Search

When cells are exposed to freezing temperatures, high concentrations of cryoprotective agents (CPA) prevent ice crystal formation, thus enhancing cell survival. However, high concentrations of CPAs can also cause cell toxicity. Exopolysaccharides (EPSs) from polar marine environments exhibit lower toxicity and display effects similar to traditional CPA. In this study, we sought to address these issues by i) selecting strains that produce EPS with novel cryoprotective activity, and ii) optimizing culture conditions for EPS production. Sixty-six bacteria producing mucous substances were isolated from the Ross Sea (Antarctic Ocean) using solid marine agar plates. Among them, Pseudoalteromonas sp. RosPo-2 was ultimately selected based on the rheological properties of the produced EPS (p-CY02). Cryoprotective activity experiments demonstrated that p-CY02 exhibited significantly cryoprotective activity at a concentration of 0.8% (w/v) on mammalian cells (HaCaT). This activity was further improved when combined with various concentrations of dimethyl sulfoxide (DMSO) compared to using DMSO alone. Moreover, the survival rate of HaCaT cells treated with 5% (v/v) DMSO and 0.8% (w/v) p-CY02 was measured at 87.9 ± 2.8% after freezing treatment. This suggests that p-CY02 may be developed as a more effective, less toxic, and novel non-permeating CPA. To enhance the production of EPS with cryoprotective activity, Response Surface Methodology (RSM) was implemented, resulting in a 1.64-fold increase in production of EPS with cryoprotective activity.

Exopolysaccharides (EPSs) are carbohydrate polymers produced and secreted by microorganisms. In a changing marine environment, EPS secretion can reduce damage from external environmental disturbances to microorganisms. Meanwhile, EPSs have promising application prospects in the fields of food, cosmetics, and pharmaceuticals. Changes in external environmental pH have been shown to affect the synthesis of EPSs in microorganisms. In this study, we analyzed the effects of different initial fermentation pHs on the production, monosaccharide composition, and antioxidant activity of the EPSs of Pseudoalteromonas agarivorans Hao 2018. In addition, the transcriptome sequence of P. agarivorans Hao 2018 under different initial fermentation pH levels was determined. GO and KEGG analyses showed that the differentially expressed genes were concentrated in the two-component regulatory system and bacterial chemotaxis pathways. We further identified the expression of key genes involved in EPS synthesis during pH changes. In particular, the expression of genes encoding the glucose/galactose MFS transporter, phosphomannomutase, and mannose-1-phosphate guanylyltransferase was upregulated when the environmental pH increased, thus promoting EPS synthesis. This study not only contributes to elucidating the environmental adaptation mechanisms of P. agarivorans , but also provides important theoretical guidance for the directed development of new products using biologically active polysaccharides.

In recent years, the wide application of exopolysaccharides (EPSs) in food, cosmetics, medicine, and other fields has drawn tremendous attention. In this study, an EPS produced by Pseudoalteromonas agarivorans Hao 2018 was isolated and purified, and its fermentation conditions were optimized. Its structure and biological functions were also studied. The purity and molecular weight of EPS were determined by high performance liquid chromatography (HPLC), and the EPS exhibited a number average of 2.26 × 10 5 and a weight average of 2.84 × 10 5 . EPS has good adsorption for Cu 2+ and Pb 2+ . The adsorption rates can reach up to 69.79% and 82.46%, respectively. The hygroscopic property of EPS was higher than that of chitosan, but slightly lower than that of sodium hyaluronate. However, the water-retaining activity of EPS was similar to that of chitosan and sodium hyaluronate. EPS has strong ability to scavenge free radicals, including OH radical and O 2- radical. Further, its activity on O 2- radicals has similarities with that of vitamin C. EPS has broad application prospects in many fields, such as cosmetics, environmental protection.

Bacterial capsular polysaccharides (CPSs) participate in environmental adaptation in diverse bacteria species. However, the role and regulation of CPS production in marine bacteria have remained largely unexplored. We previously reported that both wrinkled and translucent Pseudoalteromonas lipolytica variants with altered polysaccharide production were generated in pellicle biofilm-associated cells. In this study, we observed that translucent variants were generated at a rate of ∼20% in colony biofilms of P. lipolytica cultured on HSLB agar plates for 12 days. The DNA sequencing results revealed that nearly 90% of these variants had an IS5-like element inserted within the coding or promoter regions of nine genes in the cps operon. In contrast, IS5 insertion into the cps operon was not detected in planktonic cells. Furthermore, we demonstrated that the IS5 insertion event inactivated CPS production, which leads to a translucent colony morphology. The CPS-deficient variants showed an increased ability to form attached biofilms but exhibited reduced resistance to sublethal concentrations of antibiotics. Moreover, deleting the DNA repair gene recA significantly decreased the frequency of occurrence of CPS-deficient variants during biofilm formation. Thus, IS insertion into the cps operon is an important mechanism for the production of genetic variants during biofilm formation of marine bacteria., (© FEMS 2019.)

The EPS-producing Pseudoalteromonas sp. MER144 was selected among 606 isolates from Antarctic seawater due to its evident slimy appearance on agar plates. The production of EPSs was enhanced by a step-by-step approach varying the carbon source, substrate and NaCl concentrations, temperature, and pH. Optimal conditions for the EPS production resulted at temperature of 4 °C and pH 7, with addition of 2% sucrose (w/v) and 3% NaCl (w/v). EPSs produced under optimal conditions were chemically characterized, resulting in a moderate carbohydrate content (35%), uronic acids (14%), and proteins (12%). Monosaccharide composition was estimated to be Glu:Man:GluN:Ara:GluA:GalA:Gal (1:0.36:0.26:0.06:0.06:0.05:0.03), while the estimated molecular weight was about 250 kDa. The addition of sucrose in the culture medium, by stimulating the EPS production, allowed MER144 to tolerate higher concentrations of mercury and cadmium. This finding was probably dependent on the presence of uronic acids and sulfate groups, which can bind cations, in the extracted EPSs. Monitoring EPS production under optimal conditions at different concentrations of mercury and cadmium revealed that EPS amounts increased at increasing heavy metal concentrations, indicating an adaptation to the stress conditions tested.

Exopolysaccharides (EPS) are polymers excreted by some microorganisms with interesting properties and used in many industrial applications. A new Pseudoalteromonas sp. strain, MD12-642, was isolated from marine sediments and cultivated in bioreactor in saline culture medium containing glucose as carbon source. Its ability to produce EPS under saline conditions was demonstrated reaching an EPS production of 4.4g/L within 17hours of cultivation, corresponding to a volumetric productivity of 0.25g/Lh, the highest value so far obtained for Pseudoalteromonas sp. strains. The compositional analysis of the EPS revealed the presence of galacturonic acid (41-42mol%), glucuronic acid (25-26mol%), rhamnose (16-22mol%) and glucosamine (12-16mol%) sugar residues. The polymer presents a high molecular weight (above 1000kDa). These results encourage the biotechnological exploitation of strain MD12-642 for the production of valuable EPS with unique composition, using saline by-products/wastes as feedstocks., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Twenty-two bacterial strains that secrete exopolysaccharides (EPS) were isolated from marine samples obtained from the Chukchi Sea in the Arctic Ocean; of these, seven strains were found to be capable of producing cryoprotective EPS. The ArcPo 15 strain was isolated based on its ability to secrete large amounts of EPS, and was identified as Pseudoalteromonas elyakovii based on 16S rDNA analysis. The EPS, P-ArcPo 15, was purified by protease treatment and gel filtration chromatography. The purified EPS (P-ArcPo 15) had a molecular mass of 1.7 × 10(7) Da, and its infrared spectrum showed absorption bands of hydroxyl and carboxyl groups. The principal sugar components of P-ArcPo 15 were determined to be mannose and galacturonic acid, in the ratio of 3.3:1.0. The cryoprotective properties of P-ArcPo 15 were characterized by an Escherichia coli viability test. In the presence of 0.5% (w/v) EPS, the survival percentage of E. coli cells was as high as 94.19 ± 7.81% over five repeated freeze-thaw cycles. These biochemical characteristics suggest that the EPS P-ArcPo 15 may be useful in the development of cryoprotectants for biotechnological purposes, and we therefore assessed the utility of this novel cryoprotective EPS.

This study investigated soluble (Sol-EPS), loosely bound (LB-EPS), and tightly bound extracellular polymeric substances (TB-EPS) harvested from biofilm and planktonic cultures of the marine bacterium Pseudoalteromonas ulvae TC14. The aim of the characterization (colorimetric methods, FTIR, GC-MS, NMR, HPGPC, and AFM analyses) was to identify new anti-biofilm compounds; activity was assessed using the BioFilm Ring Test®. A step-wise separation of EPS was designed, based on differences in water-solubility and acidity. An acidic fraction was isolated from TB-EPS, which strongly inhibited biofilm formation by marine bacterial strains in a concentration-dependent manner. The main constituents of this fraction were characterized as two glucan-like polysaccharides. An active poly(glutamyl-glutamate) fraction was also recovered from TB-EPS. The distribution of these key EPS components in Sol-EPS, LB-EPS, and TB-EPS was distinct and differed quantitatively in biofilm vs planktonic cultures. The anti-biofilm potential of the fractions emphasizes the putative antifouling role of EPS in the environment.

The structure and ecological roles of the exopolysaccharides (EPSs) from sea ice microorganisms are poorly studied. Here we show that strain SM20310, with an EPS production of 567 mg liter(-1), was screened from 110 Arctic sea ice isolates and identified as a Pseudoalteromonas strain. The EPS secreted by SM20310 was purified, and its structural characteristics were studied. The predominant repeating unit of this EPS is a highly complicated α-mannan with a molecular mass greater than 2 × 10(6) Da. The backbone of the EPS consists of 2-α-, 6-α-mannosyl residues, in which a considerable part of the 6-α-mannosyl residues are branched at the 2 position with either single t-mannosyl residues or two mannosyl residues. The structure of the described EPS is different from the structures of EPSs secreted by other marine bacteria. Analysis of the ecological roles of the identified EPS showed that the EPS could significantly enhance the high-salinity tolerance of SM20310 and improve the survival of SM20310 after freeze-thaw cycles. These results suggest that the EPS secreted by strain SM20310 enables the strain to adapt to the sea ice environment, which is characterized by low temperature, high salinity, and repeated freeze-thaw cycles. In addition to its functions in strain SM20310, this EPS also significantly improved the tolerance of Escherichia coli to freeze-thaw cycles, suggesting that it may have a universal impact on microorganism cryoprotection.

Objective: Fungal keratitis (FK) is a vision-threatening infection, whose treatment requires more effective and safer anti-fungal agent exploitation urgently. With this aim, we focused on the effect of an extracellular polysaccharide on fungal adhesion to human corneal epithelial cells., Methods: We performed the cytotoxicity assays of the extracellular polysaccharide EPS-II from an antarctic bacterium Pseudoaltermonas and evaluated its inhibitory effect on Candida albicans cells' adherence to human corneal epithelial cells (HCECs)., Results: EPS-II, which displayed minor cytotoxicity but also promoted proliferation of HCECs, could inhibit the adherence of yeast cells to HCECs in a dose-dependent manner. EPS-II could also suppress the subsequent PI3K/AKT signaling pathway, and thereby decrease the expression of early inflammatory cytokines., Conclusions: Extracellular polysaccharide EPS-II was suggested as a new natural agent for attenuating FK.

High-molecular-mass polysaccharides were released by mild acid degradation of the lipopolysaccharides of two wild-type Vibrio vulnificus strain, a flagellated motile strain CECT 5198 and a non-flagellated non-motile strain S3-I2-36. Studies by sugar analysis and partial acid hydrolysis along with (1)H and (13)C NMR spectroscopies showed that the polysaccharides from both strains have the same trisaccharide repeating unit of the following structure: -->4)-beta-d-GlcpNAc3NAcylAN-(1-->4)-alpha-l-GalpNAmA-(1-->3)-alpha-d-QuipNAc-(1--> where QuiNAc stands for 2-acetamido-2,6-dideoxyglucose, GalNAmA for 2-acetimidoylamino-2-deoxygalacturonic acid, GlcNAc3NAcylAN for 2-acetamido-3-acylamino-2,3-dideoxyglucuronamide and acyl for 4-d-malyl ( approximately 30%) or 2-O-acetyl-4-d-malyl ( approximately 70%). The structure of the polysaccharide studied resembles much that of a marine bacterium Pseudoalteromonas rubra ATCC 29570 reinvestigated in this work. The latter differs in (i) the absolute configuration of malic acid (l vs d), (ii) 3-O-acetylation of GalNAmA and (iii) replacement of QuiNAc with its 4-keto biosynthetic precursor.

Biosorption of Pb2+ and Cu2+ by exopolysaccharide (EPS) from the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913 was investigated. The influence of EPS dosage, pH, equilibrium time and coexisted-ions on adsorption property and the adsorption isotherm of this EPS were studied. The results show that the equilibrium adsorption capacity (Qe) decreases with the increase of EPS dosage. The optimum pH for the adsorption of Pb2+ is 4.5-5.5, and for the adsorption of Cu2+ is 4.5-6.0. The adsorption equilibrium of Cu2+ onto the EPS is attained within 90 min, while the Pb2+ adsorption equilibrium time is 180 min. Qe of Pb2+ decreases with the addition of coexisted-ions such as Ca2+, Mg2+, Na+ and K+. Addition of Ca2+, Mg2+ also decreases the Qe of Cu2+, but low dosage of Na+ and tested dosage of K+ increase the Qe of Cu2+. Both Freundlich and Dubinin-Radushkevich isotherm equations could well describe the thermodynamics process of Pb2+ and Cu2+ adsorbed onto the EPS. The maximum adsorption capacity of the EPS determined by Dubinin-Radushkevich isotherm equation for Pb2+ and Cu2+ are 243.3 mg/g (10 degrees C) and 36.7 mg/g (40 degrees C), respectively. IR analysis demonstrates that the group of C--O--C, acetyl and hydroxyl of polysaccharide are the main functional groups for binding metal ions.

Aim: The major objective of the present study was the partial characterization of the exopolysaccharides (EPS) produced by a marine biofilm-forming bacterium Pseudoalteromonas ruthenica under shake culture conditions., Methods and Results: EPS-producing bacterial cultures were isolated from the sea water collected from the vicinity of coastal electric power station. Zobell marine broth medium was used for growth of the cultures and the EPS produced was quantified using phenol sulfuric acid method. Chemical characterization of the EPS was carried out using Fourier transform infrared spectroscopy (FTIR), and capillary gas chromatography (GC). Further, viscosity and rheological properties of the purified EPS were studied. The FTIR spectrum revealed prominent peaks of various groups of OH and CH(3) bending. GC analysis showed the presence of eight individual sugars. Rheological studies of the aqueous EPS showed good shearing property., Conclusions: Pseudoalteromonas ruthenica isolated from marine environment produced copious amount of EPS under shake culture conditions. GC analysis of the EPS revealed the presence of eight individual sugars and the EPS had good shearing property., Significance and Impact of the Study: The EPS produced by P. ruthenica is pseudoplastic in nature and is stable at higher pH levels. These properties suggest that the EPS may have potential applications in the oil, textiles and food industries.

Twenty-five bacterial strains that secrete mucous materials were isolated from sediment obtained from King George Island, Antarctica. Seven of these strains proved capable of producing cryoprotective exopolysaccharides. The strain KOPRI 21653 was selected for the further study of an anti-ice-nucleating polysaccharide (ANP), which originated from a polar region. KOPRI 21653 was identified as Pseudoalteromonas arctica as the result of 16S rRNA analysis. The exopolysaccharide, P-21653, was purified completely from the KOPRI 21653 cell culture via column chromatography and protease treatment. The principal sugar components of P-21653 were determined to be galactose and glucose, at a ratio of 1:1.5, via GC-MS analysis. The cryoprotective activity of P-21653 was characterized via an E. coli viability test. In the presence of 0.1% (w/v) P-21653, the survival ratio of E. coli cells was as high as 82.6% over three repeated freeze-thaw cycles. The survival ratio decreased drastically to 71.5 and 48.1%, respectively, in five and seven repeated cycle conditions; however, the survival ratios were greater over three (96.6-92.1%) to seven (100.5-91.6%) freeze-thaw cycles in the presence of 0.5 and 1.0% (w/v) P-21653. In addition, at much lower concentrations (0.1-1.0%), P-21653 resulted in survival ratios (83.1-98.4%) similar to those of two commercially available cryoprotectants (VEG plus X-1000, 92.9% and VM3, 95.3%), which were utilized at the recommended concentrations (90%). The biochemical characteristics of exopolysaccharide P-21653 reflect that this compound may be developed as a useful cryoprotectant for use in medical applications and in the food industry.

Pseudoalteromonas sp. SM9913 is a psychrotolerant bacterium isolated from deep-sea sediment. The structural characterization and ecological roles of the exopolysaccharide (EPS) secreted by this strain were studied in this work. The yield of the EPS increased as the culture temperature decreased in the range 30-10 degrees C, and it reached 5.25 g l(-1) (dry weight) under optimal growth conditions (15 degrees C, 52 h). EPS fraction was purified and its structure was identified by the combination of NMR spectra, high-resolution mass spectrometry (HRMS) analysis and methylation analysis. The ratio of the sugar units, the acetyl group and the ethoxyl group was close to 4 : 5 : 1. The major sugar unit of the EPS was 6-linked glucose (61.8 %); other sugar units present included terminal arabinofuranosyl (11.0 %) and glucopyranosyl (11.2 %) residues and a small amount of other sugar derivatives. Its structure was different from EPSs reported for other marine bacteria. Besides the structural elucidation of the EPS, its ecological roles were studied. This EPS could enhance the stability of the cold-adapted protease MCP-01 secreted by the same strain through preventing its autolysis. It could bind many metal ions, including Fe(2+), Zn(2+), Cu(2+), Co(2+). It was also a very good flocculating agent and could conglomerate colloidal and suspended particles. 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. This is believed to be the first report on the structure of the EPS secreted by a deep-sea psychrotolerant bacterium and its ecological roles. According to these results and other studies, a schematic diagram of the lifestyle of the deep-sea psychrotolerant strain SM9913 is suggested.

The structure of an acidic polysaccharide from Pseudoalteromonas atlantica strain 14165 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid (di-N-acetylpseudaminic acid, Pse5Ac7Ac) has been elucidated. The polysaccharide was studied by 1H and 13C NMR spectroscopy, including 2D experiments, along with sugar and methylation analyses. After a selective hydrolysis a modified polysaccharide devoid of its side chain could be isolated. It was found that the polysaccharide has pentasaccharide repeating units with following structure: [structure: see text].

Aims: This study was undertaken to examine and characterize Antarctic marine bacterial isolates and the exopolysaccharides (EPS) they produce in laboratory culture., Methods and Results: Two EPS-producing bacterial strains CAM025 and CAM036 were isolated from particulate material sampled from seawater and sea ice in the southern ocean. Analyses of 16S rDNA sequences placed these isolates in the genus Pseudoalteromonas. In batch culture, both strains produced EPS. The yield of EPS produced by CAM025 was 30-fold higher at -2 and 10 degrees C than at 20 degrees C. Crude chemical analyses showed that these EPS were composed primarily of neutral sugars and uronic acids with sulphates. Gas chromatographic analysis of monosaccharides confirmed these gross compositional findings and molar ratios of monosaccharides revealed differences between the two EPS., Conclusions: The EPS produced by Antarctic bacterial isolates examined in this study appeared to be polyanionic and, therefore, 'sticky' with respect to cations such as trace metals., Significance and Impact of the Study: As the availability of iron as a trace metal is of critical importance in the southern ocean where it is know to limit primary production, the role of these bacterial EPS in the Antarctic marine environment has important ecological implications.

The chemical structures of polysaccharides and LPS core oligosaccharides, isolated from various Gram-negative marine bacteria from the genera Pseudoalteromonas and Shewanella belonging to the Alteromonadaceae family and gamma-subclass of Proteobacteria, are reviewed. The polysaccharides are distinguished by the acidic character (e.g., due to the presence of hexuronic and aldulosonic acids and their derivatives) and the occurrence of unusual sugars, including N-acyl derivatives of 6-deoxyamino sugars, such as N-acetyl-D-quinovosamine, N-acetyl-L-fucosamine and N-acetyl-6-deoxy-L-talosamine, and higher sugars like 2,6-dideoxy-2-acetamido-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-D-galactopyranose (shewanellose). Many constituent sugars have various uncommon non-sugar substituents, such as alanine, formic, lactic and hydroxybutyric acids, sulfate, phosphate, and 2-aminopropane-1,3-diol.

In the course of the discovery of novel polysaccharides of biotechnological interest, it is now widely accepted that extremophilic microorganisms will provide a valuable resource not only for exploitation in novel biotechnological processes but also as models for investigating how biomolecules are stabilized when subjected to extreme conditions. Microbes isolated from extreme environments offer a great diversity in chemical and physical properties of their EPS as compared to anywhere else in the biosphere. Bacteria from remote areas still remain virtually unexplored and there is not doubt that extreme environments are a rich source of microorganisms of biotechnological importance. A number of interesting and unique bacterial polysaccharides have been isolated from these ecosystems and are expected to find applications in the very near future in different industrial. Further screenings are underway as well as research into understanding the structure-function relationships of these unusual polymers.