Structure, Ecological Roles And Biosynthesis Gene Cluster Of EPS From Deep-Sea Psychrotrophic Bacterium Pseudoalteromonas Sp. SM9913

The ocean covers more than 75 % of the earth's surface and this offers a great potential for the discovery of new compounds and new uses for them. There are various and abundant microorganisms in the sea. They are valuable resources for the finding of new enzymes and new medicines, and have a good potential for research and development in the future. Extremophiles are one of the research hot spots in the region of microbiology. Cold-adapted microorganisms are one kind of extremophiles and are widely distributed in the sea. Here, the exopolysaccharides (EPSs) secreted by cold-adapted bacteria from sea, especially from deep sea, were systematically studied.Deep-sea psychrotrophic bacterial strain Pseudoaltermonas sp. SM9913 was isolated from the deep-sea sediment near the Okinawa Trough (25°56' N, 125°09'E) at a water depth of 1855 m. Cold-adapted protease MCP-01 secreted by strain SM9913. a thermolabile protease, had been purified before (Chen et al, 2002; Chen et al, 2003). Large quantities of exopolysaccharide (EPS) were also secreted by the same strain when growing on sea water-based liquid medium. In this paper we have investigated the structural features and ecological functions of this exopolysaccharide which is responsible for survival of the psychrotrophic bacteria from the deep-sea extreme environments characterized by low temperature, oligotrophy and constantly changing.In this paper, we studied effect of temperature and time on the production of the EPS secreted by strain SM9913 in order to increase EPS production by optimizing for fermentation. When strain SM9913 was cultured at 10°C, 20°C and 30°C, respectively, after a short period of decrease, the sugar amount in the culture media continuously increased to the highest level. It indicated that the carbohydrate in the culture media was utilized by strain SM9913 for growth at the beginning, and then strain SM9913 started to continuously secrete EPS. The yield of EPS increased as the temperature decreased in the tested range, indicating that the EPS production of strain SM9913 had cold-adaptation. At optimal experiment conditions (15°C, 52 h), the yield of the EPS reached 5.25 g/L (dry weight), which was higher than that of the EPSs produced by other psychrotrophic microorganisms (Mancuso Nichols et al., 2005).The growth of strain Pseudoaltermonas sp. SM9913 was carried out in sea water-based liquid medium. The rough exopolysaccharide was obtained after the culture was centrifuged, condensed, active carbon decolourizing, proteins removal (Sevag method), ethanol precipitation, dialyzed and lyophilized. Firstly, it was purified through a gel-filtration chromatographic column of Sephadex G-100. Two EPS peaks were eluted by 0.1 mol/L NaCl and the first fraction containing EPS was collected when determined with Phenol-H₂SO₄ method (The latter was not collected because of limited contents). Secondly, the EPS fraction was further purified by an anion-exchange chromatography of DEAE-Sepharose Fast Flow column. The white EPS DS-EPS01 was eluted by 0-0.1 mol/L NaCl and lyophilized.Homogeneity of the EPS purified by chromatography was analyzed on the Shimadzu analytical high performance liquid chromatography (HPLC) system. Only one symmetrical acute peak was detected, which indicated that this EPS consisted of a single homogeneous component and could be used for structure analysis. The molecular mass of the EPS analyzed by the same HPLC system was 4×10⁴ Da.Glycosyl composition of the EPS was initially performed and the results showed that it was mainly glucose, with arabinose, xylose and minor peak for mannose. It did not contain any amino-residues. Linkage analysis of the EPS sample showed that it contained mainly 6-linked glucopyranosyl (6-Glc). Other linkages were terminal glucose, terminal arabinose and some minor peaks for terminal galactopyranosyl (t-Gal), 4-linked glucopyranosyl (4-Glc), 4-linked xylofuranosyl (4-Xyl) and 3,6-linked glucopyranosyl (3,6-Glc). These results showed that the main composition of the EPS was 6-Glc (61.8 %). Other components included terminal arabinofuranosyl (t-Ara f, 11.0 %) and terminal glucopyranosyl (t-Glc, 11.2 %) together with small amount of t-Gal (3.1 %), 4-Xyl (f, 3.9 %), 4-Glc (5.0 %) and 3,6-Glc (4.0 %). These results confirmed the results from the latter NMR analysis, and also provided the linkage between the repeating sugar units as anα-(1→6) linkage. The structure of DS-EPS01 was extensively investigated by one and two-dimensional NMR in D₂O, ¹H-¹H COSY (correlation spectroscopy, phase sensitive) and HSQC (heteronuclear single quantum correlation) spectra. The integration of the ¹H NMR spectrum indicated the ratio between the sugar units, the acetyl group and the ethoxyl group is close to 4:5:1. The coupling pattern and the coupling constant obtained from the phase sensitive ¹H-¹H COSY together with the chemical shift from the ¹H NMR spectra showed that no amino group was present on the sugar skeleton. The coupling pattern and coupling constants strongly suggested the repeating sugar unit has an alpha-glucose skeleton. The coupling patterns between these signals are hard to identify by 1D NMR. ¹H-¹H COSY (correlation spectroscopy, phase sensitive) was employed to resolve the coupling pattern and the coupling constants.Based on the data obtained and the analysis from the sugar composition analysis, methylation analysis, MS and 1D and 2D NMR data of DS-EPS01, the primary structure of DS-EPS01 was shown to be built up by the following tetrasaccharide repeating units characterized asα-(1,6)→Glc and high degree of acetylation:→6)-[3,6-O-acetyl]-α-D-Glcp-(1→6)-[3-O-acetyl]-α-D-Glcp-(1→6)-[3-O-acetyl ]-α-D-Glcp-(1→6)-[3-O-acetyl]-α-D-Glcp-(1→The structure of the EPS was shown in the below figure:The structure of DS-EPS01 was quite different from that of the EPSs secreted by marine bacteria from hydrothermal vents and other areas, including Antarctic Pseudoalteromonas strains (Mancuso Nichols et al, 2005, Guezennec, 2002). The EPS from strain SM9913 was an acetyl-rich exopolysaccharide with a molecular mass of 4×10⁴ Da, which was smaller than most of the EPSs secreted by the bacteria from deep-sea hydrothermal vents. Unlike most of the EPSs secreted by other marine bacteria, The EPS from strain SM9913 didn't possess uronic acid. Acetyl groups had been found in other marine EPSs such as the EPS produced by psychrotolerant strain CAM025 and CAM036 from Antarctic water and sea ice (Mancuso Nichols et al., 2004, 2005). But an EPS with so high degree of acetylation from a marine bacterium is an unusual finding. In contrast, the EPS from deep-sea hydrothermal vents bacteria have no or few acetyl groups (Guezennec, 2002). To our knowledge this is the first report on the EPS from a deep-sea psychrotolerant bacterium. While the water solubility of the exopolysaccharide was enhanced by its high degree of acetylation, the presence of acetate groups was responsible for the interesting rheological properties of the polysaccharide such as gel-like behavior. The structure of DS-EPS01 indicated that high degree of acetylation leaded to the changes of its direction finding and landscape orientation order, and ultimately changed the rheological property of the polysaccharide molecule. The introduction of acetyl groups changed its molecule flexibility, resulted in the exposure of hydroxy groups, and consequently increased the water solubility of the EPS, which further enhanced its bioactivity.Furthermore, we also made an exploration on the eclological role of EPS played in the adaptation of psychrotrophic microorganisms to the constantly changing deep-sea ecosystem with low temperature and oligotrophy.Cold-adapted protease MCP-01, the main protease secreted by strain SM9913, was thermolabile and susceptible to autolysis (Chen et al, 2002; Chen et al, 2003). Our previous study showed that autolysis of the purified protease MCP-01 was the main reason of its thermolability (Chen et al, 2003). Here, we elucidated the interaction of the EPS and cold-adapted proteases MCP-01 both secreted by the same strain. So protease MCP-01 was purified and effect of the EPS secreted by strain SM9913 on the thermostability and autolysis of protease MCP-01 was studied. The results showed that the EPS obviously enhanced the thermostability of protease MCP-01 at 40°C by the determination of enzyme activity. In the presence of the EPS (1 %, w/v), the protease activity of MCP-01 had no evident change after 150 min incubation. The autolysis process of protease MCP-01 at 40°C was monitored by capillary electrophoresis. It indicated that the DS-EPS01 prevented the autolysis of protease MCP-01 at 40°C, which could be the reason why the EPS enhanced the thermostability of protease MCP-01.The metal-binding property of the EPS was studied and the adsorption percent values (Q) of every metal were calculated. The results showed that Fe²⁺ (85.00 %), Zn²⁺ (58.15 %), Cu²⁺ (52.77 %), Co²⁺ (48.88 %) were easily absorbed by the EPS, while Mg²⁺ (30.69 %) and Mn²⁺ (25.67 %) were less adsorbed by the EPS. The worst chelating property of the EPS to Cr⁶⁺ (5.15 %) was observed through the whole test. This result indicated that the EPS might be helpful for strain SM9913 to enrich metal ions in the nutrition-scant deep-sea environment. The flocculation experiment showed that the EPS could make the colloidal and suspended particles in solution conglomerated, suggesting that the EPS was a very good flocculating agent and had a good adsorptive effect. Therefore, it might play an important role for strain SM9913 in enriching nutrient particles in the nutrition-scant and changing deep-sea environment.These results indicated that the EPS secreted by strain SM9913 might help this strain enrich the proteinaceous particles and the trace metals in the deep-sea environment, stabilize the secreted cold-adapted proteases and avoid its diffusion.On the bases of the research above, genetic and molecular mechanism of DS-EPS01 were studied on the survival of strain SM9913 in the extreme deep-sea environment. The total gene sequences involved in the biosynthesis of DS-EPS01 were successfully cloned. Upon the complete sequencing, we carried out the ORFs annotations and drew a genetic organization involved in the biosynthesis of DS-EPS01 from strain SM9913. The complete eps gene cluster is contained on a 13.062 kb chromosomal region which contains 13 ORFs, including epsPTUABCDGFEHISD genes with 35.71 % of the mean G+C %. Functions were assigned to some of the predicted gene products on the basis of homology to known sequences as follows: epsA and epsS encode a protein thought to be involved in the polymerization and. export of the exopolysaccharide; epsB,epsC,epsD,epsG,epsF and epsE encode putative sugar transferees; epsH encodes acyltransferase which can modify the acetylation of EPS. Analysis of of the epsT gene in eps gene cluster demonstrated that it encodes a UDP-glucose lipid carrier transferase; the enzyme which catalyses the first step in EPS biosynthesis in strain SM9913. Because of limited information, the functions of epsP,epsU and epsI were not found and unknown.Compared with other EPS gene cluster from other bacteria, the eps gene cluster of strain SM9913 was similar involved in biosynthesis of exopolysaccharide. To our knowledge this is the first report on the EPS gene cluster involved in the biosynthesis of EPS from a deep-sea psychrotolerant bacterium. Combined with many studies on EPS, an integrated molecular mechanism of metabolic adaptation of deep-sea psychrotrophic bacterial strain Pseudoaltermonas sp. SM9913 to the constantly changing deep-sea environment with low temperature and oligotrophy has been summarized:1. Deep-sea psychrotrophic bacterial strain SM9913 relies primarily on amino acid catabolism, rather than on saccharolytic pathways or carbohydrates fermentation, for carbon and energy.2. Many kinds of cold-adapted proteases from the deep-sea psychrotrophic bacterium may be helpful to rapidly degrade the protein particles available in the deep-sea environment with low temperature and to support the growth requirement.3. Abundance of amino acid transport an degradation enzymes, diverse peptidases, a variety of peptide and amino acid uptake systems, and versatile signal transduction machinery can be helpful to break down and adsorb effectively the limited proteinaceous particles, which can make deep-sea psychrotrophic bacterium survive in the extreme deep-sea environment with oligotrophy.4. In the marine environment, bacterial EPSs are essential in the production of aggregates, adhesion to surfaces and other organisms, biofilm formation and sequestering of nutrients, and thus provide protection and ecosystem stability. Exopolysaccharides from strain SM9913 may function in colonizing proteinaceous particles in the surrounding environment and play a role in stabilizing the proteases from the same strain in situ. In the meantime, it could concentrate the proteases secreted by this strain through preventing the protease diffusion. So the proteinaceous particles concentrated by the EPS could be effectively digested by the proteases into amino acids and oligopeptides, and they together with the concentrated metal ions were adsorbed by the strain as source for growth and energy.The results of these investigations suggest that accumulation of the EPS can enhance survival, competition abilities and stress tolerance of the psychrotrophic strain in extreme conditions prevailing in its natural deep-sea ecosystem, and it plays an important ecological role on psychrotrophic strain adapted to the dynamic deep-sea ecosystem.There are some new infers proposed based on the investigation during this work:1. Strain SM9913 is a deep-sea psychrotrophic bacterium isolated from 1855 m depth sediment. The EPS secreted by strain SM9913 was purified and its structure was elucidated in this article. This is believed to be the first report on the structure of the EPS from a deep-sea psychrotrophic bacterium. Its structure was quite different from that of the EPSs secreted by marine bacteria from other areas.2. Besides the structure elucidation of the EPS from strain SM9913, several experiments were firstly done to explain its ecological role in the cold deep-sea environment. The results in this paper showed that the EPS secreted by strain SM9913 could stabilize protease MCP-01 and effectively prevent its autolysis in vitro. This is the first report that EPS secreted by a marine bacterium has a stabilizing function on the proteases secreted by the same strain. Therefore, the EPS secreted by strain SM9913 plays important roles in the survival of this strain in the deep-sea nutrient-scarce environment.3. The total gene sequences involved in the biosynthesis of DS-EPS01 were successfully cloned. Upon the complete sequencing, we carried out the ORFs annotations and drew a genetic organization involved in the biosynthesis of DS-EPS01 from strain SM9913. The complete eps gene cluster is contained on a 13.062 kb chromosomal region which contains 13 ORFs including epsPTUABCDGFEHISD genes. Functions were assigned to some of the predicted gene products on the basis of homology to known sequences. To our knowledge this is the first report on the EPS gene cluster involved in the biosynthesis of EPS from a deep-sea psychrotrophic bacterium. Combined with many studies on EPS, an integrated mechanism of metabolic adaptation of stain SM9913 to the low temperature, organic and inorganic nutrient-scarce, constantly changing deep-sea environment has been summarized, In the meantime, a schematic diagram of lifestyle of deep-sea psychrotrophic strain SM9913 is suggested according to these experimental results and other studies (Figure I).