Halorubrum salipaludis sp. nov., isolated from the saline–alkaline soil

A novel extremely halophilic archaeon, designated WN019T, was isolated from the natural saline-alkali wetland soil of Binhai new district, Tianjin, China. Cells of WN019T were aerobic, motile, and pleomorphic rod-shaped, 0.5–0.8 µm in width and 2.0–2.5 µm in length, and the growth occurred optimally at 33–37 °C, pH 7.5–8.0, and in the presence of 15.0–20.0% (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequence comparison showed that the isolate belonged to the genus Halorubrum and exhibited moderate sequence similarity of 97.8% to Halorubrum saccharovorum JCM 8865T. The major respiratory quinones of strain WN019T were MK-8 and MK-8 (H2), and the major polar lipids were glycolipid (GL), phospholipid (PL), phosphatidylglycerol-sulphate (PGS), phosphatidylglycerol (PG) and phosphatidylglycerol-phosphate-methyl ester (Me-PGP). The DNA G + C content of the strain was 67.4 mol%. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) value based on whole genome sequences of strain WN019T and Halorubrum saccharovorum JCM 8865T were 87.5% and 35.4%, respectively. Phenotypic, chemotaxonomic, phylogenetic, and genomic analyses suggested that strain WN019T represents a novel species of the genus Halorubrum, for which the name Halorubrum salipaludis sp. nov. is proposed. The type strain is WN019T (= KCTC 4269T = ACCC 19977T).


Introduction
The genus Halorubrum was first proposed by McGenity and Grant belonging to the family Halorubraceae, and Halorubrum saccharovorum JCM 8865 T is the type strain of this genus (McGenity and Grant 1995). At the time of writing, the genus Halorubrum consists of 37 validly published species that are phylogenetically and phenotypically heterogeneous listed on the LPSN (http:// www. bacte rio. net/). It has been reported that the genus Halorubrum are widely distributed in various hypersaline environments, such as salt lakes, hypersaline soda lakes, coastal sabkhas, salt mine, saline soil, salt-fermented seafood, and marine salterns (Chen et al. 2017a, b;de la Haba et al. 2018;Ochsenreiter et al. 2002). Halorubrum species, which grow fast and are active in the conversion of different kinds of substrates, may play a major role in carbon and nitrogen cycling of several hypersaline environments (Feng et al. 2004). Salinealkali land is a special and major resource with high salinity and alkalinity. In this study, we reported the novel strain WN019 T of the genus Halorubrum, which can live in the saline-alkaline habitat, according to their phenotypic, biochemical, and genotypic characteristics. N, 117° 13′ E). The strain obtained by culturing on the modified R2A (MR2A) medium using the traditional dilution inoculation method, for 1 L medium containing the following ingredients: 0.5 g casamino acids (Difo), 0.5 g of yeast extract (Difo), 0.5 g of sodium pyruvate, 0.5 g of peptone, 0.5 g of glucose, 3.0 g of trisodium citrate, 2.0 g of KCl, 0.3 g of K 2 HPO 4 , 0.5 g of CaCl 2 , 20.0 g of MgSO 4 •7H 2 O, 180 g of NaCl, pH 7.0-7.5, agar 15.0 g, 121 °C, autoclaving 20 min (Reasoner and Geldreich 1985). And colonies were selected according to appearance characteristics. A single colony was purified and identified by 16S rRNA gene sequencing.

Chemotaxonomic characterization
To characterize respiratory quinones and polar lipids of all strains, cells were grown in medium mentioned above (37 °C) and harvested during late exponential growth phase. 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). Polar lipids were extracted using the widely used chloroform-methanol system and detected using two-dimensional thin layer chromatography (2D-TLC) on an aluminium backed silica gel 60 plates (20 × 20 cm, Merck) (Minnikin et al. 1984). Analysis of respiratory quinones and polar lipid were carried out by the Identification Service, DSMZ, Braunschweig. Germany.

Molecular characterization
Genomic DNA was extracted using a commercial kit (TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver. 3.0) based on the manufacture's protocol. The amplification of the 16S rRNA fragment was done by PCR with 4aF (5'-TCC GGT TGA TCC TGC CRG -3') and 1391R (5'-GAC GGG CRG TGW GTRCA0-3') as primers (Klatt et al. 2013) and genomic DNA as template. The genome was sequenced on the Illumina HiSeq2000 platform at Shanghai Personal Biotechnology Co., Ltd. China. Filtering and trimming of the genomic raw data were done with PRINSEQ v0.20.4 (Schmieder and Edwards 2011), and the trimmed reads were assembled using SOAPdenovo v.2.3 (Li et al. 2008(Li et al. , 2010 with default parameters. The genome completeness (100%) was assessed using CheckM (version 1.03) (Parks et al. 2015). Protein-coding open reading frames were predicted by Glimmer (version 3.02) (Delcher et al. 2007). For RNA prediction, rRNAs were predicted by RNAmmer (version 1.2) (Lagesen et al. 2007), and tRNAs were predicted by tRNAscan-SE (version 1.21) (Lowe and Eddy 1997). Pairwise sequence identity values for the 16S rRNA genes were calculated on EzBioCloud server (https:// www. ezbio cloud. net/) and related sequences of the type strains of the genus Halorubrum were retrieved. Multiple sequence alignments of strain WN019 T and the most closely related taxa were carried out using CLUSTALXv1.81 (Thompson et al. 1997). Phylogenetic trees were constructed with the maximumlikelihood (ML) method using MEGA v7.0 (Kumar et al. 2016), and neighbour-joining (NJ) and maximum parsimony (MP) phylogenetic trees were also constructed to confirm the phylogenetic position of the strain WN019 T . The resultant tree topologies were evaluated by bootstrap analyses (1000 replications).
Average nucleotide identity (ANI) values of the total genomic sequences shared between the genomic sequences of strain WN019 T and closely related genomic sequences from GenBank were performed using the ANI-BLAST (ANIb) and ANI-MUMmer (ANIm) algorithms in JSpe-ciesWS (https:// jspec ies. riboh ost. com/ jspec iesws/) (Richter et al. 2016). As a proposed complement to ANI values, digital DNA-DNA hybridization (dDDH) values were calculated using Genome-to-Genome Distance Calculator (GGDC2.1) (Meier-Kolthoff et al. 2013) using the BLAST + method. Results were recommended based on the recommended formula 2 (identities/HSP length), which was useful when dealing with incomplete draft genomes. As a further extension of genome-based phylogeny, the Genome Taxonomy Database Toolkit (GTDB-Tk) (Chaumeil et al. 2020) v1.7.0 was used to compare a set of 122 archaeal marker genes between strain WN019 T and other related Halorubrum species, and Phylogenetic trees were constructed with the maximum-likelihood (ML) method using IQ-TREE v2.1.4 (Minh et al. 2020).

Molecular characteristics
The 16S rRNA gene sequence of strain WNO19 T is 1465 bp long and has been deposited to NCBI under the accession number MF782426. The genome assembly of strain WN019 T has been deposited at DDBJ/EMBL/ GenBank under the accession NSKC00000000 as presented here is 3,506,649 bp in size, and the G + C content is 67.4 mol%. We predicted a total of 3217 proteins, 48 tRNAs, 5 rRNAs, 2 ncRNAs for strain WN019 T , and it has only one copy of the 16S partial rRNA gene. Based on the phylogenetic analysis of the 16S rRNA gene retrieved from genome, strain WN019 T was clearly affiliated with the Halorubrum clade in the family Halorubraceae (Fig. 1,  Fig. S3 and S4). The similarities of 16S rRNA gene between strain WN019 T and Halorubrum saccharovorum JCM 8865 T , Halorubrum persicum JCM 30541 T were 97.8%, 97.7%, and other neighbouring type strains are all below 97.7%. The levels of 16S rRNA gene sequence similarity between strain WN019 T and other type strains of the genus Halorubrum were all below 98.7% (Chun et al. 2018), which suggesting strain WN019 T maybe represents a novel species distinct from all other members of the genus Halorubrum.
The GTDB-based phylogenomic tree, employing a set of 122 marker genes commonly present in all archaeal genomes, indicated that strain WN019 T clustered with the species of Halorubrum but formed an independent branch clearly separated from the other species that constitute the genus (Fig. 2)