Halomonas Maris Sp. Nov., a Moderately Halophilic Bacterium Isolated From Marine Sediment in the Southwest Indian Ocean

A halophilic, Gram-staining-negative, rod-shaped, agellated and motile bacterium, strain QX-1 T , was isolated from deep-sea sediment at a depth of 3332 m in the southwestern Indian Ocean. Strain QX-1 T growth was observed at 4–50 °C (optimum 37 °C), pH 5.0–11.0 (optimum pH 7.0), 3%–25% NaCl (w/v; optimum 7%), and it did not grow without NaCl. A phylogenetic analysis based on the 16S rRNA gene placed strain QX-1 T in the genus Halomonas and most closely related to Halomonas suldaeris (97.90%), Halomonas zhaodongensis (97.80%), Halomonas songnenensis (97.59%), Halomonas hydrothermalis (97.37%), Halomonas subterranea (97.25%), Halomonas salicampi (97.09%), and Halomonas arcis (97.01%). DNA–DNA hybridization (< 26.50%) and average nucleotide identity values (< 83.54%) between strain QX-1 T and the related type strains meet the accepted criteria for a new species. The principal fatty acids (> 10%) of strain QX-1 T are C 16:0 (25.50%), C 17:0 cyclo (14.02%), C 19:0 cyclo ω8c (18.72%), and summed feature 8 (C 18:1 ω7c and/or C 18:1 ω6c, 18.08%). The polar lipids of strain QX-1 T are mainly diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unidentied phospholipid, unidentied aminophospholipid, and ve unidentied lipids. The main respiratory quinone is Q-9. The G+C content of its chromosomal DNA is 54.4 mol%. Its fatty acid prole, respiratory quinones, and G+C content also support the placement of QX-1 T in the genus Halomonas. These phylogenetic, phenotypic, and chemotaxonomic analyses indicate that QX-1 T is a novel species, for which the name Halomonas maris is proposed. The type strain is QX-1 T (=MCCC 1A17875 T = KCTC 82198 T = NBRC 114670 T ). H. arcis 1.6494 T . All strains are Gram-staining-negative, aerobic, halophilic, and rod-shaped. The data obtained with API 20NE, API 20E, API ZYM, API 50CH, and GEN III for strain QX-1 T and ve related type strains were examined in this study. Characteristics are scored as: +, positive; −, negative; w, weakly positive. All data were obtained in this study, unless otherwise indicated.

Halomonas is described as containing halophilic or salt-tolerant Gram-staining-negative bacilli. Most species of Halomonas have been isolated from salt lakes, marine environments, or saline soils (Guan et al. 2010;Ming et al. 2020; Poli et al. 2013). Halomonas has a unique structure and special physiological mechanisms, and has high research and utilization value. Some scholars have studied Halomonas strains isolated from marine sediments, nding that Halomonas is strongly adapted to the extreme environment of the deep sea. This is mainly re ected in its tolerance of heavy metal stress and its strong adaptability to changes in temperature, salinity, pressure, and oxygen concentration.
In this study, we report the characterization of a novel bacterium of the genus Halomonas that was isolated from deep-sea sediment in the southwestern Indian Ocean.

Morphological, Physiological, and Biochemical Analyses
To investigate whether strain QX-1 T can grow under anaerobic conditions, we created an anaerobic environment by placing 10 ml of MB medium into a 60 ml anaerobic ask and adding 10 mg/l azurol solution in the ratio of 1000:1 as the oxygen indicator. The pH was adjusted to 7.2 at room temperature.
The anaerobic bottle was pumped with N 2 gas to remove the oxygen in the bottle. During this process, 4 ml 0.5 M Na 2 S.9H 2 O was added to the bottle, and the solution turned weakly red. After the end of ventilation, the anaerobic bottle was immediately sealed with an anaerobic bottle stopper and an aluminum cap, and then sterilized with high-pressure steam. Sterile l-cysteine hydrochloride solution (20 g/l; Hopebiol, China) was added to the sterilized anaerobic MB medium in a ratio of 1%, and the solution turned colorless, indicating that the oxygen in the bottle had been exhausted. The medium was inoculated with strain QX-1 T in the ratio of 1:100, and incubated at 37 °C. API ZYM, API 20NE, API 20E, and API 50CH reagent strips (BioMérieux) and a Gen III MicroPlate (Biolog Inc.) were used to detect the enzyme production, hydrolysis, and substrate utilization of the strain, respectively, according to the manufacturers' instructions, with the single modi cation of adjusting the NaCl concentration to 3.0% for all tests. Seven related type strains were tested at the same time. In the Gen III MicroPlate experiment, IF-A, Gen III Inoculating Fluid was used for the matching test. The turbidity meter was calibrated with the standard turbid tube (85% turbidity), and the IF-A inoculum was initially adjusted to 100% turbidity. Fresh strain QX-1 T was scraped from the MA plate, IF-A inoculum was added to form a bacterial suspension, well-mixed, and the optical density was controlled at 95% turbidity. The prepared bacterial suspension (100 μl) was added to each well of the Gen III plate, which was placed at 37 °C.
To observe the hydrolysis of starch, cellulose, and Tween 20, 40, 60, and 80 by strain QX-1 T , 0.2% (w/v) soluble starch, 0.8% (w/v) cellulose, or 0.5% (v/v) Tween 20, 40, 60, or 80 was added to MA, respectively (Dong and Cai 2001). Oxidase activity was determined with tetramethyl p-phenylenediamine. If the reaction turned purple immediately, it was oxidase positive; otherwise, it was negative. Catalase activity was determined by adding 3% H 2 O 2 to the colony. If a large number of bubbles were generated immediately, the colony was positive for catalase activity; if a small number of bubbles was generated within 1 min, it was weakly positive; if no bubbles were generated, it was catalase negative.  (Felsenstein 1981), and minimum evolution (ME) clustering methods were applied (Rzhetsky and Nei 1992). Bootstrap values were calculated based on 1000 replications. The sequences of related taxa were obtained from the GenBank database and EzBioCloud (Yoon et al. 2017).

Molecular Analysis
DNA-DNA hybridization (DDH) and average nucleotide identity (ANI) are considered the gold standard techniques for the delineation of bacterial species (Chun et al. 2018). To compare strain QX-1 T with other strains, we calculated DDH using the web-based Genome-to-Genome Calculator (GGDC 2.1) (http//ggdc.dsmz.de/ggdc.php) (Oguntoyinbo et al. 2018), and used the EZGenome website to calculate the ANI between two genomes (Goris et al. 2007).

Chemotaxonomic Characterization
The fatty acids of QX-1 T were extracted with the standard Sherlock™ Microbial Identi cation System, version 6.0B (MIDI). Strain QX-1 T and related type strains were cultured on MA at 37 °C for 48 h, and the fatty acids were saponi ed, methylated, and extracted from the whole cells. The fatty acids were analyzed with gas chromatography (Agilent Technologies 6850) and identi ed with the TSBA6.0 database of the Microbial Identi cation System (Sasser 1990).
The polar lipids of strain QX-1 T were extracted with the chloroform-methanol system and analyzed with one-dimensional and two-dimensional thin layer chromatography (TLC) on a Merck silica gel 60 F254 aluminum-backed thin layer plate (Kates 1986). The two-dimensional development of the dot sample plate was performed with chloroform-methanol-water in a volumetric ratio of 65:25:4 as the rst solvent and chloroform-methanol-acetic acid-water in a volumetric ratio of 85:12:15:4 as the second solvent.
The total lipid substances were then detected with molybdenum phosphoric acid, and the speci c functional groups were detected with spray reagents for speci c functional groups.
Quinones were extracted with silica gel TLC, divided into different categories, and analyzed with HPLC (Tindall 1990a;Tindall 1990b).
Biochemical analyses showed that strain QX-1 T produces catalase and oxidase, but does not hydrolyze Tween 20, 40, 60, or 80, starch, or cellulase. It uses d-trehalose, sucrose, d-mannose and other compounds as carbon sources. The phenotypic differences between strain QX-1 T and related type strains are shown in In determining the QX-1 T genome sequence, 1 Gbp of clean data was generated, achieving a ~200-fold The almost full-length 16S rRNA (1451 nt) and the gyrB (2421 nt) and rpoD (1851 nt) gene sequences were obtained from the draft genome of strain QX-1 T . Sequence alignment showed that there was only difference in sequencing coverage between the 16S rRNA sequences isolated from the genome and those determined by PCR.
On a phylogenetic tree constructed with the NJ method, strain QX-1 T formed a clade with H. sul daeris ATCC BAA-803 T (Fig. S1). The maximum-likelihood and minimum-evolution trees of the 16S rRNA gene sequences presented similar topologies to that of the NJ tree, so they were condensed into the NJ tree. Phylogenetic trees were also constructed from the gyrB and rpoD genes ( Fig. S3 and S4), and the 16S rRNA, gyrB, and rpoD genes all indicated that the novel strain QX-1 T is closely related to members of the genus Halomonas.  (Table S2). These DDH values were signi cantly lower than the recommended value of 70% that is considered to de ne a new species (Wayne et al. 1987 (Table S3), all of which met the standard ANI criterion for novel species identity (< 95%-96%) ( Table S1. The polar lipids of strain QX-1 T are mainly diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unidenti ed phospholipid, unidenti ed aminophospholipid, and ve unidenti ed lipids) (Fig. S2). The results of the respiratory quinone test showed that the main component of strain QX-1 T is Q-9. This is consistent with the main respiratory quinone of Halomonas (Dobson and Franzmann 1996).
According to the annotation of the draft genome, strain QX-1 T encodes 60 RNAs, and contains 4473 protein-coding genes. Fifty-nine protein-coding genes in the QX-1 T genome are related to Na + transport (eight genes), K + transport (six genes), trehalose synthesis/metabolism (six genes), ectoine synthesis/metabolism (10 genes), or betaine synthesis/metabolism (29 genes), which may be key elements in the adaptation of QX-1 T to high-salinity environments (Table S4). Strain QX-1 T is a halophilic bacterium, and its normal growth depends on a high concentration of Na + . The genes nhaC and nahD encode Na + /H + antiporters (Ventosa et al. 1998;Yang et al. 2006), and the gene sstT encodes a serine/threonine-Na + symporter, which maintains the stability of the intracellular Na + concentration and prevents the toxic effects of high intracellular Na + . Similarly, there is a K + -regulatory mechanism in halophilic bacteria, which balances the osmotic pressure inside and outside the cell by accumulating high concentrations of K + in the cell. The genes trkA and trkH in the QX-1 T genome are related to the Trk-like K + transport system, which has been reported in H. elongate DSM 2581 T (Kraegeloh et al. 2005). As mentioned above, there are also genes related to trehalose, ectoine, and betaine biosynthesis/metabolism in the strain QX-1 T genome. Under high-salinity conditions, halophilic microorganisms can improve their intracellular water activity by the uptake, synthesis, and accumulation of compatible substances, such as sugars, amino acids, ectoine, betaine, and trehalose (Ben-Amotz and Avron 1983; Oren 2008). The genes doeC, doeX, teaA, teaB, and teaC may be related to the anabolism of ectoine (Grammann et al. 2002;Schwibbert et al. 2011); the gene betT encodes and controls the transformation of choline to betaine under high osmotic pressure (Csonka 1989); and the genes otaB and otaC encode betaine transporters. There are also other protein-coding genes related to trehalose, ectoine, and betaine synthesis, metabolism, and transport in the strain QX-1 T genome, which may guarantee its adaptation to a high-salt environment.

Conclusion
Phenotypic, phylogenetic, and chemotaxonomic analyses have shown that strain QX-1 T belongs to the genus Halomonas. However, it differ in some respects from related type strains. QX-1 T also showed low DDH and ANI values when compared with related type strains. These results con rm that strain QX-1 T is a novel species of the genus Halomonas, for which the name Halomonas maris sp. nov. is proposed.

Taxonomic and Nomenclatural Proposals
Description of Halomonas maris sp. nov.
The type strain, QX-1 T (=MCCC 1A17875 T = KCTC 82198 T = NBRC 114670 T ), was isolated from a deepsea sediment sample at 3332 m in the southwestern Indian Ocean.

Declarations
Author Contributions Xu Qiu performed the technical characterization on strain QX-1 and drafted the manuscript. Xiaorong Cao, Guangxin Xu and Huangming Wu conceived the study and aided to draft the manuscript. Xixiang Tang conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the nal manuscript.

Con icts of interest
The authors declare that there is no con ict of interest.