Pectin Medium Research Articles

Inducible production of alcohol oxidase and catalase in a pectin medium by Thermoascus aurantiacus IFO 31693

Thermoascus aurantiacus showed the best growth on medium containing pectin as a carbon source. The enzyme involved in the production of catalase in the fungus was alcohol oxidase. Formaldehyde dehydrogenase and formate dehydrogenase, in addition to alcohol oxidase and catalase, were detected in the cells grown on pectin. Alcohol oxidase was alkali resistant (pH 7 to 11), and was comparatively heat stable (55 degrees C).

Functional analysis ofCLPT1, a Rab/GTPase required for protein secretion and pathogenesis in the plant fungal pathogenColletotrichum lindemuthianum

In eukaryotic cells, Rab/GTPases are major regulators of vesicular trafficking and are involved in essential processes including exocytosis, endocytosis and cellular differentiation. To investigate the role of these proteins in fungal pathogenicity, a dominant-negative mutant allele of CLPT1, a Rab/GTPase of the bean pathogen Colletotrichum lindemuthianum, was expressed in transgenic strains. This mutated gene encodes the amino-acid substitution N123I analogous to the N133I substitution in a known trans-dominant inhibitor of the Sec4 Rab/GTPase from Saccharomyces cerevisiae. A pectinase gene promoter was used to drive the CLPT1(N123I) allele in C. lindemuthianum, allowing the expression of the foreign gene on pectin medium and during pathogenesis, but not on glucose. The same strategy was used to overexpress the wild-type CLPT1 allele. During growth on pectin medium, production of extracellular pectinases was strongly impaired only in CLPT1(N123I)-expressing strains. Cytological analysis revealed that CLPT1(N123I) strains accumulated intracellular aggregates only on pectin, resulting from the fusion of vesicles containing polysaccharides or glycoproteins. Moreover, these strains showed a severe reduction of pathogenesis and were unable to penetrate the host cells. These results indicated that the Rab/GTPase CLPT1 is essential for fungal pathogenesis by regulating the intracellular transport of secretory vesicles involved in the delivery of proteins to the extracellular medium and differentiation of infectious structures.

Cloning and characterization of a gene rpg1 encoding polygalacturonase of Rhizopus oryzae

The polygalacturonase (PG)-encoding gene (rpg1) of Rhizopus oryzae, the causal pathogen of rhizopus rot of mulberry, was cloned and sequenced. PGs were partially purified from incubation mixture of 2% pectin medium and their N-terminal amino acid sequences were determined by a gas-phase protein sequencer. RT-PCR was performed using degenerate primers designed from the amino acid sequences, which resulted in part of a PG-encoding gene being obtained. By 3'-RACE and TAIL-PCR analyses, the entire region of the PG-encoding gene was cloned and sequenced. The structural gene comprised 1199 bp coding for 383 amino acids with a putative signal peptide of 26 amino acids, and the open reading frame was interrupted by single intron of 47 bp. Phylogenetic analysis using the deduced amino acid sequence revealed that R. oryzae RPG1 belonged to a clade consisting of exo-PGs of ascomycete fungi.

Interaction of Sclerotinia sclerotiorum with a resistant Brassica napus cultivar: expressed sequence tag analysis identifies genes associated with fungal pathogenesis

Sclerotinia sclerotiorum is a ubiquitous necrotrophic fungal pathogen capable of infecting a wide range of plants. To identify genes involved in fungal development and pathogenesis we generated 2232 expressed sequence tags (ESTs) from two cDNA libraries constructed using either mycelia grown in pectin medium or tissues from infected Brassica napus stems. A total of 774 individual fungal genes were identified of which 39 were represented only among the infected plant EST collection. Annotation of 534 unigenes was possible following the categories applied to Saccharomyces cerevisiae and the Universal Gene Ontology scheme. cDNAs were identified that encoded potential pathogenicity factors including four endopolygalacturonases, two exopolygalacturonases, and several metabolite transporters. The potential role of these genes, as well as those encoding signal transduction factors, in the infection process is discussed. Index Descriptors: Sclerotinia sclerotiorum; cDNA library; Expressed sequence tags; Pathogenesis; Fungal development; Signal transduction

Germination of spore and decomposition of apple fruit tissue by hypha in Penicillium expansum

On Penicillium expansum, germination of the spore (conidia), growth using fruit components, production of various enzymes and enzyme action for apple fruit were examined. Xylan- and pectin-degrading enzymes were easily liberated in the buffer solution from the spore. The levels of these enzyme activities were largely affected by carbon sources in the sporogenesis culture medium. The spore harvested from the culture of the xylan medium showed the high xylanase activity and the spore from the pectin medium showed the high pectinase activity. The carbon source of germination medium and the enzyme level of the spore were related to the elongation of germ tube. The mold grew well in the medium containing xylan or pectin, and abundantly formed the spore. Unlike this, its growth using soluble sugars (glucose, fructose, sucrose) was poor. The mold produced gluconate from glucose, and glucose oxidase and catalase were produced in the culture broth. The fruit tissue remarkably browned, when it was incubated with glucose oxidase and catalase. This browning reaction was promoted in the presence of xylan-degrading enzyme. Two xylanases and β-xylosidase were purified from the culture filtrate as homogeneous proteins. The reducing sugar was liberated from the fruit when it was incubated with these enzymes.

Production of a cell wall-associated endopolygalacturonase by Colletotrichum lindemuthianum and pectin degradation during bean infection

The bean pathogen Colletotrichum lindemuthianum expresses two endopolygalacturonase genes, CLPG1 and CLPG2, during interaction with its host plant. However, only CLPG1 was found to be secreted to the extracellular medium during saprophytic growth of the fungus on pectin. To localize CLPG2, a FLAG epitope sequence was inserted in the C-terminal sequence of CLPG2 and the modified gene was introduced into C. lindemuthianum. Western blot analysis using a FLAG monoclonal antibody allowed the detection of CLPG2 in intracellular protein extracts and in the cell wall fraction, but not in the culture medium. Indirect immunofluorescence microscopy was performed to detect CLPG2 during saprophytic or parasitic growth. According to the expression pattern of CLPG2, it was found that CLPG2 accumulates in the fungal cell wall during growth on pectin medium and during appressorium formation, both in vitro and during interaction with the plant. Pectin degradation was not detected around the infection peg using the monoclonal antibody JIM7, specific for methyl-esterified galacturonan. However, extensive pectin dissolution was observed during the development of secondary hyphae.

Disruption of Botrytis cinerea pectin methylesterase gene Bcpme1 reduces virulence on several host plants.

The pectinolytic enzyme pectin methylesterase (PME) hydrolyses pectin in methanol and polygalacturonic acid. In the expressed sequence tag library of Botrytis cinerea T4, we identified a 1,041 bp Bcpme1 cDNA potentially encoding a 346-amino acid protein of 37 kDa showing 46.8% identity with Aspergillus sp. PMEs. Bcpme1 is a single copy gene and is similarly expressed in glucose and pectin containing media. To evaluate the role of Bcpme1 in Botrytis cinerea virulence, a mutant in Bcpme1 was generated by gene disruption. The Bcpme1 mutant showed similar growth on rich medium but reduced growth on pectin medium. Two isozymes of pI 7.4 and 7.1 were detected in pectin liquid-culture supernatants of wild-type strain Bd90 analyzed by isoelectric focusing-polyacrylamide gel electrophoresis, while those of Bcpme1 mutant possessed only the pI 7.1 isozyme. BCPME1, the pI 7.4 isozyme, is the major PME activity, as PME activity is 75% reduced in Bcpme1 mutant. Moreover, the Bcpme1 mutant was less virulent on apple fruits, grapevine, and Arabidopsis thaliana leaves. Those phenotypes were complemented by reintroducing a Bcpme1 copy in the Bcpme1 mutant. These results showed that B. cinerea possessed more than one PME-encoding gene and that BCPME1 is an important determinant of B. cinerea virulence.

Pectic Enzymes Produced In vitro and In vivo by Erwinia spp. Isolated from Carrot and Pepper in Egypt

AbstractThirty soft‐rot Erwinia strains were isolated from diseased carrot (Daucus carrota) roots and pepper (Capsicum annuum) fruits. Pathogenicity tests showed that these strains are able to induce soft‐rot disease symptoms in different vegetable species indicating a wide host range. Although pectin methyl esterase and polygalacturonase activities were not detected in culture supernatants, they were found in extracts of carrot roots and pepper fruits infected with three strains representing three soft‐rot Erwinias. On the other hand, culture supernatants and extracts of infected plant tissues contained pectic lyase activity. The enzyme from either sodium polypectate or pectin medium degraded the former more actively than the latter indicating pectate rather than pectin lyase activity. In contrast, the enzyme produced in infected tissue showed higher activity on pectin than sodium polypectate. Moreover, the enzyme activity on pectin was not reduced in the absence of Ca2+, which might suggest that, in addition to pectate lyases, pectin lyases could be produced in plant tissues by Erwinia species.

CP/MAS 13C NMR and electron diffraction study of bacterial cellulose structure affected by cell wall polysaccharides

Acetobacter xylinum was cultured in Schramm–Hestrin medium containing pectin (pectin medium), xylan (xylan medium), or glucomannan (mannan medium). X-ray diffractometry revealed that xylan and glucomannan affected the size of the cellulose crystals and their d-spacing values. Solid-state cross polarization magic angle spinning carbon-13 nuclear magnetic resonance spectroscopy indicated that the ratio of cellulose Iα was reduced by the addition of polysaccharides. These effects were more remarkable on the cellulose in the mannan medium than that in the xylan medium, and were scarcely observed in the pectin medium. Electron diffraction analysis revealed that these effects on hemicelluloses along cellulose microfibrils are continuous in the mannan medium and discontinuous in the xylan medium. These findings suggest that the uronic acid in the polysaccharides prevents interactions with cellulose leading to alterations of the structure of the cellulose crystal.

Cellulose synthesized by Acetobacter xylinum in the presence of plant cell wall polysaccharides

Acetobacter xylinum was cultured in Schramm–Hestrin (SH) medium containing glucuronoxylan (xylan medium) or pectin (pectin medium). Loose bundles of cellulose microfibrils were formed in the xylan medium. In contrast the cellulose ribbons formed in the pectin medium were the same as those normally formed in SH medium.The periodic acidthiocarbohydrazidesilver proteinate method indicated that positive reacted substances located along cellulose microfibrils formed in both mediums. Freeze-fracture and deepetching electron microscopy also revealed that polysaccharides exist around cellulose microfibrils. X-ray diffractometry and Fourier Transform In-frared spectroscopy demonstrated that the addition of xylan induced a change in the ratio of cellulose Iα and Iβ. Electron diffraction analysis revealed that xylan discontinuously affected the crystalline structure of cellulose microfibrils. Pectin did not have this effect. Glucuronoxylan in the medium prevented the assembly of cellulose

Screening and genetic improvement of pectinolytic fungi for degumming of textile fibers

Aiming at contributing to technological improvements in plant fiber processing methods, this paper reports research work on the obtainment of more efficient pectinase-producing fungi strains. More specifically, this work reports the analysis of 18 strains of filamentous fungi, with the purpose of obtaining enzymes for textile fibers degumming. The strains were evaluated for production of pectinolytic enzymes under several growth conditions (culture medium and growth temperature). Production of pectinases was measured by an enzymatic index (EI) in solid pectin medium. Among the tested strains, Penicillium chrysogenum IFO 4626 (Q 176) showed the best performance. Genetic improvement of this strain was carried out to increase its pectinase production, while keeping cellulase activity down to a negligible level, since cellulases are known to decrease the resistance of the fiber. Variability was induced through several cycles of mutation and selection by exposing conidea to ultra-violet light (UV). We selected 39 out of 390 isolated colonies. Resulting mutants produced nine times more pectin lyase (PL) than the original strain in terms of PL specific activity, and five times more in terms of PL activity (i.e. mmoles liberated per minute of reaction per mL of medium). Periodically, mutant performance was evaluated in solid pectin medium. Genetic stability was maintained for four years after isolation.

Microbiological and biochemical study of coffee fermentation.

The coffee fermentation microflora were rich and mainly constituted of aerobic Gram-negative bacilli, with Erwinia and Klebsiella genuses at the highest frequencies. The best population increase was observed with lactic acid bacteria and yeasts, whereas those microorganisms that counted on a pectin medium remained constant during the fermentation step. Qualitatively, lactic acid bacteria belonged mainly to Leuconostoc mesenteroides species but the others microflora were relatively heterogeneous. The microorganisms isolated on pectin medium were Enterobacteriaceae, identified as Erwinia herbicola and Klebsiella pneumoniae, not reported as strong pectolytic strains. Throughout coffee fermentation, 60% of the simple sugars were degraded by the total microflora and not specifically by pectolytic microorganisms.

Pectinolytic and cellulolytic activities of heat resistant fungi and their macerating effects on mango and African mango

Five heat resistant fungi (HRF), Neosartorya fischeri, N fischeri var spinosa, N quadricincta, Paecilomyces varioti and Byssochlamys nivea, were studied for production of pectinolytic and cellulolytic activities. All isolates produced considerable hydrolase, lyase and pectinesterase activities. Hydrolase activities were significantly higher in fruit tissue (mango and African mango) media than in pectin medium (P < 0.01) when assayed by both cup plate and viscometric methods. Activities produced in both fruit media were comparable in N fischeri, N fischeri var spinosa and P varioti but not in N quadricincta and B nivea. All isolates produced greater lyase activities in pectin medium than in fruit tissue media except for N quadricincta, while the converse was the case for pectinesterase. P varioti did not utilise carboxymethyl cellulose or produce cellulase activity. Other isolates produced cellulase with B nivea producing the greatest activity. Each isolate caused considerable maceration of artificially inoculated mango and African mango fruits, which is not directly related to measurable pectinase or cellulase. The possibility of co-operation between pectinase and cellulase activities in the disintegration of fruit tissues is discussed. © 1999 Society of Chemical Industry

Carbon and nitrogen utilization and acid production by mycelia of the ectomycorrhizal fungus Tricholoma bakamatsutake in vitro

Mycelial growth of an isolate of T. bakamatsutake was tested in media with C/N ratio ranging from 0 to 50 and with 32 carbon and 12 nitrogen sources. The isolate grew best at the C/N ratio of 30. It utilized the monosaccharides d-glucose, d-mannose, and d-fructose, the disaccharide trehalose, and polysaccharide pectin among the carbon sources; and yeast extract, l-glutamic acid, and ammonium compounds among the nitrogen sources. The growth of ten isolates and secretion of gluconic and oxalic acids were compared in d-glucose, trehalose, and pectin media. The utilization of d-glucose, trehalose, and pectin differed among the ten isolates, but all the isolates secreted gluconic acid in the d-glucose media and oxalic acid in the pectin media.

Some Pectinolytic and Cellulolytic Enzyme Activities of Fungi Causing Rots of Cocoyams

Representative isolates of Aspergillus niger, Botryodiplodia theobromae, Corticium rolfsii, Geotrichum candidum, Fusarium oxysporum and F solani recovered from rotten cocoyams were studied for the production of pectinolytic and cellulolytic enzymes. All the isolates elaborated high levels of hydrolase, lyase and pectinesterase in cocoyam tissue medium and lyase and pectinesterase in pectin medium. There were no significant differences in the overall levels of lyase and pectinesterase activities produced by all the isolates in both media. The level of hydrolase, lyase and pectinesterase activities individually produced by A niger, B theobromae and C rolfsii in both media was significantly higher than that of any other isolate. The highest hydrolase activity was produced by C rolfsii in cocoyam medium while A niger produced the highest lyase activity in pectin medium. Maximum pectinesterase activity was obtained from B theobromae and C rolfsii in pectin medium. All the test isolates produced cellulase in a medium containing carboxymethyl cellulose with C rolfsii showing significantly high activity followed by F oxysporum and A niger. © 1997 SCI.

3-hydroxy fatty acids in a lipopolysaccharide-like material from Treponema denticola strain FM.

Treponemes are associated with major oral diseases such as apical and marginal periodontitis. Lipopolysaccharide (LPS) is regarded as an important virulence factor in these diseases. It is unclear whether LPS is present in oral treponemes. Therefore, the present study was undertaken to examine if a common oral treponeme-Treponema denticola-possesses LPS. A modified Westphal method (phenol water-ethanol-hexane extraction) was used to extract LPS-like material from T. denticola, reference strain FM. It was cultivated in prereduced anaerobically sterilized pectin medium. Gas chromatography and gas chromatography-mass spectrometry of the extract detected three hydroxy fatty acids: C3-OH-i-13:0, C3-OH-i-15:0, and C3-OH-16:0 which constituted 12% of its fatty acid content. These acids, commonly regarded as markers of LPS, suggested the presence of LPS in T. denticola.

A New Method for Routine Isolation of Oral Treponemes using U-Tubes and Pectin Medium

Determination of the composition of the oral microflora has traditionally been based on cultivation. Treponemes are prevalent in many oral infections but, unfortunately, are not regularly cultured. In this study a new method was established for routine isolation of oral treponemes from clinical samples. Bacterial samples from 47 periodontal pockets and 4 endodontic infections were incubated anaerobically under nitrogen atmosphere at 37°C in U-tubes containing pectin medium. In the U-tube a 'bacterial sample side' and a 'sterile medium side' were established on separate sides of a membrane filter and an agar plug. Using this method we were able to isolate viable treponemes from all bacterial samples. This was in contrast to previously established methods such as the agar dilution technique, the technique involving the membrane filter placed on the surface of solid agar media and the well in agar plate technique. We believe that the 'U-tube method' is a valuable supplement to previously described techniques in routine isolation of treponemes from clinical samples.

Capillary zone electrophoresis as a new tool in the chemotaxonomy of oral treponemes.

The existing taxonomy of oral treponemes is not satisfactory. This is due to the fact that these strict anaerobic bacteria are not easily cultivated or differentiated. Therefore, new techniques that can contribute to improved cultivation, classification, and identification of these fastidious organisms should be welcomed. In the present study capillary zone electrophoresis (CZE) was used to distinguish oral treponemes by their metabolic product patterns generated in a liquid medium. To our knowledge this technique has not previously been used in bacterial chemotaxonomy. Reference strains of Treponema denticola, Treponema pectinovorum, Treponema vincentii, Treponema socranskii subspecies buccale and Treponema socranskii subspecies socranskii were cultured anaerobically in duplicate on different days in Pectin medium for 4 days at 37 degrees C under nitrogen atmosphere. Treponemal cells were harvested by centrifugation. Thereafter, their supernatants were filtered through 0.22-micron Millipore filters and subjected to CZE. The resulting electropherograms clearly distinguished T. denticola, T. pectinovorum and T. vincentii. Minor differences were detected between T. socranskii subspecies buccale and T. socranskii subspecies socranskii. Subspecies were clearly different from species. It seems that CZE of culture metabolites, which showed high resolution and good reproducibility, may be a valuable tool in the chemotaxonomy of oral treponemes, even at the subspecies level.

Characterization of the Aspergillus niger pelB gene: structure and regulation of expression.

The nucleotide sequence of pelB, a member of the Aspergillus niger pectin lyase multigene family, has been determined. The pelB gene product, PLB, shares 65% amino acid identity with pectin lyase A (PLA) and 60% with pectin lyase D (PLD). Although growth of pelB multicopy transformants on pectin-containing media results in elevated pelB mRNA levels, pectin lyase B (PLB) is barely detectable. This is probably due to degradation of PLB by acid proteases, since multicopy transformants grown on pectin medium with a high concentration of phosphate, leading to a less rapid decline in pH, secrete detectable amounts of PLB. To produce PLB in high amounts under conditions where few other extracellular enzymes are present, we tried two strategies. Firstly, heterologous expression of the pelB gene in A. nidulans, and secondly, expression of the pelB gene under control of the constitutive A. niger pki promoter.

Pectinolytic activity and isozymes in European Armillaria species

Pectinolytic activities in Armillaria ostoyae, Armillaria mellea, Armillaria gallica, Armillaria borealis, and Armillaria cepistipes were assessed by measuring the ability of fungal strains to reduce the viscosity of their pectic growth media. Isozyme patterns of pectin esterases and polygalacturonases were determined directly from the culture filtrate. A total of 94 strains, representing isolations from various parts of Europe, were analyzed for their isozyme patterns. Armillaria mellea and A. borealis caused a 50% reduction in viscosity within 7 and 9 days, respectively. Growth medium from the other species were slower to reach the 50% level, i.e., means were 13 days for A. ostoyae and 17 days for A. cepistipes and A. gallica. All species produced more isozymes on spruce wood than on citrus pectin medium, and pectic isozyme patterns differed between media. The pectic isozyme pattern for A. mellea differed distinctly from those of the other four species by having two bands of polygalacturonase not found in the others. The pectic isozyme patterns of the other four species were separated using multivariate analysis. The value of such analyses for use in distinguishing between European Armillaria species is discussed, as is the relation between enzyme activity and fungal pathogenicity. Key words: root rot, diagnostic tests, Agaricales, polygalacturonase, pectin esterase.