Introduction

The genus Oceanobacillus belongs to the family Bacillaceae within the phylum Bacillota, and it is a large taxonomic entity that was firstly described by Lu et al. [1] with Oceanobacillus iheyensis as the type species. The members of the genus Oceanobacillus are widely distributed in various habitats, such as seawater [2], chironomid egg mass [3], saline−alkali soil [4], salt lakes [5], fermented polygonum indigo [6], the skin of rainbow trout [7] and other environments. All strains of this genus are Gram-staining positive, rod-shaped, motile bacteria and extremely halotolerant or halophilic. At the time of writing, the genus comprised 28 species with validly published names (https://lpsn.dsmz.de/genus/oceanobacillus). The saline−alkaline soils located in Heilongjiang Province of China are the representatives of naturally occurring salt and alkali environment. Isolating the pure cultures of strains obtained from saline alkaline soils can help us better understand the ‘microbial dark matter’ [8,9,10]. The species isolated from saline alkaline habitats have been studied in depth because of their ecological significance and potential applications for the biotechnological and industrial purposes [11,12,13]. During a study on the cultivable microbial diversity of saline−alkaline soils in Heilongjiang, a novel strain designated as APA_H-1(4)T was isolated and has now been shown to represent a novel species of the genus Oceanobacillus. The present study was conducted to establish the taxonomic position of strain APA_H-1(4)T.

Methods and Materials

Bacterial Isolation and Cell Growth

Sample was collected from saline−alkaline soil, located in Zhaodong (46°01′44.1″N, 125°50′05.9″E), China. Strain APA_H-1(4)T was isolated using the standard dilution plate method on APA medium [14]. After incubating for one week at 37 °C, colonies were picked and re-streaked several times to obtain axenic cultures, and then stored as glycerol suspensions (20%, w/v) concentration at −80 °C for further use. Biomass for chemical and molecular studies was obtained by cultivation on APA medium at 37 °C for 3–10 days. O. damuensis KCTC 33146T [4] and ‘O. massiliensis’ DSM 24644 [15] were used as reference strains, and they were cultured under the optimum conditions as appropriate for specific comparative tests.

Molecular Characterization

The extraction of genomic DNA was carried out according to our previously standardized protocol [16]. 16S rRNA gene was amplified using the bacterial universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGYTACCTTGTTACGACTT-3′). The PCR amplicon was sequenced by Sangon Biotech (Guangzhou, China). The obtained 16S rRNA gene sequence was compared with available sequences of cultured species at EzBioCloud server [17]. The 16S rRNA gene sequences of strain APA_H-1(4)T and related type strains were aligned using ClustalW [18]. Phylogenetic trees were constructed on MEGA version X [19] using neighbor-joining [20], maximum-likelihood [21] and maximum-parsimony [22] methods. The stability of relationships was assessed by performing bootstrap analyses with 1000 replications [23]. The genome of the strain APA_H-1(4)T was sequenced using the Illumina HiSeq X platform at Majorbio (Shanghai, China). The paired-end reads were assembled using SOAPdenovo (v2.04). The completeness and contamination of strain APA_H-1(4)T genome were calculated using CheckM [24]. Average nucleotide identity (ANI) among the genomes of the genus Oceanobacillus was calculated using the pyANI with blast method [25]. The digital DNA–DNA hybridization (dDDH) analysis was performed using the DSMZ Genome-to-Genome Distance Calculator platform (http://ggdc.dsmz.de/distcalc2.php) [26]. Phylogenomic tree was constructed according to the methods of [8, 9]. The DNA G + C content was determined from the genomic sequences. Genome sequences were annotated using the KEGG databases and Prokka [27, 28].

Physiology and Morphology

The morphological properties of the strain APA_H-1(4)T were observed by transmission electron microscopy (JEM-1400FLASH; JEOL). The presence of endospores was investigated using the Schaeffer−Fulton staining method [29]. Cell motility was tested by the development of turbidity in a tube containing semi-solid APA medium. The Gram-staining reaction was performed by the Burke method [29] and the results were confirmed by the KOH test [30]. The growth was tested at temperatures ranging from 4 to 65 °C (4, 15, 20, 29, 37, 45, 55, 65 °C) on APA medium by incubating the cultures for 10 days. The ability of the strain to grow at different pH values (6.0–13.0, at intervals of 1.0 pH unit using the buffer system described by [31] and NaCl concentrations (0–20%, w/v) was examined on APA medium at 37 °C for 10 days. Catalase activity was detected by the formation of bubbles on the addition of a drop of 3% (v/v) H2O2, while oxidase activity was determined by observing color shift with oxidase reagent (bioMerieux, SA) according to the manufacturer’s instructions.

Hydrolysis of cellulose, starch and Tweens (20, 40, 60 and 80), milk peptonization and coagulation, H2S production, methyl red and Voges−Proskauer tests were performed as described by Smibert and Krieg [32]. The determination of other enzyme activities and biochemical characteristics were used API ZYM and API 20NE systems (bioMérieux) and substrate utilization was tested using Biolog GEN III Micro plate according to the manufacturer’s instructions. Antibiotic susceptibility tests were performed on APA medium containing 2% (w/v) NaCl using discs impregnated with various antimicrobial compounds.

Biochemical Characteristics

Biomass for chemical characteristics was obtained by cultivation on APA medium containing 2% NaCl at 37 °C. Analysis of amino acids of whole-cell hydrolysate was performed according to the procedures described by Lechevalier and Lechevalier [33] and Hasegawa et al. [34]. Polar lipids were extracted as described by Minnikin et al. [35] and examined by two-dimensional TLC on 10 × 10 cm silica gel G60 plates (Merck). The polar lipid profile was identified using the described procedures [36, 37]. Menaquinones were isolated according to Collins et al. [38] and separated by HPLC [39]. The biomass for fatty acid analysis was harvested when the population quantity was half of maximum value. For fatty acid analysis, strain APA_H-1(4)T was cultured on APA medium containing 2% NaCl at 37 °C for 3 days. Cellular fatty acid methyl ester (FAME) profiles were determined by GC (7890B; Agilent) according to the standard protocol of the Microbial Identification System (Sherlock Version 6.2; MIDI database: TSBA6).

Results and Discussion

Molecular Characteristics

An almost complete 16S rRNA gene sequence (1591 bp) of strain APA_H-1(4)T was obtained. The GenBank accession number for the 16S rRNA gene of strain APA_H-1(4)T was ON077165. Analyses of the 16S rRNA gene sequence of strain APA_H-1(4)T using the EzBioCloud server showed that they were moderately related to O. damuensis KCTC 33146T (98.35%, similarity) and ‘O. massiliensis’ DSM 24644 (98.32%). 16S rRNA gene-based phylogeny using the neighbor-joining method (Fig. 1 and Fig. S3) showed strain APA_H-1(4)T form a clade with O. damuensis KCTC 33146T, ‘O. massiliensis’ DSM 24644 and O. endoradicis py1294T. The stabilities of trees were further confirmed by maximum-likelihood and maximum-parsimony methods (Fig. S1 and Fig. S2). The affiliation of the strain APA_H-1(4)T to the genus Oceanobacillus was also supported by the phylogenomic tree (Fig. 2) based on the concatenated alignment of 120 marker genes. The completeness and contamination of the genome of strain APA_H-1(4)T were 100.00 and 1.61%, respectively. The genome had a total of 4082 genes, including 3985 protein-coding genes, 13 rRNA genes and 84 tRNA genes. The most abundant KEGG function pathway in strain APA_H-1(4)T was carbohydrate metabolism, followed by overview and amino acid metabolism (Table S2). Amino acids (e.g., glutamine, glutamate and proline) are widely distributed compatible solutes in prokaryotes [40]. The accumulation of compatible solutes can be beneficial for halophilic microorganisms to overcome osmotic pressure in high salt environment [41]. According to the annotation results of the KEGG automatic annotation server (KAAS), strain APA_H-1(4)T contains glutamine biosynthesis genes (glnA and GLUL) and proline biosynthesis genes (proB and proC), which can enhance the ability of cells to withstand high osmotic pressure. The genomic DNA G + C content of strain APA_H-1(4)T was 38.44%. The ANI values between strain APA_H-1(4)T and O. damuensis KCTC 33146T (GenBank accession no. GCA_001618145.1) and ‘O. massiliensis’ DSM 24644 (GenBank accession no. GCA_000285495.1) were 81.82% and 76.43%, respectively. The dDDH values of strain APA_H-1(4)T with O. damuensis KCTC 33146 T and ‘O. massiliensis’ DSM 24644 were 13.60 and 17.60%, respectively. These data supported the finding that strain APA_H-1(4)T represents a different genomic species of the genus Oceanobacillus. O. damuensis KCTC 33146T, ‘O. massiliensis’ DSM 24644, were used as reference strains.

Fig. 1
figure 1

Neighbor-joining phylogenetic tree based on the 16S rRNA gene sequences of strain APA_H-1(4)T and its closest relatives. Effusibacillus lacus DSM 27172T was selected as the outgroup. Bootstrap values are shown at the branch points. A number at nodes are bootstrap percentages based on the 1000 replications, only values > 50% are shown at branch points. Asterisks denote topologies that were also recovered in trees generated with the maximum-likelihood and maximum-parsimony methods. Bar, 0.02 substitutions per nucleotide position

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Fig. 2
figure 2

The phylogenomic tree based on the 120 marker genes showing the relationship of strain APA_H-1(4)T with representative members of the genus Oceanobacillus. Effusibacillus lacus DSM 27172T (SLZX00000000) was selected as the outgroup. Bootstrap values are shown at the branch points. Bar, 0.1 substitutions per nucleotide position

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Physiology and Morphology

Strain APA_H-1(4)T was Gram-staining positive, motile, aerobic and can produced oval terminal endospores. Cells were rods with a width of 0.5–0.7 μm and a length of 1.8–2.6 μm (Fig. S4). The growth of strain APA_H-1(4)T was observed in a wide range of temperature 4–55 °C with optimal growth at 37 °C. The pH range for growth was pH 7.0–10.0 with an optimum pH (8.0–9.0). The NaCl tolerance was up to 15.0% (w/v) with optimal growth at 2–10% NaCl (w/v). The strain APA_H-1(4)T was positive for the production of catalase, while strain APA_H-1(4)T was negative for oxidase, nitrate reduction, milk peptonization and coagulation, methyl red and Voges−Proskauer test. H2S was not produced. Tweens (20, 40, 60 and 80) and cellulose were hydrolyzed, but gelatin and starch were not. Differential characteristics of strain APA_H-1(4)T and the closely related type strains are listed in Table 1. The detailed physiological characteristics of the strain are given in the species description and all negative traits of strain APA_H-1(4)T observed with commercial kits, including API ZYM, API 20 NE and Biolog Gen III were listed in Table S3.

Table 1 Different characteristics of strain APA_H-1(4)T and its reference strains
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Biochemical Characteristics

Cell wall amino acids of strain APA_H-1(4)T contained meso-diaminopimelic acid as the diagnostic diamino acid. The phospholipids consisted of diphosphatidylglycerol, phosphatidylglycerol, an unidentified phospholipid and an unidentified polar lipid (Fig. S5). The respiratory quinone was identified as menaquinone-7 (MK-7), and the cellular fatty acid profile contained anteiso-C15:0 (66.02%) as the major fatty acid, anteiso-C17:0 (9.91%) iso-C16:0 (6.31%), iso-C14:0 (5.60%), C16:0 (5.14%) and iso-C15:0 (2.15%) as minor fatty acids.

Conclusion

Based on the phenotypic, genotypic, phylogenetic and chemotaxonomic data, strain APA_H-1(4)T belongs to the genus Oceanobacillus. Furthermore, the analyses of 16S rRNA gene sequences, ANI values, dDDH values and other properties, notably, enzyme activities, the sensitivity to antibiotics and utilization of carbon sources and nitrogen sources, indicate that strain APA_H-1(4)T could be distinguished from the species of O. damuensis KCTC 33146T and ‘O. massiliensis’ DSM 24644. Based on the data described above, strain APA_H-1(4)T represents a novel species of the genus Oceanobacillus, for which the name Oceanobacillus saliphilus sp. nov. is proposed.

Description of Oceanobacillus saliphilus sp.nov.

Oceanobacillus saliphilus (sa.li’phi.lus. L. masc. n. sal, salt; Gr. masc. adj. philos, loving; N.L. masc. adj. saliphilus, loving salt).

Gram-staining positive, motile, aerobic, spore-forming rods (0.5–0.7 × 1.8–2.6 μm) with flagella. Endospores are ellipsoid and terminally positioned. Colonies are circular, smooth, and creamy yellow. Growth occurs at 4–55 °C (optimum 37 °C), at pH 7.0–10.0 (optimum pH 8.0–9.0) and in the presence of 0–15% (w/v) NaCl (optimum 2–10% NaCl). Positive for the production of catalase. Negative for the oxidase, nitrate reduction, H2S production, milk peptonization and coagulation, methyl red and Voges−Proskauer test. Tweens (20, 40, 60 and 80) and cellulose are hydrolyzed, but gelatin, starch are not. In the GEN III Microplate (Biolog) system, the following substrates can be utilized: acetoacetic acid, acetic acid, formic acid, glucuron-amide, 3-methyl-glucose, D-mannose, D-fructose, D-galactose, D-fucose, D-galacturonic acid, D-glucuronic acid, L-fucose and L-rhamnose, α-D-glucose. According to the API ZYM, cystine arylamidase, esterase (C4), leucine arylamidase, naphthol-AS-BI-phosphohydrolase, valine arylamidase, α-chymotrypsin, α-glucosidase and β-glucuronidase are positive. In the API 20NE test, positive for glucosidase and galactosidase. Sensitive to amikacin, ampicillin, bacitracin, chloramphenicol, erythromycin, furantoin, kanamycin, norfloxacin, rifampicin, sulfamethoxazole, streptomycin, tetracycline, tobramycin. The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, an unidentified phospholipid and an unidentified polar lipid. The major fatty acid (>10%) is anteiso-C15:0. The predominant menaquinone is MK-7. The whole-cell hydrolysates contain meso-diaminopimelic acid as the diagnostic diamino acid. The genomic DNA G + C content is 38.44%.

The type strain is APA_H-1(4)T(=GDMCC 1.2239T = KCTC 43254T), isolated from saline−alkaline surface soil (0–10 cm), Zhaodong, Heilongjiang Province, China.

The GenBank accession numbers for 16S rRNA gene sequence and draft genome sequence of the strain APA_H-1(4)T are ON077165 and JALCZU000000000, respectively.