Phenotypic and physiological characterization
The cells of strain TRM 85114T was Gram-negative, short rod-shaped, aerobic, and non-motile bacteria (Fig. 1). Colonies were cream-white and smooth with entire margins after incubated at 30 ℃ on 12% MGM plates for 4 days. Strain TRM 85114T grew occurred at 4–35 ℃ (optimum, 30 ℃), NaCl 1–10% (w/v) (optimum, 3%), and pH 6.0–9.0 (optimum, 6.0). Physiological and biochemical characteristics of strain TRM 85114T are additionally described in the species descriptions and compared with those of closely related Halomonas type strains in Table 1.
The predominate respiratory quinone identified in strain TRM 85114T was Q-9 (34.8%), followed by Q-8 (15.2%), Q-6 (4.7%) and an unknown quinone (45.2%), 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 85114T were C16:0 (18.0%) and C19:0 cyclo ω8c (10.3%), in that C10:0 (0.8%), C11:0 3-OH (0.3%), and C20:2 ω6,9c (0.3%) were generally similar to those detected in other closely related strains. Moreover, strain TRM 85114T 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. S1). 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 85114T.
Phylogenetic and phylogenomic analyses
Based on the EzBioCloud analysis, the 16S rRNA gene sequence of strain TRM 85114T (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 XK1T (97.3%), followed by H. tibetensis pyc13T (96.4%), H. urumqiensis BZ-SZ-XJ27T (96.1%), H. daqiaonensis CGMCC 1.9150T (96.0%). However, the sequence similarity was less than 96.0% with other species of genera within the family Halomonadaceae. Above all analysis of 16S rRNA gene sequences of strain TRM 85114T, it was considered as a possible novel species in the genus Halomonas.
A NJ tree based on 16S rRNA gene (1504 bp) (Fig. 2) showed that strain TRM 85114T clustered tightly with H. korlensis XK1T 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. S2 and S3). A NJ tree based on the concatenated gene sequences (16S rRNA; gyrB 472 bp; rpoD 1243 bp) phylogenetic tree (Supplementary Fig. S4) showed that strain TRM 85114T forming an independent cluster separating from other group members. To further elucidate the phylogenetic relationship of strain TRM 85114T, 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 85114T (Fig. 3), which was grouped into a new clade comprising H. korlensis XK1T, which was consistent with the MLSA phylogenetic trees, supporting the view that strain TRM 85114T represent a novel member of the genus Halomonas.
Genomic features and analysis
The obtained draft genome of TRM 85114T (accession no. JAHCLU000000000) was 4,126,476 bp in length with G+C content of 61.6 mol%, comprising of 60 contigs (N50 = 190,960 bp) and 52 scaffolds (N50 = 208,915 bp). The annotated genome encodes 3902 genes, including 1666 CDSs, 57 tRNAs and 3 rRNAs (one copy of 5S, one copy of 16S, and one copy of 23S rRNA gene). The general features of the genomes of strains of TRM 85114T, including genome size, DNA G + C contents, and the numbers of rRNA and tRNA genes, were similar with those of closely related reference strains (Table 3). The ANI and dDDH values between strain TRM 85114T and the type strains of H. korlensis XK1T, H. urumqiensis BZ-SZ-XJ27T, H. daqiaonensis CGMCC 1.9150T were significantly lower than the the criteria for the prokaryotic species delineation thresholds for ANI (95–96%) (Kim et al. 2014) and dDDH (70%) (Wayne et al. 1987) (Table 3), suggesting that strain TRM 85114T represents a novel species of the genus Halomonas.
To survive and compete in diverse environmental habitats, Halomonas species evolved a variety of metabolic pathways. Based on the KEGG orthology-based annotation, 3544 genes (90.8%) were annotated and assigned to putative functions, of which 1389 genes were related to metabolism associated pathways, and 249 genes belonged to environmental information processing pathways. What’s more, 44 ORFs were detected for aromatic hydrocarbon degradation (Supplementary Table S2), in which contig15_2804 (frmA, ADH5, adhC) were comment as naphthalene degradation. The above data indicated taht TRM 85114T is a novel strain of the genus Halomonas with naphthalene degradation ability.
1-Naphthylamine degradation rate
The biodegradation efficiency of 1-naphthylamine by strain TRM 85114T was further verified and confirmed in this study. Results indicated that the retention time of 1-naphthylamine was at the 26th min, and the absorption peak area of it decreased significantly over time (Supplementary Fig. S5). According to the standard curve of 1-naphthylamine (Supplementary Fig. S6), the degradation rates of strain TRM 85114T on day 4 and 14 was calculated to be 21.6 mg/L and 32.0 mg/L, respectively (Supplementary Fig. S7). These findings indicated that TRM 85114T has a strong ability to degrade 1-naphthylamine, which may be applied to treat sewage containing 1-naphthylamine in printing and dyeing plants. As a result, strain TRM 85114T has important environmental remediation ability could contribute for bioremediation of water and/or soil systems.
The results of phylogenetic analyses based on 16S rRNA gene, housekeeping gene, and core gene analyses illustrate that strain TRM 85114T was a member of the genus Halomonas. Furthermore, phenotypic and chemotaxonomic traits showed that strain TRM 85114T 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 85114T was distinguishable from its closest phylogenetic neighbour H. korlensis XK1T. In conclusion, a new species, Halomonas jincaotanensis sp. nov., is proposed as the type strain.
Description of Halomonas jincaotanensis sp. nov.
Halomonas jincaotanensis (jin.caotan.en´ sis. NL fem. adj. jincaotanensis pertaines to Jincaotan, Pamir Plateau, Xinjiang, China, from where the type strain was isolated).
Cells are Gram-strain-negative, short rod-shaped, non-motile, and aerobic (0.9–1.0 × 0.4–0.5 μm in size). Colonies are circular, cream-white, convex, and smooth. It grows between 4–35 ℃ (optimum, 30 ℃), tolerate for 1-10% (w/v) NaCl (optimum, 3%), and pH of 6.0–9.0 (optimum, pH 6.0). This strain has the ability to hydrolyze starch Starch, aesculin, Tween 20, 40, and 60, but not Tween 80, casein, DNA, gelatin, and urea. Positive for catalase, oxidase, phenylalanine deaminase, reduce nitrate to nitrite, ferment D-glucose to produce acid, respirate on nitrate and nitrite, produce H2S from L-cysteine, produce poly-β-hydroxyalkanoate, and methyl red test, but negative for lysine and ornithine decarboxylases, o-Nitrophenyl-β-D-galactopyranosidase, produce indole, produce exopolysaccharides and Voges-Proskauer test. Growth without Mg2+, SO42-, and K+. Acid is produced from D-glucose, rhamnose, and arabinose, but not from mannitol, inositol, sorbitol, sucrose, melibiose, and amygdalin. D-Mannose and malic acid are assimilated instead of D-glucose, arabinose, mannose, mannitol, maltose, gluconate, hydroxydecanoate, citric acid, adipic acid, and phenylacetic acid. D-glucose, D-fructose, mannitol, sorbitol, glycerin, inositol, glucuronic acid, maltose, and fumaric acid can be used as sole carbon source, individually. Susceptible to most antibiotics including vancomycin, penicillin, piperacillin, cefradine, ceftriaxone, cefoperazone, gentamicin, kanamycin, neomycin, erythromycin, norfloxacin, ofloxacin, ciprofloxacin, polymyxin B, and Bactrim, but resistant to ampicillin, carbenicillin, oxacillin, cefalexin, tetracycline, minocycline, clindamycin, and deoxytetracycline. The predominant respiratory quinone is Q-9. The major fatty acids of the cells are C16:0 and C19:0 cyclo ω8c. The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phospholipids of unknown structure containing glucosamine, unidentified aminophospholipids, unidentified lipids, and three unidentified phospholipids. The genomic G+C content of the type strain is 61.6 mol%. The degradation rate of 1-naphthylamine is 32.0 mg/L in 14 days.
The type strain, TRM 85114T (= CCTCC AB 2021006T =LMG 32311T), was isolated from the wetland soil of Jincaotan in the Pamir Plateau. The GenBank accession numbers for the 16S rRNA gene and the draft genome sequences of strain TRM 85114T are MW584241 and JAHCLU000000000, respectively.