IJSEM Papers in Press. Published December 16, 2014 as doi:10.1099/ijs.0.000029
International Journal of Systematic and Evolutionary Microbiology Geobacillus icigianus sp. nov., a new thermophilic bacterium isolated from Valley of Geysers, Kamchatka --Manuscript Draft-Manuscript Number:
IJSEM-D-14-00342
Full Title:
Geobacillus icigianus sp. nov., a new thermophilic bacterium isolated from Valley of Geysers, Kamchatka
Short Title:
Geobacillus icigianus sp. nov., a new thermophilic bacterium isolated from Valley of Geysers, Kamchatka
Article Type:
Note
Section/Category:
New taxa - Firmicutes and related organisms
Keywords:
Geobacillus; Geobacillus icigianus; phylogenetic analysis; novel species; Valley of Geysers
Corresponding Author:
Alla Bryanskaya, Ph.D. Institute of Cytology and Genetics SB RAS RUSSIAN FEDERATION
First Author:
Alla Bryanskaya, Ph.D.
Order of Authors:
Alla Bryanskaya, Ph.D. Alexey Rozanov Nikolay Slynko Sergey Shekhovtsov Sergey Peltek
Manuscript Region of Origin:
RUSSIAN FEDERATION
Abstract:
A novel thermophilic spore-forming strain, G1w1, was isolated from hot spring of Valley of Geysers, Kamchatka (Russia). Based on a phylogenetic analysis of 16S rRNA gene and gene spo0A sequences strain represents a new species of the Geobacillus genus. Based on polyphasic taxonomic data the G1w1 strain is assigned to Geobacillus icigianus sp. nov. The type strain is G1w1 (VKM - B-2853T; DSM- 28325T).
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Text with table and fig legends.doc
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Geobacillus icigianus sp. nov., a new thermophilic bacterium isolated from Valley of Geysers, Kamchatka Alla V. Bryanskaya, Alexey S. Rozanov, Nikolay M. Slynko, Sergey V. Shekhovtsov and Sergey E. Peltek Laboratory of Molecular Biotechnology, The Institute of Cytology and Genetics SB RAS, Prospekt Lavrentyeva 10, Novosibirsk, 630090, Russian Federation Correspondence Alla Bryanskaya [email protected] Running title: Geobacillus icigianus sp. nov., a new thermophile Contents category: New taxa - Firmicutes and related organisms A novel thermophilic spore-forming strain, G1w1, was isolated from hot spring of Valley of Geysers, Kamchatka (Russia). Based on a phylogenetic analysis of 16S rRNA gene and gene spo0A sequences strain represents a new species of the Geobacillus genus. Based on polyphasic taxonomic data the G1w1 strain is assigned to Geobacillus icigianus sp. nov. The type strain is G1w1 (VKM – B-2853T; DSM- 28325T).1 Keywords: Geobacillus, Geobacillus icigianus, phylogenetic analysis, novel species, Valley of Geysers The genus Geobacillus was proposed by (Nazina et al., 2001) and major discoveries and descriptions of new species followed after the description of the genus (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). Currently the genus Geobacillus contains 15 species with validly published names. Within the research of unique microbial communities living in Valley of Geysers there were selected microbiological samples of vaporing hydrothermal outlets’ bottom sediments. The novel thermophilic strain of spore-forming bacteria was isolated from those assays. The aim of this study is a comprehensive characterisation of phenotypic, biochemical and molecular genetic properties of the isolated bacterial strain. As the result of the strain detailed analysis it was concluded that the strain belongs to Geobacillus genus by using 16S rRNA sequences, but it was not among previously described species of the genus. We referred to the stain as the new species Geobacillus icigianus sp. nov. The stain G1w1 was isolated from sludge samples of unnamed vaporing hydrothermal (97 0С) outlets situated in the geyser Troinoy region (Valley of Geysers, Kronotsky Nature Reserve). A microbial community which is clearly visible without a microscope represented a deep-green incrustation on pink sludge sediment. Water and sludge samples were collected using a sterile The GenBank accession number for the 16S rRNA gene and spo0A gene sequences of G1w1T strain is KF631430.1 and KF631431.1, respectively. Full genome sequencing of G1w1T strain has been deposited at DDBJ/EMBL/GenBank under the accession JPYA00000000.
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95
sampler into sterile containers and have been stored at 4ºC. Some samples were fixed using equal volume of 96% ethanol. Fermentation of the microbial community was carried out at the medium Luria Bertani (LB) at 45-70 ºC within 1-3 days (Gerhardt et al., 1994). Isolation and purification of the strain were conducted on LB agar medium at 55-65 °C. The reference strains used in this study were obtained from the Leibniz-Institut DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Geobacillus stearothermophilus DSM 22T, Geobacillus thermoglucosidasius DSM 2542T) and from the AllRussian Collection of Microorganisms-VKM (Geobacillus jurassicus B-2301T, Geobacillus thermocatenulatus B-1259T). Strains were grown on LB agar medium at 55-65 °C. Biomass for chemical and molecular systematic studies was obtained by cultivating the organisms in shake flasks containing liquid LB at 55-60 °C for 12-24 h. Bacterial growth in medium was monitored by measuring the optical density of the culture at 600 nm using an automatic multifunctional Epoch Microplate Spectrophotometr (BioTek). Medium was supplemented with 5 mg MnSO4 per liter to encourage sporulation. The shape and size of cells were determined by light and electron microscopes Axioskop 2 Plus, Axioskop А1 all produced by Carl Zeiss, in the Interinstitutional Shared Center for Microscopic Analysis of Biological Objects SB RAS. Bacteria samples were prepared by conventional methods (Netrusov et al., 2005). The Gram reaction was determined using a Gram stain kit (Sintakon, Russia) according to the manufacturer's recommended protocol. Such characteristic of the stain G1w1 as temperature and pH ranges, 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; Logan & De Vos, 2009). Most of the tests were carried out using reagents and kits produced by companies LACHEMA, DIA-M, SIGMA. Tests were performed in triplicate. Microbial DNA was extracted by standard method with phenol (Maniatis et al., 1982). Amplification of 16S rRNA was conducted with universal bacterial primers 16S-8-f-B (5'AGRGTTTGATCCTGGCTCA-3') and 16S-1350-r-B (5'-GACGGGCGGTGTGTACAAG-3'). The fragment of spo0A gene was amplified with GEOSPO-20F73 (5'CAGCCGGACATGGAAGTGAT-3') and GEOSPO-20R696 (5'GACCGTATAGCCGAACAGCG-3') primers (Kuisiene et al., 2009). Reactor feed contained 1.5 mM of MgCl2, 65 mM of Tris-HCl (pH 8.8), 16 mM of (NH4)2SO4 , 0.05% of Tween-20, 0.2 mM of dNTP, 0.3 mM of primers and 1 amu of recombinant Taq polymerase (SibEnzyme, Novosibirsk). DNA sequencing was performed in the Genomics Core Facility SB RAS. Sequences of the genus Geobacillus species’ reference strains were taken from the website StrainInfo (www.straininfo.net). The BLAST program was used for the search of the similar sequences in the nucleotide databases (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Alignment was performed using the program ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2). Phylogenetic trees were constructed using the Minimum Evolution (ME) algorithm of the program Mega v. 5.0 (Tamura et al., 2011). The 16S rRNA gene sequence determined for strain G1w1 was 1306 nucleotides long. Full genome sequencing of the strain G1w1 genome was performed after sequencing the 16S rRNA and spo0A genes; it allowed us to precisely define G+C content and to analyze full-length sequences of the aforementioned genes. To estimate taxonomic affinity of the G1w1 we used the Average Nucleotide Identity method (ANI value). ANI calculator (http://enveomics.ce.gatech.edu/ani/) was used to compare the genomes of G1w1 and three type strains of the genus Geobacillus (G. stearothermophilus ATCC 7953T, G. kaustophilus NBRC 102445T, G. thermodenitrificans DSM 465T) taken from NCBI (http://www.ncbi.nlm.nih.gov/genome/).
96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147
The analysis of fatty acid methyl ethers was performed including standardization of incubation temperature (60 oC) and incubation time (12 h). Lyophilized cells were prepared according to (Jenkins et al., 1977). After fats 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 analyzed by gas chromatography method on chromatograph Agilent Technologies 6890N with quadrupolar mass spectrometer Agilent Technologies 5973N and fused-quartz capillary column DB-1. Chromatographic-mass spectrometric analysis of the solutions was carried out by both measuring total ion current in scan mode, in the mass range 10 - 800 amu and in the selective ion monitoring mode measuring analyte molecular ion. Identification of the fatty acid methyl esters was carried out by using the base The NIST Mass Spectral Search Program for the NIST / EPA / NIH Mass Spectral Library Version 2.0a. The stain under investigation forms round cream-colored colonies with smooth and lightly curved edges. Morphology of colonies is slightly convex and sizes of colonies are in range from 3 to 5 mm. Cells of the G1w1 stain are motile, rod-shaped with rounded ends; sizes are 0.5-1.0 x 3.5-6.0 microns (Fig. S1). The cell wall is Gram-positive. Sporulation is observed in very rare cases even in conditions of growing stimulation by MnSO4. Endospores are terminal or subterminal, ellipsoidal, 1.5x3.0 micron in size. Swollen sporangia are not observed. Physiological characteristics of strain G1w1 are given in Table 1. The stain G1w1 is thermophile, since it could grow within the temperature range 50–75 oC with an optimum growth temperature of 60–65 oC. Stain growth active both at pH 5 and at pH 9, with an optimum at pH for growth of 6.5–7.0. Rapid growth was observed in medium with NaCl 1%. The stain G1w1 was able to utilize a variety of sugars, carboxylic acids and hydrocarbons. The main fatty acid of strain G1w1 is iso-C15:0 (40%). The fatty acid profile of strain G1w1 comprised iso-C15:0 (39.7%), iso-C17:0 (26.9 %), anteiso-C17:0 (11.6%), C16:0 (7.7%), anteiso-C15:0 (6.8 %), anteiso-C14:0 (2.4 %), C18:0 (1.1 %). Presence of other fatty acids is not more than 1%. A phylogenetic tree was constructed using GenBank database genus Geobacillus reference strain’s 16S rRNA sequences, which were the most related to the G1w1stain’s sequence (Fig. 1). Thus by G1w1 16S rRNA gene sequence the stain can’t be associated with any presently known species of Geobacillus genus. The DNA G+C content of strain G1w1 is 52 mol%. This value is in accordance with the G+C content of the genus Geobacillus, which is 49.0–58.0 mol% (Nazina et al., 2001). For number of strains belonging to the genus Geobacillus, full genomic sequences are known, but the molecular identification of this genus is limited to use of the 16S rRNA sequences, as data on other sequences are unavailable for most type strains. Kuisiene et al. (2009) made an attempt to study the taxonomy of the genus Geobacillus using spo0A gene sequences - the main regulator of the sporulation process, which controls more than 500 genes. The authors showed that by using this marker it is possible to identify reliably only G. thermodenitrificans, G. stearothermophilus and G. jurassicus species. Thus, the conclusion, that spo0A gene sequences cannot be used as a phylogenetic marker within this genus (Kuisiene et al., 2009), was made. Despite this, for quite a number species of the genus Geobacillus the spo0A still remains the only sequenced gene. We have constructed phylogenetic trees based on spo0A gene sequences (Fig. 2). It is observed on the tree that the strain G1w1 markedly spaced from all other representatives of the genus. The following ANI scores were obtained by comparison of G1w1 with Geobacillus type strains: 86.41% (for G. stearothermophilus ATCC 7953T), 87.75% (for G. kaustophilus NBRC 102445T), and 85.47% (for G. thermodenitrificans DSM 465T). All these values are far below the threshold value of 95-96%, which indicates that G. icigianus is a distinct species (Kim et al., 2014). Thus, we can concluded that strain obtained belongs to the genus Geobacillus, but it is markedly different from all species of the genus by biochemical and molecular genetic characteristics.
148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199
Therefore, we considered it reasonable to assign the stain into a new species G. icigianus sp. nov. Description of the new species is given below. Description of Geobacillus icigianus sp. nov. Geobacillus icigianus [i.ci.gi.a'nus. N.L. masc. adj. icigianus, referring to the Institute of Cytology and Genetics (ICiG) at Novosibirsk where the type strain was isolated and described]. Description is based upon a single strain. Gram-positive, motile rods, 0.5-1.0 x 3.5-6.0 microns, with terminal ellipsoidal or cylindrical endospores of 1.5 x 3.0 sizes. Sporulation is extremely rare. Swollen sporangia are not observed. Aerobic or facultatively anaerobic. After 24 h incubation at 60 оC forms circular cream-colored colonies with smooth or slightly curved edges, 3-5 mm in diameter. Grows at 50 and 75 оC, and optimally at 60-65 оC. Grows between pH 5.0 and 9.0; optimum is pH 6.5-7.0. Growth is rapid in medium with NaCl 1%, and is inhibited by 5% NaCl. Oxidase-negative, catalaze- and urease-positive. Aesculin and casein are hydrolysed, 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, arginine, lysine, ornithine, malonate production are negative. Utilization of the adonitol, sorbitol, dulcitol is weak. Acid is produced from D-xylose, no acids are produced from lactose and inositol. Acids producing from cellobiose, glucose, mannitol, melibiose, raffinose, rhamnose, sucrose, trehalose is weak. The type strain is G1w1 (VKM – B-2853T; DSM- 28325T), which was originally isolated from watersludge sample of the hydrothermal (97 0С) outlets, located in the geyser Troinoy region (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.58.1.3. The authors would like to express the 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, 2197–2201. Coorevits, A., Dinsdale, A. E., Halket, G., Lebbe, L., De Vos, P., Van Landschoot, A. & Logan, N. A. (2012). Taxonomic revision of the genus Geobacillus: emendation of Geobacillus, G. stearothermophilus, G. jurassicus, G. toebii, G. thermodenitrificans and G. thermoglucosidans (nom. corrig., formerly ‘thermoglucosidasius’); transfer of Bacillus thermantarcticus to the genus as G. thermantarcticus comb. nov.; proposal of Caldibacillus debilis gen. nov., comb. nov.; transfer of G. tepidamans to Anoxybacillus as A. tepidamans comb. nov.; and proposal of Anoxybacillus caldiproteolyticus sp. nov. Int J Syst Evol Microbiol 62, 1470–1485. Dinsdale, A. E., Halket, G., Coorevits, A., Van Landschoot, A., Busse, H. J., De Vos, P. & Logan, N. A. (2011). Emended descriptions of Geobacillus thermoleovorans and Geobacillus thermocatenulatus. Int J Syst Evol Microbiol 61, 1802–1810. Fortina, M. G., Mora, D., Schumann, P., Parini, C., Manachini, P. L. & Stackebrandt, E. (2001). Reclassification of Saccharococcus caldoxylosilyticus as Geobacillus caldoxylosilyticus (Ahmad et al. 2000) comb. nov. Int J Syst Evol Microbiol 51, 2063–2071.
200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251
Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology. Jenkins, C. L., Kuhn, D. A. & Daly, K. R. (1977). Fatty acid composition of Simonsiella strains. Arch Microbiol 113, 209-213. Kim, M., Oh, H. S., Park, S. C. & Chun, J. (2014). Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes Int J Syst Evol Microbiol 64, 346-351 Kuisiene, N., Raugalas, J. & Chitavichius, D. (2004). Geobacillus lituanicus sp. nov. Int J Syst Evol Microbiol 54, 1991–1995. Kuisiene, N., Raugalas, J. & Chitavichius, D. (2009). Phylogenetic, inter, and intraspecific sequence analysis of spo0A gene of the genus Geobacillus. Curr Microbiol 58, 547–553. Logan, N. A. & De Vos, P. (2009). Genus Bacillus Cohn 1872. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 3, pp. 21–128. Edited by P. De Vos, G. Garrity, D. Jones, N. R. Krieg, W. Ludwig, F. A. Rainey, K.-H. Schleifer & W. B. Whitman. New York: SpringerVerlag. Maniatis, T., Fritsch, E.F. & Sambrook, J. (1982). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Miñana-Galbis, D., Pinzón, D. L., Lorén, J. G., Manresa, Á . & Oliart-Ros, R. M. (2010). Reclassification of Geobacillus pallidus (Scholz et al. 1988) Banat et al. 2004 as Aeribacillus pallidus gen. nov., comb. nov. Int J Syst Evol Microbiol 60, 1600–1604. Nazina, T. N., Tourova, T. P., Poltaraus, A. B., Novikova, E. V., Grigoryan, A. A., Ivanova, A. E., Lysenko, A. M., Petrunyaka, V. V., Osipov, G. A. & other authors (2001). Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans. Int J Syst Evol Microbiol 51, 433–446. Nazina, T. N., Lebedeva, E. V., Poltaraus, A. B., Tourova, T. P., Grigoryan, A. A., Sokolova, D. Sh., Lysenko, A. M. & Osipov, G. A. (2004). Geobacillus gargensis sp. nov., a novel thermophile from a hot spring, and the reclassification of Bacillus vulcani as Geobacillus vulcani comb. nov. Int J Syst Evol Microbiol 54, 2019–2024. Netrusov, A. I., Egorova, M. A., Zakharchuk, L. M., Kolotilova, N. N., Kotova, I. B., Semenova, E. V., Tatarinova, N. Yu., Ugol'nikova, N. V., Tsavkelova, E. A. & other authors (2005). Praktikum po mikrobiologii: Ucheb. posobie dlya stud. vyssh. ucheb. zavedenii. Moscow: Izdatel'skii tsentr Akademiya. Poli, A., Laezza, G., Gul-Guven, R., Orlando, P., Nicolaus, B. (2011). Geobacillus galactosidasius sp. nov., a new thermophilic galactosidase-producing bacterium isolated from compost. Syst Appl Microbiol 34, 419– 423.
252 253 254 255 256 257 258 259 260 261 262 263 264
Schäffer, C., Franck, W. L., Scheberl, A., Kosma, P., McDermott, T. R. & Messner, P. (2004). Classification of isolates from locations in Austria and Yellowstone National Park as Geobacillus tepidamans sp. nov. Int J Syst Evol Microbiol 54, 2361–2368. Sung, M. H., Kim, H., Bae, J. W., Rhee, S. K., Jeon, C. O., Kim, K., Kim, J. J., Hong, S. P., Lee, S. G. & other authors (2002). Geobacillus toebii sp. nov., a novel thermophilic bacterium isolated from hay compost. Int J Syst Evol Microbiol 52, 2251–2255. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
265 266 267 268 269 270 271 272 273 274
Table 1. Characteristics that differentiate Geobacillus icigianus sp. nov. from the closely related species of the genus Geobacillus Strains: 1, G1w1; 2, G. jurassicus B-2301T; 3, G. uzenensis; 4, G. subterraneus; 5, G. thermodenitrificans (3-5 - data were taken from (Coorevits et al., 2012)); 6, G. thermoglucosidasius DSM 2542T; 7, G. stearothermophilus DSM 22T; 8, G. thermocatenulatus B-1259T; 9, G. gargensis (data were taken from Nazina et al., 2004)). Symbols: +, positive reaction; -, negative reaction; w, weak 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. Character
1
2
3
4
5
6
7
8
9
Swollen sporangia Spores: Cylindrical Subterminal Terminal Central/paracentral Hydrolysis of: Aesculin Casein Gelatin ONPG Starch Acid from: Cellobiose Glucose Lactose Inositol Mannitol Melibiose Raffinose Rhamnose Sucrose Trehalose D-Xylose Nitrate reduction Voges–Proskauer Catalase Oxidase Urease Lysine
-
-
d
-
-
-
-
-
w
+ + + -
+ -
+ -
+ v -
+ + -
v + + -
+ + -
+ -
+ -
+ + + -
+ + +
+ + +
+ w +
+ d +/w
+ + w
+ + + +
+ -
+ + ND +
w w w w w w w w + + w + + -
w + w w w w + w + w + -
+ ND + ND + + + ND ND ND ND
+ ND + + ND + + + + + + ND ND
+ ND w d ND d d w +/d +/d + ND ND
w w w w w w w + + + w w -
w w + w w w w + w -
w w w w + w w w w + w + -
+ ND + ND ND ND ND + ND ND ND
Ornithine Arginine Simmons citrate Malonate Inositol Adonitol Sorbitol
w w w
w w w
ND ND ND ND ND ND ND
ND ND ND ND ND ND ND
ND ND ND ND ND ND ND
w w w
w w w
w w w
ND ND ND ND ND ND
Dulcitol Anaerobic growth Growth at/in: pH 5 pH 9 1% NaCl (w/v) 5% NaCl (w/v) Temperature range (oC)
w -
w +
ND +
ND +
ND +
w w
w -
w -
ND -
+ + + 50-75
+ w + w 45-65
+ 45-65
+ 37-60
+ 50-70
+ w 40-60
+ + 30-70
+ w + + 37-80
+ 45-70
275 276 277 278 279 280 281 282 283 284
Fig. 1. Phylogenetic tree constructed based on 16S rRNA gene sequences by ME method. Figures near the branches indicate bootstrap support of the ME algorithm. Fig. 2. Phylogenetic tree constructed on the basis of spo0A gene sequences by ME method. Figures near the branches indicate bootstrap support of the ME algorithm.
Figure 1 without legend Click here to download Figure: Figure 1 - no legend.doc
Figure 2 without legend Click here to download Figure: Figure 2-no legend.doc
Supplementary PDF Click here to download Supplementary Material Files: Figure S1.pdf
International Journal of Systematic and Evolutionary Microbiology Geobacillus icigianus sp. nov., a new thermophilic bacterium isolated from Valley of Geysers, Kamchatka --Manuscript Draft-Manuscript Number:
IJSEM-D-14-00342
Full Title:
Geobacillus icigianus sp. nov., a new thermophilic bacterium isolated from Valley of Geysers, Kamchatka
Short Title:
Geobacillus icigianus sp. nov., a new thermophilic bacterium isolated from Valley of Geysers, Kamchatka
Article Type:
Note
Section/Category:
New taxa - Firmicutes and related organisms
Keywords:
Geobacillus; Geobacillus icigianus; phylogenetic analysis; novel species; Valley of Geysers
Corresponding Author:
Alla Bryanskaya, Ph.D. Institute of Cytology and Genetics SB RAS RUSSIAN FEDERATION
First Author:
Alla Bryanskaya, Ph.D.
Order of Authors:
Alla Bryanskaya, Ph.D. Alexey Rozanov Nikolay Slynko Sergey Shekhovtsov Sergey Peltek
Manuscript Region of Origin:
RUSSIAN FEDERATION
Abstract:
A novel thermophilic spore-forming strain, G1w1, was isolated from hot spring of Valley of Geysers, Kamchatka (Russia). Based on a phylogenetic analysis of 16S rRNA gene and gene spo0A sequences strain represents a new species of the Geobacillus genus. Based on polyphasic taxonomic data the G1w1 strain is assigned to Geobacillus icigianus sp. nov. The type strain is G1w1 (VKM - B-2853T; DSM- 28325T).
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Text with table and fig legends.doc
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Geobacillus icigianus sp. nov., a new thermophilic bacterium isolated from Valley of Geysers, Kamchatka Alla V. Bryanskaya, Alexey S. Rozanov, Nikolay M. Slynko, Sergey V. Shekhovtsov and Sergey E. Peltek Laboratory of Molecular Biotechnology, The Institute of Cytology and Genetics SB RAS, Prospekt Lavrentyeva 10, Novosibirsk, 630090, Russian Federation Correspondence Alla Bryanskaya [email protected] Running title: Geobacillus icigianus sp. nov., a new thermophile Contents category: New taxa - Firmicutes and related organisms A novel thermophilic spore-forming strain, G1w1, was isolated from hot spring of Valley of Geysers, Kamchatka (Russia). Based on a phylogenetic analysis of 16S rRNA gene and gene spo0A sequences strain represents a new species of the Geobacillus genus. Based on polyphasic taxonomic data the G1w1 strain is assigned to Geobacillus icigianus sp. nov. The type strain is G1w1 (VKM – B-2853T; DSM- 28325T).1 Keywords: Geobacillus, Geobacillus icigianus, phylogenetic analysis, novel species, Valley of Geysers The genus Geobacillus was proposed by (Nazina et al., 2001) and major discoveries and descriptions of new species followed after the description of the genus (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). Currently the genus Geobacillus contains 15 species with validly published names. Within the research of unique microbial communities living in Valley of Geysers there were selected microbiological samples of vaporing hydrothermal outlets’ bottom sediments. The novel thermophilic strain of spore-forming bacteria was isolated from those assays. The aim of this study is a comprehensive characterisation of phenotypic, biochemical and molecular genetic properties of the isolated bacterial strain. As the result of the strain detailed analysis it was concluded that the strain belongs to Geobacillus genus by using 16S rRNA sequences, but it was not among previously described species of the genus. We referred to the stain as the new species Geobacillus icigianus sp. nov. The stain G1w1 was isolated from sludge samples of unnamed vaporing hydrothermal (97 0С) outlets situated in the geyser Troinoy region (Valley of Geysers, Kronotsky Nature Reserve). A microbial community which is clearly visible without a microscope represented a deep-green incrustation on pink sludge sediment. Water and sludge samples were collected using a sterile The GenBank accession number for the 16S rRNA gene and spo0A gene sequences of G1w1T strain is KF631430.1 and KF631431.1, respectively. Full genome sequencing of G1w1T strain has been deposited at DDBJ/EMBL/GenBank under the accession JPYA00000000.
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95
sampler into sterile containers and have been stored at 4ºC. Some samples were fixed using equal volume of 96% ethanol. Fermentation of the microbial community was carried out at the medium Luria Bertani (LB) at 45-70 ºC within 1-3 days (Gerhardt et al., 1994). Isolation and purification of the strain were conducted on LB agar medium at 55-65 °C. The reference strains used in this study were obtained from the Leibniz-Institut DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Geobacillus stearothermophilus DSM 22T, Geobacillus thermoglucosidasius DSM 2542T) and from the AllRussian Collection of Microorganisms-VKM (Geobacillus jurassicus B-2301T, Geobacillus thermocatenulatus B-1259T). Strains were grown on LB agar medium at 55-65 °C. Biomass for chemical and molecular systematic studies was obtained by cultivating the organisms in shake flasks containing liquid LB at 55-60 °C for 12-24 h. Bacterial growth in medium was monitored by measuring the optical density of the culture at 600 nm using an automatic multifunctional Epoch Microplate Spectrophotometr (BioTek). Medium was supplemented with 5 mg MnSO4 per liter to encourage sporulation. The shape and size of cells were determined by light and electron microscopes Axioskop 2 Plus, Axioskop А1 all produced by Carl Zeiss, in the Interinstitutional Shared Center for Microscopic Analysis of Biological Objects SB RAS. Bacteria samples were prepared by conventional methods (Netrusov et al., 2005). The Gram reaction was determined using a Gram stain kit (Sintakon, Russia) according to the manufacturer's recommended protocol. Such characteristic of the stain G1w1 as temperature and pH ranges, 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; Logan & De Vos, 2009). Most of the tests were carried out using reagents and kits produced by companies LACHEMA, DIA-M, SIGMA. Tests were performed in triplicate. Microbial DNA was extracted by standard method with phenol (Maniatis et al., 1982). Amplification of 16S rRNA was conducted with universal bacterial primers 16S-8-f-B (5'AGRGTTTGATCCTGGCTCA-3') and 16S-1350-r-B (5'-GACGGGCGGTGTGTACAAG-3'). The fragment of spo0A gene was amplified with GEOSPO-20F73 (5'CAGCCGGACATGGAAGTGAT-3') and GEOSPO-20R696 (5'GACCGTATAGCCGAACAGCG-3') primers (Kuisiene et al., 2009). Reactor feed contained 1.5 mM of MgCl2, 65 mM of Tris-HCl (pH 8.8), 16 mM of (NH4)2SO4 , 0.05% of Tween-20, 0.2 mM of dNTP, 0.3 mM of primers and 1 amu of recombinant Taq polymerase (SibEnzyme, Novosibirsk). DNA sequencing was performed in the Genomics Core Facility SB RAS. Sequences of the genus Geobacillus species’ reference strains were taken from the website StrainInfo (www.straininfo.net). The BLAST program was used for the search of the similar sequences in the nucleotide databases (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Alignment was performed using the program ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2). Phylogenetic trees were constructed using the Minimum Evolution (ME) algorithm of the program Mega v. 5.0 (Tamura et al., 2011). The 16S rRNA gene sequence determined for strain G1w1 was 1306 nucleotides long. Full genome sequencing of the strain G1w1 genome was performed after sequencing the 16S rRNA and spo0A genes; it allowed us to precisely define G+C content and to analyze full-length sequences of the aforementioned genes. To estimate taxonomic affinity of the G1w1 we used the Average Nucleotide Identity method (ANI value). ANI calculator (http://enveomics.ce.gatech.edu/ani/) was used to compare the genomes of G1w1 and three type strains of the genus Geobacillus (G. stearothermophilus ATCC 7953T, G. kaustophilus NBRC 102445T, G. thermodenitrificans DSM 465T) taken from NCBI (http://www.ncbi.nlm.nih.gov/genome/).
96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147
The analysis of fatty acid methyl ethers was performed including standardization of incubation temperature (60 oC) and incubation time (12 h). Lyophilized cells were prepared according to (Jenkins et al., 1977). After fats 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 analyzed by gas chromatography method on chromatograph Agilent Technologies 6890N with quadrupolar mass spectrometer Agilent Technologies 5973N and fused-quartz capillary column DB-1. Chromatographic-mass spectrometric analysis of the solutions was carried out by both measuring total ion current in scan mode, in the mass range 10 - 800 amu and in the selective ion monitoring mode measuring analyte molecular ion. Identification of the fatty acid methyl esters was carried out by using the base The NIST Mass Spectral Search Program for the NIST / EPA / NIH Mass Spectral Library Version 2.0a. The stain under investigation forms round cream-colored colonies with smooth and lightly curved edges. Morphology of colonies is slightly convex and sizes of colonies are in range from 3 to 5 mm. Cells of the G1w1 stain are motile, rod-shaped with rounded ends; sizes are 0.5-1.0 x 3.5-6.0 microns (Fig. S1). The cell wall is Gram-positive. Sporulation is observed in very rare cases even in conditions of growing stimulation by MnSO4. Endospores are terminal or subterminal, ellipsoidal, 1.5x3.0 micron in size. Swollen sporangia are not observed. Physiological characteristics of strain G1w1 are given in Table 1. The stain G1w1 is thermophile, since it could grow within the temperature range 50–75 oC with an optimum growth temperature of 60–65 oC. Stain growth active both at pH 5 and at pH 9, with an optimum at pH for growth of 6.5–7.0. Rapid growth was observed in medium with NaCl 1%. The stain G1w1 was able to utilize a variety of sugars, carboxylic acids and hydrocarbons. The main fatty acid of strain G1w1 is iso-C15:0 (40%). The fatty acid profile of strain G1w1 comprised iso-C15:0 (39.7%), iso-C17:0 (26.9 %), anteiso-C17:0 (11.6%), C16:0 (7.7%), anteiso-C15:0 (6.8 %), anteiso-C14:0 (2.4 %), C18:0 (1.1 %). Presence of other fatty acids is not more than 1%. A phylogenetic tree was constructed using GenBank database genus Geobacillus reference strain’s 16S rRNA sequences, which were the most related to the G1w1stain’s sequence (Fig. 1). Thus by G1w1 16S rRNA gene sequence the stain can’t be associated with any presently known species of Geobacillus genus. The DNA G+C content of strain G1w1 is 52 mol%. This value is in accordance with the G+C content of the genus Geobacillus, which is 49.0–58.0 mol% (Nazina et al., 2001). For number of strains belonging to the genus Geobacillus, full genomic sequences are known, but the molecular identification of this genus is limited to use of the 16S rRNA sequences, as data on other sequences are unavailable for most type strains. Kuisiene et al. (2009) made an attempt to study the taxonomy of the genus Geobacillus using spo0A gene sequences - the main regulator of the sporulation process, which controls more than 500 genes. The authors showed that by using this marker it is possible to identify reliably only G. thermodenitrificans, G. stearothermophilus and G. jurassicus species. Thus, the conclusion, that spo0A gene sequences cannot be used as a phylogenetic marker within this genus (Kuisiene et al., 2009), was made. Despite this, for quite a number species of the genus Geobacillus the spo0A still remains the only sequenced gene. We have constructed phylogenetic trees based on spo0A gene sequences (Fig. 2). It is observed on the tree that the strain G1w1 markedly spaced from all other representatives of the genus. The following ANI scores were obtained by comparison of G1w1 with Geobacillus type strains: 86.41% (for G. stearothermophilus ATCC 7953T), 87.75% (for G. kaustophilus NBRC 102445T), and 85.47% (for G. thermodenitrificans DSM 465T). All these values are far below the threshold value of 95-96%, which indicates that G. icigianus is a distinct species (Kim et al., 2014). Thus, we can concluded that strain obtained belongs to the genus Geobacillus, but it is markedly different from all species of the genus by biochemical and molecular genetic characteristics.
148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199
Therefore, we considered it reasonable to assign the stain into a new species G. icigianus sp. nov. Description of the new species is given below. Description of Geobacillus icigianus sp. nov. Geobacillus icigianus [i.ci.gi.a'nus. N.L. masc. adj. icigianus, referring to the Institute of Cytology and Genetics (ICiG) at Novosibirsk where the type strain was isolated and described]. Description is based upon a single strain. Gram-positive, motile rods, 0.5-1.0 x 3.5-6.0 microns, with terminal ellipsoidal or cylindrical endospores of 1.5 x 3.0 sizes. Sporulation is extremely rare. Swollen sporangia are not observed. Aerobic or facultatively anaerobic. After 24 h incubation at 60 оC forms circular cream-colored colonies with smooth or slightly curved edges, 3-5 mm in diameter. Grows at 50 and 75 оC, and optimally at 60-65 оC. Grows between pH 5.0 and 9.0; optimum is pH 6.5-7.0. Growth is rapid in medium with NaCl 1%, and is inhibited by 5% NaCl. Oxidase-negative, catalaze- and urease-positive. Aesculin and casein are hydrolysed, 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, arginine, lysine, ornithine, malonate production are negative. Utilization of the adonitol, sorbitol, dulcitol is weak. Acid is produced from D-xylose, no acids are produced from lactose and inositol. Acids producing from cellobiose, glucose, mannitol, melibiose, raffinose, rhamnose, sucrose, trehalose is weak. The type strain is G1w1 (VKM – B-2853T; DSM- 28325T), which was originally isolated from watersludge sample of the hydrothermal (97 0С) outlets, located in the geyser Troinoy region (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.58.1.3. The authors would like to express the 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, 2197–2201. Coorevits, A., Dinsdale, A. E., Halket, G., Lebbe, L., De Vos, P., Van Landschoot, A. & Logan, N. A. (2012). Taxonomic revision of the genus Geobacillus: emendation of Geobacillus, G. stearothermophilus, G. jurassicus, G. toebii, G. thermodenitrificans and G. thermoglucosidans (nom. corrig., formerly ‘thermoglucosidasius’); transfer of Bacillus thermantarcticus to the genus as G. thermantarcticus comb. nov.; proposal of Caldibacillus debilis gen. nov., comb. nov.; transfer of G. tepidamans to Anoxybacillus as A. tepidamans comb. nov.; and proposal of Anoxybacillus caldiproteolyticus sp. nov. Int J Syst Evol Microbiol 62, 1470–1485. Dinsdale, A. E., Halket, G., Coorevits, A., Van Landschoot, A., Busse, H. J., De Vos, P. & Logan, N. A. (2011). Emended descriptions of Geobacillus thermoleovorans and Geobacillus thermocatenulatus. Int J Syst Evol Microbiol 61, 1802–1810. Fortina, M. G., Mora, D., Schumann, P., Parini, C., Manachini, P. L. & Stackebrandt, E. (2001). Reclassification of Saccharococcus caldoxylosilyticus as Geobacillus caldoxylosilyticus (Ahmad et al. 2000) comb. nov. Int J Syst Evol Microbiol 51, 2063–2071.
200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251
Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology. Jenkins, C. L., Kuhn, D. A. & Daly, K. R. (1977). Fatty acid composition of Simonsiella strains. Arch Microbiol 113, 209-213. Kim, M., Oh, H. S., Park, S. C. & Chun, J. (2014). Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes Int J Syst Evol Microbiol 64, 346-351 Kuisiene, N., Raugalas, J. & Chitavichius, D. (2004). Geobacillus lituanicus sp. nov. Int J Syst Evol Microbiol 54, 1991–1995. Kuisiene, N., Raugalas, J. & Chitavichius, D. (2009). Phylogenetic, inter, and intraspecific sequence analysis of spo0A gene of the genus Geobacillus. Curr Microbiol 58, 547–553. Logan, N. A. & De Vos, P. (2009). Genus Bacillus Cohn 1872. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 3, pp. 21–128. Edited by P. De Vos, G. Garrity, D. Jones, N. R. Krieg, W. Ludwig, F. A. Rainey, K.-H. Schleifer & W. B. Whitman. New York: SpringerVerlag. Maniatis, T., Fritsch, E.F. & Sambrook, J. (1982). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Miñana-Galbis, D., Pinzón, D. L., Lorén, J. G., Manresa, Á . & Oliart-Ros, R. M. (2010). Reclassification of Geobacillus pallidus (Scholz et al. 1988) Banat et al. 2004 as Aeribacillus pallidus gen. nov., comb. nov. Int J Syst Evol Microbiol 60, 1600–1604. Nazina, T. N., Tourova, T. P., Poltaraus, A. B., Novikova, E. V., Grigoryan, A. A., Ivanova, A. E., Lysenko, A. M., Petrunyaka, V. V., Osipov, G. A. & other authors (2001). Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans. Int J Syst Evol Microbiol 51, 433–446. Nazina, T. N., Lebedeva, E. V., Poltaraus, A. B., Tourova, T. P., Grigoryan, A. A., Sokolova, D. Sh., Lysenko, A. M. & Osipov, G. A. (2004). Geobacillus gargensis sp. nov., a novel thermophile from a hot spring, and the reclassification of Bacillus vulcani as Geobacillus vulcani comb. nov. Int J Syst Evol Microbiol 54, 2019–2024. Netrusov, A. I., Egorova, M. A., Zakharchuk, L. M., Kolotilova, N. N., Kotova, I. B., Semenova, E. V., Tatarinova, N. Yu., Ugol'nikova, N. V., Tsavkelova, E. A. & other authors (2005). Praktikum po mikrobiologii: Ucheb. posobie dlya stud. vyssh. ucheb. zavedenii. Moscow: Izdatel'skii tsentr Akademiya. Poli, A., Laezza, G., Gul-Guven, R., Orlando, P., Nicolaus, B. (2011). Geobacillus galactosidasius sp. nov., a new thermophilic galactosidase-producing bacterium isolated from compost. Syst Appl Microbiol 34, 419– 423.
252 253 254 255 256 257 258 259 260 261 262 263 264
Schäffer, C., Franck, W. L., Scheberl, A., Kosma, P., McDermott, T. R. & Messner, P. (2004). Classification of isolates from locations in Austria and Yellowstone National Park as Geobacillus tepidamans sp. nov. Int J Syst Evol Microbiol 54, 2361–2368. Sung, M. H., Kim, H., Bae, J. W., Rhee, S. K., Jeon, C. O., Kim, K., Kim, J. J., Hong, S. P., Lee, S. G. & other authors (2002). Geobacillus toebii sp. nov., a novel thermophilic bacterium isolated from hay compost. Int J Syst Evol Microbiol 52, 2251–2255. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
265 266 267 268 269 270 271 272 273 274
Table 1. Characteristics that differentiate Geobacillus icigianus sp. nov. from the closely related species of the genus Geobacillus Strains: 1, G1w1; 2, G. jurassicus B-2301T; 3, G. uzenensis; 4, G. subterraneus; 5, G. thermodenitrificans (3-5 - data were taken from (Coorevits et al., 2012)); 6, G. thermoglucosidasius DSM 2542T; 7, G. stearothermophilus DSM 22T; 8, G. thermocatenulatus B-1259T; 9, G. gargensis (data were taken from Nazina et al., 2004)). Symbols: +, positive reaction; -, negative reaction; w, weak 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. Character
1
2
3
4
5
6
7
8
9
Swollen sporangia Spores: Cylindrical Subterminal Terminal Central/paracentral Hydrolysis of: Aesculin Casein Gelatin ONPG Starch Acid from: Cellobiose Glucose Lactose Inositol Mannitol Melibiose Raffinose Rhamnose Sucrose Trehalose D-Xylose Nitrate reduction Voges–Proskauer Catalase Oxidase Urease Lysine
-
-
d
-
-
-
-
-
w
+ + + -
+ -
+ -
+ v -
+ + -
v + + -
+ + -
+ -
+ -
+ + + -
+ + +
+ + +
+ w +
+ d +/w
+ + w
+ + + +
+ -
+ + ND +
w w w w w w w w + + w + + -
w + w w w w + w + w + -
+ ND + ND + + + ND ND ND ND
+ ND + + ND + + + + + + ND ND
+ ND w d ND d d w +/d +/d + ND ND
w w w w w w w + + + w w -
w w + w w w w + w -
w w w w + w w w w + w + -
+ ND + ND ND ND ND + ND ND ND
Ornithine Arginine Simmons citrate Malonate Inositol Adonitol Sorbitol
w w w
w w w
ND ND ND ND ND ND ND
ND ND ND ND ND ND ND
ND ND ND ND ND ND ND
w w w
w w w
w w w
ND ND ND ND ND ND
Dulcitol Anaerobic growth Growth at/in: pH 5 pH 9 1% NaCl (w/v) 5% NaCl (w/v) Temperature range (oC)
w -
w +
ND +
ND +
ND +
w w
w -
w -
ND -
+ + + 50-75
+ w + w 45-65
+ 45-65
+ 37-60
+ 50-70
+ w 40-60
+ + 30-70
+ w + + 37-80
+ 45-70
275 276 277 278 279 280 281 282 283 284
Fig. 1. Phylogenetic tree constructed based on 16S rRNA gene sequences by ME method. Figures near the branches indicate bootstrap support of the ME algorithm. Fig. 2. Phylogenetic tree constructed on the basis of spo0A gene sequences by ME method. Figures near the branches indicate bootstrap support of the ME algorithm.
Figure 1 without legend Click here to download Figure: Figure 1 - no legend.doc
Figure 2 without legend Click here to download Figure: Figure 2-no legend.doc
Supplementary PDF Click here to download Supplementary Material Files: Figure S1.pdf
