Halomonas jincaotanensis sp. nov., isolated from the Pamir Plateau degrading polycyclic aromatic hydrocarbon

A Gram-strain-negative, rod-shaped, aerobic bacterium, designated strain TRM 85114T, was isolated from the Jincaotan wetland in the Pamir Plateau of China. This strain grew optimally at 30 °C and pH 6.0 in the presence of 3% (w/v) NaCl. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain TRM 85114T was affiliated with the genus Halomonas, and shared high sequence similarity with Halomonas korlensis XK1T (97.3%) and Halomonas tibetensis pyc13T (96.4%). Strain TRM 85114T contained C16:0 and C19:0 cyclo ω8c as primary cellular fatty acids, Q-9 as predominate respiratory quinone, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phospholipids of unknown structure containing glucosamine, unidentified aminophospholipids, unidentified lipids and three unidentified phospholipids as the major polar lipids. The complete genome of TRM 85114T comprised 3,902 putative genes with a total of 4,126,476 bp and a G + C content of 61.6%. The average nucleotide identity and digital DNA–DNA hybridization values between strain TRM 85114T and related type Halomonas strains of H. korlensis XK1T, H. tibetensis pyc13T, Chromohalobacter salexigens DSM 6768T, and Halomonas urumqiensis BZ-SZ-XJ27T were 75.4–88.9% and 22.9–39.2%, respectively. Based on phenotypic, chemotaxonomic, and molecular features, strain TRM 85114T represents a novel species of the genus Halomonas, for which the name is proposed as Halomonas jincaotanensis sp. nov.. The type strain is TRM 85114T (CCTCC AB 2021006T = LMG 32311T). The amount of 1-naphthylamine degradation by strain TRM 85114T reached up to 32.0 mg/L in 14 days.


Introduction
Halophilic microorganisms, particularly Halomonas spp., usually accumulate polyphosphate (Nguyen et al. 2012), produce biodegradable polyhydroxyalkanoates (Jiang et al. 2018;Tuma et al. 2020) and various metabolic chemicals (Du et al. 2020;Jiang et al. 2021). Based on the advantages of energy-saving, water-saving, time-saving, less investment in equipment, high concentration of final product, and simplification of separation process, Halomonas spp. have been used as platform strains in the field of synthetic biology to produce multiple products in Next Generation Industrial Biotechnology (NGIB) (Yu et al. 2019). The genus Halomonas initially proposed by Vreeland et al. (1980) belongs to the family Halomonadaceae of the phylum Proteobacteria. At present, this genus contains more than 160 validly named species. Members of the genus Halomonas have been isolated from diverse terrestrial and aquatic habitats, such as lake water (Kazemi et al. 2021), saline-alkali land (Dou et al. 2015), tidal flat (Koh et al. 2017), Arctic marine (Williamson et al. 2016), deep-sea sediment (Xu et al. 2013), and hypersaline wetlands (Ramezani et al. 2020). The Jincaotan wetland in Pamir Plateau is an environment characterised with saline and high altitude, in which extremophiles could be abundant.
Polycyclic aromatic hydrocarbon (PAH) is one of the most common pollutants in the soil and/or water environment and have become a matter of urgency due to their negative impacts on human health (Premnath et al. 2021 priority contaminants and carcinogens (Hu et al. 2011), significant accumulation in the soil and subsequential migration into the aquatic system have led to chronic exposure, which is associated with cancerous diseases in humans and aquatic animals and enhanced mutagenicity of soil and/or sediments. The genus of Halomonas has shown an eminent capacity to degrade polycyclic aromatic hydrocarbons (Al Farraj et al. 2020;Govarthanan et al. 2020), such as anthracene, phenanthrene, pyrene, naphthalene, and benzo [a]pyrene, and therefore have tremendous potential for environmental remediation. In this study, we obtained a novel species of Halomonas from the Pamir Plateau, designated TRM 85114 T , explored its taxonomic characterisation and ability to degrade 1-naphthylamine, which has yet to be reported.

Chemotaxonomic characterization
The respiratory quinones of strain TRM 85114 T was extracted from freeze-dried biomass (Collins et al. 1977), and subsequently confirmed using HPLC (Collins and Jones 1981). For the analysis of cellular fatty acids, strain TRM 85114 T and four reference strains were cultured on 12% MGM plates at 30 °C and the microbial cells were harvested at the 4th day. The fatty acids were extracted from fresh cells (Athalye et al. 1985), and analyzed by gas chromatography referred to the Microbial Identification System (Sherlock version 6.1; MIDI database: RTSBA6). The compositions of polar lipids were extracted, separated using two-dimensional TLC (Parsons and Patton 1967) and determined by spraying with four dyes of 10% ethanolic molybdatophosphoric acid (for total lipids), ninhydrin (for aminolipids), molybdenum blue (for phospholipids), and anisaldehyde (for glycolipids), respectively.

Genome sequencing and analysis
The whole genome of strain TRM 85114 T was sequenced on the Illumina Hiseq platform and assembled using the ABySS (version 2.0) assembler (Jackman et al. 2017), with a depthsequencing coverage of 456×. We used the software CheckM to evaluate the completeness and contamination of the genome (Parks et al. 2015), the online resource prodigal to predict assembled genome (http:// compb io. ornl. gov/ prodi gal/) (Hyatt et al. 2010), software tRNA scan-SE to predict the tRNAs in the genome (Lowe and Eddy 1997), and the software Infernal 1.1 (Nawrocki and Eddy 2013) to predict the rRNAs in the genome based on the Rfam database (Nawrocki et al. 2015). Genome annotation was performed by Personal Biotechnology Co., Ltd (Shanghai, China). The genomic DNA G + C content was calculated based on the entire genome sequence. Prediction of the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were estimated using the ChunLab's online ANI Calculator (www. ezbio cloud. net/ tools/ ani) (Lee et al. 2016) and the Genome-to-Genome Distance Calculator (http:// ggdc. dsmz. de/ distc alc2. php) (Meier-Kolthoff et al. 2013), respectively. To investigate the details of the phylogenetic placement of TRM 85114 T , we constructed a phylogenetic tree based on single-copy orthologues identified by grouping orthologous protein sequences from strain TRM 85114 T and 19 other related species of Halomonas, whose genome has been acquired from the NCBI database. Based on all amino acid sequences of the selected species, software Orthofinder (Emms and Kelly 2019) was used to cluster gene family, Blastp to compare, and software Muscle (Edgar 2004) to compare multiple protein sequences in each single-copy gene family. In addition, software Trimal (Capella-Gutierrez et al. 2009) was used to filter the comparison results, merge the filtered comparison results and connect them into super genes. Finally, software RAxML (Stamatakis 2014) was used to construct a maximum-likelihood phylogenetic tree based on core genes.

Detection of 1-naphthylamine degradation capacity
Batch experiments were performed to study the ability of TRM 85114 T on 1-naphthylamine biodegradation (Govarthanan et al. 2020). The isolate grown in 12% MGM medium for 4 days were harvested, washed, and resuspended in 12% MGM medium (A 600 , 0.6). The resuspension was (1 mL) inoculated into 250-mL Erlenmeyer flasks containing 100 mL of 12% MGM medium. Fifty mg/L of 1-naphthylamine was added into the culture flasks and incubated in dark at 180 rpm for 14 days at 30 °C. Samples (1 mL) were withdrawn at days 0, 2, 4, 6, 8, 10, 12 and 14, and 1-naphthylamine degradation efficiency of the culture was determined using HPLC. The methanol solution was used for gradient elution, the concentration lasted from 10 to 100% for 40 min, and the absorption peak of 1-naphthylamine was detected at 222 nm. Furthermore, the degradation content of 1-naphthylamine was calculated based on a standard curve, which was constructed according to different contents of 1-naphthylamine solutions (20, 40, 60, 80, 100, and 120 mg/L). The isolates were treated without 1-naphthylamine were used as control. All experiments were performed in triplicate, and mean values after subtracted blank controls (10.2 mg/L) were reported.

Phenotypic and physiological characterization
The cells of strain TRM 85114 T was Gram-stain-negative, short rod-shaped, aerobic, and non-motile bacteria ( Supplementary Fig. S1). Colonies were cream-white and smooth with entire margins after incubated at 30 °C on 12% MGM plates for 4 days. Strain TRM 85114 T grew occurred at 4-35 °C (optimum, 30 °C), NaCl 1-13% (w/v) (optimum, 3%), and pH 6.0-9.0 (optimum, 7.0). Physiological and biochemical characteristics of strain TRM 85114 T are additionally described in the species descriptions and compared with those of closely related Halomonas type strains in Table 1.

Chemotaxonomic characteristics
The predominate respiratory quinone identified in strain TRM 85114 T was Q-9 (34.8%), followed by Q-8 (15.2%), and Q-6 (4.7%), which were coincide to members detected in other strains of the genus Halomonas (Qu et al. 2011;Zhang et al. 2016;Lu et al. 2018). As shown in Supplementary Table 1, the primary cellular fatty acids in strain TRM 85114 T were C 16:0 (18.0%) and C 19:0 cyclo ω8c (10.3%), in that C 10:0 (0.8%), C 11:0 3-OH (0.3%), and C 20:2 ω6,9c (0.3%) were generally similar to those detected in other closely related strains. Moreover, strain TRM 85114 T was different from the four reference strains in terms of the types of polar lipids present, including diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phospholipids of unknown structure containing glucosamine (NPG), unidentified aminophospholipids, unidentified lipids, and three unidentified phospholipids ( Supplementary Fig.  S2). Two components of PG and PE were detected in most Halomonas spp., but NPG were evidently different from other strains of the genus Halomonas (Zhong et al. 2016;Lu et al. 2018;Ramezani et al. 2020). Based on the information of genome annotation, a process of UDP-N-acetylglucosamine biosynthetic was searched, which suggests that the presence of NPG in strain TRM 85114 T .

Phylogenetic and phylogenomic analyses
Based on EzBioCloud analysis, the 16S rRNA gene sequence of strain TRM 85114 T (GenBank accession no. MW584241) has highly similar with members of the genus Halomonas. The highest similarity was shown with the type strain of H. korlensis XK1 T (97.3%), followed by H. tibetensis pyc13 T (96.4%), Chromohalobacter israelensis ATCC 43985 T (96.2%), C. salexigensDSM 3043 T (96.1%), H. urumqiensis BZ-SZ-XJ27 T (96.1%), and H. daqiaonensis CGMCC 1.9150 T (96.0%). However, the sequence similarity was less than 96.0% with other species of genera within the family Halomonadaceae. The results suggested that strain TRM 85114 T constitutes a possible novel species in the genus Halomonas.
A NJ tree based on 16S rRNA gene (1504 bp) (Fig. 1) showed that strain TRM 85114 T clustered tightly with H. korlensis XK1 T with strong bootstrap support (> 99%) forming an independent sub-cluster with the members of the genus Halomoans, which was in good agreement with the results of the ML and MP trees (Supplementary Fig.  S3 and S4). A NJ tree based on the concatenated gene sequences (16S rRNA; gyrB 472 bp; rpoD 1243 bp) phylogenetic tree (Supplementary Fig. S5) showed that strain TRM 85114 T forming an independent cluster separating from other group members. To further elucidate the phylogenetic relationship of strain TRM 85114 T , we compared its proteome with 19 related species to investigate evolutionary specialization. A total of 8,805 orthologous gene families consisting of 71,909 genes were identified across these species, including a core set of 1140 genes shared among them. The ML tree based on these core gene sequences revealed the position of strain TRM 85114 T (Fig. 2), which was grouped into a new clade comprising H. korlensis XK1 T , which was consistent with the MLSA phylogenetic trees, supporting the view that strain TRM 85114 T represent a novel member of the genus Halomonas.

1-Naphthylamine degradation rate
The biodegradation efficiency of 1-naphthylamine by strain TRM 85114 T was verified and confirmed in this study. Results indicated that the retention time of 1-naphthylamine was at the 26 th min, and the absorption peak area of it decreased significantly over time ( Supplementary  Fig. S6). According to the standard curve of 1-naphthylamine ( Supplementary Fig. S7). The amount of 1-naphthylamine degradation by strain TRM 85114 T on days 4 and 14 were calculated for 21.6 mg/L and 32.0 mg/L, respectively ( Supplementary Fig. S8). The result indicated that strain TRM 85114 T was capable of degrading 1-naphthylamine. Thus, strain TRM 85114 T could be of potential value in the bioremediation of water and/or soil systems contained with 1-naphthylamine.

Taxonomical conclusion
The results of phylogenetic analyses based on 16S rRNA gene, core gene and housekeeping gene, phylogenetic trees (Figs. 1, 2, Supplementary Fig. S5) illustrated that strain TRM 85114 T was a member of the genus Halomonas. Furthermore, phenotypic and chemotaxonomic traits showed that strain TRM 85114 T was distinct from several closely related species in the genus Halomonas. In addition, the genomic relatedness (ANI and dDDH values) also suggested that strain TRM 85114 T was distinguishable from its closest phylogenetic neighbour H. korlensis XK1 T . In conclusion, a new species, Halomonas jincaotanensis sp. nov., is proposed as the type strain.