scholarly journals Glucose metabolism and acetate switch in archaea: Analysis of the enzymes involved in Haloferax volcanii

2021 ◽  
Author(s):  
Tom Kuprat ◽  
Marius Ortjohann ◽  
Ulrike Johnsen ◽  
Peter Schönheit
Keyword(s):  
Phylogenetic Analyses ◽  
Comprehensive Analysis ◽  
Haloferax Volcanii ◽  
Phosphoglycerate Mutase ◽  
Deletion Mutants ◽  
Aerobic Growth ◽  
Acetyl Coa ◽  
Functional Involvement ◽  
Growth Experiments

The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. Following our previous studies on key enzymes of this pathway, we now focus on the characterization of enzymes involved in 3-phosphoglycerate conversion to pyruvate, in anaplerosis and in acetyl-CoA formation from pyruvate. These enzymes include phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenolpyruvate carboxylase and pyruvate: ferredoxin oxidoreductase. The essential function of these enzymes were shown by transcript analyses and growth experiments with respective deletion mutants. Further, it is shown that H. volcanii - during aerobic growth on glucose - excreted significant amounts of acetate, which was consumed in the stationary phase (acetate switch). The enzyme catalyzing the conversion of acetyl-CoA to acetate as part of the acetate overflow mechanism, an ADP-forming acetyl-CoA synthetase (ACD), was characterized. The functional involvement of ACD in acetate formation and of AMP-forming acetyl-CoA synthetases (ACSs) in activation of excreted acetate was proven by using respective deletion mutants. Together, the data provide a comprehensive analysis of enzymes of the spED pathway, of anaplerosis, and report first genetic evidence of the functional involvement of enzymes of the acetate switch in archaea. Importance: In this work we provide a comprehensive analysis of glucose degradation via the semiphosphorylative Entner-Doudoroff pathway in the haloarchaeal model organism Haloferax volcanii. The study includes transcriptional analyses, growth experiments with deletion mutants and characterization of all enzymes involved in the conversion of 3-phosphoglycerate to acetyl-CoA and in anaplerosis. Phylogenetic analyses of several enzymes indicate various lateral gene transfer events from bacteria to haloarchaea. Further, we analyzed the key players involved in the acetate switch, i.e in the formation (overflow) and subsequent consumption of acetate during aerobic growth on glucose. Together, the data provide novel aspects on glucose degradation, anaplerosis and acetate switch in H. volcanii and thus expand our understanding of the unusual sugar metabolism in archaea.

scholarly journals Acetate Metabolism in Archaea: Characterization of an Acetate Transporter and of Enzymes Involved in Acetate Activation and Gluconeogenesis in Haloferax volcanii

2020 ◽  
Vol 11 ◽  
Author(s):  
Tom Kuprat ◽  
Ulrike Johnsen ◽  
Marius Ortjohann ◽  
Peter Schönheit
Keyword(s):  
Isocitrate Lyase ◽  
Glyoxylate Cycle ◽  
Phylogenetic Analyses ◽  
Haloferax Volcanii ◽  
Acetate Metabolism ◽  
Knock Out ◽  
Functional Involvement ◽  
Acetate Activation ◽  
Growth Experiments

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.

D-galactose catabolism in archaea: operation of the DeLey–Doudoroff pathway in Haloferax volcanii

2020 ◽  
Vol 367 (1) ◽  
Author(s):  
Julia-Beate Tästensen ◽  
Ulrike Johnsen ◽  
Andreas Reinhardt ◽  
Marius Ortjohann ◽  
Peter Schönheit
Keyword(s):  
Transcriptional Regulator ◽  
High Specificity ◽  
Comprehensive Analysis ◽  
Haloferax Volcanii ◽  
Knock Out ◽  
Genes Encoding ◽  
Functional Involvement ◽  
Genome Analyses ◽  
Galactose Dehydrogenase ◽  
Substrate Binding Protein

ABSTRACT The haloarchaeon Haloferax volcanii was found to grow on D-galactose as carbon and energy source. Here we report a comprehensive analysis of D-galactose catabolism in H. volcanii. Genome analyses indicated a cluster of genes encoding putative enzymes of the DeLey–Doudoroff pathway for D-galactose degradation including galactose dehydrogenase, galactonate dehydratase, 2-keto-3-deoxygalactonate kinase and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase. The recombinant galactose dehydrogenase and galactonate dehydratase showed high specificity for D-galactose and galactonate, respectively, whereas KDPGal aldolase was promiscuous in utilizing KDPGal and also the C4 epimer 2-keto-3-deoxy-6-phosphogluconate as substrates. Growth studies with knock-out mutants indicated the functional involvement of galactose dehydrogenase, galactonate dehydratase and KDPGal aldolase in D-galactose degradation. Further, the transcriptional regulator GacR was identified, which was characterized as an activator of genes of the DeLey–Doudoroff pathway. Finally, genes were identified encoding components of an ABC transporter and a knock-out mutant of the substrate binding protein indicated the functional involvement of this transporter in D-galactose uptake. This is the first report of D-galactose degradation via the DeLey–Doudoroff pathway in the domain of archaea.

scholarly journals Characterization of Copy Number Control of Two Haloferax volcanii Replication Origins Using Deletion Mutants and Haloarchaeal Artificial Chromosomes

2017 ◽  
Vol 200 (1) ◽  
Author(s):  
Sandy Maurer ◽  
Katharina Ludt ◽  
Jörg Soppa
Keyword(s):  
Copy Number ◽  
Haloferax Volcanii ◽  
Deletion Mutants ◽  
Genome Copy ◽  
Content Type ◽  
Artificial Chromosomes ◽  
Chromosome Copy Number ◽  
The Core ◽  
Genome Copy Number

ABSTRACT Haloferax volcanii is polyploid and contains about 20 genome copies under optimal conditions. However, the chromosome copy number is highly regulated and ranges from two during phosphate starvation to more than 40 under conditions of phosphate surplus. The aim of this study was the characterization of the influence of two replication origins on the genome copy number. The origin repeats and the genes encoding origin recognition complex (ORC) proteins were deleted. The core origin oriC1-orc1 (ori1) deletion mutant had a lower genome copy number and a higher level of fitness than the wild type, in stark contrast to the oriC2-orc5 (ori2) deletion mutant. The genes adjacent to ori1 could not be deleted, and thus, at least two of them are probably essential, while deletion of the genes adjacent to ori2 was possible. Various fragments of and around the origins were cloned into a suicide plasmid to generate haloarchaeal artificial chromosomes (HACs). The copy number of the oriC1-orc1 HAC was much higher than that of the oriC2-orc5 HAC. The addition of adjacent genes influenced both the HAC copy number and the chromosome copy number. The results indicate that the origins of H. volcanii are not independent but that the copy number is regulated via a network of genes around the origins. IMPORTANCE Several species of archaea have more than one origin of replication on their major chromosome and are thus the only known prokaryotic species that allow the analysis of the evolution of multiorigin replication. The widely studied Haloferax volcanii H26 strain has a major chromosome with four origins of replication. Two origins, ori1 and ori2, were chosen for an in-depth analysis using deletion mutants and haloarchaeal artificial chromosomes. The analysis was not restricted to the core origin regions; origin-adjacent genes were also included. Because H. volcanii is polyploid, the effects on the chromosome copy number were of specific importance. The results revealed extreme differences between the two origins.

scholarly journals Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase

2016 ◽  
Vol 198 (16) ◽  
pp. 2251-2262 ◽  
Author(s):  
Jan-Moritz Sutter ◽  
Julia-Beate Tästensen ◽  
Ulrike Johnsen ◽  
Jörg Soppa ◽  
Peter Schönheit
Keyword(s):  
Glucose Dehydrogenase ◽  
Haloferax Volcanii ◽  
Recombinant Enzymes ◽  
Prolonged Incubation ◽  
Content Type ◽  
Key Enzymes ◽  
Knockout Mutants ◽  
Functional Involvement

ABSTRACTThe halophilic archaeonHaloferax volcaniihas been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. So far, the key enzymes of this pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C4epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified fromH. volcanii, and the encoding gene,gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the Δgadmutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for thein vivooperation of the spED pathway for glucose degradation inH. volcanii.IMPORTANCEThe work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff pathway in the haloarchaeonHaloferax volcanii. The functional involvement of the three enzymes was proven by analyses of the corresponding knockout mutants. These results provide evidence for thein vivooperation of the semiphosphorylative Entner-Doudoroff pathway in haloarchaea and thus expand our understanding of the unusual sugar degradation pathways in the domainArchaea.

scholarly journals Functional and Molecular Characterization of the Halomicrobium sp. IBSBa Inulosucrase

2021 ◽  
Vol 9 (4) ◽  
pp. 749
Author(s):  
Gülbahar Abaramak ◽  
Jaime Ricardo Porras-Domínguez ◽  
Henry Christopher Janse van Rensburg ◽  
Eveline Lescrinier ◽  
Ebru Toksoy Öner ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Phylogenetic Analyses ◽  
Halophilic Archaea ◽  
Maximum Activity ◽  
Physiological Relevance ◽  
Health Related ◽  
Specific Alternative ◽  
Binding Groove ◽  
Native Host

Fructans are fructose-based (poly)saccharides with inulin and levan being the best-known ones. Thanks to their health-related benefits, inulin-type fructans have been under the focus of scientific and industrial communities, though mostly represented by plant-based inulins, and rarely by microbial ones. Recently, it was discovered that some extremely halophilic Archaea are also able to synthesize fructans. Here, we describe the first in-depth functional and molecular characterization of an Archaeal inulosucrase from Halomicrobium sp. IBSBa (HmcIsc). The HmcIsc enzyme was recombinantly expressed and purified in Escherichia coli and shown to synthesize inulin as proven by nuclear magnetic resonance (NMR) analysis. In accordance with the halophilic lifestyle of its native host, the enzyme showed maximum activity at very high NaCl concentrations (3.5 M), with specific adaptations for that purpose. Phylogenetic analyses suggested that Archaeal inulosucrases have been acquired from halophilic bacilli through horizontal gene transfer, with a HX(H/F)T motif evolving further into a HXHT motif, together with a unique D residue creating the onset of a specific alternative acceptor binding groove. This work uncovers a novel area in fructan research, highlighting unexplored aspects of life in hypersaline habitats, and raising questions about the general physiological relevance of inulosucrases and their products in nature.

scholarly journals Isolation and characterization of the acetyl-CoA synthetase from Penicillium chrysogenum. Involvement of this enzyme in the biosynthesis of penicillins.

1992 ◽  
Vol 267 (8) ◽  
pp. 5474-5481
Author(s):  
H Martínez-Blanco ◽  
A Reglero ◽  
M Fernández-Valverde ◽  
M.A. Ferrero ◽  
M.A. Moreno ◽  
...  
Keyword(s):  
Penicillium Chrysogenum ◽  
Acetyl Coa ◽  
Isolation And Characterization

scholarly journals The Underlying Order Induced by Orthogonality and the Quantum Speed Limit

2021 ◽  
Vol 3 (3) ◽  
pp. 376-388
Author(s):  
Francisco J. Sevilla ◽  
Andrea Valdés-Hernández ◽  
Alan J. Barrios
Keyword(s):  
Energy Spectrum ◽  
Energy Distribution ◽  
Finite Time ◽  
Complete Characterization ◽  
Comprehensive Analysis ◽  
Orthogonality Condition ◽  
Speed Limit ◽  
Physical Quantities

We perform a comprehensive analysis of the set of parameters {ri} that provide the energy distribution of pure qutrits that evolve towards a distinguishable state at a finite time τ, when evolving under an arbitrary and time-independent Hamiltonian. The orthogonality condition is exactly solved, revealing a non-trivial interrelation between τ and the energy spectrum and allowing the classification of {ri} into families organized in a 2-simplex, δ2. Furthermore, the states determined by {ri} are likewise analyzed according to their quantum-speed limit. Namely, we construct a map that distinguishes those ris in δ2 correspondent to states whose orthogonality time is limited by the Mandelstam–Tamm bound from those restricted by the Margolus–Levitin one. Our results offer a complete characterization of the physical quantities that become relevant in both the preparation and study of the dynamics of three-level states evolving towards orthogonality.

scholarly journals Electrophysiological characterization of a diverse group of sugar transporters from Trichoderma reesei

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sami Havukainen ◽  
Jonai Pujol-Giménez ◽  
Mari Valkonen ◽  
Ann Westerholm-Parvinen ◽  
Matthias A. Hediger ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Scientific Literature ◽  
Large Body ◽  
Comprehensive Analysis ◽  
Transport Function ◽  
Sugar Transporters ◽  
New Knowledge ◽  
Electrophysiological Characterization ◽  
Shed Light

AbstractTrichoderma reesei is an ascomycete fungus known for its capability to secrete high amounts of extracellular cellulose- and hemicellulose-degrading enzymes. These enzymes are utilized in the production of second-generation biofuels and T. reesei is a well-established host for their production. Although this species has gained considerable interest in the scientific literature, the sugar transportome of T. reesei remains poorly characterized. Better understanding of the proteins involved in the transport of different sugars could be utilized for engineering better enzyme production strains. In this study we aimed to shed light on this matter by characterizing multiple T. reesei transporters capable of transporting various types of sugars. We used phylogenetics to select transporters for expression in Xenopus laevis oocytes to screen for transport activities. Of the 18 tested transporters, 8 were found to be functional in oocytes. 10 transporters in total were investigated in oocytes and in yeast, and for 3 of them no transport function had been described in literature. This comprehensive analysis provides a large body of new knowledge about T. reesei sugar transporters, and further establishes X. laevis oocytes as a valuable tool for studying fungal sugar transporters.

scholarly journals Past and future trends of Egypt’s water consumption and its sources

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Catherine A. Nikiel ◽  
Elfatih A. B. Eltahir
Keyword(s):  
Economic Growth ◽  
Virtual Water ◽  
Comprehensive Analysis ◽  
Future Trends ◽  
Nile Water ◽  
The Gift ◽  
Per Capita ◽  
The 1960S

AbstractFor millennia the Nile supplied Egypt with more water than needed. As the population grew and the economy expanded, demand on water increased accordingly. Here, we present a comprehensive analysis to reconstruct how total demand on water outstripped supply of the Nile water in the late 1970s, starting from a surplus of about 20 km3 per year in the 1960s leading to a deficit of about 40 km3 per year by the late 2010s. The gap is satisfied by import of virtual water. The role of economic growth in driving per capita demand on water is quantified based on detailed analysis of water use by agriculture and other sectors. We develop and test an empirical model of water demand in Egypt that relates demand on water to growth rates in the economy and population. Looking forward, we project that within this decade of the 2020 s, under nominal scenarios of population and economic growth, Egypt is likely to import more virtual water than the water supplied by the Nile, bringing into question the historical characterization of Egypt as “the gift of the Nile”.

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