Halomonas Jincaotanensis sp. nov., Isolated From the Pamir Plateau Degrading Polycyclic Aromatic Hydrocarbon

doi:10.21203/rs.3.rs-1352270/v1

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Halomonas Jincaotanensis sp. nov., Isolated From the Pamir Plateau Degrading Polycyclic Aromatic Hydrocarbon

https://doi.org/10.21203/rs.3.rs-1352270/v1

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A Gram-strain-negative, rod-shaped, aerobic bacterium, designated strain TRM 85114^T, was isolated from the Jincaotan wetland in the Pamir Plateau of China. We identified this isolate was a novel species with an ability to degrade 1-naphthylamine. Strain grew optimally at 30 ℃ and pH 6.0 in the presence of 3% (w/v) NaCl. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain TRM 85114^T was affiliated with the genus Halomonas, and shared high sequence similarity (97.3%) with Halomonas korlensis XK1^T. Strain TRM 85114^T contained C_16:0 and C_19:0 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 85114^T comprises 3,902 putative genes with a total of 4,126,476 bp and a G + C content of 61.6 mol%. The average nucleotide identity and digital DNA–DNA hybridization values between strain TRM 85114^T and related type Halomonas strains was 78.6–88.9% and 25.7–39.2%, respectively. Based on phenotypic, chemotaxonomic, and molecular features, strain TRM 85114^T represents a novel species of the genus Halomonas, duo to which the name Halomonas jincaotanensis sp. nov. is proposed. The type strain is TRM 85114^T (CCTCC AB 2021006^T = LMG 32311^T). Degradation rate of 1-naphthylamine by strain TRM 85114^T reached up to 32.0 mg/L in 14 days.

Halomonas jincaotanensis

Polyphasic taxonomy

Pamir Plateau

Halophilic

1-Naphthylamine degradation

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 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 previously used as platform strains 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). 1-Naphthylamine is a derivative of PAHs, as one of the top 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 Halomonas-like strain isolated from the Pamir Plateau, designated TRM 85114^T, explored its taxonomic characterisation and ability to degrade 1-naphthylamine, which has yet to be reported.

Sample collection, isolation, and preservation

A soil sample was collected from the Jincaotan wetland (37°47´N, 75°16´E) in the Pamir Plateau, 3100 m above sea level. During the isolation process, 100 μL of mixed water and soil sample was spread onto 12% modified growth medium (MGM) agar plates (Dyall-Smith 2015), and incubated at 15 ℃ for 2 weeks. Colonies showing different morphologies were picked and purified, among which strain TRM 85114^T was routinely grown on 12% MGM agar. Colonies were preserved in 12% MGM containing 50% (v/v) glycerol at -80 °C. The reference strains H. korlensis CGMCC 1.6981^T, Halomonas urumqiensis CGMCC 1.12917^T, and Halomonas daqiaonensis CGMCC 1.9150^T were obtained from the China General Microbiological Culture Collection Center (CGMCC), while Halomonas tibetensis KCTC 52660^T was obtained from the Korean Collection for Type Cultures (KCTC). All strains were cultured under the same conditions, as specified, for comparative purposes.

Phenotypic and physiological identification

Cellular morphology was observed under light microscopy (Leica DM1000) and scanning electron microscopy (SEM) (Quanta; FEI) using cells grown on 12% MGM agar plates at 30 ℃ for 4 days. Anaerobic growth was carried out using a MGC AnaeroPack Series (MGC) on 12% MGM agar plates, which replaced O₂ with CO₂, and motility was tested on 0.5% 12% MGM agar. The temperature range (4, 8, 16, 24, 28, 30, 32, 35, 37, 40, and 45 ℃), pH range (5.0-11.0, at 1.0 pH unit intervals) [50 mM MES (pH 5; 6), HEPES (pH 7), TAPS (pH 8), CAPSO (pH 9; 10), and CAPS (pH 11)], and NaCl tolerance (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, and 30 w/v%) for growth were evaluated in 12% MGM broth at 30 ℃ for 3 days. Tolerance to different NaCl concentrations were tested in basic 12% MGM liquid medium without NaCl. Growth requirements of Mg²⁺, SO4^2-, K⁺ as well as production of exopolysaccharide and polyhydroxyalkanoate (PHA) were determined as described previously (Poli et al. 2013). Physiological tests of oxidation/fermentation of D-glucose, respiration of nitrate and nitrite, production of H₂S from L-cysteine, reduction of nitrate to nitrite, hydrolysis of gelatin, casein, starch, Tween (20, 40, 60, and 80), aesculin, and DNA, production of indole, test of methyl red and Voges-Proskauer, activity of catalase, oxidase, urease, phenylalanine deaminase, lysine and ornithine decarboxylases, and o-nitrophenyl-β-D-galactopyranosidase were performed according to the ways proposed previously (Mata et al. 2002; Arahal et al. 2007). Rapid identification systems, including API 20E, API 20NE, and API 50CH (bioMérieux), were applied to detect acid production and substrate utilization in three replicates at 30 ℃ for 72 h, in accordance with the manufacturer’s instructions. Antibiotic susceptibility was detected by filter paper discs containing the following antimicrobial compounds (content per disc): ampicillin (10 μg), vancomycin (30 μg), carbenicillin (100 μg), penicillin (1 μg), oxacillin (1 μg), piperacillin (100 μg), cefalexin (30 μg), cefradine (30 μg), ceftriaxone (30 μg), cefoperazone (75 μg), gentamicin (10 μg), kanamycin (30 μg), neomycin (30 μg), tetracycline (30 μg), deoxytetracycline (30 μg), minocycline (30 μg), erythromycin (15 μg), norfloxacin (10 μg), ofloxacin (5 μg), ciprofloxacin (5 μg), polymyxin B (300 μg), Bactrim (25 μg), or clindamycin (2 μg) on 12% MGM agar plates with inoculated strain TRM 85114^T. The inhibition zones were measured as described previously by Zhong et al. (2016), after incubation at 30 ℃ for 2 days. The test was conducted in triplicate and included only results exhibiting the same resistant pattern in all the plates.

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 ℃ and the microbial cells were harvested at the 4^th 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 (Jain and Subrahmanyam 1978) 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.

Sequence similarity and phylogenetic analysis

Genomic DNA of strain TRM 85114^T was extracted by using a TIANGEN (Beijing, China) bacterial DNA extraction kit. The 16S rRNA gene of strain TRM 85114^T was amplified and sequenced using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACCTTGTTACGACTT-3′). Primers gyrB216F (5′-GARGTBATCATGACSGTGCT-3′) and gyrB1419R (5′-GCRTCSGTCATGATGATSAY-3′), rpoD88F (5′-ATGATYAACGACATGGGYAT-3′) and rpoD1321R (5′-TTSAKCTTRTTGATGGTCTC-3′) were applied to amplified and sequenced the housekeeping genes gyrB and rpoD, respectively. The 16S rRNA gene sequence was then compared with the available sequences using EzBioCloud database (https://www.ezbiocloud.net/identify) (Yoon et al. 2017) and the NCBI database (https://blast.ncbi.nlm.nih.gov) (Altschul et al. 1990). Phylogenetic trees based on neighbor-joining (NJ) (Saitou and Nei 1987), maximum-likelihood (ML) (Felsenstein 1981), and maximum-parsimony (MP) (Czelusniak et al. 1990) algorithms with bootstrap values (1,000 replications) (Felsenstein 1985), and based on Kimura two-parameter model (Kimura 1980) to calculated evolutionary distance, were constructed using MEGA version 7.0 software (Kumar et al. 2016). The 16S rRNA, gyrB, and rpoD gene sequences of strain TRM 85114^T and the most closely species were aligned using CLUSTAL_X software (Thompson et al. 1997), and made multilocus sequence analysis (MLSA) (de la Haba et al. 2012) to infer the phylogenetic relationships of strain TRM 85114^T.

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 depth-sequencing 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://compbio.ornl.gov/prodigal/) (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.ezbiocloud.net/tools/ani) (Lee et al. 2016) and the Genome-to-Genome Distance Calculator (http://ggdc.dsmz.de/distcalc2.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) 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 supergene.

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). Isolate TRM 85114^T was inoculated into 250 mL Erlenmeyer flasks containing 100 mL 12% MGM broth with 1-naphthylamine (5 mg), then incubated in 180 rpm at 30 ℃ for 14 days. Samples (1 mL) were collected on days 0, 2, 4, 6, 8, 10, 12 and 14, and detected the content of 1-naphthylamine using HPLC at 222 nm, based on methanol as gradient elution ranging from 10% to 100% for 40 min. Furthermore, the content of 1-naphthylamine in the samples 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). All the experiments were performed in triplicate, and mean values were reported. The isolates without 1-naphthylamine treatment were used as control.

Phenotypic and physiological characterization

The cells of strain TRM 85114^T 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 85114^T 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 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%), 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 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. 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 85114^T.

Phylogenetic and phylogenomic analyses

Based on the 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%), H. urumqiensis BZ-SZ-XJ27^T (96.1%), 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. Above all analysis of 16S rRNA gene sequences of strain TRM 85114^T, 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 85114^T clustered tightly with H. korlensis XK1^Twith 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 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. 3), 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.

Genomic features and analysis

The obtained draft genome of TRM 85114^T (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 85114^T, 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 85114^T and the type strains of H. korlensis XK1^T, H. urumqiensis BZ-SZ-XJ27^T, H. daqiaonensis CGMCC 1.9150^T 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 85114^T 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 85114^T 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 85114^T was further 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. S5). According to the standard curve of 1-naphthylamine (Supplementary Fig. S6), the degradation rates of strain TRM 85114^T on day 4and 14was calculated to be 21.6 mg/L and 32.0 mg/L, respectively (Supplementary Fig. S7). These findings indicated that TRM 85114^T 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 85114^T has important environmental remediation ability could contribute for bioremediation of water and/or soil systems.

Taxonomical conclusion

The results of phylogenetic analyses based on 16S rRNA gene, housekeeping gene, and core gene analyses illustrate that strain TRM 85114^Twas 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.

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⁺, SO4^2-, 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 C_16:0 and C_19:0 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 85114^T (= CCTCC AB 2021006^T =LMG 32311^T), 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 85114^T are MW584241 and JAHCLU000000000, respectively.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (31900007), Bingtuan Science and Technology Program (2018BC008), the Microbial Resources Utilization Innovation Team in Key Field of Xin Jiang Production and Construction Corps (2017CB014), and the principal fund (TDHNLH201604).

Author contributions

XB performed the experiments, analyzed the data, and drafted the manuscript. ZL conducted the degradation test of 1-naphthylamine. ZX, CW, and MR critically revised the manuscript. LZ contributed to the creation. All authors read and approved the manuscript. We thank HC and PX for finding and providing Halomonas tibetensis pyc13^T.

Conflict of interests

All authors declare no conflict of interest.

Ethical approval

No specific ethical or institutional permission was required for sampling, and our experimental studies did not involve endangered or protected species.

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Table 1

Differential characteristics of strain TRM 85114^T and related species of the genus *Halomonas.*
Characteristic	1	2	3	4	5
Colony pigmentation	Cream–white	Brown	Brown	Cream–yellow	Cream–yellow
Cell morphology	Short rods	Rods or short^ǂ rods^ǂ	Rods^†	Short rod^⸸	Rods^‡
Cell size (µm)	0.4–0.5×0.9–1.0	0.8–1.0×1.3–1.7^ǂ	0.4×0.6–1.2^†	1.0×1.5–2.0^⸸	0.3–0.6×1.2–2.2^‡
Mobility	–	–	–	+	+
Temperature range (℃)	4–35 (30)	4–37 (30)	8–37 (30)	4–40 (37)	4–40 (37)
Salinity range (%, w/v)	1–10 (3)	1–20 (7)	1–15 (6)	1–25 (8)	1–15 (8)
pH range	6–9 (6)	6–9 (7)	6–10 (8)	6–9 (8)	6–9 (8)
Hydrolysis of:
Starch	+	+	–	–	–
Tween40	+	–	–	–	–
Tween60	+	–	–	–	–
H₂S production	+	+	–	–	–
Nitrate reduction	–	+	–	+	+
Voges-Proskauer test	+	+	+	+	–
Urease activity	–	+	–	–	–
Phenylalanine deaminase activity	+	–	–	–	+
Exopolysaccharides	–	+	+	–	–
Acid from (API 20E):
D-Glucose	+	+	–	+	–
Sucrose	–	+	–	+	–
Rhamnose	+	–	–	+	–
Arabinose	+	+	–	–	–
Melibiose	–	+	–	–	–
Assimilation of (API 20NE):
D-Glucose	–	–	–	+	–
D-Mannose	+	+	–	+	–
Mannitol	–	–	–	+	–
Maltose	–	–	–	+	–
Gluconate	–	+	–	+	–
Citric acid	–	+	–	+	–
Adipic acid	–	+	–	–	–
Malic acid	+	+	–	+	–
Utilization as sole carbon source (API 50CH):
D-Galactose	+	+	–	–	+
D-Lactose	–	+	+	+	–
D-Mannose	+	+	–	+	–
D-Arabinose	+	+	+	+	–
D-Ribose	+	+	+	+	–
D-Xylose	–	+	–	+	–
D-Melibiose	–	+	–	+	–
D-Gentiose	–	+	–	–	–
D-Fucose	–	+	–	–	–
Sensitivity to:
Ampicillin	–	+	+	–	+
Vancomycin	+	–	–	–	+
Carbenicillin	–	–	+	–	+
Cefalexin	–	+	+	+	+
Tetracycline	–	–	–	–	+
Erythromycin	+	+	+	–	–
Neomycin	+	–	+	–	–
Oxacillin	–	–	–	–	+
Doxycycline	–	–	+	–	+
Clindamycin	–	–	–	+	–
Polymyxin B	+	+	+	–	+
Taxa: 1, Strain TRM 85114^T; 2, H. korlensis CGMCC 1.6981^T; 3, H. tibetensis KCTC 52660^T; 4, H. urumqiensis CGMCC 1.12917^T; 5, H. daqiaonensis CGMCC 1.9150^T. Except special note parts, data are from this study. +, positive; –, negative; ND, not determined.
^ǂ Data from Li et al. (2008); ^† Data from Lu et al. (2018); ^⸸Data from Zhang et al. (2016); ^‡ Data from Qu et al. (2011)

Table 2 Genomic characteristics^† of strain TRM 85114^T and closely related species.

Attribute		1	2	3	4
Genome size (Mb)		4.13	3.62	3.98	3.72
DNA G + C content (%)		61.6	62.6	61.7±0.8	63.7
Coting		60	108	22	49
Total genes		3,892	3,795	3,597	3,512
Protein coding genes		3,738	3,346	3,504	3,351
RNA genes		137	79	65	71
rRNA genes (5S/16S/23S)		3	7	6	13
tRNA genes		57	54	55	54
Other RNAs		77	18	4	4
GenBank accession number		JAHCLU000000000	FPBP00000000	PYUD00000000	FOBC00000000
		dDDH
ANI value (%)^‡	1	−	39.2	25.7	28.1
	2	88.9	−	21.6	22.4
	3	78.6	77.6	−	21.8
	4	80.5	79.2	80.0	−

Taxa: 1, strain TRM 85114^T; 2, H. korlensis XK1^T; 3, H. urumqiensis BZ-SZ-XJ27^T; 4, H. daqiaonensis CGMCC 1.9150^T.

^†The bioinformatic analysis of the genomes was carried out using the NCBI prokaryotic genome annotation pipeline (www.ncbi.nlm.nih.gov/genome/annotation_prok/).

^‡ANI, average nucleotide identity.

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Editorial decision: Major revisions
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Halomonas Jincaotanensis sp. nov., Isolated From the Pamir Plateau Degrading Polycyclic Aromatic Hydrocarbon

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