Introduction

Alkalibacillus as a novel genus was proposed by Jeon et al. [1], based on a reclassification of [Bacillus] [2]. Seven species have been described in this genus till now, which were isolated from salt lake, hypersaline soil, water of a mineral pool, non-saline forest soil, marine solar saltern, and inland solar saltern [1,2,3,4,5,6,7]. Most species of the genus Alkalibacillus are halophilic bacteria that grow optimally in media containing 6–20% (w/v) NaCl and show considerable industrial benefits, particularly for the production of enzymes (proteases, xylanases, glycosidases, etc.) [2, 8]. Strain YIM 98829T was isolated from Aiding Lake in Xinjiang province, North-West China within the process of exploring microbial resources in extreme environments. The present paper reports a polyphasic characterization of the strain and its classification as a novel species of the genus Alkalibacillus.

Methods and Materials

Bacterial Isolation

Strain YIM 98829T was isolated from hypersaline sediment of the Aiding Lake in Xinjiang province, North-West China (42 68′66″ N, 89 33′07″ E). The isolation medium contained the following components, 10% (w/v) NaCl, 7.5 g casein, 10 g yeast extract, 3 g sodium citrate, 10 g MgSO4∙7H2O, 2 g KCl, 1 ml 4.98% FeSO4,15 g agar, and 1000 ml H2O, pH 7.4. Stock cultures of strain YIM 98829T were prepared in Tryptone soya agar (TSA) medium (10%, w/v NaCl) with 20% glycerol(w/v) and stored at − 80 °C. Biomass for chemical analysis and molecular studies were obtained by cultivation in TSA without agar (10%, w/v NaCl; pH 7.4) at 37 °C and 150 rpm for 1 week.

Physiological, Morphological, and Biochemical Tests

Cell morphology, size, shape, and flagellation were examined by light microscopy (BX41,Olympus) and transmission electron microscope (Model JEM1010, JEOL), using cells from exponentially growing cultures after incubation of 3 days in TSA containing 10% (w/v) NaCl and stored in refrigerator for 1 week to stimulate endospore formation. The Gram stain reaction was carried out by the Burke method [9] and the result was confirmed by the KOH test [10]. Growth at various temperatures (4, 10, 15, 20, 25, 28, 30, 35, 37, 40, 45, 50, and 55 °C) were evaluated in TSA (10%, w/v NaCl) by incubating the cultures for 7 days. A series of pH conditions (4.0–11.0, at 0.5 intervals) using the buffer system described by Xu et al. [11] and salt tolerance (0–30%, w/v, at 5% intervals) was examined at 37 °C for 14 days. Growth under anaerobic condition was determined using the GasPak anaerobic system (BBL) according to the manufacturer’s instructions.

Analysis of catalase and urease activities, nitrate reduction, H2S and melanin production, and production of indole followed procedures as described by Smibert and Krieg [12]. Hydrolysis of Tweens was examined as described by Harrigan and McCance [13]. Carbon source utilization and enzyme activities of strain YIM 98829T was analyzed using API 20E, API 20NE, and API ZYM kits according to the manufacturer (bioMérieux, France) instructions. The acid production from carbohydrates was determined using the API 50CH system (bioMérieux) according to the manufacturer’s instructions. Antibiotic susceptibility tests were performed on TSA containing 10% (w/v) NaCl using disks impregnated with various antimicrobial compounds [14]. The data from strains of species A. haloalkaliphilus DSM 5271T, A. silvisoli BM2T, and A. filiformis 4AGT were used as references.

16S rRNA Gene Sequence, Phylogenetic Analysis, DNA–DNA Hybridization, and G + C Content

The extraction of genomic DNA, PCR amplification, and sequencing of the 16S rRNA gene were carried out as described by Yang et al. [15] and Li et al. [16]. The values for sequence similarity among the most closely related strains were determined using the NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) [17]. Multiple alignments with sequences of the most closely related bacteria were carried out using the CLUSTAL X 1.8 program [18]. Phylogenetic trees were reconstructed by the neighbor-joining [19], maximum-parsimony [20], and maximum-likelihood [21] tree-making algorithms using the software packages MEGA version 7.0 [22]. The significant level of branch stability was assessed by performing bootstrap analyses with 1000 resamplings [23]. The genome of YIM 98829T was sequenced using an HiSeq X-ten at BGI.tec (Shenzhen, China). The sequenced reads were assembled using SOAPdenovo software version 1.05 (https://soap.genomics.org.cn/soapd enovo.html). The DNA–DNA hybridization (DDH) similarity between strains was calculated in silico with the Genome-to-Genome distance calculator server version 2.1 [24]. Average nucleotide identity (ANI) was calculated with OrthoANI [25]. Strain A. haloalkaliphilus DSM 5271T (GCA_007991275.1) was used as the reference strain in the ANI value calculation and digital DDH [26]. Genome tree was constructed using RAxML [26] and fast bootstrapping [27] was used to generate the support values in the tree. The DNA G + C mol% value was obtained from the genomic sequence.

Biochemical Characteristics

Peptidoglycan was purified and hydrolyzed according to Schumann [28] and was analyzed by HPLC–MS as described previously [29]. Amino acid enantiomers present in the total hydrolysate were analyzed on an Infinity Poroshell Chiral T column and detected by mass spectrometry (Fig. S5). The sugars of whole-cell hydrolysates were detected by Hasegawa et al. [30] Polar lipids were extracted and then examined by two-dimensional TLC and identified using previously described procedures [31, 32]. The isolate and separate menaquinones were isolated according to Collins et al. [33] and separated by HPLC [34]. For cellular fatty acid analysis, strain YIM 98829T was grown on tryptic soya broth with 10% NaCl (TSB, Difco) at 37 °C and harvested after 7 days. Fatty acid methyl esters were extracted, methylated, and analyzed by using the Microbial Identification System (Sherlock version 6.1, MIDI database, TSBA6) according to the manufacturer’s instructions.

Results and Discussion

Physiological, Morphological, and Biochemical Tests

Strain YIM 98829T was motile, aerobic, produced terminal endospores in swollen sporangia. Cells were long rods with a width of 0.3–0.5 μm and length of 2–5 μm (Fig. S4). Strain YIM 98829T grew at NaCl concentrations between 5.0 and 20.0% (w/v), with optimal growth occurring at 5.0–10.0% (w/v). The temperature range for growth extended from 10 to 50 °C; with an optimum at 37 °C, strain YIM 98829T grew well in the slightly alkaline conditions of pH 7.5–8.0 in TSA containing 10% (w/v) NaCl, positive for production of catalase and nitrate reduction and negative for urease, indole, and hydrolysis of aesculin. Tweens 20, 40, and 80 were not hydrolyzed. All the above characteristics are consistent with those of genus Alkalibacillus. However, strain YIM 98829T stained Gram negative, which was very different from other published species of the genus Alkalibacillus. Other physiological characteristics and biochemical characteristics are given in Table 1 and in the species description.

Table 1 The biochemical and physiological characteristics of strain YIM 98829T and the reference strains of genus Alkalibacillus
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16S rRNA Gene Sequence and Phylogenetic Analysis

An almost complete 16S rRNA gene sequence (1553 bp) of strain YIM 98829T was obtained. The closest relative of strain YIM 98829T was Alkalibacillus. haloalkaliphilus DSM 5271T with 97.0% 16S rRNA gene sequence similarity. The phylogenetic trees based on the 16S rRNA gene sequences showed that strain YIM 98829T clustered with strain DSM 5271T, A. silvisoli BM2T (96.9% similarity), and A. filiformis 4AGT (96.9% similarity) under the high bootstrap values (Fig. 1; Figs S1 and S2). Genome tree shows that YIM 98829T is steadily clustered in a branch with the type strain of this genus under the 1000 bootstrap values (Fig. S3). However, the level of DNA–DNA relatedness between strain YIM 98829T and A. haloalkaliphilus DSM 5271T (GenBank accession no. GCA 007991275.1) was only 16.9%, which was well lower than the threshold value of 70.0% recommended for recognition of separate species [35]. The ANI value between these two strains was 73.2%. The low similarity and phylogenetic results clearly demonstrated that strain YIM 98829T represents a different genomic species of genus Alkalibacillus and is distantly related to the type species of the genus as well as to the species A. almallahensis and A. salilacus. The latter two show a 16S rRNA gene sequence similarity value of 95.6% only as compared to the type species A. haloalkaliphilus. The DNA G + C content of strain YIM 98829T was 38.0 mol%.

Fig. 1
figure 1

The neighbor-joining (NJ) tree based on 16S rRNA gene sequence analysis showing phylogenetic relationships of strain YIM 98829T and members of genus Alkalibacillus. Bootstrap values more than 50% based on 1000 replications are shown at branching points. Scale bar, 0.005 substitutions per nucleotide position

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Biochemical Characteristics

The cellular fatty acid profile contained iso-C15:0 (60.0%) and anteiso-C15:0 (11.4%) as major fatty acids (> 10%), iso-C17:0 (7.4%), iso-C16:0 (8.0%), iso-C14:0 (2.8%), C16:0 (2.1%) and anteiso-C17:0 (5.1%) as minor fatty acids (< 10%). Meso-, LL-, DL-diaminopimelic acid were absent in peptidoglycan purified from YIM 98829T (Fig. S5) and l-ornithine was present as the diagnostic diamino acid. Besides d-glutamic acid and alanine, l-serine and d-aspartic acid could be detected. In a partial hydrolysate of the peptidoglycan, the dipeptides l-Ala—d-Glu, d-Glu—l-Orn, l-Orn—l-Ser and l-Orn—d-Ala and the longer peptides d-Glu—l-Orn—l-Ser, l-Orn—l-Ser—d-Asp, l-Orn—l-Ser—d-Asp—d-Ala, and l-Orn—l-Ser—d-Asp—d-Ala—l-Orn were detectable. Accordingly, the amino acid composition of the peptidoglycan of strain YIM 98829T corresponds to the peptidoglycan type A4β l-Orn—l-Ser—d-Asp (A21.7, www.peptidoglycan-types.info). Xylose (34.8%), glucose (23.2%), galactose (15.6%), and mannose (13.2%) were detected as the major whole-cell sugars; minor amounts of ribose (7.0%) and fucose (6.1%) were also detected. The phospholipids were diphosphatidylglycerol, phosphatidylglycerol, one unknown phospholipid, and two unidentified aminophospholipid (Fig. S6). And the predominant isoprenoid quinone in strain YIM 98829T was MK-7 (100%). The chemical characteristics are similar to those of other species of genus Alkalibacillus except the presence of l-ornithine as the diamino acid of the peptidoglycan.

Conclusion

Some obvious differences in physiological and biochemical characteristics, such as Gram stain reaction, absence of meso-diaminopimelic acid, and low level of DNA–DNA relatedness, distinguish strain YIM 98829T from other species. Based on the polyphasic analysis it can be concluded that strain YIM 98829T should belong to the genus Alkalibacillus, even though that there is evidence to reclassify the A. almallahensis cluster in the future. The data described above also indicated that strain YIM 98829 T represents a novel species of the genus Alkalibacillus, for which the name Alkalibacillus aidingensis sp. nov. is proposed.

Description of Alkalibacillus aidingensis sp. nov.

Alkalibacillus aidingensis (ai.ding.en′sis; N.L. masc. adj. aidingensis of or belonging to Aiding lake, a salt lake in China, where the type strain was isolated).

Gram-stain-negative; results from API 50CH tests showed that acids was produced from d-galactose, d-mannitol, d-sorbitol, d-turanose, d-lyxose, d-tagatose, d-fucose, l-fucose, d-arabitol, l-arabitol, potassium gluconate, and potassium 5-ketogluconate. Sensitive to ampicillin (10 μg), bacitracin (10 μg), tetracycline (30 μg), novobiocin (5 μg), neomycin (30 μg), cefoxitin (30 μg), cefotaxime/clavulanic acid (30 μg), carbenicillin (100 μg), chloramphenicol (30 μg), gentamicin (10 μg), rifampicin (5 μg), streptomycin (10 μg), erythromycin (5 μg), and kanamycin (5 μg). Positive for alkaline phosphatase, esterase C4, esterase lipase C8, valine arylamidase, trypsin, acid phosphatase, and naphthol-AS-BI-phosphohydrolase. The cell wall peptidoglycan contained ornithine, serine, aspartic acid, glutamic acid, and alanine. Mannose, galactose, glucose, and xylose are the major whole-cell sugars. The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, unknown phospholipid and two unidentified aminophospholipids. The predominant menaquinone is MK-7. The major fatty acids are iso-C15:0 and anteiso-C15:0. The DNA G + C content of the type strain is 38.0 mol%. The type strain is YIM 98829T (= NBRC 114103T = CGMCC 1.17260T = DSM 112470T), isolated from the Aiding Lake in Xinjiang province, North-West China.

Emended Description of the Genus Alkalibacillus Jeon et al. 2005

Alkalibacillus (Al.ka.li.ba.cil′lus. N.L. n. alkali; L. n.bacillus rod; N.L. masc. n. Alkalibacillus bacillus living under alkaline conditions).

Cells are Gram positive or negative, spore-forming rods. Spherical endospores are produced terminally in swollen sporangia. Strictly aerobic and moderately halophilic. The whole-cell hydrolysates contain meso-diaminopimelic acid, or the cell wall peptidoglycan type is of the A4β type with l-Orn as the diagnostic diamino acid. Major isoprenoid quinone is MK-7. DNA G + C content is 38.0–41 mol% (HPLC). Predominant cellular fatty acids are iso-C15:0, anteiso-C15:0, and anteiso-C17:0. Phylogenetically, the genus belongs to the family Bacillaceae. The type species of the genus is Alkalibacillus haloalkaliphilus.