Standards in Genomic Sciences (2011) 4:131-143 DOI:10.4056/sigs.1463589 Complete genome sequence of Hydrogenobacter thermophilus type strain (TK-6T) Ahmet Zeytun1,3, Johannes Sikorski2, Matt Nolan1, Alla Lapidus1, Susan Lucas1, James Han1, Hope Tice1, Jan-Fang Cheng1, Roxanne Tapia1,3, Lynne Goodwin1,3, Sam Pitluck1, Konstantinos Liolios1, Natalia Ivanova1, Konstantinos Mavromatis1, Natalia Mikhailova1, Galina Ovchinnikova1, Amrita Pati1, Amy Chen4, Krishna Palaniappan4, Olivier D. Ngatchou-Djao5, Miriam Land1,6, Loren Hauser1,6, Cynthia D. Jeffries1,6, Cliff Han1,3, John C. Detter1,3, Susanne Übler7, Manfred Rohde5, Brian J. Tindall2, Markus Göker2, Reinhard Wirth7, Tanja Woyke1, James Bristow1, Jonathan A. Eisen1,8, Victor Markowitz4, Philip Hugenholtz1,9, Hans-Peter Klenk2, and Nikos C. Kyrpides1* 1 DOE Joint Genome Institute, Walnut Creek, California, USA 2 DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany 3 Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA 4 Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory, Berkeley, California, USA 5 HZI – Helmholtz Centre for Infection Research, Braunschweig, Germany 6 Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA 7 Archaea Centre - University of Regensburg, Regensburg, Germany 8 University of California Davis Genome Center, Davis, California, USA 9 Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia *Corresponding author: Nikos C. Kyrpides Keywords: strictly thermophilic, obligately chemolithoautotrophic, Gram-negative, aerobic, hydrogen-oxidizing, nonmotile, non sporeforming, rod shaped, Aquificaceae, Aquificae, GE- BA Hydrogenobacter thermophilus Kawasumi et al. 1984 is the type species of the genus Hydro- genobacter. H. thermophilus was the first obligate autotrophic organism reported among aerobic hydrogen-oxidizing bacteria. Strain TK-6T is of interest because of the unusually effi- cient hydrogen-oxidizing ability of this strain, which results in a faster generation time com- pared to other autotrophs. It is also able to grow anaerobically using nitrate as an electron acceptor when molecular hydrogen is used as the energy source, and able to aerobically fix CO2 via the reductive tricarboxylic acid cycle. This is the fifth completed genome sequence in the family Aquificaceae, and the second genome sequence determined from a strain de- rived from the original isolate. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,742,932 bp long genome with its 1,899 protein-coding and 49 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project. Introduction Strain TK-6T (= DSM 6534 = JCM 7687 = NBRC based on page priority, a later heterotypic synonym 102181) is the type strain of Hydrogenobacter of Hydrogenobacter Kawasumi et al. 1984 [3], be- thermophilus, which in turn is the type species of cause of similar genetic, phenotypic and biochemical the genus Hydrogenobacter [1]. Currently, there are properties between the type strains of H. thermophi- four species in the genus Hydrogenobacter, one of lus and Calderobacterium hydrogenophilum. De- which has subsequently been reclassified as Hydro- spite the relatively high degree of 16S rRNA gene genobaculum acidophilum. Strain TK-6T was pre- sequence similarity between the two species, DNA- viously isolated by Kawasumi in 1980 [2]. The ge- DNA hybridization [4] indicates that they may be nus name Calderobacterium Kryukov et al. 1984 is, considered to be different species within the genus The Genomic Standards Consortium Hydrogenobacter thermophilus type strain (TK-6T) Hydrogenobacter [3]. The genus name Hydrogeno- yielded hits were 'hot' (6.5%), 'yellowstone' (5.8%), bacter is derived from the Latin words hydroge- 'spring' (5.6%), 'national/park' (5.4%) and num, meaning ‘that which produces water’ and bac- 'microbial' (3.9%). These keywords corroborate ter, referring to a rod that forms water when ex- with what is known from the ecology and physiology posed to oxygen. The species epithet thermophilus of strain TK-6T [1,2]. The two most frequent key- derives from the Greek words therme, heat, and words within the labels of environmental samples philus, loving, meaning a heat-loving organism. which yielded hits of a higher score than the highest Strain TK-6T was isolated from hot springs located scoring species were 'aquificales' (34.1%) and on the Izu peninsula in Japan [1]. Some strains of H. 'hot/spring' (32.9%). thermophilus were also isolated from a geothermal Figure 1 shows the phylogenetic neighborhood of spring in Tuscany, Italy [5,6]. Other strains similar H. thermophilus TK-6T in a 16S rRNA based tree. to H. thermophilus have been isolated from differ- The sequence of the single 16S rRNA gene in the ent environments, including a saline hot spring in genome differs by one nucleotide from the pre- Japan for 'H. halophilus' [7], and a volcanic area in viously published 16S rRNA sequence (Z30214), Iceland for Hydrogenobacter strain H-1 [8], strains which contains 31 ambiguous base calls. T3, T13 and T171 [5]. Until 1985, H. thermophilus Cells of strain TK-6T are Gram-negative, nonmotile was the only obligate autotroph among all aerobic straight rods of 0.3 to 0.5 µm by 2.0 to 3.0 µm oc- hydrogen-oxidizing bacteria reported so far [9,10]. curring singly or in pairs [1] (Figure 2 and Table 1). The activities of enzymes such as NADH:ferredoxin Molecular oxygen is used as an electron acceptor reductase (EC 1.18.1.3) and NAD-reducing hydro- for respiratory metabolism [1]. However, strain genase (EC 1.12.1.2) were studied extensively in TK-6T can grow anaerobically on nitrate as an elec- strain TK-6T [11]. Another genome sequence of a tron acceptor when molecular hydrogen is used as strain derived from the original isolate, presumably an energy source [33]. Strain TK-6T does not form held in the lab of one of the co-authors, has been colonies on agar plates, but does form colonies on published recently without much metadata [12]. plates solidified with GELRITE, a polysaccharide Here we present a summary classification and a set produced by Pseudomonas species [34]. The optim- of features for H. thermophilus strain TK-6T, togeth- al temperature for autotrophic growth on H -O - er with the description of the complete genomic 2 2 CO was between 70ºC and 75°C, no growth being sequencing and annotation. 2 observed at 37°C or 80°C [1]. A neutral pH 7.2 was suitable for growth of the strain TK-6T [1]. One im- Classification and features portant feature of the strain TK-6T is a generation The 16S rRNA gene sequence of the strain TK-6T time that is faster by about 1h compared to other (Z30214) shows the highest degree of sequence autotrophs, suggesting that this strain has an effi- identity, 97%, to the type strain of H. hydrogenophi- cient hydrogen-oxidizing ability [35]. No spore lus [6]. Further analysis shows 96% 16S rRNA gene formation was observed [1]. Strain TK-6T assimi- sequence identity with an uncultured Aquificales lates carbon dioxide via the reductive tricarboxylic bacterium clone pKA (AF453505) from a near- acid cycle [10,36,37]. This is also true when the neutral thermal spring in Kamchatka, Russia. The strain TK-6T grows anaerobically on nitrate [10]. single genomic 16S rRNA sequence of H. thermophi- Cytochromes b and c were found in strain TK-6T lus was compared with the most recent release of [1]. Interestingly, cytochrome C552 of H. thermophi- the Greengenes database [13] using NCBI BLAST lus TK-6T is extremely thermostable and can re- under default values and the relative frequencies of store its conformation even after being autoclaved taxa and keywords, weighted by BLAST scores, were for 10 minutes at 121ºC [30]. One of the denitrifica- determined. The five most frequent genera were tion enzymes of the strain TK-6T, cytochrome cd1 Hydrogenobacter (52.4%), Thermocrinis (18.8%), nitrite reductase has been isolated and analyzed Aquifex (10.3%), Sulfurihydrogenibium (6.2%) and [38]. Optimum temperature for the activity of this Hydrogenivirga (5.7%). Regarding hits to sequences enzyme was found to range between 70ºC-75ºC from other members of the genus, the average iden- [38]. Moreover, this enzyme was found to be of the tity within HSPs (high-scoring segment pairs) was heme cd1-type [33]. Ammonium and nitrate were 96.1%, whereas the average coverage by HSPs was utilized as nitrogen sources [1,33], but not urea and 93.5%. The species yielding the highest score was H. N2. Growth was inhibited by nitrite [1]. Nitrate re- hydrogenophilus. The five most frequent keywords duction and peroxidase were positive, while urease within the labels of environmental samples which 132 Standards in Genomic Sciences Zeytun et al. was negative [1]. Strain TK-6T could not utilize any in the genome. These metabolic deficits were con- of the following as sole sources of energy or carbon: sidered to be partially responsible for the obligate glucose, fructose, galactose, maltose, sucrose, xylose, autotrophy of the strain TK-6T [44]. Activities of raffinose, L-rhamnose, D-mannose, D-trehalose, phosphoenolpyruvate synthetase and pyruvate mannitol, starch, formate, acetate, propionate, pyru- carboxylase were also detected [10]. The reverse vate, succinate, malate, citrate, fumarate, maleate, reactions (dehydrogenase reacti - glycolate, gluconate, DL- -ketoglutarate, p- ketoglutarate synthase and pyruvate synthase hydroxybenzoate, DL-polyhydroxybutyrate, betaine, could be detected by using methyl viologenons) of as anα methanol, ethanol, methylamine,lactate, α dimethylamine, electron acceptor [10]. Cloning experiments of the trimethylamine, glycine, L-glutamate, L-aspartate, L- hydrogenase genes from the strain TK-6T revealed serine, L-leucine, L-valine, L-tryptophan, L-histidine, that this strain has at least four clusters of hydro- L-alanine, L-lysine, L-proline, L-arginine, nutrient genase genes [35]. Strain TK-6T assimilates ammo- broth, yeast extract-malt extract medium, and brain nium using glutamine synthetase (GS type I) [45].