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

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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).

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Table 2. Comparison of enzyme activities of strain L5T and 1492R as described by Fan et al. (2008). The PCR product type strains of the most closely related Bacillus species was cloned into pGEM-T easy vector (Promega) for

T T sequencing. The nearly complete 16S rRNA gene sequence Strains: 1, L5 ;2,B. asahii JCM 12112 (Yumoto et al., 2004); 3, T T of strain L5 (1472 bp) was compared with sequences B. kribbensis DSM 17871 (Lim et al., 2007); 4, B. deserti KCTC available in the EzTaxon-e server (Kim et al., 2012) and T + 13246 (Zhang et al., 2011). All data are from this study. , Positive; NCBI GenBank database. Multiple alignments of sequences 2, negative. were carried out using CLUSTAL X (Thompson et al., 1997) within MEGA 6.0 (Tamura et al., 2013). Phylogenetic trees Enzyme activity 1 2 3 4 were reconstructed using the maximum-likelihood Alkaline phosphatase + 22+ (Guindon et al., 2010), neighbour-joining (Saitou & Nei, Acid phosphatase 222+ 1987) and maximum-parsimony (Fitch, 1971) algorithms Trypsin 2 + 22 in the MEGA 6.0 software. Distance calculations and boot- Cystine arylamidase 222+ strap analysis were performed according to Kimura’s Valine arylamidase 22+ 2 two-parameter method (Kimura, 1980) and 1000 replications (Felsenstein, 1985), respectively. Based on 16S rRNA gene sequencing analysis (Ash et al., T T 1991), strain L5 was related most closely to members of The different characteristics of strain L5 and related type T the genus Bacillus and clustered with B. asahii MA001 strains are given in Tables 1 and 2. Cell morphology is (96.5%)(thehighest16SrRNAgenesequencesimilarity), shown in Fig. 1. The flagella and spores are shown in Fig. T T B. kribbensis BT080 , Bacillus circulans ATCC 4513 and S1 (available in the online Supplementary Material). Strain B. deserti ZLD-8T. In the maximum-likelihood tree (Fig. 2), L5T was a Gram-reaction-positive, rod-shaped, spore-form- strain L5T was located within a group containing ing bacterium that was motile by means of polar flagella. B. kribbensis BT080T and B. deserti ZLD-8T, adjacent to These features are typical characteristics of the genus Bacil- B. asahii MA001T,whileB. circulans ATCC 4513T was located lus. However, compared with other Bacillus strains, such in another cluster. The neighbour-joining and maximum- as Bacillus asahii JCM 12112T (Yumoto et al.,2004),Bacillus T parsimony trees showed similar phylogenetic positions (Figs kribbensis DSM 17871 (Lim et al., 2007) and Bacillus deserti T T T S2 and S3). B. asahii JCM 12112 , B. kribbensis DSM KCTC 13246 (Zhang et al., 2011), strain L5 has some dis- T T 17871 and B. deserti KCTC 13246 were then used as refer- tinctive characteristics. Colonies of strain L5T were pink, ence strains for further analyses (see below). whereas those of the other strains were white. Strain L5T T was unable to grow on nutrient-rich media such as LB For fatty acid analysis, strain L5 and the three most closely agar and TSA. In addition, the growth temperature of related type strains were cultivated on R2A agar and col- strain L5T was lower than the other Bacillus strains, possibly lected in the exponential phase based on the quadrant related to the cave environment from where it was found. streak pattern described in the MIS operating manual (2008). Whole-cell fatty acids were analysed by GC (6890; The nearly full-length 16S rRNA gene sequence of strain T Hewlett Packard) according to the standard protocol of L5 was amplified using 16S rRNA gene primers 27F and the Sherlock Microbial Identification System (MIDI Sher- lock version 6.1, MIDI database TSA40 4.10) (Kroppen- stedt, 1985; Sasser, 1990). The major whole-cell fatty T acids of strain L5 were iso-C14 : 0 (35.9 %), anteiso- C15 : 0 (24.9 %) and iso-C16 : 0 (17.8 %), indicating that strain L5T belonged to the genus Bacillus, but there were differences in content compared with B. asahii JCM 12112T, B. kribbensis DSM 17871T, B. deserti KCTC 13246T, B. circulans ATCC 4513T and B. subtilis NRRL NRS-744T (Table 3). The menaquinone was extracted from lyophilized cells and then analysed by the HPLC method as described by Xie & Yokota (2003). Polar lipids were detected and identified by two-dimensional TLC (Collins & Jones, 1980). The DNA G+C content was determined by HPLC according to the method of Tamaoka & Komagata (1984). The diagnostic diamino acid in the cell-wall peptidoglycan was identified by the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) according to a published protocol (Schumann, 2011). HPLC analyses revealed that the DNA T Fig. 1. Scanning electron micrograph of cells of strain L5T after G+C content of strain L5 was 45.6 mol% and the growth for 2 days. Bar, 1 mm. major menaquinone was MK-7. The polar lipids were

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87 T Bacillus jeotgali YKJ-10 (AF221061) *56 T 0.005 Bacillus boroniphilus JCM 21738 (BAUW01000204) *97 Bacillus subterraneus DSM 13966T (FR733689) *79 Bacillus thioparans BMP-1T (DQ371431)

Bacillus foraminis CV53T (AJ717382) Bacillus canaveralius KSC SF8bT (DQ870688) Bacillus pocheonensis Gsoil 420T (AB245377)

*64 Bacillus novalis LMG 21837T (AJ542512) Bacillus niacini IFO 15566T (AB021194) Bacillus kochii WCC 4582T (FN995265) 76 Bacillus horneckiae DSM 23495T (FR749913) Bacillus purgationiresistens DS22T (FR666703) *99 'Bacillus mesophilum' IITR-54 (JN210567-5) Bacillus gottheilii WCC 4585T (FN995266) Bacillus infantis SMC 4352-1T (AY904032) 62 Bacillus oceanisediminis H2T (GQ292772)

*59 Bacillus firmus NCIMB 9366T (X60616) Bacillus benzoevorans DSM 5391T (D78311) *67 Bacillus circulans ATCC 4513T (AY724690-3)

*98 Bacillus nealsonii DSM 15077T (EU656111) *99 Bacillus kribbensis BT080T (DQ280367-2) *62 Bacillus deserti ZLD-8T (GQ465041-4) Bacillus cavernae L5T (AB 2015055) 84 Bacillus asahii MA001T (AB109209-1) T 96 Bacillus huizhouensis GSS03 (KJ464756) T 85 Bacillus psychrosaccharolyticus ATCC 23296 (AB021195) T 92 Bacillus simplex NBRC 15720 (AB363738) LMG 20238T (AJ628748) 99 Bacillus muralis Brevibacterium frigoritolerans DSM 8801T (AM747813)

Bacillus shackletonii LMG 18435T (AJ250318) Virgibacillus pantothenticus IAM 11061T (D16275) Pullulanibacillus naganoensis ATCC 53909T (AB021193)

Fig. 2. Maximum-likelihood phylogenetic tree based on partial 16S rRNA gene sequences (1472 nt) showing the position of strain L5T. Bootstrap values (.50 %) based on 1000 replications are shown at branch nodes. Nodes that were also recovered in trees reconstructed with both the maximum-likelihood and the maximum-parsimony methods are indicated by asterisks. Bar, 0.005 substitutions per nucleotide position. <= diphosphatidylglycerol, phosphatidylglycerol, phosphatidy- other members of the genus Bacillus such as being Gram- lethanolamine, aminophospholipid and several unidentiﬁed reaction-positive, spore-forming, motile, strictly aerobic phospholipids (Fig. 3). The diagnostic diamino acid in the and rod-shaped. In addition, the respiratory quinone, cell-wall peptidoglycan was meso-diaminopimelic acid. fatty acids, polar lipids and diagnostic diamino acid T In summary, strain L5T has characteristics similar to those (meso-diaminopimelic acid) indicate that strain L5 is a of the type species B. subtilis NRRL NRS-744T and the member of the genus Bacillus. However, based on its 16S

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Table 3. Cellular fatty acid compositions of strain L5T and the type strains of closely related Bacillus species

Strains: 1, L5T;2,B. asahii JCM 12112T;3,B. kribbensis DSM 17871T;4, B. deserti KCTC 13246T;5,B. circulans ATCC 4513T (Venkateswaran et al., 2003); 6, B. subtilis NRRL NRS-744T (type species; Rooney et al., 2009). Data for taxa 1–4 are from this study. Values are percen- tages of total fatty acids; –, trace amount (,1 %) or not detected. DPG PL1 PE Fatty acid 1 2 3 4 5 6 PL2 Saturated PG PL3 C14 : 0 2.2 2.4 – – 2.9 – C15 : 0 – – – 1.4 1.0 – PL4 C16 : 0 3.5 3.9 3.2 1.7 2.7 1.9 PL5 APL iso-C14 : 0 35.9 23.6 5.4 11.3 4.0 – iso-C15 : 0 1.6 23.1 4.2 4.6 13.9 19.4 iso-C16 : 0 17.8 1. 8 2.8 2.1 4.4 2.5 PL6 anteiso-C15 : 0 24.9 15.5 70.0 56.3 58.4 38.2 anteiso-C17 : 0 1.9 – 8.0 2.9 4.8 12.1 PL7 Others

C16 : 1v7c alcohol 2.7 7.4 – 5.3 – – C16 : 1v11c 1. 7 13.1 – 4.8 2.6 1.6

Fig. 3. Two-dimensional TLC of the polar lipid proﬁle of strain T rRNA gene sequence, strain L5T shares less than 97 % simi- L5 detected with molybdophosphoric acid spraying reagent. larity with other Bacillus members. Moreover, strain L5T Components were visualized by staining with 5 % molybdopho- has differential characteristics such as colony colour, sphoric acid in ethanol followed by heating of the plates at 8 lower growth temperature and low nutrient requirement. 130 C. DPG, diphosphatidylglycerol; PE, phosphatidylethanola- Taking these data together, strain L5T is thus considered mine; PG, phosphatidylglycerol; APL, aminophospholipid; PL1–7, to represent a novel species of the genus Bacillus, for unknown phospholipids. which the name Bacillus cavernae sp. nov. is proposed.

valerate, glycogen, cellobiose, sodium glutamate, sodium Description of Bacillus cavernae sp. nov. caprylate, sodium butyrate, L-serine, L-phenylalanine, L-sarco- Bacillus cavernae (ca.ver9nae. L. gen. n. cavernae of a cavern). sine and L-threonine. Gelatin and Tweens 40, 60 and 80 are hydrolysed, but not starch, casein or Tween 20. Negative reac- Cells are Gram-stain-positive, rod-shaped (0.3–0.8 mm|1.0– tions for citrate utilization, H S production, nitrate reduction, 6.0 mm), round-ended or occasionally slightly tapered, occur 2 indole production, Voges–Proskauer test and methyl red in chains and sometimes singly or in pairs and are motile by test.UnabletogrowonLBagar,MacConkeyagarorTSA. means of polar ﬂagella. Oval spores are formed centrally or Signiﬁcantly, produces pink pigment and is able to grow at paracentrally in swollen sporangia. Colonies on R2A agar 4 uC, in contrast to closely related Bacillus strains. The major medium are pink, smooth, convex, non-transparent and menaquinoneisMK-7.Themajorfattyacidsareiso-C , circular. Strictly aerobic. The temperature range for growth 14 : 0 anteiso-C and iso-C . The major polar lipids are is 4–32 uC(optimum,28uC) and the pH range is 6.0–9.0 15 : 0 16 : 0 diphosphatidylglycerol, phosphatidylglycerol and phosphati- (optimum, pH 7.0). Growth occurs with 0–1 % (w/v) NaCl dylethanolamine. The diagnostic diamino acid in the cell- (optimum, 0 %). Catalase is positive but oxidase is negative. wall peptidoglycan is meso-diaminopimelic acid. Enzyme activities are observed for alkaline phosphatase, esterase (C4), esterase lipase (C8) and naphthol-AS-BI- The type strain is L5T (5KCTC 33637T5CCTCC AB phosphohydrolase, but no activities are observed for leucine 2015055T), isolated from soil of Tenglong cave, Hubei, arylamidase, acid phosphatase, trypsin, cystine arylamidase, China. The DNA G+C content of the type strain is valine arylamidase, a-glucosidase, b-galactosidase, arginine 45.6 mol% (HPLC). dihydrolase, lipase (C14), valine arylamidase, a-chymotryp- sin, a-galactosidase, b-glucuronidase, b-glucosidase, b-fucosi- dase, N-acetyl-b-glucosaminidase or a-mannosidase. Acid is Acknowledgements produced from D-glucose, D-mannitol and aesculin but not This work was supported by the National Natural Science Foundation D-ribose, glycerol, D-arabinose, D-xylose, D-fructose or of China (31170106) and the Natural Science Foundation of Hubei maltose. Assimilates D-arabinose, D-glucose, D-mannose, Province (2014CFB931). The cell-wall peptidoglycan was analysed L-lactate, maltose, D-fructose, lactose, D-ribose, D-sorbitol, by the DSMZ.

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References Kroppenstedt, R. M. (1985). Fatty acid and menaquinone analysis of actinomycetes and related organisms. In Chemical Methods in Arden Jones, M. P., McCarthy, A. J. & Cross, T. (1979). Taxonomic Bacterial Systematics (Society for Applied Bacteriology Technical Series and serologic studies on Micropolyspora faeni and Micropolyspora no. 20), pp. 173–199. Edited by M. Goodfellow & D. E. Minnikin. strains from soil bearing the specific epithet rectivirgula. J Gen London: Academic Press. Microbiol 115, 343–354. Li, J., Yang, G., Wu, M., Zhao, Y. & Zhou, S. (2014). Bacillus Ash, C., Farrow, J. A. E., Wallbanks, S. & Collins, M. D. (1991). huizhouensis sp. nov., isolated from a paddy field soil. Antonie van Phylogenetic heterogeneity of the genus Bacillus revealed by Leeuwenhoek 106, 357–363. comparative analysis of small-subunit-ribosomal RNA sequences. Lim, J. M., Jeon, C. O., Lee, J. R., Park, D. J. & Kim, C. J. (2007). Bacillus Lett Appl Microbiol 13, 202–206. kribbensis sp. nov., isolated from a soil sample in Jeju, Korea. Int J Syst Claus, D. & Berkeley, R. C. W. (1986). Genus Bacillus.InBergey’s Evol Microbiol 57, 2912–2916. Manual of Systematic Bacteriology, pp. 1105–1139. Edited by Logan,N.A.,Berge,O.,Bishop,A.H.,Busse,H.-J.,DeVos,P.,Fritze,D., P. H. Sneath, N. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Heyndrickx, M., Ka¨mpfer, P., Rabinovitch, L. & other authors (2009). Williams & Wilkins. Proposed minimal standards for describing new taxa of aerobic, Cohn, F. (1872). Untersuchungenu¨ber Bakterien. Beitrage zur endospore-forming bacteria. Int J Syst Evol Microbiol 59, 2114–2121. Biologieder Pflanzen, 1875 1 (Heft 2) 1, 127–224. Nakamura, L. K., Roberts, M. S. & Cohan, F. M. (1999). Relationship of Collins, M. D. & Jones, D. (1980). Lipids in the classification and Bacillus subtilis clades associated with strains 168 and W23: a proposal identification of coryneform bacteria containing peptidoglycans for Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus subtilis based on 2, 4-diaminobutyric acid. J Appl Bacteriol 48, 459–470. subsp. spizizenii subsp. nov. Int J Syst Bacteriol 49,1211–1215. Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Pettersson, B., de Silva, S. K., Uhle´ n, M. & Priest, F. G. (2000). Medical Bacteria. London: Cambridge University Press. Bacillus siralis sp. nov., a novel species from silage with a higher order structural attribute in the 16S rRNA genes. Int J Syst Evol Delaporte, B. & Sasson, A. (1967). [Study of bacteria from arid soils Microbiol 50, 2181–2187. of Morocco: Brevibacterium haloterans n. sp. and Brevibacterium frigoritolerans n. sp]. C R Acad Sci Hebd Seances Acad Sci D 264, Priest, F. G., Goodfellow, M. & Todd, C. (1988). A numerical 2257–2260. classification of the genus Bacillus. J Gen Microbiol 134, 1847–1882. Dong, X. Z. & Cai, M. Y. (2001). Determinative Manual for Routine Rheims, H., Fru¨ hling, A., Schumann, P., Rohde, M. & Stackebrandt, E. Bacteriology. Beijing: Scientific Press. (1999). Bacillus silvestris sp. nov., a new member of the genus Bacillus that contains lysine in its cell wall. Int J Syst Bacteriol 49,795–802. Fan, H., Su, C., Wang, Y., Yao, J., Zhao, K., Wang, Y. & Wang, G. (2008). Sedimentary arsenite-oxidizing and arsenate-reducing Rooney, A. P., Price, N. P. J., Ehrhardt, C., Swezey, J. L. & Bannan, J. D. bacteria associated with high arsenic groundwater from Shanyin, (2009). Phylogeny and molecular taxonomy of the Bacillus subtilis Northwestern China. J Appl Microbiol 105, 529–539. species complex and description of Bacillus subtilis subsp. inaquosorum subsp. nov. Int J Syst Evol Microbiol 59,2429–2436. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Ryu, E. (1938). On the Gram-differentiation of bacteria by the simplest method. J Jpn Soc Vet Sci 17, 205–207. Fitch, W. M. (1971). Towards defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406–416. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, Guindon, S., Dufayard, J. F., Lefort, V., Anisimova, M., Hordijk, W. & 406–425. Gascuel, O. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of Sasser, M. (1990). Identification of bacteria by gas chromatography of PhyML 3.0. Syst Biol 59, 307–321. cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc. Heyrman, J., Logan, N. A., Rodrı´guez-Dıáz, M., Scheldeman, P., Schumann, P. (2011). Peptidoglycan structure. Methods Microbiol 38, Lebbe, L., Swings, J., Heyndrickx, M. & De Vos, P. (2005). Study of 101–129. mural painting isolates, leading to the transfer of ‘Bacillus Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. maroccanus’ and ‘Bacillus carotarum’toBacillus simplex, emended In Methods for General and Molecular Bacteriology, pp. 607–654. description of Bacillus simplex, re-examination of the strains Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & previously attributed to ‘Bacillus macroides’ and description of N. R. Krieg. Washington, DC: American Society for Microbiology. Bacillus muralis sp. nov. Int J Syst Evol Microbiol 55, 119–131. Sonalkar, V. V., Mawlankar, R., Venkata Ramana, V., Joseph, N., Holt, J. G., Kreig, N. R., Sneath, P. H. A., Stanley, J. T. & Williams, S. T. Shouche, Y. S. & Dastager, S. G. (2015). Bacillus filamentosus sp. nov., (1994). Bergey’s Manual of Determinative Bacteriology. Baltimore: isolated from sediment sample. Antonie van Leeuwenhoek 107,433–441. Williams and Wilkins. Tamaoka, J. & Komagata, K. (1984). Determination of DNA base Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., composition by reversed-phase high-performance liquid chromatography. Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e: FEMS Microbiol Lett 25, 125–128. a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013). 716–721. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30, 2725–2729. Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & sequences. J Mol Evol 16, 111–120. Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible Kosowski, K., Schmidt, M., Pukall, R., Hause, G., Ka¨ mpfer, P. & strategies for multiple sequence alignment aided by quality analysis Lechner, U. (2014). Bacillus pervagus sp. nov. and Bacillus tools. Nucleic Acids Res 25, 4876–4882. andreesenii sp. nov., isolated from a composting reactor. Int J Syst Venkateswaran, K., Kempf, M., Chen, F., Satomi, M., Nicholson, W. & Evol Microbiol 64, 88–94. Kern, R. (2003). Bacillus nealsonii sp. nov., isolated from a

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International Journal of Systematic and Evolutionary Microbiology ijsem000794.3d 19/12/2015 10:41:25 Bacillus cavernae sp. nov.

spacecraft-assembly facility, whose spores are c-radiation resistant. Int novel bacterium isolated from soil with the ability to deodorize the J Syst Evol Microbiol 53, 165–172. bad smell generated from short-chain fatty acids. Int J Syst Evol Weeger, W., Lie` vremont, D., Perret, M., Lagarde, F., Hubert, J. C., Microbiol 54, 1997–2001. Leroy, M. & Lett, M. C. (1999). Oxidation of arsenite to arsenate by Zhang, L., Wu, G. L., Wang, Y., Dai, J. & Fang, C. X. (2011). Bacillus a bacterium isolated from an aquatic environment. Biometals 12, deserti sp. nov., a novel bacterium isolated from the desert of 141–149. Xinjiang, China. Antonie van Leeuwenhoek 99, 221–229. Xie, C. H. & Yokota, A. (2003). Phylogenetic analyses of Lampropedia Zhang, Y. Z., Chen, W. F., Li, M., Sui, X. H., Liu, H.-C., Zhang, X. X. & hyalina based on the 16S rRNA gene sequence. J Gen Appl Microbiol Chen, W. X. (2012). Bacillus endoradicis sp. nov., an endophytic 49, 345–349. bacterium isolated from soybean root. Int J Syst Evol Microbiol 62, You, Z. Q., Li, J., Qin, S., Tian, X. P., Wang, F. Z., Zhang, S. & Li, W. J. 359–363. (2013). Bacillus abyssalis sp. nov., isolated from a sediment of the Zhao, F., Feng, Y.-Z., Chen, R.-R., Zhang, H.-Y., Wang, J.-H. & Lin, South China Sea. Antonie van Leeuwenhoek 103, 963–969. X.-G. (2014). Bacillus fengqiuensis sp. nov., isolated from a typical Yumoto, I., Hirota, K., Yamaga, S., Nodasaka, Y., Kawasaki, T., sandy loam soil under long-term fertilization. Int J Syst Evol Matsuyama, H. & Nakajima, K. (2004). Bacillus asahii sp. nov., a Microbiol 64, 2849–2856.

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