Physiological and genomic features of Paraoceanicella profunda gen. nov., sp. nov., a novel piezophile isolated from deep seawater of the Mariana Trench

Abstract A novel piezophilic alphaproteobacterium, strain D4M1T, was isolated from deep seawater of the Mariana Trench. 16S rRNA gene analysis showed that strain D4M1T was most closely related to Oceanicella actignis PRQ‐67T (94.2%), Oceanibium sediminis O448T (94.2%), and Thioclava electrotropha ElOx9T (94.1%). Phylogenetic analyses based on both 16S rRNA gene and genome sequences showed that strain D4M1T formed an independent monophyletic branch paralleled with the genus Oceanicella in the family Rhodobacteraceae. Cells were Gram‐stain‐negative, aerobic short rods, and grew optimally at 37°C, pH 6.5, and 3.0% (w/v) NaCl. Strain D4M1T was piezophilic with the optimum pressure of 10 MPa. The principal fatty acids were C18:1 ω7c/C18:1 ω6c and C16:0, major respiratory quinone was ubiquinone‐10, and predominant polar lipids were phosphatidylglycerol, phosphatidylethanolamine, and an unidentified aminophospholipid. The complete genome contained 5,468,583‐bp with a G + C content of 70.2 mol% and contained 4,855 protein‐coding genes and 78 RNA genes. Genomic analysis revealed abundant clues on bacterial high‐pressure adaptation and piezophilic lifestyle. The combined evidence shows that strain D4M1T represents a novel species of a novel genus in the family Rhodobacteraceae, for which the name Paraoceanicella profunda gen. nov., sp. nov. is proposed (type strain D4M1T = MCCC 1K03820T = KCTC 72285T).


| INTRODUC TI ON
The deep sea, accounting for approximately 75% of the total ocean volume and hosting 62% of the global biosphere (Fang, Zhang, & Bazylinski, 2010), is a reservoir of remarkably diverse archaea and bacteria. The extreme physical-chemical factors (high salinity, high pressure, and low temperature) in the deep sea may have considerable influences on microbial life. For example, high pressure, the most unique physical parameter in the deep sea, decreases membrane permeability and stability, impedes energy metabolism, and inactivates proteins (Jebbar, Franzetti, Girard, & Oger, 2015;Picard & Daniel, 2013). Thus, piezophiles must evolve physiological and genomic adaptations to grow under high-pressure conditions.
Microorganisms use different strategies to thrive in high-pressure conditions, such as synthesizing piezolytes, improving permeability and stability of cell membrane, regulating gene expression, and modifying genome features (Oger & Jebbar, 2010;Simonato et al., 2006).
Despite the fact that greater than 88% of the ocean's biosphere is above 10 MPa (water depths of 1,000 m or more), a limited number of piezophiles have been isolated and characterized (Picard & Daniel, 2013;Zhang, Wu, & Zhang, 2018).

| DNA extraction, genomic, and phylogenetic analyses
Genomic DNA was extracted from liquid cultures of strain D4M1 T after being cultivated in MB for 36 hr using the ChargeSwitch® gDNA Mini Bacteria Kit (Life Technologies) according to the manufacturer's instructions. The 16S rRNA gene of strain D4M1 T was amplified and sequenced by using conserved primers Bac8F (5′-AGAGTTTGATCATGGCTCAG-3′) and U1492R (5′-GGTTACCTTGTTACGACTT-3′), as reported previously (Cao et al., 2016). The 16S rRNA gene sequence was identified using global alignment algorithm implemented at the EzBioCloud server (https ://www.ezbio cloud.net/; (Yoon et al., 2017)). Phylogenetic analysis of 16S rRNA gene was conducted with MEGA 5.0 package (Tamura et al., 2011), using the Kimura two-parameters model with the neighbor-joining (Saitou & Nei, 1987) and maximum-likelihood (Felsenstein, 1981) algorithms, respectively. The tree topology was calculated by bootstrap analysis based on 1,000 bootstraps.
Purified genomic DNA was quantified by TBS-380 fluorometer (Turner BioSystems Inc.). The complete genome was sequenced using a combination of Pacific Biosciences (PacBio) RS and Illumina sequencing platforms (Shanghai Majorbio Bio-pharm Technology Co., Ltd.). For PacBio sequencing, 8-10 k insert whole-genome shotgun libraries were generated and sequenced on a PacBio RS instrument using standard methods. For Illumina sequencing, 500 bp pairedend library were generated and sequenced using Illumina Hiseq Xten. The genome was assembled using Velvet assembler (v1.2.09) with a kmer length of 17 and "PacBioToCA with Celera Assembler" pipeline (Chin et al., 2013;Koren et al., 2012)

| Phenotypic, physiologic, and biochemical analyses
Images of cells of strain D4M1 T were obtained with a transmission electron microscopy (JEM-1230; Jeol) after glutaraldehyde prefixation and uranyl acetate staining of cells grown on MA at 37°C for 30 hr. Growth characteristics were determined by the measurement of optical density at 600 nm (OD 600 ) using a NanoDrop 2000c spectrophotometer (Thermo Scientific). The growth temperature was evaluated at 4, 10, 20, 25, 30, 37, 40, 45, and 50°C in dupli-cates in 10 days. The salinity range (0, 0.5, and 1%-10% (intervals of 1%) of NaCl, w/v) and pH range (pH 4.0-11.0 (intervals of 1 unit), added with 20 μmol/L HOMOPIPES, MES, PIPES, HEPES and CAPS buffers, respectively) were investigated as previously described in duplicates (Lai et al., 2014). Gram-staining, oxidase, and catalase activity were carried out according to the test procedures described by Dong and Cai (2001). Growth under anaerobic condition was tested in LB liquid medium (for fermentation) and in LB supplemented with Na 2 SO 4 or NaNO 3 (10 mmol/L, for anaerobic respiration) with oxygen-free N 2 gas phase (200 kPa) in sealed sterile vials at 37°C for 7 days. Poly-β-hydroxybutyrate (PHB) production was determined by using Nile blue A staining and an upright fluorescence microscope (ECLIPSE Ni-U; Nikon) according to a previous study (Ostle & Holt, 1982). Determination of the hydrostatic pressure range for growth was carried out in hydrostatic pressure vessels under a pressure range of 0.1-80 MPa (intervals of 10 MPa) at the optimal temperature (37°C), with oxygen-saturated Fluorinert (FC-40, 3M Company. 25% of total volume) added to supply oxygen (Kato, Sato, & Horikoshi, 1995). Other biochemical tests were carried out using API 20NE, API ZYM strips (bioMérieux) and GEN III microplates by Biolog system (Biolog Microstation™) according to the manufacturer's instructions.
Some tests in API strips, such as reduction of nitrate, fermentation of D-glucose, hydrolysis of aesculin, and utilization of citrate, were also re-examined by conventional biochemical identification as described by Dong and Cai (2001).

| Chemotaxonomic analysis
The fatty acid and polar lipid profiles of strain D4M1 T were analyzed on exponential growth phase of cultures grown in MB at 37°C for 48 hr. Fatty acids in whole cells were saponified, extracted, and methylated using the standard protocol of Microbial IDentification Inc. (MIDI, Sherlock Microbial Identification System, version 6.0B).
The fatty acids were analyzed by gas chromatography (GC, Agilent Technologies 6850) and identified by using the TSBA 6.0 database of the Microbial Identification System (Sasser, 1990). Polar lipids were extracted from 100 mg of freeze-dried cells using a chloroform/methanol system, separated by two-dimensional thin-layer chromatography (TLC) on silica gel 60 F 254 plates (Merck), and then identified with molybdophosphoric acid as the spray reagent according to a previously described method (Tindall, Sikorski, Smibert, & Krieg, 2007). The fatty acid and polar lipid profiles of reference strains Oceanicella actignis DSM 22673 T and Thioclava electrotropha DSM 103712 T were performed in parallel with strain D4M1 T under the same condition. The respiratory quinone was extracted from freeze-dried cells with chloroform/methanol (2:1, v/v) and evaporated to dryness at 35°C. The extracts were resuspended in chloroform/methanol (2:1, v/v) and subsequently purified by TLC on GF 254 silica gel plates (Branch of Qingdao Haiyang Chemical Co. Ltd.) with F I G U R E 1 Neighbor-joining tree showing the phylogenetic positions of strain D4M1 T and related species, based on 16S rRNA gene sequence. Chromatocurvus halotolerans EG19 T was used as outgroup. Filled circles indicate nodes that were also recovered in the maximum-likelihood ( Figure A1) tree for the same sequences. Bootstrap values (expressed as percentages of 1,000 replications) greater than 50% are shown at branch nodes. Bar, 0.01 nucleotide substitution rate (K nuc ) units n-hexane/ether (17:3, v/v). The respiratory quinones were measured by HPLC-MS system (Agilent) (Wu et al., 2015).

| Phylogenetic and phylogenomic analyses
16S rRNA gene sequence analysis showed that strain D4M1 T had the highest 16S rRNA gene sequence similarity of 94.2% with Oceanicella actignis PRQ-67 T and Oceanibium sediminis O448 T , followed by Thioclava electrotropha ElOx9 T (94.1%). Genera are generally described as agglomerates of nodal species and internodal strains (Gillis, Vandamme, De Vos, Swings, & Kersters, 2001), for which similarity values around 94.5%-95% are commonly used for genus differentiation (Ludwig et al., 1998;Yarza et al., 2014). Based on these criteria, strain D4M1 T likely represent a novel genus in the family Rhodobacteraceae. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain D4M1 T formed an independent monophyletic branch paralleled with the genus Oceanicella within the family Rhodobacteraceae, suggesting that it may represent a novel genus within the family Rhodobacteraceae ( Figure 1 and Figure A1).
Phylogenomic analysis, previously suggested to provide a better taxonomic framework at the genus and higher levels (Chun et al., 2018), was further carried out to provide a better taxonomic characterization. A total of 2.36 Gb of clean data were generated from the genome sequencing of D4M1 T . The final assembly has 431-fold coverage for the complete genome, which contains 5,468,583-bp with a G + C content of 70.2 mol%. The complete genome consists of a circular chromosome of 4,417,125 bp and six plasmids ranging from 112,235 bp to 586,520 bp in length (Table 1 and Figure 2).
The assembled and annotated genome of D4M1 T has been deposited in GenBank (accession numbers: CP040818-CP040824) and JGI portal (GOLD ID: Gp0432545; IMG Taxon ID: 2828513066).
A whole-genome-based phylogenomic tree ( Figure 3) showed that strain D4M1 T formed an independent monophyletic branch within the family Rhodobacteraceae. This result supports that strain D4M1 T represents a genus-level taxon in agreement with the result of 16S rRNA gene phylogeny.
Growth of the novel strain occurs between pH 5.0-8.0 (optimum 6.5), 10-45°C (optimum 37°C), and in the presence of 0.5%-8.0% (w/v) NaCl (optimum 3.0%). The novel strain contains poly-β-hydroxybutyrate (PHB) inside the cells. Strain D4M1 T is piezophilic, with the optimum growth pressure of 10 MPa and tolerance up to 70 MPa ( Figure A3). Anaerobic growth was not observed in LB medium nor in LB medium supplemented with 10 mmol/L of Na 2 SO 4 or NaNO 3 . Results of carbon utilization (Biolog GEN III), API ZYM and 20NE tests are given in Table 2 and the species description below.
Strain D4M1 T is distinguishable from their closest relatives in physiological characteristics as shown in Table 2.
C 18:1 ω7c/C 18:1 ω6c were present in a much higher amount in reference strains DSM 22673 T and DSM 103712 T than in strain D4M1 T , but the amount of C 16:0 was much lower in the reference strains than in strain D4M1 T .
The major isoprenoid quinone of strain D4M1 T was ubiquinone 10 (Q-10), which was the same as its related taxa in the family Size ( several unidentified phospholipids (PL) as shown in Figure A4, which were similar to those of reference strains DSM 22673 T and DSM 103712 T , except some minor differences in unidentified phospholipids.

| Genome annotation and analysis
The genome was shown to encode 4,942 predicted genes includ- Microbes are thought to preserve membrane fluidization and functionality at high pressure and low temperature in the deep sea by increasing the proportion of unsaturated fatty acids in their membrane lipids (Cao et al., 2015;Simonato et al., 2006).

Strain D4M1 T contains high proportions of monounsaturated
fatty acids, summed feature 8 (41.7%, C 18:1 ω7c/C 18:1 ω6c), probably for improving membrane piezo-adaptation. Genomic analysis showed the presence of thirty-seven genes involved in biosynthesis of unsaturated fatty acids, including four fatty acid desaturase genes (Table A3). Pressure-induced chaperones proposed to help in maintaining protein folding (Oger & Jebbar, 2010) were also encoded adjacent to the unsaturated fatty acids biosynthesis genes in D4M1 T genome, including the OmpH which was thought to function as a nutrient transporter in nutrient-limited deep sea (Table A3).
It is well known that many piezophiles change their respiratory chains in order to adapt to pressure (Oger & Jebbar, 2010). The genome was found to contain genes encoding cytochrome bd-type quinol oxidase and cytochrome cbb protein complex (Table A3), which were involved in specific piezo-adaptations in respiratory chain (Chikuma, Kasahara, Kato, & Tamegai, 2007;Qureshi, Kato, & Horikoshi, 1998). F 1 F 0 ATP-synthase was shown to facilitate energyyielding processes in high-pressure adaptation (Souza, Creczynski-Pasa, Scofano, Graber, & Mignaco, 2004). It was remarkable that two sets of the F 1 F 0 ATP-synthase genes were identified in the genome of strain D4M1 T (Table A3).
PHB was detected in the cells of strain D4M1 T in this study, and genes that encoded the enzymes required for β-HB and PHB synthesis were present in the genome, including 1 β-HB dehydrogenase and 3 polyhydroxyalkanoate synthase genes (Table A3). The PHB inside the cells could also serve as intracellular carbon and energy reserves, which have been linked to pressure adaptation (Martin et al., 2002;Methe et al., 2005). Genes involved in biosynthesis and transport of compatible solutes, such as glycine betaine, were also identified in the genome, including genes encoding choline dehydrogenase and transcriptional repressor BetI (Table A3). It was suggested that trehalose protects proteins and cellular membranes from inactivation or denaturation caused by a variety of stress conditions, including high hydrostatic pressure (Simonato et al., 2006). Nineteen genes in the genome were predicted to encode trehalose biosynthesis and trehalose-specific transporters (Table A3), which were probably involved in pressure adaptation.
Additionally, the genome of D4M1 T has six copies of glnA, including the counterpart of the pressure-upregulated glnA (IMG Gene OID: 2828515862) in piezophile Shewanella violacea DSS12 (Ikegami, Nakasone, Kato, Nakamura, et al., 2000). Furthermore, the pressure-regulated regulator ntrBC in S. violacea DSS12 was also identified in the genome of D4M1 T (Table A3), which was predicted to play a role in activation of transcription of pressure-regulated promoters .
The increasing number of rRNA operons in a bacterial genome was previously proposed to represent a strategy for adapting to specific selective pressures from the environment (Klappenbach, Dunbar, & Schmidt, 2000). The genome of the strain was found to contain four rRNA operons (Table 1), which may correlate with the adaptation to the deep-sea environment. Pressure is thermodynamically coupled to temperature. One "universal" response to environmental pressures is the biosynthesis of stress proteins (Kültz, 2005). The genome encoded 6 heat shock protein genes and 4 cold shock protein genes (Table A3), which were previously reported to be induced when exposed to high pressure (Simonato et al., 2006). Our results suggest that hydrostatic pressure is an important environmental stress that drives the adaptation of heat shock protein genes and cold shock protein genes in deep-sea microorganisms.

Strain D4M1 T exhibits the typical characteristics of the family
Rhodobacteraceae, but it is also distinguishable from its closest relatives in the phylogenetic analysis of 16S rRNA gene sequence, the phylogenomic analysis based on whole-genome protein sequences, the fatty acids profiles, the enzyme activities, the carbon utilization, the G + C contents, and the low 16S rRNA gene sequence similarity (≤95.8%) to the type species of the closely related genera of the family Rhodobacteraceae. Therefore, from the polyphasic evidence, strain D4M1 T represents a novel species of a novel genus for which the name Paraoceanicella profunda gen. nov., sp. nov. is proposed.
Cells are aerobic, Gram-stain-negative, oxidase-and catalasepositive, short rods (1.0-1.5 × 0.6-0.8 μm). The G + C content of the genomic DNA of the type strain of the type species is 70.2 mol%.
The type species is Paraoceanicella profunda.

E TH I C S S TATEM ENT
None required.

ACK N OWLED G M ENTS
The

CO N FLI C T O F I NTE R E S T S
None declared.

AUTH O R CO NTR I B UTI O N S
JC supervised the project. PL, WD, and QL carried out the experiments. LP and JC analyzed the data. PL, JC, and JF wrote the manuscript with support from RL, YW, LW, and ZX.