Introduction

The genus Aquibacillus was proposed by Amoozegar et al. (2014) with the description of Aquibacillus halophilus as well as the transfer of Virgibacillus koreensis to Aquibacillus koreensis and Virgibacillus albus to Aquibacillus albus. Therefore, at the time writing, this genus, belonging to the family Bacillaceae, comprises three species with validly published names and the type species is A. halophilus. The type strains of all three species were isolated from saline environments. Members of the genus Aquibacillus are Gram-stain positive, rod-shaped, motile, produce endospores, moderately halophilic, catalase- and oxidase-positive. Chemotaxonomically they are characterized by having MK-7 as the predominant respiratory quinone, phosphatidylglycerol and diphosphatidylglycerol as the major polar lipids, anteiso-C15:0 as the major fatty acid and meso-diaminopimelic acid (meso-DAP) as the diagnostic diamino acid in the peptidoglycan. The DNA G+C content of members of this genus ranges from 35.8–41.0 mol%.

During our surveys on bacterial diversity of a salt mine in Wensu county, Akesu area in Xinjiang province of China, a bacterial strain named WSY08-1T was obtained. By using a polyphasic taxonomic approach, we conclude that strain WSY08-1T represents a novel species of the genus Aquibacillus, for which the name Aquibacillus salifodinae sp. nov. is proposed.

Materials and methods

Bacterial strains and culture condition

Strain WSY08-1T was isolated from a salt mine sample (salt crystal, 41°34′52″N, 80°43′37″E) in Xinjiang province, China. Marine broth 2216 (MB, Difco) was used for isolation. The medium was solidified with 2.0 % agar (MA). To enrich bacteria about 3 g salt sample was suspended in 30 ml MB at 28 °C for 2 days. The enrichment culture was diluted and spread onto MA plates which were incubated at 28 °C. After 3 days of incubation, one of the aerobic isolates, strain WSY08-1T, was observed to produce cream-pigmented, circular and elevated colonies on MA plates. One of these colonies was picked up and purified by repeated restreaking. The isolate was routinely cultured on MA plates and maintained at −80 °C with 25 % (v/v) glycerol.

Biomass for chemotaxonomic and molecular studies was obtained by cultivation in shaking flasks with MB at 28 °C for 2 days. The recommended Minimal Standards for describing new taxa of aerobic, endospore-forming bacteria were followed (Logan et al. 2009). The reference type strain A. halophilus B6BT were obtained from Korean Collection for Type Cultures (KCTC; Korea); A. koreensis JCM 12387T and A. albus JCM 17364T were obtained from the Japan Collection of Microorganisms (JCM; Japan). Reference type strains were cultured under the same conditions as strain WSY08-1T for comparative analyses.

Morphological, physiological and biochemical characterization

Cell morphology was determined by optical microscopy (BX40; Olympus) and transmission electron microscopy (JEM-1230; JEOL) (Huo et al. 2010). Motility was detected by optical microscopy using the hanging drop technique (Skerman 1967). Gram staining was performed by following the method outlined by Dussault (1955). To determine the growth conditions of strain WSY08-1T, MB-1 medium with various NaCl concentrations (0–15 %, at intervals of 1 %, w/v) was used. MB-1 medium contained (per l distilled water): peptone (Becton–Dickinson) 5.0 g, yeast extract (Becton–Dickinson) 1.0 g, Ferric citrate 0.1 g, MgCl2·6H2O 12.6 g, MgSO4·7H2O 6.64 g, CaCl2 1.8 g, KCl 0.55 g, KBr 0.08 g, SrCl 34.0 mg, Boric acid 22.0 mg and NH4NO3 2.4 mg. The ranges of pH and temperature for growth were determined by using MB medium according to Zhang et al. (2013).

Catalase and oxidase activities and nitrate and nitrite reduction were tested according to (Dong and Cai 2001). Indole, methyl red and Voges-Proskauer tests, H2S production, as well as hydrolysis of aesculin, casein, gelatin and starch were performed as described by Zhu (2011). Esterase activity was detected as outlined by (Gutiérrez and González 1972). To analyze the use of accessory electron acceptors, sodium thiosulfate (20 mM), sodium sulfate (20 mM), sodium sulfite (5 mM), sodium nitrite (5 mM) and sodium nitrate (20 mM) were respectively added to sterile MB. Oxygen was removed as described by Grishchenkov et al. (2000). The same medium lacking l-cysteine and resazurin was used as aerobic control. Other enzyme activities and physiological properties were determined by using API ZYM, API 20E and API 20NE kits, using distilled water supplemented with 4 % (w/v) NaCl to suspend the cells. Acid production tests were carried out by using API 50CH kits, using modified MB from which yeast extract and peptone were omitted and 0.05 g/L yeast extract was added. Utilization of single carbon sources for growth was performed using Biolog GP2 96-well Microplates (Biolog) by using the modified MB. All API tests were performed according to the manufacturer’s instructions (bioMérieux). Antibiotic sensitivity tests were determined on MA plates with antibiotic discs containing the following amount (μg per disc, unless indicated): amikacin (30), ampicillin (10), bacitracin (0.04 IU), chloramphenicol (30), ciprofloxacin (5), erythromycin (15), gentamicin (10), kanamycin (30), neomycin (30), norfloxacin (10), novobiocin (30), nystatin (100), penicillin G (10 IU), polymyxin (300 IU), rifampicin (5), streptomycin (10), sulfamethoxazole (300), tobramycin (10) and vancomycin (30).

Chemotaxonomic characterization

For chemotaxonomic experiments, late exponential phase cells of strains WSY08-1T, A. koreensis JCM 12387T and A. albus JCM 17364T were harvested from MB. The isomer type of the diamino acid in the cell-wall peptidoglycan and isoprenoid quinones were determined as described by Komagata and Suzuki (1987). Polar lipids of strain WSY08-1T and reference strains were extracted and separated on silica gel 60 F254 aluminium-backed thin-layer plates (10 × 10 cm, Merk 5554) and further analyzed as described by Minnikin et al. (1984). Fatty acid methyl esters were obtained from freeze-dried cells as described by Kuykendall et al. (1988) and their identification and quantification were performed using the Sherlock Microbial Identification System (MIDI) with the standard MIS Library Generation Software version 4.5 (Microbial ID).

Molecular studies

The 16S rRNA gene was amplified and analyzed as described previously (Xu et al. 2007). PCR products were cloned into vector pMD19-T (TaKaRa) and then sequenced. The sequence was compared with closely related sequences of reference species using the EzTaxon-e server (Kim et al. 2012) and BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). Multiple sequences were aligned with CLUSTAL W1.8 (Thompson et al. 1994). Phylogenetic trees were constructed by the neighbour-joining (Saitou and Nei 1987), the maximum-parsimony (Fitch 1971) and the maximum-likelihood (Felsenstein 1981) methods within the MEGA 5 program package (Tamura et al. 2011). Evolutionary distances were calculated according to the algorithm of Kimura’s two-parameter model (Kimura 1980) for the neighbour-joining method.

The DNA G+C content was determined by reversed-phase HPLC as described by Mesbah and Whitman (1989). DNA–DNA hybridizations were performed by using the thermal denaturation and renaturation method (De Ley et al. 1970) as modified by Huß et al. (1983), using a Beckman DU 800 Spectrophotometer. The experiments were carried out in quadruplicate.

Results and conclusion

Morphological, physiological and biochemical characteristics

Colonies of strain WSY08-1T on MA plates after 2 days incubation were observed to be 0.1–0.3 mm in diameter, cream-pigmented, circular and elevated. Strain WSY08-1T was found to be strictly aerobic. Cells were observed to be Gram-stain positive, rod-shaped (0.4–0.6 × 5.0–8.1 μm), motile by means of peritrichous flagella and endospore-forming. Spherical to ellipsoidal endospores were observed to be formed terminal in swollen sporangia (Fig. S1). The physiological and biochemical characteristics of strain WSY08-1T that differentiate it from the reference strains are shown in Table 1. The detailed physiological and biochemical characteristics of the type strain WSY08-1T are given in the species description.

Table 1 Differential characteristics among strains WSY08-1T, A. albus YIM 93642T, A. koreensis BH30097T and A. halophilus B6BT
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The type strain was found to be sensitive to ampicillin, bacitracin, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, kanamycin neomycin, norfloxacin, novobiocin, penicillin G, rifampicin, sulfamethoxazole, tobramycin and vancomycin; and resistant to amikacin, nystatin, polymyxin and streptomycin.

Chemotaxonomy results

The cell wall peptidoglycan of strain WSY08-1T was found to contain meso-diaminopimelic acid as the diamino-acid. The isoprenoid quinones were identified as MK-7 (94 %) and MK-6 (6 %). The polar lipids were found to include phosphatidylglycerol, diphosphatidylglycerol, an unknown glycolipid, two unknown phospholipids and an unknown lipid (Fig. S2). The major fatty acids were identified as anteiso-C15:0 (72.7 %) and anteiso-C17:0 (10.0 %). The complete fatty acid profiles of strain WSY08-1T and the reference type strains are summarized in Table 2.

Table 2 Cellular fatty acids for strain WSY08-1T together with type strains of related species
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Molecular characterization

The nearly complete 16S rRNA gene sequence (1,528 bp) of strain WSY08-1T was determined (GenBank accession number AB859945). Based on 16S rRNA gene sequence analysis, the validly named species that shared highest sequence similarities to strain WSY08-1T were identified as A. halophilus B6BT (97.6 %), followed by A. koreensis BH30097T (96.9 %), A. albus YIM 93624T (96.5 %), Sediminibacillus halophilus En8dT (96.4 %) and Virgibacillus carmonensis JCM 16508T (96.0 %). Type strains of other species shared lower sequence similarities (<96 %) to strain WSY08-1T. Phylogenetic trees revealed that strain WSY08-1T clustered with A. halophilus B6BT, A. koreensis BH30097T and A. albus YIM 93624T in a separate clade (Fig. 1; Fig. S3 and Fig. S4). DNA–DNA hybridization between strains WSY08-1T and A. halophilus B6BT showed a low relatedness value of 31.4 %.

Fig. 1
figure 1

Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences showed the relationships of strain WSY08-1T and related species. Bootstrap values based on 1,000 replications are listed as percentages at branching points. Bootstrap values higher than 50 % are indicated at branch-points. Enterococcus faecium JCM 5803T was used as outgroup. Bar 1 % sequence divergence

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The genomic G+C content of strain WSY08-1T was determined to be 36.9 mol%, which is in the range for the members of the genus Aquibacillus.

Taxonomic conclusion

Based on its characterisation as Gram-positive, rod-shaped, endospore-forming, moderately halophilic cells; positive for catalase and oxidase activities; the major fatty acid (anteiso-C15:0); the major polar lipids (phosphatidylglycerol and diphosphatidylglycerol); the predominant menaquinone (MK-7); the cell-wall peptidoglycan diamino acid (meso-diaminopimelic acid); the G+C content (36.9 mol%); and the phylogenetic analyses, all these data suggest that strain WSY08-1T should belong to the genus Aquibacillus. However, strain WSY08-1T showed notable differences in comparison to the type strains of the most closely related species A. halophilus B6BT, A. koreensis BH30097T and A. albus YIM 93642T with regard to morphological characteristics, growth ranges (temperature, pH and NaCl tolerance), nitrate reduction, Voges–Proskauer, hydrolysis of starch and Tween 80, and acid production from carbohydrates (Table 1). The cellular fatty acid profile of strain WSY08-1T was qualitatitively similar to that of the reference strains but differences in the proportions of some fatty acids were noted (Table 2). Differences in the polar lipid profile also existed compared to the reference strains (Fig. S4). Strain WSY08-1T shared low 16S rRNA gene sequence similarities to the type strains A. halophilus B6BT, A. koreensis BH30097T and A. albus YIM 93624T (97.6, 96.9 and 96.5 %, respectively). DNA–DNA hybridization data (31.4 %) clearly differentiated strain WSY08-1T from strain A. halophilus B6BT.

Based on the phenotypic, chemotaxonomic and genotypic characteristics described above, strain WSY08-1T was identified as a novel species within the genus Aquibacillus, for which the name Aquibacillus salifodinae sp. nov. is proposed.

Description of Aquibacillus salifodinae sp. nov

Aquibacillus salifodinae (sa.li.fo.di’nae. L. n. sal, salt; L. n. fodina, a pit; N.L. gen. n. salifodinae of a saltpit, salt mine).

Cells are Gram-positive, strictly aerobic, motile by means of peritrichous flagella and rod-shaped (0.4–0.6 × 5.0–8.1 μm). Spherical to ellipsoidal endospores are formed in terminal swollen sporangia. Colonies are 0.1–0.3 mm in diameter, cream-pigmented, circular and elevated. Growth occurs at 20–45 °C (optimum 37 °C), 0–10 % (w/v) NaCl (optimum 4 %, w/v) and pH 6.0–9.0 (optimum 7.0). Catalase and oxidase activities are positive. Nitrate is reduced to nitrite but not to nitrogen. Indole, methyl red, Voges–Proskauer, H2S production, hydrolysis of starch, casein and gelatin, as well as the production of arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and urease are negative. Clearly positive in the Biolog GP2 MicroPlate system for the oxidation of acetic acid, N-acetylglucosamine, N-acetyl-l-glurtamic acid, adenosine-5′-monophosphate, l-alaninamide, l-alanyl-glycine, amygdalin, l-asparagine, l-arabinose, arbutin, d-cellobiose, α-cyclodextrin, 2′-deoxy adenosine, l-fructose, d-fructose, d-fructose-6-phosphate, d-galactose, gentiobiose, d-gluconic acid, d-glucose, α-d-glucose-1-phosphate, d-glucose-6-phosphate, l-glutamic acid, d-l-α-glycerol phosphate, glycyl-l-glutamic acid, glycerol, glycogen, γ-hydroxybutyric acid, m-inositol, inosine, inulin, α-ketoglutaric acid, α-ketovaleric acid, lactamide, α-d-lactose, lactulose, d-malic acid, l-malic acid, maltose, mannan, d-mannose, d-melezitose, d-melibiose, α-methyl-d-galactoside, β-methyl-d-galactoside, 3-methyl-glucose, α-methyl-d-glucoside, α-methyl-d-mannoside, palatinose, propionic acid, d-psicose, pyruvic acid, l-pyroglutamic acid, d-raffinose, l-rhamnose, d-ribose, salicin, sedoheptulosan, d-sorbitol, succinamic acid, succinic acid, succinic acid mono-methyl ester, sucrose, d-tagatose, thymidine, thymidine-5′-monophosphate, trehalose, Tween 40 and 80, uridine-5′-monophosphate, xylitol and d-xylose. Other substrates in the GP2 MicroPlate are not utilized as sole carbon source. According to the API 50 CH system, positive for acid production from N-acetylglucosamine, d-adonitol, aesculin, amygdalin, l-arabinose, l-arabitol, arbutin, cellobiose, d-fructose, gentiobiose, d-glucose, 2-ketogluconate, 5-ketogluconate, d-lyxose, maltose, d-mannose, β-methyl-d-xylopyranoside, d-ribose, salicin, sucrose, trehalose and d-xylose; Weakly positive of glycerol, l-sorbose, d-tagatose and xylitol; other substrates of the API 50 CH system are not converted to acid. With API ZYM systems, alkaline phosphatase, esterase (C4), esterase lipase (C8), β-glucosidase, β-glucuronidase, naphtol-AS-BI-phosphohydrolase are positive; acid phosphohydrolase is weakly positive; lipase (C14), leucine arylamidase, valine arylamidase, cysteine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, α-glucosidase. N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase are negative. The cell-wall peptidoglycan contains meso-diaminopimelic acid as the diamino-acid. MK-7 is the predominant menaquinone and MK-6 is also present. The polar lipids are phosphatidylglycerol, diphosphatidylglycerol, an unknown glycolipid, two unknown phospholipids and an unknown lipid. The major fatty acids are anteiso-C15:0 and anteiso-C17:0. The DNA G+C content of the type strain is 36.9 mol% (as determined by HPLC).

The type strain WSY08-1T (= JCM 19761T = CGMCC 1.12849T) was isolated from a salt mine taken from Wensu county, Xinjiang province, China. The GenBank accession number of the 16S rRNA gene sequence of the type strain is AB859945.