Introduction

The genus Salipaludibacillus was first proposed by Sultanpuram and Mothe (2016) for an isolate from a saline–alkaline lake. At the time of writing, this genus comprises 4 species with validly published names: Salipaludibacillus aurantiacus (Sultanpuram and Mothe 2016), Salipaludibacillus neizhouensis (Sultanpuram and Mothe 2016; Chen et al. 2009), Salipaludibacillus agaradhaerens (Sultanpuram and Mothe 2016; Nielsen et al. 1995), and Salipaludibacillus halalkaliphilus (Amoozegar et al. 2018). Members of the genus Salipaludibacillus are generally characterised to be Gram-stain positive, non-motile, rod shaped, aerobic or facultatively anaerobic, form oval or ellipsoidal endospores at the sub-terminal position, and have anteiso-C15:0, C16:0 and iso-C15:0 as their major fatty acids, MK-7 as their predominant isoprenoid quinone with minor traces of MK-6, and phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol as the major polar lipids, and show relatively low G + C contents (39.3–42.4 mol%) (Sultanpuram and Mothe 2016).

Saline–alkaline lakes represent a unique ecosystem with extremely high pH and salinity (Sorokin et al. 2011). These haloalkaliphiles under double stress play essential roles and functions in biogeochemical processes and the ecological function (Sorokin et al. 2011). Furthermore, the unique metabolic pathways of haloalkaliphiles can be applied in the biodegradation and (or) biotransformation of a broad range of toxic industrial pollutants, and in the biofuel industry (Zhao et al. 2014). Therefore, it is of great importance to discover novel extremophiles. In the course of surveying the microbial community of the Keqin Lake, Heilongjiang Province, China (46°18′32′′N, 123°25′58′′E), a novel strain, strain KQ-12T, was isolated. As a result of testing using different taxonomic approaches, we consider the strain to represent a novel species of the genus Salipaludibacillus, and here name it Salipaludibacillus keqinensis sp. nov.

Materials and methods

Strain and culture conditions

KQ-12T was isolated from mixed water and sediment samples collected from Keqin Lake (28 mM Na+, 0.47 mM Mg2+, pH 8.7) in Heilongjiang Province, China (46°18′32′′N, 123°25′58′′E). Collected samples were transferred immediately to sterile serum bottles, tightly sealed with blue butyl-rubber stoppers, kept at room temperature during transportation and subsequently stored at 4–8 °C for up to 2 weeks until ready for use. While studying the cultivable bacterial diversity of saline ecosystems of Keqin Lake, KQ-12T was isolated using serial dilutions up to 10−5 from the mixed water and sediment sample on solid medium. The isolation medium contained (l−1): NaCl (100 g), NH4Cl (1.0 g), KCl (K+, 13.4 mM) (1.0 g), KH2PO4 (K+, 2.2 mM) (0.3 g), MgSO4·7H2O (0.1 g), Na2CO3 (0.1283 M Na+) (6.8 g), NaHCO3 (0.0452 M Na+) (3.8 g), Yeast extract (Difco) (4 g), Casamino acids (Difco) (0.5 g). The medium was adjusted to pH 9.2 with NaHCO3/Na2CO3 buffer (100 mM in deionized water; pH 9.2) at room temperature and 2% agar was added. After autoclaving at 121 °C for 45 min, 0.2% (w/v) filter-sterilized glucose was added to the medium before pouring plates. KQ-12T was maintained on slant tubes at 4–6 °C and preserved as 15% (w/v) glycerol suspensions at − 80 °C. Unless otherwise stated, cells for physiological and biochemistry analyses were obtained through cultivation in shake flasks at 150 rpm with the aforementioned liquid culture medium at 25 °C for 48 h.

Phenotypic characteristics

General cell morphology was examined by light microscopy (BH-2, Olympus Co., Japan) and transmission electron microscopy (Hitachi H-600, Japan) using cells from exponentially growing cultures. Gram-staining test was examined according to the methods described by Smibert and Krieg (1994), in parallel with the KOH lysis method (Gregersen 1978). Motility was observed by stab-culture in semi-solid medium according to the procedure of Gerhardt et al. (1981). The ISCC-NBS colour charts (Kelly 1964) were used to assess the colony colour. Growth at different temperatures (4–55 °C) and NaCl tolerance (0–30% (w/v)) were tested using LB as the basal medium. The pH range (pH 5.5–11.5, with intervals of 0.5, with MES buffer for pH 5.5–6.5, HEPES buffer for pH 7.0–8.0, TAPS buffer for pH 8.0–9.0, CHES buffer for pH 9.0–10.0 and CAPS buffer for pH 10.0–11.5). Anaerobic growth test was performed according to previously described method (Zhang et al. 2016). Hydrolysis of aesculin, casein, cellulose, gelatin, starch, tweens 20 and 80, citrate utilization, methyl-red reaction, production of indole and H2S, and observation of endospores were tested as described by Dong and Cai (2001). The Voges–Proskauer reaction, reduction of nitrate, and urease activity were determined according to the methods described by Pettersson et al. (1996). Catalase activity was assessed by a bubble production in 3.0% (v/v) H2O2 (Ohta and Hattori 1983). Oxidase activity was determined with 1% (w/v) tetramethyl-p-phenylenediamine (Cappuccino and Sherman 2002). DNase test was conducted with DNase test agar (Difco). Other enzyme activities and substrate oxidation patterns were assayed using the API ZYM kits (bioMérieux) and GP2 MicroPlates (Biolog), respectively, according to the manufacturer’s instructions with 8% (w/v) NaCl and pH 9.0.

Phylogenetic analysis

Extraction of genomic DNA and amplification of the 16S rRNA gene were carried out as previously reported by Wang et al. (2018). Amplification products were cloned into the vector pMD 19-T (TaKaRa) and then sequenced. The 16S rRNA gene sequence was compared with those of Salipaludibacillus and Bacillus species available in the EzBioCloud server (www.ezbiocloud.net/) (Yoon et al. 2017). Multiple alignments with closely related sequences were performed using the clustal_w program integrated in the mega 7.0 software (Kumar et al. 2016). Phylogenetic trees were reconstructed by the neighbour-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and minimum-evolution (Rzhetsky and Nei 1992) methods with bootstrap values based on 1000 replications. Evolutionary distances among the related taxa were calculated according to Kimura’s two-parameter model (Kimura 1980).

The draft genome of KQ-12T and S. neizhouensis KCTC 13187T were sequenced using the Hiseq 4000 sequencing platform with paired-end read length of 2 × 150 bp and de novo assembled using MicrobeTrakr plus v. 0.9.1 (http://www.microbetrakr.com). The obtained genomes were submitted to the GenBank database, and the DNA G + C content was gained directly from the genome sequence. The level of pairwise genome-based similarity was evaluated using average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values, which were achieved by using the Orthologous Average Nucleotide Identity Tool (www.ezbiocloud.net/tools/orthoani) and Genome-to-Genome Distance Calculator software version 2.1 (http://ggdc.dsmz.de/) with Formula 2, respectively.

Chemotaxonomy

For cellular fatty acid analysis, KQ-12T and the three related reference strains were cultured on LB medium at pH 9.0, 25 °C and 8% (w/v) NaCl for 48 h. Fatty acids were purified, identified and quantified by GC using the Sherlock Microbial Identification System (MIDI) (Kämpfer and Kroppenstedt 1996). MIDI Sherlock version 6.0 and the TSBA6 database were employed for this analysis. Isoprenoid quinones were extracted from lyophilized cells, purified by thin-layer chromatography (TLC) and investigated by HPLC (Collins 1985) using the menaquinones of the reference type strains as standards. Preparation of cell walls and determination of peptidoglycan structure were analysed as described by Hasegawa et al. (1983). Polar lipids were extracted following Minnikin et al. (1984), separated by two-dimensional TLC and detected by spraying individual plates with: molybdophosphoric acid, molybdenum blue, ninhydrin, p-anisaldehyde.

Results and discussion

Cells of KQ-12T were observed to be Gram-stain positive, aerobic, motile, producing endospores which are ellipsoidal and located sub-terminally, rod-shaped and 0.7–0.9 × 1.5–2.4 µm in size (Fig. S1). Other phenotypic and physiological characteristics are presented in the species description. Differential characteristics between KQ-12T and the closely related species in the genus Salipaludibacillus are given in Table 1.

Table 1 Differential characteristics between KQ-12T and the type strains of closely related species of the genus Salipaludibacillus
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The almost-complete 16S rRNA gene sequence (1491 bp) of KQ-12T has been deposited as MH939198 in the GenBank/EMBL/DDBJ databases. Identification using the EzTaxon server revealed that KQ-12T is closely related to S. aurantiacus S9T (98.1%, with 16S rRNA gene sequence similarity), followed by S. neizhouensis JSM 071004T (97.7%), S. agaradhaerens DSM 8721T (97.6%), S. halalkaliphilus GASy1T (96.6%). These values are at the level suggested to allocate this strain to a new species (Kim et al. 2014). The neighbour-joining tree demonstrated that KQ-12T formed a separate branch with S. neizhouensis JSM 071004T and S. halalkaliphilus GASy1T, and is closely related to other members of the genus Salipaludibacillus (Fig. 1). The same cluster was recovered when the trees were reconstructed using minimum-evolution (Fig. S2) and maximum-likelihood (Fig. S3) algorithms. Phylogenetic analysis showed KQ-12T is a member of the genus Salipaludibacillus.

Fig. 1
figure 1

Neighbor-joining tree showing the phylogenetic position of the novel species based on 16S rRNA gene sequences. Bootstrap values more than 50% based on 1000 replications are shown at branching points. Marinococcus halophilus DSM 20408T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position

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The draft genome size of KQ-12T is 4,150,426  bp with a G + C content of 39.6 mol%. The draft genome size of S. neizhouensis KCTC 13187T is 5,397,042 bp with a G + C content of 37.2 mol%. The genomic G + C content (39.6 mol%) of KQ-12T is within the range of the genus salipaludibacillus (39.3–42.4 mol%) (1). The ANI values between strain KQ-12T (GeneBank: PDOD00000000) and its related species S. aurantiacus S9T (FOGT00000000), S. neizhouensis KCTC 13187T (PDOE00000000) and S. agaradhaerens DSM 8721T (MTIU00000000) were 72.5, 71.6 and 72.0%, respectively, which are much lower than the accepted ANI species cut-off value of 94–96% (Richter and Rosselló-Móra 2009). Furthermore, the dDDH values of KQ-12T with the selected reference strains S. aurantiacus S9T, S. neizhouensis KCTC 13187T and S. agaradhaerens DSM 8721T were 19.6, 20.5 and 22.3%, respectively, well below the threshold of 70% (Wayne et al. 1987), indicating that KQ-12T does not belong to any of these related species.

Chemotaxonomic characteristics of KQ-12T also supported its classification as a member of the genus salipaludibacillus. The peptidoglycan cell wall of KQ-12T contained meso-diaminopimelic acid (m-DAP) as the diagnostic diamino acid, which is consistent with the results reported for members of the genus Salipaludibacillus (Sultanpuram and Mothe 2016; Amoozegar et al. 2018). The major cellular fatty acids (content ≥ 5%) of KQ-12T were anteiso-C15:0 (47.1%), anteiso-C17:0 (12.1%), iso-C15:0 (6.7%), and C16:0 (5.5%). The fatty acid profile of KQ-12T was similar to those of the three selected reference strains in genus Salipaludibacillus (Table 2). However, some minor differences were observed between KQ-12T and the reference strains, which included the presence of Summed feature 4 (anteiso-C 17:1ω7c and/or iso I), low percentage of Summed feature 3 (C 16:1ω6c and/or C 16:1ω7c;) and a high percentage of anteiso-C 17:0 compared to S. aurantiacus KCTC 33633T (Table 2). As can be seen, KQ-12T showed a similar fatty acid profile to other species of the genus Salipaludibacillus. (Table 2).The isoprenoid quinone profile of KQ-12T was characterised by the predominance of MK-7 (approx. 100%), which was similar to that of S. halalkaliphilus GASy1T (Amoozegar et al. 2018). The polar lipids of KQ-12T were identified as diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine followed by an unidentified phospholipid (Fig. S4); similar profiles were also reported in the descriptions of the genus Salipaludibacillus (Sultanpuram and Mothe 2016; Amoozegar et al. 2018).

Table 2 Fatty acid composition (%) of KQ-12T and related species of the genus Salipaludibacillus
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To summarize, KQ-12T shared high 16S rRNA gene sequence similarities with respect to the type strains of the genus Salipaludibacillus, phylogenetic analysis exhibited that the isolate grouped with Salipaludibacillus species, and it should be assigned to this genus. Furthermore, the chemotaxonomic data (the major fatty acids, the predominant menaquinone, the polar lipids and the diagnostic diamino acid) support the affiliation of KQ-12T to the genus Salipaludibacillus. Also, the new isolate can be clearly distinguished from the other recognized species of the genus Salipaludibacillus based on genomic relatedness (ANI and dDDH), and morphological and physiological properties (Table 1). Accordingly, it is evident that KQ-12T should be considered to represent a novel species of the genus Salipaludibacillus, for which the name S. keqinensis sp. nov., is proposed. The Digital Protologue database (Rosselló-Móra et al. 2017) TaxoNumber for strain KQ-12T is TA00789.

Description of Salipaludibacillus keqinensis sp. nov.

Salipaludibacillus keqinensis (ke.qin.en’sis. N.L. masc. adj. keqinensis pertaining to salt lake Keqin in Heilongjiang Province, China, where the type strain was isolated).

Cells are Gram-stain positive, aerobic, motile short rods (0.7–0.9 × 1.5–2.4 μm). Colonies are circular, smooth, convex, light yellow in colour and 1.0–2.0 mm in diameter after 48 h of incubation at 25 °C. Growth is observed at 4–40 °C, pH 8.0–10.0 and with up to 16% (w/v) NaCl. Optimal growth occurs at 25 °C, pH 9.0 and in the presence of 8% (w/v) NaCl. Aesculin, casein, cellulose, DNA, gelatin, starch Tween 20 and 80 are not hydrolysed. Positive for catalase activity, nitrate reduction, and Voges-Proskauer test, but negative for oxidase, urease, citrate utilization, methyl red test, indole and H2S production. Enzyme activities are detected for esterase (C4), esterase lipase (C8), α-chymotrypsin, naphthol-AS-BI-phosphohydrolase, β-galactosidase and α-glucosidase; No activity is detected for alkaline phosphatase, lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, α-galactosidase, β-glucuronidase, β-glucosidase, N-acetul-β-glucosaminidase, α-mannosidase or α-fucosidase (API ZYM test strips). In Biolog GP2 microplates (48 h incubation), the following substrates yield positive reactions for substrate oxidation: l-arabinose, palatinose, d-psicose, d-ribose and d-xylose; the other substrates are not. Major cellular fatty acids (content ≥ 5%) are anteiso-C15:0, anteiso-C17:0, iso-C15:0 and C16:0. The predominant menaquinone is MK-7. The peptidoglycan cell wall contains meso-diaminopimelic acid. The polar lipids include diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and one unidentified phospholipid.

The type strain, KQ-12T (=  ACCC 60430T = KCTC 33935T), was isolated from Keqin lake in Heilongjiang Province, China. The GenBank/EMBL/DDJB accession number for the 16S rRNA gene sequence and the whole genome sequence of KQ-12T are MH939198 and PDOD00000000, respectively.