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Li S, Nilsson E, Seidel L, Ketzer M, Forsman A, Dopson M, Hylander S. Baltic Sea coastal sediment-bound eukaryotes have increased year-round activities under predicted climate change related warming. Front Microbiol 2024; 15:1369102. [PMID: 38596378 PMCID: PMC11002985 DOI: 10.3389/fmicb.2024.1369102] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2024] [Accepted: 03/05/2024] [Indexed: 04/11/2024] Open
Abstract
Climate change related warming is a serious environmental problem attributed to anthropogenic activities, causing ocean water temperatures to rise in the coastal marine ecosystem since the last century. This particularly affects benthic microbial communities, which are crucial for biogeochemical cycles. While bacterial communities have received considerable scientific attention, the benthic eukaryotic community response to climate change remains relatively overlooked. In this study, sediments were sampled from a heated (average 5°C increase over the whole year for over 50 years) and a control (contemporary conditions) Baltic Sea bay during four different seasons across a year. RNA transcript counts were then used to investigate eukaryotic community changes under long-term warming. The composition of active species in the heated and control bay sediment eukaryotic communities differed, which was mainly attributed to salinity and temperature. The family level RNA transcript alpha diversity in the heated bay was higher during May but lower in November, compared with the control bay, suggesting altered seasonal activity patterns and dynamics. In addition, structures of the active eukaryotic communities varied between the two bays during the same season. Hence, this study revealed that long-term warming can change seasonality in eukaryotic diversity patterns. Relative abundances and transcript expression comparisons between bays suggested that some taxa that now have lower mRNA transcripts numbers could be favored by future warming. Furthermore, long-term warming can lead to a more active metabolism in these communities throughout the year, such as higher transcript numbers associated with diatom energy production and protein synthesis in the heated bay during winter. In all, these data can help predict how future global warming will affect the ecology and metabolism of eukaryotic community in coastal sediments.
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Affiliation(s)
- Songjun Li
- Centre for Ecology and Evolution in Microbial Model Systems, Linnaeus University, Kalmar, Sweden
| | - Emelie Nilsson
- Centre for Ecology and Evolution in Microbial Model Systems, Linnaeus University, Kalmar, Sweden
| | - Laura Seidel
- Centre for Ecology and Evolution in Microbial Model Systems, Linnaeus University, Kalmar, Sweden
- Department of Ecology, Environment and Plant Sciences, Stockholm University, Stockholm, Sweden
| | - Marcelo Ketzer
- Department of Biology and Environmental Sciences, Linnaeus University, Kalmar, Sweden
| | - Anders Forsman
- Centre for Ecology and Evolution in Microbial Model Systems, Linnaeus University, Kalmar, Sweden
| | - Mark Dopson
- Centre for Ecology and Evolution in Microbial Model Systems, Linnaeus University, Kalmar, Sweden
| | - Samuel Hylander
- Centre for Ecology and Evolution in Microbial Model Systems, Linnaeus University, Kalmar, Sweden
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Koendjbiharie JG, van Kranenburg R, Kengen SWM. The PEP-pyruvate-oxaloacetate node: variation at the heart of metabolism. FEMS Microbiol Rev 2021; 45:fuaa061. [PMID: 33289792 PMCID: PMC8100219 DOI: 10.1093/femsre/fuaa061] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2020] [Accepted: 11/18/2020] [Indexed: 12/15/2022] Open
Abstract
At the junction between the glycolysis and the tricarboxylic acid cycle-as well as various other metabolic pathways-lies the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node (PPO-node). These three metabolites form the core of a network involving at least eleven different types of enzymes, each with numerous subtypes. Obviously, no single organism maintains each of these eleven enzymes; instead, different organisms possess different subsets in their PPO-node, which results in a remarkable degree of variation, despite connecting such deeply conserved metabolic pathways as the glycolysis and the tricarboxylic acid cycle. The PPO-node enzymes play a crucial role in cellular energetics, with most of them involved in (de)phosphorylation of nucleotide phosphates, while those responsible for malate conversion are important redox enzymes. Variations in PPO-node therefore reflect the different energetic niches that organisms can occupy. In this review, we give an overview of the biochemistry of these eleven PPO-node enzymes. We attempt to highlight the variation that exists, both in PPO-node compositions, as well as in the roles that the enzymes can have within those different settings, through various recent discoveries in both bacteria and archaea that reveal deviations from canonical functions.
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Affiliation(s)
- Jeroen G Koendjbiharie
- Laboratory of Microbiology, Wageningen University, Stippeneng4, 6708 WE Wageningen, The Netherlands
| | - Richard van Kranenburg
- Laboratory of Microbiology, Wageningen University, Stippeneng4, 6708 WE Wageningen, The Netherlands
- Corbion, Arkelsedijk 46, 4206 AC Gorinchem, The Netherlands
| | - Servé W M Kengen
- Laboratory of Microbiology, Wageningen University, Stippeneng4, 6708 WE Wageningen, The Netherlands
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Microbe-Mineral Interaction and Novel Proteins for Iron Oxide Mineral Reduction in the Hyperthermophilic Crenarchaeon Pyrodictium delaneyi. Appl Environ Microbiol 2021; 87:AEM.02330-20. [PMID: 33419739 PMCID: PMC8105010 DOI: 10.1128/aem.02330-20] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2020] [Accepted: 12/17/2020] [Indexed: 11/24/2022] Open
Abstract
Understanding iron reduction in the hyperthermophilic crenarchaeon Pyrodictium delaneyi provides insight into the diversity of mechanisms used for this process and its potential impact in geothermal environments. The ability of P. delaneyi to reduce Fe(III) oxide minerals through direct contact potentially using a novel cytochrome respiratory complex and a membrane-bound molybdopterin respiratory complex sets iron reduction in this organism apart from previously described iron reduction processes. Dissimilatory iron reduction by hyperthermophilic archaea occurs in many geothermal environments and generally relies on microbe-mineral interactions that transform various iron oxide minerals. In this study, the physiology of dissimilatory iron and nitrate reduction was examined in the hyperthermophilic crenarchaeon type strain Pyrodictium delaneyi Su06. Iron barrier experiments showed that P. delaneyi required direct contact with the Fe(III) oxide mineral ferrihydrite for reduction. The separate addition of an exogenous electron shuttle (anthraquinone-2,6-disulfonate), a metal chelator (nitrilotriacetic acid), and 75% spent cell-free supernatant did not stimulate growth with or without the barrier. Protein electrophoresis showed that the c-type cytochrome and general protein compositions of P. delaneyi changed when grown on ferrihydrite relative to nitrate. Differential proteomic analyses using tandem mass tagged protein fragments and mass spectrometry detected 660 proteins and differential production of 127 proteins. Among these, two putative membrane-bound molybdopterin-dependent oxidoreductase complexes increased in relative abundance 60- to 3,000-fold and 50- to 100-fold in cells grown on iron oxide. A putative 8-heme c-type cytochrome was 60-fold more abundant in iron-grown cells and was unique to the Pyrodictiaceae. There was also a >14,700-fold increase in a membrane transport protein in iron-grown cells. For flagellin proteins and a putative nitrate reductase, there were no changes in abundance, but a membrane nitric oxide reductase was more abundant on nitrate. These data help to elucidate the mechanisms by which hyperthermophilic crenarchaea generate energy and transfer electrons across the membrane to iron oxide minerals. IMPORTANCE Understanding iron reduction in the hyperthermophilic crenarchaeon Pyrodictium delaneyi provides insight into the diversity of mechanisms used for this process and its potential impact in geothermal environments. The ability of P. delaneyi to reduce Fe(III) oxide minerals through direct contact potentially using a novel cytochrome respiratory complex and a membrane-bound molybdopterin respiratory complex sets iron reduction in this organism apart from previously described iron reduction processes. A model is presented where obligatory H2 oxidation on the membrane coupled with electron transport and either Fe(III) oxide or nitrate reduction leads to the generation of a proton motive force and energy generation by oxidative phosphorylation. However, P. delaneyi cannot fix CO2 and relies on organic compounds from its environment for biosynthesis.
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Kuprat T, Johnsen U, Ortjohann M, Schönheit P. Acetate Metabolism in Archaea: Characterization of an Acetate Transporter and of Enzymes Involved in Acetate Activation and Gluconeogenesis in Haloferax volcanii. Front Microbiol 2020; 11:604926. [PMID: 33343547 PMCID: PMC7746861 DOI: 10.3389/fmicb.2020.604926] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2020] [Accepted: 11/13/2020] [Indexed: 02/01/2023] Open
Abstract
The haloarchaeon Haloferax volcanii grows on acetate as sole carbon and energy source. The genes and proteins involved in uptake and activation of acetate and in gluconeogenesis were identified and analyzed by characterization of enzymes and by growth experiments with the respective deletion mutants. (i) An acetate transporter of the sodium: solute-symporter family (SSF) was characterized by kinetic analyses of acetate uptake into H. volcanii cells. The functional involvement of the transporter was proven with a Δssf mutant. (ii) Four paralogous AMP-forming acetyl-CoA synthetases that belong to different phylogenetic clades were shown to be functionally involved in acetate activation. (iii) The essential involvement of the glyoxylate cycle as an anaplerotic sequence was concluded from growth experiments with an isocitrate lyase knock-out mutant excluding the operation of the methylaspartate cycle reported for Haloarcula species. (iv) Enzymes involved in phosphoenolpyruvate synthesis from acetate, namely two malic enzymes and a phosphoenolpyruvate synthetase, were identified and characterized. Phylogenetic analyses of haloarchaeal malic enzymes indicate a separate evolutionary line distinct from other archaeal homologs. The exclusive function of phosphoenolpyruvate synthetase in gluconeogenesis was proven by the respective knock-out mutant. Together, this is a comprehensive study of acetate metabolism in archaea.
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Affiliation(s)
- Tom Kuprat
- Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität, Kiel, Germany
| | - Ulrike Johnsen
- Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität, Kiel, Germany
| | - Marius Ortjohann
- Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität, Kiel, Germany
| | - Peter Schönheit
- Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität, Kiel, Germany
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Ren Y, Li J, Guo L, Liu JN, Wan H, Meng Q, Wang H, Wang Z, Lv L, Dong X, Zhao W, Zeng Q, Ou J. Full-length transcriptome and long non-coding RNA profiling of whiteleg shrimp Penaeus vannamei hemocytes in response to Spiroplasma eriocheiris infection. FISH & SHELLFISH IMMUNOLOGY 2020; 106:876-886. [PMID: 32800983 DOI: 10.1016/j.fsi.2020.06.057] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/01/2020] [Revised: 06/24/2020] [Accepted: 06/28/2020] [Indexed: 06/11/2023]
Abstract
Spiroplasma eriocheiris (S. eriocheiris) infection causes a significant economic loss in Penaeus vannamei (P. vannamei) culture industry. However, the response of P. vannamei hemocytes to S. eriocheiris infection has not been extensively studied. In this study, we conducted full-length transcriptome and long non-coding RNA (lncRNA) analyses of P. vannamei hemocytes by a challenge test with S. eriocheiris. Following assembly and annotation, there were 8077 high-quality unigenes. A total of 1168 differentially expressed genes (DEGs) were obtained, including 792 up-regulated and 376 down-regulated genes by differential expression analysis. Gene ontology (GO) enrichment analysis showed that the up-regulated DEGs were mainly clustered into immune system process, defense response, cell cycle and organelle organization. On the other hand, the down-regulated DEGs included that genes that were mainly clustered into metabolic processes related to organic compounds, metabolic process and cellular metabolic process. Protein-protein interaction (PPI) network analysis of DEGs indicated that the pivotal gene interactions were connected to stress response, immune system process and cell cycle. The lncRNA analysis identified multiple lncRNAs, which were highly co-expressed with the immune-related genes, such as lncRNA transcript-12631 and transcript-12631, suggesting that lncRNAs may be involved in the regulation of immune defense in shrimp hemocytes. Additionally, 20 hub unigenes and putative lncRNAs related to immune system were validated by quantitative real-time PCR (qRT-PCR), validating the reliability of RNA-Seq. This study revealed a close connection between the immune and metabolic systems of S. eriocheiris infected P. vannamei.
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Affiliation(s)
- Yaoqing Ren
- Jiangsu Key Laboratory of Biochemistry and Biotechnology of Marine Wetland, School of Marine and Biological Engineering, Yancheng Institute of Technology, Yancheng, 224051, China
| | - Jingyu Li
- Jiangsu Key Laboratory of Biochemistry and Biotechnology of Marine Wetland, School of Marine and Biological Engineering, Yancheng Institute of Technology, Yancheng, 224051, China
| | - Liang Guo
- Jiangsu Key Laboratory of Biochemistry and Biotechnology of Marine Wetland, School of Marine and Biological Engineering, Yancheng Institute of Technology, Yancheng, 224051, China
| | - Jian Ning Liu
- KeGene Science & Technology Co. Ltd, Nantianmen Middle Road, Tai'an, 271018, China
| | - Hui Wan
- Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing, 210023, China
| | - Qingguo Meng
- Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing, 210023, China
| | - Hui Wang
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an, 271018, China
| | - Zisheng Wang
- Jiangsu Key Laboratory of Biochemistry and Biotechnology of Marine Wetland, School of Marine and Biological Engineering, Yancheng Institute of Technology, Yancheng, 224051, China
| | - Linlan Lv
- Jiangsu Key Laboratory of Biochemistry and Biotechnology of Marine Wetland, School of Marine and Biological Engineering, Yancheng Institute of Technology, Yancheng, 224051, China
| | - Xuexing Dong
- Jiangsu Key Laboratory of Biochemistry and Biotechnology of Marine Wetland, School of Marine and Biological Engineering, Yancheng Institute of Technology, Yancheng, 224051, China
| | - Weihong Zhao
- Jiangsu Key Laboratory of Biochemistry and Biotechnology of Marine Wetland, School of Marine and Biological Engineering, Yancheng Institute of Technology, Yancheng, 224051, China
| | - Qifan Zeng
- Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Science, Ocean University of China, Qingdao, 266003, China.
| | - Jiangtao Ou
- Jiangsu Key Laboratory of Biochemistry and Biotechnology of Marine Wetland, School of Marine and Biological Engineering, Yancheng Institute of Technology, Yancheng, 224051, China.
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Unusual Phosphoenolpyruvate (PEP) Synthetase-Like Protein Crucial to Enhancement of Polyhydroxyalkanoate Accumulation in Haloferax mediterranei Revealed by Dissection of PEP-Pyruvate Interconversion Mechanism. Appl Environ Microbiol 2019; 85:AEM.00984-19. [PMID: 31350314 DOI: 10.1128/aem.00984-19] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2019] [Accepted: 07/08/2019] [Indexed: 12/21/2022] Open
Abstract
Phosphoenolpyruvate (PEP)/pyruvate interconversion is a major metabolic point in glycolysis and gluconeogenesis and is catalyzed by various sets of enzymes in different Archaea groups. In this study, we report the key enzymes that catalyze the anabolic and catabolic directions of the PEP/pyruvate interconversion in Haloferax mediterranei The in silico analysis showed the presence of a potassium-dependent pyruvate kinase (PYKHm [HFX_0773]) and two phosphoenol pyruvate synthetase (PPS) candidates (PPSHm [HFX_0782] and a PPS homolog protein named PPS-like [HFX_2676]) in this strain. Expression of the pyk Hm gene and pps Hm was induced by glycerol and pyruvate, respectively; whereas the pps-like gene was not induced at all. Similarly, genetic analysis and enzyme activities of purified proteins showed that PYKHm catalyzed the conversion from PEP to pyruvate and that PPSHm catalyzed the reverse reaction, while PPS-like protein displayed no function in PEP/pyruvate interconversion. Interestingly, knockout of the pps-like gene led to a 70.46% increase in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production. The transcriptome sequencing (RNA-Seq) and quantitative reverse transcription-PCR (qRT-PCR) results showed that many genes responsible for PHBV monomer supply and for PHBV synthesis were upregulated in a pps-like gene deletion strain and thereby improved PHBV accumulation. Additionally, our phylogenetic evidence suggested that PPS-like protein diverged from PPS enzyme and evolved as a distinct protein with novel function in haloarchaea. Our findings attempt to fill the gaps in central metabolism of Archaea by providing comprehensive information about key enzymes involved in the haloarchaeal PEP/pyruvate interconversion, and we also report a high-yielding PHBV strain with great future potentials.IMPORTANCE Archaea, the third domain of life, have evolved diversified metabolic pathways to cope with their extreme habitats. Phosphoenol pyruvate (PEP)/pyruvate interconversion during carbohydrate metabolism is one such important metabolic process that is highly differentiated among Archaea However, this process is still uncharacterized in the haloarchaeal group. Haloferax mediterranei is a well-studied haloarchaeon that has the ability to produce polyhydroxyalkanoates (PHAs) under unbalanced nutritional conditions. In this study, we identified the key enzymes involved in this interconversion and discussed their differences with their counterparts from other members of the Archaea and Bacteria domains. Notably, we found a novel protein, phosphoenolpyruvate synthetase-like (PPS-like), which exhibited high homology to PPS enzyme. However, PPS-like protein has evolved some distinct sequence features and functions, and strikingly the corresponding gene deletion helped to enhance poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) synthesis significantly. Overall, we have filled the gap in knowledge about PEP/pyruvate interconversion in haloarchaea and reported an efficient strategy for improving PHBV production in H. mediterranei.
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Haferkamp P, Tjaden B, Shen L, Bräsen C, Kouril T, Siebers B. The Carbon Switch at the Level of Pyruvate and Phosphoenolpyruvate in Sulfolobus solfataricus P2. Front Microbiol 2019; 10:757. [PMID: 31031731 PMCID: PMC6474364 DOI: 10.3389/fmicb.2019.00757] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2019] [Accepted: 03/26/2019] [Indexed: 01/26/2023] Open
Abstract
Sulfolobus solfataricus P2 grows on different carbohydrates as well as alcohols, peptides and amino acids. Carbohydrates such as D-glucose or D-galactose are degraded via the modified, branched Entner–Doudoroff (ED) pathway whereas growth on peptides requires the Embden–Meyerhof–Parnas (EMP) pathway for gluconeogenesis. As for most hyperthermophilic Archaea an important control point is established at the level of triosephophate conversion, however, the regulation at the level of pyruvate/phosphoenolpyruvate conversion was not tackled so far. Here we describe the cloning, expression, purification and characterization of the pyruvate kinase (PK, SSO0981) and the phosphoenolpyruvate synthetase (PEPS, SSO0883) of Sul. solfataricus. The PK showed only catabolic activity [catalytic efficiency (PEP): 627.95 mM-1s-1, 70°C] with phosphoenolpyruvate as substrate and ADP as phosphate acceptor and was allosterically inhibited by ATP and isocitrate (Ki 0.8 mM). The PEPS was reversible, however, exhibited preferred activity in the gluconeogenic direction [catalytic efficiency (pyruvate): 1.04 mM-1s-1, 70°C] and showed some inhibition by AMP and α-ketoglutarate. The gene SSO2829 annotated as PEPS/pyruvate:phosphate dikinase (PPDK) revealed neither PEPS nor PPDK activity. Our studies suggest that the energy charge of the cell as well as the availability of building blocks in the citric acid cycle and the carbon/nitrogen balance plays a major role in the Sul. solfataricus carbon switch. The comparison of regulatory features of well-studied hyperthermophilic Archaea reveals a close link and sophisticated coordination between the respective sugar kinases and the kinetic and regulatory properties of the enzymes at the level of PEP-pyruvate conversion.
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Affiliation(s)
- Patrick Haferkamp
- Molecular Enzyme Technology and Biochemistry, Biofilm Centre, Centre for Water and Environmental Research, Faculty of Chemistry, University of Duisburg-Essen, Essen, Germany
| | - Britta Tjaden
- Molecular Enzyme Technology and Biochemistry, Biofilm Centre, Centre for Water and Environmental Research, Faculty of Chemistry, University of Duisburg-Essen, Essen, Germany
| | - Lu Shen
- Molecular Enzyme Technology and Biochemistry, Biofilm Centre, Centre for Water and Environmental Research, Faculty of Chemistry, University of Duisburg-Essen, Essen, Germany
| | - Christopher Bräsen
- Molecular Enzyme Technology and Biochemistry, Biofilm Centre, Centre for Water and Environmental Research, Faculty of Chemistry, University of Duisburg-Essen, Essen, Germany
| | - Theresa Kouril
- Molecular Enzyme Technology and Biochemistry, Biofilm Centre, Centre for Water and Environmental Research, Faculty of Chemistry, University of Duisburg-Essen, Essen, Germany.,Department of Biochemistry, University of Stellenbosch, Stellenbosch, South Africa
| | - Bettina Siebers
- Molecular Enzyme Technology and Biochemistry, Biofilm Centre, Centre for Water and Environmental Research, Faculty of Chemistry, University of Duisburg-Essen, Essen, Germany
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Qian X, Zhang Y, Lun DS, Dismukes GC. Rerouting of Metabolism into Desired Cellular Products by Nutrient Stress: Fluxes Reveal the Selected Pathways in Cyanobacterial Photosynthesis. ACS Synth Biol 2018; 7:1465-1476. [PMID: 29617123 DOI: 10.1021/acssynbio.8b00116] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Boosting cellular growth rates while redirecting metabolism to make desired products are the preeminent goals of gene engineering of photoautotrophs, yet so far these goals have been hardly achieved owing to lack of understanding of the functional pathways and their choke points. Here we apply a 13C mass isotopic method (INST-MFA) to quantify instantaneous fluxes of metabolites during photoautotrophic growth. INST-MFA determines the globally most accurate set of absolute fluxes for each metabolite from a finite set of measured 13C-isotopomer fluxes by minimizing the sum of squared residuals between experimental and predicted mass isotopomers. We show that the widely observed shift in biomass composition in cyanobacteria, demonstrated here with Synechococcus sp. PCC 7002, favoring glycogen synthesis during nitrogen starvation is caused by (1) increased flux through a bottleneck step in gluconeogenesis (3PG → GAP/DHAP), and (2) flux overflow through a previously unrecognized hybrid gluconeogenesis-pentose phosphate (hGPP) pathway. Our data suggest the slower growth rate and biomass accumulation under N starvation is due to a reduced carbon fixation rate and a reduced flux of carbon into amino acid precursors. Additionally, 13C flux from α-ketoglutarate to succinate is demonstrated to occur via succinic semialdehyde, an alternative to the conventional TCA cycle, in Synechococcus 7002 under photoautotrophic conditions. We found that pyruvate and oxaloacetate are synthesized mainly by malate dehydrogenase with minimal flux into acetyl coenzyme-A via pyruvate dehydrogenase. Nutrient stress induces major shifts in fluxes into new pathways that deviate from historical metabolic pathways derived from model bacteria.
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Affiliation(s)
- Xiao Qian
- Waksman Institute, Rutgers University, New Brunswick, New Jersey 08854, United States
| | - Yuan Zhang
- Waksman Institute, Rutgers University, New Brunswick, New Jersey 08854, United States
| | - Desmond S. Lun
- Center for Computational and Integrative Biology, Rutgers University, Camden, New Jersey 08102, United States
- Department of Computer Science, Rutgers University, Camden, New Jersey 08102, United States
- Department of Plant Biology, Rutgers University, New Brunswick, New Jersey 08901, United States
| | - G. Charles Dismukes
- Waksman Institute, Rutgers University, New Brunswick, New Jersey 08854, United States
- Department of Chemistry & Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, United States
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Taillefer M, Sparling R. Glycolysis as the Central Core of Fermentation. ADVANCES IN BIOCHEMICAL ENGINEERING/BIOTECHNOLOGY 2017; 156:55-77. [PMID: 26907549 DOI: 10.1007/10_2015_5003] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The increasing concerns of greenhouse gas emissions have increased the interest in dark fermentation as a means of productions for industrial chemicals, especially from renewable cellulosic biomass. However, the metabolism, including glycolysis, of many candidate organisms for cellulosic biomass conversion through consolidated bioprocessing is still poorly understood and the genomes have only recently been sequenced. Because a variety of industrial chemicals are produced directly from sugar metabolism, the careful understanding of glycolysis from a genomic and biochemical point of view is essential in the development of strategies for increasing product yields and therefore increasing industrial potential. The current review discusses the different pathways available for glycolysis along with unexpected variations from traditional models, especially in the utilization of alternate energy intermediates (GTP, pyrophosphate). This reinforces the need for a careful description of interactions between energy metabolites and glycolysis enzymes for understanding carbon and electron flux regulation.
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Affiliation(s)
- M Taillefer
- Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada, R3T 2N2
| | - R Sparling
- Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada, R3T 2N2.
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10
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Santiago-Martínez MG, Encalada R, Lira-Silva E, Pineda E, Gallardo-Pérez JC, Reyes-García MA, Saavedra E, Moreno-Sánchez R, Marín-Hernández A, Jasso-Chávez R. The nutritional status of Methanosarcina acetivorans regulates glycogen metabolism and gluconeogenesis and glycolysis fluxes. FEBS J 2016; 283:1979-99. [PMID: 27000496 DOI: 10.1111/febs.13717] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2015] [Revised: 03/10/2016] [Accepted: 03/17/2016] [Indexed: 11/27/2022]
Abstract
Gluconeogenesis is an essential pathway in methanogens because they are unable to use exogenous hexoses as carbon source for cell growth. With the aim of understanding the regulatory mechanisms of central carbon metabolism in Methanosarcina acetivorans, the present study investigated gene expression, the activities and metabolic regulation of key enzymes, metabolite contents and fluxes of gluconeogenesis, as well as glycolysis and glycogen synthesis/degradation pathways. Cells were grown with methanol as a carbon source. Key enzymes were kinetically characterized at physiological pH/temperature. Active consumption of methanol during exponential cell growth correlated with significant methanogenesis, gluconeogenic flux and steady glycogen synthesis. After methanol exhaustion, cells reached the stationary growth phase, which correlated with the rise in glycogen consumption and glycolytic flux, decreased methanogenesis, negligible acetate production and an absence of gluconeogenesis. Elevated activities of carbon monoxide dehydrogenase/acetyl-CoA synthetase complex and pyruvate: ferredoxin oxidoreductase suggested the generation of acetyl-CoA and pyruvate for glycogen synthesis. In the early stationary growth phase, the transcript contents and activities of pyruvate phosphate dikinase, fructose 1,6-bisphosphatase and glycogen synthase decreased, whereas those of glycogen phosphorylase, ADP-phosphofructokinase and pyruvate kinase increased. Therefore, glycogen and gluconeogenic metabolites were synthesized when an external carbon source was provided. Once such a carbon source became depleted, glycolysis and methanogenesis fed by glycogen degradation provided the ATP supply. Weak inhibition of key enzymes by metabolites suggested that the pathways evaluated were mainly transcriptionally regulated. Because glycogen metabolism and glycolysis/gluconeogenesis are not present in all methanogens, the overall data suggest that glycogen storage might represent an environmental advantage for methanosarcinales when carbon sources are scarce. Also, the understanding of the central carbohydrate metabolism in methanosarcinales may help to optimize methane production.
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Affiliation(s)
| | - Rusely Encalada
- Departamento de Bioquímica, Instituto Nacional de Cardiología, México DF, México
| | - Elizabeth Lira-Silva
- Departamento de Bioquímica, Instituto Nacional de Cardiología, México DF, México
| | - Erika Pineda
- Departamento de Bioquímica, Instituto Nacional de Cardiología, México DF, México
| | | | | | - Emma Saavedra
- Departamento de Bioquímica, Instituto Nacional de Cardiología, México DF, México
| | | | | | - Ricardo Jasso-Chávez
- Departamento de Bioquímica, Instituto Nacional de Cardiología, México DF, México
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McCormick NE, Jakeman DL. On the mechanism of phosphoenolpyruvate synthetase (PEPs) and its inhibition by sodium fluoride: potential magnesium and aluminum fluoride complexes of phosphoryl transfer. Biochem Cell Biol 2015; 93:236-40. [DOI: 10.1139/bcb-2014-0153] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Phosphoenolpyruvate synthase (PEPs) catalyzes the conversion of pyruvate to phosphoenolpyruvate (PEP) using a two-step mechanism invoking a phosphorylated-His intermediate. Formation of PEP is an initial step in gluconeogenesis, and PEPs is essential for growth of Escherichia coli on 3-carbon sources such as pyruvate. The production of PEPs has also been linked to bacterial virulence and antibiotic resistance. As such, PEPs is of interest as a target for antibiotic development, and initial investigations of PEPs have indicated inhibition by sodium fluoride. Similar inhibition has been observed in a variety of phospho-transfer enzymes through the formation of metal fluoride complexes within the active site. Herein we quantify the inhibitory capacity of sodium fluoride through a coupled spectrophotometric assay. The observed inhibition provides indirect evidence for the formation of a MgF3−complex within the enzyme active site and insight into the phospho-transfer mechanism of PEPs. The effect of AlCl3on PEPs enzyme activity was also assessed and found to decrease substrate binding and turnover.
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Affiliation(s)
- Nicole E. McCormick
- College of Pharmacy, Dalhousie University, 5968 College St., Halifax, NS B3H 4R2, Canada
| | - David L. Jakeman
- College of Pharmacy, Dalhousie University, 5968 College St., Halifax, NS B3H 4R2, Canada
- Department of Chemistry, Dalhousie University, 6274 Coberg Rd., Halifax, NS B3H 4R2, Canada
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Reassessment of the transhydrogenase/malate shunt pathway in Clostridium thermocellum ATCC 27405 through kinetic characterization of malic enzyme and malate dehydrogenase. Appl Environ Microbiol 2015; 81:2423-32. [PMID: 25616802 DOI: 10.1128/aem.03360-14] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
Clostridium thermocellum produces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role in C. thermocellum metabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with a kcat of 45.8 s(-1) or 14.9 s(-1), respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with a kcat of 520.8 s(-1). The malic enzyme used NADP(+) as a cofactor along with NH4 (+) and Mn(2+) as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with a Ki of 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH4 (+) based on the characterization of the malate dehydrogenase and malic enzyme.
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Martin-Cuadrado AB, Garcia-Heredia I, Moltó AG, López-Úbeda R, Kimes N, López-García P, Moreira D, Rodriguez-Valera F. A new class of marine Euryarchaeota group II from the Mediterranean deep chlorophyll maximum. ISME JOURNAL 2014; 9:1619-34. [PMID: 25535935 DOI: 10.1038/ismej.2014.249] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/26/2014] [Revised: 11/14/2014] [Accepted: 11/19/2014] [Indexed: 11/09/2022]
Abstract
We have analyzed metagenomic fosmid clones from the deep chlorophyll maximum (DCM), which, by genomic parameters, correspond to the 16S ribosomal RNA (rRNA)-defined marine Euryarchaeota group IIB (MGIIB). The fosmid collections associated with this group add up to 4 Mb and correspond to at least two species within this group. From the proposed essential genes contained in the collections, we infer that large sections of the conserved regions of the genomes of these microbes have been recovered. The genomes indicate a photoheterotrophic lifestyle, similar to that of the available genome of MGIIA (assembled from an estuarine metagenome in Puget Sound, Washington Pacific coast), with a proton-pumping rhodopsin of the same kind. Several genomic features support an aerobic metabolism with diversified substrate degradation capabilities that include xenobiotics and agar. On the other hand, these MGIIB representatives are non-motile and possess similar genome size to the MGIIA-assembled genome, but with a lower GC content. The large phylogenomic gap with other known archaea indicates that this is a new class of marine Euryarchaeota for which we suggest the name Thalassoarchaea. The analysis of recruitment from available metagenomes indicates that the representatives of group IIB described here are largely found at the DCM (ca. 50 m deep), in which they are abundant (up to 0.5% of the reads), and at the surface mostly during the winter mixing, which explains formerly described 16S rRNA distribution patterns. Their uneven representation in environmental samples that are close in space and time might indicate sporadic blooms.
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Affiliation(s)
- Ana-Belen Martin-Cuadrado
- Evolutionary Genomics Group, Departamento de Producción Vegetal y Microbiología, Universidad Miguel Hernández, San Juan de Alicante, Alicante, Spain
| | - Inmaculada Garcia-Heredia
- Evolutionary Genomics Group, Departamento de Producción Vegetal y Microbiología, Universidad Miguel Hernández, San Juan de Alicante, Alicante, Spain
| | - Aitor Gonzaga Moltó
- Evolutionary Genomics Group, Departamento de Producción Vegetal y Microbiología, Universidad Miguel Hernández, San Juan de Alicante, Alicante, Spain
| | - Rebeca López-Úbeda
- Evolutionary Genomics Group, Departamento de Producción Vegetal y Microbiología, Universidad Miguel Hernández, San Juan de Alicante, Alicante, Spain
| | - Nikole Kimes
- Evolutionary Genomics Group, Departamento de Producción Vegetal y Microbiología, Universidad Miguel Hernández, San Juan de Alicante, Alicante, Spain
| | - Purificación López-García
- Unité d'Ecologie, Systématique et Evolution, UMR CNRS 8079, Université Paris-Sud, Orsay Cedex, France
| | - David Moreira
- Unité d'Ecologie, Systématique et Evolution, UMR CNRS 8079, Université Paris-Sud, Orsay Cedex, France
| | - Francisco Rodriguez-Valera
- Evolutionary Genomics Group, Departamento de Producción Vegetal y Microbiología, Universidad Miguel Hernández, San Juan de Alicante, Alicante, Spain
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Carbohydrate metabolism in Archaea: current insights into unusual enzymes and pathways and their regulation. Microbiol Mol Biol Rev 2014; 78:89-175. [PMID: 24600042 DOI: 10.1128/mmbr.00041-13] [Citation(s) in RCA: 200] [Impact Index Per Article: 18.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
The metabolism of Archaea, the third domain of life, resembles in its complexity those of Bacteria and lower Eukarya. However, this metabolic complexity in Archaea is accompanied by the absence of many "classical" pathways, particularly in central carbohydrate metabolism. Instead, Archaea are characterized by the presence of unique, modified variants of classical pathways such as the Embden-Meyerhof-Parnas (EMP) pathway and the Entner-Doudoroff (ED) pathway. The pentose phosphate pathway is only partly present (if at all), and pentose degradation also significantly differs from that known for bacterial model organisms. These modifications are accompanied by the invention of "new," unusual enzymes which cause fundamental consequences for the underlying regulatory principles, and classical allosteric regulation sites well established in Bacteria and Eukarya are lost. The aim of this review is to present the current understanding of central carbohydrate metabolic pathways and their regulation in Archaea. In order to give an overview of their complexity, pathway modifications are discussed with respect to unusual archaeal biocatalysts, their structural and mechanistic characteristics, and their regulatory properties in comparison to their classic counterparts from Bacteria and Eukarya. Furthermore, an overview focusing on hexose metabolic, i.e., glycolytic as well as gluconeogenic, pathways identified in archaeal model organisms is given. Their energy gain is discussed, and new insights into different levels of regulation that have been observed so far, including the transcript and protein levels (e.g., gene regulation, known transcription regulators, and posttranslational modification via reversible protein phosphorylation), are presented.
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The Gluconeogenic Pathway in a Soil Mycobacterium Isolate with Bioremediation Ability. Curr Microbiol 2012; 66:122-31. [DOI: 10.1007/s00284-012-0248-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2012] [Accepted: 09/23/2012] [Indexed: 11/26/2022]
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Auger C, Appanna V, Castonguay Z, Han S, Appanna VD. A facile electrophoretic technique to monitor phosphoenolpyruvate-dependent kinases. Electrophoresis 2012; 33:1095-101. [PMID: 22539312 DOI: 10.1002/elps.201100517] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Phosphoenolpyruvate (PEP)-dependent kinases are central to numerous metabolic processes and mediate the production of adenosine triphosphate (ATP) by substrate-level phosphorylation (SLP). While pyruvate kinase (PK, EC: 2.7.1.40), the final enzyme of the glycolytic pathway is critical in the anaerobic synthesis of ATP from ADP, pyruvate phosphate dikinase (PPDK, EC: 2.7.9.1), and phosphoenolpyruvate synthase (PEPS, EC: 2.7.9.2) help generate ATP from AMP coupled to PEP as a substrate. Here we demonstrate an inexpensive and effective electrophoretic technology to determine the activities of these enzymes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). The generation of pyruvate is linked to exogenous lactate dehydrogenase (LDH), and the oxidation of reduced nicotinamide adenine dinucleotide (NADH) coupled to 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium chloride (INT) results in a formazan precipitate which is easily quantifiable. The selectivity of the enzymes is ensured by including either AMP or ADP and pyrophosphate (PP(i) ) or inorganic phosphate (P(i) ). Activity bands were readily obtained after incubation in the respective reaction mixtures for 20-30 min. Cell-free extract concentrations as low as 20 μg protein equivalent yielded activity bands and substrate levels were manipulated to optimize sensitivity of this analytical technique. High-pressure liquid chromatography (HPLC), two-dimensional (2-D) SDS-PAGE (where SDS is sodium dodecyl sulfate), and immunoblot studies of the excised activity band help further characterize these PEP-dependent kinases. Furthermore, these enzymes were readily identified on the same gel by incubating it sequentially in the respective reaction mixtures. This technique provides a facile method to elucidate these kinases in biological systems.
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Affiliation(s)
- Christopher Auger
- Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada
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Schut GJ, Boyd ES, Peters JW, Adams MWW. The modular respiratory complexes involved in hydrogen and sulfur metabolism by heterotrophic hyperthermophilic archaea and their evolutionary implications. FEMS Microbiol Rev 2012; 37:182-203. [PMID: 22713092 DOI: 10.1111/j.1574-6976.2012.00346.x] [Citation(s) in RCA: 95] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2012] [Revised: 05/30/2012] [Accepted: 06/08/2012] [Indexed: 12/01/2022] Open
Abstract
Hydrogen production is a vital metabolic process for many anaerobic organisms, and the enzyme responsible, hydrogenase, has been studied since the 1930s. A novel subfamily with unique properties was recently recognized, represented by the 14-subunit membrane-bound [NiFe] hydrogenase from the archaeon Pyrococcus furiosus. This so-called energy-converting hydrogenase links the thermodynamically favorable oxidation of ferredoxin with the formation of hydrogen and conserves energy in the form of an ion gradient. It is therefore a simple respiratory system within a single complex. This hydrogenase shows a modular composition represented by a Na(+)/H(+) antiporter domain (Mrp) and a [NiFe] hydrogenase domain (Mbh). An analysis of the large number of microbial genome sequences available shows that homologs of Mbh and Mrp tend to be clustered within the genomes of a limited number of archaeal and bacterial species. In several instances, additional genes are associated with the Mbh and Mrp gene clusters that encode proteins that catalyze the oxidation of formate, CO or NAD(P)H. The Mbh complex also shows extensive homology to a number of subunits within the NADH quinone oxidoreductase or complex I family. The respiratory-type membrane-bound hydrogenase complex appears to be closely related to the common ancestor of complex I and [NiFe] hydrogenases in general.
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Affiliation(s)
- Gerrit J Schut
- Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA 30602, USA
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Comparative analysis of diatom genomes reveals substantial differences in the organization of carbon partitioning pathways. ALGAL RES 2012. [DOI: 10.1016/j.algal.2012.04.003] [Citation(s) in RCA: 89] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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Mi S, Song J, Lin J, Che Y, Zheng H, Lin J. Complete genome of Leptospirillum ferriphilum ML-04 provides insight into its physiology and environmental adaptation. J Microbiol 2011; 49:890-901. [DOI: 10.1007/s12275-011-1099-9] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2011] [Accepted: 07/27/2011] [Indexed: 12/23/2022]
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Matsubara K, Yokooji Y, Atomi H, Imanaka T. Biochemical and genetic characterization of the three metabolic routes in Thermococcus kodakarensis linking glyceraldehyde 3-phosphate and 3-phosphoglycerate. Mol Microbiol 2011; 81:1300-12. [PMID: 21736643 DOI: 10.1111/j.1365-2958.2011.07762.x] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
In the classical Embden-Meyerhof (EM) pathway for glycolysis, the conversion between glyceraldehyde 3-phosphate (GAP) and 3-phosphoglycerate (3-PGA) is reversibly catalysed by phosphorylating GAP dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK). In the Euryarchaeota Thermococcus kodakarensis and Pyrococcus furiosus, an additional gene encoding GAP:ferredoxin oxidoreductase (GAPOR) and a gene similar to non-phosphorylating GAP dehydrogenase (GAPN) are present. In order to determine the physiological roles of the three routes that link GAP and 3-PGA, we individually disrupted the GAPOR, GAPN, GAPDH and PGK genes (gor, gapN, gapDH and pgk respectively) of T. kodakarensis. The Δgor strain displayed no growth under glycolytic conditions, confirming its proposed function to generate reduced ferredoxin for energy generation in glycolysis. Surprisingly, ΔgapN cells also did not grow under glycolytic conditions, suggesting that GAPN plays a key role in providing NADPH under these conditions. Disruption of gor and gapN had no effect on gluconeogenic growth. Growth experiments with the ΔgapDH and Δpgk strains indicated that, unlike their counterparts in the classical EM pathway, GAPDH/PGK play a major role only in gluconeogenesis. Biochemical analyses indicated that T. kodakarensis GAPN did not recognize aldehyde substrates other than d-GAP, preferred NADP(+) as cofactor and was dramatically activated with glucose 1-phosphate.
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Affiliation(s)
- Kohei Matsubara
- Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
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Novel metabolic pathways in Archaea. Curr Opin Microbiol 2011; 14:307-14. [PMID: 21612976 DOI: 10.1016/j.mib.2011.04.014] [Citation(s) in RCA: 57] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2011] [Accepted: 04/18/2011] [Indexed: 11/24/2022]
Abstract
The Archaea harbor many metabolic pathways that differ to previously recognized classical pathways. Glycolysis is carried out by modified versions of the Embden-Meyerhof and Entner-Doudoroff pathways. Thermophilic archaea have recently been found to harbor a bi-functional fructose-1,6-bisphosphate aldolase/phosphatase for gluconeogenesis. A number of novel pentose-degrading pathways have also been recently identified. In terms of anabolic metabolism, a pathway for acetate assimilation, the methylaspartate cycle, and two CO2-fixing pathways, the 3-hydroxypropionate/4-hydroxybutyrate cycle and the dicarboxylate/4-hydroxybutyrate cycle, have been elucidated. As for biosynthetic pathways, recent studies have clarified the enzymes responsible for several steps involved in the biosynthesis of inositol phospholipids, polyamine, coenzyme A, flavin adeninedinucleotide and heme. By examining the presence/absence of homologs of these enzymes on genome sequences, we have found that the majority of these enzymes and pathways are specific to the Archaea.
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Labeling and enzyme studies of the central carbon metabolism in Metallosphaera sedula. J Bacteriol 2010; 193:1191-200. [PMID: 21169486 DOI: 10.1128/jb.01155-10] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Metallosphaera sedula (Sulfolobales, Crenarchaeota) uses the 3-hydroxypropionate/4-hydroxybutyrate cycle for autotrophic carbon fixation. In this pathway, acetyl-coenzyme A (CoA) and succinyl-CoA are the only intermediates that can be considered common to the central carbon metabolism. We addressed the question of which intermediate of the cycle most biosynthetic routes branch off. We labeled autotrophically growing cells by using 4-hydroxy[1-¹⁴C]butyrate and [1,4-¹³C₁]succinate, respectively, as precursors for biosynthesis. The labeling patterns of protein-derived amino acids verified the operation of the proposed carbon fixation cycle, in which 4-hydroxybutyrate is converted to two molecules of acetyl-CoA. The results also showed that major biosynthetic flux does not occur via acetyl-CoA, except for the formation of building blocks that are directly derived from acetyl-CoA. Notably, acetyl-CoA is not assimilated via reductive carboxylation to pyruvate. Rather, our data suggest that the majority of anabolic precursors are derived from succinyl-CoA, which is removed from the cycle via oxidation to malate and oxaloacetate. These C₄intermediates yield pyruvate and phosphoenolpyruvate (PEP). Enzyme activities that are required for forming intermediates from succinyl-CoA were detected, including enzymes catalyzing gluconeogenesis from PEP. This study completes the picture of the central carbon metabolism in autotrophic Sulfolobales by connecting the autotrophic carbon fixation cycle to the formation of central carbon precursor metabolites.
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Menon AL, Poole FL, Cvetkovic A, Trauger SA, Kalisiak E, Scott JW, Shanmukh S, Praissman J, Jenney FE, Wikoff WR, Apon JV, Siuzdak G, Adams MWW. Novel multiprotein complexes identified in the hyperthermophilic archaeon Pyrococcus furiosus by non-denaturing fractionation of the native proteome. Mol Cell Proteomics 2008; 8:735-51. [PMID: 19043064 DOI: 10.1074/mcp.m800246-mcp200] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Virtually all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes, the compositions of which are largely undefined. They cannot be predicted solely from bioinformatics analyses nor are there well defined techniques currently available to unequivocally identify protein complexes (PCs). To address this issue, we attempted to directly determine the identity of PCs from native microbial biomass using Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100 degrees C, as the model organism. Novel PCs were identified by large scale fractionation of the native proteome using non-denaturing, sequential column chromatography under anaerobic, reducing conditions. A total of 967 distinct P. furiosus proteins were identified by mass spectrometry (nano LC-ESI-MS/MS), representing approximately 80% of the cytoplasmic proteins. Based on the co-fractionation of proteins that are encoded by adjacent genes on the chromosome, 106 potential heteromeric PCs containing 243 proteins were identified, only 20 of which were known or expected. In addition to those of unknown function, novel and uncharacterized PCs were identified that are proposed to be involved in the metabolism of amino acids (10), carbohydrates (four), lipids (two), vitamins and metals (three), and DNA and RNA (nine). A further 30 potential PCs were classified as tentative, and the remaining potential PCs (13) were classified as weakly interacting. Some major advantages of native biomass fractionation for PC identification are that it provides a road map for the (partial) purification of native forms of novel and uncharacterized PCs, and the results can be utilized for the recombinant production of low abundance PCs to provide enough material for detailed structural and biochemical analyses.
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Affiliation(s)
- Angeli Lal Menon
- Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA
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Martin W, Russell MJ. On the origin of biochemistry at an alkaline hydrothermal vent. Philos Trans R Soc Lond B Biol Sci 2007; 362:1887-925. [PMID: 17255002 PMCID: PMC2442388 DOI: 10.1098/rstb.2006.1881] [Citation(s) in RCA: 396] [Impact Index Per Article: 22.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
A model for the origin of biochemistry at an alkaline hydrothermal vent has been developed that focuses on the acetyl-CoA (Wood-Ljungdahl) pathway of CO2 fixation and central intermediary metabolism leading to the synthesis of the constituents of purines and pyrimidines. The idea that acetogenesis and methanogenesis were the ancestral forms of energy metabolism among the first free-living eubacteria and archaebacteria, respectively, stands in the foreground. The synthesis of formyl pterins, which are essential intermediates of the Wood-Ljungdahl pathway and purine biosynthesis, is found to confront early metabolic systems with steep bioenergetic demands that would appear to link some, but not all, steps of CO2 reduction to geochemical processes in or on the Earth's crust. Inorganically catalysed prebiotic analogues of the core biochemical reactions involved in pterin-dependent methyl synthesis of the modern acetyl-CoA pathway are considered. The following compounds appear as probable candidates for central involvement in prebiotic chemistry: metal sulphides, formate, carbon monoxide, methyl sulphide, acetate, formyl phosphate, carboxy phosphate, carbamate, carbamoyl phosphate, acetyl thioesters, acetyl phosphate, possibly carbonyl sulphide and eventually pterins. Carbon might have entered early metabolism via reactions hardly different from those in the modern Wood-Ljungdahl pathway, the pyruvate synthase reaction and the incomplete reverse citric acid cycle. The key energy-rich intermediates were perhaps acetyl thioesters, with acetyl phosphate possibly serving as the universal metabolic energy currency prior to the origin of genes. Nitrogen might have entered metabolism as geochemical NH3 via two routes: the synthesis of carbamoyl phosphate and reductive transaminations of alpha-keto acids. Together with intermediates of methyl synthesis, these two routes of nitrogen assimilation would directly supply all intermediates of modern purine and pyrimidine biosynthesis. Thermodynamic considerations related to formyl pterin synthesis suggest that the ability to harness a naturally pre-existing proton gradient at the vent-ocean interface via an ATPase is older than the ability to generate a proton gradient with chemistry that is specified by genes.
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Affiliation(s)
- William Martin
- Institute of Botany, University of Düsseldorf, 40225 Düsseldorf, Germany.
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Hallam SJ, Konstantinidis KT, Putnam N, Schleper C, Watanabe YI, Sugahara J, Preston C, de la Torre J, Richardson PM, DeLong EF. Genomic analysis of the uncultivated marine crenarchaeote Cenarchaeum symbiosum. Proc Natl Acad Sci U S A 2006; 103:18296-301. [PMID: 17114289 PMCID: PMC1643844 DOI: 10.1073/pnas.0608549103] [Citation(s) in RCA: 306] [Impact Index Per Article: 16.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Crenarchaeota are ubiquitous and abundant microbial constituents of soils, sediments, lakes, and ocean waters. To further describe the cosmopolitan nonthermophilic Crenarchaeota, we analyzed the genome sequence of one representative, the uncultivated sponge symbiont Cenarchaeum symbiosum. C. symbiosum genotypes coinhabiting the same host partitioned into two dominant populations, corresponding to previously described a- and b-type ribosomal RNA variants. Although they were syntenic, overlapping a- and b-type ribotype genomes harbored significant variability. A single tiling path comprising the dominant a-type genotype was assembled and used to explore the genomic properties of C. symbiosum and its planktonic relatives. Of 2,066 ORFs, 55.6% matched genes with predicted function from previously sequenced genomes. The remaining genes partitioned between functional RNAs (2.4%) and hypotheticals (42%) with limited homology to known functional genes. The latter category included some genes likely involved in the archaeal-sponge symbiotic association. Conversely, 525 C. symbiosum ORFs were most highly similar to sequences from marine environmental genomic surveys, and they apparently represent orthologous genes from free-living planktonic Crenarchaeota. In total, the C. symbiosum genome was remarkably distinct from those of other known Archaea and shared many core metabolic features in common with its free-living planktonic relatives.
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Affiliation(s)
| | | | - Nik Putnam
- Joint Genome Institute, Walnut Creek, CA 94598
| | - Christa Schleper
- Department of Biology, University of Bergen, Jahnebakken 5, N-5020 Bergen, Norway
| | - Yoh-ichi Watanabe
- Department of Biomedical Chemistry, University of Tokyo, Tokyo 113-0033, Japan
| | - Junichi Sugahara
- Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan
| | - Christina Preston
- **Monterey Bay Aquarium Research Institute, Moss Landing, CA 95069; and
| | | | | | - Edward F. DeLong
- *Massachusetts Institute of Technology, Cambridge, MA 02139
- To whom correspondence should be addressed. E-mail:
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Imanaka H, Yamatsu A, Fukui T, Atomi H, Imanaka T. Phosphoenolpyruvate synthase plays an essential role for glycolysis in the modified Embden-Meyerhof pathway in Thermococcus kodakarensis. Mol Microbiol 2006; 61:898-909. [PMID: 16879645 DOI: 10.1111/j.1365-2958.2006.05287.x] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
We have carried out a genetic analysis on pyruvate kinase (PykTk) and phosphoenolpyruvate synthase (PpsTk) in the hyperthermophilic archaeon, Thermococcus kodakarensis. In principle, both enzymes can catalyse the final step of the modified Embden-Meyerhof (EM) pathway found in Thermococcales, the conversion of phosphoenolpyruvate (PEP) to pyruvate, with the former utilizing ADP, while the latter is dependent on AMP and phosphate. Enzyme activities and transcript levels of both PykTk and PpsTk increased in T. kodakarensis under glycolytic conditions when compared with cells grown on pyruvate or amino acids. Using KW128, a tryptophan auxotrophic mutant with a trpE gene disruption, as a host strain, we obtained mutant strains with single gene disruptions in either the pykTk (Deltapyk strain) or ppsTk (Deltapps strain) gene. Specific growth rates and cell yields were examined in various media and compared with the host KW128 strain. The results indicated that both enzymes participated in pyruvate metabolism, but were not essential. In the presence of maltooligosaccharides, the Deltapyk strain displayed a 15% decrease in growth rate compared with the host strain, indicating that PykTk does participate in glycolysis. However an even more dramatic effect was observed in the Deltapps strain in that the strain could not grow at all on maltooligosaccharides. The results clearly indicate that, in contrast to the conventional EM pathway dependent on pyruvate kinase, PEP synthase is the essential enzyme for the glycolytic conversion of PEP to pyruvate in T. kodakarensis. The physiological roles of the two enzymes under various growth conditions are discussed.
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Affiliation(s)
- Hiroyuki Imanaka
- Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
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Hu Y, Holden JF. Citric acid cycle in the hyperthermophilic archaeon Pyrobaculum islandicum grown autotrophically, heterotrophically, and mixotrophically with acetate. J Bacteriol 2006; 188:4350-5. [PMID: 16740941 PMCID: PMC1482950 DOI: 10.1128/jb.00138-06] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The hyperthermophilic archaeon Pyrobaculum islandicum uses the citric acid cycle in the oxidative and reductive directions for heterotrophic and autotrophic growth, respectively, but the control of carbon flow is poorly understood. P. islandicum was grown at 95 degrees C autotrophically, heterotrophically, and mixotrophically with acetate, H2, and small amounts of yeast extract and with thiosulfate as the terminal electron acceptor. The autotrophic growth rates and maximum concentrations of cells were significantly lower than those in other media. The growth rates on H2 and 0.001% yeast extract with and without 0.05% acetate were the same, but the maximum concentration of cells was fourfold higher with acetate. There was no growth with acetate if 0.001% yeast extract was not present, and addition of H2 to acetate-containing medium greatly increased the growth rates and maximum concentrations of cells. P. islandicum cultures assimilated 14C-labeled acetate in the presence of H2 and yeast extract with an efficiency of 55%. The activities of 11 of 19 enzymes involved in the central metabolism of P. islandicum were regulated under the three different growth conditions. Pyruvate synthase and acetate:coenzyme A (CoA) ligase (ADP-forming) activities were detected only in heterotrophically grown cultures. Citrate synthase activity decreased in autotrophic and acetate-containing cultures compared to the activity in heterotrophic cultures. Acetylated citrate lyase, acetate:CoA ligase (AMP forming), and phosphoenolpyruvate carboxylase activities increased in autotrophic and acetate-containing cultures. Citrate lyase activity was higher than ATP citrate synthase activity in autotrophic cultures. These data suggest that citrate lyase and AMP-forming acetate:CoA ligase, but not ATP citrate synthase, work opposite citrate synthase to control the direction of carbon flow in the citric acid cycle.
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Affiliation(s)
- Yajing Hu
- Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA
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Hakobyan M, Poladyan A, Bagramyan K. Energy transformation coupled to formate oxidation during anaerobic fermentation. Biophysics (Nagoya-shi) 2006. [DOI: 10.1134/s0006350906030122] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
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Tjaden B, Plagens A, Dörr C, Siebers B, Hensel R. Phosphoenolpyruvate synthetase and pyruvate, phosphate dikinase of Thermoproteus tenax: key pieces in the puzzle of archaeal carbohydrate metabolism. Mol Microbiol 2006; 60:287-98. [PMID: 16573681 DOI: 10.1111/j.1365-2958.2006.05098.x] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The interconversion of phosphoenolpyruvate and pyruvate represents an important control point of the Embden-Meyerhof-Parnas (EMP) pathway in Bacteria and Eucarya, but little is known about this site of regulation in Archaea. Here we report on the coexistence of phosphoenolpyruvate synthetase (PEPS) and the first described archaeal pyruvate, phosphate dikinase (PPDK), which, besides pyruvate kinase (PK), are involved in the catalysis of this reaction in the hyperthermophilic crenarchaeote Thermoproteus tenax. The genes encoding T. tenax PEPS and PPDK were cloned and expressed in Escherichia coli, and the enzymic and regulatory properties of the recombinant gene products were analysed. Whereas PEPS catalyses the unidirectional conversion of pyruvate to phosphoenolpyruvate, PPDK shows a bidirectional activity with a preference for the catabolic reaction. In contrast to PK of T. tenax, which is regulated on transcript level but exhibits only limited regulatory potential on protein level, PEPS and PPDK activities are modulated by adenosine phosphates and intermediates of the carbohydrate metabolism. Additionally, expression of PEPS is regulated on transcript level in response to the offered carbon source as revealed by Northern blot analyses. The combined action of the differently regulated enzymes PEPS, PPDK and PK represents a novel way of controlling the interconversion of phosphoenolpyruvate and pyruvate in the reversible EMP pathway, allowing short-term and long-term adaptation to different trophic conditions. Comparative genomic analyses indicate the coexistence of PEPS, PPDK and PK in other Archaea as well, suggesting a similar regulation of the carbohydrate metabolism in these organisms.
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Affiliation(s)
- Britta Tjaden
- Department of Microbiology, Universität Duisburg-Essen, 45117 Essen, Germany.
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Snijders APL, Walther J, Peter S, Kinnman I, de Vos MGJ, van de Werken HJG, Brouns SJJ, van der Oost J, Wright PC. Reconstruction of central carbon metabolism inSulfolobus solfataricus using a two-dimensional gel electrophoresis map, stable isotope labelling and DNA microarray analysis. Proteomics 2006; 6:1518-29. [PMID: 16447154 DOI: 10.1002/pmic.200402070] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
In the last decade, an increasing number of sequenced archaeal genomes have become available, opening up the possibility for functional genomic analyses. Here, we reconstructed the central carbon metabolism in the hyperthermophilic crenarchaeon Sulfolobus solfataricus (glycolysis, gluconeogenesis and tricarboxylic acid cycle) on the basis of genomic, proteomic, transcriptomic and biochemical data. A 2-DE reference map of S. solfataricus grown on glucose, consisting of 325 unique ORFs in 255 protein spots, was created to facilitate this study. The map was then used for a differential expression study based on (15)N metabolic labelling (yeast extract + tryptone-grown cells (YT) vs. glucose-grown cells (G)). In addition, the expression ratio of the genes involved in carbon metabolism was studied using DNA microarrays. Surprisingly, only 3 and 14% of the genes and proteins, respectively, involved in central carbon metabolism showed a greater than two-fold change in expression level. All results are discussed in the light of the current understanding of central carbon metabolism in S. solfataricus and will help to obtain a system-wide understanding of this organism.
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Affiliation(s)
- Ambrosius P L Snijders
- Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, Sheffield, UK
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Siebers B, Schönheit P. Unusual pathways and enzymes of central carbohydrate metabolism in Archaea. Curr Opin Microbiol 2005; 8:695-705. [PMID: 16256419 DOI: 10.1016/j.mib.2005.10.014] [Citation(s) in RCA: 156] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2005] [Accepted: 10/13/2005] [Indexed: 11/29/2022]
Abstract
Sugar-utilizing hyperthermophilic and halophilic Archaea degrade glucose and glucose polymers to acetate or to CO2 using O2, nitrate, sulfur or sulfate as electron acceptors. Comparative analyses of glycolytic pathways in these organisms indicate a variety of differences from the classical Emden-Meyerhof and Entner-Doudoroff pathways that are operative in Bacteria and Eukarya, respectively. The archaeal pathways are characterized by the presence of numerous novel enzymes and enzyme families that catalyze, for example, the phosphorylation of glucose and of fructose 6-phosphate, the isomerization of glucose 6-phosphate, the cleavage of fructose 1,6-bisphosphate, the oxidation of glyceraldehyde 3-phosphate and the conversion of acetyl-CoA to acetate. Recent major advances in deciphering the complexity of archaeal central carbohydrate metabolism were gained by combination of classical biochemical and genomic-based approaches.
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Affiliation(s)
- Bettina Siebers
- Universität Duisburg-Essen, Campus Essen, FB Biologie und Geografie, Mikrobiologie, Universitätsstr.5, D-45117 Essen, Germany
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Ronimus RS, Morgan HW. Distribution and phylogenies of enzymes of the Embden-Meyerhof-Parnas pathway from archaea and hyperthermophilic bacteria support a gluconeogenic origin of metabolism. ARCHAEA-AN INTERNATIONAL MICROBIOLOGICAL JOURNAL 2005; 1:199-221. [PMID: 15803666 PMCID: PMC2685568 DOI: 10.1155/2003/162593] [Citation(s) in RCA: 97] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Enzymes of the gluconeogenic/glycolytic pathway (the Embden-Meyerhof-Parnas (EMP) pathway), the reductive tricarboxylic acid cycle, the reductive pentose phosphate cycle and the Entner-Doudoroff pathway are widely distributed and are often considered to be central to the origins of metabolism. In particular, several enzymes of the lower portion of the EMP pathway (the so-called trunk pathway), including triosephosphate isomerase (TPI; EC 5.3.1.1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12/13), phosphoglycerate kinase (PGK; EC 2.7.2.3) and enolase (EC 4.2.1.11), are extremely well conserved and universally distributed among the three domains of life. In this paper, the distribution of enzymes of gluconeogenesis/glycolysis in hyperthermophiles--microorganisms that many believe represent the least evolved organisms on the planet--is reviewed. In addition, the phylogenies of the trunk pathway enzymes (TPIs, GAPDHs, PGKs and enolases) are examined. The enzymes catalyzing each of the six-carbon transformations in the upper portion of the EMP pathway, with the possible exception of aldolase, are all derived from multiple gene sequence families. In contrast, single sequence families can account for the archaeal and hyperthermophilic bacterial enzyme activities of the lower portion of the EMP pathway. The universal distribution of the trunk pathway enzymes, in combination with their phylogenies, supports the notion that the EMP pathway evolved in the direction of gluconeogenesis, i.e., from the bottom up.
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Affiliation(s)
- Ron S Ronimus
- Thermophile Research Unit, Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, New Zealand.
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Fukui T, Atomi H, Kanai T, Matsumi R, Fujiwara S, Imanaka T. Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes. Genome Res 2005; 15:352-63. [PMID: 15710748 PMCID: PMC551561 DOI: 10.1101/gr.3003105] [Citation(s) in RCA: 357] [Impact Index Per Article: 17.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2004] [Accepted: 12/21/2004] [Indexed: 01/27/2023]
Abstract
The genus Thermococcus, comprised of sulfur-reducing hyperthermophilic archaea, belongs to the order Thermococcales in Euryarchaeota along with the closely related genus Pyrococcus. The members of Thermococcus are ubiquitously present in natural high-temperature environments, and are therefore considered to play a major role in the ecology and metabolic activity of microbial consortia within hot-water ecosystems. To obtain insight into this important genus, we have determined and annotated the complete 2,088,737-base genome of Thermococcus kodakaraensis strain KOD1, followed by a comparison with the three complete genomes of Pyrococcus spp. A total of 2306 coding DNA sequences (CDSs) have been identified, among which half (1165 CDSs) are annotatable, whereas the functions of 41% (936 CDSs) cannot be predicted from the primary structures. The genome contains seven genes for probable transposases and four virus-related regions. Several proteins within these genetic elements show high similarities to those in Pyrococcus spp., implying the natural occurrence of horizontal gene transfer of such mobile elements among the order Thermococcales. Comparative genomics clarified that 1204 proteins, including those for information processing and basic metabolisms, are shared among T. kodakaraensis and the three Pyrococcus spp. On the other hand, among the set of 689 proteins unique to T. kodakaraensis, there are several intriguing proteins that might be responsible for the specific trait of the genus Thermococcus, such as proteins involved in additional pyruvate oxidation, nucleotide metabolisms, unique or additional metal ion transporters, improved stress response system, and a distinct restriction system.
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Affiliation(s)
- Toshiaki Fukui
- Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
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Daniel RM, van Eckert R, Holden JF, Truter J, Crowan DA. The stability of biomolecules and the implications for life at high temperatures. THE SUBSEAFLOOR BIOSPHERE AT MID-OCEAN RIDGES 2004. [DOI: 10.1029/144gm03] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
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Verhees CH, Kengen SWM, Tuininga JE, Schut GJ, Adams MWW, De Vos WM, Van Der Oost J. The unique features of glycolytic pathways in Archaea. Biochem J 2003; 375:231-46. [PMID: 12921536 PMCID: PMC1223704 DOI: 10.1042/bj20021472] [Citation(s) in RCA: 195] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2002] [Revised: 05/16/2003] [Accepted: 08/18/2003] [Indexed: 11/17/2022]
Abstract
An early divergence in evolution has resulted in two prokaryotic domains, the Bacteria and the Archaea. Whereas the central metabolic routes of bacteria and eukaryotes are generally well-conserved, variant pathways have developed in Archaea involving several novel enzymes with a distinct control. A spectacular example of convergent evolution concerns the glucose-degrading pathways of saccharolytic archaea. The identification, characterization and comparison of the glycolytic enzymes of a variety of phylogenetic lineages have revealed a mosaic of canonical and novel enzymes in the archaeal variants of the Embden-Meyerhof and the Entner-Doudoroff pathways. By means of integrating results from biochemical and genetic studies with recently obtained comparative and functional genomics data, the structure and function of the archaeal glycolytic routes, the participating enzymes and their regulation are re-evaluated.
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Affiliation(s)
- Corné H Verhees
- Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands
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Schut GJ, Brehm SD, Datta S, Adams MWW. Whole-genome DNA microarray analysis of a hyperthermophile and an archaeon: Pyrococcus furiosus grown on carbohydrates or peptides. J Bacteriol 2003; 185:3935-47. [PMID: 12813088 PMCID: PMC161589 DOI: 10.1128/jb.185.13.3935-3947.2003] [Citation(s) in RCA: 105] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The first complete-genome DNA microarray was constructed for a hyperthermophile or a nonhalophilic archaeon by using the 2,065 open reading frames (ORFs) that have been annotated in the genome of Pyrococcus furiosus (optimal growth temperature, 100 degrees C). This was used to determine relative transcript levels in cells grown at 95 degrees C with either peptides or a carbohydrate (maltose) used as the primary carbon source. Approximately 20% (398 of 2065) of the ORFs did not appear to be significantly expressed under either growth condition. Of the remaining 1,667 ORFs, the expression of 125 of them (8%) differed by more than fivefold between the two cultures, and 82 of the 125 (65%) appear to be part of operons, indicating extensive coordinate regulation. Of the 27 operons that are regulated, 5 of them encode (conserved) hypothetical proteins. A total of 18 operons are up-regulated (greater than fivefold) in maltose-grown cells, including those responsible for maltose transport and for the biosynthesis of 12 amino acids, of ornithine, and of citric acid cycle intermediate products. A total of nine operons are up-regulated (greater than fivefold) in peptide-grown cells, including those encoding enzymes involved in the production of acyl and aryl acids and 2-ketoacids, which are used for energy conservation. Analyses of the spent growth media confirmed the production of branched-chain and aromatic acids during growth on peptides. In addition, six nonlinked enzymes in the pathways of sugar metabolism were regulated more than fivefold--three in maltose-grown cells that are unique to the unusual glycolytic pathway and three in peptide-grown cells that are unique to gluconeogenesis. The catalytic activities of 16 metabolic enzymes whose expression appeared to be highly regulated in the two cell types correlated very well with the microarray data. The degree of coordinate regulation revealed by the microarray data was unanticipated and shows that P. furiosus can readily adapt to a change in its primary carbon source.
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Affiliation(s)
- Gerrit J Schut
- Department of Biochemistry and Molecular Biology and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA
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Sapra R, Bagramyan K, Adams MWW. A simple energy-conserving system: proton reduction coupled to proton translocation. Proc Natl Acad Sci U S A 2003; 100:7545-50. [PMID: 12792025 PMCID: PMC164623 DOI: 10.1073/pnas.1331436100] [Citation(s) in RCA: 204] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Oxidative phosphorylation involves the coupling of ATP synthesis to the proton-motive force that is generated typically by a series of membrane-bound electron transfer complexes, which ultimately reduce an exogenous terminal electron acceptor. This is not the case with Pyrococcus furiosus, an archaeon that grows optimally near 100 degrees C. It has an anaerobic respiratory system that consists of a single enzyme, a membrane-bound hydrogenase. Moreover, it does not require an added electron acceptor as the enzyme reduces protons, the simplest of acceptors, to hydrogen gas by using electrons from the cytoplasmic redox protein ferredoxin. It is demonstrated that the production of hydrogen gas by membrane vesicles of P. furiosus is directly coupled to the synthesis of ATP by means of a proton-motive force that has both electrochemical and pH components. Such a respiratory system enables rationalization in this organism of an unusual glycolytic pathway that was previously thought not to conserve energy. It is now clear that the use of ferredoxin in place of the expected NAD as the electron acceptor for glyceraldehyde 3-phosphate oxidation enables energy to be conserved by hydrogen production. In addition, this simple respiratory mechanism readily explains why the growth yields of P. furiosus are much higher than could be accounted for if ATP synthesis occurred only by substrate-level phosphorylation. The ability of microorganisms such as P. furiosus to couple hydrogen production to energy conservation has important ramifications not only in the evolution of respiratory systems but also in the origin of life itself.
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Affiliation(s)
- Rajat Sapra
- Department of Biochemistry and Molecular Biology, Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602-7229, USA
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Wu YQ, Jiang PH, Fan CS, Wang JG, Shang L, Huang WD. Co-expression of five genes in E coli for L-phenylalanine in Brevibacterium flavum. World J Gastroenterol 2003; 9:342-6. [PMID: 12532463 PMCID: PMC4611343 DOI: 10.3748/wjg.v9.i2.342] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the effect of co-expression of ppsA, pckA, aroG, pheA and tyrB genes on the production of L-phenylalanine, and to construct a genetic engineering strain for L-phenylalanine.
METHODS: ppsA and pckA genes were amplified from genomic DNA of E. coli by polymerase chain reaction, and then introduced into shuttle vectors between E coli and Brevibacterium flavum to generate constructs pJN2 and pJN5. pJN2 was generated by inserting ppsA and pckA genes into vector pCZ; whereas pJN5 was obtained by introducing ppsA and pckA genes into pCZ-GAB, which was originally constructed for co-expression of aroG, pheA and tyrB genes. The recombinant plasmids were then introduced into B. flavum by electroporation and the transformants were used for L-phenylalanine fermentation.
RESULTS: Compared with the original B. flavum cells, all the transformants were showed to have increased five enzyme activities specifically, and have enhanced L-phenylalanine biosynthesis ability variably. pJN5 transformant was observed to have the highest elevation of L-phenylalanine production by a 3.4-fold. Co-expression of ppsA and pckA increased activity of DAHP synthetase significantly.
CONCLUSION: Co-expression of ppsA and pckA genes in B. flavum could remarkably increase the expression of DAHP synthetase; Co-expression of ppsA, pckA, aroG, pheA and tyrB of E. coli in B. flavum was a feasible approach to construct a strain for phenylalanine production.
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Affiliation(s)
- Yong-Qing Wu
- Department of Microbiology, School of Life Science, Fudan University, 220 Han Dan Road, Shanghai 200433, China
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Bhuiya MW, Sakuraba H, Ohshima T. Temperature dependence of kinetic parameters for hyperthermophilic glutamate dehydrogenase from Aeropyrum pernix K1. Biosci Biotechnol Biochem 2002; 66:873-6. [PMID: 12036066 DOI: 10.1271/bbb.66.873] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The temperature dependence of the steady-state kinetic parameters for a glutamate dehydrogenase from Aeropyrum pernix K1 was investigated. The enzyme showed a biphasic kinetic characteristic for L-glutamate and a monophasic one for NADP at 50-90 degrees C. At low concentrations of L-glutamate the Km decreased from 2.02 to 0.56 mM and the catalytic efficiency (Vmax/Km) markedly increased (4-150 micromol x mg(-1) x mM(-1)) along with the increase of temperature from 50 to 90 degrees C. At high concentrations of the substrate the Km was fairly high and approximately constant (around 225 mM), and the catalytic efficiency was low and its temperature-dependent change was small. The Km (0.039 mM) for NADP did not change with the increase of temperature. In the reductive amination, the Kms for 2-oxoglutarate (1.81 and 9.37 mM at low and high levels of ammonia, respectively) were independent on temperature, but the Kms for ammonia and NADPH rose from 86 to 185 mM and 0.050 to 0.175 mM, respectively.
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Affiliation(s)
- Mohammad W Bhuiya
- Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Japan
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Schut GJ, Zhou J, Adams MW. DNA microarray analysis of the hyperthermophilic archaeon Pyrococcus furiosus: evidence for anNew type of sulfur-reducing enzyme complex. J Bacteriol 2001; 183:7027-36. [PMID: 11717259 PMCID: PMC95549 DOI: 10.1128/jb.183.24.7027-7036.2001] [Citation(s) in RCA: 80] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2001] [Accepted: 09/21/2001] [Indexed: 01/01/2023] Open
Abstract
DNA microarrays were constructed by using 271 open reading frame (ORFs) from the genome of the archaeon Pyrococcus furiosus. They were used to investigate the effects of elemental sulfur (S(primary)) on the levels of gene expression in cells grown at 95 degrees C with maltose as the carbon source. The ORFs included those that are proposed to encode proteins mainly involved in the pathways of sugar and peptide catabolism, in the metabolism of metals, and in the biosynthesis of various cofactors, amino acids, and nucleotides. The expression of 21 ORFs decreased by more than fivefold when cells were grown with S(primary) and, of these, 18 encode subunits associated with three different hydrogenase systems. The remaining three ORFs encode homologs of ornithine carbamoyltransferase and HypF, both of which appear to be involved in hydrogenase biosynthesis, as well as a conserved hypothetical protein. The expression of two previously uncharacterized ORFs increased by more than 25-fold when cells were grown with S(primary). Their products, termed SipA and SipB (for sulfur-induced proteins), are proposed to be part of a novel S(primary)-reducing, membrane-associated, iron-sulfur cluster-containing complex. Two other previously uncharacterized ORFs encoding a putative flavoprotein and a second FeS protein were upregulated more than sixfold in S(primary)-grown cells, and these are also thought be involved in S(primary) reduction. Four ORFs that encode homologs of proteins involved in amino acid metabolism were similarly upregulated in S(primary)-grown cells, a finding consistent with the fact that growth on peptides is a S(primary)-dependent process. An ORF encoding a homolog of the eukaryotic rRNA processing protein, fibrillarin, was also upregulated sixfold in the presence of S(primary), although the reason for this is as yet unknown. Of the 20 S(primary)-independent ORFs that are the most highly expressed (at more than 20 times the detection limit), 12 of them represent enzymes purified from P. furiosus, but none of the products of the 34 S(primary)-independent ORFs that are not expressed above the detection limit have been characterized. These results represent the first derived from the application of DNA microarrays to either an archaeon or a hyperthermophile.
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Affiliation(s)
- G J Schut
- Department of Biochemistry and Molecular Biology and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA
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Adams MW, Holden JF, Menon AL, Schut GJ, Grunden AM, Hou C, Hutchins AM, Jenney FE, Kim C, Ma K, Pan G, Roy R, Sapra R, Story SV, Verhagen MF. Key role for sulfur in peptide metabolism and in regulation of three hydrogenases in the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol 2001; 183:716-24. [PMID: 11133967 PMCID: PMC94929 DOI: 10.1128/jb.183.2.716-724.2001] [Citation(s) in RCA: 128] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100 degrees C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S(0)). Growth rates were highest on media containing peptides and S(0), with or without maltose. Growth did not occur on the peptide medium without S(0). S(0) had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S(0) with or without maltose were the same, suggesting that S(0) is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S(0) in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S(0)-reducing enzyme in this organism and the mechanism of the S(0) dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.
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Affiliation(s)
- M W Adams
- Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229, USA.
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