Introduction

Halomonas is the type genus of the family of Halomonadaceae. Currently the genus includes more than 90 species with validly published names (https://lpsn.dsmz.de/genus/halomonas). A prominent feature of the genus Halomonas is halophily and/or halotolerance and the species of Halomonas typically occur in saline or hypersaline environments. From the variety of habitats and phenotypic heterogeneity, members of Halomonas may be regarded as ubiquitous, versatile chemoheterotrophs [1]. They are important candidates to make significant contributions to a wide array of biotechnological applications [2, 3]. In this study we determine the taxonomic position of a novel Halomonas isolate DX6T.

Materials and Methods

Strains and Culture Conditions

The Gobi soil sample was collected from Bachu County, which locates on the northwest of the Taklimakan Desert, China (39°53΄10˝N, 78°27΄8˝E). The soil sample was suspended in 0.9% physiological saline solution, serially diluted and spread onto Luria-Bertani (LB) agar. After 2–3 days of incubation at 30 °C, individual colonies of distinct morphology were picked and purified. The isolates were routinely cultured on LB agar aerobically at 30 °C and maintained as glycerol suspensions (15%, v/v) at −80 °C. The 16S rRNA genes of all the isolates were sequenced and analyzed (as described below) and a potential novel species closely related to Halomonas desiderata was further identified in this study. H. desiderata DSM 9502T (= FB2T) was obtained from the Leibniz Institute DSMZ and used as a reference strain.

Phylogenetic Analysis

PCR amplification of the 16S rRNA gene was performed using a bacterial universal primer set (27F and 1540R) [4]. The 16S rRNA gene was sequenced using the Sanger dideoxy sequencing method by TsingKe Biological Technology Company, Beijing, China. The isolate was identified using the EzBiocloud server on the basis of 16S rRNA sequence data [5]. Additionally, the GenBank database was used to search for gyrB and rpoD gene sequences in type species from the Halomonas genus and the sequence data of the two genes were download. Phylogenetic trees were constructed using the maximum composite likelihood model and the neighbor-joining statistical method (1000 bootstrap replicates) in the MEGA X program [6].

Genomic Analysis

Genomic DNA from strain DX6T was extracted using TIANamp Bacteria DNA Kit from TIANGEN Biotech (Beijing) Co., Ltd. Genome sequencing of strain DX6T and H. desiderata DSM 9502T was performed using the Illumina HiSeq platform with paired-end reads of 2 × 150 bp by Personal Biotechnology Co., Ltd., Shanghai (China). Average nucleotide identity (ANI) values were calculated using Kostas Konstantinidis lab’s online ANI calculator (http://enve-omics.ce.gatech.edu/ani/) [7]. DNA–DNA hybridization (DDH) experiments were carried out using the method described by De Ley et al. [8] with a Beckman DU 800 spectrophotometer. The digital DNA-DNA hybridization (dDDH) values between strain DX6T and its closest type strains were determined using Genome-to-Genome Distance Calculator (GGDC) version 2.1 (https://ggdc.dsmz.de/ggdc.php#) and formula 2 was applied [9].

Phenotypic Analysis

Strain DX6T was grown on LB agar medium at 37 °C for 12 h and cell morphology was examined and observed by transmission electron microscopy (JEM-1400; Jeol USA Inc., Peabody, MA) after uranyl acetate staining. Gram-staining was carried out by using the standard Gram reaction [10]. The growth of strain DX6T at various NaCl concentrations was also investigated in LB medium supplemented with 0–20% of NaCl and incubated for 3–5 days. The temperature range for growth was determined in LB medium by incubating at 4, 10, 20, 25, 30, 35, 37, 43, 45, and 50 °C for 5 days. pH tolerance (pH 4.0–12.0, at intervals of 1 pH unit) was determined in LB medium pre-adjusted to various pH values by the addition of HCl or NaOH. Carbon source utilization was tested in a medium containing 2.5 g L−1 (NH4)2SO4, 0.5 g L−1 NaH2PO4·2H2O, 0.5 g L−1 K2HPO4, 0.5 g L−1 MgSO4·7H2O, 0.1 g L−1 CaCl2·2H2O and 1.0 g L−1 carbohydrate. Other physiological and biochemical properties were performed using the HBI Microbial Biochemical Identification Tubes from Hopebio-Technology Co., Ltd., Qingdao (China) according to the manufacturer’s instructions. Antimicrobial susceptibility testing was performed according to the conventional Kirby–Bauer method [11].

Chemotaxonomic Analysis

Respiratory quinones were extracted and analyzed using reversed-phase HPLC (LC20AD system, Shimadzu) according to a previously described method [12]. Cellular fatty acid methyl esters were extracted, separated and identified according to the instructions of the Microbial Identification System (MIDI, Microbial ID). Polar lipids were extracted, separated using two-dimensional TLC and identified according to published procedures [13].

Results and Discussion

Phylogenetic Analysis

Comparative analysis of 16S rRNA gene sequence indicated that strain DX6T (GenBank accession No. MT180568) was closely related to H. desiderata DSM 9502T (X92417, 98.3%), H. kenyensis AIR-2T (AY962237, 97.7%), H. daqingensis DQD2-30T (EF121854, 97.6%), H. saliphila LCB169T (KX008964, 97.4%) and H. endophytica MC28T (MF850257, 96.2%). As shown in Fig. 1, strain DX6T formed a distinct lineage in the clade of the genus Halomonas. Strain DX6T showed 82.0–86.5% gyrB and 82.6–86.6% rpoD gene sequence similarity to the above five most closely related type strains of the Halomonas species. A neighbor-joining tree based on a concatenated alignment of 16S rRNA, gyrB and rpoD gene sequences was also reconstructed (Fig. 2) and the phylogenetic analysis also revealed that strain DX6T clustered within the genus Halomonas and formed a distinct lineage.

Fig. 1
figure 1

Neighbor-joining tree based on 16S rRNA gene sequences showing the phylogenetic position of strain DX6T with respect to species of the genus Halomonas. Accession numbers are given in parentheses. Numbers at branching points represent bootstrap values (%) from 1000 replicates; only values >50% are shown. Bar, 0.01 substitutions per nucleotide position

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Fig. 2
figure 2

Neighbor-joining tree based on concatenated 16S rRNA, gyrB and rpoD gene sequences, showing the position of DX6T among members of the genus Halomonas. Numbers at branching points represent bootstrap values from 1000 replications; only values 50% are shown. Accession numbers are given in parentheses. Bar, 0.02 substitutions per nucleotide position

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Genomic Analysis

Draft genome sequencing of strain DX6T (GenBank accession No. JAAQTO000000000) yielded a genome of 4,701,666 bp in length with 80 contigs (all >1049 bp, N50 was 106,214 bp). 4289 protein coding genes, 3 complete rRNA genes, 51 tRNA genes, and 4 ncRNAs genes were predicted. The 16S rRNA gene sequence extracted from the genome shared 99.9% similarity with the 16S rRNA gene sequenced by the Sanger dideoxy sequencing method. In contrast, the draft genome size of H. desiderata DSM 9502T (GenBank accession No. JAAQTN000000000) was 4,886,954 bp with 106 contigs (all >1082 bp, N50 was 96,043 bp). 4405 protein coding genes, 3 complete rRNA genes, 54 tRNA genes, and 5 ncRNAs genes were predicted. The read size or the depth coverage of strain DX6T and H. desiderata DSM 9502T was 284x and 334x, respectively. The DDH value between DX6T and H. desiderata DSM 9502T was 35.4%. The ANI and the dDDH results between strain DX6T and the five closely related Halomonas species (Table 1) were far below the 95% (ANI) and 70% (dDDH) threshold values suggested for the description of bacterial species [14, 15], indicating that strain DX6T belongs to a novel species of the genus Halomonas.

Table 1 The dDDH and ANI values between strain DX6T and its closest type species of the genus Halomonas
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Phenotypic Characterization

Strain DX6T was Gram-stain-negative and cells were short rods (0.4–0.5 × 0.8–1.5 μm). Colonies were creamy, circular, smooth and 1–2 mm in diameter after 2 days of incubation at 30 °C on LB agar. Growth of strain DX6T was observed at 10–45 °C (optimum 37 °C), pH 5.0–10.0 (optimum pH 7.0–9.0) and 1–15% (w/v) NaCl (optimum 2–10%). Antimicrobial susceptibility testing results showed that strain DX6T was susceptible to (amounts per disc) gentamicin (10 μg), kanamycin (30 μg), rifampicin (5 μg), tetracycline (30 μg) and chloramphenicol (30 μg), resistant to carbenicillin (100 μg), penicillin (6 μg) and ampicillin (10 μg). Other detailed physiological and biochemical characteristics are summarized in the species description and a comparison of DX6T and related type strains is given in Table 2.

Table 2 Characteristics of strain DX6T and the related Halomonas species. Strains: 1, DX6T; 2, H. desiderata DSM 9502T; 3, H. daqingensis DQD2-30T [16]; 4, H. saliphila LCB169T [17]; 5, H. endophytica MC28T [3]; 6, H. kenyensis AIR-2T [18]. Data for strain DX6T and H. desiderata DSM 9502T are from the present study. +, positive; −, negative; NA, no data available
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Chemotaxonomic Characterization

Results of chemotaxonomic analyses revealed that Q-9 (78.5%) and Q-8 (21.5%) were the predominant respiratory quinones in strain DX6T, which was consistent with the ubiquinone systems of members of the genus Halomonas. As shown in Table 3, strain DX6T differed from related bacteria in the composition of cellular fatty acids. The major cellular fatty acids of strain DX6T were summed feature 8 (C18:1ω7c and/or C18:1ω6c), summed feature 3 (C16:1ω7c and/or C16:1ω6c) and C16:0. The predominant polar lipids found in cells of strain DX6T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids, an unidentified phosphatidylglycolipid, and four unidentified lipids.

Table 3 Cellular fatty acids compositions of strain DX6T and other closely related species of the genus Halomonas. Strains: 1, DX6T; 2, H. desiderata DSM 9502T; 3, H. saliphila LCB169T; 4, H. daqingensis DQD2-30T; 5, H. kenyensis AIR-2T; H. endophytica MC28T. Values shown are percentages of total fatty acids; −, Percentage of fatty acids was less than 1.0% or not detected. Fatty acids representing to 10% or more of the total fatty acids are in bold
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Taxonomic Conclusion

Based on the distinct phylogenetic, phenotypic, biochemical, and chemotaxonomic characteristics, strain DX6T represents a novel species within the genus Halomonas, for which the name Halomonas bachuensis sp. nov. is proposed.

Description of Halomonas bachuensis sp. nov.

Halomonas bachuensis (ba.chu.en’sis. N.L. fem. Adj. bachuensis pertaining to Bachu, north-western China, where the strain was isolated).

Cells are Gram-stain-negative, aerobic, rod-shaped, 0.4–0.5 μm wide and 0.8–1.5 μm long. Colonies are creamy, circular, smooth and 1–2 mm in diameter after 2 days of incubation at 30 °C on LB agar. Growth occurs at 10–45 °C (optimum 37 °C), pH 5.0–10.0 (optimum pH 7.0–9.0) and 1–15% (w/v) NaCl (optimum 2–10%). Positive for nitrate reduction, oxidase, D-glucose fermentation, methyl-red test, and hydrolysis of aesculin, ONPG, citric acid, gelatin. Negative for hydrolysis of myo-inositol and starch. D-galactose, D-glucose, D-mannose, maltose, D-ribose, D-xylose, L-rhamnose and sucrose are utilized as sole carbon and energy source but D-lactose, and D-arabinose are not utilized. Sensitive to (amounts per disc) gentamicin (10 μg), kanamycin (30 μg), rifampicin (5 μg), tetracycline (30 μg) and chloramphenicol (30 μg), but resistant to carbenicillin (100 μg), penicillin (6 μg) and ampicillin (10 μg). The respiratory quinone is Q-9 and Q-8. The predominant fatty acids are summed feature 8 (C18:1ω7c and/or C18:1ω6c), summed feature 3 (C16:1ω7c and/or C16:1ω6c) and C16:0. The polar lipid profile is composed of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids, an unidentified phosphatidylglycolipid, and four unidentified lipids. The genomic DNA G + C content is 63.6 mol%. The type strain is DX6T (= CCTCC AB 2020094T = KCTC 82196T), isolated from a Gobi soil sample collected in Bachu County, Xinjiang province, China. The GenBank accession number for the 16S rRNA gene sequence of strain DX6T is MT180568. The draft genome sequence accession numbers for strain DX6T is JAAQTO000000000.