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

Strain WRN001T, 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 WRN001T 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 affiliated to the genus Halomonas, and the highest 16S rRNA gene sequence similarity of strain WRN001T to its closest relative Halomonas qiaohouensis DSM 26770 T was 97.5%. 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 strainWRN001T was Q-9, and the dominant fatty acids were summed feature 8, summed feature 3, C10:0, C12:0, C12:0 3-OH, C16:0, and C17:0 cyclo. The major polar lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phophatidylcholine (PC), two phospholipids (PL), aminolipid (AL), and three unidentified lipids (L). These data combined with the low digital DDH values between strain WRN001T and the close relative, Halomonas alkalitolerans CGMCC 1.9129 T (42.2%) and based on comparisons with currently available genomes, the highest average nucleotide identity (ANIm) value was 91.4% to Halomonas alkalitolerans CGMCC 1.9129 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 WRN001T = KCTC 52853 T = ACCC 19974 T).


Introduction
Halomonas, as a large genus, was first 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 (https:// www. bacte rio. net/). At the time of writing, the genus Halomonas includes 96 species with validly published names (http:// www. bacte rio. net/ halom onas. html). In this paper, we described the isolation, identification, and physio-biochemical characteristics of novel strain WRN001 T and proposed the name Halomonas salipaludis for this bacterium.

Isolation and culture conditions
Strain WRN001 T was isolated from the natural salinealkali wetland soil of Binhai new district, Tianjin,China (38°46' N,117°13' E) in June 2015. 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 Communicated by Erko Stackebrandt. heterotrophic microorganisms, 1.0 g of soil was placed in sterile 30 ml glass tube for enrichment using Difco ™ marine 2216 amended with final concentration of 10% (w/v) NaCl and cultivated for 3 days and subsequently purified into single colonies.

Morphological, physiological and biochemical characterization
Cell size, morphology and motility of strain WRN001 T were established 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 ™ marine 2216 agar (8.0% NaCl, w/v) and shaking in Difco ™ marine 2216 liquid medium (8.0% NaCl, w/v) 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 final pH to 5. 0, 5.5, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 (at 8.0% NaCl, w/v, 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 at 33 °C with 8.0% NaCl (w/v) in the tubes with the butyl rubber stopper and screw cap.
For all physiological experiments, Halomonas qiaohouensis DSM 26770 T and the closely related strain Halomonas pantelleriensis DSM 9661 T were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) as the reference strains, and Halomonas socia CCTCC AB 2011033 T was obtained from China Center for Type Culture Collection (CCTCC). Unless otherwise stated, all the strains mentioned above were incubated at 30 °C in Difco ™ marine 2216 medium amended with final concentration of 8.0% NaCl (w/v) for strain WRN001 T and final concentration of 10.0% NaCl (w/v) for reference organisms.

Chemotaxonomic characterization
Cells of strain WRN001 T and the reference strains were harvested during the late exponential growth phase in Difco ™ marine 2216 liquid medium (8% NaCl for strain WRN001 T and 10% 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 purified using high-performance liquid chromatography (HPLC) (Minnikin et al. 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 6890 N 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), modified 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 thinlayer chromatography. The first 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 specific functional groups detected using spray reagents specific for defined functional groups (Tindall 2007).

Molecular characterization
The 16S rRNA gene was amplified from chromosomal DNA using the universal bacterial primer set 27F and 1492R (Lane 1991;Weisburg et al. 1991). The PCR product was purified using the PCR purification kits (MinElute PCR Purification 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 maximum-likelihood method, and Neighborjoining (NJ) and 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).
DNA-DNA hybridization (DDH) between strain WRN001 T and the type strains of the phylogenetically most closely related Halomonas species were performed as described by Ezaki et al. (1989). In addition to DDH, 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
Cells of Strain WRN001 T are aerobic, Gram-staining-negative, size with 0.3-0.5 × 1.5-2.5 μm (Fig. S1), and was sensitive to chloramphenicol, erythromycin, streptomycin, vancomycin and polymyxin B, but resistant to ampicillin, kanamycin, penicillin G, tetracycline, and gentamicin. Strain WRN001 T and Halomonas socia CCTCC AB 2011033 T were negative for nitrate reduction, which distinguished them from Halomonas pantelleriensis DSM 9661 T and Halomonas qiaohouensis DSM 26770 T . Moreover, compared with the three related type strains, strain WRN001 T can hydrolyse starch, Tween 80, and pectin. The physiological and biological characteristics of Strain WRN001 T are summarized in the species description and Table S1. And further selective and comparative characteristics of strain WRN001 T and the related type strains are shown in Table 1.

Molecular characteristics
Strain WRN001 T has only one copy of the 16S rRNA gene (1528 bp). Based on the 16S rRNA phylogenetic analysis, strain WRN001 T was affiliated with the Halomonas clade in the family Halomonadaceae (Fig. 1, Fig. S3 and S4). The 16S rRNA gene of strain WRN001 T was most closely related to Halomonas qiaohouensis DSM 26770 T (97.47%), Halomonas socia CCTCC AB 2011033 T (96.65%), and Halomonas pantelleriensis DSM 9661 T (95.96%). The levels of 16S rRNA gene sequence similarity between strain WRN001 T and other type strains of the genus Halomonas were below 98.7% (Chun et al. 2018), which suggests that strain WRN001 T represents a novel species distinct from all other members of the genus Halomonas.
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 4940 ORFs, 65 tRNAs, and 10 rRNAs. Digital DDH values between strain WRN001 T and its close relatives, Halomonas alkalitolerans CGMCC 1.9129 T , Halomonas pantelleriensis DSM 9661 T and Halomonas shengliensis CGMCC 1.6444 T , were 42.2%, 35.1%, and 23.3%, respectively. And ANI values between strain WRN001 T and its close relatives, Halomonas alkalitolerans CGMCC 1.9129 T , Halomonas pantelleriensis DSM 9661 T and Halomonas shengliensis CGMCC 1.6444 T , were 88.7%, 87.0%, and 77.7%, respectively (Table S2). These add evidence that strain WRN001 T represents a novel species of the genus Halomonas, based on the recommended minimum relatedness DDH value is 70% and ANI value is 95% for strains of the same species (Graham 1991;Wayne et al. 1987;Richter and Rosselló-Móra 2009).  (Romano et al. 1996) b Data from (Wang et al. 2014) c Data from (Cao et al. 2013), and all other data were obtained from this study   Scale bar, 0.01 substitutions per nucleotide position. Zymobacter palmae T109 T (D14555) was used as an outgroup. GenBank accession numbers are indicated for each strain