IJSEM Papers in Press. Published June 6, 2014 as doi:10.1099/ijs.0.059766-0

1 Geothermomicrobium terrae gen. nov., sp. nov., a novel member

2 of the family Thermoactinomycetaceae

3

4 En-Min Zhou1†, Tian-Tian Yu1†, Lan Liu1, Hong Ming1,2, Yi-Rui Yin1, Lei Dong1,

5 Min Tseng3, Guo-Xing Nie4, Wen-Jun Li1,5*

6

7 1Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, Yunnan

8 Institute of Microbiology, Yunnan University, Kunming, 650091, PR China

9 2Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang,

10 453003, PR China

11 3Bio-resource Collection and Research Center, Food Industry Research and Development

12 Institute, HsinChu, 300, Taiwan

13 4 College of Fisheries, Henan Normal University, Xinxiang, 453007, PR China

14 5State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources,

15 School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, PR China 16 17 18 19 Author for correspondence: Wen-Jun Li 20 Tel & Fax: +86 871 65033335 21 E-Mail: [email protected]; [email protected]

22

23 †These authors contributed equally to this work. 24 25 Running title: Geothermomicrobium terrae gen. nov., sp. nov. 26 Category: New Taxa – Firmicutes and Related Organisms 27 28 The DDBJ/EMBL/GenBank accession numbers for the 16S rRNA gene sequences of 29 strains YIM 77562T and YIM 77580 are AB859256 and AB859257, respectively. 30 Strains YIM 77562T and YIM 77580, two novel Gram-staining positive, 31 filamentous bacterial isolates were recovered from the Rehai Geothermal Field, 32 Tengchong, Yunnan province, south-west China. Good growth was observed at 33 50-55 °C and pH 7.0. Aerial mycelium was absent on all the media tested. 34 Substrate mycelia were well-developed, long, with moderately flexuous and 35 formed abundant single warty ornamented endospores. Phylogenetic analysis of 36 the 16S rRNA gene sequences of the two strains indicated that they belong to the 37 family Thermoactinomycetaceae. Similarity levels between the 16S rRNA gene 38 sequences of the two strains and those of the type strains of 39 Thermoactinomycetaceae members were 88.33-93.24 %; the highest sequence 40 similarity was with Hazenella coriacea DSM 45707T. In both strains, the 41 predominant menaquinone was MK-7, the diagnostic diamino acid was

42 meso-diaminopimelic acid, and the major cellular fatty acids were iso-C14:0,

43 iso-C15:0, and iso-C16:0. The major polar lipids were diphosphatidylglycerol, 44 phosphatidylmethylethanolamine, unidentified polar lipid and unidentified 45 phospholipid. The G+C contents of the genomic DNAs of strains YIM 77562T 46 and YIM 77580 were 45.5 and 44.2 mol%, respectively. DNA-DNA relatedness 47 data suggest that the two isolates represent a single species. Based on 48 phylogenetic analyses and physiological and biochemical characteristics, it is 49 proposed that the two strains represent a novel species in a new genus, 50 Geothermomicrobium terrae gen. nov., sp. nov. and the type strain is YIM 77562T 51 (= CCTCC AA 2011022T = JCM 18057T). 52 53 Key words: Geothermomicrobium terrae, Rehai Geothermal Field, Tengchong, 54 Thermoactinomycetaceae 55 56 The genus Thermoactinomyces was firstly proposed by Tsilinsky (1899) and was long 57 recognized as actinomycetes because of their morphological characteristics, forming 58 aerial and substrate mycelia. However, the genus has now been placed within the 59 order Bacillales (Park et al., 1993; Stackebrandt & Woese, 1981; Yoon & Park, 2000; 60 Yoon et al., 2000) due to similarities in the development, heat resistance, and other 61 properties of the endospores to those of Bacillus strains (Cross et al., 1971) and due to 62 lower DNA G+C contents, and phylogenetic differences from actinomycetes. Based 63 on further 16S rRNA gene sequence analysis, as well as chemotaxonomic and 64 physiological characterization, this genus was divided into four genera, 65 Thermoactinomyces sensu stricto, Laceyella, Thermoflavimicrobium and Seinonella 66 (Yoon et al., 2005). Subsequently, the family Thermoactinomycetaceae was described 67 by Matsuo et al. (2006) to accommodate four genera together with Planifilum 68 (Hatayama et al., 2005) and Mechercharimyces (Matsuo et al., 2006). Other genera 69 have been described and included in the family, such as Shimazuella (Park et al., 70 2007), Desmospora (Yassin et al., 2009), Kroppenstedtia (Von Jan et al., 2011), 71 Melghirimyces (Addou et al., 2012), Lihuaxuella (Yu et al., 2012), Marininema (Li et 72 al., 2012), Polycladomyces (Tsubouchi et al., 2013) and Hazenella (Buss et al., 2013). 73 And the description of this family has recently been emended by Yassin et al. (2009), 74 Von Jan et al. (2011) and Li et al.(2012). The members of this family are 75 characterized by the formation of a single, non-stalked spore on the aerial or substrate 76 hyphae, or consecutive spores on straight or branched sporephores. And they are 77 aerobic, Gram-positive and thermophilic, except for five species, Seinonella 78 peptonophila (Nonomura & Ohara, 1971; Yoon et al., 2005), Mechercharimyces 79 asporophorigenens, Mechercharimyces mesophilus (Matsuo et al., 2006), 80 Shimazuella kribbensis (Park et al., 2007), Marininema mesophilum (Li et al., 2012) 81 and Hazenella coriacea (Buss et al., 2013), which are mesophilic, growing at below 82 45 oC. 83 84 The Rehai Geothermal Field, located within the Indo-Burma Range near the border 85 between China and Myanmar within Tengchong County，in central-western Yunnan 86 Province, is the largest and most intensively studied geothermal field in China. During 87 our investigating thermophilic microorganisms in this Geothermal Field, three 88 members of the family Thermoactinomycetaceae had been described in the previous 89 study (Chen et al., 2012; Yu et al., 2012; Zhang et al., 2010) and Planifilum 90 yunnanense described by Zhang et al. (2007) was also isolated from this area. Here 91 we characterize another new member of the family Thermoactinomycetaceae on the 92 basis of phylogenetic and polyphasic biochemical studies. 93 94 Strains YIM 77562T and YIM 77580 were isolated from a geothermal soil sample (N 95 24.95014°, W 98.43742°) collected from Rehai National Park, Tengchong, Yunnan 96 Province, south-west China, by the serial dilution technique using R2A medium (BD; 97 Becton, Dickinson and Company) after 1 week incubation at 55 °C. Then the purified 98 isolate was routinely cultured on International Streptomyces Project medium 2 (ISP 2) 99 agar (Shirling & Gottlieb, 1966) at 50 °C and maintained as a glycerol suspension (20 100 %, w/v) at -80 °C. 101 102 Gram staining was carried out by using the standard Gram reaction and was 103 confirmed by using the KOH lysis test method (Cerny, 1978). For cultural 104 characterization, strain YIM 77562T and YIM 77580 were grown for 1-7 days at 50 105 ºC on ISP media 2, 3, 4 and 5 (all prepared as described by Shirling & Gottlieb, 1966), 106 Nutrient agar (NA; 67 Difco), Czapek’s agar, potato-glucose agar prepared as 107 described by Dong and Cai (2001), and tryptic soya agar (TSA). Colours were 108 determined by using colour chips from the ISCC-NBS colour charts (standard 109 samples, no. 2106) (Kelly, 1964). In order to determine cell morphology, strains YIM 110 77562T and YIM 77580 were grown on ISP 2 agar media at 50 °C for 3 days and then 111 observed by using light (BH-2; Olympus) and scanning electron (QUANTA 200; FEI) 112 microscopes. The temperature range (25 to 70 ºC) and NaCl concentrations (0 to 10% 113 added, w/v) for growth were determined on ISP 2 agar by incubating the cultures at 50 114 ºC for 15 days. The pH range (pH 4 to 10, using the buffer system described by Xu et 115 al. 2005) for growth were tested at 50 ºC for 28 days by culturing the strains in ISP 2 116 broth. Utilization of carbohydrates as sole carbon sources was tested by the methods 117 described by Shirling & Gottlieb (1966) and Locci (1989). Nitrogen source utilization 118 was determined according to Williams (1989). Oxidase activity was determined from 119 the oxidation of tetramethyl-p-phenylenediamine. Catalase activity was determined 120 with hydrogen peroxide. Test of hydrolysis of gelatin, cellulose, starch, Tweens 20, 121 40, 60 and 80, milk coagulation and peptonization, reduction of nitrate, and urease 122 activity were determined as described by Gonzalez et al. (1978) with incubation for 2 123 weeks at 50 ºC. 124 125 Good growth of the strains YIM 77562T and YIM 77580 were observed on ISP 2, ISP 126 3 and Nutrient agar, and moderate growth on ISP 4, ISP 5 and Czapek’s agar media 127 while no growth occurred on TSA or potato-glucose agar media. Aerial mycelium and 128 diffusible pigment were not observed on any of the test media. The two strains formed 129 white substrate mycelium on ISP 4, ISP 5 and Czapek’s agar media and formed gray 130 yellow substrate mycelium on ISP 2 and nutrient agar media. Strain YIM 77562T 131 formed white substrate mycelium on ISP 3 media while strain YIM 77580 form 132 yellow white substrate mycelium. Microscopic studies revealed that cells of the two 133 strains were long and with moderately flexuous hyphae, forming single warty 134 ornamented endospores (Fig. 1 a, b). Growth of strains YIM 77562T and YIM 77580 135 were observed at temperatures between 30 and 60 ºC, with an optimum growth at 136 50-55 °C; at pH 5.0-8.0, with optimum at pH 7.0. The two strains can tolerate to 1.0 137 % (w/v) NaCl concentration and exhibited optimum growth with no added NaCl. The 138 detailed physiological properties of the strains are shown in the Table 1 and given in 139 the species description. 140 141 The biomass used for chemotaxonomic analyses was obtained from cultures grown in 142 the stationary growth phases. The two strains were inoculated into ISP2 medium and 143 incubated at 50 ºC on a rotary shaker (200 r.p.m.) for 3 days. The isomer of 144 diaminopimelic acid and whole-cell sugars were analysed according to the procedures 145 developed by Hasegawa et al. (1983) and Tang et al. (2009). Polar lipids were 146 extracted as described by Minnikin et al. (1979) and identified by two-dimensional 147 TLC followed by spraying with specific reagents (Collins & Jones, 1980). The 148 respiratory quinones were extracted and purified as described by Collins et al. (1977) 149 and analysed by HPLC (Groth et al., 1997). Cellular fatty acid methyl esters were 150 prepared as described by Sasser (1990) and analysed according to the manufacturer’s 151 instructions of the Microbial Identification System (Sherlock Version 6.1; MIDI 152 database: TSBA6). The gas chromatograph was Agilent Technologies 7890A GC 153 System. The G+C content of the genomic DNA was determined by using the HPLC 154 method (Mesbah et al., 1989). 155 156 The two strains contained meso-diaminopimelic acid as the diagnostic diamino acid in 157 the cell wall peptidoglycan, and the whole-cell sugars detected were mannose, ribose, 158 rhamnose, glucose and galactose, but no diagnostic sugars. MK-7 was the only 159 menaquinone of the two strains. The polar lipid profile of strain YIM 77562T was 160 diphosphatidylglycerol (DPG), phosphatidylmethylethanolamine (PME), 161 phosphatidylglycerol (PG), phosphatidylethanolamine (PE), four unidentified polar 162 lipids (UL1-UL4), two unidentified phospholipid (PL1 and PL2), and an unidentified 163 aminophospholipid (APL) (Table 1 and Fig. S1a). The polar lipid characters of strain 164 YIM 77580 were similar to strain YIM 77562T except that one unidentified 165 phospholipids (PL3) were present (Fig. S1b). The cellular fatty acid (>0.5 % of total 166 fatty acids) profiles of strains YIM 77562T and YIM 77580 were composed of

167 iso-C14:0 (34.1 and 34.0 %, respectively), iso-C15:0 (33.6 and 36.9 %, respectively),

168 iso-C16:0 (21.2 and 16.9 %, respectively), anteiso-C15:0 (3.6 and 4.1 %, respectively),

169 iso-C13:0 (0.9 and 0.6 %, respectively), iso-C17:0 (1.5 and 1.4 %, respectively), iso

170 H-C16:1(1.7 and 1.2 %, respectively), C14:0 (1.0 and 1.7 %, respectively), C16:0 (1.0 and 171 1.1 %, respectively), summed Feature 1 (0.6 and 0.5 %, respectively; summed feature

172 1 comprises iso H-C15:1 and/or 3OH-C13:0) and summed feature 4 (0.5 and 0.5 %,

173 respectively; summed feature 4 comprised iso I/anteiso B-C17:1). The DNA G+C 174 contents of strains YIM 77562T and YIM 77580 were 45.5 and 44.2 mol% 175 respectively. 176 177 Extraction of genomic DNA and PCR amplification of the 16S rRNA gene sequence 178 were performed as described by Li et al. (2007). The resulting 16S RNA gene 179 sequences of the two strains were compared with available 16S rRNA gene sequences 180 of cultured species from GenBank via the BLAST program and the EzTaxon-e server 181 (http://eztaxon-e.ezbiocloud.net/, Kim et al. 2012). Multiple alignments with 182 sequences of closely related taxa were carried out using CLUSTAL_X (Thompson et 183 al., 1997). Gaps at the 5’ and 3’ ends of the alignment were omitted from further 184 analysis. A phylogenetic analysis was performed using the software package MEGA 185 (Molecular Evolutionary Genetics Analysis) version 5.0 (Tamura et al., 2011). 186 Evolutionary distances were calculated using the distance options according to 187 Kimura two-parameter model. Clustering using the neighbour-joining (Saitou & Nei, 188 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 189 1981) methods were determined by using bootstrap values based on 1000 replicates. 190 DNA-DNA hybridization between strain YIM 77562T and YIM 77580 was carried 191 out by applying the fluorometric micro-well method (Ezaki et al., 1989; He et al., 192 2005) at the optimal hybridization temperature (37 °C). The experiments were 193 performed with 5 replications and the DNA-DNA relatedness value was expressed as 194 the means of the five values. 195 196 The almost complete 16S rRNA gene sequences of strains YIM 77562T (1506 bp) and 197 YIM 77580 (1506 bp) were determined in this study. 16S rRNA gene sequence 198 comparisons revealed that the two strains were most closely related to the species 199 Hazenella coriacea DSM 45707T (93.24 and 93.10 %, respectively), Lihuaxuella 200 thermophila YIM 77831T (92.93 and 92.79 %, respectively), Shimazuella kribbensis 201 KCTC 9933T (92.76 and 92.55 %, respectively), Thermoflavimicrobium 202 dichotomicum KCTC 3667T (92.22 and 91.94 %, respectively), Seinonella 203 peptonophila KCTC 9740T (91.24 and 91.17 %, respectively), Planifilum yunnanense 204 LA5T (91.64 and 91.23 %, respectively) and to the type strains of members of the 205 genera Laceyella (92.40-92.71% similarity), Thermoactinomyces (91.46-92.22% 206 similarity), and Marininema (91.29-91.95% similarity). Phylogenetic analysis of the 207 16S rRNA gene sequence of strains YIM 77562T and YIM 77580 revealed that the 208 two strains belonged to a separate line within the family Thermoactinomycetaceae 209 that is distinct from all other genera within the family and formed a well-supported 210 clade (supported by a 97% bootstrap value, see Fig.2) with Shimazuella kribbensis 211 DSM 45090T. The topologies of the tree generated with the maximum-parsimony and 212 maximum-likelihood algorithm were similar (Supplementary Fig. S2 and Fig. S3). 213 The 16S rRNA gene sequence similarities between strains YIM 77562T and YIM 214 77580 was 99.5 %, and the determined DNA-DNA relatedness value was 79.2 ± 2.8 215 %. In the current bacterial systematics, a species is defined when DNA-DNA 216 relatedness between the two strains is below 70% (Wayne et al., 1987). Therefore, 217 strains YIM 77562T and YIM 77580 should be considered as members of the same 218 species. 219 220 Strains YIM 77562T and YIM 77580 are Gram-positive, filamentous growth, heat 221 resistance and singly endospores producing along mycelia. The two strains contained 222 meso-diaminopimelic acid in the cell wall and the predominant menaquinone was 223 MK-7. These results were consistent with that obtained previously for other members 224 of the family Thermoactinomycetaceae. The phylogenetic analysis also indicated the 225 two strains fell within the family Thermoactinomycetaceae but belonged to a separate 226 line. In this study, in spite of the BLAST results showed the two strains had the highest 227 gene sequence similarity with the genus Hazenella, the two strains formed a coherent 228 clade with genera Shimazuella and Seinonella in the phylogenetic tree (Fig.2). The 229 two novel isolates could be clearly distinguished from the three genus as well as other 230 genera of the family Thermoactinomycetaceae (Table 1). The absence of aerial 231 mycelium differentiates the two strains from the all genera in the family 232 Thermoactinomycetaceae, with the exception of Hazenella, Lihuaxuella, Planifilum 233 and Marininema. The two strains could not hydrolyse gelatin further differs from 234 almost all members of this family except members of the genera Shimazuella, 235 Seinonella, and Marininema. The higher optimal growth temperature differentiates 236 them from members of the genera Shimazuella, Seinonella, Mechercharimyces, 237 Marininema, Hazenella, and Kroppenstedtia. The predominant menaquinone 238 differentiates them from members of the genera Shimazuella, Laceyella and 239 Mechercharimyces. The presence of meso-DAP in the cell-wall peptidoglycan 240 differentiates them from members of the genera Marininema, Melghirimyces, and 241 Kroppenstedtia. These results suggested that strains YIM 77562T and YIM 77580 242 represent a novel species in a new genus, for which the name Geothermomicrobium 243 terrae gen. nov., sp. nov. is proposed. 244 245 Description of Geothermomicrobium gen. nov. 246 Geothermomicrobium (Ge.o.ther'mo. mi.cro'bi.um. Gr. n. ge, the earth; Gr. n. thermos, 247 heat; Gr. adj. micros small; Gr. n. bios life; N.L. neut. n. Geothermomicrobium, a 248 microbe from hot earth). 249 Cells are Gram-positive, aerobic and thermotolerant. Aerial mycelium is not produced. 250 The substrate mycelia are well-developed, long, with moderately flexuous. Single 251 warty ornamented endospores are formed on the substrate mycelium. The cell-wall 252 peptidoglycan contains meso-diaminopimelic acid and the whole-cell sugars detected 253 were mannose, ribose, rhamnose, glucose and galactose, but no characteristic sugars.

254 The predominant menaquinone is MK-7. Major fatty acids are iso-C14:0, iso-C15:0 and

255 iso-C16:0. The major polar lipids are diphosphatidylglycerol, 256 phosphatidylmethylethanolamine, unidentified polar lipid and unidentified 257 phospholipid. The G+C content is about 44-46 mol%. The type species is 258 Geothermomicrobium terrae. 259 260 Description of Geothermomicrobium terrae sp. nov. 261 Geothermomicrobium terrae (ter’rae. L. gen. n. terrae of the soil, the source of the 262 type strain). 263 In addition to the genus description, exhibits the following characteristics. Good 264 growth is observed on ISP 2, ISP 3 and Nutrient media, gray yellow substrate 265 mycelium is formed on ISP 2 media. Soluble pigment is not produced on any of the 266 tested media. Growth occurs at 30-60 °C, pH 5.0-8.0, and at NaCl concentrations not 267 more than 1%. The optimal growth occurs at 50-55 ºC, pH 7.0 and no NaCl added. 268 Positive for oxidase, catalase, milk coagulation and peptonization. Nitrate reduction, 269 gelatin liquefaction, urea, starch and cellulose hydrolysis are negative. Tweens 40, 60, 270 80 is hydrolyzed, but Tween 20 is not. Raffinose, glucose, sucrose, galactose, 271 cellobiose, mannose can be utilized as sole carbon sources, but ribose, fructose, 272 trehalose, xylitol, lactose, maltose, rhamnose, xylose, mannitol, glycerin, and 273 L-arabinose are not utilized. L-histidine, L-asparagines, L-cysteine, L-tryptophan, 274 L-tyrosine and L-phenylalanine are utilized as sole nitrogen sources, but not 275 L-hydroxyproline, L-cystine L-threonine, L-valine, L-alanine, L-lysine, L-proline, 276 L-arginine, L-ornithine, L-methionine, L-serine, and L-glycine. The cell wall contains 277 meso-DAP, and whole-cell hydrolysates contain mannose, ribose, rhamnose, glucose 278 and galactose. The polar lipids are diphosphatidylglycerol, 279 phosphatidylmethylethanolamine, phosphatidylglycerol, phosphatidylethanolamine, 280 four unidentified polar lipids, two unidentified phospholipid, and an unidentified 281 aminophospholipid. The only menaquinone is MK-7. The major cellular fatty acid T 282 profile of strain YIM 77562 is composed of iso-C14:0, iso-C15:0, and iso-C16:0. The 283 G+C content of the DNA of the type strain is 45.5 mol%. The type strain, YIM 284 77562T (= CCTCC AA 2011022T= JCM 18057T), was isolated from a geothermal soil 285 sample collected at Rehai National Park, Tengchong, Yunnan Province, south-west 286 China. 287 288 Acknowledgments 289 The authors are grateful to Mr. Yong-Jun Liu for his help on scanning electron 290 microscopic observation. This research was supported by the Key Project of 291 International Cooperation of Ministry of Science & Technology (MOST) (No. 292 2013DFA31980), National Science Foundation grant (OISE-0968421) and Key 293 Project of Yunnan Provincial Natural Science Foundation (2013FA004). 294 295 References

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425 thermoactinomycete isolated from a hot spring. Int J Syst Evol Microbiol 57, 1851-1854. 426 427 Legends to Figures 428 (b)(a) 429 Fig.1 Scanning electron micrographs of strain YIM 77562T grown on ISP2 medium 430 for 3 days at 50 °C. Bars: a, 5 µm; b, 3 µm. 431 432 Fig.2 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences 433 showing the position of strains YIM 77562T and YIM 77580 within the family 434 Thermoactinomycetaceae. Bacillus subtilis NCDO 1769T and Geobacillus 435 stearothermophilus DSM 22T were used as outgroups. Bootstrap values greater than 436 50% are given at branch points. Bar, 0.02 substitutions per nucleotide position. 437 Asterisks denote nodes that were also recovered using the maximum-parsimony and 438 maximum-likelihood methods. Table 1. Characteristics differentiating Geothermomicrobium terrae gen. nov., sp. nov. from other members of the genera classified in the family Thermoactinomycetaceae Taxa are indicated as: 1, Geothermomicrobium terrae gen. nov., sp. nov. (data from this study); 2, Shimazuella (Park et al., 2007); 3, Seinonella (Yoon et al., 2005); 4, Hazenella (in press, Buss et al., 2013); 5, Lihuaxuella (Yu et al., 2012); 6, Laceyella (Lacey & Cross, 1989; Yoon et al., 2005); 7, Thermoactinomyces (Lacey & Cross, 1989; Yoon et al., 2005); 8, Thermoflavimicrobium (Lacey & Cross, 1989; von Jan et al., 2010; Yoon et al., 2005); 9, Marininema (Li et al., 2012); 10, Desmospora (von Jan et al., 2010; Yassin et al., 2009); 11, Planifilum (Hatayama et al., 2005; von Jan et al., 2010; Zhang et al., 2007); 12, Polycladomyces (Tsubouchi et al., 2012); 13, Melghirimyces (Addou et al., 2012); 14, Mechercharimyces (Matsuo et al., 2006); 15, Kroppenstedtia (von Jan et al., 2010); +, positive reaction; -, negative reaction; ND, not detected.

Characteristic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Colour of aerial ND white white ND ND white white yellow ND yellow ND White yellow white white mycelium Degradation of:

gelatin - - - + + + + + - + + + + + +

starch - + - - - + - - - + + - - - - Optimal 50-55 32 35 42-45 50 48-55 50-55 55 30 30-50 55-70 55-60 40-55 30 45 temperature DNA G+C 44-46 39.4 40 37.8 55.6 48.0-49.0 48 43 46.5 49.3 56.8-60.3 55.1 47.3 45 54.6 content mol % Predominant MK-7 MK-9 MK-7 MK-7 MK-7 MK-9 MK-7 MK-7 MK-7 MK-7 MK-7 MK-7 MK-7 MK-9 MK-7 Menaquinone

MK-8, MK-7 or Other detected MK-8, MK-8 or MK-6, ND MK-10 MK-9, ND MK-8 or ND ND ND ND MK-8 MK-8 ND menaquinones MK-6 MK-9 MK-8 MK-10 MK-10

anteiso-C15:0 iso-C17:0 iso-C15:0 iso-C14:0 iso-C16:0 iso-C15:0 iso-C15:0 iso-C15:0 iso-C15:0 iso-C15:0 Major cellular iso-C14:0 iso-C15:0 anteiso-C15:0 anteiso-C17:0 iso-C17:0 iso-C15:0 iso-C15:0 iso-C15:0 C16:0 anteiso-C15:0 anteiso-C15:0 iso-C17:0 anteiso-C15:0 iso-C17:0 iso-C15:0 fatty acids anteiso-C15:0 anteiso-C15:0 iso-C15:0 iso-C15:0 or iso-C16:0 anteiso-C15:0 anteiso-C15:0 iso-C16:0 iso-C15:0 iso-C13:0 anteiso-C17:0 anteiso-C15:0 iso-C16:0 C16:0 C16:0 C16:0 anteiso-C17:0 Diaminopime meso-DAP meso-DAP meso-DAP meso-DAP meso-DAP meso-DAP meso-DAP meso-DAP LL-DAP meso-DAP meso-DAP meso-DAP LL-DAP meso-DAP LL-DAP lic acid DPG, DPG, Polar lipids DPG, PG, DPG, PG, DPG, PG, DPG, PG, DPG, PG, DPG, PG, DPG, PG, PME, PL, PE ND PG,PE, PI, ND ND ND ND components PE, PME, PE PME, PE PME, PE PME, PE PME, PE PE UL PIM

a b

Fig.1. Scanning electron micrographs of strain YIM 77562T grown on ISP2 medium for 3 days at 50 oC. Bars: a, 5 μm; b, 3 μm.

100* YIM 77562T 97* YIM 77580 56* Shimazuella kribbensis KCTC 9933T (AB049939) 54* Seinonella peptonophila KCTC 9740T (AF138735) * Hazenella coriacea 23436T (JQ798970) 58* Laceyella sacchari KCTC 9790T (AF138737)

T 91* 80 Thermoactinomyces vulgaris KCTC 9076 (AF138739) Lihuaxuella thermophila YIM 77831T (JQ750619) Thermoflavimicrobium dichotomicum KCTC 3667T (AF138733)

* 98* Desmospora activa IMMIB L-1269T (AM940019) 74* Marininema mesophilum SCSIO 10219T (JN006758) Mechercharimyces mesophilus YM3-251T (AB239529)

77* Kroppenstedtia eburnea JFMB-ATET (FN665656) 67 Melghirimyces algeriensis NariEXT (HQ383683) 98 Planifilum fimeticola H0165T (AB088364) Polycladomyces abyssicola JIR-001T (AB688114)

100* Geobacillus stearothermophilus DSM 22T (AJ294817) Bacillus subtilis NCDO 1769T (X60646)

0.01 Fig. 2. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of strains YIM 77562T and YIM 77580 within the family Thermoactinomycetaceae. Bacillus subtilis NCDO 1769T and Geobacillus stearothermophilus DSM 22T were used as outgroups. Bootstrap values greater than 50% are given at branch points. Bar, 0.01 substitutions per nucleotide position. Asterisks denote nodes that were also recovered using the maximum- parsimony and maximum-likelihood methods.