Polysaccharides production by Rhizobium phaseoli and the typing of their excreted anionic polysaccharides

Abstract The pattern of polysaccharide production amongst strains of Rhizobium phaseoli appear very varied: some strains produce anionic exopolysaccharides (EPS) as major polysaccharides (EPS) as major polymer without any other product, but most strains exhibit greater polysaccharide diversity. Apart from EPS they excrete capsular polysaccharides (CPS) and accumulate poly-β-hydroxybutyric acid (PHB) and/or glycogen in their cells. The latter can then be used as C-sources for further synthesis of EPS and CPS. Some strains are only very poor producers or do not produce at all. Nine strains of R. phaseoli have been analysed and shown to possess the K-36 type of polysaccharide (EPS), as do strains of R. leguminosarum (6 strains) and R. trifolli (9 strains). Three strains of R. phaseoli have been found to possess the K-87 type of polysaccharide and types K-38 and K-44 polysaccharides have only been found in their own type strains.     

Immune Modulation Capability of Exopolysaccharides Synthesised by Lactic Acid Bacteria and Bifidobacteria

During recent years, the exopolysaccharides (EPS) produced by some strains of lactic acid bacteria and bifidobacteria have attracted the attention of researchers, mainly due to their potential technological applications. However, more recently, it has been observed that some of these EPS present immunomodulatory properties, which suggest a potential effect on human health. Whereas EPS from lactic acid bacteria have been studied in some detail, those of bifidobacteria largely remain uncharacterized in spite of the ubiquity of EPS genes in Bifidobacterium genomes. In this review, we have analysed the data collected in the literature about the potential immune-modulating capability of EPS produced by lactic acid bacteria and bifidobacteria. From this data analysis, as well as from results obtained in our group, a hypothesis relating the physicochemical characteristics of EPS with their immune modulation capability was highlighted. We propose that EPS having negative charge and/or small size (molecular weight) are able to act as mild stimulators of immune cells, whereas those polymers non-charged and with a large size present a suppressive profile.     

Rhizobium meliloti exopolysaccharides: genetic analyses and symbiotic importance.

Genetic experiments have indicated that succinoglycan (EPS I), the acidic Calcofluor-binding exopolysaccharide, of the nitrogen-fixing bacterium Rhizobium meliloti strain Rm1021 is required for nodule invasion and possibly for later events in nodule development on alfalfa and other hosts. Fourteen exo loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. We have identified two loci, exoR and exoS, that are involved in the regulation of EPS I synthesis in the free-living state. Certain exo mutations which completely abolish EPS I production are lethal in an exoR95 or exoS96 background. Histochemical analyses of the expression of exo genes during nodulation using exo::TnphoA fusions have indicated that the exo genes are expressed most strongly in the invasion zone. In addition, we have discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for exopolysaccharides in symbiosis are discussed.     

Genetic analyses of Rhizobium meliloti exopolysaccharides

We have recently obtained strong genetic evidence that the acidic Calcofluor-binding exopolysaccharide (EPS I) of Rhizobium meliloti Rm1021 is required for nodule invasion and possibly for later events in nodule development. Thirteen loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. exoH mutants fail to succinylate their EPS I and form empty Fix- nodules. We have identified two unlinked regulatory loci, exoR and exoS, whose products play negative roles in the regulation of expression of the exo genes. We have recently discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for Rhizobium exopolysaccharides in nodulation are discussed.     

A gene cluster required for coordinated biosynthesis of lipopolysaccharide and extracellular polysaccharide also affects virulence of Pseudomonas solanacearum.

Bacterial cell surface components can be important determinants of virulence. At least three gene clusters important for extracellular polysaccharide (EPS) biosynthesis have been previously identified in the plant pathogen Pseudomonas solanacearum. We have found that one of these gene clusters, named ops, is also required for lipopolysaccharide (LPS) biosynthesis. Mutations in any complementation unit of this cluster decreased EPS production, prevented the binding of an LPS-specific phage, and altered the mobility of purified LPS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, restoration of LPS biosynthesis alone was not sufficient to restore virulence to the wild-type level, suggesting that EPS is important for pathogenesis.     

Direct visualization of the interaction between pilin and exopolysaccharides of Myxococcus xanthus with eGFP-fused PilA protein

Type IV pili (TFP) and exopolysaccharides (EPS) are important components for social behaviors in Myxococcus xanthus, including gliding motility and fruiting body formation. Although specific interactions between TFP and EPS have been proposed, there have as yet been no direct observations of these interactions under native conditions. In this study, we found that a truncated PilA protein (PilACt) containing only the C-terminal domain (amino acids 32-208) is sufficient for EPS binding in vitro. Furthermore, an enhanced green fluorescent protein (eGFP) and PilACt fusion protein were constructed and used to label the native EPS in M. xanthus. Under confocal laser scanning microscope, the eGFP-PilACt-bound fruiting bodies, trail structures and biofilms exhibited similar patterns as the wheat germ agglutinin lectin-labeled EPS structures. This study showed that eGFP-PilACt fusion protein was able efficiently to label the EPS of M. xanthus, providing evidence for the first time of the direct interaction between the PilA protein and EPS under native conditions.     

Molecular-mechanics modelling of drug-DNA structures; the effects of differing dielectric treatment on helix parameters and comparison with a fully solvated structural model

This study analyses the influence that the nature of the dielectric constant has on the final structures obtained from in vacuo molecular mechanics calculations on a drug-DNA complex and compares these structures with the energy minimised complex including explicit solvent molecules. Minimisations have been performed on a proflavine-decanucleotide structure, where the drug was intercalated at the d(CpG) site of the d(GpApTpApCpGpApTpApC) decamer duplex, using two expressions for the dielectric constant: a distance-independent, epsilon ij = EPS, and a distance-dependent, epsilon ij = EPS*Rij, form and for values of EPS from 1 to 8. Significantly different structures are obtained for the distance-independent and the distance-dependent expressions of the dielectric constant. The use of a distance-independent dielectric constant leads to distorted structures, which are very sensitive to slight changes in the value of EPS. The use of a distance-dependent dielectric constant leads to less distorted and more stable structures. The effects on helical parameters are analysed in detail. The structures obtained for different values of EPS (within the distance-dependent formalism) seem to converge for values of EPS equal to 4 or greater. Based on these results a distance-dependent form of the dielectric with an EPS value of 4 is recommended in order to produce reliable refined nucleic acid structures by molecular mechanics. These conclusions have been supported by molecular-mechanics minimisation of the same structure with the inclusion of explicit water molecules and counter-ions.     

Towards a better production of bacterial exopolysaccharides by controlling genetic as well as physico-chemical parameters

Bacterial extracellular polymeric substances, which are basically bacterial metabolites, have currently become a subject of great concern of modern day microbiologists and biotechnologists. Among these metabolites, bacterial exopolysaccharides or EPS, in particular, have gained a significant importance. EPS are formed by the bacteria in their late exponential or stationary phase of growth under special situations for specific purposes. They take part in the formation of bacterial biofilms. There is a great diversity in the types of EPS. Strikingly enough, a same species of bacterium can produce different types of EPS under different situations. The importance of EPS is largely because of their different applications in various industries. Now that the bacterial EPS has got the potentiality to become an upcoming tool in various futuristic applications of human benefit, the focus currently develops towards how better they can be produced in the laboratory by promoting the favorable factors for their production. While studying with different EPS forming bacteria, both the intrinsic factors like genetic configuration of the bacteria and the extrinsic factors like culture conditions under the influence of different physico-chemical parameters in order to maximize the EPS production have been taken into consideration. Both the factors have proved their worth. Hence, towards a better outcome for EPS production, it is indicated that a genetic manipulation of the bacteria should be synchronized with a proper selection of its culture condition by controlling different physico-chemical parameters.

     

Controlled activity of mouse astrocytes on electrospun PCL nanofiber containing polysaccharides from brown seaweed

The central nervous system (CNS), once injured, rarely recovers original function mainly due to its limited regeneration ability. Astrocytes are cells that play critical roles in neural regeneration. Several biomaterials have been studied to replace and regenerate lost tissues within injured CNS. Seaweeds have extracellular polymeric substances (EPS) with bioactive properties such as antiviral and antioxidant properties. In this study, astrocyte activity was assessed, after being cultured on an electrospun polycaprolactone (PCL) nanofibrous mat containing a brown seaweed EPS. Laminarin and fucoidan, two main components of EPS extract from the brown seaweed, were concluded to increase or decrease astrocyte activity with respect to their concentration. When the concentration was under 10 ??g/ml, the astrocytes tended to increase their viability. In contrast, over 10 ??g/ml EPS in media suppressed the viability of astrocytes. In addition, when contained in PCL nanofiber, the EPS extract was also proven to influence astrocyte activity in the same way as the case when astrocytes were exposed to EPS in solution. This implies that the brown seaweed EPS?CPCL nanofiber mat can be used for temporal control of astrocyte activity by EPS concentration. Through this research, we propose that the electrospun EPS?CPCL nanofiber could be used as a nanomedicine or scaffold to treat CNS injuries.     

Changes in Intestinal Microbiota Due to the Expanded Polystyrene Diet of Mealworms (Tenebrio molitor)

Expanded polystyrene (EPS), which is difficult to decompose, is usually buried or incinerated, causing the natural environment to be contaminated with microplastics and environmental hormones. Digestion of EPS by mealworms has been identified as a possible biological solution to the problem of pollution, but the complete degradation mechanism of EPS is not yet known. Intestinal microorganisms play a significant role in the degradation of EPS by mealworms, and relatively few other EPS degradation microorganisms are currently known. This study observed significant differences in the intestinal microbiota of mealworms according to the dietary results of metagenomics analysis and biodiversity indices. We have proposed two new candidates of EPS-degrading bacteria, Cronobacter sakazakii and Lactococcus garvieae, which increased significantly in the EPS feeding group population. The population change and the new two bacteria will help us understand the biological mechanism of EPS degradation and develop practical EPS degradation methods.     

Potential of Unicellular Cyanobacteria from Saline Environments as Exopolysaccharide Producers

Fifteen Cyanothece strains isolated from saline environments have been characterized with regard to exopolysaccharide (EPS) production. The polymers contained six to eight monosaccharides, with one or two acidic sugars. In some EPS samples, the additional presence of acetyl, pyruvyl, and/or sulfate groups was also detected.     

Common loci for Agrobacterium tumefaciens and Rhizobium meliloti exopolysaccharide synthesis and their roles in plant interactions.

Mutants of Rhizobium meliloti have been isolated which are deficient in exopolysaccharide (EPS) production and effective nodulation of alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We isolated approximately 100 analogous EPS-deficient (Exo) mutants of the closely related plant pathogen Agrobacterium tumefaciens, including strains whose EPS deficiencies were specifically complemented by each of five cloned R. meliloti exo loci. We also cloned A. tumefaciens genes which complemented EPS defects in three of the R. meliloti Exo mutants. In two of these cases, symbiotic defects were also complemented. All of the A. tumefaciens Exo mutants formed normal crown gall tumors on four different plant hosts, except ExoC mutants, which were nontumorigenic and unable to attach to plant cells in vitro. Like their R. meliloti counterparts, A. tumefaciens Exo mutants were deficient in production of succinoglycan, the major acidic EPS species produced by both genera. A. tumefaciens ExoC mutants also produced extremely low levels of another major EPS, cyclic 1,2-beta-D-glucan. This deficiency has been noted previously in a different set of nontumorigenic, attachment-defective A. tumefaciens mutants.     

Exopolysaccharides produced by Lactobacillus plantarum: technological properties,biological activity,and potential application in the food industry

Some lactic acid bacteria are capable of producing capsular or extracellular polysaccharides, with desirable technological properties and biological activities. Such polysaccharides produced by lactic acid bacteria are called exopolysaccharides and can be used to alter rheological properties, acting in processes involving viscosity, emulsification, and flocculation, among others. They may also be involved in prebiotic, probiotic, and biological activities, as well as having potential application in the food industry. In this mini-review, the objectives were to present some beneficial properties of exopolysaccharides (EPS) produced by Lactobacillus plantarum that have not been commercially explored. For that, the article focused to summarize revision of current publications within the following topics: (1) rheological properties, (2) prebiotic properties, (3) biological activities, and (4) potential application in the food industry. EPS produced by Lb. plantarum can be used as gelling agent, emulsifier, or stabilizer for food products. The glucan nature of the produced EPS enhances probiotic properties of this LAB species. Lactobacillus plantarum EPS has antioxidant, antibiofilm, and antitumor activities. Finally, there is an improvement in texture of fermented food products where Lb. plantarum is used as starter culture which is related to EPS production in situ. Therefore, EPS produced by Lb. plantarum have important and desirable properties to be explored for several applications, including health and food areas.     

Step-by-step strategy for protein enrichment and proteome characterisation of extracellular polymeric substances in wastewater treatment systems

Extracellular polymeric substances (EPS) are keys in biomass aggregation and settleability in wastewater treatment systems. In membrane bioreactors (MBR), EPS are an important factor as they are considered to be largely responsible for membrane fouling. Proteins were shown to be the major component of EPS produced by activated sludge and to be correlated with the properties of the sludge, like settling, hydrophobicity and cell aggregation. Previous EPS proteomic studies of activated sludge revealed several problems, like the interference of other EPS molecules in protein analysis. In this study, a successful strategy was outlined to identify the proteins from soluble and bound EPS extracted from activated sludge of a lab-scale MBR. EPS samples were first subjected to pre-concentration through lyophilisation, centrifugal ultrafiltration or concentration with a dialysis membrane coated by a highly absorbent powder of polyacrylate-polyalcohol, preceded or not by a dialysis step. The highest protein concentration factors were achieved with the highly absorbent powder method without previous dialysis step. Four protein precipitation methods were then tested: acetone, trichloroacetic acid (TCA), perchloric acid and a commercial kit. Protein profiles were compared in 4–12 % sodium dodecyl sulphate polyacrylamide gel electrophoresis gels. Both acetone and TCA should be applied for the highest coverage for soluble EPS proteins, whereas TCA was the best method for bound EPS proteins. All visible bands of selected profiles were subjected to mass spectrometry analysis. A high number of proteins (25–32 for soluble EPS and 17 for bound EPS) were identified. As a conclusion of this study, a workflow is proposed for the successful proteome characterisation of soluble and bound EPS from activated sludge samples.     

Impact of Azotobacter exopolysaccharides on sustainable agriculture

Recently, increasing attention have lead to search other avenue of biofertilizers with multipurpose activities as a manner of sustainable soil health to improve the plant productivity. Azotobacter have been universally accepted as a major inoculum used in biofertilizer to restore the nitrogen level into cultivated field. Azotobacter is well characterized for their profuse production of exopolysaccharides (EPS). Several reviews on biogenesis and multifunctional role of Azotobacter EPS have been documented with special emphasis on industrial applications. But the impact of Azotobacter EPS in plant growth promotion has not received adequate attention. This review outlines the evidence that demonstrates not only the contribution of Azotobacter EPS in global nutrient cycle but also help to compete successfully in different adverse ecological and edaphic conditions. This also focuses on new insights and concepts of Azotobacter EPS which have positive effects caused by the biofilm formation on overall plant growth promotion with other PGPRs. In addition, their potentials in agricultural improvement are also discussed. Recent data realized that Azotobacter EPS have an immense agro-economical importance including the survivability and maintenance of microbial community in their habitat. This leads us to confirm that the next generation Azotobacter inoculum with high yielding EPS and high nitrogen fixing ability can be utilized to satisfy the future demand of augmented crop production attributed to increase plant growth promoting agents.     

Microbial influence on dolomite and authigenic clay mineralisation in dolocrete profiles of NW Australia

Dolomite (CaMg(CO₃)₂) precipitation is kinetically inhibited at surface temperatures and pressures. Experimental studies have demonstrated that microbial extracellular polymeric substances (EPS) as well as certain clay minerals may catalyse dolomite precipitation. However, the combined association of EPS with clay minerals and dolomite and their occurrence in the natural environment are not well documented. We investigated the mineral and textural associations within groundwater dolocrete profiles from arid northwest Australia. Microbial EPS is a site of nucleation for both dolomite and authigenic clay minerals in this Late Miocene to Pliocene dolocrete. Dolomite crystals are commonly encased in EPS alveolar structures, which have been mineralised by various clay minerals, including montmorillonite, trioctahedral smectite and palygorskite-sepiolite. Observations of microbial microstructures and their association with minerals resemble textures documented in various lacustrine and marine microbialites, indicating that similar mineralisation processes may have occurred to form these dolocretes. EPS may attract and bind cations that concentrate to form the initial particles for mineral nucleation. The dolomite developed as nanocrystals, likely via a disordered precursor, which coalesced to form larger micritic crystal aggregates and rhombic crystals. Spheroidal dolomite textures, commonly with hollow cores, are also present and may reflect the mineralisation of a biofilm surrounding coccoid bacterial cells. Dolomite formation within an Mg-clay matrix is also observed, more commonly within a shallow pedogenic horizon. The ability of the negatively charged surfaces of clay and EPS to bind and dewater Mg²⁺, as well as the slow diffusion of ions through a viscous clay or EPS matrix, may promote the incorporation of Mg²⁺ into the mineral and overcome the kinetic effects to allow disordered dolomite nucleation and its later growth. The results of this study show that the precipitation of clay and carbonate minerals in alkaline environments may be closely associated and can develop from the same initial amorphous Ca–Mg–Si-rich matrix within EPS. The abundance of EPS preserved within the profiles is evidence of past microbial activity. Local fluctuations in chemistry, such as small increases in alkalinity, associated with the degradation of EPS or microbial activity, were likely important for both clay and dolomite formation. Groundwater environments may be important and hitherto understudied settings for microbially influenced mineralisation and for low-temperature dolomite precipitation.     

Exopolysaccharide biosynthesis genes required for social motility in Myxococcus xanthus

Social (S)-motility in Myxococcus xanthus is a flagellum-independent gliding motility system that allows bacteria to move in groups on solid surfaces. S-motility has been shown to require type IV pili (TFP), exopolysaccharide (EPS; a component of fibrils) and lipopolysaccharide (LPS). Previously, information concerning EPS biogenesis in M. xanthus was lacking. In this study, we screened 5000 randomly mutagenized colonies for defects in S-motility and EPS and identified two genetic regions essential for EPS biogenesis: the EPS synthesis (eps) region and the EPS-associated (eas) region. Mutants with insertions in the eps and eas regions were defective in S-motility and fruiting body formation. These mutants failed to bind the dye calcofluor white, indicating that they lacked EPS; however, they retained normal TFP and LPS. Analysis of the eps locus showed several open reading frames (ORFs) that encode homologues to glycosyltransferases, glucanases and EPS transporters as well as regulatory proteins; the eas locus contains two ORFs: one exhibits homology to hypothetical proteins with a conserved domain of unknown function and the other displays no apparent homology to other proteins in the database. Further genetic mutagenesis analysis indicates that the whole eps region is involved in the biosynthesis of fibrils and fibril EPS. The operon at the proximal end of the eps region was analysed by generating in-frame deletion mutations. These mutants showed varying degrees of defects in the bacterium's ability to produce EPS or perform EPS-related functions, confirming the involvement of these genes in M. xanthus EPS biogenesis.     

Kinetics and physico-chemical characterization of exopolysaccharides produced by the cyanobacterium Oscillatoria formosa

Exopolysaccharides of the cyanobacterium Oscillatoria formosa have been physico-chemically characterized and kinetics of their production studied. The organism produced 334.8 μg EPS per ml culture in 24 days with the maximum rate of production obtained during initial days of growth. HPLC analysis of the EPS hydrolysate revealed that besides three unidentified sugars, EPS contained ribose, mannose, and galacturonic acid. FT-IR spectrum of EPS revealed the presence of methyl, carboxyl and C–N groups. Elemental analysis indicated the presence of 4.7% nitrogen in EPS. The organism produced 75.6% more EPS when incubated at 35°C compared to cultures at 28°C. Under varied nutritional conditions, though the growth of the organism was less yet it produced enhanced amounts of EPS. Aqueous dispersions of EPS showed non-Newtonian, pseudoplastic behaviour. The viscosity of the aqueous solution of EPS was quite stable over a wide range of pH and temperature but it was observed to be affected by CaCl_2.     

Extracellular Polysaccharide of Erwinia amylovora: a Correlation with Virulence

The extracellular polysaccharides produced as slime or capsule layers by bacterial pathogens of animals and plants have been often implicated as factors essential to pathogenesis. In the present study, virulence of the plant pathogen Erwinia amylovora was correlated with the ability to produce extracellular polysaccharide (EPS). EPS production by a series of field isolates and bacterio-phage-resistant mutants differing in the extent to which they cause symptoms in host tissues was examined by quantitation with a modified Laurell rocket immunoelectrophoresis assay. The amount of EPS produced as an easily removed capsular layer or slime on solid nutrient agar approximated the capacity to exhibit symptoms in host inoculation tests. Features common to the virulent isolates are mucoid colony morphology, sensitivity to EPS-specific bacteriophages [S3 and PEal(h)], and ability to produce a characteristic EPS. Mutants selected for resistance to S3 or nonmucoid colony morphology are deficient in EPS production and have lost the ability to multiply in host tissue and cause symptoms. We conclude that EPS may be directly involved in symptom expression and provide a function essential to the growth of the pathogen in host tissues.     

Extraction and optimization of exopolysaccharide from Lactobacillus sp. using response surface methodology and artificial neural networks

Abstract

The microbial polysaccharides secreted and produced from various microbes into their extracellular environment is known as exopolysaccharide. These polysaccharides can be secreted from the microbes either in a soluble or insoluble form.Lactobacillus sp. is one of the organisms that have been found to produce exopolysaccharide. Exo-polysaccharides (EPS) have various applications such as drug delivery, antimicrobial activity, surgical implants and many more in different fields. Medium composition is one of the major aspects for the production of EPS from Lactobacillus sp., optimization of medium components can help to enhance the synthesis of EPS . In the present work, the production of exopolysaccharide with different medium composition was optimized by response surface methodology (RSM) followed by tested for fitting with artificial neural networks (ANN). Three algorithms of ANN were compared to investigate the highest yeild of EPS. The highest yeild of EPS production in RSM was achieved by the medium composition that consists of (g/L) dextrose 15, sodium dihydrogen phosphate 3, potassium dihydrogen phosphate 2.5, triammonium citrate 1.5, and, magnesium sulfate 0.25. The output of 32 sets of RSM experiments were tested for fitting with ANN with three algorithms viz. Levenberg–Marquardt Algorithm (LMA), Bayesian Regularization Algorithm (BRA) and Scaled Conjugate Gradient Algorithm (SCGA) among them LMA found to have best fit with the experiments as compared to the SCGA and BRA.