Characterization of exopolysaccharides produced by three moderately halophilic bacteria belonging to the family Alteromonadaceae

Aims:  To study the exopolysaccharides (EPSs) produced by three novel moderately halophilic species belonging to the family Alteromonadaceae to optimize EPS yields, characterize their physical and chemical properties and evaluate possible biotechnological applications for these polymers.
Methods and Results:  EPSs synthesized by Idiomarina fontislapidosi F32^T, Idiomarina ramblicola R22^T and Alteromonas hispanica F23^T were collected and analysed under optimum conditions: MY medium supplemented with 7·5% (w/v) salts; 32°C; and 1% (w/v) glucose. Polymers were synthesized mainly during the early stationary growth phase with yields ranging from 1 to 1·5 g l⁻¹. The Idiomarina species each produced an anionic EPS composed mainly of glucose, mannose and galactose. A. hispanica synthesized an anionic EPS composed mainly of glucose, mannose and xylose. Solutions of all the polymers were low in viscosity and pseudoplastic in their behaviour. They showed emulsifying activity and the capacity to bind some metals.
Conclusions:  The Alteromonadaceae species studied in this work produced EPSs with physical and chemical properties different from those produced by other halophilic and nonhalophilic bacteria, suggesting that the wide diversity of micro-organisms being encountered nowadays in hypersaline environments offers enormous potential resources for biotechnological applications.
Significance and Impact of the Study:  We have optimized the EPS production and analysed new biopolymers produced by some recently described, moderately halophilic bacteria. These biopolymers are chemically and physically different from others already in use in biotechnology and offer hopes for new applications, especially in the case of A. hispanica , which may prove to be a viable source of xylo-oligosaccharides.     

Biological characterization of lead-enhanced exopolysaccharide produced by a lead resistant Enterobacter cloacae strain P2B

A lead resistant bacterial strain isolated from effluent of lead battery manufacturing company of Goa, India has been identified as Enterobacter cloacae strain P2B based on morphological, biochemical characters, FAME profile and 16S rDNA sequence data. This bacterial strain could resist lead nitrate up to 1.6?mM. Significant increase in exopolysaccharide (EPS) production was observed as the production increased from 28 to 108?mg/L dry weight when exposed to 1.6?mM lead nitrate in Tris buffered minimal medium. Fourier-transformed infrared spectroscopy of this EPS revealed presence of several functional groups involved in metal binding viz. carboxyl, hydroxyl and amide groups along with glucuronic acid. Gas chromatography coupled with mass spectrometry analysis of alditol-acetate derivatives of acid hydrolysed EPS produced in presence of 1.6?mM lead nitrate demonstrated presence of several neutral sugars such as rhamnose, arabinose, xylose, mannose, galactose and glucose, which contribute to lead binding hydroxyl groups. Scanning electron microscope coupled with energy dispersive X-ray spectrometric analysis of this lead resistant strain exposed to 1.6?mM lead nitrate interestingly revealed mucous EPS surrounding bacterial cells which sequestered 17?% lead (as weight %) extracellularly and protected the bacterial cells from toxic effects of lead. This lead resistant strain also showed multidrug resistance. Thus these results significantly contribute to better understanding of structure, function and environmental application of lead-enhanced EPSs produced by bacteria. This lead-enhanced biopolymer can play a very important role in bioremediation of several heavy metals including lead.     

Flocculating performances of exopolysaccharides produced by a halophilic bacterial strain cultivated on agro-industrial waste

This study reports the first systematic investigation of the flocculation dynamics of exopolysaccharides (EPSs) produced by a halophilic bacterial strain grown on pretreated molasses as fermentation substrate. The potential use of these EPSs as an easily biodegradable, natural alternative for synthetic polyelectrolytes which are widely used and contain toxic and carcinogenic monomers was investigated. Flocculating activities of the EPS samples in synthetic water, synthetic sea water and natural sea water media which were used as model raw waters were monitored via the Photometric Dispersion Analyser (PDA 2000) instrument and removals were determined by measuring residual turbidities. One of the six EPS specimens, which formed the largest flocs thus performed highest turbidity removal, exhibited flocculation performance and particle removal efficiency comparable with commercial cationic, nonionic and anionic synthetic polyelectrolytes.     

Stable-Isotope Probing Identifies Uncultured Planctomycetes as Primary Degraders of a Complex Heteropolysaccharide in Soil

The exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced by Gluconacetobacter xylinus or the EPS produced by Beijerinckia indica. The latter is a heteropolysaccharide comprised primarily of l-guluronic acid, d-glucose, and d-glycero-d-mannoheptose. ¹³C-labeled EPS and ¹³C-labeled cellulose were purified from bacterial cultures grown on [¹³C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from ¹³C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However, B. indica EPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylum Planctomycetes. In one incubation, members of the Planctomycetes made up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo- or monosaccharides, the predominance of Planctomycetes suggested that they were primary degraders of EPS. Other bacteria assimilating B. indica EPS included members of the Verrucomicrobia, candidate division OD1, and the Armatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species.     

Sugar composition and FT-IR analysis of exopolysaccharides produced by microbial isolates from paper mill slime deposits

Thirty exopolysaccharides (EPS) produced by bacteria isolated from biofilms or slimelayers from different paper and board mills in Finland, France and Spain were subjected to size exclusion chromatography and sugar compositional analysis. High performance size exclusion chromatography (HPSEC) analysis revealed that some samples were composed of several molecular weight populations. These samples were fractionated by size exclusion chromatography and pooled accordingly. Principal components analysis (PCA) of the sugar compositions of the different pools indicated the presence of glucans and mannans caused by insufficient removal of the carbon or nitrogen source (yeast extract) from the bacteria growth medium leading to an overestimation of the glucose and mannose level in the sample, respectively. From the point of view of slime problems the EPS populations are the most important for multivariate analysis. Four groups of EPSs have been recognized by PCA analysis: a group of EPSs produced by Enterobacter and related genera similar to the regularly reported colanic acid; a group of Methylobacterium EPSs having high galactose and pyruvate levels and two groups that showed less dense clusters produced by Bacillus and related genera, showing high mannose and/or glucose levels and Klebsiella EPSs that showed galactose with rhamnose as major characteristic sugar moieties. Fourier transform infrared spectroscopy (FT-IR) of the same samples followed by discriminant partial least squares regression (DPLS) and linear discriminant analysis (LDA) showed that, when used with a well-defined training set, FT-IR could be used clustering instead of time-consuming sugar composition analysis. The Enterobacter and Methylobacetrium EPS groups could be recognized clearly. However the fact that this could hardly be done for the other two groups in the dataset indicates the importance of a larger and well-defined training or calibration set. The potential to use FT-IR, as a tool for pattern recognition and clustering with respect to EPS structures produced by micro organisms isolated from a paper mill environment is discussed.     

Phylogenetic analysis and screening of antimicrobial and cytotoxic activities of moderately halophilic bacteria isolated from the Weihai Solar Saltern (China)

A total of 45 moderately halophilic bacteria was isolated from sediment and saline water collected from the Weihai Solar Saltern (China). The phylogenetic position of all the isolated strains was determined by 16S rRNA sequencing. The halophilic strains were tested for their antimicrobial activity. Cytotoxicity assay was performed to determine which of the halophilic strains could inhibit proliferation of human hepatocellular carcinoma Bel 7402 cells. Our results showed that all of the isolated 45 strains displayed moderately halophilic characteristics. Phylogenetic analysis indicated that 17 of the isolated strains were related to the phylum Firmicutes and belonged to four genera, Bacillus, Halobacillus, Planococcus and Salinicoccus. The other strains identified as genus of Halomonas belonged to phylum γ-Proteobacteria. Most of the halophilic bacterial strains showed potent activities against Gram-positive bacteria, human pathogenic fungi and plant pathogenic fungi. In addition, the crude extracts from 14 halophilic bacterial strains showed cytotoxic activity against tumor cells Bel 7402, and five of them showed remarkable activities with IC₅₀ less than 40 μg ml⁻¹. Our results suggest that the moderately halophilic bacteria may be developed as promising sources for the discovery of novel bioactive substances.     

Resistance of the oil-oxidizing microorganism <Emphasis Type="Italic">Dietzia</Emphasis> sp. to hyperosmotic shock in reconstituted biofilms

A number of halotolerant and halophilic bacterial strains were isolated from the Romashkinskoe oil field (Tatarstan) stratal waters having a salinity of up to 100 g/l. The isolation of pure cultures involved biofilm reconstitution on M9 medium with paraffins. The associations obtained were dispersed and reinoculated onto solid media that contained either peptone and yeast extract (PY medium) or paraffins. It was shown that such associations included both oil-oxidizing bacteria and accompanying chemoheterotrophic bacteria incapable of oil oxidation. The pure cultures that were isolated were used for creating binary biofilms. In these biofilms, interactions between halophilic and nonhalophilic bacteria under hypo-and hyperosmotic shocks were investigated. We conducted a detailed study of a biofilm obtained from an oil-oxidizing halotolerant species (with an upper growth limit of 10–12% NaCl) identified as Dietzia sp. and an extremely halophilic gram-negative bacterium (growing within the 5–20% NaCl concentration range) of the genus Chromohalobacter that did not oxidize paraffins. If these microorganisms were grown in a mixed suspension (planktonic) culture that was not supplemented with an additional amount of NaCl, no viable cells of the halophilic microorganism were detected after reinoculation. In contrast, only halophilic cells were detected at a NaCl concentration of 15%. Thus, no mutual protective influence of the microorganisms manifested itself in suspension culture, either under hypoor under hyperosmotic shock. Neither could halophile cells be detected after reinoculating a biofilm obtained on a peptone medium without the addition of NaCl. However, biofilms produced at a NaCl concentration of 15% contained approximately equal numbers of cells of the halophilic and halotolerant organisms. Thus, the halophile in biofilms sustaining a hyperosmotic shock exerts a protective influence on the halotolerant microorganism. Preliminary data suggest that this effect is due to release by the halophile of osmoprotective substances (ectoine and glutamate), which are taken up by the halotolerant species. Such substances are diluted by a large medium volume in suspension cultures, whereas, in biofilms, their diffusion into the medium is apparently hampered by their interaction with the intercellular polymer matrix.     

Bacterial polysaccharides suppress induced innate immunity by calcium chelation

Bacterial pathogens and symbionts must suppress or negate host innate immunity. However, pathogens release conserved oligomeric and polymeric molecules or MAMPs (Microbial Associated Molecular Patterns), which elicit host defenses [1], [2] and [3]. Extracellular polysaccharides (EPSs) are key virulence factors in plant and animal pathogenesis, but their precise function in establishing basic compatibility remains unclear [4], [5], [6] and [7]. Here, we show that EPSs suppress MAMP-induced signaling in plants through their polyanionic nature [4] and consequent ability to chelate divalent calcium ions [8]. In plants, Ca2+ ion influx to the cytosol from the apoplast (where bacteria multiply [4], [5] and [9]) is a prerequisite for activation of myriad defenses by MAMPs [10]. We show that EPSs from diverse plant and animal pathogens and symbionts bind calcium. EPS-defective mutants or pure MAMPs, such as the flagellin peptide flg22, elicit calcium influx, expression of host defense genes, and downstream resistance. Furthermore, EPSs, produced by wild-type strains or purified, suppress induced responses but do not block flg22-receptor binding in Arabidopsis cells. EPS production was confirmed in planta, and the amounts in bacterial biofilms greatly exceed those required for binding of apoplastic calcium. These data reveal a novel, fundamental role for bacterial EPS in disease establishment, encouraging novel control strategies.     

Colanic acid is an exopolysaccharide common to many enterobacteria isolated from paper-machine slimes

In this study, polysaccharide-producing bacteria were isolated from slimes collected from two Finnish and one Spanish paper mill and the exopolysaccharides (EPSs) produced by 18 isolates were characterised. Most of the isolates, selected on the bases of slimy colony morphology, were members of the family Enterobacteriaceae most frequently belonging to the genera Enterobacter and Klebsiella including Raoultella. All of the EPSs analysed showed the presence of charged groups in the form of uronic acid or pyruvate revealing the polyanionic nature of these polysaccharides. Further results of the carbohydrate analysis showed that the EPS produced by nine of the enterobacteria was colanic acid.     

Exopolysaccharide-overproducing Lactobacillus paracasei KB28 induces cytokines in mouse peritoneal macrophages via modulation of NF-κβ and MAPKs

Exopolysaccharides (EPSs) are microbial polysaccharides that are released outside of the bacterial cell wall. There have been few studies on EPS-producing lactic acid bacteria that can enhance macrophage activity and the underlying signaling mechanism for cytokine expression. In the current study, EPS-overproducing Lactobacillus (L.) paracasei KB28 was isolated from kimchi and cultivated in conditioned media containing glucose, sucrose, and lactose. The whole bacterial cells were obtained with their EPS being attached, and the cytokine-inducing activities of these cells were investigated. Gas chromatography analysis showed the presence of glucose, galactose, mannose, xylose, arabinose, and rhamnose in EPS composition. EPS-producing L. paracasei KB28 induced the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12 in mouse macrophages. This strain also caused the degradation of IκBα and phosphorylation of the major MAPKs: Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK)1/2. The use of pharmacological inhibitors showed that different signaling pathways were involved in the induction of TNF-α, IL-6 and IL-12 by L. paracasei KB28. Our results provide information for a better understanding of molecular mechanisms of the immunomodulatory effect of food-derived EPS-producing lactic acid bacteria.     

Acute lymphoblastic leukemia-derived extracellular vesicles affect quiescence of hematopoietic stem and progenitor cells

Patient-derived xenografted (PDX) models were generated through the transplantation of primary acute lymphoblastic leukemia (ALL) cells into immunodeficient NSG mice. We observed that ALL cells from mouse bone marrow (BM) produced extracellular vesicles (EVs) with specific expression of inducible heat shock protein HSP70, which is commonly activated in cancer cells. Taking advantage of this specific expression, we designed a strategy to generate fluorescent HSP70-labeled ALL EVs and monitor the impact of these EVs on endogenous murine BM cells ex vivo and in vivo. We discovered that hematopoietic stem and progenitor cells (HSPC) were mainly targeted by ALL EVs, affecting their quiescence and maintenance in the murine BM environment. Investigations revealed that ALL EVs were enriched in cholesterol and other metabolites that contribute to promote the mitochondrial function in targeted HSPC. Furthermore, using CD34⁺ cells isolated from cord blood, we confirmed that ALL EVs can modify quiescence of human HSPC. In conclusion, we have discovered a new oncogenic mechanism illustrating how EVs produced by proliferative ALL cells can target and compromise a healthy hematopoiesis system during leukemia development.Subject terms: Acute lymphocytic leukaemia, Cancer models, Cancer metabolism     

Isolation and characterization of <Emphasis Type="Italic">Pseudoalteromonas ruthenica</Emphasis> (SBT033), an EPS-producing biofilm bacterium from the seawater intake point of a tropical power station

Biofilm formation in bacteria is closely linked with production of exopolysaccharides (EPS). This study examined the quantitative variations in EPS production and biofilm-forming ability among bacteria isolated from the seawater intake point of a power station located on the east coast of India. Of the 233 isolates obtained from the intake site, 71 bacterial isolates displayed different colony morphological characteristics. Thirteen isolates that produced wide and thick mucoid colonies were further tested for their ability to attach and form biofilms by microtitre plate assay and confocal microscopy. EPS production among the selected bacterial isolates ranged from 826 to 1838 μg ml⁻¹. Strain SBT033, which produced the maximum amount of EPS also displayed the maximum biofilm-forming ability among the 13 isolates. This strain was selected for further characterization using biochemical and molecular methods. The pale orange-pigmented isolate was a Gram negative, aerobic, short rod-shaped and grew well only in the presence of 2% NaCl. On the basis of phenotypic characteristics the isolate SBT033 is shown to belong to the genus Pseudoalteromonas. Analysis of 16S rRNA of the isolate revealed 99% homology with Pseudoalteromonas ruthenica.     

Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure

AIMS: To compare galactose-negative strains of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus isolated from fermented milk products and known to produce exopolysaccharides (EPSs). METHODS AND RESULTS: The structures of the EPSs were determined using nuclear magnetic resonance (NMR) and their genetic relationships determined using restriction endonuclease analysis (REA) and random amplification of polymorphic DNA (RAPD). Similar groupings were apparent by REA and RAPD, and each group produced an EPS with a particular subunit structure. CONCLUSION: Although none of the strains assimilated galactose, all inserted a high proportion of galactose into their EPS when grown in skimmed milk, and fell into three distinct groups. Significance and Impact of the Study: This information should help in an understanding of genetic exchanges in lactic acid bacteria.     

Small-scale analysis of exopolysaccharides from Streptococcus thermophilus grown in a semi-defined medium

Background  

Exopolysaccharides (EPSs) produced by lactic acid bacteria are important for the texture of fermented foods and have received a great deal of interest recently. However, the low production levels of EPSs in combination with the complex media used for growth of the bacteria have caused problems in the accurate analysis of the EPS. The purpose of this study was to find a growth medium for physiological studies of the lactic acid bacterium Streptococcus thermophilus, and to develop a simple method for qualitative and quantitative analysis of EPSs produced in this medium.     

四川冬菜中细菌群落组成及多样性

【目的】了解腌制4年的四川南充冬菜中细菌群落组成及多样性。【方法】通过16S rDNA多样性分析样品细菌落组成;采用16S rDNA-RFLP方法分析从样品中分离出的纯培养细菌。【结果】16S rDNA多样性分析结果表明,样品中细菌主要属于变形杆菌门(Proteobacteria)和厚壁菌门(Firmicutes),分别占克隆文库的87.9%、7.1%,其中包括Virgibacillus kekensis,Marinococcus albus,Salinicoccus sp.,Lactobacillus halophilus和Halomonas等中度嗜盐菌,仅有5%属于放线菌门(Actinobacteria)。通过纯培养方法从冬菜中分离到35株菌,16S rDNA-RFLP分析结果表明,34株属于厚壁菌门(Firmicutes),包括Virgibacillus,Bacillus megaterium和Gracilibacillus saliphilus等中度嗜盐菌,1株属于放线菌门(Actinobacteria)。【结论】冬菜中细菌群落多样性较低,以中度嗜盐菌为主。     

Diversity of Heterotrophic Halophilic Bacteria Isolated from Coastal Solar Salterns,Bulgaria and Their Ability to Synthesize Bioactive Molecules with Biotechnological Impact

Investigations on the microbial life in several coastal solar salterns have revealed the presence of novel organisms and synthesis of unusual molecules active in extreme conditions which might be useful in different biotechnological industries. Biodiversity of heterotrophic aerobic bacteria isolated from two salterns, Pomorie salterns and Burgas salterns located at Burgas Bay, Black Sea coast, Bulgaria, as well as ability of the isolates to synthesize biotechnologically valuable compounds were investigated. The results revealed high taxonomic and metabolic bacterial diversity—we isolated 20 morphologically different moderately halophilic and two halotolerant strains affiliated with 11 species from eight genera referred to the phyla Proteobacteria, Firmicutes, and Actinobacteria. Gram-negative bacteria belonged to the genera Halomonas, Chromohalobacter, Salinivibrio, Cobetia, and Nesiotobacter, and gram-positive strains were representatives of the genera Virgibacillus, Salinicoccus, and Brevibacterium. All isolates were found to be alkalitolerant, and 41% of them were psychrotolerant. The strains degraded nine of the tested 18 substrates; polygalacturonase, catalase, phytase, and lipase producers were predominant. This is the first reported detection of xanthan lyase, gellan lyase, arabinase, and phytase activities in halophilic bacteria. Nine of the strains belonging to five different genera were found to produce exopolysaccharides (EPS). The highest level of EPS was observed in Chromohalobacter canadensis strain 28. More than a half of the strains displayed antimicrobial activity against one to five test bacteria and yeasts. The present study is the first report on halophilic bacteria isolated from salterns at the Black Sea coast indicating that the investigated area is an untapped resource of halophilic bacteria with biotechnological potential.     

Microbial exopolysaccharides: Main examples of synthesis, excretion, genetics and extraction

Exopolysaccharides (EPSs) produced by microorganisms represent an industrially untapped market. Some microorganisms can produce and excrete over 40 g L⁻¹ of EPS in simple but costly production conditions.Approximately thirty strains of eukaryotic and prokaryotic microorganisms are notable for their EPS production. EPSs are produced in response to biotic and abiotic stress factors and/or to adapt to an extreme environment. The main function of EPSs is to aid in protection against environmental pressures.Heteropolysaccharides and some homopolysaccharides are synthesised in microbial cells and then secreted into the extracellular environment. More currently, homopolysaccharide synthesis occurs outside of the cells after specific enzymes are exuded.Although natural secretory mechanisms exist in microorganisms, it is often necessary to resort to physical or chemical extraction methods to improve the yield of EPSs at an industrial level.In light of growing interest, our basic understanding of microbial EPSs needs to be improved.     

Exopolysaccharides Isolated from Hydrothermal Vent Bacteria Can Modulate the Complement System

The complement system is involved in the defence against bacterial infection, or in the elimination of tumour cells. However, disturbances in this system contributes to the pathogenesis of various inflammatory diseases. The efficiency of therapeutic anti-tumour antibodies is enhanced when the complement system is stimulated. In contrast, cancer cells are able to inhibit the complement system and thus proliferate. Some marine molecules are currently being developed as new drugs for use in humans. Among them, known exopolyssacharides (EPSs) generally originate from fungi, but few studies have been performed on bacterial EPSs and even fewer on EPSs extracted from deep-sea hydrothermal vent microbes. For use in humans, these high molecular weight EPSs must be depolymerised. Furthermore, the over-sulphation of EPSs can modify their biological activity. The aim of this study was to investigate the immunodulation of the complement system by either native or over-sulphated low molecular weight EPSs isolated from vent bacteria in order to find pro or anti-activators of complement.     

Biodegradability of Food-Associated Extracellular Polysaccharides

Exopolysaccharides (EPSs) produced by lactic acid bacteria, which are common in fermented foods, are claimed to have various beneficial physiological effects on humans. Although the biodegradability of EPSs is important in relation to the bioactive properties, knowledge on this topic is limited. Therefore, the biodegradability of eight EPSs, six of which were produced by lactic acid bacteria, was compared with microorganisms from human feces or soil. EPS-degradation was determined from the decrease in polysaccharide-sugar concentration and by high-performance size exclusion chromatography (HPSEC). Xanthan, clavan, and the EPSs produced by Streptococcus thermophilus SFi 39 and SFi 12 were readily degraded, in contrast to the EPSs produced by Lactococcus lactis ssp. cremoris B40, Lactobacillus sakei 0-1, S. thermophilus SFi20, and Lactobacillus helveticus Lh59. Clearly, the susceptibility of exopolysaccharides to biological breakdown can differ greatly, implying that the physiological effects of these compounds may also vary a lot. Received: 23 August 1999 / Accepted: 5 October 1999