Thermogladius calderae gen. nov., sp. nov., an anaerobic, hyperthermophilic crenarchaeote from a Kamchatka hot spring

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1 International Journal of Systematic and Evolutionary Microbiology (2016), 66, DOI /ijsem Thermogladius calderae gen. nov., sp. nov., an anaerobic, hyperthermophilic crenarchaeote from a Kamchatka hot spring Tatiana V. Kochetkova, 1 Ilya V. Kublanov, 1 Stepan V. Toshchakov, 2 Magdalena R. Osburn, 3 Andrei A. Novikov, 4 Elizaveta A. Bonch-Osmolovskaya 1 and Anna A. Perevalova 1 Correspondence Tatiana V. Kochetkova slepysh@gmail.com 1 Winogradsky Institute of Microbiology, Research Center of Biotechnology RAS, 33/2 Leninsky prospect, Moscow, Russia 2 Immanuel Kant Baltic Federal University, Botkina str. 3, , Kaliningrad, Russia 3 Department of Earth and Planetary Sciences, Northwestern University, Evanston, IL, USA 4 Gubkin Russian State University of Oil and Gas, Leninskiy Prospect 65, Moscow, Russia An obligately anaerobic, hyperthermophilic, organoheterotrophic archaeon, strain 1633 T, was isolated from a terrestrial hot spring of the Uzon Caldera (Kamchatka Peninsula, Russia). Cells were regular cocci, mm in diameter, with one flagellum. The temperature range for growth was C, with an optimum at 84 8C. Strain 1633 T grew on yeast extract, beef extract, peptone, cellulose and cellobiose. No growth was detected on other sugars or carbohydrates, organic acids, or under autotrophic conditions. The only detected growth products were CO 2, acetate, and H 2. The growth rate was stimulated by elemental sulfur, which was reduced to hydrogen sulfide. The in silico-calculated G+C content of the genomic DNA of strain 1633 T was mol%. 16S rrna gene sequence analysis placed strain 1633 T together with the non-validly published Thermogladius shockii strain WB1 in a separate genus-level cluster within the family Desulfurococcaceae. Average nucleotide identity (ANI) results revealed % identity between strain 1633 T and Thermogladius shockii WB1. Based on these results we propose a novel genus and species with the name Thermogladius calderae gen. nov., sp. nov. The type strain of the type species is 1633 T (5DSM T 5VKM B-2946 T ). The vast majority of cultured Crenarchaeota have been isolated from terrestrial geothermal environments (Huber & Stetter, 2006). Located in Far East of Russia, Kamchatka Peninsula is a volcanically active area with hundreds of associated volcanoes and areas with geothermal activity, among which Uzon Caldera is the largest. Molecular studies have documented the presence of a significant number of cultured and uncultured Crenarchaeota in Uzon Caldera thermal pools. It was reported that Crenarchaeota inhabited not only high-temperature springs (Wemheuer et al., 2013; Chernyh et al., 2015), but also springs Abbreviations: ANI, average nucleotide identity; GDGT, glycerol dibiphytanyl glycerol tetraether. The GenBank/EMBL/DDBJ accession number for the complete genome sequence of Thermogladius calderae 1633 T is CP The genome sequencing project of Thermogladius shockii WB1 is available in the GenBank/EMBL/DDBJ databases under accession number PRJEB with moderate temperatures (Perevalova et al., 2008; Burgess et al., 2012; Menzel et al., 2015). At the time of writing, many thermophilic and hyperthermophilic crenarchaeotes have been isolated from hot springs of Uzon Caldera, including members of genera Desulfurococcus (Bonch-Osmolovskaya et al., 1988; Perevalova et al., 2005; Kublanov et al., 2009), Thermoproteus (Bonch- Osmolovskaya et al., 1990), Acidilobus (Prokofeva et al., 2000; 2009), Vulcanisaeta (Prokofeva et al., 2005), Fervidicoccus (Perevalova et al., 2010) and Pyrobaculum (Slobodkina et al., 2015). Here we report the isolation of strain 1633 T obtained from a hot spring of the Uzon Caldera, and found to be closely related to Thermogladius shockii, a species with a name that was effectively, but never validly, published (Osburn & Amend, 2011). Strain 1633 T was isolated from a sample of mixed water and black mud from a freshwater hot spring ( N E) located in the West Thermal Field of Uzon Caldera. The in situ water temperature and G 2016 IUMS Printed in Great Britain 1407

2 T. V. Kochetkova and others ph were 86 8C and 5.5, respectively. The following basal medium was used for enrichment and isolation of the novel strain (g l 21 ): 0.66 NH 4 Cl, 0.16 MgCl 2.6H 2 O, 0.1 CaCl 2.6H 2 O, 0.33 KCl and 0.5 KH 2 PO 4. After boiling, the medium was flushed with N 2 and upon cooling the following compounds were added (l 21 ): 1 ml trace element solution (Kevbrin & Zavarzin, 1992) and vitamin solution (Wolin et al., 1963); 0.5 g NaHCO 3, 0.5 g Na 2 S.9H 2 O, 0.2 g yeast extract and 0.2 g cysteine hydrochloride. Finally, the ph was adjusted to with 6 M HCl. Ten-millilitre portions of the medium were dispensed into 18 ml Hungate tubes with butyl rubber stoppers under a N 2 atmosphere, and 10 % (v/v) inoculum (a mix of sediment and water) was added. The tubes were incubated at 82 8C with carboxymethyl-cellulose (Sigma, 2gl 21 ) as sole carbon substrate, and penicillin ( mg ml 21 ). After incubation for 5 days, single regular coccoid cells dominated the cultures. To isolate strain 1633 T, flat ml bottles (Bellco) containing 3 ml basal medium supplemented with peptone (1 g l 21 ), glucose (1 g l 21 ) and yeast extract (1 g l 21 ) and solidified with 1.2 % (w/v) Gelrite (Gifco) and MgSO 4 (1 g l 21 ) were used. Individual colonies were picked under a stream of % N 2 and transferred to liquid medium. The purity of the isolate was tested by cultivation on basal medium with various complex carbon sources (e.g. yeast extract, beef extract, peptone) and electron acceptors and subsequent monitoring of the cultures by light and electron microscopy, as well as by 16S rrna gene sequencing. For phase-contrast light microscopy an Olympus CX41RF microscope was used. For electron microscopy (negative staining), cultures were fixed as described previously (Sokolova et al., 2002) and examined under a JEM-B microscope (JEOL). Cells of strain 1633 T were cocci, mm in diameter, occasionally with straight thin protrusions and one long flagellum (Fig. 1a). Virus-like particles associated with strain 1633 T were detected by electron microscopy before isolation of the strain on solid medium (Fig. 1b, c). Single- and occasionally two-tailed (data not shown) spindle-shaped particles were observed in cultures, maintained over 10 weeks with successive dilutions every 3 4 days. No evidence was found for virus-induced cell lysis. No virus-like particles were detected by electron microscopy in cultures after transferring from colonies. Growth of the novel isolate was registered by direct cell counts using phase-contrast microscopy. All growth experiments were conducted in triplicates. The novel isolate is an obligate anaerobe, growing only under anoxic conditions with a reductant (Na 2 S). The organism is a hyperthermophile and neutrophile growing in the temperature range from 80 to 95 8C (optimum 84 8C), and in the ph range from 6.5 to 8.2 (optimum ph 7.1). No growth was observed at 75 8C and 98 8C, or at ph 6.2 and ph 8.6. Strain 1633 T grew only at low NaCl concentrations, up to 2.5 g l 21, and the best growth was obtained in the absence of NaCl. The doubling time of growth on yeast extract (1 g l 21 ) under optimal conditions was 7.7 h. (a) (b) (c) Fig. 1. Electron micrographs of negatively stained cells of a pure culture of strain 1633 T (a) and infected by virus-like particles (b, c). Bars, 0.5 mm. Strain 1633 T grew well on yeast extract (YE), beef extract and peptone (all at 2.0 g l 21 ) with the final cell yield ranging from cells ml 21. Weaker growth was 1408 International Journal of Systematic and Evolutionary Microbiology 66

3 Thermogladius calderae gen. nov., sp. nov. observed on cellobiose and various celluloses [filter paper, microcrystalline cellulose (Chemapol, Czech Republic), carboxymethyl cellulose (Sigma)] (all at 2.0 g l 21 ). For growth on cellulose, the addition of 1.0 g l 21 YE was essential. No growth was detected on glucose, xylose, fructose, sucrose, maltose, lactose, agarose, starch, amorphous cellulose, keratin, xylan, chitin, formate, pyruvate, lactate, fumarate, glycerine, methanol, ethanol, under a H 2 /CO 2 (4 : 1, v/v), N 2 /CO (19 : 1, 5 : 1, 1 : 1, v/v) or % CO atmosphere. Elemental sulfur (at final concentration 10 g l 21 ) stimulated growth of strain 1633 T and was reduced to hydrogen sulfide. SO 2-4 and S 2 O 2-3, added as sodium salts (20 mm) did not stimulate growth of the strain whereas sodium sulfite or nitrate (20 mm) inhibited growth. Under optimal growth conditions, the final cell yields of the strain were cells ml 21 on the medium supplemented with 1.0 g l 21 YE; cells ml 21 on the medium with 1.0 g l 21 YE and S8; and cells ml 21 with 1.0 g l 21 YE, S8 and 2.0 g l 21 cellulose. Strain 1633 T was not sensitive to kanamycin, chloramphenicol or penicillin ( mg ml 21 ). The main end-products of growth were determined by GLC using a Stayer chromatograph with UV/VIS detector at 220 nm and REZEX ROA mm column (Phenomenex), maintained at 35 8C, and using 0.2 % H 3 PO 4 solution as a solvent at a flow rate of 0.5 ml min 21. The only growth products with YE as the carbon substrate were CO 2, acetate and H 2. During growth on cellulose, neither acetate nor ethanol was formed. Cells of strain 1633 T were lyophilized for lipid analysis via hydrolysis in 5 % HCl/MeOH (Pitcher et al., 2011). Core lipid extracts were injected into an HPLC-APCI-MS system (HPLC Agilent 1290 series with Econosphere NH 2 column; Agilent 6530 Q-TOF detector). The results showed that the majority (89.9 %) of the archaeal lipids were glycerol dibiphytanyl glycerol tetraethers (GDGTs) that contained zero to four cyclopentyl rings; the remaining small proportion (11.1 %) consisted of the glycerol diether (m/z ), probably the macrocyclic archaeol found in Methanocaldococcus jannaschii (Sprott, 1992) and Methanococcus igneus (Trincone et al., 1992). Of the key GDGT compounds, GDGT-4 was the most abundant (52.7 %), followed by GDGT-3 (16.9 %), GDGT-2 (7.4 %), GDGT-1 (4.1 %) and GDGT-0 (3.0 %). Small amounts of isomers GDGT-4 iso (3.9 %) and GDGT-3 iso (0.9 %) were detected, probably the antiparallel regioisomers based on their MS- MS spectra being identical to those of GDGT-4 and GDGT-3 (Sinninghe Damsté et al., 2002). No crenarchaeol was detected in the lipids of strain 1633 T. The G+C content (55.64 mol%) of the genomic DNA of strain 1633 T was calculated in silico using the whole genome sequence (GenBank accession no. NC_017954, Mardanov et al., 2012). The genome has a single copy of the 16S rrna gene, which was used for phylogenetic analysis. BLAST analysis against species with validly published names using the EzBioCloud/EzTaxon server (Kim et al., 2012) revealed the nearest sequences belonged to representatives of the crenarchaeal genus Staphylothermus. However, the most similar sequence to strain 1633 T belonged to another member of the family Desulfurococcaceae, Thermogladius shockii WB1 (Osburn & Amend, 2011). In order to reconstruct the phylogenetic position of strain 1633 T and T. shockii WB1, their 16S rrna gene sequences as well as sequences of other members of the family Desulfurococcaceae with validly published names were aligned (total 19 sequences) using MUSCLE (Edgar, 2004) and analysed. Phylogenetic analysis was performed in the MEGA 6 package (Tamura et al., 2013). Evolutionary history was inferred using the maximum-likelihood method with 0 repetitions [bootstraps, Felsenstein (1985)]. Evolutionary distances were calculated using the General Time Reversible [G+I, 4 categories, Nei & Kumar (2000)] model. The phylogenetic tree with the highest log-likelihood ( ) placed the novel isolate into a cluster with T. shockii WB1, Staphylothermus hellenicus DSM T, Staphylothermus marinus F1 T (Fig. 2). The distances calculated using the above model suggested that the closest relatives of strain 1633 T were T. shockii (98.7 %), Staphylothermus hellenicus (96.1 %) and Staphylothermus marinus (95.9 %). The genome of T. shockii WB1 was sequenced and assembled into a single chromosome in the course of this work (data not shown). Evaluation of the average nucleotide identity (ANI) between genomes of strain 1633 T and T. shockii WB1 using ANI calculator ( ce.gatech.edu/ani/) revealed the value to be %, suggesting these strains represent two different species. The closest cultivated relative of strain 1633 T, Thermogladius shockii strain WB1 (Osburn & Amend, 2011), was isolated from Boomerang Pool in Yellowstone National Park, USA. Two strains (2412Fs and 2412Pk) also very closely related to strain 1633 T with 99 % 16S rrna gene similarity, were isolated from Burlyaschy hot spring of Uzon Caldera recently (Bidzhieva et al., 2014). All isolates are anaerobic, hyperthermophilic, organotrophic cocci, able to grow without sulfur, but stimulated by its presence. This is in contrast to related species of the genus Staphylothermus, which are obligately dependent on elemental sulfur (Table 1). Unlike other closely related isolates, strain 1633 T is able to grow not only on proteinaceous substrates, but also on cellulose (filter paper, microcrystalline and carboxymethyl-cellulose). However, the complete genome analysis did not reveal the presence of genes encoding glycoside hydrolases or known exo/endobeta-1,4-glucanases (Mardanov et al., 2012), suggesting either the existence of novel mechanisms of cellulose hydrolysis in strain 1633 T or the utilization of unidentified thermal degradation products of cellulose by this organism. The ability to grow on biopolymers (proteins and carbohydrates) was previously reported for several representatives of the family Desulfurococcaceae (Bonch- Osmolovskaya et al., 1988; Sako et al., 1996; Perevalova et al., 2005; Kublanov et al., 2009; Bidzhieva et al., 2014)

4 T. V. Kochetkova and others Desulfurococcus kamchatkensis 1221n T (NC_ ) Desulfurococcus amylolyticus Z-533 T (NR_ ) 99 Desulfurococcus fermentans DSM T (NR_ ) Desulfurococcus mobilis DSM 2161 T (M ) Desulfurococcus mucosus DSM 2162 T (NC_ ) 37 Thermosphaera aggregans M11TL T (X99556) 51 Sulfophobococcus zilligii K1 T (X98064) Thermogladius calderae 1633 T (NC_ ) 92 Thermogladius shockii WB1 (EU ) Staphylothermus hellenicus DSM T (NR_ ) 99 Staphylothermus marinus F1 T (X99560) Ignicoccus hospitalis KIN4/I T (NC_ ) Ignicoccus islandicus Kol8 T (X99562) 97 Ignicoccus pacificus LPC33 T (NR_ ) Stetteria hydrogenophila 4ABC T (Y07784) Desulfurococcaceae 98 Thermodiscus maritimus S2 T (X99554) Aeropyrum pernix K1 T (D83259) Aeropyrum camini SY1 T (AB ) Fig. 2. Maximum-likelihood 16S rrna gene phylogenetic tree of strain 1633 T, Thermogladius shockii WB1 and all representatives of the family Desulfurococcaceae with validly published names. Branch lengths (see scale) correspond to the number of substitutions per site with corrections, associated with the model. All positions with,95 % site coverage were eliminated, that is,,5 % alignment gaps, missing data, and ambiguous bases were allowed at any position. There were 1259 positions in the final alignment of 19 sequences. Numbers at nodes indicate bootstrap values of 0 repetitions. Hyperthermus butylicus DSM 5456 T (GenBank accession no. NC_ ) was chosen as an outgroup (not shown). Strain 1633 T is a novel member of the order Thermoproteales that shares this capability. The low level of genomic DNA identity between strains 1633 T and Thermogladius shockii WB1 (75.72 %), 16S rrna gene-based phylogenetic analysis and phenotypic differences indicate that the strains represent two different species (Goris et al., 2007). Based on the previous characterization of T. shockii (Osburn & Amend, 2011) and phylogenetic and physiological differences, we propose a novel genus and species represented by strain 1633 T, with the name Thermogladius calderae gen. nov., sp. nov. This species will be the type species of the genus Thermogladius after valid publication of the genus name, instead of the earlier proposed Thermogladius shockii, a name that was never validated. Description of Thermogladius gen. nov. Thermogladius (Ther.mo.gla9di.us. Gr. adj. thermos hot; L. n. gladius sword; N.L. masc. n. Thermogladius the hot sword). Cells are regular to slightly irregular cocci, with occasional straight, thin protrusions. The cell envelope consists of an S-layer covering the cytoplasmic membrane. Obligately anaerobic, showing organoheterotrophic metabolism with the ability to ferment a variety of complex organic substrates, including cellulose and keratin. Facultative S 0 -reducer. Cells can thrive in high temperatures and in a broad ph range. Known strains have been isolated from terrestrial hot springs. The type species is Thermogladius calderae. Description of Thermogladius calderae sp. nov. Thermogladius calderae (cal.de9rae. N.L. gen. n. calderae of a caldera, referring to the isolation of the type strain from the Uzon Caldera). Displays the following properties in addition to those in the genus description. Cells are regular cocci, mm in diameter, with one long flagellum. The temperature range for growth is uc, with an optimum at 84 uc. The ph range for growth at 84 uc is ph (optimum ph 7.1). Grows on yeast extract, beef extract, peptone and cellulose (filter paper, microcrystalline cellulose, carboxymethyl-cellulose) and cellobiose. No growth is 1410 International Journal of Systematic and Evolutionary Microbiology 66

5 Thermogladius calderae gen. nov., sp. nov. Table 1. Characteristics of strain 1633 T in comparison with those of the closest phylogenetic relatives Strains: 1, 1633 T (data from this study); 2, Thermogladius shockii WB1 (Osburn & Amend, 2011); 3, Staphylothermus marinus (Fiala et al., 1986); 4, Staphylothermus hellenicus (Arab et al., 2000). +, Positive; 2, negative; ND, no data available (min./opt./max.). Characteristic Cell shape and size (mm) Regular cocci, Cocci, Cocci, Irregular cocci, Flagellum Growth temperature (8C) 80/84/95 64/84/93 65/92/98 70/85/90 (min./opt./max.) Optimum doubling time (h) 7.7* 4.9* Growth ph (min./opt./max.) 6.5/7.1/ /5 6/ /6.5/ /6.0/7.0 NaCl range (%, w/v) 0/0.25 0/0.46 1/3.5 2/4/8 Growth substrates Yeast extract, beef extract, peptone and cellulose Yeast extract, peptone, casein Yeast extract, peptone Yeast extract, peptone (filter paper, microcrystalline cellulose, carboxymethyl-cellulose), cellobiose Effect of sulfur Stimulated No effect Required Required DNA G+C content (mol%) Resistance to antibiotics Kanamycin, chloramphenicol, penicillin *Doubling time was measured in the absence of elemental sulfur in medium. Kanamycin, rifampicin, chloramphenicol, streptomycin Kanamycin, chloramphenicol, streptomycin ND detected on glucose, xylose, fructose, sucrose, maltose, lactose, agarose, starch, amorphous cellulose, chitin, xylan, keratin, formate, pyruvate, lactate, fumarate, glycerine, methanol, ethanol, under a H 2 /CO 2 (4 : 1, v/v), N 2 /CO (19 : 1, 5 : 1, 1 : 1, v/v) or % CO atmosphere. Growth is stimulated by elemental sulfur, which is reduced to hydrogen sulfide. The only growth products found are CO 2, acetate and H 2. Does not show sensitivity to kanamycin, chloramphenicol or penicillin. The type strain is 1633 T (5DSM T 5VKM B-2946 T ), isolated from a hot spring of the West Thermal Field of the Uzon Caldera, Kamchatka. The G+C content of the genomic DNA of the type strain is mol%. Acknowledgements We are grateful to Nadezhda Kostrikina (Research Center of Biotechnology RAS) for the electron microscopy and Salima Bidzhieva (Research Center of Biotechnology RAS) for checking growth of the novel isolate on keratin. The work of A. P. and A. N. was supported by RFBR grant number (lipid analysis), A. P. and T. K. by , by the Molecular and Cell Biology Program of the Russian Academy of Sciences, and by the grant of RF President NSh References Arab, H., Völker, H. & Thomm, M. (2000). Thermococcus aegaeicus sp. nov. and Staphylothermus hellenicus sp. nov., two novel hyperthermophilic archaea isolated from geothermally heated vents off Palaeochori Bay, Milos, Greece. Int J Syst Evol Microbiol 50, Bidzhieva, S. Kh, Derbikova, K. S., Kublanov, I. V. & Bonch- Osmolovskaya, E. A. (2014). [Capacity of hyperthermophilic Crenarchaeota for decomposition of refractory protiens (a- and b-keratins)]. Mikrobiologiia 83, (in Russian). Bonch-Osmolovskaya, E. A., Slesarev, A. I., Miroshnichenko, M. L., Svetlichnaya, T. P. & Alekseev, V. A. (1988). Characterization of Desulfurococcus amylolyticus n. sp. a novel extremely thermophilic archaebacterium isolated from Kamchatka and Kurils hot springs. Microbiology (English translation of Mikrobiologiia) 57, Bonch-Osmolovskaya, E. A., Miroshnichenko, M. L., Kostrikina, N. A., Chernyh, N. A. & Zavarzin, G. A. (1990). Thermoproteus uzoniensis sp. nov., a new extremely thermophilic archaebacterium from Kamchatka continental hot springs. Arch Microbiol 154, Burgess, E. A., Unrine, J. M., Mills, G. L., Romanek, C. S. & Wiegel, J. (2012). Comparative geochemical and microbiological characterization of two thermal pools in the Uzon Caldera, Kamchatka, Russia. Microb Ecol 63, Chernyh, N. A., Mardanov, A. V., Gumerov, V. M., Miroshnichenko, M. L., Lebedinsky, A. V., Merkel, A. Y., Crowe, D., Pimenov, N. V., Rusanov, I. I. & other authors (2015). Microbial life in Bourlyashchy, the hottest thermal pool of Uzon Caldera, Kamchatka. Extremophiles 19, Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32, Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, Fiala, G., Stetter, K. O., Jannasch, H. W., Langworthy, T. A. & Madon, J. (1986). Staphylothermus marinus sp. nov. represents a novel genus

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