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Geothermomicrobium Terrae Gen. Nov., Sp. Nov., a Novel Member Of 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.
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