has only been provided for Aspergillus nidulans and Aspergillus fumigatus. In this study we demonstrate that pathogenic Fusarium species also possess a functional methylcitrate cycle. Fusarium solani appears highly adapted to saprophytic growth as it utilized propionate with high efficiency, whereas Fusarium verticillioides grew poorly on this carbon source. In order to elucidate the mechanisms of propionyl-CoA detoxification, we first identified the genes coding for methylcitrate synthase from both species. Despite sharing 96 % amino acid sequence identity, analysis of the two purified enzymes demonstrated that their biochemical properties differed in several respects. Both methylcitrate synthases exhibited low K m values for propionyl-CoA, but that of F. verticillioides displayed significantly higher citrate synthase activity and greater thermal stability. Activity determinations from cell-free extracts of F. solani revealed a strong methylcitrate synthase activity during growth on propionate and to a lesser extent on Casamino acids, whereas activity by F. verticillioides was highest on Casamino acids. Further phenotypic analysis confirmed that these biochemical differences were reflected in the different growth behaviour of the two species on propionyl-CoA-generating carbon sources.

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Yan, Yuxin, Huan Wang, Siyi Zhu, Jing Wang, Xiaohong Liu, Fucheng Lin, and Jianping Lu. "The Methylcitrate Cycle is Required for Development and Virulence in the Rice Blast Fungus Pyricularia oryzae." Molecular Plant-Microbe Interactions® 32, no. 9 (September 2019): 1148–61. http://dx.doi.org/10.1094/mpmi-10-18-0292-r.

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The methylcitrate cycle metabolizes propionyl-CoA, a toxic metabolite, into pyruvate. Pyricularia oryzae (syn. Magnaporthe oryzae) is a phytopathogenic fungus that causes a destructive blast disease in rice and wheat. We characterized the essential roles of the methylcitrate cycle in the development and virulence of P. oryzae using functional genomics. In P. oryzae, the transcript levels of MCS1 and MCL1, which encode a 2-methylcitrate synthase and a 2-methylisocitrate lyase, respectively, were upregulated during appressorium formation and when grown on propionyl-CoA-producing carbon sources. We found that deletion of MCS1 and MCL1 inhibited fungal growth on media containing both glucose and propionate, and media using propionate or propionyl-CoA-producing amino acids (valine, isoleucine, methionine, and threonine) as the sole carbon or nitrogen sources. The Δmcs1 mutant formed sparse aerial hyphae and did not produce conidia on complete medium (CM), while the Δmcl1 mutant showed decreased conidiation. The aerial mycelium of Δmcs1 displayed a lowered NAD+/NADH ratio, reduced nitric oxide content, and downregulated transcription of hydrophobin genes. Δmcl1 showed reduced appressorium turgor, severely delayed plant penetration, and weakened virulence. Addition of acetate recovered the growth of the wild type and Δmcs1 on medium containing both glucose and propionate and recovered the conidiation of both Δmcs1 and Δmcl1 on CM by reducing propionyl-CoA formation. Deletion of MCL1 together with ICL1, an isocitrate lyase gene in the glyoxylate cycle, greatly reduced the mutant’s virulence as compared with the single-gene deletion mutants (Δicl1 and Δmcl1). This experimental evidence provides important information about the role of the methylcitrate cycle in development and virulence of P. oryzae by detoxification of propionyl-CoA and 2-methylisocitrate.

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Dolan, Stephen K., Andre Wijaya, Stephen M. Geddis, David R. Spring, Rafael Silva-Rocha, and Martin Welch. "Loving the poison: the methylcitrate cycle and bacterial pathogenesis." Microbiology 164, no. 3 (March 1, 2018): 251–59. http://dx.doi.org/10.1099/mic.0.000604.

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Claes, Wilfried A., Alfred Pühler, and Jörn Kalinowski. "Identification of Two prpDBC Gene Clusters in Corynebacterium glutamicum and Their Involvement in Propionate Degradation via the 2-Methylcitrate Cycle." Journal of Bacteriology 184, no. 10 (May 15, 2002): 2728–39. http://dx.doi.org/10.1128/jb.184.10.2728-2739.2002.

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ABSTRACT Genome sequencing revealed that the Corynebacterium glutamicum genome contained, besides gltA, two additional citrate synthase homologous genes (prpC) located in two different prpDBC gene clusters, which were designated prpD1B1C1 and prpD2B2C2. The coding regions of the two gene clusters as well as the predicted gene products showed sequence identities of about 70 to 80%. Significant sequence similarities were found also to the prpBCDE operons of Escherichia coli and Salmonella enterica, which are known to encode enzymes of the propionate-degrading 2-methylcitrate pathway. Homologous and heterologous overexpression of the C. glutamicum prpC1 and prpC2 genes revealed that their gene products were active as citrate synthases and 2-methylcitrate synthases. Growth tests showed that C. glutamicum used propionate as a single or partial carbon source, although the beginning of the exponential growth phase was strongly delayed by propionate for up to 7 days. Compared to growth on acetate, the specific 2-methylcitrate synthase activity increased about 50-fold when propionate was provided as the sole carbon source, suggesting that in C. glutamicum the oxidation of propionate to pyruvate occurred via the 2-methylcitrate pathway. Additionally, two-dimensional gel electrophoresis experiments combined with mass spectrometry showed strong induction of the expression of the C. glutamicum prpD2B2C2 genes by propionate as an additional carbon source. Mutational analyses revealed that only the prpD2B2C2 genes were essential for the growth of C. glutamicum on propionate as a sole carbon source, while the function of the prpD1B1C1 genes remains obscure.

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Feng, Jiao, Liya He, Xing Xiao, Zhiwen Chen, Chunmei Chen, Jieming Chu, Sha Lu, Xiqing Li, Eleftherios Mylonakis, and Liyan Xi. "Methylcitrate cycle gene MCD is essential for the virulence of Talaromyces marneffei." Medical Mycology 58, no. 3 (July 9, 2019): 351–61. http://dx.doi.org/10.1093/mmy/myz063.

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Abstract Talaromyces marneffei (T. marneffei), which used to be known as Penicillium marneffei, is the causative agent of the fatal systemic mycosis known as talaromycosis. For the purpose of understanding the role of methylcitrate cycle in the virulence of T. marneffei, we generated MCD deletion (ΔMCD) and complementation (ΔMCD+) mutants of T. marneffei. Growth in different carbon sources showed that ΔMCD cannot grow on propionate media and grew slowly on the valerate, valine, methionine, isoleucine, cholesterol, and YNB (carbon free) media. The macrophage killing assay showed that ΔMCD was attenuated in macrophages of mice in vitro, especially at the presence of propionate. Finally, virulence studies in a murine infection experiment revealed attenuated virulence of the ΔMCD, which indicates MCD is essential for T. marneffei virulence in the host. This experiment laid the foundation for the further study of the specific mechanisms underlying the methylcitrate cycle of T. marneffei and may provide suitable targets for new antifungals.

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Lee, Seung-Ho, You-Kyoung Han, Sung-Hwan Yun, and Yin-Won Lee. "Roles of the Glyoxylate and Methylcitrate Cycles in Sexual Development and Virulence in the Cereal Pathogen Gibberella zeae." Eukaryotic Cell 8, no. 8 (June 12, 2009): 1155–64. http://dx.doi.org/10.1128/ec.00335-08.

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ABSTRACT The glyoxylate and methylcitrate cycles are involved in the metabolism of two- or three-carbon compounds in fungi. To elucidate the role(s) of these pathways in Gibberella zeae, which causes head blight in cereal crops, we focused on the functions of G. zeae orthologs (GzICL 1 and GzMCL1) of the genes that encode isocitrate lyase (ICL) and methylisocitrate lyase (MCL), respectively, key enzymes in each cycle. The deletion of GzICL1 (ΔGzICL1) caused defects in growth on acetate and in perithecium (sexual fruiting body) formation but not in virulence on barley and wheat, indicating that GzICL1 acts as the ICL of the glyoxylate cycle and is essential for self-fertility in G. zeae. In contrast, the ΔGzMCL1 strains failed to grow on propionate but exhibited no major changes in other traits, suggesting that GzMCL1 is required for the methylcitrate cycle in G. zeae. Interestingly, double deletion of both GzICL1 and GzMCL1 caused significantly reduced virulence on host plants, indicating that both GzICL1 and GzMCL1 have redundant functions for plant infection in G. zeae. Thus, both GzICL1 and GzMCL1 may play important roles in determining major mycological and pathological traits of G. zeae by participating in different metabolic pathways for the use of fatty acids.

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Zheng, Cao, Zhaoqing Yu, Cuiying Du, Yujing Gong, Wen Yin, Xinfeng Li, Zhou Li, Ute Römling, Shan‐Ho Chou, and Jin He. "2‐Methylcitrate cycle: a well‐regulated controller of Bacillus sporulation." Environmental Microbiology 22, no. 3 (December 28, 2019): 1125–40. http://dx.doi.org/10.1111/1462-2920.14901.

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Zhang, Yong-Qiang, and Nancy P. Keller. "Blockage of methylcitrate cycle inhibits polyketide production in Aspergillus nidulans." Molecular Microbiology 52, no. 2 (March 4, 2004): 541–50. http://dx.doi.org/10.1111/j.1365-2958.2004.03994.x.

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Rocco, Christopher J., and Jorge C. Escalante-Semerena. "In Salmonella enterica, 2-Methylcitrate Blocks Gluconeogenesis." Journal of Bacteriology 192, no. 3 (November 30, 2009): 771–78. http://dx.doi.org/10.1128/jb.01301-09.

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ABSTRACT Strains of Salmonella enterica serovar Typhimurium LT2 lacking a functional 2-methylcitric acid cycle (2-MCC) display increased sensitivity to propionate. Previous work from our group indicated that this sensitivity to propionate is in part due to the production of 2-methylcitrate (2-MC) by the Krebs cycle enzyme citrate synthase (GltA). Here we report in vivo and in vitro data which show that a target of the 2-MC isomer produced by GltA (2-MCGltA) is fructose-1,6-bisphosphatase (FBPase), a key enzyme in gluconeogenesis. Lack of growth due to inhibition of FBPase by 2-MCGltA was overcome by increasing the level of FBPase or by micromolar amounts of glucose in the medium. We isolated an fbp allele encoding a single amino acid substitution in FBPase (S123F), which allowed a strain lacking a functional 2-MCC to grow in the presence of propionate. We show that the 2-MCGltA and the 2-MC isomer synthesized by the 2-MC synthase (PrpC; 2-MCPrpC) are not equally toxic to the cell, with 2-MCGltA being significantly more toxic than 2-MCPrpC. This difference in 2-MC toxicity is likely due to the fact that as a si-citrate synthase, GltA may produce multiple isomers of 2-MC, which we propose are not substrates for the 2-MC dehydratase (PrpD) enzyme, accumulate inside the cell, and have deleterious effects on FBPase activity. Our findings may help explain human inborn errors in propionate metabolism.

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London, Robert E., Devon L. Allen, Scott A. Gabel, and Eugene F. DeRose. "Carbon-13 Nuclear Magnetic Resonance Study of Metabolism of Propionate by Escherichia coli." Journal of Bacteriology 181, no. 11 (June 1, 1999): 3562–70. http://dx.doi.org/10.1128/jb.181.11.3562-3570.1999.

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ABSTRACT We have evaluated the use of [1,2-13C2]propionate for the analysis of propionic acid metabolism, based on the ability to distinguish between the methylcitrate and methylmalonate pathways. Studies using propionate-adapted Escherichia coli MG1655 cells were performed. Preservation of the13C-13C-12C carbon skeleton in labeled alanine and alanine-containing peptides involved in cell wall recycling is indicative of the direct formation of pyruvate from propionate via the methylcitrate cycle, the enzymes of which have recently been demonstrated in E. coli. Additionally, formation of 13C-labeled formate from pyruvate by the action of pyruvate-formate lyase is also consistent with the labeling of pyruvate C-1. Carboxylation of the labeled pyruvate leads to formation of [1,2-13C2]oxaloacetate and to multiply labeled glutamate and succinate isotopomers, also consistent with the flux through the methylcitrate pathway, followed by the tricarboxylic acid (TCA) cycle. Additional labeling of TCA intermediates arises due to the formation of [1-13C]acetyl coenzyme A from the labeled pyruvate, formed via pyruvate-formate lyase. Labeling patterns in trehalose and glycine are also interpreted in terms of the above pathways. The information derived from the [1,2-13C2]propionate label is contrasted with information which can be derived from singly or triply labeled propionate and shown to be more useful for distinguishing the different propionate utilization pathways via nuclear magnetic resonance analysis.

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Schlachter, Caleb R., Vincent Klapper, Taylor Radford, and Maksymilian Chruszcz. "Comparative studies of Aspergillus fumigatus 2-methylcitrate synthase and human citrate synthase." Biological Chemistry 400, no. 12 (December 18, 2019): 1567–81. http://dx.doi.org/10.1515/hsz-2019-0106.

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Abstract Aspergillus fumigatus is a ubiquitous fungus that is not only a problem in agriculture, but also in healthcare. Aspergillus fumigatus drug resistance is becoming more prominent which is mainly attributed to the widespread use of fungicides in agriculture. The fungi-specific 2-methylcitrate cycle is responsible for detoxifying propionyl-CoA, a toxic metabolite produced as the fungus breaks down proteins and amino acids. The enzyme responsible for this detoxification is 2-methylcitrate synthase (mcsA) and is a potential candidate for the design of new anti-fungals. However, mcsA is very similar in structure to human citrate synthase (hCS) and catalyzes the same reaction. Therefore, both enzymes were studied in parallel to provide foundations for design of mcsA-specific inhibitors. The first crystal structures of citrate synthase from humans and 2-methylcitrate synthase from A. fumigatus are reported. The determined structures capture various conformational states of the enzymes and several inhibitors were identified and characterized. Despite a significant homology, mcsA and hCS display pronounced differences in substrate specificity and cooperativity. Considering that the active sites of the enzymes are almost identical, the differences in reactions catalyzed by enzymes are caused by residues that are in the vicinity of the active site and influence conformational changes of the enzymes.

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Upton, Anna M., and John D. McKinney. "Role of the methylcitrate cycle in propionate metabolism and detoxification in Mycobacterium smegmatis." Microbiology 153, no. 12 (December 1, 2007): 3973–82. http://dx.doi.org/10.1099/mic.0.2007/011726-0.

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Munoz-Elias, Ernesto J., Anna M. Upton, Joseph Cherian, and John D. McKinney. "Role of the methylcitrate cycle in Mycobacterium tuberculosis metabolism, intracellular growth, and virulence." Molecular Microbiology 60, no. 5 (June 2006): 1109–22. http://dx.doi.org/10.1111/j.1365-2958.2006.05155.x.

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Brock, Matthias. "Generation and Phenotypic Characterization of Aspergillus nidulans Methylisocitrate Lyase Deletion Mutants: Methylisocitrate Inhibits Growth and Conidiation." Applied and Environmental Microbiology 71, no. 9 (September 2005): 5465–75. http://dx.doi.org/10.1128/aem.71.9.5465-5475.2005.

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ABSTRACT Propionate is a very abundant carbon source in soil, and many microorganisms are able to use this as the sole carbon source. Nevertheless, propionate not only serves as a carbon source for filamentous fungi but also acts as a preservative when added to glucose containing media. To solve this contradiction between carbon source and preservative effect, propionate metabolism of Aspergillus nidulans was studied and revealed the methylcitrate cycle as the responsible pathway. Methylisocitrate lyase is one of the key enzymes of that cycle. It catalyzes the cleavage of methylisocitrate into succinate and pyruvate and completes the α-oxidation of propionate. Previously, methylisocitrate lyase was shown to be highly specific for the substrate (2R,3S)-2-methylisocitrate. Here, the identification of the genomic sequence of the corresponding gene and the generation of deletion mutants is reported. Deletion mutants did not grow on propionate as sole carbon and energy source and were severely inhibited during growth on alternative carbon sources, when propionate was present. The strongest inhibitory effect was observed, when glycerol was the main carbon source, followed by glucose and acetate. In addition, asexual conidiation was strongly impaired in the presence of propionate. These effects might be caused by competitive inhibition of the NADP-dependent isocitrate dehydrogenase, because the Ki of (2R,3S)-2-methylisocitrate, the product of the methylcitrate cycle, on NADP-dependent isocitrate dehydrogenase was determined as 1.55 μM. Other isomers had no effect on enzymatic activity. Therefore, methylisocitrate was identified as a potential toxic compound for cellular metabolism.

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Serafini, Agnese, Lendl Tan, Stuart Horswell, Steven Howell, Daniel J. Greenwood, Deborah M. Hunt, Minh‐Duy Phan, et al. "Mycobacterium tuberculosis requires glyoxylate shunt and reverse methylcitrate cycle for lactate and pyruvate metabolism." Molecular Microbiology 112, no. 4 (August 23, 2019): 1284–307. http://dx.doi.org/10.1111/mmi.14362.

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Textor, Susanne, Volker F. Wendisch, Albert A. De Graaf, U. Müller, Monica I. Linder, Dietmar Linder, and W. Buckel. "Propionate oxidation in Escherichia coli : evidence for operation of a methylcitrate cycle in bacteria." Archives of Microbiology 168, no. 5 (October 15, 1997): 428–36. http://dx.doi.org/10.1007/s002030050518.

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Darley, Dan J, Thorsten Selmer, William Clegg, Ross W Harrington, Wolfgang Buckel, and Bernard T Golding. "Stereocontrolled Synthesis of (2R,3S)-2-Methylisocitrate, a Central Intermediate in the Methylcitrate Cycle." Helvetica Chimica Acta 86, no. 12 (December 2003): 3991–99. http://dx.doi.org/10.1002/hlca.200390332.

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Eoh, Hyungjin, and Kyu Y. Rhee. "Methylcitrate cycle defines the bactericidal essentiality of isocitrate lyase for survival ofMycobacterium tuberculosison fatty acids." Proceedings of the National Academy of Sciences 111, no. 13 (March 17, 2014): 4976–81. http://dx.doi.org/10.1073/pnas.1400390111.

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Limenitakis, Julien, Rebecca D. Oppenheim, Darren J. Creek, Bernardo J. Foth, Michael P. Barrett, and Dominique Soldati‐Favre. "The 2‐methylcitrate cycle is implicated in the detoxification of propionate in T oxoplasma gondii." Molecular Microbiology 87, no. 4 (January 11, 2013): 894–908. http://dx.doi.org/10.1111/mmi.12139.

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Ruetz, Markus, Gregory C. Campanello, Meredith Purchal, Hongying Shen, Liam McDevitt, Harsha Gouda, Shoko Wakabayashi, et al. "Itaconyl-CoA forms a stable biradical in methylmalonyl-CoA mutase and derails its activity and repair." Science 366, no. 6465 (October 31, 2019): 589–93. http://dx.doi.org/10.1126/science.aay0934.

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Itaconate is an immunometabolite with both anti-inflammatory and bactericidal effects. Its coenzyme A (CoA) derivative, itaconyl-CoA, inhibits B12-dependent methylmalonyl-CoA mutase (MCM) by an unknown mechanism. We demonstrate that itaconyl-CoA is a suicide inactivator of human and Mycobacterium tuberculosis MCM, which forms a markedly air-stable biradical adduct with the 5′-deoxyadenosyl moiety of the B12 coenzyme. Termination of the catalytic cycle in this way impairs communication between MCM and its auxiliary repair proteins. Crystallography and spectroscopy of the inhibited enzyme are consistent with a metal-centered cobalt radical ~6 angstroms away from the tertiary carbon-centered radical and suggest a means of controlling radical trajectories during MCM catalysis. Mycobacterial MCM thus joins enzymes in the glyoxylate shunt and the methylcitrate cycle as targets of itaconate in pathogen propionate metabolism.

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Luttik, Marijke A. H., Peter Kötter, Florian A. Salomons, Ida J. van der Klei, Johannes P. van Dijken, and Jack T. Pronk. "The Saccharomyces cerevisiae ICL2 Gene Encodes a Mitochondrial 2-Methylisocitrate Lyase Involved in Propionyl-Coenzyme A Metabolism." Journal of Bacteriology 182, no. 24 (December 15, 2000): 7007–13. http://dx.doi.org/10.1128/jb.182.24.7007-7013.2000.

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ABSTRACT The Saccharomyces cerevisiae ICL1 gene encodes isocitrate lyase, an essential enzyme for growth on ethanol and acetate. Previous studies have demonstrated that the highly homologousICL2 gene (YPR006c) is transcribed during the growth of wild-type cells on ethanol. However, even when multiple copies are introduced, ICL2 cannot complement the growth defect oficl1 null mutants. It has therefore been suggested thatICL2 encodes a nonsense mRNA or nonfunctional protein. In the methylcitrate cycle of propionyl-coenzyme A metabolism, 2-methylisocitrate is converted to succinate and pyruvate, a reaction similar to that catalyzed by isocitrate lyase. To investigate whetherICL2 encodes a specific 2-methylisocitrate lyase, isocitrate lyase and 2-methylisocitrate lyase activities were assayed in cell extracts of wild-type S. cerevisiae and of isogenicicl1, icl2, and icl1 icl2 null mutants. Isocitrate lyase activity was absent in icl1 andicl1 icl2 null mutants, whereas in contrast, 2-methylisocitrate lyase activity was detected in the wild type and single icl mutants but not in the icl1 icl2mutant. This demonstrated that ICL2 encodes a specific 2-methylisocitrate lyase and that the ICL1-encoded isocitrate lyase exhibits a low but significant activity with 2-methylisocitrate. Subcellular fractionation studies and experiments with an ICL2-green fluorescent protein fusion demonstrated that theICL2-encoded 2-methylisocitrate lyase is located in the mitochondrial matrix. Similar to that of ICL1, transcription of ICL2 is subject to glucose catabolite repression. In glucose-limited cultures, growth with threonine as a nitrogen source resulted in a ca. threefold induction ofICL2 mRNA levels and of 2-methylisocitrate lyase activity in cell extracts relative to cultures grown with ammonia as the nitrogen source. This is consistent with an involvement of the 2-methylcitrate cycle in threonine catabolism.

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Wilson, Kirkland A., Yong Han, Miaoqi Zhang, Jeremy P. Hess, Kimberly A. Chapman, Gary W. Cline, Gregory P. Tochtrop, Henri Brunengraber, and Guo-Fang Zhang. "Inter-relations between 3-hydroxypropionate and propionate metabolism in rat liver: relevance to disorders of propionyl-CoA metabolism." American Journal of Physiology-Endocrinology and Metabolism 313, no. 4 (October 1, 2017): E413—E428. http://dx.doi.org/10.1152/ajpendo.00105.2017.

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Propionate, 3-hydroxypropionate (3HP), methylcitrate, related compounds, and ammonium accumulate in body fluids of patients with disorders of propionyl-CoA metabolism, such as propionic acidemia. Although liver transplantation alleviates hyperammonemia, high concentrations of propionate, 3HP, and methylcitrate persist in body fluids. We hypothesized that conserved metabolic perturbations occurring in transplanted patients result from the simultaneous presence of propionate and 3HP in body fluids. We investigated the inter-relations of propionate and 3HP metabolism in perfused livers from normal rats using metabolomic and stable isotopic technologies. In the presence of propionate, 3HP, or both, we observed the following metabolic perturbations. First, the citric acid cycle (CAC) is overloaded but does not provide sufficient reducing equivalents to the respiratory chain to maintain the homeostasis of adenine nucleotides. Second, there is major CoA trapping in the propionyl-CoA pathway and a tripling of liver total CoA within 1 h. Third, liver proteolysis is stimulated. Fourth, propionate inhibits the conversion of 3HP to acetyl-CoA and its oxidation in the CAC. Fifth, some propionate and some 3HP are converted to nephrotoxic maleate by different processes. Our data have implications for the clinical management of propionic acidemia. They also emphasize the perturbations of the liver intermediary metabolism induced by supraphysiological, i.e., millimolar, concentrations of labeled propionate used to trace the intermediary metabolism, in particular, inhibition of CAC flux and major decreases in the [ATP]/[ADP] and [ATP]/[AMP] ratios.

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Martini, Wenjun Z., William C. Stanley, Hazel Huang, Christine Des Rosiers, Charles L. Hoppel, and Henri Brunengraber. "Quantitative assessment of anaplerosis from propionate in pig heart in vivo." American Journal of Physiology-Endocrinology and Metabolism 284, no. 2 (February 1, 2003): E351—E356. http://dx.doi.org/10.1152/ajpendo.00354.2002.

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Normal cardiac metabolism requires continuous replenishment (anaplerosis) of catalytic intermediates of the citric acid cycle. Little is known about the quantitative aspects of propionate as a substrate of in vivo anaplerosis; therefore, we measured the rate of propionate entry into the citric acid cycle in hearts of anesthetized pigs. [U-13C3]propionate (0.25 mM) was infused in a coronary artery branch for 1 h via an extracorporeal perfusion circuit, and cardiac biopsies were analyzed for the mass isotopomer distribution of citric acid cycle intermediates. Infusion of propionate did not affect myocardial oxygen consumption, heart rate, or contractile function. In the infused territory, propionate infusion did not affect uptake of glucose and lactate but decreased free fatty acid uptake by one-half ( P < 0.05). Propionate extraction and uptake were 57.4 ± 3.3% and 0.078 ± 0.009 μmol · min−1 · g−1. Anaplerosis from propionate, calculated from the mass isotopomer distribution of succinate, accounted for 8.9 ± 1.3% of the citric acid cycle flux. Propioylcarnitine release accounted for only 0.033 ± 0.002% of propionate uptake. Methylcitrate did not accumulate. Thus administration of a low concentration of propionate appears to be a convenient and safe way to boost anaplerosis in the heart.

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Tsoukalas, Dimitris, Vassileios Fragoulakis, Evangelos Papakonstantinou, Maria Antonaki, Athanassios Vozikis, Aristidis Tsatsakis, Ana Maria Buga, Mihaela Mitroi, and Daniela Calina. "Prediction of Autoimmune Diseases by Targeted Metabolomic Assay of Urinary Organic Acids." Metabolites 10, no. 12 (December 8, 2020): 502. http://dx.doi.org/10.3390/metabo10120502.

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Autoimmune diseases (ADs) are chronic disorders characterized by the loss of self-tolerance, and although being heterogeneous, they share common pathogenic mechanisms. Self-antigens and inflammation markers are established diagnostic tools; however, the metabolic imbalances that underlie ADs are poorly described. The study aimed to employ metabolomics for the detection of disease-related changes in autoimmune diseases that could have predictive value. Quantitative analysis of 28 urine organic acids was performed using Gas Chromatography-Mass Spectrometry in a group of 392 participants. Autoimmune thyroiditis, inflammatory bowel disease, psoriasis and rheumatoid arthritis were the most prevalent autoimmune diseases of the study. Statistically significant differences were observed in the tricarboxylate cycle metabolites, succinate, methylcitrate and malate, the pyroglutamate and 2-hydroxybutyrate from the glutathione cycle and the metabolites methylmalonate, 4-hydroxyphenylpyruvate, 2-hydroxyglutarate and 2-hydroxyisobutyrate between the AD group and the control. Artificial neural networks and Binary logistic regression resulted in the highest predictive accuracy scores (66.7% and 74.9%, respectively), while Methylmalonate, 2-Hydroxyglutarate and 2-hydroxybutyrate were proposed as potential biomarkers for autoimmune diseases. Urine organic acid levels related to the mechanisms of energy production and detoxification were associated with the presence of autoimmune diseases and could be an adjunct tool for early diagnosis and prediction.

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Palacios, Sergio, and Jorge C. Escalante-Semerena. "2-Methylcitrate-dependent activation of the propionate catabolic operon (prpBCDE) of Salmonella enterica by the PrpR protein." Microbiology 150, no. 11 (November 1, 2004): 3877–87. http://dx.doi.org/10.1099/mic.0.27299-0.

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The function of the PrpR protein of Salmonella enterica serovar Typhimurium LT2 was studied in vitro and in vivo. The PrpR protein is a sensor of 2-methylcitrate (2-MC), an intermediate of the 2-methylcitric acid cycle used by this bacterium to convert propionate to pyruvate. PrpR was unresponsive to citrate (a close structural analogue of 2-MC) and to propionate, suggesting that 2-MC, not propionate, is the metabolite that signals the presence of propionate in the environment to S. enterica. prpR alleles encoding mutant proteins with various levels of 2-MC-independent activity were isolated. All lesions causing constitutive PrpR activity were mapped to the N-terminal domain of the protein. Removal of the entire sensing domain resulted in a protein (PrpRc) with the highest 2-MC-independent activity. Residue A162 is critical to 2-MC sensing, since the mutant PrpR protein PrpRA162T was as active as the PrpRc protein in the absence of 2-MC. DNA footprinting studies identified the site in the region between prpR and the prpBCDE operon to which the PrpR protein binds. Analysis of the binding-site sequence revealed two sites with dyad symmetry. Results from DNase I footprinting assays suggested that the PrpR protein may have higher affinity for the site proximal to the PprpBCDE promoter.

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Dubey, Mukesh K., Anders Broberg, Dan Funck Jensen, and Magnus Karlsson. "Role of the methylcitrate cycle in growth, antagonism and induction of systemic defence responses in the fungal biocontrol agent Trichoderma atroviride." Microbiology 159, Pt_12 (December 1, 2013): 2492–500. http://dx.doi.org/10.1099/mic.0.070466-0.

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Plassmeier, Jens, Aiko Barsch, Marcus Persicke, Karsten Niehaus, and Jörn Kalinowski. "Investigation of central carbon metabolism and the 2-methylcitrate cycle in Corynebacterium glutamicum by metabolic profiling using gas chromatography–mass spectrometry." Journal of Biotechnology 130, no. 4 (July 2007): 354–63. http://dx.doi.org/10.1016/j.jbiotec.2007.04.026.

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Mikosa, Malgorzata, Marta Sochacka-Pietal, Jadwiga Baj, and Dariusz Bartosik. "Identification of a transposable genomic island of Paracoccus pantotrophus DSM 11072 by its transposition to a novel entrapment vector pMMB2." Microbiology 152, no. 4 (April 1, 2006): 1063–73. http://dx.doi.org/10.1099/mic.0.28603-0.

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A novel shuttle entrapment vector, pMMB2, was used to identify a large transposable element, TnPpa1 (44·3 kb), of Paracoccus pantotrophus DSM 11072. TnPpa1 has a composite structure with divergently oriented copies of a cryptic transposon, Tn3434 (Tn3 family), located at both termini. The core region of the element contains a large set of putative genes, whose products show similarity to enzymes involved in central intermediary metabolism (e.g. tricarboxylic acid cycle or 2-methylcitrate cycle), transporters, transcriptional regulators and conserved proteins of unknown function. A 4·2 kb DNA segment of TnPpa1 is homologous to a region of chromosome cII of Rhodobacter sphaeroides 2.4.1, which exemplifies the mosaic structure of this element. TnPpa1 is bordered by 5 bp long directly repeated sequences and is located within a mega-sized replicon, pWKS5, in the DSM 11072 genome. Spontaneous inversion of the core region of TnPpa1 was detected in the host genome. Analysis of the distribution of TnPpa1 in three other strains of P. pantotrophus revealed that this element was present exclusively within DSM 11072, which suggests its relatively recent acquisition by lateral transfer. The identification of TnPpa1 (which may be considered a transposable genomic island) provides evidence for the transposition and lateral transfer of large DNA segments of chromosomal origin (carrying various housekeeping genes), which may have a great impact on the evolution of bacterial genomes.

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Caterino, Marianna, Margherita Ruoppolo, Guglielmo Rosario Domenico Villani, Emanuela Marchese, Michele Costanzo, Giovanni Sotgiu, Simone Dore, Flavia Franconi, and Ilaria Campesi. "Influence of Sex on Urinary Organic Acids: A Cross-Sectional Study in Children." International Journal of Molecular Sciences 21, no. 2 (January 16, 2020): 582. http://dx.doi.org/10.3390/ijms21020582.

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The characterization of urinary metabolome, which provides a fingerprint for each individual, is an important step to reach personalized medicine. It is influenced by exogenous and endogenous factors; among them, we investigated sex influences on 72 organic acids measured through GC-MS analysis in the urine of 291 children (152 males; 139 females) aging 1–36 months and stratified in four groups of age. Among the 72 urinary metabolites, in all age groups, 4-hydroxy-butirate and homogentisate are found only in males, whereas 3-hydroxy-dodecanoate, methylcitrate, and phenylacetate are found only in females. Sex differences are still present after age stratification being more numerous during the first 6 months of life. The most relevant sex differences involve the mitochondria homeostasis. In females, citrate cycle, glyoxylate and dicarboxylate metabolism, alanine, aspartate, glutamate, and butanoate metabolism had the highest impact. In males, urinary organic acids were involved in phenylalanine metabolism, citrate cycle, alanine, aspartate and glutamate metabolism, butanoate metabolism, and glyoxylate and dicarboxylate metabolism. In addition, age specifically affected metabolic pathways, the phenylalanine metabolism pathway being affected by age only in males. Relevantly, the age-influenced ranking of metabolic pathways varied in the two sexes. In conclusion, sex deeply influences both quantitatively and qualitatively urinary organic acids levels, the effect of sex being age dependent. Importantly, the sex effects depend on the single organic acid; thus, in some cases the urinary organic acid reference values should be stratified according the sex and age.

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Murray, Sandra L., and Michael J. Hynes. "Metabolic and Developmental Effects Resulting from Deletion of the citA Gene Encoding Citrate Synthase in Aspergillus nidulans." Eukaryotic Cell 9, no. 4 (February 19, 2010): 656–66. http://dx.doi.org/10.1128/ec.00373-09.

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ABSTRACT Citrate synthase is a central activity in carbon metabolism. It is required for the tricarboxylic acid (TCA) cycle, respiration, and the glyoxylate cycle. In Saccharomyces cerevisiae and Arabidopsis thaliana, there are mitochondrial and peroxisomal isoforms encoded by separate genes, while in Aspergillus nidulans, a single gene, citA, encodes a protein with predicted mitochondrial and peroxisomal targeting sequences (PTS). Deletion of citA results in poor growth on glucose but not on derepressing carbon sources, including those requiring the glyoxylate cycle. Growth on glucose is restored by a mutation in the creA carbon catabolite repressor gene. Methylcitrate synthase, required for propionyl-coenzyme A (CoA) metabolism, has previously been shown to have citrate synthase activity. We have been unable to construct the mcsAΔ citAΔ double mutant, and the expression of mcsA is subject to CreA-mediated carbon repression. Therefore, McsA can substitute for the loss of CitA activity. Deletion of citA does not affect conidiation or sexual development but results in delayed conidial germination as well as a complete loss of ascospores in fruiting bodies, which can be attributed to loss of meiosis. These defects are suppressed by the creA204 mutation, indicating that McsA activity can substitute for the loss of CitA. A mutation of the putative PTS1-encoding sequence in citA had no effect on carbon source utilization or development but did result in slower colony extension arising from single conidia or ascospores. CitA-green fluorescent protein (GFP) studies showed mitochondrial localization in conidia, ascospores, and hyphae. Peroxisomal localization was not detected. However, a very low and variable detection of punctate GFP fluorescence was sometimes observed in conidia germinated for 5 h when the mitochondrial targeting sequence was deleted.

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Tang, Su, Nathan D. Hicks, Yu-Shan Cheng, Andres Silva, Sarah M. Fortune, and James C. Sacchettini. "Structural and functional insight into the Mycobacterium tuberculosis protein PrpR reveals a novel type of transcription factor." Nucleic Acids Research 47, no. 18 (August 26, 2019): 9934–49. http://dx.doi.org/10.1093/nar/gkz724.

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Abstract The pathogenicity of Mycobacterium tuberculosis depends upon its ability to catabolize host cholesterol. Upregulation of the methylcitrate cycle (MCC) is required to assimilate and detoxify propionyl-CoA, a cholesterol degradation product. The transcription of key genes prpC and prpD in MCC is activated by MtPrpR, a member of a family of prokaryotic transcription factors whose structures and modes of action have not been clearly defined. We show that MtPrpR has a novel overall structure and directly binds to CoA or short-chain acyl-CoA derivatives to form a homotetramer that covers the binding cavity and locks CoA tightly inside the protein. The regulation of this process involves a [4Fe4S] cluster located close to the CoA-binding cavity on a neighboring chain. Mutations in the [4Fe4S] cluster binding residues rendered MtPrpR incapable of regulating MCC gene transcription. The structure of MtPrpR without the [4Fe4S] cluster-binding region shows a conformational change that prohibits CoA binding. The stability of this cluster means it is unlikely a redox sensor but may function by sensing ambient iron levels. These results provide mechanistic insights into this family of critical transcription factors who share similar structures and regulate gene transcription using a combination of acyl-CoAs and [4Fe4S] cluster.

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Cabecas Segura, Paloma, Quentin De Meur, Audrey Tanghe, Rob Onderwater, Laurent Dewasme, Ruddy Wattiez, and Baptiste Leroy. "Effects of Mixing Volatile Fatty Acids as Carbon Sources on Rhodospirillum rubrum Carbon Metabolism and Redox Balance Mechanisms." Microorganisms 9, no. 9 (September 21, 2021): 1996. http://dx.doi.org/10.3390/microorganisms9091996.

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Rhodospirillum rubrum has a versatile metabolism, and as such can assimilate a broad range of carbon sources, including volatile fatty acids. These carbon sources are gaining increasing interest for biotechnological processes, since they reduce the production costs for numerous value-added compounds and contribute to the development of a more circular economy. Usually, studies characterizing carbon metabolism are performed by supplying a single carbon source; however, in both environmental and engineered conditions, cells would rather grow on mixtures of volatile fatty acids (VFAs) generated via anaerobic fermentation. In this study, we show that the use of a mixture of VFAs as carbon source appears to have a synergy effect on growth phenotype. In addition, while propionate and butyrate assimilation in Rs. rubrum is known to require an excess of bicarbonate in the culture medium, mixing them reduces the requirement for bicarbonate supplementation. The fixation of CO2 is one of the main electron sinks in purple bacteria; therefore, this observation suggests an adaptation of both metabolic pathways used for the assimilation of these VFAs and redox homeostasis mechanism. Based on proteomic data, modification of the propionate assimilation pathway seems to occur with a switch from a methylmalonyl-CoA intermediate to the methylcitrate cycle. Moreover, it seems that the presence of a mixture of VFAs switches electron sinking from CO2 fixation to H2 and isoleucine production.

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de Meeûs d’Argenteuil, Constance, Berit Boshuizen, Maarten Oosterlinck, Don van de Winkel, Ward De Spiegelaere, Cornelis Marinus de Bruijn, Klara Goethals, Katrien Vanderperren, and Cathérine John Ghislaine Delesalle. "Flexibility of equine bioenergetics and muscle plasticity in response to different types of training: An integrative approach, questioning existing paradigms." PLOS ONE 16, no. 4 (April 13, 2021): e0249922. http://dx.doi.org/10.1371/journal.pone.0249922.

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Equine bioenergetics have predominantly been studied focusing on glycogen and fatty acids. Combining omics with conventional techniques allows for an integrative approach to broadly explore and identify important biomolecules. Friesian horses were aquatrained (n = 5) or dry treadmill trained (n = 7) (8 weeks) and monitored for: evolution of muscle diameter in response to aquatraining and dry treadmill training, fiber type composition and fiber cross-sectional area of the M. pectoralis, M. vastus lateralis and M. semitendinosus and untargeted metabolomics of the M. pectoralis and M. vastus lateralis in response to dry treadmill training. Aquatraining was superior to dry treadmill training to increase muscle diameter in the hindquarters, with maximum effect after 4 weeks. After dry treadmill training, the M. pectoralis showed increased muscle diameter, more type I fibers, decreased fiber mean cross sectional area, and an upregulated oxidative metabolic profile: increased β-oxidation (key metabolites: decreased long chain fatty acids and increased long chain acylcarnitines), TCA activity (intermediates including succinyl-carnitine and 2-methylcitrate), amino acid metabolism (glutamine, aromatic amino acids, serine, urea cycle metabolites such as proline, arginine and ornithine) and xenobiotic metabolism (especially p-cresol glucuronide). The M. vastus lateralis expanded its fast twitch profile, with decreased muscle diameter, type I fibers and an upregulation of glycolytic and pentose phosphate pathway activity, and increased branched-chain and aromatic amino acid metabolism (cis-urocanate, carnosine, homocarnosine, tyrosine, tryptophan, p-cresol-glucuronide, serine, methionine, cysteine, proline and ornithine). Trained Friesians showed increased collagen and elastin turn-over. Results show that branched-chain amino acids, aromatic amino acids and microbiome-derived xenobiotics need further study in horses. They feed the TCA cycle at steps further downstream from acetyl CoA and most likely, they are oxidized in type IIA fibers, the predominant fiber type of the horse. These study results underline the importance of reviewing existing paradigms on equine bioenergetics.

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dos Santos, Ana Cristina Doria, Victor Hugo de Souza Marinho, Pedro Henrique de Aviz Silva, Barbarella de Matos Macchi, Mara Silvia Pinheiro Arruda, Edilene Oliveira da Silva, José Luiz Martins do Nascimento, and Chubert Bernardo Castro de Sena. "Microenvironment ofMycobacterium smegmatisCulture to Induce Cholesterol Consumption Does Cell Wall Remodeling and Enables the Formation of Granuloma-Like Structures." BioMed Research International 2019 (April 15, 2019): 1–13. http://dx.doi.org/10.1155/2019/1871239.

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Pathogenic species of mycobacteria are known to use the host cholesterol during lung infection as an alternative source of carbon and energy. Mycobacteria culture in minimal medium (MM) has been used as anin vitroexperimental model to study the consumption of exogenous cholesterol. Once in MM, different species of mycobacteria start to consume the cholesterol and initiate transcriptional and metabolic adaptations, upregulating the enzymes of the methylcitrate cycle (MCC) and accumulating a variety of primary metabolites that are known to be important substrates for cell wall biosynthesis. We hypothesized that stressful pressure of cultures in MM is able to induce critical adaptation for the bacteria which win the infection. To identify important modifications in the biosynthesis of the cell wall, we cultured the fast-growing and nonpathogenicMycobacterium smegmatisin MM supplemented with or without glycerol and/or cholesterol. Different from the culture in complete medium Middlebrook 7H9 broth, the bacteria when cultured in MM decreased growth and changed in the accumulation of cell wall molecules. However, the supplementation of MM with glycerol and/or cholesterol recovered the accumulation of phosphatidylinositol mannosides (PIMs) and other phospholipids but maintained growth deceleration. The biosynthesis of lipomannan (LM) and of lipoarabinomannan (LAM) was significantly modulated after culture in MM, independently of glycerol and/or cholesterol supplementation, where LM size was decreased (LM13-25KDa) and LAM increased (LAM37-100KDa), when compared these molecules after bacteria culture in complete medium (LM17-25KDaandLAM37-50KDa). These changes modified the cell surface hydrophobicity and susceptibility against H2O2. The infection of J774 macrophages withM. smegmatis,after culture in MM, induced the formation of granuloma-like structures, while supplementation with cholesterol induced the highest rate of formation of these structures. Taken together, our results identify critical changes in mycobacterial cell wall molecules after culture in MM that induces cholesterol accumulation, helping the mycobacteria to increase their capacity to form granuloma-like structures.

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Denburg, Michelle R., Yunwen Xu, Alison G. Abraham, Josef Coresh, Jingsha Chen, Morgan E. Grams, Harold I. Feldman, et al. "Metabolite Biomarkers of CKD Progression in Children." Clinical Journal of the American Society of Nephrology 16, no. 8 (August 2021): 1178–89. http://dx.doi.org/10.2215/cjn.00220121.

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Background and objectivesMetabolomics facilitates the discovery of biomarkers and potential therapeutic targets for CKD progression.Design, setting, participants, & measurementsWe evaluated an untargeted metabolomics quantification of stored plasma samples from 645 Chronic Kidney Disease in Children (CKiD) participants. Metabolites were standardized and logarithmically transformed. Cox proportional hazards regression examined the association between 825 nondrug metabolites and progression to the composite outcome of KRT or 50% reduction of eGFR, adjusting for age, sex, race, body mass index, hypertension, glomerular versus nonglomerular diagnosis, proteinuria, and baseline eGFR. Stratified analyses were performed within subgroups of glomerular/nonglomerular diagnosis and baseline eGFR.ResultsBaseline characteristics were 391 (61%) male; median age 12 years; median eGFR 54 ml/min per 1.73 m2; 448 (69%) nonglomerular diagnosis. Over a median follow-up of 4.8 years, 209 (32%) participants developed the composite outcome. Unique association signals were identified in subgroups of baseline eGFR. Among participants with baseline eGFR ≥60 ml/min per 1.73 m2, two-fold higher levels of seven metabolites were significantly associated with higher hazards of KRT/halving of eGFR events: three involved in purine and pyrimidine metabolism (N6-carbamoylthreonyladenosine, hazard ratio, 16; 95% confidence interval, 4 to 60; 5,6-dihydrouridine, hazard ratio, 17; 95% confidence interval, 5 to 55; pseudouridine, hazard ratio, 39; 95% confidence interval, 8 to 200); two amino acids, C-glycosyltryptophan, hazard ratio, 24; 95% confidence interval 6 to 95 and lanthionine, hazard ratio, 3; 95% confidence interval, 2 to 5; the tricarboxylic acid cycle intermediate 2-methylcitrate/homocitrate, hazard ratio, 4; 95% confidence interval, 2 to 7; and gulonate, hazard ratio, 10; 95% confidence interval, 3 to 29. Among those with baseline eGFR <60 ml/min per 1.73 m2, a higher level of tetrahydrocortisol sulfate was associated with lower risk of progression (hazard ratio, 0.8; 95% confidence interval, 0.7 to 0.9).ConclusionsUntargeted plasma metabolomic profiling facilitated discovery of novel metabolite associations with CKD progression in children that were independent of established clinical predictors and highlight the role of select biologic pathways.

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Carter, Michael S., and Birgit E. Alber. "Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides." Journal of Bacteriology 197, no. 19 (July 13, 2015): 3048–56. http://dx.doi.org/10.1128/jb.00402-15.

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ABSTRACTPropionyl coenzyme A (propionyl-CoA) assimilation byRhodobacter sphaeroidesproceeds via the methylmalonyl-CoA pathway. The activity of the key enzyme of the pathway, propionyl-CoA carboxylase (PCC), was upregulated 20-fold during growth with propionate compared to growth with succinate. Because propionyl-CoA is an intermediate in acetyl-CoA assimilation via the ethylmalonyl-CoA pathway, acetate growth also requires the methylmalonyl-CoA pathway. PCC activities were upregulated 8-fold in extracts of acetate-grown cells compared to extracts of succinate-grown cells. The upregulation of PCC activities during growth with propionate or acetate corresponded to increased expression of thepccBgene, which encodes a subunit of PCC. PccR (RSP_2186) was identified to be a transcriptional regulator required for the upregulation ofpccBtranscript levels and, consequently, PCC activity: growth substrate-dependent regulation was lost whenpccRwas inactivated by an in-frame deletion. In thepccRmutant,lacZexpression from a 215-bp plasmid-bornepccBupstream fragment including 27 bp of thepccBcoding region was also deregulated. A loss of regulation as a result of mutations in the conserved motifs TTTGCAAA-X4-TTTGCAAA in the presence of PccR allowed the prediction of a possible operator site. PccR, together with homologs from other organisms, formed a distinct clade within the family ofshort-chainfatty acyl coenzyme Aregulators (ScfRs) defined here. Some members from other clades within the ScfR family have previously been shown to be involved in regulating acetyl-CoA assimilation by the glyoxylate bypass (RamB) or propionyl-CoA assimilation by the methylcitrate cycle (MccR).IMPORTANCEShort-chain acyl-CoAs are intermediates in essential biosynthetic and degradative pathways. The regulation of their accumulation is crucial for appropriate cellular function. This work identifies a regulator (PccR) that prevents the accumulation of propionyl-CoA by controlling expression of the gene encoding propionyl-CoA carboxylase, which is responsible for propionyl-CoA consumption byRhodobacter sphaeroides. Many otherProteobacteriaandActinomycetalescontain one or several PccR homologs that group into distinct clades on the basis of the pathway of acyl-CoA metabolism that they control. Furthermore, an upstream analysis of genes encoding PccR homologs allows the prediction of conserved binding motifs for these regulators. Overall, this study evaluates a single regulator of propionyl-CoA assimilation while expanding the knowledge of the regulation of short-chain acyl-CoAs in many bacterial species.

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Gould, Ty A., Helmus van de Langemheen, Ernesto J. Munoz-Elias, John D. McKinney, and James C. Sacchettini. "Dual role of isocitrate lyase 1 in the glyoxylate and methylcitrate cycles in Mycobacterium tuberculosis." Molecular Microbiology 61, no. 4 (August 2006): 940–47. http://dx.doi.org/10.1111/j.1365-2958.2006.05297.x.

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Freitas e Silva, Kleber Santiago, Raisa Melo Lima, Patrícia de Sousa Lima, Lilian Cristiane Baeza, Roosevelt Alves da Silva, Célia Maria de Almeida Soares, and Maristela Pereira. "Interaction of Isocitrate Lyase with Proteins Involved in the Energetic Metabolism in Paracoccidioides lutzii." Journal of Fungi 6, no. 4 (November 23, 2020): 309. http://dx.doi.org/10.3390/jof6040309.

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Background: Systemic mycosis is a cause of death of immunocompromised subjects. The treatment directed to evade fungal pathogens shows severe limitations, such as time of drug exposure and side effects. The paracoccidioidomycosis (PCM) treatment depends on the severity of the infection and may last from months to years. Methods: To analyze the main interactions of Paracoccidioides lutzii isocitrate lyase (ICL) regarding the energetic metabolism through affinity chromatography, we performed blue native PAGE and co-immunoprecipitation to identify ICL interactions. We also performed in silico analysis by homology, docking, hot-spot prediction and contact preference analysis to identify the conformation of ICL complexes. Results: ICL interacted with 18 proteins in mycelium, 19 in mycelium-to-yeast transition, and 70 in yeast cells. Thirty complexes were predicted through docking and contact preference analysis. ICL has seven main regions of interaction with protein partners. Conclusions: ICL seems to interfere with energetic metabolism of P. lutzii, regulating aerobic and anaerobic metabolism as it interacts with proteins from glycolysis, gluconeogenesis, TCA and methylcitrate cycles, mainly through seven hot-spot residues.

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Liu, Wei-Bing, Xin-Xin Liu, Meng-Jia Shen, Guo-Lan She, and Bang-Ce Ye. "The Nitrogen Regulator GlnR Directly Controls Transcription of theprpDBCOperon Involved in Methylcitrate Cycle inMycobacterium smegmatis." Journal of Bacteriology 201, no. 8 (February 11, 2019). http://dx.doi.org/10.1128/jb.00099-19.

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ABSTRACTMycobacterium tuberculosisutilizes fatty acids of the host as the carbon source. Metabolism of odd-chain fatty acids byMycobacterium tuberculosisproduces propionyl coenzyme A (propionyl-CoA). The methylcitrate cycle is essential for mycobacteria to utilize the propionyl-CoA to persist and grow on these fatty acids. InM. smegmatis, methylcitrate synthase, methylcitrate dehydratase, and methylisocitrate lyase involved in the methylcitrate cycle are encoded byprpC,prpD,and prpB, respectively, in operonprpDBC. In this study, we found that the nitrogen regulator GlnR directly binds to the promoter region of theprpDBCoperon and inhibits its transcription. The binding motif of GlnR was identified by bioinformatic analysis and validated using DNase I footprinting and electrophoretic mobility shift assays. The GlnR-binding motif is separated by a 164-bp sequence from the binding site of PrpR, a pathway-specific transcriptional activator of methylcitrate cycle, but the binding affinity of GlnR toprpDBCis much stronger than that of PrpR. Deletion ofglnRresulted in faster growth in propionate or cholesterol medium compared with the wild-type strain. The ΔglnRmutant strain also showed a higher survival rate in macrophages. These results illustrated that the nitrogen regulator GlnR regulates the methylcitrate cycle through direct repression of the transcription of theprpDBCoperon. This finding not only suggests an unprecedented link between nitrogen metabolism and the methylcitrate pathway but also reveals a potential target for controlling the growth of pathogenic mycobacteria.IMPORTANCEThe success of mycobacteria survival in macrophage depends on its ability to assimilate fatty acids and cholesterol from the host. The cholesterol and fatty acids are catabolized via β-oxidation to generate propionyl coenzyme A (propionyl-CoA), which is then primarily metabolized via the methylcitrate cycle. Here, we found a typical GlnR binding box in theprpoperon, and the affinity is much stronger than that of PrpR, a transcriptional activator of methylcitrate cycle. Furthermore, GlnR repressed the transcription of theprpoperon. Deletion ofglnRsignificantly enhanced the growth ofMycobacterium tuberculosisin propionate or cholesterol medium, as well as viability in macrophages. These findings provide new insights into the regulatory mechanisms underlying the cross talk of nitrogen and carbon metabolisms in mycobacteria.

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Pei, Jin-Feng, Nan Qi, Yu-Xin Li, Jing Wo, and Bang-Ce Ye. "RegX3-Mediated Regulation of Methylcitrate Cycle in Mycobacterium smegmatis." Frontiers in Microbiology 12 (February 2, 2021). http://dx.doi.org/10.3389/fmicb.2021.619387.

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Mycobacterium tuberculosis is a global human pathogen that infects macrophages and can establish a latent infection. Emerging evidence has established the nutrients metabolism as a key point to study the pathogenesis of M. tuberculosis and host immunity. It was reported that fatty acids and cholesterol are the major nutrient sources of M. tuberculosis in the period of infection. However, the mechanism by which M. tuberculosis utilizes lipids for maintaining life activities in nutrient-deficiency macrophages is poorly understood. Mycobacterium smegmatis is fast-growing and generally used to study its pathogenic counterpart, M. tuberculosis. In this work, we found that the phosphate sensing regulator RegX3 of M. smegmatis is required for its growing on propionate and surviving in macrophages. We further demonstrated that the expression of prpR and related genes (prpDBC) in methylcitrate cycle could be enhanced by RegX3 in response to the phosphate-starvation condition. The binding sites of the promoter region of prpR for RegX3 and PrpR were investigated. In addition, cell morphology assay showed that RegX3 is responsible for cell morphological elongation, thus promoting the proliferation and survival of M. smegmatis in macrophages. Taken together, our findings revealed a novel transcriptional regulation mechanism of RegX3 on propionate metabolism, and uncovered that the nutrients-sensing regulatory system puts bacteria at metabolic steady state by altering cell morphology. More importantly, since we observed that M. tuberculosis RegX3 also binds to the prpR operon in vitro, the RegX3-mediated regulation might be general in M. tuberculosis and other mycobacteria for nutrient sensing and environmental adaptation.

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Bhusal, Ram Prasad, Wanting Jiao, Brooke X. C. Kwai, Jóhannes Reynisson, Annabelle J. Collins, Jonathan Sperry, Ghader Bashiri, and Ivanhoe K. H. Leung. "Acetyl-CoA-mediated activation of Mycobacterium tuberculosis isocitrate lyase 2." Nature Communications 10, no. 1 (October 11, 2019). http://dx.doi.org/10.1038/s41467-019-12614-7.

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Abstract Isocitrate lyase is important for lipid utilisation by Mycobacterium tuberculosis but its ICL2 isoform is poorly understood. Here we report that binding of the lipid metabolites acetyl-CoA or propionyl-CoA to ICL2 induces a striking structural rearrangement, substantially increasing isocitrate lyase and methylisocitrate lyase activities. Thus, ICL2 plays a pivotal role regulating carbon flux between the tricarboxylic acid (TCA) cycle, glyoxylate shunt and methylcitrate cycle at high lipid concentrations, a mechanism essential for bacterial growth and virulence.

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Qi, Nan, Guo-Lan She, Wei Du, and Bang-Ce Ye. "Mycobacterium smegmatis GlnR Regulates the Glyoxylate Cycle and the Methylcitrate Cycle on Fatty Acid Metabolism by Repressing icl Transcription." Frontiers in Microbiology 12 (February 3, 2021). http://dx.doi.org/10.3389/fmicb.2021.603835.

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Mycobacterium smegmatis (Msm), along with its pathogenic counterpart Mycobacterium tuberculosis (Mtb), utilizes fatty acids and cholesterol as important carbon and energy sources during the persistence within host cells. As a dual-functional enzyme in the glyoxylate cycle and the methylcitrate cycle, isocitrate lyase (ICL, encoded by icl or MSMEG_0911) is indispensable for the growth of Msm and Mtb on short-chain fatty acids. However, regulation of icl in mycobacteria in response to nutrient availability remains largely unknown. Here, we report that the global nitrogen metabolism regulator GlnR represses icl expression by binding to an atypical binding motif in the icl promoter region under nitrogen-limiting conditions. We further show that GlnR competes with PrpR, a transcriptional activator of icl, and dominantly occupies the co-binding motif in the icl promoter region. In the absence of GlnR or in response to the excess nitrogen condition, Msm cells elongate and exhibit robust growth on short-chain fatty acids due to the PrpR-mediated activation of icl, thereby inducing enhanced apoptosis in infected macrophages. Taken together, our findings reveal the GlnR-mediated repression of icl on fatty acid metabolism, which might be a general strategy of nutrient sensing and environmental adaptation employed by mycobacteria.

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Mackenzie, Jared S., Dirk A. Lamprecht, Rukaya Asmal, John H. Adamson, Khushboo Borah, Dany J. V. Beste, Bei Shi Lee, et al. "Bedaquiline reprograms central metabolism to reveal glycolytic vulnerability in Mycobacterium tuberculosis." Nature Communications 11, no. 1 (November 30, 2020). http://dx.doi.org/10.1038/s41467-020-19959-4.

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AbstractThe approval of bedaquiline (BDQ) for the treatment of tuberculosis has generated substantial interest in inhibiting energy metabolism as a therapeutic paradigm. However, it is not known precisely how BDQ triggers cell death in Mycobacterium tuberculosis (Mtb). Using 13C isotopomer analysis, we show that BDQ-treated Mtb redirects central carbon metabolism to induce a metabolically vulnerable state susceptible to genetic disruption of glycolysis and gluconeogenesis. Metabolic flux profiles indicate that BDQ-treated Mtb is dependent on glycolysis for ATP production, operates a bifurcated TCA cycle by increasing flux through the glyoxylate shunt, and requires enzymes of the anaplerotic node and methylcitrate cycle. Targeting oxidative phosphorylation (OXPHOS) with BDQ and simultaneously inhibiting substrate level phosphorylation via genetic disruption of glycolysis leads to rapid sterilization. Our findings provide insight into the metabolic mechanism of BDQ-induced cell death and establish a paradigm for the development of combination therapies that target OXPHOS and glycolysis.

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Panyushkina, Anna E., Vladislav V. Babenko, Anastasia S. Nikitina, Oksana V. Selezneva, Iraida A. Tsaplina, Maria A. Letarova, Elena S. Kostryukova, and Andrey V. Letarov. "Sulfobacillus thermotolerans: new insights into resistance and metabolic capacities of acidophilic chemolithotrophs." Scientific Reports 9, no. 1 (October 21, 2019). http://dx.doi.org/10.1038/s41598-019-51486-1.

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Abstract The first complete genome of the biotechnologically important species Sulfobacillus thermotolerans has been sequenced. Its 3 317 203-bp chromosome contains an 83 269-bp plasmid-like region, which carries heavy metal resistance determinants and the rusticyanin gene. Plasmid-mediated metal resistance is unusual for acidophilic chemolithotrophs. Moreover, most of their plasmids are cryptic and do not contribute to the phenotype of the host cells. A polyphosphate-based mechanism of metal resistance, which has been previously unknown in the genus Sulfobacillus or other Gram-positive chemolithotrophs, potentially operates in two Sulfobacillus species. The methylcitrate cycle typical for pathogens and identified in the genus Sulfobacillus for the first time can fulfill the energy and/or protective function in S. thermotolerans Kr1 and two other Sulfobacillus species, which have incomplete glyoxylate cycles. It is notable that the TCA cycle, disrupted in all Sulfobacillus isolates under optimal growth conditions, proved to be complete in the cells enduring temperature stress. An efficient antioxidant defense system gives S. thermotolerans another competitive advantage in the microbial communities inhabiting acidic metal-rich environments. The genomic comparisons revealed 80 unique genes in the strain Kr1, including those involved in lactose/galactose catabolism. The results provide new insights into metabolism and resistance mechanisms in the Sulfobacillus genus and other acidophiles.

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Luo, Tao, Peng Xu, Yangyi Zhang, Jessica L. Porter, Marwan Ghanem, Qingyun Liu, Yuan Jiang, et al. "Population genomics provides insights into the evolution and adaptation to humans of the waterborne pathogen Mycobacterium kansasii." Nature Communications 12, no. 1 (May 3, 2021). http://dx.doi.org/10.1038/s41467-021-22760-6.

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AbstractMycobacterium kansasii can cause serious pulmonary disease. It belongs to a group of closely-related species of non-tuberculous mycobacteria known as the M. kansasii complex (MKC). Here, we report a population genomics analysis of 358 MKC isolates from worldwide water and clinical sources. We find that recombination, likely mediated by distributive conjugative transfer, has contributed to speciation and on-going diversification of the MKC. Our analyses support municipal water as a main source of MKC infections. Furthermore, nearly 80% of the MKC infections are due to closely-related M. kansasii strains, forming a main cluster that apparently originated in the 1900s and subsequently expanded globally. Bioinformatic analyses indicate that several genes involved in metabolism (e.g., maintenance of the methylcitrate cycle), ESX-I secretion, metal ion homeostasis and cell surface remodelling may have contributed to M. kansasii’s success and its ongoing adaptation to the human host.

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Zhang, Yang, Xiuling Zhou, Xuemei Wang, Lu Wang, Menglei Xia, Jianmei Luo, Yanbing Shen, and Min Wang. "Improving phytosterol biotransformation at low nitrogen levels by enhancing the methylcitrate cycle with transcriptional regulators PrpR and GlnR of Mycobacterium neoaurum." Microbial Cell Factories 19, no. 1 (January 28, 2020). http://dx.doi.org/10.1186/s12934-020-1285-8.

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Kappelmann, Jannick, Bianca Klein, Mathias Papenfuß, Julian Lange, Bastian Blombach, Ralf Takors, Wolfgang Wiechert, Tino Polen, and Stephan Noack. "Comprehensive Analysis of C. glutamicum Anaplerotic Deletion Mutants Under Defined d-Glucose Conditions." Frontiers in Bioengineering and Biotechnology 8 (January 20, 2021). http://dx.doi.org/10.3389/fbioe.2020.602936.

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Wild-type C. glutamicum ATCC 13032 is known to possess two enzymes with anaplerotic (C4-directed) carboxylation activity, namely phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx). On the other hand, C3-directed decarboxylation can be catalyzed by the three enzymes phosphoenolpyruvate carboxykinase (PEPCk), oxaloacetate decarboxylase (ODx), and malic enzyme (ME). The resulting high metabolic flexibility at the anaplerotic node compromises the unambigous determination of its carbon and energy flux in C. glutamicum wild type. To circumvent this problem we performed a comprehensive analysis of selected single or double deletion mutants in the anaplerosis of wild-type C. glutamicum under defined d-glucose conditions. By applying well-controlled lab-scale bioreactor experiments in combination with untargeted proteomics, quantitative metabolomics and whole-genome sequencing hitherto unknown, and sometimes counter-intuitive, genotype-phenotype relationships in these mutants could be unraveled. In comparison to the wild type the four mutants C. glutamiucm Δpyc, C. glutamiucm Δpyc Δodx, C. glutamiucm Δppc Δpyc, and C. glutamiucm Δpck showed lowered specific growth rates and d-glucose uptake rates, underlining the importance of PCx and PEPCk activity for a balanced carbon and energy flux at the anaplerotic node. Most interestingly, the strain C. glutamiucm Δppc Δpyc could be evolved to grow on d-glucose as the only source of carbon and energy, whereas this combination was previously considered lethal. The prevented anaplerotic carboxylation activity of PEPCx and PCx was found in the evolved strain to be compensated by an up-regulation of the glyoxylate shunt, potentially in combination with the 2-methylcitrate cycle.

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Gifford, Scott M., Jamie W. Becker, Oscar A. Sosa, Daniel J. Repeta, and Edward F. DeLong. "Quantitative Transcriptomics Reveals the Growth- and Nutrient-Dependent Response of a Streamlined Marine Methylotroph to Methanol and Naturally Occurring Dissolved Organic Matter." mBio 7, no. 6 (November 22, 2016). http://dx.doi.org/10.1128/mbio.01279-16.

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ABSTRACT The members of the OM43 clade of Betaproteobacteria are abundant coastal methylotrophs with a range of carbon-utilizing capabilities. However, their underlying transcriptional and metabolic responses to shifting conditions or different carbon substrates remain poorly understood. We examined the transcriptional dynamics of OM43 isolate NB0046 subjected to various inorganic nutrient, vitamin, and carbon substrate regimes over different growth phases to (i) develop a quantitative model of its mRNA content; (ii) identify transcriptional markers of physiological activity, nutritional state, and carbon and energy utilization; and (iii) identify pathways involved in methanol or naturally occurring dissolved organic matter (DOM) metabolism. Quantitative transcriptomics, achieved through addition of internal RNA standards, allowed for analyses on a transcripts-per-cell scale. This streamlined bacterium exhibited substantial shifts in total mRNA content (ranging from 1,800 to 17 transcripts cell −1 in the exponential and deep stationary phases, respectively) and gene-specific transcript abundances (>1,000-fold increases in some cases), depending on the growth phase and nutrient conditions. Carbon metabolism genes exhibited substantial dynamics, including those for ribulose monophosphate, tricarboxylic acid (TCA), and proteorhodopsin, as well as methanol dehydrogenase ( xoxF ), which, while always the most abundant transcript, increased from 5 to 120 transcripts cell −1 when cultures were nutrient and vitamin amended. In the DOM treatment, upregulation of TCA cycle, methylcitrate cycle, vitamin, and organic phosphorus genes suggested a metabolic route for this complex mixture of carbon substrates. The genome-wide inventory of transcript abundances produced here provides insight into a streamlined marine bacterium’s regulation of carbon metabolism and energy flow, providing benchmarks for evaluating the activity of OM43 populations in situ . IMPORTANCE Bacteria exert a substantial influence on marine organic matter flux, yet the carbon components targeted by specific bacterial groups, as well as how those groups’ metabolic activities change under different conditions, are not well understood. Gene expression studies of model organisms can identify these responses under defined conditions, which can then be compared to environmental transcriptomes to elucidate in situ activities. This integration, however, is limited by the data’s relative nature. Here, we report the fully quantitative transcriptome of a marine bacterium, providing a genome-wide survey of cellular transcript abundances and how they change with different states of growth, nutrient conditions, and carbon substrates. The results revealed the dynamic metabolic strategies this methylotroph has for processing both simple one-carbon compounds and the complex multicarbon substrates of naturally derived marine organic matter and provide baseline quantitative data for identifying their in situ activities and impact on the marine carbon cycle.

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Journal articles on the topic 'Methylcitrate cycle'

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