Article

# Genetic Evidence Identifying the True Gluconeogenic Fructose-1,6-Bisphosphatase in Thermococcus kodakaraensis and Other Hyperthermophiles

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
(Impact Factor: 2.81). 10/2004; 186(17):5799-807. DOI: 10.1128/JB.186.17.5799-5807.2004
Source: PubMed

ABSTRACT

Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes in gluconeogenesis. Although FBPase activity has been detected
in several hyperthermophiles, no orthologs corresponding to the classical FBPases from bacteria and eukaryotes have been identified
in their genomes. An inositol monophosphatase (IMPase) from Methanococcus jannaschii which displayed both FBPase and IMPase activities and a structurally novel FBPase (FbpTk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 have been proposed as the “missing” FBPase. For this study, using T. kodakaraensis, we took a genetic approach to elucidate which candidate is the major gluconeogenic enzyme in vivo. The IMPase/FBPase ortholog
in T. kodakaraensis, ImpTk, was confirmed to possess high FBPase activity along with IMPase activity, as in the case of other orthologs. We therefore
constructed Δfbp and Δimp strains by applying a gene disruption system recently developed for T. kodakaraensis and investigated their phenotypes. The Δfbp strain could not grow under gluconeogenic conditions while glycolytic growth was unimpaired, and the disruption resulted
in the complete abolishment of intracellular FBPase activity. Evidently, fbpTk is an indispensable gene for gluconeogenesis and is responsible for almost all intracellular FBPase activity. In contrast,
the endogenous impTk gene could not complement the defect of the fbp deletion, and its disruption did not lead to any detectable phenotypic changes under the conditions examined. These facts
indicated that impTk is irrelevant to gluconeogenesis, despite the high FBPase activity of its protein product, probably due to insufficient transcription.
Our results provide strong evidence that the true FBPase for gluconeogenesis in T. kodakaraensis is the FbpTk ortholog, not the IMPase/FBPase ortholog.

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Available from: Naeem Rashid,
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• "The genome harbors pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) and proton-translocating pyrophosphatase, which have been identified in methanotrophic and thiotrophic Gammaproteobacteria [35], [55], [56]. However, typical bacterial fructose 1,6-bisphosphatases (class 1 and 2) and their possible alternatives, such as archaeal ADP-dependent phosphofructokinase [57] and recently identified fructose 1,6 bisphosphate aldolase/phosphatase [58], were not identified. We checked the enzymatic activities of bacterial 6-phosphofructokinases and fructose 1,6-bisphosphatases in the cell-free extract prepared from cells grown chemolithoautotrophically. Consumption and production activities of fructose 6-phoshate by PPi-PFK were determined to be 0.66 and 0.15 µmol/min/mg protein, respectively, in the cell-free extract at 37°C. "
##### Article: Physiological and Genomic Features of a Novel Sulfur-Oxidizing Gammaproteobacterium Belonging to a Previously Uncultivated Symbiotic Lineage Isolated from a Hydrothermal Vent
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ABSTRACT: Strain Hiromi 1, a sulfur-oxidizing gammaproteobacterium was isolated from a hydrothermal vent chimney in the Okinawa Trough and represents a novel genus that may include a phylogenetic group found as endosymbionts of deep-sea gastropods. The SSU rRNA gene sequence similarity between strain Hiromi 1 and the gastropod endosymbionts was approximately 97%. The strain was shown to grow both chemolithoautotrophically and chemolithoheterotrophically with an energy metabolism of sulfur oxidation and O2 or nitrate reduction. Under chemolithoheterotrophic growth conditions, the strain utilized organic acids and proteinaceous compounds as the carbon and/or nitrogen sources but not the energy source. Various sugars did not support growth as a sole carbon source. The observation of chemolithoheterotrophy in this strain is in line with metagenomic analyses of endosymbionts suggesting the occurrence of chemolithoheterotrophy in gammaproteobacterial symbionts. Chemolithoheterotrophy and the presence of homologous genes for virulence- and quorum sensing-related functions suggest that the sulfur-oxidizing chomolithotrophic microbes seek animal bodies and microbial biofilm formation to obtain supplemental organic carbons in hydrothermal ecosystems.
PLoS ONE 08/2014; 9(8):e104959. DOI:10.1371/journal.pone.0104959 · 3.23 Impact Factor
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• "Disruption of the reverse gyrase gene indicated that the enzyme provides an advantage to cells when grown at temperatures of 80 • C or higher (Atomi et al., 2004). In terms of metabolism, genes examined include those involved in glycolysis (Imanaka et al., 2006; Matsubara et al., 2011), gluconeogenesis (Sato et al., 2004), pentose metabolism (Orita et al., 2006), as well as coenzyme A (Yokooji et al., 2009), polyamine (Morimoto et al., 2010), and compatible solute biosynthesis (Borges et al., 2010). Gene disruption of three putative hydrogenase gene clusters and phenotypic analyses indicated that the cytosolic hydrogenase Hyh and the membrane-bound oxidoreductase complexes Mbh and Mbx are involved in H 2 consumption, H 2 generation, and H 2 S generation, respectively (Kanai et al., 2011). "
##### Article: Overview of the genetic tools in the Archaea
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ABSTRACT: This section provides an overview of the genetic systems developed in the Archaea. Genetic manipulation is possible in many members of the halophiles, methanogens, Sulfolobus, and Thermococcales. We describe the selection/counterselection principles utilized in each of these groups, which consist of antibiotics and their resistance markers, and auxotrophic host strains and complementary markers. The latter strategy utilizes techniques similar to those developed in yeast. However, Archaea are resistant to many of the antibiotics routinely used for selection in the Bacteria, and a number of strategies specific to the Archaea have been developed. In addition, examples utilizing the genetic systems developed for each group will be briefly described.
Frontiers in Microbiology 10/2012; 3:337. DOI:10.3389/fmicb.2012.00337 · 3.99 Impact Factor
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• "New avenues, based on advances in T. kodakarensis genetics, permit direct characterization of innumerable archaeal enzymes and their chemistries (Atomi et al., 2001, 2004a; Shiraki et al., 2003; Fukuda et al., 2004, 2008; Rashid et al., 2004; Sato et al., 2004, 2007; Imanaka et al., 2006; Murakami et al., 2006; Orita et al., 2006; Kanai et al., 2007, 2010, 2011; Danno et al., 2008; Fujiwara et al., 2008; Louvel et al., 2009; Yokooji et al., 2009; Borges et al., 2010; Kobori et al., 2010; Morimoto et al., 2010; Matsubara et al., 2011), provide industrially relevant alternative biofuel platforms(Kanai et al., 2005, 2011; Chou et al., 2008; Kim et al., 2010; Atomi et al., 2011; Santangelo et al., 2011; Bae et al., 2012; Davidova et al., 2012), unlock the largely untapped reservoir of archaeal encoded natural products (Atomi, 2005; Kim and Peeples, 2006; Littlechild, 2011; Matsumi et al., 2011; Sato and Atomi, 2011), and offer the opportunity to dissect eukaryotic-like information processing systems composed of minimal components (Yamamoto et al., 2003; Santangelo and Reeve, 2006, 2010a; Kanai et al., 2007; Santangelo et al., 2007, 2008a, 2009; Hirata et al., 2008; Dev et al., 2009; Yamaji et al., 2009; Fujikane et al., 2010; Li et al., 2010, 2011; Ishino et al., 2011; Nunoura et al., 2011; Pan et al., 2011; Santangelo and Artsimovitch, 2011). "
##### Article: Genetics Techniques for Thermococcus kodakarensis
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ABSTRACT: Thermococcus kodakarensis (T. kodakarensis) has emerged as a premier model system for studies of archaeal biochemistry, genetics, and hyperthermophily. This prominence is derived largely from the natural competence of T. kodakarensis and the comprehensive, rapid, and facile techniques available for manipulation of the T. kodakarensis genome. These genetic capacities are complemented by robust planktonic growth, simple selections, and screens, defined in vitro transcription and translation systems, replicative expression plasmids, in vivo reporter constructs, and an ever-expanding knowledge of the regulatory mechanisms underlying T. kodakarensis metabolism. Here we review the existing techniques for genetic and biochemical manipulation of T. kodakarensis. We also introduce a universal platform to generate the first comprehensive deletion and epitope/affinity tagged archaeal strain libraries.
Frontiers in Microbiology 06/2012; 3:195. DOI:10.3389/fmicb.2012.00195 · 3.99 Impact Factor