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PubMed1998

Conditional mutations in OutE and OutL block exoenzyme secretion across the Erwinia carotovora outer membrane.

The phytopathogen Erwinia carotovora subspecies carotovora secretes pectinases and cellulase via the general secretory pathway, a process requiring at least 13 proteins encoded by the out gene cluster. By exploiting delta::Tn5, a generalised transducing phage (psi KP) and localised mutagenesis of the out gene cluster, we have produced a histidine auxotroph and 19 new secretory mutants, including two (HJN1003 and HJN1004) which were conditional (temperature sensitive) for secretion. All of the mutants accumulated pectinases and cellulase in the periplasm, but in the case of HJN1003 and HJN1004, only at the restrictive temperature. HJN1003 and HJN1004 were complemented by the outE and outL wild-type genes, respectively, and both mutant alleles were cloned and sequenced to reveal single missense substitutions. HJN1003 carries an Arg166 to His alteration in OutE and HJN1004 carries a Pro159 to Leu alteration in OutL. Topology mapping of OutL using a beta-lactamase probe confirmed that OutL is a type II bitopic trans-inner membrane protein and that the mutated Pro159 residue in HJN1004 is located in the cytoplasmic domain of OutL. Hence, the secretion of exoenzymes across the outer membrane is critically dependent on the conformation of secretory components located at the cytoplasmic face of the inner membrane.

Bacterial Outer Membrane Proteins↗
PubMed2003

Impala, a transposon from Fusarium oxysporum, is active in the genome of Penicillium griseoroseum.

An autonomous impala transposon trapped in Fusarium oxysporum by insertion within the niaD gene encoding nitrate reductase was introduced in the genome of the fungus Penicillium griseoroseum, a producer of pectinase enzymes. Through a phenotypic assay, we demonstrate that this element is able to excise from the niaD gene and to reinsert at new genomic positions. As in the original host, impala inserts into a TA site and footprints left by impala excisions are generally 5 bp. The fact that impala is able to transpose in P. griseoroseum offers the opportunity to develop a gene-tagging system based on this element with the objective to detect and clone genes related in pectinase production.

Blotting, Southern↗
PubMed1997

Identification and retting efficiencies of fungi isolated from dew-retted flax in the United States and europe.

Seven strains of filamentous fungi and one yeast were isolated from flax that was dew retted in the United States. These filamentous fungi were subcultured to purity and identified, and six appear not to have been reported earlier as isolates from dew-retted flax. Five of the purified U.S. strains, two fungi isolated from flax that was dew retted in Europe, and a laboratory culture of Aspergillus sojae were tested for their ability to ret flax stems. The monocultures were evaluated for the degree of retting, fiber strength, dry weight loss, and tactile response (i.e., feel of softness) as reflected in the retted fiber. Structural modifications of representative samples of the retted flax were assessed by scanning electron microscopy. All of the filamentous fungi were able to carry out some retting, whereas the isolated yeast could not. All organisms produced pectinases when they were cultivated in shake flasks on ball-milled flax as the sole carbon source. Some fungi also produced cellulases, mannanases, and xylanases. Rhizomucor pusillus and Fusarium lateritium were noteworthy as retting organisms by their high level of pectinase activity, ability to attack noncellulosic cell types without attacking cellulose, capacity to penetrate the cuticular surface of the stem, and efficient fiber release from the core. The results indicated that these organisms deserve further study as potential organisms for retting of bast fibers in industrial applications.

Journal Article↗
PubMed1999

The exopolygalacturonate lyase PelW and the oligogalacturonate lyase Ogl, two cytoplasmic enzymes of pectin catabolism in Erwinia chrysanthemi 3937.

Erwinia chrysanthemi 3937 secretes into the external medium several pectinolytic enzymes, among which are eight isoenzymes of the endo-cleaving pectate lyases: PelA, PelB, PelC, PelD, and PelE (family 1); PelI (family 4); PelL (family 3); and PelZ (family 5). In addition, one exo-cleaving pectate lyase, PelX (family 3), has been found in the periplasm of E. chrysanthemi. The E. chrysanthemi 3937 gene kdgC has been shown to exhibit a high degree of similarity to the genes pelY of Yersinia pseudotuberculosis and pelB of Erwinia carotovora, which encode family 2 pectate lyases. However, no pectinolytic activity has been assigned to the KdgC protein. After verification of the corresponding nucleotide sequence, we cloned a longer DNA fragment and showed that this gene encodes a 553-amino-acid protein exhibiting an exo-cleaving pectate lyase activity. Thus, the kdgC gene was renamed pelW. PelW catalyzes the formation of unsaturated digalacturonates from polygalacturonate or short oligogalacturonates. PelW is located in the bacterial cytoplasm. In this compartment, PelW action could complete the degradation of pectic oligomers that was initiated by the extracellular or periplasmic pectinases and precede the action of the cytoplasmic oligogalacturonate lyase, Ogl. Both cytoplasmic pectinases, PelW and Ogl, seem to act in sequence during oligogalacturonate depolymerization, since oligomers longer than dimers are very poor substrates for Ogl but are good substrates for PelW. The estimated number of binding subsites for PelW is three, extending from subsite -2 to +1, while it is probably two for Ogl, extending from subsite -1 to +1. The activities of the two cytoplasmic lyases, PelW and Ogl, are dependent on the presence of divalent cations, since both enzymes are inhibited by EDTA. In contrast to the extracellular pectate lyases, Ca2+ is unable to restore the activity of PelW or Ogl, while several other cations, including Co2+, Mn2+, and Ni2+, can activate both cytoplasmic lyases.

Amino Acid Sequence↗
PubMed1999

Regulation of pelD and pelE, encoding major alkaline pectate lyases in Erwinia chrysanthemi: involvement of the main transcriptional factors.

The main virulence factors of the phytopathogenic bacterium Erwinia chrysanthemi are pectinases which attack pectin, the major constituent of the plant cell wall. Of these enzymes, the alkaline isoenzyme named PelD in strain 3937 and PelE in strain EC16 has been described as being particularly important, based on virulence studies of plants. Expression of the pelD and pelE genes is tightly modulated by various regulators, including the KdgR repressor and the cyclic AMP-cyclic AMP receptor protein (CRP) activator complex. The use of a lacZ reporter gene allowed us to quantify the repression of E. chrysanthemi 3937 pelD expression exerted by PecS, another repressor of pectinase synthesis. In vitro DNA-protein interaction experiments, centered on the pelD and pelE wild-type or pelE mutated promoter regions, allowed us to define precisely the sequences involved in the binding of these three regulators and of RNA polymerase (RNAP). These studies revealed an unusual binding of the KdgR repressor and suggested the presence of a UP (upstream) element in the pelD and pelE genes. Investigation of the simultaneous binding of CRP, KdgR, PecS, and the RNAP to the regulatory region of the pelD and pelE genes showed that (i) CRP and RNAP bind cooperatively, (ii) PecS partially inhibits binding of the CRP activator and of the CRP-RNAP complex, and (iii) KdgR stabilizes the binding of PecS and prevents transcriptional initiation by RNAP. Taken together, our data suggest that PecS attenuates pelD and pelE expression rather than acting as a true repressor like KdgR. Overall, control of the pelD and pelE genes of E. chrysanthemi appears to be both complex and novel.

Alkalies↗
PubMed2004

Characterization, regulation, and phylogenetic analyses of the Penicillium griseoroseum nitrate reductase gene and its use as selection marker for homologous transformation.

Penicillium griseoroseum has been studied because of its efficient pectinases production. In this work, the Penicillium griseoroseum nitrate reductase gene was characterized, transcriptionally analyzed in different nitrogen sources, and used to create a phylogenetic tree and to develop a homologous transformation system. The regulatory region contained consensus signals involved in nitrogen metabolism and the structural region was possibly interrupted by 6 introns coding for a deduced protein with 864 amino acids. RT-PCR analysis revealed high amounts of niaD transcript in the presence of nitrate. Transcription was repressed by ammonium, urea, and glutamine showing an efficient turnover of the niaD mRNA. Phylogenetics analysis showed distinct groups clearly separated in accordance with the classical taxonomy. A mutant with a 122-bp deletion was used in homologous transformation experiments and showed a transformation frequency of 14 transformants/microg DNA. All analyzed transformants showed that both single- and double-crossover recombination occurred at the niaD locus. The establishment of this homologous transformation system is an essential step for the improvement of pectinase production in Penicillium griseoroseum.

Amino Acid Sequence↗
PubMed2006

Molecular characterization and expression profile of pectin-lyase-encoding genes from Penicillium griseoroseum.

Penicillium griseoroseum has been studied by our group because of its good pectinase production. Attempts have been done to clone pectinolytic genes, aiming to obtain pectinase-overproducing strains for industrial purposes. Here, two genes coding for pectin lyase were isolated from the P. griseoroseum genome. The plg1 gene has an open reading frame of 1341 bp coding for a putative protein of 374 amino acids with a calculated molecular mass of 40.1 kDa. The plg2 gene is characterized by an open reading frame of 1400 nucleotides and codes for a polypeptide of 383 amino acids. The plg1 gene 5'-flanking region contains putative binding sites for the transcription factors involved in regulation by ambient pH and catabolite repression. The primary structure of Plg1 and Plg2 proteins showed a relatively high homology (varying between 32.4% and 74.8%) to fungal pectin lyases characterized to date. Southern blotting analysis revealed that both genes are present as single copies in the fungus genome. Expression studies revealed a differing pattern of gene expression of plg1 and plg2 when mycelium was cultivated on medium containing different pectic components. Citric pectin followed by apple pectin were the carbon sources that best induced plg1 expression, and transcripts were detected from 24 to 76 h. The expression of the plg2 gene was monitored by reverse transcriptase - polymerase chain reaction, since Northern analysis failed to detect hybridization signals. The differential expression of these genes may provide means for the fungus to adapt to various growth conditions.

Amino Acid Sequence↗
PubMed2004

Functional analysis of CLPT1, a Rab/GTPase required for protein secretion and pathogenesis in the plant fungal pathogen Colletotrichum 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.

Alleles↗
PubMed1983

Biochemical and physicochemical characterization of a mitogen obtained from an oriental crude drug, Tohki (Angelica actiloba Kitagawa).

Chemical composition and physicochemical properties of an immunomodulator, which is a non-dialyzable and acetone precipitable material(s) extracted with hot water from Angelica actiloba KITAGAWA (Yamato Tohki) (AIP), were investigated. AIP was composed of about 90% sugar and 10% protein. The major polysaccharide was identified as pectic substance(s) because its main component sugars were found to be arabinose, glucose, and galacturonic acid by gas liquid chromatographic analysis. The pectic substance(s) was not concerned with the mitogenicity of AIP since the activity was similar before and after pectinase(endo-polygalacturonase) treatment. More than half of the mitogenicity was destroyed by acid or alkali treatment. With pronase treatment, the activity was not affected, but the molecular weight of the mitogen was lowered. In addition, the mitogenic substance was partially purified from AIP by pectinase treatment and Westphal's phenol/water fractionation. The partially purified mitogenic substance(s) was rich in protein. These facts suggest that the mitogenicity of AIP was carried by a heat stable and protease resistant protein.

Amino Acids↗
PubMed2002

Relationship between conidial enzymes and germination of the apple blue mold, Penicillium expansum.

The relationship between conidial enzymes of Penicillium expansum and spore germination was examined. The activities of xylanase and pectinase, but not of cellulase and amylase, were detected in the conidia. The levels of xylanase and pectinase were greatly enhanced by xylan and pectin as respective carbon sources in the basal medium. No conidia germinated in the basal medium without a carbon source. The type of carbon source and the enzyme levels of the conidia did not affect the rate of germination. However, a relationship was found between the enzyme levels and the elongation of the germ tubes.

Penicillium↗
PubMed2004

Dietary supplementation with multienzyme preparations improves nutrient utilization and growth performance in weaned pigs.

Two experiments with young pigs (25 d of age) were conducted to investigate the effect of multienzyme preparations on nutrient digestibility, growth performance, and P utilization and excretion. In Exp. 1, 24 pigs (six pigs per treatment) were used in a 28-d performance and digestibility trial using four diets: control (no enzyme) and control supplemented with enzyme preparation A, B, or C. The control diet was formulated to meet 95% of NRC (1998) nutrient specifications (except for available P, which was at 44% NRC) and composed of corn, wheat, wheat by-products, barley, soybean meal, canola meal, and peas. All three enzyme preparations contained xylanase, glucanase, amylase, protease, invertase, and phytase activities and differed in the type of plant cell wall-degrading activities; Enzyme A contained cellulase, galactanase, and mannanase; Enzyme B contained cellulase and pectinase; and Enzyme C contained cellulase, galactanase, mannanase, and pectinase. Pigs fed enzyme-supplemented diets had higher ADG (P = 0.02) and G:F (P = 0.01) than those fed the control diet. On average, and when compared with control diet, enzyme supplementation improved (P = 0.001 to 0.04) ileal digestibility of DM (60 vs. 66%), GE (62.8 vs. 70.4%), CP (62 vs. 72%), starch (86.7 vs. 94.2%), nonstarch polysaccharides (NSP; 10.1 vs. 17.6%), and phytate (59 vs. 70%). Compared with the control, total-tract digestibility of nutrients was increased (P = 0.001 to 0.01) owing to enzyme supplementation, with Enzyme C showing the highest improvement in DM, GE, CP, starch, NSP, phytate, and P utilization. Pigs fed enzyme-supplemented diets had decreased (P = 0.04) fecal P excretion. The benefit from improved nutrient utilization with enzyme supplementation was further substantiated in a 38-d growth performance study with 48 pigs. The control and Enzyme C-supplemented diets (same as Exp. 1) were assigned to six replicate pens (four pigs per pen). The study was conducted in three phases (Phase 1 = d 0 to 7; Phase 2 = d 7 to 21; Phase 3 = d 21 to 38). Individual BW and pen feed disappearance were monitored. Average daily gain and G:F were 231 and 257 g (P = 0.01), and 0.56 and 0.63 (P = 0.001) for the control and enzyme-supplemented diets, respectively. It is evident from this study that the use of enzyme preparations may allow for cost-effective and environmentally friendly formulation of young pig diets.

Animal Feed↗
PubMed2000

Enzyme, bacterial inoculant, and formic acid effects on silage composition of orchardgrass and alfalfa.

We evaluated the effects of cellulase (from Trichoderma longibrachiatum) application rates on neutral detergent fiber (NDF) concentration and fermentation products of orchardgrass (Dactylis glomerata L.) and alfalfa (Medicago sativa L.) silages harvested with decreasing dry matter (DM) digestibility. Additionally, the impacts of inoculant (Lactobacillus plantarum and Pediococcus cerevisiae), pectinase (from Aspergillus niger), or formic acid on silage composition were studied. Forages wilted to a DM content of about 320 g/kg were ensiled in laboratory silos for 60 d. Cellulase, combined with inoculant, was applied at 2, 10, and 20 ml/kg of herbage (at least 2500 IU/ml). Cellulase at 10 ml/kg was also applied alone or in combination with pectinase and inoculant or formic acid. The NDF concentration of orchardgrass silage decreased with increasing cellulase up to 20 ml/kg, at which NDF content was decreased by 30%. The NDF concentration of alfalfa silage decreased with increasing cellulase application up to 10 ml/kg, at which NDF content was decreased by 13%. Immature plants were more responsive to cellulase treatment than mature plants. Cellulase at 2 ml/kg combined with inoculant improved fermentation characteristics of the silages but generally, there was no effect on silage fermentation by higher cellulase applications, resulting in an accumulation of sugar. The improved fermentation of orchardgrass treated with cellulase and inoculant was mostly related to the effect of inoculant, whereas cellulase alone improved fermentation characteristics of alfalfa silage and this effect was enhanced by addition of inoculant. Decreased NDF and increased sugar concentrations did not improve the in vitro DM digestibility of cellulase-treated silages.

Aspergillus niger↗
PubMed1981

Effect of dietary cereal grain, citrus pectin, and guar gum on liver fat in laying hens and young chicks.

One laying hen and three chick experiments were conducted to study the effect of cereal grains, citrus pectin, and guar gum on liver fat of chicks and hens and on serum cholesterol of chicks. Feeding of wheat, rye, or 2% pectin in place of corn reduced liver fat content. The decrease in liver fat of hens fed wheat or rye was larger than that of those fed 2% pectin (P less than .05). The smallest amount of liver fat was found in livers of hens fed the rye diet. Wheat or pectin did not affect egg production of egg weight, but rye caused a significant decline in egg production in comparison with other treatments. Addition of 4% pectin or 2% guar gum to the corn control diet lowered liver fat, serum cholesterol (P less than .05), and body weight in chicks (P less than .05). Penicillin added to the diets containing 2% guar gum gave an increase in body weight (P less than .05) but had no effect on body weight of chicks fed diets containing 4% pectin. In contrast, pectinase prevented growth depression by pectin and guar gum. Pectinase also increased liver fat and serum cholesterol of birds fed the diet containing pectin or guar gum. When the corn control diet was pair-fed to the pectin diet, growth of the pair-fed chicks was better than that of the pectin-fed birds but was lower than that of birds fed the control diet ad libitium. Pectin-fed birds again had lower liver fat the serum cholesterol than the control birds. The reduced feed intake of chicks pair-fed the corn control diet had no effect on liver fat content.

Adipose Tissue↗
PubMed2002

A useful protocol for in situ RT-PCR on plant tissues.

This study describes an effective method of in situ RT-PCR (RT-ISPCR) that was developed to localize gene expression in plant tissues. This RT-PCR technique was performed on sectioned tissues of female buds of the cucumber GY3 inbred line. The CUS1 gene, encoding the MADS-box type (agamus-like) protein, the expression pattern of which was described earlier, was used as a marker gene for optimisation of steps in the in situ RT-PCR inside the cells. For the identification of RT-PCR products inside the cells of the female buds, they were fixed in FAA solution, embedded in Paraplast Plus and cut into 7 microm thick sections which were dewaxed by immersion in HistoClear and dehydrated with ethanol. They were washed in water, then in 0.02M HCl, 2xSSC and PBS buffer. In the next step of tissue pretreatment, the sections were digested with 1% pectinase. As shown, the pectinase treatment proved to be a crucial step in the tissue preparation procedure to get successful RT-PCR products. After washing in PBS buffer, the sections were digested with protease K followed by incubation with RNase-free DNase I, and subsequently washed in 2xSSC, 1xSSC and 0.5xSSC and finally in DEPC-treated water. Then the sections were covered with 50 microl of the RT-PCR reaction mixture supplemented with 0.5 microM digoxigenin dUTP and sealed with a coverslip. After amplification in situ the PCR products were identified with anti-digoxigenin antibody (Roche Molecular Biochemicals), conjugated with alkaline phosphatase. The data obtained showed that specific signals reflecting CUS1 gene expression were detected in the female flower buds of cucumber. The specificity of the in situ RT-PCR protocol was confirmed by dot blot hybridization of RT-PCR products with CUS1 cDNA probe.

Cucumis sativus↗
PubMed2002

Effect of leachate recycling and inoculation on the biochemical characteristics of municipal refuse in landfill bioreactors.

Activity development of key groups of enzymes involved in municipal refuse decomposition was measured in laboratory landfill bioreactors with and without leachate recycling and inoculation for about 210 days. The results showed that the enzymes (amylase, protease, cellulase, lipase and pectinase) were present in fresh refuse but at low values and positively affected by leachate recycling and refuse inoculation. The total average of cellulase activity in digesters D3 operated with leachate recycling but no inoculation, D4 and D5 operated with leachate recycling and inoculation was much higher than that in digesters D1 and D2 without leachate recycling and inoculation by 88%-127%, 117%-162% and 64%-98%. The total average of protease activity was higher in digester D4 than that in digesters D1, D2, D3 and D5 by 63%, 39%, 24% and 24%, respectively, and the positive effect of leachate recycling and inoculation on protease activity of landfilled refuse mainly was at the first two months. The total average of amylase activity was higher in digesters D3, D4 and D5 than that in digesters D1 and D2 by 83%-132%, 96%-148% and 81%-129%. During the early phase of incubation, the stimulatory effect of inoculation on lipase activity was measured, but refuse moisture was the main factor affecting lipase activity of landfilled refuse. The inoculation, initial and continuous inoculation of microorganisms existing in leachate, was the mainly stimulatory factor affecting pectinase activity of landfilled refuse.

Bioreactors↗
PubMed1968

Cell wall dissolution and enzyme secretion during leaf abscission.

Cell wall changes leading to the formation of the separation layer during abscission of unifoliate (Phaseolus vulgaris) leaves are reviewed. Based on evidence from explants and intact plants, dissolution of pectic substances between cells of adjacent tissue regions (petiole and pulvinus) is necessary and may be sufficient to form the separation layer. Initially, the abscission zone is not structurally weak. The decline in break strength accompanying formation of the separation layer correlates with the appearance of pectinase activity. Pectinase activity is not detectable in freshly harvested explants but increases to about 0.09 mug per abscission zone at the time of 50% separation. At the same time, water extractable pectin fractions increase with a corresponding decline in the pectin fraction extractable with dilute acid. Separation is aided by internal shear forces generated by differential growth and hydrostatic pressure or both.

Journal Article↗
PubMed1991

Improvement of the biodegradation of some cellulosic wastes by acid pretreatment.

Acid pretreatment of cellulosic wastes improved their susceptibility to Fusarium acuminatum enzymes. The effectiveness of acid pretreatment was demonstrated with an increase in both fungal growth and enzyme activities. A growth yield of 0.15 g/100 ml was achieved on medium containing 5% acid pretreated pods of bean for 60 minutes. Avicelase (C1), carboxymethylcellulase (Cx) and B-glucosidase (C2) reached their maximal biosynthesis on acid pretreated wheat bran, sugar-cane bagasse and sawdust-containing media, respectively. Xylanase and pectinase attained their highest accumulation on pretreated pods of bean media. A mixture of free sugars has been released by acid pretreatment. O.199 g dry mycelium was obtained when the fungus was grown on 100 ml of medium containing hydrolysate of 10% H2SO4 pretreated pods of bean for 30 min. No cellulase enzymes could be detected on hydrolysate medium at the time that low contents of both xylanase and pectinase were accumulated.

Biodegradation, Environmental↗
PubMedDate not supplied

[Semicontinuous cultivation of fungi of the genus Aspergillus, producers of hydrolases].

The production of exohydrolases (alpha-amylase and pectinase) by fungi belonging to the genus Aspergillus was studied in the course of batch cultivation and, if immobilized cells were used, in the semicontinuous regime of growth. The cells were immobilized on a fixed filtering plate and on floating, in the growth medium, polyhedrons. Such a cultivation of immobilized microbial cells in the semicontinuous regime of growth on submerged polyhedrons freely floating in the nutrient medium makes it possible to cultivate the cells for 1.5 months with the active production of exocellular hydrolases. Under these conditions, Aspergillus oryzae 3-9-15 produces more alpha-amylase and A. awamori synthesizes more pectinases.

Aspergillus↗