Geobacillus icigianus sp. nov., a thermophilic bacterium isolated from a hot spring

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1 International Journal of Systematic and Evolutionary Microbiology (2015), 65, DOI /ijs Geobacillus icigianus sp. nov., a thermophilic bacterium isolated from a hot spring Alla V. Bryanskaya, Alexey S. Rozanov, Nikolay M. Slynko, Sergey V. Shekhovtsov and Sergey E. Peltek Correspondence Alla V. Bryanskaya bal412003@mail.ru Laboratory of Molecular Biotechnology, The Institute of Cytology and Genetics SB RAS, Prospekt Lavrentyeva 10, Novosibirsk, , Russian Federation A Gram-reaction-positive, motile, thermophilic spore-forming strain, G1w1 T, was isolated from a hot spring of the Valley of Geysers, Kamchatka (Russia). Based on data from the present polyphasic taxonomic study, including phylogenetic analysis of 16S rrna and spo0a gene sequences, the strain is considered to represent a novel species of the genus Geobacillus, for which the name Geobacillus icigianus sp. nov. is proposed. The type strain is G1w1 T (5VKM B-2853 T 5DSM T ). The genus Geobacillus was proposed by Nazina et al. (2001), since when descriptions of several novel species have been published (Fortina et al., 2001; Sung et al., 2002; Banat et al., 2004; Nazina et al., 2004; Kuisiene et al., 2004; Schäffer et al., 2004; Miñana-Galbis et al., 2010; Dinsdale et al., 2011; Poli et al., 2011; Coorevits et al., 2012). At the time of writing, the genus Geobacillus comprises 15 species with validly published names. During the course of research into unique microbial communities living in the Valley of Geysers, Kamchatka, Russia, we selected microbiological samples from bottom sediments of hydrothermal outlets. One novel thermophilic strain of spore-forming bacteria was isolated. The aim of this study was to provide a comprehensive characterization of the phenotypic, biochemical and molecular genetic properties of the isolated bacterial strain. As a result of this detailed analysis, we conclude that the strain belongs to the genus Geobacillus and represents a novel species. Strain G1w1 T was isolated from sludge samples of an unnamed explosive hydrothermal (97 uc) outlet situated near the Troinoy geyser (Valley of Geysers, Kronotsky Nature Reserve). A microbial community comprising a deep-green incrustation on pink sludge sediment was clearly visible to the naked eye. Water and sludge samples were collected using a sterile sampler into sterile containers and stored at 4 uc. Some samples were fixed using an equal volume of 96 % ethanol. Fermentation of the microbial Abbreviations: ANI, average nucleotide identity; ME, minimum-evolution. The GenBank/EMBL/DDBJ accession numbers for the 16S rrna and spo0a gene sequences of strain G1w1 T are KF and KF , respectively. The full genome sequence of strain G1w1 T has been deposited at DDBJ/EMBL/GenBank under accession number JPYA One supplementary figure is available with the online Supplementary Material. community was carried out on Luria Bertani (LB) medium at uc for 1 3 days (Gerhardt et al., 1994). Isolation and purification of the strain were conducted on LB agar medium at uc. The reference strains used in this study were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Geobacillus stearothermophilus DSM 22 T, Geobacillus thermoglucosidasius DSM 2542 T ) and from the All-Russian Collection of Microorganisms-VKM (Geobacillus jurassicus B T, Geobacillus thermocatenulatus B-1259 T ). Strains were grown on LB agar medium at uc. Biomass for chemical and molecular systematic studies was obtained by cultivating the organisms in shake flasks containing liquid LB medium at uc for h. Bacterial growth in the medium was monitored by measuring the optical density of the culture at 600 nm using an automatic multifunctional Epoch Microplate Spectrophotometer (BioTek). Medium was supplemented with 5 mg MnSO 4 l 1 to encourage sporulation. The shape and size of cells were determined using light and electron microscopes (Axioskop 2 Plus, Axioskop A1; Zeiss), at the Interinstitutional Shared Center for Microscopic Analysis of Biological Objects SB RAS. Bacterial samples were prepared by conventional methods (Netrusov et al., 2005). The Gram reaction was determined using a Gram stain kit (Sintakon) according to the manufacturer s recommended protocol. Characteristics of strain G1w1 T such as temperature and ph ranges for growth, NaCl tolerance, catalase, urease and oxidase activity, anaerobic growth, starch and casein degradation, gelatin liquefaction, citrate utilization, nitrate reduction, acid production and growth with various carbohydrates were tested according to Netrusov et al. (2005) and Logan & De Vos (2009). Most of the tests were carried out using reagents and kits produced by Lachema, DIA-M and Sigma. Tests were performed in triplicate G 2015 IUMS Printed in Great Britain

2 Geobacillus icigianus sp. nov. Microbial DNA was extracted using standard methods with phenol (Maniatis et al., 1982). Amplification of the 16S rrna gene was conducted with universal bacterial primers 16S-8-f-B (59-AGRGTTTGATCCTGGCTCA-39) and 16S r-B (59-GACGGGCGGTGTGTACAAG-39). The fragment of the spo0a gene was amplified with primers GEOSPO-20F73 (59-CAGCCGGACATGGAAGTGAT-39) and GEOSPO-20R696 (59-GACCGTATAGCCGAACAG- CG-39) (Kuisiene et al., 2009). The reactor feed contained 1.5 mm MgCl 2, 65 mm Tris/HCl (ph 8.8), 16 mm (NH 4 ) 2 SO 4, 0.05 % Tween 20, 0.2 mm dntp, 0.3 mm of primers and 1 Da of recombinant Taq polymerase (SibEnzyme). DNA sequencing was performed in the Genomics Core Facility SB RAS. Sequences of reference strains of the genus Geobacillus were taken from StrainInfo ( The BLAST program ( ncbi.nlm.nih.gov/blast.cgi) was used to search for similar sequences in the nucleotide databases. Alignment was performed using the program CLUSTAL W ( ac.uk/tools/msa/clustalw2). Phylogenetic trees were reconstructed using the minimum-evolution (ME) algorithm of the program MEGA version 5.0 (Tamura et al., 2011). The 16S rrna gene sequence determined for strain G1w1 T was 1306 nt long. Full sequencing of the strain G1w1 T genome was performed after sequencing of the 16S rrna and spo0a genes; this allowed us to precisely define the DNA G+C content and to analyse full-length sequences of the aforementioned genes. To determine the taxonomic affinity of strain G1w1 T we used the average nucleotide identity method (ANI value). An ANI calculator ( was used to compare the genomes of strain G1w1 T and those of G. stearothermophilus ATCC 7953 T, Geobacillus kaustophilus NBRC T and Geobacillus thermodenitrificans DSM 465 T taken from NCBI ( genome/). The analysis of fatty acid methyl ethers was performed, including standardization of incubation temperature (60 uc) and incubation time (12 h). Lyophilized cells were prepared according to Jenkins et al. (1977). After fast alkaline hydrolysis acids were extracted by hexane and methylated by HCl methanolic solution according to Schäffer et al. (2004). A mixture of fatty acid methyl esters was analysed by GC on an Agilent Technologies 6890N chromatograph with an Agilent Technologies 5973N quadrupolar mass spectrometer and fused-quartz capillary column DB-1. GC-MS analysis of the solutions was carried out by measuring both total ion current in scan mode, in the mass range Da, and in the selective ion monitoring mode measuring analyte molecular ions. Identification of the fatty acid methyl esters was carried out by using the NIST mass spectral search program for the NIST/EPA/NIH Mass Spectral Library version 2.0a. Strain G1w1 T formed round cream-coloured colonies with smooth and lightly curved edges. Colonies were slightly convex and ranged in size from 3 to 5 mm. Cells of strain G1w1 T were motile, rod-shaped with rounded ends and mm in size (Fig. S1, available in the online Supplementary Material). The cell wall was Gram-reactionpositive. Sporulation was observed in very rare cases even under conditions of growth stimulation by MnSO 4. Terminal or subterminal ellipsoidal endospores were observed that were mm in size. Swollen sporangia were not observed. The physiological characteristics of strain G1w1 T are given in Table 1. Strain G1w1 T was thermophilic; it could grow within the temperature range uc with an optimum growth temperature of uc. Growth was observed both at ph 5 and at ph 9, with an optimum ph for growth of Rapid growth was observed in medium with 1 % (w/v) NaCl. Strain G1w1 T was able to utilize a variety of sugars, carboxylic acids and hydrocarbons. The main fatty acid of strain G1w1 T was iso-c 15 : 0. The fatty acid profile of strain G1w1 T included iso-c 15 : 0 (39.7 %), iso-c 17 : 0 (26.9 %), anteiso-c 17 : 0 (11.6 %), C 16 : 0 (7.7 %), anteiso-c 15 : 0 (6.8 %), anteiso-c 14 : 0 (2.4 %) and C 18 : 0 (1.1 %). No other fatty acids were present at more than 1 %. A phylogenetic tree was reconstructed using 16S rrna gene sequences of members of the genus Geobacillus from the GenBank database (Fig. 1). Based on 16S rrna gene sequences, strain G1w1 T is not a member of any recognized species of the genus Geobacillus. The DNA G+C content of strain G1w1 was 52 mol%. This value is in accordance with values reported for the genus Geobacillus, which are in the range mol% (Nazina et al., 2001). For a number of strains belonging to the genus Geobacillus, full genomic sequences are known, although the molecular identification of this genus has been limited to use of 16S rrna gene sequences, as data on other sequences are unavailable for most type strains. Kuisiene et al. (2009) studied the taxonomy of the genus Geobacillus based on spo0a gene sequences this is the main regulator of the sporulation process, which controls more than 500 genes. The authors showed that by using this marker it was possible to identify reliably only G. thermodenitrificans, G. stearothermophilus and G. jurassicus. They therefore concluded that spo0a gene sequences cannot be used as a phylogenetic marker within this genus (Kuisiene et al., 2009). Despite this, for a number species of the genus Geobacillus the spo0a gene remains the only one to be sequenced. We have reconstructed a phylogenetic trees based on spo0a gene sequences (Fig. 2). This again shows that strain G1w1 T is separate from all other representatives of the genus. The following ANI scores were obtained when comparing strain G1w1 T with the type strains of Geobacillus species: % for G. stearothermophilus ATCC 7953 T, % for G. kaustophilus NBRC T and % for G. thermodenitrificans DSM 465 T. These values are well below the threshold range of %, indicating that strain G1w1 T represents a distinct species (Kim et al., 2014)

3 A. V. Bryanskaya and others Table 1. Characteristics that differentiate strain G1w1 T from closely related species of the genus Geobacillus Strains: 1, G1w1 T ;2,G. jurassicus B-2301 T (data for strains 1 and 2 are from the present study); 3, Geobacillus uzenensis DSM T ;4,Geobacillus subterraneus DSM T ;5,G. thermodenitrificans (data for taxa 3 5 were taken from Coorevits et al., 2012); 6, G. thermoglucosidasius DSM 2542 T ; 7, G. stearothermophilus DSM 22 T ;8,G. thermocatenulatus B-1259 T ;9,Geobacillus gargensis DSM T (data for taxa 9 were taken from Nazina et al., 2004). +, Positive reaction; 2, negative reaction; W, weakly positive reaction; +/W, positive or weakly positive reaction; D, different strains give different reactions; +/D, usually positive, but different strains give different reactions; V, result varies; ND, not determined. Characteristic Swollen sporangia 2 2 D W Spores: Cylindrical V Subterminal Terminal V Central/paracentral Hydrolysis of: Aesculin Casein Gelatin W D ONPG ND Starch /W W Acid production from: Cellobiose W W W 2 W + Glucose W + ND ND ND W W W ND Lactose W Inositol Mannitol W W + + W W 2 W + Melibiose W D 2 W W ND Raffinose W ND Rhamnose W W ND ND ND W W W 2 Sucrose W W + + D W W W ND Trehalose W W + + D W W W ND D-Xylose W Nitrate reduction + W + + +/D Voges Proskauer reaction W /D 2 W W 2 Catalase + + ND ND Oxidase 2 W ND 2 2 W 2 W ND Urease + + ND ND ND W W + 2 Lysine 2 2 ND ND ND ND Ornithine 2 2 ND ND ND ND Arginine 2 2 ND ND ND ND Simmons citrate 2 2 ND ND ND Malonate 2 2 ND ND ND ND Inositol W W ND ND ND W W W ND Adonitol W W ND ND ND W W W ND Sorbitol W W ND ND ND W W W ND Dulcitol W W ND ND ND W W W ND Anaerobic growth W Growth at/in: ph ph 9 + W W 2 1 % NaCl (w/v) W % NaCl (w/v) 2 W Temperature range (uc) Thus, we concluded strain G1w1 T is a member of the genus Geobacillus, but differs markedly based on biochemical and molecular genetic characteristics. Therefore, we suggest that strain G1w1 T represents a novel species of the genus Geobacillus, for which the name Geobacillus icigianus sp. nov. is proposed. 866 International Journal of Systematic and Evolutionary Microbiology 65

4 Geobacillus icigianus sp. nov Geobacillus thermoleovorans DSM 5366 T (Z26923) 97 Geobacillus gargensis DSM T (FR749979) Geobacillus stearothermophilus IFO T (AB021196) Geobacillus jurassicus DS1 T (AY312404) 99 Geobacillus uzenensis U T (AF276304) Geobacillus sp. TC-S8 (EU740981) 99 Geobacillus lituanicus N-3 T (AY044055) Geobacillus kaustophilus NCIMB 8547 T (X60618) Geobacillus vulcani 3S-1 T (AJ293805) Geobacillus thermocatenulatus NBRC T (AB680835) Geobacillus icigianus G1w1 T (KF ) Geobacillus subterraneus 34 T (AF276306) Geobacillus thermodenitrificans R T (FN538993) Geobacillus thermoglucosidasius ATCC T (AB021197) Geobacillus thermantarcticus DSM 9572 T (FR749957) Geobacillus caldoxylolyticus ATCC T (AF067651) Geobacillus toebii SK-1 T (AF326278) Geobacillus galactosidasius CF1B T (AM408559) Anoxybacillus tepidamans R T (FN428691) Aeribacillus pallidus DSM 3670 T (Z26930) Caldibacillus debilis R T (FN428699) Fig. 1. ME phylogenetic tree reconstructed based on 16S rrna gene sequences. Numbers near the branches are ME bootstrap support values. Bar, changes per nucleotide position Geobacillus gargensis DSM T (FJ226592) Geobacillus caldoxylosilyticus DSM T (FJ226591) 83 Geobacillus thermocatenulatus DSM 730 T (FJ226598) Geobacillus thermoglucosidasius DSM 2542 T (FJ226600) 100 Geobacillus toebii DSM (FJ226602) Geobacillus thermoleovorans DSM 5366 T (FJ226601) Geobacillus kaustophilus DSM 7263 T (FJ226594) 98 Geobacillus vulcani 22 (FJ226605) Geobacillus vulcani DSM T (FJ226604) 71 Geobacillus lituanicus DSM T (FJ226595) Geobacillus jurassicus DSM T (FJ226593) Geobacillus icigianus G1w1 T (KF ) Geobacillus thermodenitrificans DSM 465 T (FJ226599) Geobacillus uzenensis DSM T (FJ226603) Geobacillus subterraneus DSM T (FJ226597) Geobacillus stearothermophilus 17 (FJ226607) Geobacillus stearothermophilus 9 (FJ226606) Geobacillus stearothermophilus (AY672766) Geobacillus stearothermophilus DSM 22 T (FJ226596) Geobacillus stearothermophilus (AJ002297) Fig. 2. ME phylogenetic tree reconstructed based on spo0a gene sequences. Numbers near the branches are ME bootstrap support values. Bar, 0.02 changes per nucleotide position

5 A. V. Bryanskaya and others Description of Geobacillus icigianus sp. nov. Geobacillus icigianus [i.ci.gi.a9nus. N.L. masc. adj. icigianus referring to the Institute of Cytology and Genetics (ICiG) at Novosibirsk where the type strain was isolated and described]. The description is based on a single strain. Cells are Gramreaction-positive, motile rods, mm, with terminal ellipsoidal or cylindrical endospores mm in size. Sporulation is extremely rare. Swollen sporangia are not observed. Aerobic or facultatively anaerobic. After 24 h of incubation at 60 uc forms circular cream-coloured colonies with smooth or slightly curved edges, 3 5 mm in diameter. Grows at 50 and 75 uc, and optimally at uc. Grows between ph 5.0 and 9.0; optimum growth at ph Growth is rapid in medium with 1 % (w/v) NaCl and is inhibited in the presence of 5 % NaCl. Oxidase-negative, but catalase- and urease-positive. Aesculin and casein are hydrolysed, but starch and ONPG are not. Growth occurs on yeast extract; glucose and xylose can be utilized as sole carbon sources. Gelatin hydrolysis and nitrate reduction are positive and the Voges Proskauer reaction is weak, but citrate utilization, and arginine, lysine, ornithine and malonate production are negative. Utilization of adonitol, sorbitol and dulcitol is weak. Acid is produced from D- xylose but not from lactose or inositol. Production of acids from cellobiose, glucose, mannitol, melibiose, raffinose, rhamnose, sucrose and trehalose is weak. The type strain is G1w1 T (5VKM B-2853 T 5DSM T ), which was originally isolated from a water sludge sample of the hydrothermal (97 uc) outlets of the Troinoy geyser (Valley of Geysers, Kamchatka). The DNA G+C content of the type strain is 52 mol%. Acknowledgements This work was financially supported by SB RAS integration projects 94 and 93; Budget Project VI The authors would like to express their gratitude to the staff of the Kronotsky State Biosphere Reserve for assistance in the work in Uzon caldera. References Banat, I. M., Marchant, R. & Rahman, T. J. (2004). Geobacillus debilis sp. nov., a novel obligately thermophilic bacterium isolated from a cool soil environment, and reassignment of Bacillus pallidus to Geobacillus pallidus comb. nov. Int J Syst Evol Microbiol 54, Coorevits, A., Dinsdale, A. E., Halket, G., Lebbe, L., De Vos, P., Van Landschoot, A. & Logan, N. A. (2012). 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