Draft genome sequence of Sanguibacter species strain 25GB23B1, cultivated from arctic surface seawater.

We report a draft genome sequence for Sanguibacter species strain 25GB23B1, isolated from arctic surface seawater off the coast of Alaska. The whole genome sequence will provide knowledge of the bacteria's relationship to its environment and possibly a new species of Sanguibacter.

Photoperiod and temperature interactions drive the latitudinal distribution of Laminaria hyperborea (Laminariales, Phaeophyceae) under climate change.

Due to global rises in temperature, recent studies predict marine species shifting toward higher latitudes. We investigated the impact of interacting abiotic drivers on the distribution potential of the temperate kelp Laminaria hyperborea. The ecosystem engineering species is widespread along European coasts but has not yet been observed in the High Arctic, although it can survive several months of low temperatures and darkness. To investigate its ability to extend northward in future, we conducted a long-term multifactorial experiment with sporophytes from Porsangerfjorden, Norway-close to the species' documented northernmost distribution margin. The samples were exposed to three different photoperiods (PolarDay, LongDay, and PolarNight) at 0°C, 5°C, and 10°C for 3 months. Optimum quantum yield of photosynthesis (Fv/Fm), dry weight, pigments, phlorotannins, and storage carbohydrates were monitored. Both physiological and biochemical parameters revealed that L. hyperborea was strongly influenced by the different photoperiods and their interaction with temperature, while temperature alone exerted only minor effects. The Fv/Fm data were integrated into a species distribution model to project a possible northward expansion of L. hyperborea. The combination of extended day lengths and low temperatures appeared to be the limiting reason for northward spread of L. hyperborea until recently. However, with water temperatures reaching 10°C in summer, this kelp will be able to thrive also in the High Arctic. Moreover, no evidence of stress to Arctic winter warming was observed. Consequently, L. hyperborea has a high potential for spreading northward with further warming which may significantly affect the structure and function of Arctic ecosystems.

Decomposition of brown algae in the ocean by microbiota: biological insights for recycling blue carbon.

Brown algae are one of the most abundant biomasses on Earth. To recycle them as blue carbon sources, an effective decomposition system is necessary. This study focused on microorganisms present in seawater that decompose brown algae which contain laminarin and alginate. Where Undaria and Sargassum spp. were present, genera Psychromonas, Psychrobacter, and Pseudoalteromonas were predominant in seawater, while genera Arcobacter and Fusobacterium increased in abundance during the process of decomposition. The inoculation of Undaria samples into laminarin-minimal media led to a predominance of Pseudoalteromonas species. A Pseudoalteromonas isolate, identified as Pseudoalteromonas distincta, possesses genes encoding a putative laminarinase, polysaccharide lyase family 6 (PL6) alginate lyases, and a PL7 alginate lyase. The culture media of P. distincta contained no monosaccharides, suggesting the rapid conversion of polysaccharides to metabolites. These findings indicated that Pseudoalteromonas species play a major role in the decomposition of brown algae and affect the microbiota associated with them.

Expression of C/EBP and Kr-h1 transcription factors under immune stimulation in the noble crayfish.

Transcription factors (TFs) have an important role in the regulation of the gene expression network. The role of TFs in the immune response of freshwater crayfish is poorly understood, but leveraging the regulatory mechanisms of immune response could augment the resistance against the invasive oomycete pathogen, Aphanomyces astaci. Previous studies indicated that the TFs CCAAT/enhancer-binding protein (C/EBP) and putative Krüppel homolog-1 protein (Kr-h1) might play a role in immune and stress response of the noble crayfish (Astacus astacus). Here, we aimed to further characterise these two gene products to gain a better understanding of their evolutionary origin, domain organisation and expression patterns across different crayfish tissues. Furthermore, we conducted an immune stimulation experiment to observe the potential changes in the gene expression of C/EBP and Kr-h1 under immune challenge in different crayfish tissues. Our results showed that both C/EBP and Kr-h1 are closely related to other C/EBPs and Kr-h1s in Malacostraca. Gene expression analysis revealed that both TFs are present in all analysed tissues, with higher expression of C/EBP in the gills and Kr-h1 in the abdominal muscle. Immune stimulation with laminarin (mimicking ß-1-3-glucan in the oomycete cell wall) showed an activation of the crayfish immune system, with an overall increase in the total haemocyte count (THC) compared to untreated control and crayfish buffered saline (CBS) treatment. On the gene expression level, an up-regulation of the C/EBP gene was detected in the laminarin treated group in hepatopancreas and heart, while no changes were observed for the Kr-h1 gene. Our results indicate an early change in C/EBP expression in multiple tissues during immune stimulation and suggest its involvement in the immune response of the noble crayfish.

Laminarin-modulated osmium nanozymes with high substrate-affinity and selective peroxidase-like behavior engineered colorimetric assay for hydroxyl radical scavenging capacity estimation.

Hydroxyl radical (·OH) scavenging capacity (HOSC) estimation is essential for evaluating antioxidants, natural extracts, or drugs against clinical diseases. While nanozymes offer advantages in related applications, they still face limitations in activity and selectivity. In response, this work showcases the fabrication of laminarin-modulated osmium (laminarin-Os) nanoclusters (1.45 ± 0.05 nm), functioning as peroxidase-like nanozymes within a colorimetric assay tailored for rational HOSC estimation. This study validates both the characterization and remarkable stability of laminarin-Os. By leveraging the abundant surface negative charges of laminarin-Os and the surface hydroxyls of laminarin, oxidation reactions are facilitated, augmenting laminarin-Os's affinity for 3,3',5,5'-tetramethylbenzidine (TMB) (KM = 0.04 mM). This enables the laminarin-Os-based colorimetric assay to respond to ·OH more effectively than citrate-, albumin-, or other polysaccharides-based Os. In addition, experimental results also validate the selective peroxidase-like behavior of laminarin-Os under acidic conditions. Antioxidants like ascorbic acid, glutathione, tannic acid, and cysteine inhibit absorbance at 652 nm in the colorimetric platform using laminarin-Os's peroxidase-like activity. Compared with commercial kits, this assay demonstrates superior sensitivity (e.g., responds to ascorbic acid 0.01-0.075 mM, glutathione 1-15 µg/mL, tannic acid 0.5-5 µM, and monoammonium glycyrrhizinate cysteine 1.06-10.63 µM) and HOSC testing for glutathione, tannic acid, and monoammonium glycyrrhizinate cysteine. Overall, this study introduces a novel Os nanozyme with exceptional TMB affinity and ·OH selectivity, paving the way for HOSC estimation in biomedical research, pharmaceutical analysis, drug quality control, and beyond.

No colonization resistance to Campylobacter jejuni in broilers fed brown algal extract-supplemented diets.

Introduction: Campylobacter jejuni gastroenteritis is the most commonly reported zoonosis within the EU, with poultry products regarded as the primary source of transmission to humans. Therefore, finding strategies to reduce Campylobacter colonization in broilers holds importance for public health. Recent studies suggest that supplementation of broiler feed with brown algal extracts, particularly laminarin, can provide beneficial effects on broiler gut health, growth performance, and gut microbiota. However, its effect on gut microbiota development and subsequent reduction of Campylobacter loads in broiler caeca during the later stages of the birds' lives remains unclear. Methods: Experimental colonization of Ross 308 broilers with two different strains of C. jejuni was conducted, with groups fed either a basal diet or the same basal diet supplemented with 725 ppm algal extract from Saccharina latissima to provide 290 ppm laminarin. Fecal samples were collected for bacterial enumeration, and caecal samples were obtained before and after the C. jejuni challenge for the determination of microbiota development. Results and discussion: No significant differences in fecal C. jejuni concentrations between the groups fed different diets or exposed to different C. jejuni strains were observed. This suggests that both strains colonized the birds equally well and that the laminarin rich algal extract did not have any inhibitory effect on C. jejuni colonization. Notably, 16S rRNA amplicon sequencing revealed detailed data on the caecal microbiota development, likely influenced by both bird age and C. jejuni colonization, which can be valuable for further development of broiler feed formulations aimed at promoting gut health.

Impact of dietary Laminaria digitata with alginate lyase or carbohydrase mixture on nutrient digestibility and gut health of weaned piglets.

Laminaria digitata is a brown seaweed rich in prebiotic polysaccharides, mainly laminarin, but its alginate-rich cell wall could compromise nutrient access. Carbohydrase supplementation, such as individual alginate lyase and carbohydrases mixture (Rovabio® Excel AP), could enhance nutrient digestibility and prebiotic potential. This study aimed to evaluate the effect of these enzymes on nutrient digestibility and gut health of weaned piglets fed with 10% L. digitata. Diets did not affect growth performance (P > 0.05). The majority of the feed fractions had similar digestibility across all diets, but the supplementation of alginate lyase increased hemicellulose digestibility by 3.3% compared to the control group (P = 0.047). Additionally, we observed that algal zinc was more readily available compared to the control group, even without enzymatic supplementation (P < 0.001). However, the increased digestibility of some minerals, such as potassium, raises concerns about potential mineral imbalance. Seaweed groups had a higher abundance of beneficial bacteria in colon contents, such as Prevotella, Oscillospira and Catenisphaera. Furthermore, the addition of alginate lyase led to a lower pH in the colon (P < 0.001) and caecum (P < 0.001) of piglets, which is possibly a result of released fermentable laminarin, and is consistent with the higher proportion of butyric acid found in these intestinal compartments. L. digitata is a putative supplement to enhance piglet gut health due to its prebiotic polysaccharides. Alginate lyase supplementation further improves nutrient digestibility and prebiotic potential. These results suggest the potential use of L. digitata and these enzymatic supplements in commercial piglet-feeding practices.

Physiochemical changes, metabolite discrepancies of brown seaweed-derived sulphated polysaccharides in the upper gastrointestinal tract and their effects on bioactive expression.

Brown seaweed-derived polysaccharides, notably fucoidan and laminarin, are known for their extensive array of bioactivities and physicochemical properties. However, the effects of upper digestive tract modification on the bioactive performance of fucoidan and laminarin fractions (FLFs) sourced from Australian native species are largely unknown. Here, the digestibility and bioaccessibility of FLFs were evaluated by tracking the dynamic changes in reducing sugar content (CR), profiling the free monosaccharide composition using LC-MS, and comparing high-performance gel permeation chromatography profile variation via LC-SEC-RI. The effects of digestive progression on bioactive performance were assessed by comparing the antioxidant and antidiabetic potential of FLFs and FLF digesta. We observed that molecular weight (Mw) decreased during gastric digestion indicating that FLF aggregates were disrupted in the stomach. During intestinal digestion, Mw gradually decreased and CR increased indicating cleavage of glycosidic bonds releasing free sugars. Although the antioxidant and antidiabetic capacities were not eliminated by the digestion progression, the bioactive performance of FLFs under a digestive environment was reduced contrasting with the same concentration level of the undigested FLFs. These data provide comprehensive information on the digestibility and bioaccessibility of FLFs, and shed light on the effects of digestive progression on bioactive expression.

Laminarin ameliorates iodoacetamide-induced functional dyspepsia via modulation of 5-HT3 receptors and the gut microbiota.

Visceral and somatic hypersensitivity is a common cause of functional dyspepsia. Marine bioactive components have been revealed to possess numerous valuable abilities. However, as a kind of polysaccharide extracted from brown algae, the study focused on the biological properties of laminarin is still limited, especially in gastrointestinal disorders. In our study, indicators associated with visceral sensational function and gastrointestinal microecology were determined to investigate the modulatory effects of laminarin on functional dyspepsia induced by iodoacetamide. Mice with visceral hypersensitivity were orally administrated with laminarin (50 and 100 mg per kg bw) for fourteen days. The results indicated that laminarin partly alleviated the dysfunction by regulating corticosterone secretion, the expression of 5HT3 receptors at both protein and mRNA levels, and mechanical transduction through the PIEZO2-EPAC1 axis. Furthermore, laminarin administration moderated the imbalanced gut microbial profile, including modulating the abundance of Bacteroidetes and Firmicutes. Our findings revealed that laminarin may restore the overexpression of 5HT3 receptors, the abnormal mechanical transduction, and impaired gut microecology. In conclusion, we provide evidence to support the utilization of laminarin as the ingredient of complementary and alternative medicine of regulating visceral and somatic hypersensitivity.

Essential bioactive competence of laminarin (ß-glucan)/ laminaran extracted from Padina tetrastromatica and Sargassum cinereum biomass.

Marine algae-based drug discovery has recently received a lot of attention. This study was conducted to extract laminarin-enriched solvent extracts from Padina tetrastromatica and Sargassum cinereum and to evaluate their anticancer activity against the HeLa cell line in vitro (MTT assay). Furthermore, their toxicity was determined through a zebra fish model study. P. tetrastromatica and S. cinereum biomasses have a higher concentration of essential biomolecules such as carbohydrates, protein, and crude fiber, as well as essential minerals (Na, Mg, K, Ca, and Fe) and secondary metabolites. Methanol extracts, in particular, contain a higher concentration of vital phytochemicals than other solvent extracts. The laminarin quantification assay states that methanol extracts of P. tetrastromatica and S. cinereum are rich in laminarin, which is primarily confirmed by FTIR analysis. In an anticancer study, laminarin-MeE from P. tetrastromatica and S. cinereum at concentrations of 750 and 1000 µg mL-1 demonstrated 100% activity against HeLa cells. The Zebra fish model-based toxicity study revealed that the laminarin-enriched MeE of P. tetrastromatica and S. cinereum is non-toxic. These findings revealed that the laminarin-enriched MeE of P. tetrastromatica and S. cinereum has significant anticancer activity without causing toxicity.

Antibiofilm and antivirulence activities of laminarin-gold nanoparticles in standard and host-mimicking media.

The rapidly rising antimicrobial resistance (AMR) in pathogenic bacteria has become one of the most serious public health challenges, with a high death rate. Most pathogenic bacteria have been recognized as a source of AMR and a primary barrier to antimicrobial treatment failure due to the development of biofilms and the production of virulence factors. In this work, nanotechnology was employed as a substitute method to control the formation of biofilms and attenuate virulence features in Pseudomonas aeruginosa and Staphylococcus aureus. We synthesized biocompatible gold nanoparticles from marine-derived laminarin as potential biofilm and virulence treatments. Laminarin-gold nanoparticles (Lam-AuNPs) have been identified as spherical, 49.84 ± 7.32 nm in size and - 26.49 ± 1.29 mV zeta potential. The MIC value of Lam-AuNPs against several drug-resistant microbial pathogens varied from 2 to 1024 µg/mL in both standard and host-mimicking media. Sub-MIC values of Lam-AuNPs were reported to effectively reduce the production of P. aeruginosa and S. aureus biofilms in both standard and host-mimicking growth media. Furthermore, the sub-MIC of Lam-AuNPs strongly reduced hemolysis, pyocyanin, pyoverdine, protease, and several forms of flagellar and pili-mediated motility in P. aeruginosa. Lam-AuNPs also inhibited S. aureus hemolysis and the production of amyloid fibrils. The Lam-AuNPs strongly dispersed the preformed mature biofilm of these pathogens in a dose-dependent manner. The Lam-AuNPs would be considered an alternative antibiofilm and antivirulence agent to control P. aeruginosa and S. aureus infections. KEY POINTS: • Lam-AuNPs were biosynthesized to control biofilm and virulence. • Lam-AuNPs show effective biofilm inhibition in standard and host-mimicking media. • Lam-AuNPs suppress various virulence factors of P. aeruginosa and S. aureus.

Effects of laminarin on growth performance and resistance against Pseudomonas plecoglossicida of large yellow croaker （Larimichthys crocea）.

Laminarin (LAM) is widely used as an immunopotentiator in aquaculture, but its protective mechanism is still unclear. In this study, the effects of LAM on the growth performance and resistance against Pseudomonas plecoglossicida of large yellow croaker were studied in vitro and in vivo. The 42 d-feeding trial in large yellow croaker showed that dietary LAM could obviously promote the fish growth by improving the weight gain rate (WGR), specific growth rate (SGR), and feed conversion rate (FCR). Dietary LAM could also improve the survival rate of large yellow croakers subjected to P. plecoglossicida infection, and 500 mg/kg LAM produced the highest relative percent survival (RPS) of 35.00 %. LAM improved fish antioxidant level by enhancing serum total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity, and reducing malondialdehyde (MDA) content. In addition, LAM also improved fish innate immunity by increasing serum acid phosphatase (ACP) and alkaline phosphatase (AKP) activities and complement 3 (C3) content under P. plecoglossicida infection. What is more, on 9 d post P. plecoglossicida challenge, LAM could significantly decrease the bacteria load in head kidneys, spleens and livers of fish, and the lowest bacterial load was found in 500 mg/kg LAM group. In vitro, LAM exerted a protective role against inactivated P. plecoglossicida-triggered inflammatory injury in primary head kidney macrophages (PKM) of large yellow croaker by recovering cell viability, suppressing NO production, and reversing pro-inflammatory cytokine expression (IL-1ß, IL-6, and IL-8). All these findings therefore will provide insights into the protection mechanism of LAM in fish, facilitating its application in prevention and control of fish bacteriosis.

Sequential extraction of fucoidan, laminarin, mannitol, alginate and protein from brown macroalgae Ascophyllum nodosum and Fucus vesiculosus.

The study involves development of a green biorefinery process for obtaining fucoidan, laminarin, mannitol, alginate and protein from dry and fresh Fucus vesiculosus and Ascophyllum nodosum using hydrochloric acid and a green extraction solvent. After the extraction of fucoidan which was the targeted biomolecule, an extract and by-product (residual biomass) were obtained. The extract was passed through an ultrafiltration membrane, where fucoidan was obtained in the ultrafiltration retentate while ultrafiltration permeate was analysed for laminarin and mannitol. The residual biomass was used for obtaining alginate using ultrasound (20 kHz, 64 % amplitude and 32 min, optimum parameters for alginate extraction based on our previous study). All the samples, showed good results for alginate, laminarin and mannitol, indicating that the by-products can be utilised using this green extraction process. The comparison of both dry and fresh seaweed is relevant from an industry perspective, as fresh seaweed can directly be used for extraction, avoiding drying which adds significantly to the cost of the process. Life cycle impact assessment of the complete seaweed value chain has been carried out to identify the energy demand and key environmental hotspots. This biorefinery process can be used by industry to improve their processes and utilise the by-products generated efficiently.

Laminarin Reduces Cholesterol Uptake and NPC1L1 Protein Expression in High-Fat Diet (HFD)-Fed Mice.

Aberrantly high dietary cholesterol intake and intestinal cholesterol uptake lead to dyslipidemia, one of the risk factors for cardiovascular diseases (CVDs). Based on previous studies, laminarin, a polysaccharide found in brown algae, has hypolipidemic activity, but its underlying mechanism has not been elucidated. In this study, we investigated the effect of laminarin on intestinal cholesterol uptake in vitro, as well as the lipid and morphological parameters in an in vivo model of high-fat diet (HFD)-fed mice, and addressed the question of whether Niemann-Pick C1-like 1 protein (NPC1L1), a key transporter mediating dietary cholesterol uptake, is involved in the mechanistic action of laminarin. In in vitro studies, BODIPY-cholesterol-labeled Caco-2 cells were examined using confocal microscopy and a fluorescence reader. The results demonstrated that laminarin inhibited cholesterol uptake into Caco-2 cells in a concentration-dependent manner (EC50 = 20.69 µM). In HFD-fed C57BL/6J mice, laminarin significantly reduced the serum levels of total cholesterol (TC), total triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C). It also decreased hepatic levels of TC, TG, and total bile acids (TBA) while promoting the excretion of fecal cholesterol. Furthermore, laminarin significantly reduced local villous damage in the jejunum of HFD mice. Mechanistic studies revealed that laminarin significantly downregulated NPC1L1 protein expression in the jejunum of HFD-fed mice. The siRNA-mediated knockdown of NPC1L1 attenuated the laminarin-mediated inhibition of cholesterol uptake in Caco-2 cells. This study suggests that laminarin significantly improves dyslipidemia in HFD-fed mice, likely by reducing cholesterol uptake through a mechanism that involves the downregulation of NPC1L1 expression.

Effects of algal supplementation in feed to broiler breeders on transfer of nutrients and antibodies to chicks and quality of hatchlings.

Breeder nutrition is an important factor for chick quality since the chick embryo relies on nutrients available in the egg for growth and development. In addition, the egg is providing the chick with important antibodies that are vital during the first weeks of life. Brown algae contains several bioactive compounds, and dietary supplementation with algal extracts have shown improved gut health and immune responses in both pigs and poultry. The aim of this study was to investigate if feeding the brown algae Saccharina latissima, intact or as an extract, to broiler breeders can affect breeder hens' antibody responses to vaccination, egg quality and transfer of antibodies and nutrients to the egg and thereby improve the quality of newly hatched chicks. Forty-five hens and nine roosters of the parent lines of the fast-growing broiler Ross 308 were included in the experiment where hens were 31 weeks at the start. The hens were housed individually and fed one of three dietary treatments for seven weeks; (a) control, (b) addition of 0.6% algal meal or (c) addition of 0.08% algal extract. The hens were given a booster vaccination against infectious bronchitis virus (IBV) 21 days after the start of experiment. During experimental days 32-42, hens were naturally mated every 5th day and hatching eggs were collected. A total of 255 chicks were hatched, and chick quality was assessed. Moreover, on chick day three, blood was collected from 48 focal chickens and total immunoglobulin Y levels and specific titres to IBV in serum were determined. The results showed that feeding the brown algae Saccharina latissima, intact or as an extract to broiler breeders did not affect egg production, egg quality, antibody responses to vaccination or transfer of antibodies from hen to chick. However, feeding intact algae significantly increased the levels of iodine and decreased the level of selenium in the eggs and resulted in a lower proportion of chicks with maximum quality score. Interestingly, algal feeding, both intact and as an extract, increased the abdominal fat pad in broiler breeders by about 17% without affecting BW. In conclusion, supplementation of broiler breeder diets with algal extract from Saccharina latissima, but not intact algal meal is a promising dietary strategy to increase the abdominal fat pad without causing any adverse effects on nutrient level in eggs or chick quality.

Chitin and laminarin additively trigger wheat reactive oxygen species but not resistance to Fusarium head blight.

Plants respond to fungal infections by activating defense genes including producing reactive oxygen species (ROS). The fungus Fusarium graminearum causes Fusarium head blight (FHB), a serious disease of wheat and barley. FHB results in crop yield loss and contaminates grain with mycotoxins. In a prior study, we discovered that chitin induces tissue-specific ROS burst in wheat. However, it is unknown whether other fungal cell wall components could induce defense response in wheat. Therefore, we evaluated ROS and defense gene responses in different wheat tissues that had been treated with chitin, laminarin, or both. Different ROS patterns were induced in wheat treated with laminarin or chitin. Furthermore, we found that ROS were enhanced in wheat tissues treated with both chitin and laminarin. This study provides novel information for enhancing plat immunity to increase plant resistance.

Dietary supplementation of laminarin improves the reproductive performance of sows and the growth of suckling piglets.

BACKGROUND: Maternal nutrition is essential in keeping a highly efficient production system in the pig industry. Laminarin has been shown to improve antioxidant capacity, reduce the inflammatory response, and favor the homeostasis of intestinal microbiota. However, the effect of dietary supplementation of laminarin on the reproductive performance of sows and the growth of suckling offspring remains unknown. METHODS: A total of 40 Landrace × Yorkshire multiparous sows on d 85 of gestation, similar in age, body weight (BW), parity and reproductive performance, were randomly divided into four dietary treatments with 10 sows per treatment, receiving a control diet (basal pregnancy or lactating diets) and a basal diet supplemented with 0.025%, 0.05% and 0.10% laminarin, respectively. The experiment lasted from d 85 of gestation to d 21 of lactation. RESULTS: Laminarin supplementation linearly increased number born alive per litter (P = 0.03), average daily feed intake (ADFI, P < 0.01), and total milk yield of sows during the lactation of 1-21 d (P = 0.02). Furthermore, maternal laminarin supplementation increased the average daily gain (ADG) of piglets while tending to reduce the culling and death rate before weaning. In addition, alterations to the composition of colostrum and milk, as well as to serum inflammatory cytokines and immunoglobulins of sows were observed. The fecal microbiota profile of sows supported the improvement of reproductive performance in sows and the growth performance in suckling offspring. CONCLUSIONS: Dietary supplementation of laminarin during late pregnancy and lactation could significantly improve reproductive performance of sows and growth performance of piglets.

Molecular mechanism for endo-type action of glycoside hydrolase family 55 endo-ß-1,3-glucanase on ß1-3/1-6-glucan.

The glycoside hydrolase family 55 (GH55) includes inverting exo-ß-1,3-glucosidases and endo-ß-1,3-glucanases, acting on laminarin, which is a ß1-3/1-6-glucan consisting of a ß1-3/1-6-linked main chain and ß1-6-linked branches. Despite their different modes of action toward laminarin, endo-ß-1,3-glucanases share with exo-ß-1,3-glucosidases conserved residues that form the dead-end structure of subsite -1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-ß-1,3-glucanase MnLam55A, identified from Microdochium nivale. MnLam55A, like other endo-ß-1,3-glucanases, degraded internal ß-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. ß1-3-Glucans lacking ß1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal ß1-3-linkage of the laminarioligosaccharide moiety at the reducing end side of the main chain ß1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but kcat/Km values to laminarioligosaccharides (≤4.21 s-1 mM-1) were much lower than to laminarin (5920 s-1 mM-1). These results indicate that ß-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites -1 and -2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-ß-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-ß-1,3-glucanases.

Plant Resistance Inducers Affect Multiple Epidemiological Components of Plasmopara viticola on Grapevine Leaves.

Plant resistance inducers (PRIs) harbor promising potential for use in downy mildew (DM) control in viticulture. Here, the effects of six commercial PRIs on some epidemiological components of Plasmopara viticola (Pv) on grapevine leaves were studied over 3 years. Disease severity, mycelial colonization of leaf tissue, sporulation severity, production of sporangia on affected leaves, and per unit of DM lesion were evaluated by inoculating the leaves of PRI-treated plants at 1, 3, 6, 12, and 19 days after treatment (DAT). Laminarin, potassium phosphonate (PHO), and fosetyl-aluminium (FOS) were the most effective in reducing disease severity as well as the Pv DNA concentration of DM lesions on leaves treated and inoculated at 1 and 3 DAT; PHO and FOS also showed long-lasting effects on leaves established after treatment (inoculations at 6 to 19 DAT). PRIs also prevented the sporulation of Pv on lesions; all the PRI-treated leaves produced fewer sporangia than the nontreated control, especially in PHO-, FOS-, and cerevisane-treated leaves (>75% reduction). These results illustrate the broader and longer effect of PRIs on DM epidemics. The findings open up new perspectives for using PRIs in a defense program based on single, timely, and preventative field interventions.

Identification and Characterization of Two Novel Extracellular ß-Glucanases from Chaetomium globosum against Fusarium sporotrichioides.

Chaetomium globosum can inhibit the growth of fusarium by means of their extracellular proteins. Two novel ß-glucanases, designated Cgglu17A and Cgglu16B, were separated from the supernatant of C. globosum W7 and verified to have the ability to hydrolyze cell walls of Fusarium sporotrichioides MLS-19. Cgglu17A (397 amino acids) was classified as glycoside hydrolase family 17 while Cgglu16B belongs to the family16 (284 amino acids). Recombinant protein Cgglu17A was successfully expressed in Escherichia coli, and the enzymes were purified by affinity chromatography. Maximum activity of Cgglu17A appeared at the pH 5.5 and temperature 50 °C, but Cgglu16B shows the maximum activity at the pH 5.0 and temperature 50 °C. Most of heavy metal ions had inhibition effect on the two enzymes, but Cgglu17A and Cgglu16B were respectively activated by Ba2+ and Mn2+. Cgglu17A exhibited high substrate specificity, almost only catalyzing the cleavage of ß-1,3-glycosidic bond, in various polysaccharose, to liberate glucose. However, Cgglu16B showed high catalytic activities to both ß-1,3-glycosidic and ß-1,3-1,4-glycosidic bonds. Cgglu17A was an exo-glucanase, but Cgglu16B was an endo-glucanase based on hydrolytic properties assay. Both of two enzymes showed potential antifungal activity, and the synergistic effect was observed in the germination experiment of pathogenic fungus. In conclusion, Cgglu17A (exo-1,3-ß-glucanase) and Cgglu16B (endo-1,3(4)-ß-glucanase) were confirmed to play a key role in the process of C. globosum controlling fusarium and have potential application value on industry and agriculture for the first time.