Bacillus Cihuensis Sp. Nov., Isolated from Rhizosphere Soil of a Plant in the Cihu Area of Taiwan

Antonie van Leeuwenhoek (2014) 106:1147–1155 DOI 10.1007/s10482-014-0284-3

ORIGINAL PAPER

Bacillus cihuensis sp. nov., isolated from rhizosphere soil of a plant in the Cihu area of Taiwan

Bo Liu • Guo-Hong Liu • Cetin Sengonca • Peter Schumann • Ming-Kuang Wang • Jian-Yang Tang • Mei-Chun Chen

Received: 12 July 2014 / Accepted: 11 September 2014 / Published online: 26 September 2014 Ó Springer International Publishing Switzerland 2014

Abstract A Gram-positive, moderately halotoler- muralis DSM 16288T (97.6 %) and Bacillus simplex ant, rod-shaped, spore forming bacterium, designated DSM 1321T (97.5 %). Levels of DNA–DNA related- strain FJAT-14515T was isolated from a soil sample in ness between strain FJAT-14515T and the reference Cihu area, Taoyuan County, Taiwan. The strain grew strains of B. muralis DSM 16288T and B. simplex at 10–35 °C (optimum at 30 °C), pH 5.7–9.0 (opti- DSM 1321T were 27.9 % ± 3.32 and 44.1 % ± 0.57, mum at pH 7.0) and at salinities of 0–5 % (w/v) NaCl respectively. Therefore, on the basis of phenotypic, (optimum at 1 % w/v). The diagnostic diamino acid of chemotaxonomic and genotypic properties, strain the peptidoglycan of the isolated strain was meso- FJAT-14515T represents a novel species of the genus diaminopimelic acid and major respiratory isoprenoid Bacillus, for which the name Bacillus cihuensis sp. quinone was MK-7. Major cellular fatty acids were nov. is proposed. The type strain is FJAT-14515T T T anteiso-C15:0 (40.6 %), iso-C15:0 (20.7 %) and the (=DSM 25969 = CGMCC 1.12697 ). DNA G?C content of strain FJAT-14515T was 37.1 mol %. A phylogenetic analysis based on 16S Keywords Bacillus cihuensis DNA–DNA rRNA gene sequences indicated that strain FJAT- relatedness Phylogenetic analysis Polyphasic 14515T belongs to the genus Bacillus, and was most taxonomy closely related to the reference strains of Bacillus

The GenBank accession number for the 16S rRNA gene Introduction sequence of strain FJAT-14515T is JX262264. The genus Bacillus was ﬁrst described by Cohn in Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0284-3) contains supple- 1872 (Cohn 1872). Members of the genus Bacillus can mentary material, which is available to authorized users.

B. Liu (&) G.-H. Liu J.-Y. Tang M.-C. Chen P. Schumann Agricultural Bio-resource Institute, Fujian Academy of Leibniz Institute DSMZ-German Collection of Agricultural Sciences, Fuzhou 350003, Fujian, China Microorganisms and Cell Cultures, Inhoffenstraße7B, e-mail: [email protected] 38124 Braunschweig, Germany

C. Sengonca M.-K. Wang Institute of Crop Sciences and Resource Conservation Department of Agricultural Chemistry, National Taiwan (INRES), University of Bonn, Meckenheimer Allee 166A, University, Taipei 10617, Taiwan 53115 Bonn, Germany 123 1148 Antonie van Leeuwenhoek (2014) 106:1147–1155 occupy diverse ecological niches and have been was investigated by using the Schaeffer–Fulton stain- isolated from various habitats, such as from a soil ing method (Murray et al. 1994). For examination of (Logan et al. 2004), a pond (Albuquerque et al. 2008) strain growth, colony morphology was examined after and some clinical samples (Seiler et al. 2013). Some 3 days incubation at 30 °C in nutrient broth (NB). Bacillus-like species have been found in Taiwan, e.g. Strain FJAT-14515T was cultured over the tempera- Paenibacillus taiwanensis isolated from a farmland ture range of 5–50 °C (at intervals of 5 °C) and the pH soil (Lee et al. 2007), Paenibacillus taichungensis range of 5.0–10.0 (at intervals of 1 pH unit) in NB. from a soil (Lee et al. 2008), Paenibacillus fonticola Growth at various NaCl concentrations was tested from a warm spring (Chou et al. 2007), Virgibacillus over the range of 0.5–10 % (w/v) NaCl [0.5 % (w/v) chiguensis from a former commercial saltern (Wang and 1–10 % (w/v) at intervals of 1 %] by incubating et al. 2008), Virgibacillus soli from a mountain soil under conditions of 30 °C and pH 7.0 (Atlas 1993). (Ka¨mpfer et al. 2011) and Allobacillus halotolerans Growth under anaerobic condition was determined from shrimp paste (Sheu et al. 2011). However, at the after incubation in a CO2 incubator on anaerobically time of writing, no new species in the genus Bacillus prepared maintenance medium. have been found in Taiwan. The aim of the present The Gram reaction was determined using the KOH study was to elucidate the taxonomic position, using a method as described by Buck (1982). Motility was polyphasic taxonomic approach, of a Gram-positive, examined on motility agar (Chen et al. 2007). Oxygen endospore-forming bacterium designated FJAT- requirement, activities of catalase, urease and oxidase, 14515T, isolated from the rhizosphere soil of a Acacia hydrolysis of casein, starch, Tweens 20, 40 and 80, confusa plant in the Cihu area of Taiwan. aesculin, nitrate reduction, Voges–Proskauer test,

indole and H2S production were performed according to the conventional methods described by Cowan and Materials and methods Steel (1965) and Smibert and Krieg (1994). The utilization of sole carbon and nitrogen sources was Strains isolation and culture determined according to the method described by Gao et al. (1994). API 20E and API 50CH kits (bio- Strain FJAT-14515T was isolated from an air-dried soil Me´rieux) were used according to the manufacturer’s sample collected in the rhizosphere soil (0–20 cm) of instructions to further determine the physiological and an A. confusa plant in the Cihu area of Taoyuan County biochemical characteristics of strain FJAT-14515T. in Taiwan (121°17089.0300E and 24°50045.7300N). For isolation, the sample was suspended in sterilized water, Chemotaxonomic characterization serially diluted, spread on nutrient agar (NA) and incubated at 30 °C for 48 h (Atlas 1993). Pure cultures For measurement of chemotaxonomic characteristics, were obtained by several successive single colony biomass of strain FJAT-14515T and reference strains isolations. The strain was stored both on NA slants at were harvested from cultures after incubation on NA 4 °C and as suspensions in Luria–Bertani broth (LB) medium at 30 °C for 48 h. The physiological age at with 20 % (v/v) glycerol at -80 °C. The reference the point of harvest of the bacterial strains tested was strains Bacillus muralis DSM 16288T and Bacillus the logarithmic growth phase. For cell wall chemo- simplex DSM 1321T were obtained from the culture taxonomy, the peptidoglycan diamino acid test was collections indicated and used as controls in the carried out according to the method described by phenotypic tests. Schleifer (1985). Cell-wall hydrolysates were sepa- rated by one-dimensional chromatography on micro- Phenotypic and physiological characterization cellulose thin layers. Menaquinones were analyzed as described by Collins (Collins et al. 1977) using For investigation of cell morphology, strain FJAT- reverse-phase HPLC (Groth et al. 1996). Extraction 14515T was investigated after cultivation on NA plates and analysis of polar lipids by two-dimensional TLC for 16 h. The endospores were detected after 48 h by was performed according to Minnikin et al. (1979)by using a phase-contrast optical microscope (Leica the Identification Service of DSMZ, Germany. For DMI3000B, Germany). The presence of endospores determination of cellular fatty acids, experiments were 123 Antonie van Leeuwenhoek (2014) 106:1147–1155 1149 performed according to the standard protocol of the De Ley et al. (1970) and (Huß et al. 1983), using a UV/ Sherlock Microbial Identification System version 6.0 VIS spectrometer equipped with a temperature pro- (MIDI), analysed by GC (model 7890; Agilent) and grammer controller (Lambda 35, Perkin-Elmer, US). identified using the TSBA6 database of the Microbial DNAs were sheared by sonication (SCIENTZ, China) at Identification System (Sasser 1990). 40 W for three periods of 5 s. The renaturation was performed in 2 9 saline-sodium citrate buffer at Molecular characterization 65.9 °C. Three replicate hybridizations were carried out.

For 16S rRNA gene sequencing and phylogenetic analysis, chromosomal DNA was extracted and puri- Results and discussion fied according to standard methods (Hopwood et al. 1985). The 16S rRNA gene sequences were amplified Phylogenetic analysis based on 16S rRNA gene by PCR with the universal primers 9F (50-GAG TTTGATCCT GGCTCAG-30) and 1542R (50-GGA An almost-complete 16S rRNA gene sequence GAAAGGAGGTGATCCAGCC-30) as described by (1439 bp, GenBank accession number JX262264) of Lee et al. (2001). The sequence of the amplified 16S strain FJAT-14515T was determined. Pairwise com- rRNA gene was determined using a DNA sequencer parisons indicated FJAT-14515T to be most closely (ABI 310 sequencer; Applied Biosystems). The resul- related to B. muralis DSM 16288T and B. simplex tant 16S rRNA gene sequence was compared with that DSM 1321T, with 16S rRNA gene sequence similar- of reference strains with validly published names using ities of 97.6 and 97.5 % respectively. In the neigh- the EzTaxon-e sever (http://www.eztaxon-e.ezbio bour-joining tree, the phylogenetic analysis positioned cloud.net/; Kim et al. 2012). For calculation of pair- strain FJAT-14515T in the genus Bacillus with the wise sequence similarity values, sequence data were closely related species B. muralis DSM 16288T, B. calculated using the global alignment algorithm simplex DSM 1321T (97.5 %) and others (Fig. 1). The obtained through the EzTaxon-e server (Kim et al. topologies of the phylogenetic trees built using ML 2012). After multiple alignments of data by CLUS- and MP methods also supported the conclusion that TAL_X (Thompson et al. 1997), phylogenetic trees strain FJAT-14515T form a stable clade with the were constructed using the neighbour-joining (NJ) reference strains B. muralis DSM 16288T, B. simplex (Saitou and Nei 1987), maximum-parsimony (MP) DSM 1321T and others (Supplementary Fig. S1 and (Fitch 1971) and maximum-likelihood (ML) (Felsen- Supplementary Fig. S2). This analysis suggested that stein 1981) method implemented with MEGA version strain FJAT-14515T may represent a distinct species 6 (Tamura et al. 2013). Evolutionary distances were within the genus Bacillus. computed according to the Jukes-Cantor model (Jukes and Cantor 1969). The reliability of each branch was DNA G?C content and DNA–DNA hybridization evaluated by bootstrap analysis based on 1,000 repli- cations (Felsenstein 1985). The 16S rRNA gene Calculation of DNA G?C content from the genome sequences used for the phylogenetic comparisons are sequence indicated that strain FJAT-14515T has a shown in the neighbour-joining phylogenetic tree with DNA G?C content of 37.1 mol %, which is within the their strain designations and accession numbers. range (35.6–44.8 %) for the genus Bacillus (Heyrman et al. 2004; Seiler et al. 2012; Ten et al. 2007; Yoon DNA G?C content and DNA–DNA hybridization et al. 2001; Zhang et al. 2010). DNA–DNA hybridizations revealed that strain FJAT-14515T exhibited For determination of DNA G?C content, genomic DNA 27.9 % ± 3.32 DNA–DNA relatedness to B. muralis was prepared using the method described by Hopwood DSM 16288T and 44.1 % ± 0.57 to B. simplex DSM et al. (1985). The G?C content was determined 1321T, values (\70 %) which are low enough to according to the genome sequence (Liu et al. 2014). distinguish a taxon at the species level (Stackebrandt For analysis of DNA–DNA hybridization, levels of and Goebel 1994; Wayne et al. 1987), supporting the DNA–DNA relatedness was performed using a modi- conclusion that strain FJAT-14515T represents a novel fication of the optical renaturation method described by species within the genus Bacillus. 123 1150 Antonie van Leeuwenhoek (2014) 106:1147–1155

T 49 Bacillus drentensis LMG 21831 (AJ542506) T 79 Bacillus soli LMG 21838 (AJ542513) Bacillus novalis LMG 21837T (AJ542512) 99 88 Bacillus vireti DSM 15602T (AJ542509) 90 Bacillus bataviensis LMG 21833T (AJ542508) 93 Bacillus pocheonensis Gsoil420T (AB245377)

Bacillus horneckiae DSM 23495T (FR749913) Bacillus asahii JCM 12112T (AB109209)

Bacillus cihuensis FJAT-14515T (JX262264) 95 Bacillus psychrosaccharolyticus DSM 13778T (AB021195) 74 Bacillus butanolivorans DSM 18926T (EF206294) 93 Bacillus muralis DSM 16288T (AJ628748) 98 Bacillus simplex T 61 DSM 1321 (AB363738) 66 ‘Brevibacterium frigoritolerans‘ DSM 8801T (AM747813) Bacillus koreensis DSM 16467T (AY667496)

59 Bacillus coahuilensis CECT 7197T (ABFU01000135) Bacillus endoradicis LMG 25492T (GU434676) Bacillus halmapalus DSM 8723T (X76447)

Bacillus herbersteinensis DSM 16534T (AJ781029) 62 Bacillus subtilis DSM 10T (AJ276351)

0.005

Fig. 1 Neighbour-joining phylogenetic tree based on the 16S branch is indicated by a bootstrap value calculated for 1,000 rRNA gene sequence of strain FJAT-14515T and closely related subsets. Bar, 0.005 substitutions per site species within the genus Bacillus. The signiﬁcance of each

Morphological features and phenotypic xylitol, D-arabitol, maltose, lactose, glucose, trehalose characteristics and sucrose. The morphological, physiological and biochemical characteristics in detail are given in the Morphological features showed that cells of the strain species description below. The phenotypic differences FJAT-14515T are Gram-positive and rod-shaped with between the novel strain and the reference species B. a size range of 0.4–0.8 9 1.3–2.2 lm (Supplementary muralis DSM 16288T and B. simplex DSM 1321T are Fig. S3). Ellipsoidal endospores were found and the shown in Table 1. cells were observed to be motile. Growth was determined to occur at 10–35 °C, pH 6–9 and 0–5 % Chemotaxonomic characteristics (w/v) NaCl. Analysis of phenotypic characteristics indicated that catalase and urease tests are positive, but Analysis of the cell-wall peptidoglycan showed that b-galactosidase negative. Gelatin, starch and aesculin strain FJAT-14515T contained meso-diaminopimelic were found to be hydrolyzed. H2S and indole were not acid as the diagnostic diamino acid(Supplementary observed to be produced. Nitrate was found to be Fig. S4), which is characteristic of a large majority of reduced to nitrite. The Voges–Proskauer test was the members of the genus Bacillus (Priest et al. 1988). negative. Acids were found to be produced from Strain FJAT-14515T was found to contain MK-7 123 Antonie van Leeuwenhoek (2014) 106:1147–1155 1151

Table 1 Physiological characteristics of strain FJAT-14515T Table 1 continued and the close phylogenetic relatives within the genus Bacillus Characteristic B. cihuensis B. muralis B. simplex Characteristic B. cihuensis B. muralis B. simplex FJAT- DSM DSM FJAT- DSM DSM 14515T 16288T 1321T 14515T 16288T 1321T Acid production from (using API 50 CH) Temperature (°C) Glycerol --? 10 w -- Erythritol w -- 15 w - w L-arabinose --? 20 ? w ? Ribose ?-- 25 ??? D-xylose w -- 30 ??? L-xylose w -- 35 ??? Adonitol w -- 40 -?? Fructose w w ? 50 --- Mannose - w - 45 --- Rhamnose --? Range 10–35 20–40 15–40 Inositol --? Optimum 30 30 30 Mannitol w -? pH Sorbitol w -- 5 --? N-acetyl ww- 6 ??? glucosamine 7 ??? Arbutin ?-? 8 ??? Cellobiose --? 9 ??? Maltose ?-? 10 -?? Lactose ?-- Range 6–9 6–10 5–10 Sucrose ?-? Optimum 7 7 7 Trehalose ? w ? NaCl % Xylitol ?-- 0% ??? D-arabitol ?-- 1% ??? 5-keto-D-gluconate - w - 2% ??? All data are obtained from this study unless indicated 3% ??? otherwise. ?, positive; -, negative; w, weakly positive; All 4% w ?? strains could produce acid from glucose, fructose, esculin, and salicin; hydrolysis of casein, esculin, ornithine decarboxylase, 5% w ?? tryptophan deaminase, arginine dihydrolase, lysine Range 0–5 % 0–5 % 0–5 % decarboxylase and tryptophan were negative. Starch Optimum 0 0 0 hydrolysis was positive. None of strains could produce Urease ??- indole, ONPG and H2S, produce acid from D-arabinose, b-methyl-D-xyloside, galactose, sorbose, dulcitol, a-methyl-D- Mobile ??- mannose glycosides, a-methyl-D-glucoside, amygdalin, Hydrolysis of melibiose, inulin, melizitose, rafﬁnose, starch, glycogen, Voges–Proskauer -?- gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, Gelatin ??- L-fucose, L-arabitol, gluconate and 2-keto-D-gluconate Starch ??? Tween 20 --? (97.4 %) as the predominant menaquinone, with MK- Tween 40 --? 6 (1.1 %) and MK-8 (0.2 %) present as minor constituents. The polar lipids detected were diphos- Tween 80 --- phatidylglycerol, phosphatidylglycerol, phosphatidyl- Citrate utilization ?-- ethanolamine and two unknown phospholipids Nitrate reduce --? (Supplementary Fig. S5). The major fatty acids

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Table 2 Cellular fatty acid composition of strain FJAT- relatedness in current bacteriology are considered to 14515T and closely related reference strains in the genus belong to different species (Stackebrandt and Goebel, Bacillus 1994; Wayne et al. 1987). Many members of the genus Fatty acid B. cihuensis B. muralis B. simplex Bacillus with [97 % 16S rRNA gene sequence FJAT- DSM DSM similarity are considered to be representatives of 14515T 16288T 1321T separate species. For example, Chen et al. (2011) T iso-C15:0 20.7 12.1 14.8 isolated strain JSM 081004 with 97.8 % 16S rRNA anteiso-C15:0 40.6 55.3 56.9 gene sequence similarity to the nearest phylogenetic T iso-C14:0 10.0 3.2 3.3 neighbour, Bacillus lehensis JMLB2 , but proposed it

C16:0 9.4 9.5 8.4 to be a novel species, Bacillus xiaoxiensis, based on its

C14:0 4.4 2.7 2.5 different phenotypic profile and a DNA–DNA reasso- B. lehensis T iso-C16:0 4.4 2.5 2.9 ciation value of 18.6 % with JMLB2 . Similar cases occurred in the description of Bacillus anteiso-C17:0 2.7 2.9 2.8 ginsengisoli, Bacillus pocheonensis and Bacillus ba- C16:1x7c alcohol 1.3 0.8 0.7 taviensis, which all showed 98–98.3 % pairwise C18:1x9c 0.6 1.3 0 similarity but below 55 % DNA–DNA relatedness C18:0 1.5 4.3 0.9 values (Nguyen et al. 2013). More recently, Meier- C16:1x11c 1.0 1.2 2.1 Kolthoff et al. (2013) and Kim et al. (2014) have iso-C17:0 0.8 1.6 1.7 suggested threshold 16S rRNA similarity values of C12:0 0.5 1.1 0 98.2–99 % and 98.65 % respectively can be used to iso-C13:0 0.5 0 0 delineate new bacterial species. Strain FJAT-14515T Summed feature 4 0 1.2 0 exhibits similarity values well below these levels. All data were obtained from this study. Data are percentages of Furthermore, Goodfellow et al. (1998) reported that the total fatty acid content. Summed feature 4 meant anteiso- the DNA–DNA relatedness provides a reliable way of C17:1 B and/or iso-C17:1 I distinguishing between representatives of species that share high 16S rRNA gene sequence similarity. In the present in strain FJAT-14515T were identified as iso- present study, levels of DNA–DNA relatedness T T C15:0 (40.6 %), anteiso-C15:0 (20.7 %), iso-C14:0 between FJAT-14515 , B. muralis DSM 16288 and T (10.0 %) and C16:0 (9.4 %), which comprised approx- B. simplex DSM 1321 were 27.9 % ± 3.32 and imately 80 % of the cellular fatty acids extracted 44.1 % ± 0.57, respectively, all which are much (Table 2). The fatty acid profile of strain FJAT- below the 70 % cut-off point for the delineation of 14515T was similar to that of the reference strains novel species. These results indicate that strain FJAT- tested, although there were differences in the propor- 14515T should be considered as representative of a tions of some fatty acid components. These data are novel species in the genus Bacillus. consistent with reports that iso- and anteiso-branched Therefore, the phenotypic (morphology, biochem- fatty acids of the 14–17 carbon series are typical of istry and chemotaxonomy) and genotypic (G?C those found in the membranes of members of the content, 16S rRNA gene sequence and DNA–DNA genus Bacillus (Albert et al. 2005;Ka¨mpfer 1994). relatedness) properties of strain FJAT-14515T support its classification in a novel species within the genus Bacillus, for which the name Bacillus cihuensis sp. Conclusion nov. is proposed.

16S rRNA gene sequence analysis indicated strain FJAT-14515T to be most closely related to B. muralis Description of Bacillus cihuensis sp. nov DSM 16288T (97.6 % sequence similarity) and B. simplex DSM 1321T (97.5 %). Generally accepted Bacillus cihuensis (ci.hu.en’sis, N.L. masc. adj. cih- criteria for delineating bacterial species state that uensis, belonging to Cihu, Taoyuan County in Taiwan, strains showing less than 97 % of 16S rRNA gene where an Acacia rhizosphere soil sample was col- sequence similarity or lower than 70 % of DNA–DNA lected for isolation of the organism). 123 Antonie van Leeuwenhoek (2014) 106:1147–1155 1153

Cells are motile, aerobic, Gram-positive rods rRNA gene sequence of strain FJAT-14515T is (0.4–0.8 9 1.3–2.2 lm) that mostly occur singly, JX262264. sometimes in pairs and short chains. Ellipsoidal endospores are formed subterminally. Colonies are Acknowledgments We thank Professor J. P. Euze´by for his 1–3 mm in diameter, pale yellow, opaque, glistening, suggestion on the spelling of the specific epithet. We thank also the Agricultural Bioresources Institute, Fujian Academy of flat with irregular margins. Grows at 10–35 °C Agricultural Sciences, PR China, and the international (optimum at 30 °C) and pH 5.7–9.0 (optimum at pH cooperation project of Chinese Ministry of Science and 7.0) and at salinities of 0–5 % (w/v) NaCl (optimum at Technology (2012DFA31120), Natural Science Foundation of 1 %, w/v). Catalase-positive and oxidase-negative. China (NSFC) (31370059), 948 project of Chinese Ministry of Agriculture (2011-G25), 973 program earlier research project Positive for citrate utilization, esculin, starch hydro- (2011CB111607) and project of agriculture science and lysis, gelatin hydrolysis, and urease but negative for technology achievement transformation (2010GB2C400220) casein, arginine dihydrolase, lysine decarboxylase, for the supporting, respectively. ornithine decarboxylase, ONPG, tryptophan deaminase, Tweens 20, 40 and 80, Voges–Proskauer test, nitrate reduction, hydrogen sulphide and indole production. 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