2 University of Chinese Academy of Sciences, Beijing 100049, China;
3 International Associated Laboratory of Evolution and Development of Magnetotactic Multicellular Organisms, CNRS-Marseille/CAS-Beijing-Qingdao-Sanya;
4 CAS Key Laboratory for Experimental Study under Deep-sea Extreme Conditions, Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences, Sanya 572000, China;
5 AMU, LCB UMR 7283, CNRS-Marseille, 143402, France
The deep-sea with depth over 1 000 m accounts for over 75% of the ocean. Our current knowledge of the microorganisms inhabiting the deep-sea environment is generally obtained by means of high-throughput sequencing, due to the difficulties in bacterial cultivation and analysis under HHP conditions. Metagenomic analysis demonstrated that Gammaproteobacteria and Alphaproteobacteria are the most abundant taxa in diverse deep-sea environments, and Deltaproteobacteria, Acidobacteria and Actinobacteria are often detected as well (DeLong et al., 2006; Sogin et al., 2006; Lauro and Bartlett, 2008; Salazar et al., 2016; Tarn et al., 2016). Stratified deep-sea bacterial community composition has been observed in different sites. Chemolithotrophic microbes are more abundant in the upper abyssal water while heterotrophic, methane and hydrogen utilizing, and sulfur-cycling microbes are enriched in the hadal water (Nunoura et al., 2015; Tarn et al., 2016). However, the molecular basis of deep-sea adaptation remains unresolved since few of the deepsea bacteria have been successively cultivated in laboratory conditions. Piezophiles are microbes that favor growth at pressure conditions higher than atmospheric pressure (0.1 MPa). The majority of piezophiles isolated and cultivated to date are affiliated to the genera of Shewanella, Photobacterium, Colwellia, Moritella and Psychromonas of Gammaproteobacteria (Fang et al., 2010). The studies confined to limited species of Shewanella and Photobacterium demonstrated that bacterial adaptation to the deep-sea environment is a multifactor event including alterations in the composition and structure of macromolecules, regulation of gene transcription and metabolic pathway (Tamegai et al., 2012; Ohke et al., 2013).
TMAO is an important component of organic nitrogen in the marine ecological environment. Its concentration in surface water reaches 76.9 nmol/dm3, higher than all the other methylamines (Gibb and Hatton, 2004; Ge et al., 2011). TMAO can be produced through oxidation of trimethylamine (TMA) by a variety of marine bacteria, phytoplankton, invertebrates and fishes (Barrett and Kwan, 1985; Seibel and Walsh, 2002; McCrindle et al., 2005). Due to the difficulties in sampling techniques and TMAO detection approaches, the concentration of TMAO in deep-sea environment remains unknown. However, highly accumulated TMAO (up to 386±18 mmol/kg) has been observed in the muscle tissue of deep-sea animals, where it functions as an osmolyte and protects the proteins from multiple stresses including low temperature, high concentration of urea and HHP (Yancey et al., 1982, 2001, 2014; Gillett et al., 1997; Saad-Nehme et al., 2001; Zou et al., 2002; He et al., 2009; Petrov et al., 2012). It is speculated that TMAO released from deep-sea animals may create a microenvironment with instant high concentration of TMAO and provides bacteria nearby precious nutrient for growth (Zhang et al., 2016).
Marine microorganisms metabolize TMAO through two different pathways. SAR11 clade and marine Roseobacter clade (MRC) bacteria could utilize TMAO as a carbon and nitrogen source (Lidbury et al., 2014, 2015). TMAO is transported into the cytoplasm through a TMAO-specific ABC transporter system and converted into DMA (dimethylamine) by TMAO demethylase. DMA is further catalyzed into MMA (monomethylamine) and ammonia, and participates into diverse metabolism pathways (Zhu et al., 2014). In addition, diverse species of marine bacteria, including Alteromonas, Campylobacter, Flavobacterium, Photobacterium, Pseudomonas and Vibrio, and most species of Enterobacteriaceae are known to use TMAO as electron acceptor of respiration under anaerobic conditions (Barrett and Kwan, 1985; Dos Santos et al., 1998; Dunn and Stabb, 2008). TMAO reductase that is responsible for TMAO anaerobic respiration has also been detected in numerous deep-sea bacterial strains. Moderately piezophiles P. profundum SS9 and P. phospherum ANT-2200 encode 3 and 4 sets of TMAO reductase (TorA), respectively. Previously, we observed that one of the TMAO reductase isozyme TorA1 is induced under HHP condition in ANT-2200 (Zhang et al., 2016). In addition, we discovered, for the first time, that TMAO improves the pressure tolerance of a piezo-sensitive strain Vibrio fluvialis QY27, and an HHP inducible TMAO reductase is involved in this process (Yin et al., 2018). Yet, whether the utilization of TMAO and the TMAO-promoted pressure tolerance is a common trait of deep-sea bacteria remains unknown.
To answer these questions, we analyzed the ability of TMAO utilization and the pressure tolerance with or without the presence of TMAO of over 200 strains isolated from the South China Sea and the Mariana Trench. Our results demonstrated that there was no apparent correlation between the depth where the bacteria inhabit and their pressure tolerance, regarding to these samples. Strains from the genera of Alteromonas, Halomonas, Marinobacter, Photobacterium and Vibrio showed capacity of TMAO utilization, but none of the isolated Acinebacter, Bacillus, Brevundimonas, Muricauda, Novosphingobium, Rheinheimera, Sphingobium and Stenotrophomonas did. Furthermore, we noticed that TMAO has greater impact on the growth of deep-sea isolates of Vibrio neocaledonicus than shallow-water isolates. For most isolates, the utilization of TMAO does not change their pressure tolerance. Therefore, the TMAO-improved pressure tolerance we recently reported might be a species-specific trait of V. fluvialis.2 MATERIAL AND METHOD 2.1 Sample collection
Seawater samples of the South China Sea were collected at different depths with Niskin bottles mounted on the rosette sampler. Seawater samples of the Mariana Trench were collected by pressureretaining sampler (Top Industrie, France) mounted on the rosette sampler or Niskin bottles carried by seabed lander. Sediment samples were collected by box sampler or push-core carried by seabed lander. Deepsea amphipods were captured using baited traps carried by seabed lander. The information of sampling sites is listed in table 1.2.2 Isolation of deep-sea bacteria
The samples collected from the South China Sea and the Mariana Trench were first incubated under high pressure vessels (Feiyu Technology Development Co. Ltd., China) for the enrichment of pressure tolerant bacteria. To specify, seawater samples were inoculated into cultural medium YPG (Martini et al., 2013), 2216E (Simon-Colin et al., 2008) and R2A (Smith et al., 2004) with inoculums of 1/10. Sediment samples were processed by mixing 10 mL medium with 1 g sediment. Regarding to the deep-sea amphipods, an individual with length of approximately 1.5 cm was incubated with 3 mL cultural medium. After incubation under high pressure condition under ambient temperature for 10 days, a series dilution was prepared for each of the samples and spread on solid medium for the purification of single colonies. Purified strains were cultured in the corresponding medium at room temperature (approximately 23– 25℃) for molecular identification and strain conservation.2.3 Phylogenetic analyses based on 16s rRNA gene sequences
The 16S rRNA sequences were amplified with 27F and 1492R primers, and BLAST against the EzBioCloud Database (https://www.ezbiocloud.net/identify) (Yoon et al., 2017). Sequence identity of 97% and 95% are used as the thresholds for classification of species and genus, respectively (Tindall et al., 2010). The phylogenetic tree was established with the sequences of 16S rRNA, using neighbor-joining (N-J) method in the software MEGA 5, in which 1 000 times repeated bootstrap analysis was used to test the credibility of the phylogenetic tree.2.4 Cultivation of deep-sea bacterial isolates
For evaluation of growth at different conditions, bacterial isolates were cultivated in 1 mL liquid medium overnight before inoculated into fresh medium with inoculums of 1:100. The cultures were then transferred into a disposable syringe and blocked with sterilized stopper. The syringes were placed in a high-pressure vessel and pressure was applied with a water pump (Top Industrie, France). After cultivation for 24 h, absorption at 600 nm was measured with a spectrophotometer (Agilent Technologies, the United States). TMAO was supplemented to a final concentration of 1% (w/v) unless otherwise mentioned. All the culture experiments were carried out at room temperature (23–25℃).
For the measurement of the growth curve, cells were cultivated under the condition of 0.1, 10, 20, 30, 40 and 50 MPa, respectively. The absorption at 600 nm was measured every 5 h to obtain the growth curve. The maximum specific growth rate μ was calculated from the logarithm of the growth curve, μ=3.322×Lg(x2/x1)/(t2–t1). The x1 and x2 are the numbers of cells at the time of t1 and t2, respectively.3 RESULT 3.1 Molecular identification of the deep-sea bacterial isolates
In total 237 strains were isolated from the seawater, sediments and deep-sea amphipods collected from the South China Sea and the Mariana Trench at the depth ranged from 500 m to over 10 000 m. Phylogenetic analysis based on the 16S rRNA gene sequences revealed their affiliation to 50 species of 23 genera, 4 phyla, using 97% and 95% sequence identity as the thresholds for classification of species and genus, respectively (Tindall et al., 2010). Proteobacteria accounted for 81.4% of all the isolates (193 strains), with the majority belonged to Gammaproteobacteria (149 strains, 77% of Proteobacteria), and the rest belonged to Alphaproteobacteria (44 strains, 23% of Proteobacteria). In addition, we also obtained 8 isolates (3.4%) affiliated to Bacteroidetes, 14 isolates (5.9%) affiliated to Firmicutes, and 22 isolates (9.3%) affiliated to Actinobacteria (Fig. 1).
Twenty-eight strains from 4 genera were isolated from sublittoral zone (200–1 000 m), dominated by Pseudoalteromonas (43%) and Vibrio (46%). Seventy-eight strains were isolated from the bathyal zone (1 000–4 000 m), consisting of Pseudomonas (24%), Halomonas (8%), Pseudoalteromonas (8%), Stenotrophomonas (8%), Photobacterium (8%) and other 12 genera. Over a hundred strains belonged to 14 genera were isolated from abyssal zone (4 000– 6