Carbohydrate specificity of lectins from luminescent bacteria

The presence of lectins on a cell surface was demonstrated for 70 cultures of luminescent bacteria using hemagglutination reactions. It was shown that hemagglutination of luminescent bacteria is inhibited by glucose, maltose, fructose, mannose, and N-acetyl-D-glucosamine. The differences in the inhibition of hemagglutination of luminescent and nonluminescent (spontaneous mutants) symbiotic cultures by N-acetyl-D-galactosamine were revealed. The fact that N-acetyl-D-galactosamine inhibits hemagglutination of the luminescent symbiotic bacteria but does not inhibit hemagglutination of the symbiotic cultures lacking luminescence suggests that lectins with N-acetyl-D-galactosamine specificity are possibly involved in the formation and functioning of the symbiosis of luminescent bacteria with marine animals possessing luminous organs.     

Carbohydrate specificity of lectins from Boletopsis leucomelas and Aralia cordate

The carbohydrate specificity of three novel lectins, Boletopsis leucomelas lectin (BLL), Aralia cordate lectin (ACL), and Wasabia japonica lectin (WJL), was examined by frontal affinity chromatography using a panel of fluorescently labeled 47 oligosaccharides. The results indicate that BLL recognizes an agalacto structure of the biantennary chain and its bisecting structure. ACL showed strong affinity for triantennary oligosaccharides, but no affinity for tetraantennary structure. WJL showed no appreciable affinity for any of the 47 glycans examined. These lectins with a unique affinity specificity might be useful for examining alterations in the glycan structures of the glycoconjugates in association with development and various diseases.     

Carbohydrate specificity of lectins from plants of the genus horsetail

Carbohydrate specificity of partially purified lectins from 4 species of plants: horse-tail genus Equisetum (Equisetum arvense L., E. sylvaticum L., E. hyemale L. and E. tempatelia Ehrh.) has been studies. The obtained lectins have similar carbohydrate specificity. Among the tested carbohydrates the best inhibitor of activity is phenyl-2-acetamido-alpha-D-glucosaminopyranoside. Lectins poorly interact with yeasty mannan and galactomannan Trigonella foenum graecum seeds. Among glycoproteins the best inhibitor of activity is ovomucoid.     

Carbohydrate and glycoprotein specificity of two endogenous cerebellar lectins

Two endogenous cerebellar mannose binding lectins have been isolated in an active form by immunoaffinity chromatography employing their respective immobilized antibodies. One of them, termed cerebellar soluble lectin (CSL), was extracted in the absence of detergents, whereas the other, called Receptor 1 (R1), was soluble only in the presence of detergents. Tests of inhibition of agglutination of erythrocytes were performed with mono-, oligo and polysaccharides, as well as glycoconjugates of known structures. On the basis of agglutinating activities these 2 lectins are different from the previously reported lectins in brain, since they were not inhibited by galactosides and lactosides and were only marginally inhibited by glycosaminoglycans. CSL and R1 were better inhibited by mannose-rich glycopeptides as compared to the corresponding oligosaccharides. The different inhibition patterns obtained with glycans of known structures indicated that these lectins are very discriminative. Although CSL and R1 have similar specificities, they differed in their binding properties towards glycopeptides of ovalbumin. Both lectins showed considerable affinity for endogenous cerebellar glycopeptides, also rich in mannose. These glycopeptides belong to a few endogenous Con A-binding cerebellar glycoprotein subunits and are not present on other endogenous Con A-binding glycoproteins. In the forebrain, where CSL and R1 were also present, at least some of the glycoproteins interacting with the lectins were different from that observed in the cerebellum. Our data overall suggest that specific cell recognition in the nervous system could be invoked via the interactions between widely distributed lectins and cell-specific glycoproteins.     

Galactosyl-binding lectins from the tunicate Didemnum candidum. Carbohydrate specificity and characterization of the combining site

The plasma of the ascidian Didemnum candidum possesses lectin activity directed toward galactosyl moieties. We report the characterization of the affinity chromatography-purified galactosyl-binding lectins from the plasma of this protochordate species in terms of their hemagglutination patterns, temperature stability, saccharide specificities, divalent cation requirements, and the comparison of the properties of their combining sites to those of other characterized lectins. The major galactosyl-specific lectin, termed DCL-I, has an apparent mass of 14,500 daltons and a minor lectin (DCL-II) has an apparent subunit mass of 15,500 daltons. The two molecules differed somewhat in their hemagglutination profiles with untreated and enzyme-treated erythrocytes: a 10-fold increase in DCL-II concentration is required to obtain agglutination titers comparable to those of DCL-I. Although both DCL-I and DCL-II will agglutinate neuraminidase-treated erythrocytes from all vertebrate species tested and most Pronase-treated erythrocytes, DCL-I will agglutinate some untreated erythrocytes which are not agglutinated by DCL-II. Both lectins required divalent cations, were inactivated by temperatures above 70 degrees C, and both exhibited optimal agglutinating activity over a wide range of pH (from 5 to 11). The DCL-I molecule was characterized for its saccharide specificity by binding and inhibition assays using characterized sugars and glycoproteins. Galactose and oligosaccharides bearing nonreducing terminal galactose were the best inhibitors. The inhibition analysis indicated that the DCL-I combining site is small, interacts only with hydroxyls on carbons 2, 3, and 4 of galactose, and exhibits moderate steric hindrance for voluminous groups on carbon 6 and the alpha-anomeric linkage. The data suggest that the combining site would be smaller than the peanut lectin combining site for galactose since DCL-I does not interact with the subterminal monosaccharide hydroxyls for C4 and C6 as does peanut agglutinin. To our knowledge, this is the first isolation and detailed characterization of a lectin from a protochordate species.     

Cellulolytic activity of luminous bacteria

Summary The luminous bacteriaVibrio harveyi andV. fischeri degrade cellulose. Cellulase activity was high when carboxymethyl cellulose and cellobiose were used as substrates but low with cellulose powder. The role of these microbes in the digestion of food material in fish gut is also discussed.

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Resumen Las bacterias luminosasVibrio harveyi yV. fischeri degradan celulosa. La actividad celulásica fue alta cuando se utilizó Carboxy-metil-celulosa o celobiosa como sustrato y baja cuando se utilizó polvo de celulosa. Se discute el papel de estos microorganismos en la digestión intestinal de los alimentos en peces.

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Résumé Les bactéries lumineusesVibrio harveyi etV. fischeri dégradent la cellulose. L'activité cellulolytique était élevée lorsque la carboxméthylcellulose et la cellobiose servaient de substrats, mais elle était faible avec la poudre de cellulose. On discute également de rôle de ces microorganismes dans la digestion du matériau alimentaire.

     

New lectins from aspergilli and their carbohydrate specificity

Lectin activity was assessed in sixteen Aspergillius species using human A, B, O, AB, rabbit, goat, pig and sheep erythrocytes. Neuraminidase and protease treated blood group O erythrocytes were also used to evaluate lectin activity from all the cultures unable to agglutinate native red blood cells. Lectin activity was revealed from Aspergillus acristatus, A. gorakhpurensis, A. panamensis and A. carbonarius extracts, while undiluted extract of A. fischeri showed weak haemagglutination. Lectin activity was expressed after 5 days of growth by A. acristatus, A. gorakhpurensis, A. panamensis and A. carbonarius and after 8 days of cultivation a sharp decline in lectin activity was observed. Higher titres were observed from these species with enzymatically modified blood type O erythrocytes. A variety of carbohydrates were used to study their minimum inhibitory concentration capable of inhibiting haemagglutination. Porcine stomach mucin was found to be the most potent inhibitor of all the lectins. A. gorakhpurensis lectin showed high specificity for chondroitin-6-sulphate and N-acetyl-D-galactosamine. Significant specificity for L-fucose, D-arabinose and 2-deoxy-D-ribose was identified with A. panamensis lectin. Low concentrations of 0.625 mM of D-galactosamine HCl and 0.12 mg/mL of chondroitin-6-sulphate were found optimal to prevent haemagglutination of A. carbonarius extract. A. carbonarius lectin was partially purified 2.75-fold using ammonium sulphate precipitation, dialysis and ultrafiltration. It was found to be stable upto 40°C and within the pH range of 7.0–8.0. Lectin activity was not affected by guanidine-HCl, while it was reduced to half after incubation with urea and thiourea after 24 h.     

Carbohydrate binding specificities of several poly-N-acetyllactosamine-binding lectins

The structural requirements for the interaction of the Asn-linked poly-N-acetyllactosamine-type oligosaccharide moieties of glycoproteins with variousN-acetylglucosamine-binding lectins were investigated by means of affinity chromatography on immobilized lectin-Sepharose columns.High molecular weight glycopeptides containing poly-N-acetyllactosamine-type oligosaccharides obtained by Pronase digestion of human erythrocyte ghosts were treated with 0.1 M trifluoroacetic acid at 100°C for 40 min and then several oligosaccharide fragments were purified with an amino-bonded silica column. Among these oligosaccharide fragments, trisaccharide Gal1-4GlcNAc1-6Galol bound to the wheat germ agglutinin (WGA)- and pokeweed mitogen (PWM)-Sepharose columns, and also showed affinity to theDatura stramonium agglutinin (DSA)-,Lycopersicon esculentum (tomato) agglutinin-andSolanum tuberosum (potato) agglutinin-Sepharose columns. Pentasaccharide Gal1-4GlcNAc1-3(Gal1-4GlcNAc1-6)Galol showed weaker affinity to the WGA- and PWM-Sepharose columns, compared to the trisaccharide. Trisaccharide GlcNAc1-3(GlcNAc1-6)Galol showed weak affinity to the WGA-Sepharose column and did not show any affinity to the other lectin-Sepharose columns. Hexasaccharide Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4GlcNAcol bound only to the DSA-Sepharose column, indicating that only DSA does not require a GlcNAc(1-6)-linkage for interaction.Abbreviations HPLC high performance liquid chromatography - WGA wheat germ agglutinin - PWM pokeweed mitogen - DSA Datura stramonium agglutinin - LEA Lycopersicon esculentum (tomato) agglutinin - STA Solanum tuberosum (potato) agglutinin - EVA Erythrina variegata agglutinin - PBS 10 mM sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl - Galol galactitol - GlcNAcol N-acetylglucosaminitol     

Permeability studies on luminous bacteria

Summary The changes in light intensity of suspensions of a luminous bacterium in solutions of different electrolytes and non-electrolytes have been watched using an electronic photometer. The rapidity of the gradual extinction of the light was used as an approximate measure of the rapidity of penetration of the substances tested.The results concerning non-electrolytes and amino-acids are summarized in Table 10. (TheP _r -values of this table represent only quite crude estimations of the permeability.)The permeability towards both cations and anions seems to be very small.On the whole, the permeability properties of the bacterium studied seem fairly similar to those of the cells of higher plants.This investigation has been supported by a grant from The State Scientific Council. The author is indebted to Dr. Veijo Wartiovaara for valuable suggestions and to Mr. Sverker Norrback for careful technical assistance.     

Flavin mononucleotide reductase of luminous bacteria

NAD(P)H: FMN oxidoreductase (flavin reductase) couples in vitro to bacterial luciferase. This reductase, which is also postulated to supply reduced flavin mononucleotide in vivo as a substrate for the bioluminescent reaction, has been partially purified and characterized from two species of luminous bacterial. From Photobacterium fischeri the enzyme has a M. W. determined by Sephadex gel filtration, of 43,000 and may have a subunit structure. The turnover number at 20 degrees C, based on a purity estimate of 20 percent, is 1.7 times 10-4 moles of NADH oxidized per min per mole of reductase. The reductase isolated from Beneckea harveyi has an apparent molecular weight of 23,000; its purity was too low to permit estimation of specific activity. Using a spectrophotometric assay at 340 nm with the P. fischeri reductase, both NADH (Km, 8 times 10-5 M) and NADPH (Km, 4 times 10-4 M) were enzymatically oxidized, the Vmax with NADH being approximately twice that of NADPH. Of the flavins tested in this assay, only FMN (Km, 7.3 times 10-5 M) and FAD (Km, 1.4 times 10-4 M) were effective, FMN having a Vmax three times that of FAD. In the coupled assay, i.e., measuring the bioluminescence intensity of the reaction with added luciferase, the optimum FMN concentration was nearly 100 times less than in the spectrophotometric assay. The studies reported suggest the existence of a functional reductase-luciferase complex.     

Taxonomy of the marine,luminous bacteria

Summary One hundred and seventy-three strains of marine, luminous bacteria isolated from sea water, surfaces and intestines of fish, as well as from the luminous organs of fish and squid were submitted to an extensive phenotypic characterization. A numerical analysis of the results grouped these strains into four clusters which were formed on the basis of overall phenotypic similarity. One cluster, which was given the designationBeneckea harveyi, consisted of strains which had a moles% GC content in their DNAs of 46.5±1.3 and a single, sheathed, polar flagellum when grown in liquid medium. Most of these strains had unsheathed, peritrichous flagella in addition to the sheathed, polar flagellum when grown on solid medium. The two phenotypically similar clusters which were assigned the species designationsPhotobacterium phosphoreum andP. mandapamensis consisted of strains which had 1–3 unsheathed, polar flagella and moles % GC contents in their DNAs of 41.5±0.7 and 42.9±0.5, respectively. The cluster designatedP. fischeri contained strains having 2–8 sheathed, polar flagella and a moles % GC content of 39.8±1.1. These four species could be further distinguished on the basis of a number of nutritional properties as well as other phenotypic traits. The assignment of the luminous, marine bacteria to four species was supported by differences in the properties of the luminous system as well as differences in the pattern of regulation of spartokinase activity which are discussed. The speciesB. harveyi was found to be phenotypically similar to a number of previously characterized, non-luminous strains ofBeneckea which should probably be assigned to this species.Non-Standard Abbreviations ASW artificial sea water - ATCC American Type Culture Collection - BM basal medium - BMA basal medium agar - GC guanine plus cytosine - LA luminous medium agar - LB luminous medium broth - MA Difco Marine Agar - NCMB National Collection of Marine Bacteria - PHB poly--hydroxybutyrate - S similarity coefficient - YEB yeast extract broth This paper is part of a dissertation submitted by the senior author to the Graduate Division of the University of Hawaii in partial fulfillment of the requirements for the Ph.D. Degree in Microbiology