Polysaccharide-degrading Activity Research Articles

Identification of a phage-derived depolymerase specific for KL64 capsule of Klebsiella pneumoniae and its anti-biofilm effect.

The increasing prevalence of Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a serious threat to global health. Phages and phage-derived enzymes gained increasing attention for controling CRKP infections. In this study, a lytic phage P510 infecting KL64 type K. pneumoniae was isolated and characterized. Whole genome analysis and electron microscopy analysis showed that phage P510 belonged to genus Przondovirus, family Autographiviridae, the order Caudovirales. The tail fiber protein of the phage was predicted to encode capsule depolymerase. Further analysis demonstrated that recombinant depolymerase P510dep had polysaccharide-degrading activity against KL64-types capsule of K. pneumoniae, and its lysis spectrum matched to host range of phage P510. Wealso demonstrated that the recombinant depolymerase was able to significantly inhibit biofilm formation. The discovery of the phage-derived depolymerase lays the foundation for controlling the spread of CRKPs.

Undaria pinnatifida exudates trigger shifts in seawater chemistry and microbial communities from Atlantic Patagonian coasts

The invasive kelp Undaria pinnatifida has spread from northeastern Asia to temperate coastal environments worldwide, with profound effects on colonized ecosystems. In this work, we analyzed the effect of exudates from U. pinnatifida on the chemical and microbial properties of seawater from a semi-enclosed gulf from Atlantic Patagonia. Exudates of U. pinnatifida, consisting mainly of carbohydrates, were released at a rate of 1.6 ± 0.8 mg C g−1 algae day−1, affecting the quality and optical properties of seawater in experimental incubations. Parallel factor analysis based on excitation-emission matrices collected from exudates revealed the presence of two humic-like and one non-humic fluorescent components. Exudate release stimulated microbial growth and polysaccharide degrading activity in seawater. After a 7-day incubation of fresh seawater with the exudates, changes in microbial community structure were analyzed by large-scale 16S rRNA gene amplicon sequencing. Copiotrophic and fermentative genera such as Spirochaeta (Spirochaetes) and Propionigenium (Fusobacteria) increased in the incubations with algal exudates. Genomic potential prediction revealed that the selected bacterial community could have higher ribosome content—an indicator of the potential for reaching higher metabolic rates—and genes for the degradation of complex organic compounds such as polysaccharides and other carbohydrates present in the exudates. Nutrient addition triggered the emergence of other microbial populations with different ecophysiological niches: unclassified Flavobacteriales, unclassified bacteria related to the recently described Phylum Kiritimatiellaeota, as well as potential pathogens such as Vibrio (Gammaproteobacteria) and Arcobacter (Epsilonproteobacteria), suggesting potential synergistic effects between invasive macroalgae and human activities.

Colorimetric determination of β-1,2-glucooligosaccharides for an enzymatic assay using 3-methyl-2-benzothiazolinonehydrazone

A colorimetric determination method measuring the reducing ends of sugars is usually used for quantitative evaluation of polysaccharide-degrading activity of endo-type enzymes. However, no appropriate colorimetric method has been established for enzymatic assay of β-1,2-glucanases, which produce β-1,2-glucooligosaccharides from β-1,2-glucans. The Anthon-MBTH method has been potentially the most adaptable for color development of β-1,2-glucooligosaccharides among various known colorimetric methods for detecting the reducing power of oligosaccharides, since the difference between sophorose and other β-1,2-glucooligosaccharides in absorbance is relatively small. Almost the same color development was obtained for β-1,2-glucooligosaccharides when the heating time with the Anthon-MBTH method was changed. The kind of base and concentration of dithiothreitol did not markedly affect the color development. Most buffer components, salts and a chelating reagent used for usual enzymatic experiments did not inhibit color development. Furthermore, assay was performed successfully for a β-1,2-glucanase using the modified MBTH method.

Polysaccharide-Degrading Activity in Marine and Terrestrial Strains of Mycelial Fungi

The activity of extracellular polysaccharide-degrading enzymes and glycosidases from mycelial fungi towards various carbohydrates and carbohydrate derivatives from plant and algal cell walls has been screened. Twenty-three strains of mycelial fungi isolated from the marine sediment and dung were grown by submerged cultivation on a plant-based substrate (a by-product of the grain processing industry) for previous screening for their biomass and protein productivity. Molecular identification allowed for the assignment of marine fungal strains to the following species: Sirastachys phyllophila, Ochroconis mirabilis, Pseudallescheria boydii, Pseudallescheria ellipsoidea, Beauveria felina, Scopulariopsis brevicaulis, Cladosporium sp., and Trichoderma sp. The terrestrial strains belonged to the species Thermomyces thermophilus, Thermomyces dupontii, Thermomyces lanuginosus, Fusarium avenaceum, Mycothermus thermophilum, and Thermothelomyces thermophila. Seven strains of thermophilic terrestrial fungal species T. thermophila, T. thermophilus, T. dupontii and M. thermophilus and two marine fungal strains of S. brevicaulis and Beauveria felina exhibited the highest protein yields and a wide range of polysaccharide-degrading activity when the cultures were cultivated at 22–25°C. The cellulolytic thermophilic strain M. thermophilus 55 isolated from dung demonstrated unusual specificity, most intensive increase of mycelial biomass, and high activity towards algal polysaccharides after seven days of cultivation. The specific activity of laminarinase was one order of magnitude higher than in the marine strains and amounted to 1180 U/mg, and the alginate lyase, carrageenase, polymannuronate lyase, agarase, and fucoidanase activity levels (from 208 to 500 U/mg) were also higher than in all marine strains. All active polysaccharide-degrading strains of thermophilic terrestrial and marine fungi identified in the present study are of considerable interest, as the potential of these fungi for polysaccharide degradation can be applied in the transformation of various agricultural and maricultural waste of plant origin and in the modification of carbohydrate-containing substances in structural research and biotechnology.

Comparative genome analysis of novel Podoviruses lytic for hypermucoviscous Klebsiella pneumoniae of K1, K2, and K57 capsular types

Hypermucoviscous (HV) strains of capsular types K1, K2 and K57 are the most virulent representatives of the Klebsiella pneumoniae species. Eight novel bacteriophages lytic for HV K. pneumoniae were isolated and characterized. Three bacteriophages, KpV41, KpV475, and KpV71 were found to have a lytic activity against mainly K. pneumoniae of capsular type K1. Two phages, KpV74, and KpV763 were lytic for K2 capsular type K. pneumoniae, and the phage KpV767 was specific to K57-type K. pneumoniae only. Two more phages, KpV766, and KpV48 had no capsular specificity. The phage genomes consist of a linear double-stranded DNA of 40,395–44,623bp including direct terminal repeats of 180–246 bp. The G + C contents are 52.3–54.2 % that is slightly lower than that of genomes of K. pneumoniae strains being used for phage propagation. According to the genome structures, sequence similarity and phylogenetic data, the phages are classified within the genus Kp32virus and Kp34virus of subfamily Autographivirinae, family Podoviridae. In the phage genomes, genes encoding proteins with putative motifs of polysaccharide depolymerase were identified. Depolymerase genes of phages KpV71 and KpV74 lytic for hypermucoviscous K. pneumoniae of K1 and K2 capsular type, respectively, were cloned and expressed in Escherichia coli, and the recombinant gene products were purified. The specificity and polysaccharide-degrading activity of the recombinant depolymerases were demonstrated.

The Cultivable Surface Microbiota of the Brown Alga Ascophyllum nodosum is Enriched in Macroalgal-Polysaccharide-Degrading Bacteria.

Bacteria degrading algal polysaccharides are key players in the global carbon cycle and in algal biomass recycling. Yet the water column, which has been studied largely by metagenomic approaches, is poor in such bacteria and their algal-polysaccharide-degrading enzymes. Even more surprisingly, the few published studies on seaweed-associated microbiomes have revealed low abundances of such bacteria and their specific enzymes. However, as macroalgal cell-wall polysaccharides do not accumulate in nature, these bacteria and their unique polysaccharidases must not be that uncommon. We, therefore, looked at the polysaccharide-degrading activity of the cultivable bacterial subpopulation associated with Ascophyllum nodosum. From A. nodosum triplicates, 324 bacteria were isolated and taxonomically identified. Out of these isolates, 78 (~25%) were found to act on at least one tested algal polysaccharide (agar, ι- or κ-carrageenan, or alginate). The isolates “active” on algal-polysaccharides belong to 11 genera: Cellulophaga, Maribacter, Algibacter, and Zobellia in the class Flavobacteriia (41) and Pseudoalteromonas, Vibrio, Cobetia, Shewanella, Colwellia, Marinomonas, and Paraglaceciola in the class Gammaproteobacteria (37). A major part represents likely novel species. Different proportions of bacterial phyla and classes were observed between the isolated cultivable subpopulation and the total microbial community previously identified on other brown algae. Here, Bacteroidetes and Gammaproteobacteria were found to be the most abundant and some phyla (as Planctomycetes and Cyanobacteria) frequently encountered on brown algae weren't identified. At a lower taxonomic level, twelve genera, well-known to be associated with algae (with the exception for Colwellia), were consistently found on all three A. nosodum samples. Even more interesting, 9 of the 11 above mentioned genera containing polysaccharolytic isolates were predominant in this common core. The cultivable fraction of the bacterial community associated with A. nodosum is, thus, significantly enriched in macroalgal-polysaccharide-degrading bacteria and these bacteria seem important for the seaweed holobiont even though they are under-represented in alga-associated microbiome studies.

Relating nutrient molar ratios of microbial attached communities to organic matter utilization in a forested stream

In Mediterranean forested streams, the large input of particulate organic matter (POM) in autumn structures the benthic microbial community into two interconnected habitats: that associated with the POM itself (leaves and branches) and that colonizing gravel and sandy substrata. Relationships were examined between microbial decomposition activities (β-glucosidase, β-xylosidase, cellobiohydrolase, phenoloxidase, peroxidase, peptidase and phosphatase) at different stream substrata (leaves, branches, sand and gravel) and total carbon (C), nitrogen (N) and phosphorus (P) content of attached microbial communities and stream water matter fractions (particulate and dissolved nutrient concentrations). Microbial communities associated to leaves and branches showed higher C:N and lower N:P molar ratios (averaging 21.7 ± 1.4 and 5.4 ± 2.1, respectively) and higher polysaccharide degrading activity (sum of β-glucosidase, β-xylosidase and cellobiohydrolase activities). Instead, biofi lms on sand and gravel, where algae accumulate and fi ne particulate material were more available, showed lower C:N and higher N:P molar ratios (averaging 10.3 ± 0.6 and 19.9 ± 3.7, respectively) and greater ligninolytic (sum of phenol oxidase and peroxidase activities) and peptidase activities. These results suggest that enzyme activities of microbial attached communities are linked to their nutrient molar ratios and, at the same time, these might be modulated by the different nature of available organic matter (OM) in each substratum and the microbial groups accumulated (algae, bacteria, fungi, microand meiofauna). However, similarities (C:N) and divergences (N:P) between stream water and nutrient molar ratios in microbial communities may also affect nutrient demands and in consequence, the expression of extracellular enzymes. Our results show a relationship between function (extracellular enzyme activities) and nutrient molar ratios of attached microbial communities.

A plate assay for simultaneous screening of polysaccharide- and protein-degrading micro-organisms

To develop a plate assay for simultaneous screening of polysaccharide-degrading and protein-degrading micro-organisms. A plate assay, based on the visible solubilization of small substrate particles and the formation of haloes on Petri dishes, containing a mixture of diversely coloured insoluble polysaccharides and dye-labelled collagen as chromogenic substrates, was developed. This method was successfully applied for isolating the diverse polysaccharide- and/or protein-degrading bacteria from soil and sludge samples. Selected strains were identified using 16S rDNA partial sequencing; most of them belong to the genera Bacillus, Cellulomonas and Cellulosimicrobium. This novel approach provides unique and valuable information for direct primary screening when the target of selection is micro-organisms exhibiting protein-degrading activity, polysaccharide-degrading activity or a specific combination of them. This plate assay is convenient and easy to perform, rapid, and more adaptable for screening of a large number of samples, compared with other existing methods in the literature.

Polysaccharide degradation by human intestinal bacteria during growth under multi-substrate limiting conditions in a three-stage continuous culture system

Human faecal microorganisms were grown on mixtures of chemically diverse polymerised C-sources (starch, pectin, xylan, mucin, arabinogalactan, inulin, guar gum) in a three-stage continuous culture model of the colon. The effects of retention time ( R=27.1 h, R=66.7 h) on bacterial populations, their expression of hydrolytic enzymes involved in substrate depolymerisation, carbohydrate utilisation and short chain fatty acid formation were investigated. Eleven bacterial marker groups were studied in the fermenters. Strictly anaerobic bacteria predominated including bacteroides, bifidobacteria, clostridia and anaerobic Gram-positive cocci. Changing system retention time from 27.1 to 66.7 h did not significantly affect the predominant bacterial populations in V1, however, enterobacterial cell numbers increased in V3, while saccharolytic anaerobe recoveries declined markedly, reflecting their greater dependence on polymerised carbon sources in the fermentation system. The majority of polysaccharide degrading activity in the colon model was cell-associated, under all culture conditions. Increasing R from 27.1 to 66.7 h did not substantially change overall polysaccharidase (amylase, polygalacturonanase, xylanase, arabinogalactanase, galactomannanase) profiles, however, synthesis of some glycosidases was enhanced (e.g. α-glucosidase, N-acetyl-β-glucosaminidase, neuraminidase), whereas reduced expression of other enzymes such as β-galactosidase, N-acetyl-α-galactosaminidase, α-fucosidase and α-arabinofuranosidase occurred. These observations demonstrate that catabolite regulation is an important control process in the colonic microbiota, with respect to the induction and repression of enzyme synthesis, and that substrate availability plays a major role in regulating bacterial metabolism. Measurements of carbohydrate utilisation demonstrated that while all polysaccharides in the feed medium were digested extensively by bacteria growing in the fermentation system, specific rates of carbohydrate utilisation were maximal at R=27.1 h. These data also provided evidence of bacterial substrate preferences in the colon model, particularly in relation to xylan and inulin digestion, demonstrating that catabolite regulatory mechanisms were also involved in controlling the assimilation of carbohydrate in the microbiota. Short chain fatty acid measurements showed that fermentation was more efficient at R=27.1 h compared to R=66.7 h, with putative conversion of carbohydrate to short chain fatty acids being approximately 60% and 40%, respectively. This was probably due to increased maintenance energy requirements at low bacterial growth rates. Differences were also observed with respect to short chain fatty acid molar ratios, with more propionate, branched chain and longer chain fatty acid production at R=66.7 h, demonstrating the increasing importance of amino acid fermentation under these culture conditions.

Polysaccharide degradation by human intestinal bacteria during growth under multi-substrate limiting conditions in a three-stage continuous culture system

Abstract Human faecal microorganisms were grown on mixtures of chemically diverse polymerised C-sources (starch, pectin, xylan, mucin, arabinogalactan, inulin, guar gum) in a three-stage continuous culture model of the colon. The effects of retention time (R=27.1 h, R=66.7 h) on bacterial populations, their expression of hydrolytic enzymes involved in substrate depolymerisation, carbohydrate utilisation and short chain fatty acid formation were investigated. Eleven bacterial marker groups were studied in the fermenters. Strictly anaerobic bacteria predominated including bacteroides, bifidobacteria, clostridia and anaerobic Gram-positive cocci. Changing system retention time from 27.1 to 66.7 h did not significantly affect the predominant bacterial populations in V1, however, enterobacterial cell numbers increased in V3, while saccharolytic anaerobe recoveries declined markedly, reflecting their greater dependence on polymerised carbon sources in the fermentation system. The majority of polysaccharide degrading activity in the colon model was cell-associated, under all culture conditions. Increasing R from 27.1 to 66.7 h did not substantially change overall polysaccharidase (amylase, polygalacturonanase, xylanase, arabinogalactanase, galactomannanase) profiles, however, synthesis of some glycosidases was enhanced (e.g. α-glucosidase, N-acetyl-β-glucosaminidase, neuraminidase), whereas reduced expression of other enzymes such as β-galactosidase, N-acetyl-α-galactosaminidase, α-fucosidase and α-arabinofuranosidase occurred. These observations demonstrate that catabolite regulation is an important control process in the colonic microbiota, with respect to the induction and repression of enzyme synthesis, and that substrate availability plays a major role in regulating bacterial metabolism. Measurements of carbohydrate utilisation demonstrated that while all polysaccharides in the feed medium were digested extensively by bacteria growing in the fermentation system, specific rates of carbohydrate utilisation were maximal at R=27.1 h. These data also provided evidence of bacterial substrate preferences in the colon model, particularly in relation to xylan and inulin digestion, demonstrating that catabolite regulatory mechanisms were also involved in controlling the assimilation of carbohydrate in the microbiota. Short chain fatty acid measurements showed that fermentation was more efficient at R=27.1 h compared to R=66.7 h, with putative conversion of carbohydrate to short chain fatty acids being approximately 60% and 40%, respectively. This was probably due to increased maintenance energy requirements at low bacterial growth rates. Differences were also observed with respect to short chain fatty acid molar ratios, with more propionate, branched chain and longer chain fatty acid production at R=66.7 h, demonstrating the increasing importance of amino acid fermentation under these culture conditions.

Polysaccharide breakdown by mixed populations of human faecal bacteria

Measurements of polysaccharide-degrading activity in different fractions of human faeces showed that bacterial polysaccharidases and glycosidases were primarily associated with the washed bacterial fractions. Amylase, pectinase and xylanase were the major polysaccharide-hydrolysing enzymes detected, whilst α-L-arabinofuranosidase, β-D-xylosidase, β-D-galactosidase and β-D-glucosidase were the most active glycosidases. Starch and 3 non-starch polysaccharides (NSP; pectin, xylan and arabinogalactan) were fermented by mixed populations of human faecal bacteria in batch culture. Detailed carbohydrate analysis demonstrated that starch and pectin were the most rapidly degraded substrates and that arabinogalactan and the relatively insoluble polysaccharide xylan were broken down more slowly. Free sugars and oligosaccharides did not accumulate in culture media with any polysaccharide tested. Time-course measurements of polysaccharide remaining in the batch culture fermentations showed that the arabinose side chains of pectin, xylan and arabinogalactan were co-utilised with the backbone sugars. In these cultures, polysaccharide-degrading activity was mainly cell-associated, but extracellular polysaccharidase activity increased as the fermentations progressed. Molar ratios of acetate, propionate and butyrate produced in these experiments were dependent upon the polysaccharide substrate tested. Molar ratios of acetate, propionate and butyrate in the starch, arabinogalactan, xylan and pectin fermentations were 50:22:29, 50:42:8, 82:15:3, and 84:14:2, respectively. The presence of starch did not inhibit the breakdown of arabinogalactan, xylan or pectin by faecal bacterial, providing evidence that multicomponent substrate utilisation occurs when complex populations of faecal bacteria are provided with mixed polysaccharide substrates.

Polysaccharide-degrading enzymes of Botrytis allii and Sclerotium cepivorum. Enzyme production in culture and the effect of the enzyme on isolated onion cell walls

Botrytis allii and Sclerotium cepivorum were grown on liquid media with purified onion cell walls as the sole or major carbon source respectively. Polysaccharide-degrading activity determined at daily intervals for 10 days after inoculation increased over the course of the experiment. For both fungi, degrading activity against sodium polypectate was the highest at all times. Xylan, galactan and citrus pectin-degrading activities appeared at similar rates to each other with some variation in relative amounts from the two fungi. Carboxymethyl cellulase activity increased markedly after 6 days and activity against araban remained relatively low. Cell walls were incubated with enzyme preparations and the release of total carbohydrate and of individual sugars was measured. Cell wall carbohydrate was solubilized by enzyme preparations in amounts which increased with the age of the culture. The pattern of release of the individual sugars showed two groups, one containing uronic acid, galactose, rhamnose and arabinose and the second xylosem glucose, fucose and mannose. The release of the first group was related to polygalacturonase activity and the second to the cellulase and possibly xylanase activities. These findings emphasize the importance of polygalacturonase activity in the solubilization of onion cell walls by both fungi.