Isolation and Culture
Strain QX-1T was isolated from deep-sea sediment at a depth of 3332 m in the southwestern Indian Ocean (China Ocean 40 voyages, leg III, 55.15°W, 34.32°S, TV grab sampling). To isolate this strain, we added the sediment to marine broth (MB; BD Difco) containing 10% NaCl (w/v). After 7 days in shaking culture at 10 °C, the culture was serially 10-fold diluted (10−1, 10−2, 10−3, 10−4, 10−5), and the diluted cultures were plated onto marine agar (MA; BD Difco) containing 10% NaCl (w/v) and placed at 10 °C. After culture for about 30 days, a large number of colonies were observed, and single colonies were picked and streaked repeatedly to obtain pure cultures. The pure bacterial liquid cultures were stored in 15% glycerol solution at −80 °C.
Morphological, Physiological, and Biochemical Analyses
A Gram-staining kit (Hangzhou Tianhe Microbial Reagent Co., Ltd) was used to test the bacterium, according to the manufacturer’s instructions. The morphology of the cells was observed with a transmission electron microscope (JEM-1230, JEOL) (Fig. S1, available in the online Supplementary Material). Cell movement was observed with the hanging drop method (Skerman 1960).
The temperature range of QX-1T growth was determined in MB by incubating cultures at 0, 4, 10, 15, 20, 25, 30, 37, 45, 50, 55, and 60 °C. The pH range for growth was determined in MB at pHs 3.0–12.0 in intervals of 1 pH unit, established with citric acid/phosphate (pH 3.0–7.0), Tris/HCl (pH 8.0–9.0), or sodium carbonate/sodium bicarbonate buffers (pH 10.0–12.0). The formula of MB was adjusted so that the salinity of the medium was 0, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or 30% (w/v), and was used to determine the salinity growth range of QX-1T.
To investigate whether strain QX-1T can grow under anaerobic conditions, we created an anaerobic environment by placing 10 ml of MB medium into a 60 ml anaerobic flask 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 N2 gas to remove the oxygen in the bottle. During this process, 4 ml 0.5 M Na2S.9H2O 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-1T 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 modification 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-1T 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-1T, 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% H2O2 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.
A bacterial genome extraction kit (SBS) was used according to the manufacturer’s instructions to extract the genomic DNA of QX-1T. The 16S rRNA gene was amplified with the universal bacterial primers 27F (5¢-AGAGTTTGATCCTGGCTCAG-3¢) and 1492R (5¢-TACGGTTACCTTGTTACGACTT-3¢) (Lane 1991) and Ex Taq DNA Polymerase in a 50 μl amplification system (Sangon Biotech, China).
The draft genome of QX-1T was determined by Shanghai Majorbio Bio-Pharm Technology Co., Ltd (Shanghai, China), using the Illumina paired-end (500 bp library) sequencing technique. The clean data were assembled with SPAdes v 3.8.1 with the default settings (Bankevich et al. 2012). Contigs longer than 1 kb and with similar read coverage were retained for further analysis. The G+C content of the chromosomal DNA of strain QX-1T was determined from the draft genomic sequence. The RAST website (https://rast.nmpdr.org/) was used to annotate the genomic data of strain QX-1T.
The 16S rRNA and gyrB and rpoD gene sequences were extracted from the draft genomic data of strain QX-1T. We used the EzBioCloud program (https://www.ezbiocloud.net) to compare the 16S rRNA gene sequences (Kim et al. 2012; Maidak et al. 2000) and analyzed the gyrB and rpoD gene sequences in the GenBank database with BLAST.
A phylogenetic analysis was performed with MEGA version X (Kumar et al. 2016). The distance option was used according to the Kimura two-parameter model, and the neighbor-joining (NJ) (Saitou and Nei 1987), maximum-likelihood (ML) (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).
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-1T 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).
The fatty acids of QX-1T were extracted with the standard Sherlock™ Microbial Identification System, version 6.0B (MIDI). Strain QX-1T and related type strains were cultured on MA at 37 °C for 48 h, and the fatty acids were saponified, methylated, and extracted from the whole cells. The fatty acids were analyzed with gas chromatography (Agilent Technologies 6850) and identified with the TSBA6.0 database of the Microbial Identification System (Sasser 1990).
The polar lipids of strain QX-1T 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 first 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 specific functional groups were detected with spray reagents for specific functional groups.
Quinones were extracted with silica gel TLC, divided into different categories, and analyzed with HPLC (Tindall 1990a; Tindall 1990b).