Halomonas salipaludis sp. nov., isolated from the saline-alkali wetland soil

Strain WRN001 T , a Gram–staining–negative, strictly aerobic, non–motile bacterium was isolated from the natural saline-alkali wetland soil of Binhai new district, Tianjin, China (38°46′N, 117°13′E). Cells of strain WRN001 T were 0.3-0.5 µm in width and 1.5-2.5 µm in length, and the growth occurred optimally at 33-37 °C, pH 7.5-8.0, and in the presence of 8-10% (w/v) NaCl. Based on 16S rRNA gene sequence analysis, the isolate could be aliated to the genus Halomonas, and the highest 16S rRNA gene sequence similarity of strain WRN001 T to its closest relative Halomonas qiaohouensis YIM QH88 T was 97.47%. The size of the genome as presented here was 5,475,884 bp with a G+C content of 63.8 mol %. The major respiratory quinone of strainWRN001 T was Q-9, and the dominant fatty acids were summed feature 8, summed feature 3, C 10:0 , C 12:0 , C 12:0 3-OH, C 16:0 , and C 17:0 cyclo. The major polar lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phophatidylcholine (PC), two phospholipids (PL), aminolipid (AL), and three unidentied lipids (L). These data mentioned above combined with the low DDH values between strain WRN001 T and the close relative, Halomonas alkalitolerans 1513 T (42.20%) and base on comparisons with currently available genomes, the highest average nucleotide identity (ANIm) value was 91.39% to Halomonas alkalitolerans 1513 T (GenBank accession No. GCA_001971685.1). Therefore, we propose a novel species in the genus Halomonas to accommodate this novel isolate: Halomonas salipaludis sp. nov. (type strain WRN001 T = KCTC 52853 T = ACCC 19974 T ).


Introduction
The Halomonas as a large genus was rstly described by Vreeland et al. (1980), with Halomonas elongata as the type species, and most of the species of the genus Halomonas were isolated from saline habitats, such as sea sediments, salt lakes, brines, salty foods, deep sea hydrothermal vent environments, as well as saline sand and soils (Arenas et al. 2009;Cao et al. 2013;Dou et al. 2015;Guan et al. 2010;Guzmán et al. 2010;Kaye et al. 2004;Kim et al. 2010;Lee et al. 2005;Reddy et al. 2003;Romano et al. 1996;Wang et al. 2012;Wang et al. 2014;Xu et al. 2007). At the time of writing, the genus Halomonas includes 96 species with validly published names (http://www.bacterio.net/halomonas.html). Some of them have been recognized for potential use in biotechnology, such as in the production of bioactive compounds (Wang et al. 2006), extreme-enzymes (Kumar et al. 2012), exopolysaccharide (Kumar et al. 2012;Martínezcheca et al. 2005;Poli et al. 2013), and hydrolytic enzymes (Sánchez-Porro et al. 2003). In this paper, we described the isolation, identi cation and physio-biochemical characteristics of novel strain WRN001 T and proposed the name Halomonas salipaludis for this bacterium.

Materials And Methods
Isolation and Culture Conditions Strain WRN001 T was isolated from the natural saline-alkali wetland soil of Binhai new district, Tianjin, China (38°46′N,117°13′E) in June 2015. Soils 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, respectively. To isolate halophilic heterotrophic microorganisms, 1.0 g of soil was placed in sterile 30 ml glass tube for enrichment using Difco TM marine 2216 amended with nal concentration of 10.0% NaCl for 3 days and subsequently puri ed into single colonies.

Morphological, Physiological and Biochemical Characterization
Cell size, morphology and motility of strain WRN001 T were established by using a Leica microscope equipped with phase-contrast optics (Leica DM 6000 B) during exponential growth phase. 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). Gram staining was performed using BD Gram staining kits according to the manufacturer's instructions. 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% H 2 O 2 solution onto bacterial colonies and observing bubble production. Reduction of nitrate and hydrolysis of starch, casein, gelatin, and Tween 80 were analyzed according to the methods of Smibert and Krieg (1994) and Dong and Cai (2001). The optimal growth temperature of strain WRN001 T was determined after incubation on Difco TM marine 2216 agar (8.0% NaCl) and shaking in Difco TM marine 2216 liquid medium (8.0% NaCl) at 4, 10, 15, 20, 25, 30, 33, 37, 40, 45, and 50 °C (at pH 7.5). Bacterial growth was measured as increase in turbidity at 600 nm, using a DU 800 spectrophotometer (Beckman Coulter). NaCl tolerance was tested on Luria-Bertani (LB) agar and in LB liquid medium amended with 0.0-25.0% NaCl (w/v). Similarly, the pH range for growth was measured by adjusting the nal 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 (Na 2 HPO 4 /NaH 2 PO 4 for pH 5.0-7.0 and Na 2 CO 3 /NaHCO 3 for pH 8.0-12.0). Anaerobic growth was determined through measuring the OD 600 nm at 33 °C with 8.0% NaCl (w/v) in the tubes with the butyl rubber stopper and screw cap.
For all physiological experiments, we selected the Halomonas qiaohouensis YIM QH88 T , Halomonas socia NY-011 T , and the closely related strain Halomonas pantelleriensis AAP T as reference organisms.
Unless otherwise stated, all the strains mentioned above were incubated at 30 °C in Difco TM marine 2216 medium amended with nal concentration of 8.0% NaCl for strain WRN001 T and nal concentration of 10.0% NaCl for reference organisms.

Chemotaxonomic Characterization
Cells of strain WRN001 T and the reference strains were harvested during the late exponential growth phase in Difco TM marine 2216 liquid medium (8.0% NaCl for strain WRN001 T and 10.0% NaCl for reference strains) at 33 °C for characterization of respiratory quinones, cellular fatty acids, and polar lipids. Respiratory quinones were extracted with chloroform/methanol (2:1) (v/v) from lyophilized cells (300 mg) and puri ed using high performance liquid chromatography (HPLC) (Minnikin DE 1984). The fatty acids were identified and quantified by the Sherlock 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 (1990). Polar lipids were extracted from 200 mg of freeze-dried cell material using a chloroform: methanol: aqueous NaCl mixture (0.3%, w/v) with the ratio of 1:2:0.8 (v/v/v), modi ed after Bligh and Dyer (Bligh and Dyer 1959), recovered into the chloroform phase by adjusting the mixture to a ratio of 1:1:0.9 (v/v/v), and separated by two-dimensional silica gel thin-layer chromatography. The rst dimension was developed in a chloroform: methanol: water (65:25:4, v/v/v) mixture and the second was developed in a chloroform: methanol: acetic acid: water (80:12:15:4, v/v/v/v) mixture. Total lipid material was detected using molybdatophosphoric acid and speci c functional groups detected using spray reagents speci c for de ned functional groups (Tindall BJ 2007). The respiratory quinones and cellular fatty acids were determined by the identi cation service of the China Center of Agricultural Culture Collection in Beijing, China, and the polar lipid analysis was performed was performed by the Identi cation Service of the DSMZ, Braunschweig, Germany.

Molecular Characterisation
The 16S rRNA gene was ampli ed from chromosomal DNA using the universal bacterial primer set 27F and 1492R (Lane 1991;Weisburg et al. 1991). The PCR product was puri ed using the PCR puri cation kits (MinElute PCR Puri cation Kit, QIAGEN) and sequenced by Sangon Biotech (Shanghai) Co., Ltd., China. The 16S rRNA gene sequence of strain WRN001 T , as determined in this study, was submitted to GenBank, and the 16S rRNA gene sequences of microorganisms related taxa were obtained from the GenBank database (http://www.ncbi.nlm.nih.gov/). Phylogenetic trees were constructed by maximumlikelihood method, and Neighbor-joining (NJ) & maximum parsimony (MP) phylogenetic trees were also constructed to corroborate the phylogenetic position of the strain WRN001 T in software MEGA 7.0 (Kumar et al. 2016).
Experiments with digital DNA-DNA hybridization (dDDH) between strain WRN001 T and the type strains of the phylogenetically most closely related Halomonas species were performed as described by Ezaki et al. (Takayuki et al. 1989). In addition to dDDH, average nucleotide identity (ANI) values between the strain WRN001 T genome and closely related genomic sequences from GenBank were also determined according to Goris et al. (2007), i.e., 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 (Kurtz et al. 2004) and ANI values were calculated using JSpecies version 1.2.1 (Goris et al. 2007).

Morphological, Physiological and Biochemical Characteristics
Colonies of strain WRN001 T on Difco TM marine 2216 (8.0% NaCl) were circular, wet, smooth, and pigmented light yellow, and around 0.35 mm in diameter after 72 h of incubation at 30 °C. Strain WRN001 T cells were aerobic, Gram-stain-negative, non-motile, rod-shaped, approximately 0.3-0.5 µm in width and 1.5-2.5 µm in length ( Supplementary Fig. S1), and cells were oxidase-positive and catalasenegative. Strain WRN001 T grew with 0.5-20.0% NaCl (optimum growth at 8.0-10.0% NaCl), at 10-45 °C (optimum 33-37 °C), and pH 5.5-11.0 (optimum pH 7.5-8.0). The novel isolate WRN001 T can utilize starch, casein and Tween 80, but gelatin was not used as carbon source, and nitrate was not reduced, and strain WRN001 T cannot grow under anaerobic conditions. The physiological and biological characteristics of Strain WRN001 T are summarized in the species description, and a comparison of the selective characteristics of strain WRN001 T and the related type strains was given in Table 1.
The genome assembly of strain WRN001 T (accession number NSKB00000000) as presented here is 5,475,884 bp in size. It has a G+C content of 63.8% and consists of 33 contigs with a 200-fold coverage.
We predicted a total of 4,940 ORFs, 65 tRNAs, and 10 rRNAs. And the dDDH values between strain WRN001 T and its close relatives, Halomonas alkalitolerans 1513 T , Halomonas pantelleriensis AAP T and Halomonas shengliensis CGMCC 1.6444 T , were 42.20%, 35.10%, and 23.30%, respectively, which adding evidence that strain WRN001 T represents a novel species of the genus Halomonas, based on the DNA-DNA hybridization as a standard method for species de nition and the recommended minimum relatedness value for strains of the same species is 70% (Graham 1991;Wayne 1987).

Taxonomic Conclusion
In this study, we isolated and described the novel strain WRN001 T from the natural saline-alkali wetland soil. Phylogenetic, phenotypic, and genetic analyses indicate that the strain WRN001 T represents a novel species of the genus Halomonas, for which we propose the name Halomonas salipaludis sp. nov.
The type strain WRN001 T (= KCTC 52853 T = ACCC19974 T ) was isolated from the natural saline-alkali wetland soil of Binhai new district, Tianjin, China (38°46′N, 117°13′E), and the genomic DNA is a single circular chromosome (5,475,884 bp) with a G + C content of 63.8%.

Declarations
Funding This work was supported by National Natural Science Foundation of China (NSFC No. 31670113).

Con icts of interest/Competing interests
The authors declare that they have no con ict of interest.

Availability of data and material
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene of Halomonas salipaludis sp. nov. strain WRN001 T is MF782428. The whole genome was deposited at GenBank/EMBL/DDBJ under the accession number NSKB00000000 for strain WRN001 T . Transmission electron micrographs (TEM) of cells of strain WRN001 T , thin-layer chromatograms of the polar lipids extracted from strain WRN001 T and closely related species, additional phylogenetic trees, and the table containing the average nucleotide identity (ANI), and dDDH values to closely related genomes are available as Supplementary Materials.

Code availability
Not applicable.
Authors' contributions JX contributed to performing the experiments and writing the initial draft. QG provided samples of experiment and participated in the isolation and cultivation of strains. GZ and JZ contributed to the guidance of experimental operations. LT and JL contributed to the morphological analyzes. HF, XW and HL performed genome analysis. GZ and JZ contributed to reagents, instrumentation, and the nancial support for this work.
Ethics approval Not applicable.

Consent to participate
All authors approved the manuscript.

Consent for publication
Written informed consent for publication was obtained from all participants. Wang YX, Xiao W, Dong MH, Zhao Q, Li ZY, Lai YH, Cui XL (2014) (10)   Halomonas socia NY-011 T . The compositions of the fatty acids that less than 1.0 % in all strains were not listed in the Table 2. All the data were taken from this study.
a Summed features represent groups of two or three fatty acids that cannot be separated by GLC with the MIDI system, summed feature 3 contains C 16:1 ω6c and/or C 16:1 ω7c; summed feature 8 comprised C 18:1 ω6c and/or C 18:1 ω7c.

Supplementary Files
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