Characterization of marine bacteria and the activity of their enzyme systems involved in degradation of the algal storage glucan laminarin

The algal storage glucan laminarin is one of the most abundant carbon sources for marine prokaryotes. Its degradation was investigated in bacteria isolated during and after a spring phytoplankton bloom in the coastal North Sea. On average, 13% of prokaryotes detected by epifluorescence counts were able to grow in Most Probable Number dilution series on laminarin as sole carbon source. Several bacterial strains were isolated from different dilutions, and phylogenetic characterization revealed that they belonged to different phylogenetic groups. The activity of the laminarin-degrading enzyme systems was further characterized in three strains of Vibrio sp. that were able to grow on laminarin as sole carbon source. At least two types of activity were detected upon degradation of laminarin: release of glucose, and release of glucans larger than glucose. The expression of laminarinase activity was dependent on the presence of the substrate, and was repressed by the presence of glucose. In addition, low levels of activity were expressed under starvation conditions. Laminarinase enzymes showed minimal activity on substrates with similar glucosidic bonds to those of laminarin, but different sizes and secondary and/or tertiary structures. The characteristics found in these enzyme systems may help to elucidate factors hampering rapid carbohydrate degradation by prokaryotes.     

Regulation of Potato Immune Responses by Laminarin

Laminarin blocks potato immune responses by inhibiting the reaction of oversensitivity, formation of phytoalexins, wound repair, and the activity of proteinase inhibitors. It was found that laminarin exhibits antielicitor activity. Addition of salicylic acid to laminarin enhances its immunosuppressing effect, which becomes systemic.     

绿僵菌侵染对椰心叶甲酚氧化酶活性的影响

对椰心叶甲Brontispa longissima(Gestro)成虫血淋巴中酚氧化酶的特性进行分析,并研究绿僵菌(Metarhizium anisopliae)侵染对血浆甲酚氧化酶活性的影响。结果显示,椰心叶甲成虫的血浆及血细胞裂解液中均检测到酚氧化酶活性,且昆布多糖及胰蛋白酶可显著提高其活性。绿僵菌MA-4侵染组在侵染后第1至第5d的血浆酚氧化酶活性高于未侵染组(P<0.05),但是椰心叶甲成虫体内注射10μg昆布多糖后,侵染组的酚氧化酶活性显著低于未侵染组(P<0.05),表明绿僵菌一方面对可激活椰心叶甲的酚氧化酶原激活系统,另一方面又可抑制昆布多糖对椰心叶甲酚氧化酶原激活系统的诱导作用。     

Beta-1,3 glucan sulfate, but not beta-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis

Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals. We show that laminarin, a beta-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana. In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst. Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems. In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation. Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis. Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins. In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent. Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated beta-1,3 glucans are active. In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance. The results open new routes to work out new molecules suitable for crop protection.     

Lipopolysaccharides of Pectobacterium atrosepticum and Pseudomonas corrugata induce different defence response patterns in tobacco, tomato, and potato

Lipopolysaccharides (LPS), ubiquitous cell surface components of Gram-negative bacteria, are directly implicated in plant/pathogen interactions. However, their perception by the plant, the subsequent signal transduction in both compatible and incompatible interactions, as well as the defence reactions induced in compatible interactions are as yet poorly understood. We focused on biochemical and physiological reactions induced in cell suspensions of three Solanaceae species (tobacco, tomato, and potato) by purified lipopolysaccharides from PECTOBACTERIUM ATROSEPTICUM (PA), a pathogen of potato, and PSEUDOMONAS CORRUGATA (PSC), a pathogen of tomato. LPS PA and LPS PSC caused a significant acidification of potato, tomato, and tobacco extracellular media, whereas laminarin (a linear beta-1,3 oligosaccharide elicitor) induced an alkalinisation in tobacco and tomato, but not in potato cell suspensions. None of the two LPS induced the formation of active oxygen species in any of the hosts, while laminarin induced H (2)O (2) production in cells of tobacco but not of tomato and potato. In tomato cells, LPS PA and LPS PSC induced a strong but transitory stimulation of lipoxygenase activity, whereas laminarin induced a stable or slightly increasing LOX activity over the first 24 h of contact. In tobacco, LOX activity was not triggered by either LPS, but significantly increased following treatment with laminarin. In potato, neither LPS nor laminarin induced LOX activity, in contrast with concentrated culture filtrate of PHYTOPHTHORA INFESTANS (CCF). These results demonstrate that LPS, as well as laminarin, are perceived in different ways by SOLANACEAE species, and possibly cultivars. They also suggest that defence responses modulated by LPS depend on plant genotypes rather than on the type of interaction.     

Structure of beta-glucan oligomer from laminarin and its effect on human monocytes to inhibit the proliferation of U937 cells

We analyzed the human monocyte-stimulating ability of laminarin from Eisenia bicyclis, lichenan from Cetraria islandica, and their oligomers depolymerized with endo-1,3-beta-glucanase from Arthrobacter sp. The respective beta-glucan oligomers with different degrees of polymerization (DP) were fractionated from hydrolytic products of laminarin and lichenan using gel-filtration chromatography. The monocyte-conditioned medium pre-cultured in the presence of a fraction of beta-glucan oligomer (DP>/=8) from laminarin exhibited inhibitory activity against the proliferation of human myeloid leukemia U937 cells, while those pre-cultured with other beta-glucan oligomers and the original laminarin and lichenan showed little or no activity. NMR analysis indicated that the beta-glucan oligomer (DP>/=8) has an average DP value of 13, and its ratio of beta-1,3- to beta-1,6-linkages in glucopyranose units was estimated to be 1.3:1. These results indicate that the beta-1,3-glucan oligomer with a higher content of beta-1,6-linkage stimulates monocytes to inhibit the proliferation of U937 cells.     

Beta-glucanases of the yeast Pichia polymorpha.

Fractionation of proteins secreted into the culture medium by intact cells and protoplasts of Pichia polymorpha showing enzyme activity against laminarin, pustulan or p-nitrophenyl-beta-D-glucopyranoside has been performed, and the results compared with those obtained with cell-free extracts and lysed protoplasts. Fractionation with DEAE Sephadex A50 has proved to be the best method, yielding at least three fractions which hydrolyse laminarin. One of these fractions was active on both laminarin and pustulan. Filtration on Sephadex G-100 column only yielded one active preparation. Evidence supporting the conclusion that there are three different beta-glucanases located in the periplasmic space is presented.     

Synthesis of 1,3-β-Glucanases in Saccharomyces cerevisiae During the Mitotic Cycle, Mating, and Sporulation

Upon fractionating Saccharomyces cerevisiae asynchronous cultures by sucrose density gradient centrifugation in a zonal rotor and examining the exo-1,3-beta-glucanase and deoxyribonucleic acid content of the cells, a periodic step increase in the activity of this enzyme was observed, indicating a discontinuous pattern of synthesis or activation of exo-1,3-beta-glucanase during the mitotic cycle at the transition from the S to the G(2) phase. Similar results were obtained for endo-1,3-beta-glucanase by assaying activity against oxidized laminarin in permeabilized cells, suggesting that the synthesis of endo-1,3-beta-glucanase is controlled in the same way. When a and alpha strains were mated, the specific activity of cell extracts against laminarin, oxidized laminarin, and pustulan remained constant while zygote formation was taking place. However, when growth resumed, active synthesis of 1,3-beta-glucanases took place as shown by the occurrence of a significant increase in the specific activity against the three substrates. Specific changes in the level of glucan degradative enzymes, not observed in a haploid parental strain, occurred when the diploid S. cerevisiae AP-1 was induced to sporulate. The sporulation process triggered the activation of first the pustulan degradative capacity and then the capacity to hydrolyze oxidized laminarin. The specific activity against this substrate was 10 times higher than that against pustulan.     

不同硫取代度昆布多糖硫酸酯的合成及理化性质初步研究

目的:对昆布多糖进行不同硫取代度的硫酸酯化修饰,并对其产物的硫酸基含量、糖含量与分子量进行检测,为研究不同硫取代度昆布多糖硫酸酯的生物活性奠定物质基础。方法:采用氯磺酸-吡啶法对昆布多糖进行硫酸化修饰,通过改变硫酸化修饰条件,来制取不同硫酸基取代度的昆布多糖硫酸酯;利用盐酸水解-硫酸钡比浊法测定昆布多糖硫酸酯的硫酸基含量,并通过公式求得其硫取代度;用苯酚-硫酸法测定昆布多糖硫酸酯的多糖含量,并使用HPGPC法测定其分子量。结果:两种不同硫取代度昆布多糖硫酸酯的硫酸基含量分别为37.8%、45.92%,取代度分别为1.07、1.51,糖含量分别为44.52%、37.19%,分子量分别为13000、16000。结论:利用氯磺酸-吡啶法对昆布多糖进行硫酸酯化修饰,该方法可以获取不同取代度产物,酯化率高。     

Novel beta-1,3-, 1,6-oligoglucan elicitor from Alternaria alternata 102 for defense responses in tobacco

A novel elicitor that induces chitinases in tobacco BY-2 cells was isolated from Alternaria alternata 102. Six other fungi, including A. alternata IFO 6587, could not induce, or weakly induce chitinase activity. The purified elicitor was soluble in 75% methanol and showed the chitinase-inducing activity when applied at concentrations of as low as 25 ng x mL(-1). Structural determination by methylation analysis, reducing-end analysis, MALDI-TOF/MS, and NMR spectroscopy indicated that the elicitor was a mixture of beta-1,3-, 1,6-oligoglucans mostly with a degree of polymerization of between 8 and 17. Periodate oxidation of the elicitor suggested that the 1,6-linked and nonreducing terminal residues are essential for the elicitor activity. Further analysis of the elicitor responses in BY-2 cells indicated that the activity of this beta-1,3-, 1,6-glucan elicitor was about 1000 times more potent than that of laminarin, which is a known elicitor of defense responses in tobacco. Analyzing the expression of defense-related genes indicated that a phenylalanine ammonia-lyase gene and a coumaroyl-CoA O-methyltransferase gene were transiently expressed by this beta-1,3-, 1,6-glucan elicitor. The elicitor induced a weak oxidative burst but did not induce cell death in the BY-2 cells. In the tissue of tobacco plants, this beta-1,3-, 1,6-glucan elicitor induced the expression of basic PR-3 genes, the phenylpropanoid pathway genes, and the sesquiterpenoid pathway genes. In comparison with laminarin and laminarin sulfate, which are reported to be potent elicitors of defense responses in tobacco, the expression pattern of genes induced by the purified beta-1,3-, 1,6-glucan elicitor was more similar to that induced by laminarin than to that induced by laminarin sulfate.     

Laminarin sulfate mimics the effects of heparin on smooth muscle cell proliferation and basic fibroblast growth factor-receptor binding and mitogenic activity

Heparin and heparin-like molecules may function, apart from their effect on hemostasis, as regulators of cell growth and neovascularization. We investigated whether similar effects are exerted by laminarin sulfate, an unrelated polysulfated saccharide isolated from the cell wall of seaweed and composed of chemically O-sulfated b?-(1,3)-linked glucose residues. Laminarin sulfate exhibits about 30% of the anticoagulant activity of heparin and is effective therapeutically in the prevention and treatment of cerebrovascular diseases. We characterized the effect of laminarin sulfate on interaction of the heparin-binding angiogenic factor, basic fibroblast growth factor (bFGF), with a naturally produced subendothelial extracellular matrix (ECM) and with cell surface receptor sites. Laminarin sulfate (1-2 m?g/ml) inhibited the binding of bFGF to ECM and to the surface of vascular smooth muscle cells (SMC) in a manner similar to that observed with heparin. Likewise, laminarin sulfate efficiently displaced both ECM-and cell-bound bFGF at concentrations as low as 1 m?g/ml. Both laminarin sulfate and heparin efficiently induced restoration of bFGF receptor binding in xylosyltransferase-deficient CHO cell mutants defective in initiation of glycosaminoglycan synthesis. Moreover, laminarin sulfate elicited bFGF receptor activation and mitogenic response in heparan sulfate(HS)-deficient, cytokine-dependent lymphoid cells. These results indicate that laminarin sulfate effectively replaced the need for heparin and HS in the induction of bFGF receptor binding and signaling. In other experiments, laminarin sulfate was found to inhibit the proliferation of vascular SMC in a manner similar to that observed with heparin. These effects of laminarin sulfate may have potential clinical applications in diverse situations such as wound healing, angiogenesis, and atherosclerosis. © 1995 Wiley-Liss, Inc.     

The role of bacterial symbionts in suppressing the defence reaction in larval hemolymph of the cotton leafworm Spodoptera littoralis (Biosd.)

Abstract

Spodoptera littoralis hemolymph exhibited a decrease in phenoloxidase activity during the first hour of exposure to alive or dead Xenorhabdus nematophilus and Photorhabdus luminescens even in the presence or absence of laminarin and α-chymotrypsin. Also, as the bacterial numbers in the hemolymph of the larvae of S. littoralis increased, the suppression of the enzyme, phenoloxidase, activity in vivo increased. On the other hand, in the in vitro incubation of the infected hemolymph with X. nematophilus for 30 min with laminarin, the decrease in phenoloxidase activity reached 92% in the infection with the highest dose (1 × 10¹⁰ cells/ml) live bacteria, and reached 100% at the same dose in the infection with live bacteria in the absence of laminarin. A two-fold decrease in enzyme activity was recorded in the case of injection of (1 × 10⁶ cells/ml) dead X. nematophilus and the absence of laminarin compared with injection of the same dose in the presence of laminarin. The same trend was also observed by the end of incubation of X. nematophilus-treated hemolymph without α-chymotrypsin. The decrease in phenoloxidase activity was highly significantly different in injection of dead P. luminescens and the absence of laminarin during incubation. In the case of injection of (1 × 10⁸ cells/ml) live P. luminescens a higher degree in the reduction of enzyme activity was recorded in the absence of α-chymotrypsin, where it reached to nearly a two-fold decrease. The results of these studies indicated that both X. nematophilus and P. luminescens alive or dead suppress the phenoloxidase activity in the presence or absence of both laminarin and α-chymotrypsin but, the suppression in absence of both during the in vitro incubation was highly comparable.     

The interplay of processivity, substrate inhibition and a secondary substrate binding site of an insect exo-beta-1,3-glucanase

Abracris flavolineata midgut contains a processive exo-beta-glucanase (ALAM) with lytic activity against Saccharomyces cerevisiae, which was purified (yield, 18%; enrichment, 37 fold; specific activity, 1.89 U/mg). ALAM hydrolyses fungal cells or callose from the diet. ALAM (45 kDa; pI 5.5; pH optimum 6) major products with 0.6 mM laminarin as substrate are beta-glucose (61%) and laminaribiose (39%). Kinetic data obtained with laminaridextrins and methylumbelliferyl glucoside suggest that ALAM has an active site with at least six subsites. The best fitting of kinetic data to theoretical curves is obtained using a model where one laminarin molecule binds first to a high-affinity accessory site, causing active site exposure, followed by the transference of the substrate to the active site. The two-binding-site model is supported by results from chemical modifications of amino acid residues and by ALAM action in MUbetaGlu plus laminarin. Low laminarin concentrations increase the modification of His, Tyr and Asp or Glu residues and MUbetaGlu hydrolysis, whereas high concentrations abolish modification and inhibit MUbetaGlu hydrolysis. Our data indicate that processivity results from consecutive transferences of substrate between accessory and active site and that substrate inhibition arises when both sites are occupied by substrate molecules abolishing processivity.     

The role of prophenoloxidase activation in non-self recognition and phagocytosis by insect blood cells

Experiments indicate that the prophenoloxidase activating system, which is responsible for melanin production, is also involved in immunorecognition in insects. Using haemocyte monolayer preparations of Blaberus craniifer, Galleria mellonella and Leucophae maderae, it was shown that laminarin, a β 1,3-glucan extracted from fungal cell walls and an activator of the prophenoloxidase system, enhanced the phagocytosis of test bacteria.Scanning electron microscopy of haemocyte monolayers showed that incubation of test bacteria with laminarin significantly increased the number of microorganisms attached to both the plasmatocytes and the granular cells. Furthermore with the granular cells, these bacteria became entrapped in an amorphous matrix. This material probably consists of the “sticky” proteins previously reported to be produced by crustacean haemocytes following prophenoloxidase activation. Pretreatment of haemocytes with laminarin abolished the stimulatory effect on ingestion, indicating that these “sticky” proteins are opsonic, since they would have been discharged from the haemocytes onto the glass monolayer leaving few molecules available for subsequent coating of the test particles.Preliminary biochemical studies on the G. mellonella prophenoloxidase system demonstrated that it was activated by trypsin, laminarin and laminarin G, a highly purified β 1,3-glucan, but not by dextran. Serine protease activities were also enhanced by adding laminarin to a haemocyte lysate supernatant, suggesting that the stimulatory mechanism may involve the proteolytic activity of such enzymes.     

根虫瘟霉不同菌株对小菜蛾幼虫血淋巴酚氧化酶原的激活作用

在对小菜蛾Plutella xylostella幼虫血淋巴酚氧化酶原的存在部位及免疫激活作用特点研究的基础上,比较了根虫瘟霉Zoophthora radicans不同菌株对酚氧化酶原激活系统的免疫激化及防御作用的差异。研究发现, 酚氧化酶原主要位于小菜蛾幼虫血细胞膜及血细胞裂解液中,极少存在于血浆中。在免疫激活剂昆布多糖存在下,分别测得小菜蛾幼虫血细胞碎片、血细胞裂解液和血浆的酚氧化酶活性为26.80 U,16.68 U和2.53 U。酚氧化酶原显著地受血浆和昆布多糖同时存在的激活,但两者单独存在时对酚氧化酶原的激活作用较弱。根虫瘟霉菌丝裂解液对酚氧化酶原有不同程度的激活作用,其激活作用在有血浆存在时显著增强,其酚氧化酶活性可提高2.9～3.4倍。各菌株间对酚氧化酶原的激活作用则以ARSEF1342菌株最强,ARSEF2699和F99101菌株次之,ARSEF1100菌株最弱。被激活的酚氧化酶可粘附于根虫瘟霉菌丝上并能产生黑化反应,各菌株间酚氧化酶粘附于ARSEF1342菌株的能力最强,粘附于ARSEF2699和F99101菌株的次之,粘附于ARSEF1100菌株的最弱。但酚氧化酶粘附于昆布多糖的能力显著强于各虫霉菌株,表明各菌株在一定程度上能逃避寄主的免疫识别;各菌株激活酚氧化酶原及酚氧化酶粘附于菌株强弱,与对小菜蛾毒力呈负相关性,表明高毒力菌株具有易逃避寄主免疫识别的趋向。     

Isolation and characterization of two types of β-1,3-glucanases from the common sea hare Aplysia kurodai

Two types of β-1,3-glucanases, AkLam36 and AkLam33 with the molecular masses of 36 kDa and 33 kDa, respectively, were isolated from the digestive fluid of the common sea hare Aplysia kurodai. AkLam36 was regarded as an endolytic enzyme (EC 3.2.1.6) degrading laminarin and laminarioligosaccharides to laminaritriose, laminaribiose, and glucose, while AkLam33 was regarded as an exolytic enzyme (EC 3.2.1.58) directly producing glucose from polymer laminarin. AkLam36 showed higher activity toward β-1,3-glucans with a few β-1,6-linked glucose branches such as Laminaria digitata laminarin (LLam) than highly branched β-1,3-glucans such as Eisenia bicyclis laminarin (ELam). AkLam33 showed moderate activity toward both ELam and LLam and high activity toward smaller substrates such as laminaritetraose and laminaritriose. Although both enzymes did not degrade laminaribiose as a sole substrate, they were capable of degrading it via transglycosylation reaction with laminaritriose. The N-terminal amino-acid sequences of AkLam36 and AkLam33 indicated that both enzymes belong to the glycosyl hydrolase family 16 like other molluscan β-1,3-glucanases.     

Intracellular and cell wall associated (1 → 3) β glucanases of Saprolegnia

Summary Fractionation of proteins of mycelial cell free extracts from Saprolegnia monoica revealed the presence of two different (1.3) glucanases. The most important fraction exhibited activity against laminarin and p. nitrophenyl BD. glucopyranoside. The other was active on both laminarin and oxidized laminarin. This endo-glucanase represented the main part of glucanase activities released during cell wall autolysis. Properties and cellular distribution of these enzymes are discussed in respect to their morphogenetic role in hyphal differentiation.     

Laminarinase (beta-glucanase) activity in Bacteroides from the human colon.

Laminarin, a beta(1 leads to 3)-glucan similar to those found in plant cell walls, is fermented by some species of anaerobic bacteria from the human colon. Laminarinase (EC 3.2.1.6) and beta-glucosidase (EC 3.2.1.21) activities were determined in strains representing Bacteroides thetaiotaomicron, Bacteroides distasonis, and an unnamed deoxyribonucleic acid homology group of Bacteroides fragilis. In all three species, laminarinase activity was inducible by laminarin and was predominantly cell bound. The products of laminarinase activity varied with each species. In the case of B. thetaiotaomicron, the major product of laminarin hydrolysis was glucose (70 to 90%), and there were small amounts of laminaribiose (G2) and oligomers of glucose as high as G4. In the case of group '0061-1,' glucose (40 to 50%) and oligomers of glucose as high as G6 were found. The laminarinase of B. distasonis differed from the laminarinases of the other two species in that it mainly produced oligomers of glucose (G2-G5). beta-Glucosidase activity was also found in all three species. beta-Glucosidase was induced by glucose-containing disaccharides as well as by laminarin. The beta-glucosidases of the three Bacteroides species differed with respect to level of activity, induction pattern, and sensitivity to inhibition by D-glucono-1,5-lactone.     

Beta-1,3-glucooligosaccharide induced activation of four enzymes responsible for N-p-coumaroyloctopamine biosynthesis in potato (Solanum tuberosum cv.) tuber tissue

Potato tuber disks, when treated with laminarin, a beta-1,3-glucooligosaccharide from Laminaria digitata, accumulate a hydroxycinnamoyl amide compound, N-p-coumaroyloctopamine (p-CO). The biosynthesis of p-CO was investigated by feeding experiments, in order to show that the precursors of N-p-coumaroyl and octopamine moieties of p-CO are L-phenylalanine and L-tyrosine, respectively. The treatment of potato tuber tissue with laminarin resulted in elevated activities of four enzymes which are putatively involved in p-CO biosynthesis: phenylalanine ammonia lyase (PAL; EC 4.3.1.5), 4-hydroxycinnamic acid:CoA ligase (4CL; EC 6.2.1.12), hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) and tyrosine decarboxylase (TyrDC; EC 4.1.1.25). Among these, the response of TyrDC was specific to laminarin treatment, thus indicating that the regulation of TyrDC activity is critical for the accumulation of p-CO in potato tuber tissue.