The T7-related Pseudomonas putida phage φ15 displays virion-associated biofilm degradation properties

Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a ‘T7-like virus’ with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10⁴ and 10⁶ pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.     

Exopolysaccharide depolymerases induced by Rhizobium bacteriophages.

Enzymes induced by two Rhizobium trifolii bacteriophages caused depolymerization of exopolysaccharides from most R. trifolii and R. leguminosarum strains tested, but did not, in general, attack the exopolysaccharides of R. meliloti, the slow-growing rhizobia, or Agrobacterium. Ca2+ and (or) Mg2+ were required for enzyme activity. In all strains tested, depolymerization of exopolysaccharide occurred when there was successful phage infection, but depolymerization also occurred with exopolysaccharides from nonsusceptible strains.     

Structural analysis of the alpha-D-glucan (EPS180) produced by the Lactobacillus reuteri strain 180 glucansucrase GTF180 enzyme

The neutral exopolysaccharide EPS180 produced from sucrose by the glucansucrase GTF180 enzyme from Lactobacillus reuteri 180 was found to be a (1-->3,1-->6)-alpha-D-glucan, with no repeating units present. Based on linkage analysis, periodate oxidation, and 1D/2D 1H and 13C NMR spectroscopy of the intact EPS180, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis of EPS180, a composite model, that includes all identified structural features, was formulated as follows: [Formula: see text].     

Marine bacterial exopolysaccharide EPS11 inhibits migration and invasion of liver cancer cells by directly targeting collagen I

Many natural polysaccharides have significant anticancer activity with low toxicity, but the complex chemical structures make in-depth studies of the involved mechanisms extremely difficult. The purpose of this study was to investigate the effect of the marine bacterial exopolysaccharide (exopolysaccharide 11 [EPS11]) on liver cancer metastasis to explore the underlying target protein and molecular mechanism. We found that EPS11 significantly suppressed cell adhesion, migration, and invasion in liver cancer cells. Proteomic analysis showed that EPS11 induced downregulation of proteins related to the extracellular matrix–receptor interaction signaling pathway. In addition, the direct pharmacological target of EPS11 was identified as collagen I using cellular thermal shift assays. Surface plasmon resonance and pull-down assays further confirmed the specific binding of EPS11 to collagen I. Moreover, EPS11 was shown to inhibit tumor metastasis by directly modulating collagen I activity via the β1-integrin–mediated signaling pathway. Collectively, our study demonstrated for the first time that collagen I could be a direct pharmacological target of polysaccharide drugs. Moreover, directly targeting collagen I may be a promising strategy for finding novel carbohydrate-based drugs.     

Biofilm formation by Escherichia coli O157:H7 on stainless steel: effect of exopolysaccharide and Curli production on its resistance to chlorine

The resistance of Escherichia coli O157:H7 strains ATCC 43895-, 43895-EPS (an exopolysaccharide [EPS]-overproducing mutant), and ATCC 43895+ (a curli-producing mutant) to chlorine, a sanitizer commonly used in the food industry, was studied. Planktonic cells of strains 43895-EPS and/or ATCC 43895+ grown under conditions supporting EPS and curli production, respectively, showed the highest resistance to chlorine, indicating that EPS and curli afford protection. Planktonic cells (ca. 9 log(10) CFU/ml) of all strains, however, were killed within 10 min by treatment with 50 microg of chlorine/ml. Significantly lower numbers of strain 43895-EPS, compared to those of strain ATCC 43895-, attached to stainless steel coupons, but the growth rate of strain 43895-EPS on coupons was not significantly different from that of strain ATCC 43895-, indicating that EPS production did not affect cell growth during biofilm formation. Curli production did not affect the initial attachment of cells to coupons but did enhance biofilm production. The resistance of E. coli O157:H7 to chlorine increased significantly as cells formed biofilm on coupons; strain ATCC 43895+ was the most resistant. Population sizes of strains ATCC 43895+ and ATCC 43895- in biofilm formed at 12 degrees C were not significantly different, but cells of strain ATCC 43895+ showed significantly higher resistance than did cells of strain ATCC 43895-. These observations support the hypothesis that the production of EPS and curli increase the resistance of E. coli O157:H7 to chlorine.     

Characterization of an exopolysaccharide produced by a marine Enterobacter cloacae

An exopolysaccharide producing marine bacterium, Enterobacter cloacae, was isolated from marine sediment collected from Gujarat coast, India. Chemical investigation of exopolysaccharide (EPS 71 a) revealed that this exopolysaccharide was an acidic polysaccliaride containing high amount of uronic acid, fucose and sulfate which is rare for bacterial exopolysaccharides. EPS 71a was found to have fucose, galactose, glucose and glucuronic acid in a molar ratio of 2: 1: 1: 1.     

Structural analysis of the alpha-D-glucan (EPS35-5) produced by the Lactobacillus reuteri strain 35-5 glucansucrase GTFA enzyme

The neutral exopolysaccharide EPS35-5 (reuteran) produced from sucrose by the glucansucrase GTFA enzyme from Lactobacillus reuteri 35-5 was found to be a (1-->4,1-->6)-alpha-D-glucan, with no repeating units present. Based on linkage analysis and 1D/2D 1H and 13C NMR spectroscopy of intact EPS35-5, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis and enzymatic hydrolysis, using pullulanase M1 (Klebsiella planticola), of EPS35-5, a composite model, that includes all identified structural elements, was formulated as follows: [Formula: see text].     

Methylobacterium sp. isolated from a Finnish paper machine produces highly pyruvated galactan exopolysaccharide

The slime-forming bacterium Methylobacterium sp. was isolated from a Finnish paper machine and its exopolysaccharide (EPS) was produced on laboratory scale. Sugar compositional analysis revealed a 100% galactan (EPS). However, FT-IR showed a very strong peak at 1611 cm(-1) showing the presence of pyruvate. Analysis of the pyruvate content revealed that, based on the sugar composition, the EPS consists of a trisaccharide repeating unit consisting of D-galactopyranose and [4,6-O-(1-carboxyethylidene)]-D-galactopyranose with a molar ratio of 1:2, respectively. Both linkage analysis and 2D homo- and heteronuclear 1H and 13C NMR spectroscopy revealed the following repeating unit: -->3)-[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)-alpha-D-Galp-(1-->. By enrichment cultures from various ground and compost heap samples a polysaccharide-degrading culture was obtained that produced an endo acting enzyme able to degrade the EPS described. The enzyme hydrolysed the EPS to a large extent, releasing oligomers that mainly consisted out of two repeating units.     

Exopolysaccharide sugars contribute to biofilm formation by Salmonella enterica serovar typhimurium on HEp-2 cells and chicken intestinal epithelium

Recently, we demonstrated that Salmonella enterica serovar Typhimurium can form biofilm on HEp-2 cells in a type 1 fimbria-dependent manner. Previous work on Salmonella exopolysaccharide (EPS) in biofilm indicated that the EPS composition can vary based upon the substratum on which the bacterial biofilm forms. We have investigated the role of genes important in the production of colanic acid and cellulose, common components of EPS. A mutation in the colanic acid biosynthetic gene, wcaM, was introduced into S. enterica serovar Typhimurium strain BJ2710 and was found to disrupt biofilm formation on HEp-2 cells and chicken intestinal tissue, although biofilm formation on a plastic surface was unaffected. Complementation of the wcaM mutant with the functional gene restored the biofilm phenotype observed in the parent strain. A mutation in the putative cellulose biosynthetic gene, yhjN, was found to disrupt biofilm formation on HEp-2 cells and chicken intestinal epithelium, as well as on a plastic surface. Our data indicate that Salmonella attachment to, and growth on, eukaryotic cells represent complex interactions that are facilitated by species of EPS.     

Exopolysaccharide production is required for biofilm formation and plant colonization by the nitrogen-fixing endophyte Gluconacetobacter diazotrophicus

The genome of the endophytic diazotrophic bacterial species Gluconacetobacter diazotrophicus PAL5 (PAL5) revealed the presence of a gum gene cluster. In this study, the gumD gene homologue, which is predicted to be responsible for the first step in exopolysaccharide (EPS) production, was insertionally inactivated and the resultant mutant (MGD) was functionally studied. The mutant MGD presented normal growth and nitrogen (N(2)) fixation levels but did not produce EPS when grown on different carbon sources. MGD presented altered colony morphology on soft agar plates (0.3% agar) and was defective in biofilm formation on glass wool. Most interestingly, MGD was defective in rice root surface attachment and in root surface and endophytic colonization. Genetic complementation reverted all mutant phenotypes. Also, the addition of EPS purified from culture supernatants of the wild-type strain PAL5 to the mutant MGD was effective in partially restoring wild-type biofilm formation and plant colonization. These data provide strong evidence that the PAL5 gumD gene is involved in EPS biosynthesis and that EPS biosynthesis is required for biofilm formation and plant colonization. To our knowledge, this is the first report of a role of EPS in the endophytic colonization of graminaceous plants by a nitrogen-fixing bacterium.     

Flocculation behavior and mechanism of an exopolysaccharide from the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913

Flocculation behavior and mechanism of the exopolysaccharide secreted by Pseudoalteromonas sp. SM9913 (EPS SM9913), a psychrophilic bacterium isolated from 1855m deep-sea sediment, has been studied in this paper. EPS SM9913 showed a peak flocculating activity of 49.3 in 1g/L kaolin suspension with 4.55mmol/L CaCl2 and the optimum pH range of 5-8. It appears that the flocculating activity of EPS SM9913 was stimulated by Ca2+ and Fe2+. This study found that EPS SM9913 showed a better flocculation performance than Al2(SO4)3 at salinity of 5-100 per thousand or temperatures of 5-15 degrees C. In addition, this EPS was effective to flocculate several other suspended solids. The measured zeta-potentials, the size of flocs formed during the flocculation process and the surface profile of flocs revealed by scan electron micrograph suggest that bridging is the main flocculation mechanism of the studied EPS. Deacetylation of EPS SM9913 resulted in a significant decrease in its flocculating activity indicating that the large number of acetyl groups in EPS SM9913 played an important role in its flocculation performance.     

红球菌HX-2所产胞外多糖的特性和对Cu2+的吸附

【背景】目前,微生物所产胞外多糖(exopolysaccharide,EPS)的理化性质及其在重金属吸附中的应用受到了广泛关注。【目的】研究红球菌HX-2所产胞外多糖的理化性质,并探究其对重金属的吸附情况。【方法】使用离子交换和凝胶色谱分离法对胞外多糖粗品进行纯化;利用苯酚硫酸法测胞外多糖中糖含量;用Bradford试剂盒检测胞外多糖中蛋白含量;使用甲醇萃取法检测胞外多糖中脂质含量;用高效液相色谱(high performance liquid chromatography,HPLC)法分析胞外多糖中单糖组成;用扫描电镜(scanningelectronmicroscopy,SEM)法观察多糖表面形态;通过等温吸附模型和动力学模型探究胞外多糖对重金属的吸附效果。【结果】测得胞外多糖主要成分EPS-G-1中总糖含量为78.43%,蛋白含量为8.31%,脂质含量为8.22%;纯化后胞外多糖中单糖组成为葡萄糖、甘露糖、半乳糖、葡萄糖醛酸和岩藻糖,质量比为27.31:26.67:24.83:15.85:4.80;通过等温吸附模型拟合得到HX-2所产胞外多糖对Cu~(2+)的最大吸附量为144.93 mg/g。【结论】红球菌HX-2所产胞外多糖对水体中Cu~(2+)具有良好的吸附作用,可用于工业废水中重金属离子的处理。     

Isolation and characterization of mucous exopolysaccharide (EPS) produced by Vibrio furnissii strain VB0S3

Marine bacterial strains were isolated from coastal regions of Goa and screened for the strains that produce the highest amount of mucous exopolysaccharide (EPS). Our screening resulted in the identification of the strain Vibrio furnissii VB0S3 (hereafter called VB0S3), as it produced the highest EPS in batch cultures during the late logarithmic growth phase. The isolate was identified as VB0S3 based on morphological and biochemical properties. Growth and EPS production were studied in mineral salts medium supplemented with NaCl (1.5%) and glucose (0.2%). The exopolymer was recovered from the culture supernatant by using three volumes of cold ethanol precipitation and dialysis procedure. Chemical analyses of EPS revealed that it is primarily composed of neutral sugars, uronic acids, and proteins. Fourier-transform infrared (FT-IR) spectroscopy revealed the presence of carboxyl, hydroxyl, and amide groups, which correspond to a typical heteropolymeric polysaccharide, and the EPS also possessed good emulsification activity. The gas chromatographic analysis of an alditol-acetate derivatized sample of EPS revealed that it was mainly composed of galactose and glucose. Minor components found were mannose, rhamnose, fucose, ribose, arabinose, and xylose. EPS was readily isolated from culture supernatants, which suggests that the EPS was a slime-like exopolysaccharide. This is the first report of exopolysaccharide characterization that describes the isolation and characterization of an EPS expressed by Vibrio furnissii strain VB0S3. The results of the study contribute significantly and go a long way towards an understanding of the correlation between growth and EPS production, chemical composition, and industrial applications of the exopolysaccharide in environmental biotechnology and bioremediation.     

The structure of a polysaccharide from infectious strains of Burkholderia cepacia.

The structure of an acidic exopolysaccharide (EPS) from eight strains of Burkholderia cepacia has been investigated by methylation and sugar analysis, periodate oxidation-Smith degradation, and partial acid-hydrolysis. An enzyme preparation obtained from the same organisms producing the EPS was also used to depolymerize the polysaccharide. Detailed NMR studies of the chemical and enzymatic degradation products showed that this EPS consists of a highly branched heptasaccharide-repeating unit with the following structure: [abstract: see text]. About three O-acetyl groups per repeating unit are present at undetermined positions.     

Selectively inducing the synthesis of a key structural exopolysaccharide in aerobic granules by enriching for <Emphasis Type="BoldItalic">Candidatus</Emphasis> “<Emphasis Type="BoldItalic">Competibacter phosphatis</Emphasis>”

A gel-forming exopolysaccharide was previously shown to play an important structural role in aerobic granules treating nutrient-rich industrial wastewater. To identify whether this exopolysaccharide performs a similar role in other granular biomass and if conditions favouring its production can be more precisely elucidated, extracellular polymeric substances (EPS) were extracted from granules grown under four different operating conditions. ¹H nuclear magnetic resonance (NMR) spectroscopy of their EPS indicated that the gel-forming exopolysaccharide was expressed in two granular sludges both enriched in Candidatus “Competibacter phosphatis”. In contrast, it was not expressed in granules performing denitrification with methanol as a carbon source and nitrate as the electron acceptor or granules enriched in Candidatus “Accumulibacter phosphatis” performing enhanced biological phosphorus removal from synthetic wastewater. In one of the first two sludges, the exopolysaccharide contained in the seeding granular sludge continued to be a major component of the granule EPS while Competibacter was being enriched. In the second sludge, a floccular sludge not containing the gel-forming exopolysaccharide initially was also enriched for Competibacter. In this sludge, an increase in particle size was detected coinciding with a yield increase of EPS. NMR spectroscopy confirmed its yield increase to be attributable to the production of this structural gel-forming exopolysaccharide. The results show that (1) the particular gel-forming exopolysaccharide previously identified is not necessarily a key structural exopolysaccharide for all granule types, and (2) synthesis of this exopolysaccharide is induced under conditions favouring the selective enrichment of Competibacter. This indicates that Competibacter may be involved in its production.     

Evidence for the Adhesive Function of the Exopolysaccharide of Hyphomonas Strain MHS-3 in Its Attachment to Surfaces

Hyphomonas strain MHS-3 (MHS-3) is a marine procaryote with a biphasic life cycle and which has prosthecate stages that adhere to submerged substrata. We found that adherent forms produced an exopolysaccharide (EPS) capsule that bound Glycine max lectin, Arachis hypogaea lectin, and Bauhinia purpurea lectin (BPA), each having affinity for N-acetyl-d-galactosamine. It also bound the dye Calcofluor. BPA and Calcofluor were tested for the ability to hinder MHS-3 adhesion to glass surfaces; they reduced attachment by >50 and >85%, respectively. Periodate treatment also reduced attachment (by >80%), but pronase treatment did not. Furthermore, an EPS(sup-) variant, Hyphomonas strain MHS-3 rad, did not attach well to surfaces. These results suggest that the MHS-3 EPS capsule is an adhesin.     

Peculiarities of ethanol metabolism in an Acinetobacter sp. mutant strain defective in exopolysaccharide synthesis

Activities of the key enzymes of ethanol metabolism were assayed in ethanol-grown cells of an Acinetobacter sp. mutant strain unable to synthesize exopolysaccharides (EPS). The original EPS-producing strain could not be used for enzyme analysis because its cells could not to be separated from the extremely viscous EPS with a high molecular weight. In Acinetobacter sp., ethanol oxidation to acetaldehyde proved to be catalyzed by the NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1.). Both NAD+ and NADP+ could be electron accepters in the acetaldehyde dehydrogenase reaction. Acetate is implicated in the Acinetobacter sp. metabolism via the reaction catalyzed by acetyl-CoA-synthetase (EC 6.2.1.1.). Isocitrate lyase (EC 4.1.3.1.) activity was also detected, indicating that the glyoxylate cycle is the anaplerotic mechanism that replenishes the pool of C4-dicarboxylic acids in Acinetobacter sp. cells. In ethanol metabolism by Acinetobacter sp., the reactions involving acetate are the bottleneck, as evidenced by the inhibitory effect of sodium ions on both acetate oxidation in the intact cells and on acetyl-CoA-synthetase activity in the cell-free extracts, as well as by the limitation of the C2-metabolism by coenzyme A. The results obtained may be helpful in developing a new biotechnological procedure for obtaining ethanol-derived exopolysaccharide ethapolan.