Bacillus Halosaccharovorans Sp. Nov., a Moderately Halophilic Bacterium from a Hypersaline Lake

International Journal of Systematic and Evolutionary Microbiology (2013), 63, 2776–2781 DOI 10.1099/ijs.0.046961-0

Bacillus halosaccharovorans sp. nov., a moderately halophilic bacterium from a hypersaline lake

Maliheh Mehrshad,1 Mohammad Ali Amoozegar,1,2 Maryam Didari,1 Maryam Bagheri,1,2 Seyed Abolhassan Shahzadeh Fazeli,2,3 Peter Schumann,4 Cathrin Spro¨er,4 Cristina Sa´nchez-Porro5 and Antonio Ventosa5

Correspondence 1Extremophiles Laboratory, Department of Microbiology, Faculty of Biology and Center of M. A. Amoozegar Excellence in Phylogeny of living organisms, College of Science, University of Tehran, Tehran, Iran [email protected] or 2Microorganisms Bank, Iranian Biological Resource Centre (IBRC), ACECR, Tehran, Iran [email protected] 3Department of Genetics, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran 4DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, 38124 Braunschweig, Germany 5Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain

A novel Gram-stain-positive, moderately halophilic bacterium, designated strain E33T, was isolated from water of the hypersaline lake Aran-Bidgol in Iran and characterized taxonomically using a polyphasic approach. Cells of strain E33T were motile rods and produced ellipsoidal endospores at a central or subterminal position in swollen sporangia. Strain E33T was a strictly aerobic bacterium, catalase- and oxidase-positive. The strain was able to grow at NaCl concentrations of 0.5–25 % (w/v), with optimum growth occurring at 5–15 % (w/v) NaCl. The optimum temperature and pH for growth were 40 6C and pH 7.5–8.0, respectively. On the basis of 16S rRNA gene sequence analysis, strain E33T was shown to belong to the genus Bacillus within the phylum Firmicutes and showed the closest phylogenetic similarity with the species Bacillus niabensis 4T19T (99.2 %), Bacillus herbersteinensis D-1-5aT (97.3 %) and Bacillus litoralis SW-211T (97.2 %). The DNA G+C content of the type strain of the novel species was T 42.6 mol%. The major cellular fatty acids of strain E33 were anteiso-C15 : 0 and iso-C15 : 0, and the polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, two unknown glycolipids, an unknown lipid and an unknown phospholipid. The isoprenoid quinones were MK-7 (97 %), MK-6 (2 %) and MK-8 (0.5 %). The peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. All these features confirm the placement of isolate E33T within the genus Bacillus. DNA–DNA hybridization experiments revealed low levels of relatedness between strain E33T and Bacillus niabensis IBRC-M 10590T (22 %), Bacillus herbersteinensis CCM 7228T (38 %) and Bacillus litoralis DSM 16303T (19 %). On the basis of polyphasic evidence from this study, a novel species of the genus Bacillus, Bacillus halosaccharovorans sp. nov. is proposed, with strain E33T (5IBRC-M 10095T5DSM 25387T) as the type strain.

The genus Bacillus in the family Bacillaceae, belonging to or halotolerant endospore-forming species have been the phylum Firmicutes, includes obligate aerobic or described (de la Haba et al., 2011). These bacteria are facultative anaerobic, endospore-forming bacilli and is frequently isolated from saline and hypersaline environ- metabolically and ecologically a diverse group. Among ments, such as saline soils and saline aquatic habitats the species of this genus, several moderately halophilic (Arahal & Ventosa, 2002; Ma´rquez et al., 2011; Ventosa, 2006; Ventosa et al., 1998). During the study of the The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene diversity of halophilic and halotolerant prokaryotes in sequence of Bacillus halosaccharovorans E33T is HQ433447. Aran-Bidgol Lake (34u 189–34u 459 N51u 339–52u 109 E, 2 A supplementary figure and a supplementary table are available with the 2400 km ), Esfehan Province, Iran, a large number of novel online version of this paper. taxa have been isolated (Amoozegar et al., 2012; Bagheri

2776 046961 G 2013 IUMS Printed in Great Britain Bacillus halosaccharovorans sp. nov.

et al., 2012; Didari et al., 2012 ; Makhdoumi-Kakhki et al., pH 7.5. Growth was monitored by turbidity (OD600) using 2012). The pH of the water of the lake is neutral (about a spectroscopic method (model UV-160 A; Shimadzu). pH 7.0–7.5) and its salinity reaches saturation. Here we Other physiological and biochemical tests were performed describe the taxonomic properties of a halophilic bacterial as described previously (Mata et al., 2002; Quesada et al., strain, designated E33T, which was isolated from this 1984; Ventosa et al., 1982). Strain E33T was Gram-positive, hypersaline lake. non-motile and strictly aerobic. Cells were rods with a width of 0.5 mm and length of 3.0–8.0 mm. Ellipsoidal The strain was isolated by plating the water sample on endospores were formed at a central or subterminal 7.5 % HM medium and incubating at 35 uC aerobically. position in swollen sporangia. When grown for 2 days at The 7.5 % HM medium contained (g l21): NaCl, 60.7; 40 uC on 7.5 % HM medium, the colonies were puncti- MgCl .6H O, 5.2; MgSO .7H O, 7.2; CaCl .2H O, 0.27; 2 2 4 2 2 2 form, convex, entire, smooth, cream and with a diameter of KCl, 1.5; NaHCO , 0.045; NaBr, 0.0195; proteose–peptone 3 2 mm. This isolate was moderately halophilic, growing in number 3, 5; yeast extract, 10 and glucose, 1; 1.5 % (w/v) media containing 0.5–25 % (w/v) NaCl and optimally in agar was added when necessary (Ventosa et al., 1982). The media containing 5–15 % (w/v) NaCl. Strain E33T grew at pH of this medium was adjusted to pH 7.5. The strain was pH 6.0–9.0 and optimally in media at pH 7.5–8.0. Strain subsequently purified three times by plating on the same E33T was sensitive to bacitracin (10 U), carbenicillin medium. It was maintained on the same medium and also (100 mg), cephalothin (30 mg), chloromphenicol (30 mg), at 280 uC in 7.5 % HM without agar and supplemented erythromycin (15 mg), gentamicin (30 mg), kanamycin with 30 % (v/v) glycerol. Bacillus niabensis IBRC-M 10590T (30 mg), nalidixic acid (30 mg), nitrofurantoin (300 mg), was obtained from the IBRC culture collection and was tetracycline (30 mg) and tobramycine (10 mg) but resistant used as reference strain for comparison in our study. It was to amoxicillin (30 mg) and polymixin B (100 U). Other cultured under the same growth conditions as strain E33T. phenotypic features are included in Table 1 and the species In order to phenotypically characterize strain E33T, description. standard phenotypic tests were performed. The recom- Genomic DNA from strain E33T was isolated using the mended Minimal Standards for describing new taxa of method described by Marmur (1961). The 16S rRNA gene aerobic, endospore-forming bacteria were followed (Logan was amplified by PCR with the forward primer 16F27 and et al., 2009). Cell morphology was examined using an the reverse primer 16R1488. The amplification was Olympus BX41 microscope equipped with phase-contrast performed by initial denaturation at 95 uC for 5 min optics using cells from exponentially growing cultures. followed by: 10 cycles of 93 uC for 1 min, 63 uC for 1 min, Gram staining was performed by the Burke method 71 uC for 1.5 min; 20 cycles of 93 uC for 1 min, 67 uC for (Murray et al., 1994). The presence of endospores was 1 min, 71 uC for 2 min; and a final extension at 71 uC for investigated by using the Schaeffer–Fulton staining method 5 min. The purified PCR product was sequenced in both (Murray et al., 1994). Motility was analysed by the wet- directions using an automated sequencer by the SeqLab mount method (Murray et al., 1994). Catalase, oxidase and Laboratory (Go¨ttingen, Germany). The 16S rRNA gene urease activities, nitrate and nitrite reduction, hydrolysis of sequence analysis was performed with the ARB software aesculin, production of indole, methyl red and Voges– package (Ludwig et al., 2004). The 16S rRNA gene Proskauer tests were tested as recommended by Smibert & sequence was aligned with the published sequences of the Krieg (1994) using media supplemented with 7.5 % NaCl. closely related bacteria and the alignment was confirmed Hydrolysis of Tween 20, 40, 60 or 80 was examined as and checked against both primary and secondary structures described by Harrigan & McCance (1976). Determination of the 16S rRNA molecule using the alignment tool of the of acid production from carbohydrates, as well as ARB software package. Phylogenetic trees were recon- utilization of carbon and energy sources, was performed structed using three different methods: maximum-par- as recommended by Ventosa et al. (1982). Antimicrobial simony (Fitch, 1971), maximum-likelihood (Felsenstein, susceptibility tests were performed on Mueller–Hinton 1981) and neighbour-joining (Saitou & Nei, 1987), agar plus 7.5 % (w/v) sea salts (Ventosa et al., 1982) seeded algorithms integrated in the ARB software for phylogenetic with a bacterial suspension containing 1.56106 c.f.u. ml21 inference. The 16S rRNA gene sequences used for phylo- using discs (HiMedia) impregnated with various antimi- genetic comparisons were obtained from the GenBank crobial compounds. The plates were incubated at 35 uC for database and their strain designations and accession 48 h and the inhibition zone was interpreted according the numbers are shown in Fig. 1. manufacturer’s manual. To determine the optimal temperature and pH for growth of the strain, broth cultures An almost-complete 16S rRNA gene sequence (1449 bp) of T were incubated at temperatures of 15–50 uC at intervals of strain E33 was obtained and used for initial BLAST searches 5 uC and at pH 5–10 at intervals of 0.5 pH units. pH values in GenBank and phylogenetic analysis. The identification below 6, pH 6–9 and pH values above 9 were obtained of phylogenetic neighbours and calculation of pairwise 16S using sodium acetate/acetic acid, Tris/HCl and glycine/ rRNA gene sequence similarity were achieved using the sodium hydroxide buffers, respectively. Growth at different EzTaxon-e server (http://www.eztaxon-e.ezbiocloud.net; NaCl concentrations (0, 0.5, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, Kim et al., 2012). Phylogenetic analysis based on the 20.0, 25.0 and 27.5 %, w/v) was tested on HM medium at maximum-parsimony algorithm revealed that strain E33T http://ijs.sgmjournals.org 2777 M. Mehrshad and others

Table 1. Characteristics that distinguish strain E33T from members of the most closely phylogenetically related species of the genus Bacillus

Strains: 1, Strain E33T;2,B. niabensis IBRC-M 10590T;3,B. herbersteinensis CCM 7228T. All data are from this study unless marked otherwise.

Characteristic 1 2 3

Colony colour Cream Yellowish-white CreamD Endospore shape Ellipsoidal Oval Oval Sporangium Swollen Swollen* Non-swollenD Endospore position Central/subterminal Terminal TerminalD NaCl range (%) 0.5–25 0–5* 0–5D Optimum NaCl (%) 5.0–15 0 0–0.5 Temperature range (uC) 20–45 15–50* 4–28D pH range 6.0–9.0 6.0–8.0* 7.0–9.0D Optimum pH 7.5–8.0 7.0 8.0 Anaerobic growth 2 + 2 Oxidase activity + 2 + Hydrolysis of: Casein + 22 Tween 20 + 22 Tween 40 + 22 Urease activity 2 + 2 Nitrate reduction 2 + 2 Acid production from: D-Glucose ++2 D-Mannitol ++2 D-Xylose ++2 Galactose + 22 Lactose + 22 Ribose + 22 DNA G+C content (mol%) 42.6 40.9* 36.2D

Major fatty acids anteiso-C15 : 0, iso-C15 : 0, anteiso-C15 : 0, anteiso-C17 : 0 iso-C15 : 0, anteiso-C15 : 0, anteiso-C17 : 0 iso-C16 : 0* iso-C16 : 0D

*Data from Kwon et al. (2007). DData from Wieser et al. (2005).

was included in a cluster constituted by species of the genus 100 Bio UV/VIS spectrophotometer equipped with a Peltier- Bacillus (Fig. 1). The most closely related species of strain thermostat-regulated 666 multicell changer and a temper- E33T were B. niabensis 4T19T (99.2 % 16S rRNA gene sequence ature controller with an in situ temperature probe (Varian). similarity), Bacillus herbersteinensis D-1-5aT (97.3 %) and The percentages of DNA–DNA reassociation of strain E33T Bacillus litoralis SW211T (97.2 %). The 16S rRNA gene to B. niabensis IBRC-M 10590T, B. herbersteinensis DSM sequence similarity between strain E33T and other species of 16534T and B. litoralis DSM 16303T were 22 %, 38 % and the genus Bacillus was lower than 96.7 %. Maximum- 19 %, respectively. According to Wayne et al. (1987), likelihood and neighbour-joining methods resulted in highly DNA–DNA relatedness lower than 70 % is considered to be similar tree topologies (Fig. 1) and confirmed the phylogenetic the threshold value for the delineation of genospecies, so cluster formed by strain E33T and strains of species of the genus the result obtained is low enough to separate strain E33T Bacillus. Based on the sequence divergence, it was evident that from B. niabensis IBRC-M 10590T and the other most strain E33T constituted a different taxon separated from other closely relates species of the genus Bacillus. species of the genus Bacillus. The DNA G+C content was determined by reversed-phase For determination of DNA base composition and DNA– HPLC of nucleosides according to the protocol of Mesbah DNA hybridization, DNA was isolated using a French et al. (1989). The G+C content of the DNA of strain E33T pressure cell (Thermo Spectronic) and was purified by was 42.6 mol%. This value is within the range for the genus chromatography on hydroxyapatite as described by Cashion Bacillus and higher than that of B. niabensis (Table 1). et al. (1977). DNA–DNA hybridization was carried out as described by De Ley et al. (1970) incorporating the Cell biomass for analysis of the peptidoglycan, isoprenoid modifications described by Huß et al. (1983), using a Cary quinones and polar lipids was obtained by cultivation on

2778 International Journal of Systematic and Evolutionary Microbiology 63 Bacillus halosaccharovorans sp. nov.

Brevibacterium frigoritolerans DSM 8801T (AM747813) 100 0.01 Bacillus simplex NBRC 15720T (AB363738) 77 Bacillus muralis LMG 20238T (AJ628748) DSM 23495T (FR749913) 98 Bacillus horneckiae Bacillus foraminis CV53T (AJ717382) 89 Bacillus humi LMG 22167T (AJ627210) Bacillus pocheonensis Gsoil 420T (AB245377) T 100 98 Bacillus novalis LMG 21837 (AJ542512) Bacillus vireti LMG 21834T (AJ542509) Bacillus soli LMG 21838T (AJ542513) 89 73 Bacillus drentensis LMG 21831T (AJ542506) 98 Bacillus bataviensis LMG 21833T (AJ542508) 77 Bacillus asahii MA001T (AB109209) 100 100 Bacillus aryabhattai B8W22T (EF114313) T 76 Bacillus flexus IFO 15715 (AB021185) Bacillus cohnii DSM 6307T (X76437) Bacillus fastidiosus DSM 91T (X60615) 96 Bacillus halosaccharovorans E33T (HQ433447) 100 84 Bacillus niabensis 4T19T (AY998119) 100 Bacillus litoralis SW-211T (AY608605) 96 D-1-5aT (AJ781029) 100 Bacillus herbersteinensis 96 Bacillus idriensis SMC 4352-2T (AY904033) Bacillus cibi JG-30T (AY550276) Bacillus indicus Sd/3T (AJ583158) Bacillus galliciensis BFLP-1T (FM162181) Bacillus subtilis subsp. subtilis DSM 10T (AJ276351)

Fig. 1. Phylogenetic tree, based on the maximum-parsimony algorithm of the 16S rRNA sequences, showing the position of strain E33T and the closely related species of the genus Bacillus. GenBank accession numbers are shown in parentheses. Bootstrap values .70 % are indicated at branch-points. Filled circles indicate branches also found in phylogenetic consensus trees generated with the maximum-likelihood, neighbour-joining methods. Bacillus subtilis subsp. subtilis DSM 10T (GenBank accession no. AJ276351) was used as outgroup. Bar, 1 % sequence divergence.

7.5 % HM agar at 35 uC. The whole-cell hydrolysate (4 M lipoquinone and peptidoglycan diamino acid of the cell HCl, 100 uC, 16 h) was analysed by chromatography on a wall of strain E33T were typical of those found in members cellulose thin-layer plate (Merck) using the solvent system of the genus Bacillus (Bagheri et al., 2012; Chen et al., 2011; of Rhuland et al. (1955). Polar lipids were analysed as Wieser et al., 2005; Xue et al., 2008). The whole-cell fatty described by Groth et al. (1996). Isoprenoid quinone acid composition was determined by GC using the analysis was carried out as described by Monciardini et al. Microbial Identification System (MIDI, Version 6.1; (2003). Identification Library TSBA40 4.1; Microbial ID). Cells were cultured on marine agar containing 5 % (w/v) NaCl at The peptidoglycan was based on meso-diaminopimelic 30 uC following the protocol of MIDI (http://www. acid and the polar lipids detected were phosphatidylgly- microbialid.com/PDF/TechNote_101.pdf). The cellular cerol, diphosphatidylglycerol, two unknown glycolipids, an fatty acid profile of strain E33T was characterized by the unknown lipid and an unknown phospholipid (Fig. S1, fatty acids anteiso-C (43.5 %), iso-C (21.0 %) and available in IJSEM Online). Strain E33T differs from the 15 : 0 15 : 0 anteiso-C (9.5 %) as the major fatty acids (Table S1). type strain of the type species, Bacillus subtilis subsp. 17 : 0 The fatty acid composition could not be compared under subtilis DSM 10T, by the absence of phosphatidylethano- standardized conditions because of different growth lamine and an aminophospholipid and the additional conditions to those used for B. niabensis IBRC-M 10590T, occurrence of an unidentified phospholipid and lipid. On since the two species have different NaCl range and the other hand, the presence of two glycolipids in strain optimum for growth, so it is not possible to compare them E33T has also been reported for the type species of the under the same optimal growth conditions. Their fatty acid genus Bacillus (Ka¨mpfer et al., 2006). The polar lipid profile is similar except for the content of iso-C and pattern of strain E33T is very similar to that of B. 15 : 0 anteiso-C . herbersteinensis (Wieser et al., 2005). Both of them are 17 : 0 lacking phosphatidylethanolamine and have two glycoli- The differences in some features, such as endospore pids with similar chromatographic motility. Their similar position and shape, oxidase test, anaerobic growth, NaCl polar lipid profile might be considered in the future for the concentration for growth (range and optimum), temper- reclassification of these taxa within a new genus when more ature and pH range and optimum for growth, nitrate data support it. The major isoprenoid quinone of strain reduction and urease activity as well as the quinone E33T was MK-7 (97.5 %), although MK-6 and MK-8 were composition and fatty acids composition can be used to also present (2 % and 0.5 %, respectively. The respiratory distinguish this strain from the phylogenetically related http://ijs.sgmjournals.org 2779 M. Mehrshad and others taxa (Tables 1 and S1). Also, the DNA–DNA hybridization Centre (IBRC) (MI-1388-04) and the International Foundation for data clearly separate strain E33T from the most closely Science (IFS) (A/4527-1) (to M. A. A.), from the Spanish Ministerio related species. Therefore, on the basis of these polyphasic de Economıá y Competitividad (CGL2010-19303) that include European Funds [European Regional Development Fund (FEDER)], taxonomic data, we propose that the novel strain represents National Science Foundation (Grant DEB-0919290) and Junta de a novel species of the genus Bacillus, for which the new name Andalucıá (P10-CVI-6226) (to A. V.). We thank Bettina Straübler for Bacillus halosaccharovorans sp. nov. is proposed. excellent technical assistance.

Description of Bacillus halosaccharovorans sp. nov. References Bacillus halosaccharovorans (ha.lo.sac.cha.ro.vo9rans, Gr. n. Amoozegar, M. A., Makhdoumi-Kakhki, A., Shahzadeh Fazeli, S. A., hals halos salt; Gr. n. sakchaˆr sugar; L. part. adj. vorans Azarbaijani, R. & Ventosa, A. (2012). Halopenitus persicus gen. nov., devouring; N.L. part.adj. halosaccharovorans sugar-devour- sp. nov., an archaeon from an inland salt lake. Int J Syst Evol Microbiol ing halophile). 62, 1932–1936. Arahal, D. R. & Ventosa, A. (2002). Moderately halophilic and Cells are Gram-positive, motile, endospore-forming rods, halotolerant species of Bacillus and related genera. In Applications and 0.5 6 3.0–8.0 mm in size. Ellipsoidal endospores are Systematics of Bacillus and Relatives, pp. 83–99. Edited by: R. Berkeley, produced at a central or subterminal position in swollen M. Heyndrickx, N. Logan & P. De Vos. Oxford: Blackwell. sporangia. 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2780 International Journal of Systematic and Evolutionary Microbiology 63 Bacillus halosaccharovorans sp. nov.

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