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Bacillus Cavernae Sp. Nov. Isolated from Cave Soil Liling Feng, Dongmei Liu, Xuelian Sun, Gejiao Wang3 and Mingshun Li3

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Bacillus Cavernae Sp. Nov. Isolated from Cave Soil Liling Feng, Dongmei Liu, Xuelian Sun, Gejiao Wang3 and Mingshun Li3 International Journal of Systematic and Evolutionary Microbiology (2015), 00, 1–7 DOI 10.1099/ijsem.0.000794 Bacillus cavernae sp. nov. isolated from cave soil Liling Feng, Dongmei Liu, Xuelian Sun, Gejiao Wang3 and Mingshun Li3 Correspondence National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Gejiao Wang Huazhong Agricultural University, Wuhan, 430070, PR China [email protected] A Gram-stain-positive, spore-forming, motile, strictly aerobic, rod-shaped bacterium, designated strain L5T, was isolated from soil of Tenglong cave, China. 16S rRNA gene sequence analysis showed that strain L5T was related most closely to Bacillus asahii MA001T (96.5 %) (the highest 16S rRNA gene sequence similarity), Bacillus kribbensis BT080T (96.4 %) and Bacillus deserti ZLD-8T (96.2 %). The DNA G+C content of strain L5T was 45.6 mol%. The major menaquinone was MK-7. The major fatty acids were iso-C14 : 0, anteiso-C15 : 0 and iso-C16 : 0, and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. In addition, strain L5T had different characteristics compared with the other Bacillus strains such as pink colony colour, low growth temperature and low nutrient requirement. The results indicate that strain L5T represents a novel species of the genus Bacillus, for which the name Bacillus cavernae sp. nov. is proposed. The type strain is L5T (5KCTC 33637T5CCTCC AB 2015055T). The genus Bacillus belongs to the family Bacillaceae,order (Claus & Berkeley, 1986; Holt et al., 1994; Rheims et al., Bacillales,phylumFirmicutes, and was first proposed by 1999; Yumoto et al., 2004; Lim et al., 2007; Zhang et al., Cohn (1872) with Bacillus subtilis as the type species. 2011; You et al., 2013; Sonalkar et al., 2015). Additional Bacillus species were then identified, and Strain L5T was isolated from soil of Tenglong cave some of them were reclassified within other genera, such (308 339 510 N 1088 989 290 E, elevation 2000 m) in as Alicyclobacillus, Aneurinibacillus, Brevibacillus, Geobacillus, Enshi city, Hubei province, China. The soil texture was Marinibacillus, Paenibacillus, Salibacillus, Ureibacillus and clay, with pH of 6.0. Five grams of soil was serially diluted Virgibacillus. At the time of writing, the genus Bacillus com- with 0.85 % (w/v) NaCl and cultured on chemical defined prised more than 300 species (http://www.bacterio.cict.fr/b/ medium (CDM) plates (solution A: MgSO ,NHCl, bacillus.html) isolated from different environments. 4 4 Na SO ,KPO .3HO, CaCl .2HO, sodium lactate; Recently described species include Bacillus fengqiuensis 2 4 2 4 2 2 2 solution B: FeSO .7HO; solution C: NaHCO – 100 ml (Zhao et al., 2014), Bacillus huizhouensis (Li et al., 2014), 4 2 3 of solution A, 2.5 ml of solution B and 10 ml of solution Bacillus filamentosus (Sonalkar et al., 2015), Bacillus pervagus C were mixed and made up to 1 litre with water, 15 g and Bacillus andreesenii (Kosowski et al., 2014), isolated agar, pH 6.0; Weeger et al., 1999). A bacterial colony was from sandy loam soil under long-term fertilization, paddy selected based on its pink colour and named L5T. Later, field soil, sediment and composting reactor, respectively. the isolate was routinely cultivated on R2A agar (Bacton) Common characteristics of members of the genus Bacillus and preserved in 25 % glycerol at 280 8C. are Gram-reaction-positive, spore-forming, rod-shaped, containing menaquinone 7 (MK-7) as the major menaqui- Analyses of morphological, physiological and biochemical none, diphosphatidylglycerol, phosphatidylglycerol and characteristics were performed based on the recommended phosphatidylethanolamine as the major polar lipids, and minimal standards for the description of new aerobic spore- meso-diaminopimelic acid as the diagnostic cell-wall dia- forming taxa (Logan et al., 2009). Strain L5T and related mino acid. Most Bacillus strains have iso-C14 : 0,iso-C15 : 0, type strains were cultivated on R2A agar at 28 8C, pH 7.0, anteiso-C15 : 0,iso-C16 : 0 or anteiso-C17 : 0 as major fatty unless otherwise mentioned. Cells were observed by scan- acids. The DNA G+C content range is 36–52 mol% ning electron microscopy (JSM-6390; JEOL). Flagella were observed by light microscopy (10006, oil; Nikon). Spores 6 3 were observed by phase-contrast microscopy (1000 , oil; These authors contributed equally to this paper. Nikon) after cultivation for 1 week. The motility of the The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene cells was detected by semi-solid puncturing in 0.3 % R2A sequence of strain L5T is KT186244. agar for 1 week. Gram staining was determined using a bio- Three supplementary figures are available with the online Supplementary Me´rieux Gram-stain kit in combination with the 3 % KOH ; Material. method (Ryu, 1938). Growth temperature was tested on 000794 G 2015 IUMS Printed in Great Britain 1 International Journal of Systematic and Evolutionary Microbiology ijsem000794.3d 19/12/2015 10:41:23 L. Feng and others R2A agar at 4, 13, 20, 28, 32, 37 and 42 8C for 1 week. Growth was tested on trypticase soy agar (TSA; Bacto), 1/10 To determine the tolerance to NaCl, the strains were TSA, R2A agar, MacConkey agar, Luria–Bertani (LB) agar grown in R2A liquid medium with different NaCl concen- (all from Difco) and CDM agar (Weeger et al., 1999). Cat- trations (0, 1, 2, 3, 5, 7, 9, 11, 15 and 20 %, w/v) for alase activity was determined using 3 % (v/v) H2O2 (Smi- 1 week. Experiments testing the pH range for growth at bert & Krieg, 1994). Oxidase activity was determined using pH 4.0–11.0 (1 pH unit interval) were performed in R2A 1 % (w/v) tetramethyl-b-phenylenediamine (Merck). liquid medium with different buffer systems (pH 4–5, Hydrolysis of starch, gelatin, casein and Tweens 20, 40, 60 0.1 M citric acid/0.1 M sodium citrate; pH 6–8, 0.1 M and 80 was tested according to Cowan & Steel (1965) and KH2PO4/0.1 M NaOH; pH 9–10, 0.1 M NaHCO3/0.1 M Arden Jones et al. (1979). Nitrate and nitrite reduction, Na2CO3; pH 11, 0.05 M Na2HPO4/0.1 M NaOH) for production of H2S and indole, and methyl red and Voges– 1 week. Growth in R2A liquid medium was determined Proskauer tests were performed according to Dong & Cai based on OD600 values with a blank culture medium as (2001). The ability to utilize sole carbon sources, acid pro- the control. Anaerobic growth was determined in an duction and enzyme activities were tested using traditional anaerobic chamber with an O2-absorbing and CO2-generat- methods (Dong & Cai, 2001) in combination with API ing agent (Anaero-Pack; Mitsubishi Gas Chemical) for 20E, API 32GN and API 50CH strips (bioMe´rieux) accord- 2 weeks. ing to the manufacturer’s instructions. Table 1. Differential phenotypic characteristics between strain L5T and the type strains of closely related species of the genus Bacillus Strains: 1, L5T;2,B. asahii JCM 12112T (Yumoto et al., 2004); 3, B. kribbensis DSM 17871T (Lim et al.,2007);4,B. deserti KCTC 13246T (Zhang et al., 2011); 5, B. circulans ATCC 4513T (Pettersson et al., 2000); 6, B. huizhouensis GSS03T (Li et al., 2014); 7, B. psychrosaccharolyticus DSM 6T (Priest et al., 1988; Zhang et al., 2012); 8, B. muralis LMG 20238T (Heyrman et al., 2005); 9, ‘B. frigoritolerans’ DSM 8801 (Delaporte & Sasson, 1967); 10, B. simplex NBRC 15720T (Li et al., 2014; Heyrman et al., 2005); 11, B. subtilis NRRL NRS-744T (type species; Nakamura et al., 1999). Data for taxa 1–4 are from this study except for the growth characters and DNA G+C contents. NG,Nogrowth;ND,nodataavailable;V,variable;+,positive;2, negative; W, weakly positive. P, pink; W, white; C-W, cream-white. Spore position: C, central or paracentral; T, terminal or subterminal. > Characteristic 1 2 3 4 5 6 7 8 9 10 11 Colony colour P W W W C-W W W P ND WW Spore shape Oval Oval Oval Oval Oval Oval ND Oval ND Oval Oval Spore position C C/T ND C ND C ND C ND C/T C Nitrate reduction 2 + 2 +* 22++ 2 ++ Growth at/with: 4 8C + 22222+ 2222 40 8C 2 +++++++ 22+ NaCl (%, w/v) 0–1 0–1 0–5 0–4 0–5 0–2 0–2 0–7 0–4 0–5 0–7 pH 6.0–9.0 6.0–9.0 5.0–9.0 6.0–9.0 5.6–6.5 6.5–8.0 5.2–8.0 7.0–9.0 6.0–10.0 5.0–9.0 5.5–5.7 Anaerobic growth 222 ++ND W ND V 2 Growth on LB agar 2 ++2 ++++ + ++ Hydrolysis of: Starch 22* + 2 + 2 ++ 2 ++ Gelatin + 2 +++222 +++ Tween 20 222+++++ + +ND Tween 80 + NG 2 + ND +++ + +ND Acid production from: D-Mannitol + 2 +++222 2 ++ D-Glucose + 2 ++++++ + ++ Starch 2 ++2 + ND ND 2 ND 2 ND D-Ribose 22++2 ND ND ND ND ND ND Glycerol 22W 2 W ++ W 22+ D-Arabinose 22+++++ W +++ D-Xylose 22W 222+ 2 +++ Maltose 22++ND ND ND + ND ND ND D-Fructose 22+ 2 ++++ + 2 ND DNA G+C content (mol%) 45.6 39.4 43.3 40.1 35.7 40.2 ND 41.2 40.8 39.4 ND *Data different from the previous studies (Yumoto et al., 2004; Lim et al., 2007; Zhang et al., 2011). 2 International Journal of Systematic and Evolutionary Microbiology 00 International Journal of Systematic and Evolutionary Microbiology ijsem000794.3d 19/12/2015 10:41:24 Bacillus cavernae sp.
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