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Basis for the susceptibility of several algae to microbial decomposition.

Partially purified cellulase and a cellulase-containing polygalacturonase but notlysozyme extensively degraded the walls of Chlamydomonas reinhardtii and Ulothrix fimbrata and converted intact cells of the algae to spheroplasts. A streptomycete cellulase cochromatographed with the enzyme system releasing glucose from walls of these organisms, and this preparation also converted the algal cells to spheroplasts. The dominant constituent in the walls was carbohydrate, and glucose and small quantities of galacturonic acid but no amino sugars were present in acid hydrolysates of the walls. Glucose accounted for essentially all of the material solobilized by the cellulase preparation. Lysozyme acted on Cylindrospermum sp. walls, and it, but not the otherenzymes, converted some of the Cylindrospermum sp. cells to spheroplasts. Streptomycete enzymes lysing Micrococcus lysodeikticus cochromatographed with the proteins releasing reducing sugars from Cylindrospermum sp. walls, and components in the active fraction converted cells of this alga into spheroplasts. X-ray diffraction revealed that the walls of C. reinhardtii and U. fimbrata but not those of Cylindrospermum sp. contained cellulose. The data suggest that the susceptibility of the first twospecies to microbial degradation in natural ecosystems results from an attack on the cellulose in their walls, and the susceptibility of the third is linked with the microbial production of a lysozyme.

Carbohydrates↗

Fungal exudates.

The exudates or liquid droplets on various structures of a number of fungi were examined. The droplets were enveloped in membranous material and were associated with actively growing mycelia, including fruiting structures. Osmium tetroxide vapour-fixed droplets of Claviceps purpurea, Myrothecium roridum, Sclerotinia sclerotiorum, Sclerotium rolfsii, and Thanathephorus cucumeris did not dry to a powder but remained intact as spheres when freeze-dried. Fractured spheres, examined with the scanning electron microscope, showed the presence of a membranous structure similar to that of rapidly frozen colloidal solutions with the ice crystals removed by sublimation. Locules or cavities within the freeze-dried droplets are thought to be due to the entrapment of air when droplets coalesce. Biochemical analyses of the exudates showed that acid phosphatase, beta-glucosidase, acid and alkaline protease. RNase polygalacturonase and cellulase enzymes as well as oxalic acid and ammonia were present.

Ammonia↗

Extracellular enzyme production by Rhizopus and Mucor species on solid media.

Solid media were employed to determine the presence and absence of extracellular enzyme production by two genera of fruit-rot fungi, Rhizopus and Mucor. The results of this investigation revealed that phosphatase was released into the cultural medium by all the fungi examined; however, only R. oryzae, R. tritici, M. mucedo, and M. piriformis showed the possibility of being high producers of the enzyme. Protease, urease, ribonuclease, pectate lyase, and polygalacturonase, at varying levels of activity, were detected, in the majority of the fungi, in the cultural medium.

Culture Media↗

Genetic and biochemical characterization of an exopolygalacturonase and a pectate lyase from Yersinia enterocolitica.

Yersinia enterocolitica, an invasive foodborne human pathogen, degrades polypectate by producing two depolymerizing enzymes, pectate lyase (PL) and polygalacturonase (PG). The gene encoding the PG activity, designated pehY, was located in a 3-kb genomic fragment of Y. enterocolitica ATCC 49397. The complete nucleotide sequence of this 3-kb fragment was determined and an open reading frame consisting of 1803 bp was predicted to encode a PG protein with an estimated M(r) of 66 kDa and pI of 6.3. The amino acid sequence of prePG showed 59 and 43% identity to that of the exopolygalacturonase (exoPG) of Erwinia chrysanthemi and Ralstonia solanacearum, respectively. The Y. enterocolitica PG overproduced in Escherichia coli was purified to near homogeneity using perfusion cation exchange chromatography. Analysis of the PG depolymerization products by high performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD) revealed the exolytic nature of this enzyme. The Y. enterocolitica PL overproduced in E. coli was also partially purified and the M(r) and pI were estimated to be 55 kDa and 5.2, respectively. HPAEC-PAD analysis of the PL depolymerization products indicated the endolytic nature of this enzyme. Southern hybridization analyses revealed that pehY and pel genes of Y. enterocolitica are possibly encoded in the chromosome rather than in the plasmid. Purified exopolygalacturonase (over 10 activity units) was unable to macerate plant tissues.

Amino Acid Sequence↗

Regulation of pectinolysis in Erwinia chrysanthemi.

Erwinia chrysanthemi is an enterobacterium that causes various plant diseases. Its pathogenicity results from the secretion of pectinolytic enzymes responsible for the disorganization of the plant cell wall. The E. chrysanthemi strain 3937 produces two pectin methylesterases, at least seven pectate lyases, a polygalacturonase, and a pectin lyase. The extracellular degradation of the pectin leads to the formation of oligogalacturonides that are catabolized through an intracellular pathway. The pectinase genes are expressed from independent cistrons, and their transcription is favored by environmental conditions such as presence of pectin and plant extracts, stationary growth phase, low temperature, oxygen or iron limitation, and so on. Moreover, transcription of the pectin lyase gene responds to DNA-damaging agents. The differential expressions of individual pectinase genes presumably reflect their role during plant infection. The regulation of pel genes requires several regulatory systems, including the KdgR repressor, which mediates the induction of all the pectinolysis genes in the presence of pectin catabolites. KdgR also controls the genes necessary for pectinase secretion and other pectin-inducible genes not yet characterized. PecS, a cytoplasmic protein homologous to other transcriptional regulators, can bind in vitro to the regulatory regions of pectinase and cellulase genes. The PecT protein, a member of the LysR family of transcriptional regulators, represses the expression of some pectinase genes and also affects other metabolic pathways of the bacteria. Other proteins involved in global regulations, such as CRP or HNS, can bind to the regulatory regions of the pectinase genes and affect their transcription.

Carbohydrate Sequence↗

Pickles, pectin, and penicillin.

My professional life has been devoted to the study of microbial products and their biosynthesis, regulation, and overproduction. These have included primary metabolites (glutamic acid, tryptophan, inosinic acid, guanylic acid, vitamin B(12), riboflavin, pantothenic acid, ethanol, and lactic acid) and secondary metabolites (penicillin, cephalosporins, streptomycin, fosfomycin, gramicidin S, rapamycin, indolmycin, microcin B17, fumagillin, mycotoxins, Monascus pigments, and tetramethylpyrazine). Other areas included microbial nutrition, strain improvement, bioconversions of statins and beta-lactams, sporulation and germination, plasmid stability, gel microdroplets, and the production of double-stranded RNA, the polymer xanthan, and enzymes (polygalacturonase, protease, cellulase). Most of the studies were carried out with me by devoted and hardworking industrial scientists for 15 years at Merck & Co. and by similarly characterized students, postdoctorals, and visiting scientists during my 32 years at the Massachusetts Institute of Technology. I owe much of my success to my mentors from academia and industry. My recent research activities with undergraduate students at the Charles A. Dana Research Institute for Scientists Emeriti (R.I.S.E.) at Drew University have been very rewarding and are allowing me to continue my career.

Anti-Bacterial Agents↗

Identification and characterization of the allergens in the tomato fruit by immunoblotting.

BACKGROUND: Although the tomato fruit (Lycopersicon esculentum) has been widely investigated for breeding purposes, there have been few studies on tomato allergenicity. We attempted to identify the tomato fruit allergens and to compare the concentrations of IgE-binding proteins among the different growth stages with sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. METHODS: An immunoblot experiment on tomato fruit extracts was performed using sera from 11 patients with oral allergy syndrome (OAS) to tomatoes. Bands reacting with IgE from more than half of the OAS patients' sera were excised and subjected to determination of N-terminal amino acid sequences using the automated Edman degradation method. Moreover, we compared the concentrations of these proteins at each growth stage of the tomato fruit with SDS-PAGE and immunoblotting. RESULTS: Four proteins binding with IgE from more than half of the OAS patients' sera were determined to be polygalacturonase 2A (PG2A), beta-fructofuranosidase, superoxide dismutase (SOD) and pectinesterase (PE). The concentrations of PG2A, beta-fructofuranosidase and PE were highest in the red ripening stage with both SDS-PAGE and immunoblotting. CONCLUSION: The concentrations of 3 of 4 tomato allergens increased during ripening.

Adolescent↗

Group 13 allergens as environmental and immunological markers for grass pollen allergy: studies by immunogold field emission scanning and transmission electron microscopy.

BACKGROUND: Polygalacturonases were recently identified as important grass pollen allergens and designated group 13 allergens. The objective of the present study was to investigate the presence of group 13 grass pollen allergens in different grass species, their release and ultrastructural location in dry and hydrated grass pollen. METHODS: Nitrocellulose-blotted allergen extracts from 12 wild and cultivated grass genera were probed with a rabbit antiserum raised against purified recombinant timothy grass pollen allergen, Phl p 13. The release kinetics of Phl p 13 from timothy grass pollen hydrated for 0.5 min to 3 h were analyzed by immunoblotting. Phl p 13 was localized in dry and hydrated grass pollen grains by immunogold field emission scanning and transmission electron microscopy. RESULTS: Group 13 allergens were detected in all 12 wild and cultivated grass genera representing the major subfamilies of the Poaceae. Ultrastructurally, the allergen was located in the wall and in the cytoplasm of timothy grass pollen grains. In the cytoplasm, Phl p 13 was associated with polysaccharide particles and as yet undescribed stacks of microtubule-like structures. After hydration in rain water, pollen grains expel cytoplasmic particles of respirable size containing Phl p 13, which becomes detectable in aqueous supernatants already after 0.5 min. CONCLUSIONS: Group 13 allergens represent one set of marker allergens which specifically occur in pollen of the major grass subfamilies and are rapidly released in association with respirable particles after pollen hydration. They may be considered as environmental markers for grass pollen exposure and group 13-specific IgE antibodies as immunological markers for genuine grass pollen sensitization.

Allergens↗

Severe immediate allergic reactions to grapes: part of a lipid transfer protein-associated clinical syndrome.

BACKGROUND: Grape allergy is considered rare; grape lipid transfer protein (LTP; Vit v 1), an endochitinase and a thaumatin-like protein (TLP) have been reported as grape allergens. A considerable number of patients have referred to our department for severe reactions to grapes, and several IgE binding proteins were detected. OBJECTIVES: The aim of this study was to identify and characterise the allergens involved in severe allergic reactions to grapes and describe the population in which they occur. METHODS: Patients with reported severe allergic reactions to grapes (n = 37) are described. Grape allergens were purified/fractionated by a combination of chromatographic techniques, identified by proteomic analysis and biochemically characterised. Immunoreactivity was assessed by blot (inhibitions) and RAST (inhibitions), and skin prick tests were performed with the isolated allergens. RESULTS: All subjects were polyallergic, sensitised and reactive to several additional foods and pollen. All patients were sensitised to grape LTP. A 28-kDa expansin, a 37.5-kDa polygalacturonase-inhibiting protein, a 39-kDa beta-1,3-glucanase and a 60-kDa protein were identified as minor grape allergens. Endochitinase and TLP did not play a role. Inhibition experiments revealed the possible cross-reactive role of LTP for clinical sensitivities to other LTP-containing plant foods, but also the involvement of cross-reactive carbohydrate determinants of minor allergens in IgE cross-reactivity. CONCLUSIONS: LTP is the major grape allergen, while additional minor allergens may contribute to clinical reactivity. Severe grape allergy presents in atopic patients who frequently react to other LTP-containing, plant-derived foods. The 'LTP syndrome' is the appropriate term to describe this condition.

Adolescent↗

Cold-active pectinolytic activity of psychrophilic-basidiomycetous yeast Cystofilobasidium capitatum strain PPY-1.

A pectinolytic and psychrophilic yeast was isolated from soil from Abashiri, Hokkaido, Japan. The phenotype and sequencing of the 28S rDNA of the isolated strain (PPY-1) indicated a taxonomic affiliation to the basidiomycetous yeast Cystofilobasidium capitatum. C. capitatum strain PPY-1 was able to grow on two pectic compounds, polygalacturonate and pectin, at below 5 degrees C. Moreover, the extracellular fraction of the strain exhibited pectin methylesterase, pectin lyase and polygalacturonase activities at 5 degrees C. Thus strain PPY-1 may produce novel enzymes that are able to degrade pectin at low temperature, although the strain has isozymes of these enzymes.

Journal Article↗

Analysis of the molecular construction of xylogalacturonan isolated from soluble soybean polysaccharides.

Soluble soybean polysaccharides (SSPS) extracted from soybean cotyledons are acidic polysaccharides, and exhibited a pectin-like structure. After digesting galacturonan with polygalacturonase, two novel galacturonan (GN) fragments, which were directly linked to xylosyl oligosaccharides, were obtained. One consisted of (beta-D-Xyl)7 branched at the C-3 site of 1,4-linked (alpha-D-GalA)4, and the other consisted of (beta-D-Xyl)4 branched at the C-3 site of 1,4-linked (alpha-D-GalA)3.

Carbohydrate Sequence↗

Structural studies by stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan.

Soybean soluble polysaccharides (SSPS) extracted from soybean cotyledons are acidic polysaccharides and have a pectin-like structure. The results of a structural analysis of SSPS by using polygalacturonase (PGase) and rhamnogalacturonase (RGase) clarified that the main backbone consisted of galacturonan (GN) and rhamnogalacturonan (RG), which were composed of the diglycosyl repeating unit, -4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-. The side chains of beta-1,4-galactans, branched with fucose and arabinose residues, were linked to the C-4 side of rhamnose residues in the RG regions. The degree of polymerization (dps) of GN, which linked the RG regions together, was estimated to be about 4-10 residues, and some were modified with xylose residues on the C-3 side of the galacturonates. The dps of GN at the reducing end of SSPS was estimated to be about 7-9 residues. Moreover, the fragment of the basic structure of the RG region, -[4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-]2-, some of which had long-chain beta-1,4-galactans branched on the C-4 side of rhamnose residues, were liberated from SSPS by the RGase treatment. The dps of the galactan side chain was estimated to be about 43-47 residues by an analysis of the digestion products from the beta-galactosidase treatment.

Carbohydrate Conformation↗

Fermentations of pectin-rich biomass with recombinant bacteria to produce fuel ethanol.

Pectin-rich residues from sugar beet processing contain significant carbohydrates and insignificant amounts of lignin. Beet pulp was evaluated for conversion to ethanol using recombinant bacteria as biocatalysts. Hydrolysis of pectin-rich residues followed by ethanolic fermentations by yeasts has not been productive because galacturonic acid and arabinose are not fermentable to ethanol by these organisms. The three recombinant bacteria evaluated in this study, Escherichia coli strain KO11, Klebsiella oxytoca strain P2, and Erwinia chrysanthemi EC 16 pLOI 555, ferment carbohydrates in beet pulp with varying efficiencies. E. coli KO11 is able to convert pure galacturonic acid to ethanol with minimal acetate production. Using an enzyme loading of 10.5 filter paper units of cellulase, 120.4 polygalacturonase units of pectinase, and 6.4 g of cellobiase (per gram of dry wt sugar beet pulp), with substrate addition after 24 h of fermentation, 40 g of ethanol/L was produced. Other recombinants exhibited lower ethanol yields with increases in acetate and succinate production.

Alcohol Dehydrogenase↗

Variation in in vitro alpha-amylase and protease activity is related to the virulence of Aspergillus flavus isolates.

Variation in the ability of Aspergillus flavus isolates to spread between cotton boll locules was previously shown to be at least partially related to variation in the production of a specific polygalacturonase (pectinase P2C). To determine if non-pectolytic hydrolase differences between low- and high-virulence isolates exist and, thus, could also potentially contribute to virulence differences, the present investigation was undertaken. Two A. flavus isolates, AF12 with low virulence and lacking pectinase P2C and AF13 with high virulence and producing pectinase P2C, were compared for production of nonpectolytic hydrolases after growth in 10% potato dextrose broth. Activity of amylases, cellulases, xylanases, and proteases was quantified using the radial diffusion/cup plate technique followed by differential staining. Culture filtrates also were subjected to native polyacrylamide gel electrophoresis. Both isolates produced amylases, proteases, and xylanases, whereas cellulases were not detected for either. AF13 produced more amylase than AF12, and this difference was supported by amylase isoform differences between isolates on native polyacrylamide gel electrophoresis gels. AF13 also produced more protease than AF12; however, isoform differences between isolates were inconclusive. These variations in other hydrolytic activities (besides pectinases) may contribute to virulence differences in cotton bolls between AF12 and AF13.

Aspergillus flavus↗

Gene expression during adventitious root formation in apple.

A model system for adventitious root formation in woody plants was used to identify transcripts that are up-regulated during this process. 1 mm stem-disks from micropropagated shoots of the apple cultivar Jork 9 can be induced to form roots by treatment with the auxin indole butyric acid (IBA). Stem discs are placed on medium containing IBA for 24 hours and then transferred to IBA-free medium. Root initials become visible after 5-6 days and root elongation occurs within 7-9 days. The first visible cell divisions, which will give rise to the adventitious roots, are detectable 48 hours following IBA treatment. We have used this system to identify transcripts that are induced during adventitious root formation. Two techniques were employed in this analysis: differential messenger RNA display (DDRT) and mRNA representational difference analysis (RDA), a technique that couples PCR and subtractive hybridisation. Using both of these techniques a number of clones have been isolated that exhibit differential expression during auxin induced root formation. Both up-regulated and down-regulated transcripts have been identified. Expression of these genes has initially been verified by reverse northern blot analysis. Northern blot analysis with individual clones has confirmed the expression pattern observed in the reverse northern analysis. Tentative identities of some of the clones have been established by sequencing the partial cDNAs. Among the up-regulated transcripts are clones that share sequence homology with polygalacturonase and MAP kinases. A full-length cDNA for the most abundant up-regulated mRNA, a 2-oxoacid dependent dioxygenase, was characterised. This mRNA is expressed between 24 and 72 hours following IBA treatment of apple stem disks.

Amino Acid Sequence↗

Recombinant cypress allergens.

Cedar/cypress/juniper pollen allergy is probably one of the most common worldwide pollen allergy. Purification, identification and characterization of allergens increased the knowledge about the IgE reactivity of allergic subjects toward defined allergenic components. Molecular biology give the opportunity to produce the allergens in virtually unlimited reproducible amount, to further characterize the IgE reactivity of the allergenic molecule, and finally how this reactivity can be modified for therapeutic purposes. The DNA recombinant technique has been applied to the study of cedar/cypress/juniper pollen allergens and several molecules have been cloned. These molecules can be divided in four groups depending on their biologic activity: he Pectate lyases (rCry j 1, rCha o 1, rJun a 1); the Polygalacturonase (rCry j 2, rCha o 2); the Thaumatin-like pathogenesis-related protein (rJun a 3); the Calcium-binding protein (rJun o 2). Main immunobiologic features of these cedar/cypress/juniper recombinant allergens will be summarized.

Allergens↗

Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate.

The systemic accumulation of both hydrogen peroxide (H(2)O(2)) and proteinase inhibitor proteins in tomato leaves in response to wounding was inhibited by the NADPH oxidase inhibitors diphenylene iodonium (DPI), imidazole, and pyridine. The expression of several defense genes in response to wounding, systemin, oligosaccharides, and methyl jasmonate also was inhibited by DPI. These genes, including those of four proteinase inhibitors and polyphenol oxidase, are expressed within 4 to 12 hr after wounding. However, DPI did not inhibit the wound-inducible expression of genes encoding prosystemin, lipoxygenase, and allene oxide synthase, which are associated with the octadecanoid signaling pathway and are expressed 0.5 to 2 hr after wounding. Accordingly, treatment of plants with the H(2)O(2)-generating enzyme glucose oxidase plus glucose resulted in the induction of only the later-expressed defensive genes and not the early-expressed signaling-related genes. H(2)O(2) was cytochemically detected in the cell walls of vascular parenchyma cells and spongy mesophyll cells within 4 hr after wounding of wild-type tomato leaves, but not earlier. The cumulative results suggest that active oxygen species are generated near cell walls of vascular bundle cells by oligogalacturonide fragments produced by wound-inducible polygalacturonase and that the resulting H(2)O(2) acts as a second messenger for the activation of defense genes in mesophyll cells. These data provide a rationale for the sequential, coordinated, and functional roles of systemin, jasmonic acid, oligogalacturonides, and H(2)O(2) signals for systemic signaling in tomato plants in response to wounding.

Acetates↗

Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants.

Excessive softening is the main factor limiting fruit shelf life and storage. Transgenic plants modified in the expression of cell wall modifying proteins have been used to investigate the role of particular activities in fruit softening during ripening, and in the manufacture of processed fruit products. Transgenic experiments show that polygalacturonase (PG) activity is largely responsible for pectin depolymerization and solubilization, but that PG-mediated pectin depolymerization requires pectin to be de-methyl-esterified by pectin methylesterase (PME), and that the PG beta-subunit protein plays a role in limiting pectin solubilization. Suppression of PG activity only slightly reduces fruit softening (but extends fruit shelf life), suppression of PME activity does not affect firmness during normal ripening, and suppression of beta-subunit protein accumulation increases softening. All these pectin-modifying proteins affect the integrity of the middle lamella, which controls cell-to-cell adhesion and thus influences fruit texture. Diminished accumulation of either PG or PME activity considerably increases the viscosity of tomato juice or paste, which is correlated with reduced polyuronide depolymerization during processing. In contrast, suppression of beta-galactosidase activity early in ripening significantly reduces fruit softening, suggesting that the removal of pectic galactan side-chains is an important factor in the cell wall changes leading to ripening-related firmness loss. Suppression or overexpression of endo-(1-->4)beta-D-glucanase activity has no detectable effect on fruit softening or the depolymerization of matrix glycans, and neither the substrate nor the function for this enzyme has been determined. The role of xyloglucan endotransglycosylase activity in softening is also obscure, and the activity responsible for xyloglucan depolymerization during ripening, a major contributor to softening, has not yet been identified. However, ripening-related expansin protein abundance is directly correlated with fruit softening and has additional indirect effects on pectin depolymerization, showing that this protein is intimately involved in the softening process. Transgenic work has shown that the cell wall changes leading to fruit softening and textural changes are complex, and involve the coordinated and interdependent activities of a range of cell wall-modifying proteins. It is suggested that the cell wall changes caused early in ripening by the activities of some enzymes, notably beta-galactosidase and ripening-related expansin, may restrict or control the activities of other ripening-related enzymes necessary for the fruit softening process.

Cell Wall↗