Introduction

The genus Halomonas was proposed by Vreeland et al. in 1980 [1] and then emended by Dobson and Franzmann in 1996 [2], it belongs to the family Halomonadaceae [3], besides from Halomonas, the family comprises of 14 different genera (https://lpsn.dsmz.de/family/halomonadaceae) [4]. At present, there are more than 113 species under genus Halomonas with validly published names (www.bacterio. net/halomonas.html) [4]. Members of this genus can grow in a wide range of saline concentrations, and they are isolated from diverse environments, such as saline and alkaline soils, saline lakes, marine and deep-sea environments, solar salterns, fermented seafood [5]. For the application of the genus Halomonas, besides the capacity to carry out denitrification [6], some of the Halomonas species show the ability to degrade aromatic compounds [7] or to produce biomolecules, hydrolytic enzymes [8], exopolysaccharide [9,10,11,12] with high industrial interest [13,14,15], that makes them a group with high biotechnological potential. During a microbial resource survey of natural saline–alkali wetland soil of Binhai new district, we found a strain which can live in the ultra-high salinity habitat. The present study determined the taxonomic status of the isolated species using polyphasic approaches. The results revealed that strain WN018T represents a novel species of the genus Halomonas.

Materials and Methods

Selective Isolation, Strains and Cultural Conditions

The strain WN018T was collected from the natural saline–alkali wetland soil of Binhai new district, Tianjin, China (38°46′N, 117°13′E) in June 2015, and then were transferred to the laboratory with ice. The in situ temperature, salinity and pH of the samples were measured as 30 °C, 4.0%-13.5% and 7.8–9.3. To isolate halophilic heterotrophic microorganisms, 1.0 g of soil was placed in sterile 30-ml glass tube for enrichment using Difco™ marine 2216 [16] with containing a final concentration of 10.0% NaCl for 3 days, and colonies were selected according to appearance characteristics. A single colony was purified and identified by 16S rRNA gene sequencing. Finally subsequently purified into single colonies. The isolates were maintained on MA medium slants and preserved at −80 °C as suspensions in marine broth 2216 (MB, Difco) containing 25.0% (v/v) glycerol. Halomonas alkaliphila DSM 16354T, Halomonas lutescens KCTC 42517T, Halomonas hydrothermalis DSM 15725T and Halomonas venusta DSM 4743T were incubated at 30 °C on marine broth 2216E agar, and used as reference strain for physiological tests and analysis of morphological, phylogenetic and chemotaxonomic characteristics.

Molecular Characterization

Genomic DNA was extracted using a commercial kit (TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver. 3.0) and subsequently sequenced on the Illumina MiSeq 2000 platform in Shanghai Personal Biotechnology Co., Ltd. China. The 16S rRNA gene was amplified from genomic DNA using the universal bacterial primer set 27F (5-AGAGTTTGATCMTGGCTCAG-3) and 1492R (5-TACGGYTACCTTGTTACGACTT-3) [17, 18]. The following cycling conditions were used: 95 °C for 7 min; followed by 35 cycles of 95 °C for 0.5 min, 55 °C for 0.5 min, and 72 °C for 1.5 min; and a final extension of 72 °C for 7 min. The PCR product was purified using a PCR purification kit (MinElute PCR Purification Kit, QIAGEN) and sequenced by Sangon Biotech (Shanghai) Co., Ltd., China. Filtering and trimming of the genomic raw data was done with PRINSEQ v0.20.4. The 16S rRNA sequence was submitted to GenBank and compared with available 16S rRNA sequences of validly described species from the GenBank database (http://www.ncbi.nlm.nih.gov/). Phylogenetic trees were constructed by the neighbor-joining method [19], maximum-parsimony method [20], and maximum-likelihood method [21] in MEGA7 [22]. The resultant tree topologies were evaluated by bootstrap analyses (1000 replications) [23]. Filtering and trimming of the genomic raw data was done with PRINSEQ v0.20.4, and the trimmed reads were assembled using SOAPdenovo v1.05 with default parameters. The genome completeness (100%) was assessed using CheckM (version 1.03). Protein-coding open reading frames were predicted by Glimmer (version 3.02). For RNA prediction, rRNAs were predicted by RNAmmer (version 1.2), and tRNAs were predicted by tRNAscan-SE (version 1.21). DNA–DNA hybridization (dDDH) values were calculated at the Genome-to-Genome Distance Calculator (GGDC) website using formula 2, as originally described by Auch et al. [24] and updated by Meier-Kolthoffet et al. [25] Average nucleotide identity (ANI) values between the strain WN018T genome and closely related genomic sequences from GenBank were also determined according to Goris et al. [26]. The whole-genome sequences in a pairwise comparison were split into consecutive 1000 bp windows, then sequences were aligned with nucmer in MUMmer version 3.23 and ANI values were calculated using JSpecies version 1.2.1.

Phenotypic and Physiological Tests

Phenotypic and physiological tests were carried out the following recommendations of the proposed minimal standards for describing new taxa of the family Halomonadaceae [27]. Gram staining was performed using BD Gram staining kits, according to the manufacturer’s instructions. Cell motility was determined using semi-MA medium (0.5% agar, w/v). Cell morphology was also assessed by transmission electron microscopy (TEM), i.e., cells were harvested from exponentially growing culture, and the cells were negatively stained with 0.5% uranyl acetate and the grids were examined at the microscope (Tecnai Spirit, FEI, Hillsboro, OR, USA).Oxidase activity was tested using the oxidase reagent kit (bioMérieux) according to the manufacturer’s instructions. Catalase activity was determined by pouring a 3.0% H2O2 solution onto bacterial colonies and observing bubble production. The optimal growth temperature of strain WN018T was determined after incubation on Difco™ marine 2216E agar (8.0% NaCl) and shaking in Difco™ marine 2216E liquid medium (8.0% NaCl) at 4, 10, 15, 20, 25, 30, 33, 37, 40, 45, and 50 °C (at pH 7.5). NaCl tolerance was tested on Luria–Bertani (LB) agar and in LB liquid medium amended with 0.0–25.0% NaCl (w/v). The pH range for growth was measured by adjusting the final pH to 5.0, 5.5, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 (at 8.0% NaCl, 33 °C) with the appropriate buffers (Na2HPO4/NaH2PO4 for pH 5.0–7.0 and Na2CO3/NaHCO3 for pH 8.0–12.0). Carbon source utilization and enzyme activities were tested using the API 20NE, API ZYM (bioMérieux), and GEN III Microplate systems (Biolog Inc.), according to the manufacturers’ instructions. Antimicrobial susceptibilities were determined on marine agar plates with antimicrobial compound disks for 1 week at 30 °C [28].

Chemotaxonomic Analysis

Chemotaxonomic analyses were performed on strain WN018T as well as on the 4 reference strains mentioned above. For the fatty acid analysis, the strains were grown on MA medium at 30 °C, OD600nm = 3.0. The fatty acids were identified and quantified by theSherlock Microbial Identification System with standard MIS Library Generation Software (VERSION 6.0 and Date 4, Microbial ID Inc., Newark, DE, USA) and a 6890N gas chromatograph (Agilent)according to the method of Sasser et al. [29]. Polar lipids were extracted from 200 mg of freeze-dried cell material using Komagata et al. method [30]. The respiratory quinones and cellular fatty acids were determined by the identification service of the China Center of Agricultural Culture Collection in Beijing, China, and the polar lipid analysis was performed was performed by the Identification Service of the DSMZ, Braunschweig, Germany. Quinone components were separated and identified by reversed-phase HPLC and photodiode array detection with internal and external quinone standards [31]. The AntiSMASH was used to predict the biosynthetic gene clusters of strain WN018T.

Results and Discussion

Morphological, Physiological and Biochemical Characteristics

Strain WN018T was found to be Gram staining-negative, halophilic, aerobic, non-motile, short rod-shaped. Colonies were observed to be circular, wet, convex and cream-colored after growth on MA at 28 °C for 4 days, and strain WN018T grew with 0.5–25.0% NaCl (optimum growth at 4.0–8.0% NaCl), at 15–45 °C (optimum 30–33 °C), and pH 5.0–12.0 (optimum pH 7.5–8.0).

Some physiological and biochemical characteristics of strain WN018T and the differential phenotypic features from the reference strains was described in Table 1 and Table S1. Strain WN018T was sensitive to chloramphenicol (30 μg), erythromycin (15 μg), streptomycin (10 μg), tetracycline (30 μg), gentamicin (10 μg) and polymyxin B (30 μg), but resistant to ampicillin (10 μg), kanamycin (30 μg), penicillin G (10 μg) and vancomycin (30 μg).

Table 1 Differential phenotypic characteristics that distinguish strain WN018T from other related species of the genus Halomonas
Full size table

Chemotaxonomic Characteristics

The major respiratory quinones of strain WN018T were Q-9 (91%) and Q-8 (9%). The major polar lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), three phospholipids (PL), aminolipid (AL), and two unidentified lipids (L) (Table 1). The dominant fatty acids were Sum in Feature 8 (57.76%), Sum in Feature 3 (11.08%), C10:0 (1.95%), C12:0 3-OH (5.34%), C14:0 (3.18%) and C16:0 (16.26%) (Table 2).

Table 2 Cellular fatty acid compositions (%) of strain WN018T and related species of the genus Halomonas
Full size table

Molecular Characteristics

The genome of strain WN018T (accession number NSKD00000000) is 4,054,120 bp in size, and the G+C content is 63.8%. Comparison of the 16S rRNA sequence of strain WN018T with those available from public databases demonstrated that strain WN018T belongs to the genus Halomonas. At the same time, it is also proven in the maximum-likelihood method (ML), and Neighbor-joining (NJ) & maximum-parsimony (MP) phylogenetic trees based on the 16S rRNA gene sequence. Strain WN018T was most closely related to Halomonas venusta DSM 4743T (Fig. 1), with high sequence similarity to Halomonas alkaliphila DSM 19434T(97.5%), Halomonas venusta DSM 4743T(97.5%), Halomonas hydrothermalis DSM 15725T(97.45%), and Halomonas lutescens KCTC 42517T (96.87%). The levels of 16S rRNA gene sequence similarity between strain WN018T and other type strains of the genus Halomonas were below 98.0%, suggested strain WN018T maybe represents a novel species distinct from all other members of the genus Halomonas [32].

Fig. 1
figure 1

Maximum-likelihood (ML) phylogenetic tree based on 16S rRNA gene sequences showing the relationships between strain WN018T and related taxa. Bootstrap values > 50% (1000 resamplings) are shown. Scale bar, 0.01 substitutions per nucleotide position. Zymobacter palmae T109 T was used as an outgroup. GenBank accession numbers are indicated for each strain

Full size image

According to the ANI analysis of the genome of strain WN018T and closely related genomes from GenBank, the highest average nucleotide identity (ANI) value was 93.02% with Halomonas hydrothermalis DSM 15725T (Supplementary Table S2). This value is lower than the widely accepted threshold range (95–96%) for species demarcation suggested by Kim [33] and Richter [34].

In addition, we calculated DNA–DNA hybridization (dDDH) tests between the new isolate WN018T and Halomonas hydrothermalis DSM 15725 T, Halomonas venusta DSM 4743T, Halomonas andesensis DSM 19434T, and Halomonas nigrificans DSM 105749T, and the values were 49.7%, 49.3%, 31.2% and 22.0%, respectively, which is well below the threshold of 70%, the threshold generally accepted for species delineation [35], the dDDH value between these two strains was determined to be 49.7% (Supplementary Table S2), which added evidence that strain WN018T represents a novel species of the genus Halomonas. The antiSMASH biosynthetic gene cluster prediction tool was used to investigate the genome sequence of strain WN081T and one crochelin, one ectoine, one mycosubtilin was detected. It is a compatible solute which serves as a protective substance to help organisms survive in extreme osmotic stress. Most halophilic and halotolerant microorganisms adapt to high environmental salinity by accumulation of ectoine. So we speculate that the ectoine gene clusters is an important reason why the strain WN018T can live in extreme environments with high salt content. In summary, the sequencing of the genome of strain WN018T further clarified the evolutionary relationship between strains and will guide the screening for active secondary metabolites.

Taxonomic Conclusion

On the basis of the phylogenetic and genomic evidence, DNA–DNA hybridization values (dDDH), fatty acid profiles, quinones and differences in phenotypic characteristics, the strain WN018T represent a novel species of the genus Halomonas, for which the name Halomonas humidisoli sp. nov. is proposed.

Description of Halomonas humidisoli Sp. Nov.

Halomonas humidisoli (hu.mi.di.soil. L. masc. adj. humidus humid; L. neut.n. solum soil; N.L. gen. n. humidisoli of wet soil).

Cells are Gram staining-negative, halophilic, aerobic, non-motile and short rod-shaped (0.3–0.4 µm wide, 0.5–1.9 µm long). Colonies were observed to be circular, wet, convex and cream-colored after growth on MA at 28 °C for 4 days. Cells grew with 0.5–25.0% NaCl (optimum growth at 4.0–8.0% NaCl), at 15–45 °C (optimum 30–33 °C), and pH 5.0–12.0 (optimum pH 7.5–8.0). Nitrate reduction, catalase and oxidase-positive are also positive. Positive for the oxidation of dextrin, d-maltose, d-trehalose, d-cellobiose, sucrose,d-turanose, n-acetyl-d-glucosamine, n-acetyl-β-d-mannosamine, d-fructose, d-fucose, l-fucose, l-rhamnose, inosine, d-serine, glycerol, d-fructose-6-po4, d-aspartic acid, d-serine, l-alanine, l-arginine, l-aspartic acid, l-glutamic acid, l-histidine, l-pyroglutamic acid, l-serine, l-galactonic acid lactone, d-gluconic acid, d-glucuronic acid, d-lactic acid methyl ester,l -lactic acid, citric acid, α-keto-glutaric acid, l-malic acid, bromo-succinic acid, β-hydroxy-d,l-butyric acid, α-keto-butyric acid, acetoacetic acid, propionic acid, acetic acid and pectin. Cells were sensitive to chloramphenicol, erythromycin, streptomycin, tetracycline, gentamicin and polymyxin B, but resistant to ampicillin, kanamycin, penicillin G and vancomycin. The major respiratory quinone of cells are Q-9 (91.0%) and Q-8 (9.0%). The major polar lipids are diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), three phospholipids (PL), aminolipid (AL), and two unidentified lipids (L). The dominant fatty acids are Sum In Feature 8 (57.76%), Sum In Feature 3 (11.08%), C10:0 (1.95%), C12:0 3-OH (5.34%), C14:0 (3.18%) C16:0 (16.26%). The genomic DNA is a single circular chromosome (5,475,884-bp) with a G+C content of 63.8%.

The type strain, WN018T (= ACCC19975T = KCTC52854T), was isolated from the natural saline–alkali wetland soil of Binhai new district, Tianjin, China (38°46′N, 117°13′E). The GenBank accession number for genomic sequence of strain WN018T is NSKA00000000.