Erwinia chrysanthemi EC16 Produces a Second Set of Plant-Inducible Pectate Lyase Isozymes

The enterobacterium Erwinia chrysanthemi causes soft-rot diseases involving extensive tissue maceration in a wide variety of plants and secretes multiple pectic enzymes that degrade plant cell walls and middle lamellae. An E. chrysanthemi mutant with directed deletions or insertions in genes pehX, pelX, pelA, pelB, pelC, and pelE, which encode exo-poly-alpha-d-galacturonosidase, exopolygalacturonate lyase, and four isozymes of pectate lyase, respectively, was constructed by the marker exchange of a cloned pehX::TnphoA fragment into E. chrysanthemi CUCPB5010, a Delta(pelA pelE) Delta(pelB pelC)::28bp Delta(pelX)Delta4bp derivative of strain EC16. This mutant, E. chrysanthemi CUCPB5012, no longer caused pitting in a standard pectate semisolid agar medium used to detect pectolytic activity in bacteria. Nevertheless, the mutant still macerated leaves of chrysanthemum (Chrysanthemum morifolium), although with reduced virulence. The mutant was found to produce significant pectate lyase activity in rotting chrysanthemum tissue and in minimal media containing chrysanthemum extracts or cell walls as the sole carbon source. Activity-stained, ultra-thin-layer isoelectric focusing gels revealed the presence in these preparations of several pectate lyase isozymes with pIs ranging from highly acidic to highly alkaline. Sterile culture fluids containing these isozymes were able to macerate chrysanthemum leaf tissue. Unlike the products of the pelA, pelB, pelC, and pelE genes in E. chrysanthemi EC16, these plant-inducible pectate lyase isozymes were not produced in minimal medium containing pectate. The results suggest that E. chrysanthemi produces two sets of independently regulated pectate lyase isozymes that are capable of macerating plant tissues.     

Physiological Basis for a Cycloheximide-induced Soft Rot of Potatoes by Pseudomonas fluorescens

Cycloheximide renders discs of potato tissue (Solanum tuberosum,cultivar Kennebec) susceptible to soft rot by a non-pathogenicisolate of Pseudomonas fluorescens. Pectate lyases (E.C. 4.2.99.3 [EC] )are the dominant extracellular macerating agents produced bythe test organism. Potato discs aged 24 h become resistant tomaceration by purified lyase preparations. Cycloheximide blocksthe development of resistance by inhibiting suberization. Thesite of inhibition is thought to be the cycloheximide-sensitivesynthesis of phenylalanine ammonia-lyase (E.C. 4.3.1.5 [EC] ) in potatodiscs. This enzyme is necessary for production of phenolic precursorsof suberin. Comparison of tissue from a number of potato cultivarscorrelates the synthesis of phenylalanine ammonia-lyase withresistance of discs to attack by the Pseudomonad. Resistance of potato tissue to pectate lyase is also affectedby intrinsic reactions not involving suberization. Resistanceincreases in fresh unsuberized discs when tubers are transferredfrom cold storage to room temperature before use. Resistancedecreases rapidly when tubers are transferred back to the cold.The intrinsic resistance appears to increase in the surfacelayer of cells in ageing discs. It is estimated that intrinsicreactions and suberization contribute equally to resistanceof aged discs to pectate lyase maceration.     

Synthesis of Mono- and Sesquiterpenoids

The synthesis of isocitrate lyase in Candida tropicalis, the growth of which was stimulated by exogenously added biotin, was released from repression by glucose under biotin-deficient conditions. Biotin deficiency reduced remarkably the levels of biotin-enzymes, pyruvate carboxylase and acetyl-Co A carboxylase, in the glucose-utilizing cells of this yeast. A marked increase in intracellular level of pyruvate was observed in the biotin-deficient cells. Acetyl-CoA-donating compounds, such as pyruvate, acetate and alkanes, stimulated the formation of isocitrate lyase in the yeast regardless of the presence or absence of biotin. On the other hand, malate and succinate did not affect the enzyme synthesis. The isocitrate lyase synthesis under biotin-sufficient conditions was repressed by not only glucose but also glucosamine and 2-deoxyglucose. This repression by glucose was not eliminated by cAMP. The stimulated synthesis of isocitrate lyase under biotin-deficient conditions was also observed in C. albicans and C. guilliermondii growing on glucose.     

Regulatory mutations affecting the synthesis of pectate lyase in Xanthomonas campestris

Four classes of Xanthomonas campestris mutants were identified with respect to pectate lyase. Pectate lyase production in the wild-type and classes I and IIb mutants was partially dependent on the growth-phase whereas in classes IIa and III it was totally dependent. Enzyme activity in some of the mutants was constitutive and resistant to catabolite repression.     

Cloning and characterization of a pectate lyase gene from Erwinia carotovora EC153

A pel gene cloned from strain EC153 of Erwinia carotovora encoded a pectate lyase that macerated plant tissue with moderate efficiency. This gene, called pel153, was sequenced and found to possess considerable homology with a pectate lyase gene from Yersinia pseudotuberculosis. The Yersinia protein, however, was truncated at the carboxyl terminal end relative to the Erwinia gene product and had a lower isoelectric point. The Erwinia pel153 gene was overexpressed in cells of Escherichia coli, and a 56-kDa protein was observed on sodium dodecyl sulfate-polyacrylamide gels. This compares with a molecular weight of 61 kDa for the mature, secreted protein as determined from sequencing data. Southern blot analysis disclosed the presence of the pel153 gene in three different strains of E. carotovora, but mutation of the gene in strain EC153 did not affect its ability to soft-rot potato tubers.     

Colletotrichum gloeosporioides pelB is an important virulence factor in avocado fruit-fungus interaction

Colletotrichum gloeosporioides is an important pathogen of tropical and subtropical fruits. The C. gloeosporioides pelB gene was disrupted in the fungus via homologous recombination. Three independent isolates, GD-14, GD-23, and GD-29, did not produce or secrete pectate lyase B (PLB) and exhibited 25% lower pectate lyase (PL) and pectin lyase (PNL) activities and 15% higher polygalacturonase (PG) activity than the wild type. The PLB mutants exhibited no growth reduction on glucose, Na polypectate, or pectin as the sole carbon source at pH 3.8 or 6.0, except for a 15% reduction on pectin at pH 6.0. When pelB mutants were inoculated onto avocado fruits, however, a 36 to 45% reduction in estimated decay diameter was observed compared with the two controls, the wild type and undisrupted transformed isolate. In addition, these pelB mutants induced a significantly higher host phenylalanine ammonia lyase activity as well as the antifungal diene, which is indicative of higher host resistance. These results suggest that PLB is an important factor in the attack of C. gloeosporioides on avocado fruit, probably as a result of its virulence factor and role in the induction of host defense mechanisms.     

Culture conditions for the production of pectinolytic enzymes by the white-rot fungus Trametes trogii on a laboratory scale

Different cultural parameters that regulate pectinolytic enzyme production in vitro by Trametes trogii were studied. When grown in a medium containing pectin, T. trogii produced extracellular polymethylgalacturonase, polygalacturonase and pectin lyase but no pectate lyase activity. No significant differences in the maximum enzyme activities measured were observed with the addition of xylan, carboxymethylcellulose or both to the medium containing pectin. The addition of glucose to that medium considerably decreases all the activities studied, and in a medium with glucose as the sole carbon source no galacturonase activity could be measured, and pectin lyase activity was at its minimum. The low synthesis of pectin lyase in cultures containing glucose suggests that this enzyme is constitutive in contrast to the polygalacturonases that were not detected. The increase in pectin concentration stimulated growth and enzyme production. The highest specific activities were attained with the greatest concentration tested (15 g/l). Casamino acids were the best nitrogen source for enzyme production. Maximum growth was measured at pH 3.3; pH values of around 4.5 stimulated enzyme production, but high pectinase activities were also detected in media with more alkaline initial pH values (6.2 for galacturonases and 6.6 for lyases), probably owing to the specific induction of particular isoforms. In the range of 23 to 28°C, good results were obtained in growth as well as in enzyme production. The addition of Tween 80 promoted growth and gave the highest yield of polymethylgalacturonase and pectin lyase (0.37 and 36.2 E.U./ml, respectively). The highest polygalacturonase activity (1.1 E.U/ml) was achieved with polyethylene glycol. Tween 20 and Triton X-100 inhibited growth and pectinase production.     

Role of the nucleoid-associated protein H-NS in the synthesis of virulence factors in the phytopathogenic bacterium Erwinia chrysanthemi

The ability of the enterobacterium Erwinia chrysanthemi to induce pathogenesis in plant tissue is strongly related to the massive production of plant-cell-wall-degrading enzymes (pectinases, cellulases, and proteases). Additional factors, including flagellar proteins and exopolysaccharides (EPS), also are required for the efficient colonization of plants. Production of these virulence factors, particularly pectate lyases, the main virulence determinant, is tightly regulated by environmental conditions. The possible involvement of the protein H-NS in this process was investigated. The E. chrysanthemi hns gene was cloned by complementation of an Escherichia coli hns mutation. Its nucleotide sequence contains a 405-bp open reading frame that codes for a protein with 85% identity to the E. coli H-NS protein. An E. chrysanthemi hns mutant was constructed by reverse genetics. This mutant displays a reduced growth rate and motility but an increased EPS synthesis and sensitivity toward high osmolarity. Furthermore, pectate lyase production is dramatically reduced in this mutant. The hns mutation acts on at least two conditions affecting pectate lyase synthesis: induction of pectate lyase synthesis at low temperatures (25 degrees C) is no longer observed in the hns mutant and induction of pectate lyase production occurs in the late stationary growth phase in the hns background, instead of in the late exponential growth phase as it does in the parental strain. Moreover, the E. chrysanthemi hns mutant displays reduced virulence on plants. Taken together, these data suggest that H-NS plays a crucial role in the expression of the virulence genes and in the pathogenicity of E. chrysanthemi.     

Production of cell wall-degrading enzymes by Aspergillus nidulans: a model system for fungal pathogenesis of plants.

The cell wall-degrading enzymes polygalacturonase and pectate lyase have been suggested to be crucial for penetration and colonization of plant tissues by some fungal pathogens. We have found that Aspergillus nidulans (= Emericella nidulans), a saprophytic Ascomycete, produces levels of these enzymes equal to those produced by soft-rotting Erwinia species. Induction of polygacturonase and pectate lyase in A. nidulans requires substrate and is completely repressed by glucose. Surprisingly, inoculation of excised plant tissues with A. nidulans conidia leads to formation of necrotic, water-soaked lesions within which the organism sporulates. Thus, A. nidulans has phytopathogenic potential. The release of glucose and other sugars from wounded tissues may repress pectolytic enzyme production and limit disease development. Therefore, we tested creA204, a mutation that relieves glucose repression of some A. nidulans carbon utilization enzymes, for its effect on production of pectolytic enzymes. creA204 failed to relieve catabolite repression of polygalacturonase or pectate lyase and had no effect on disease severity.     

Properties of Cytophaga johnsonae strains causing spoilage of fresh produce at food markets.

Two strains of gliding, orange-pigmented bacteria, isolated from fresh bell pepper and watermelon, respectively, showing soft-rot lesions, were identified as Cytophaga johnsonae. They differed from seven type strains of C. johnsonae deposited at the American Type Culture Collection (ATCC) in some properties, such as the ability to utilize glucose, xylose, trehalose, rhamnose, and sucrose. Spherical bodies resembling microcysts of Sporocytophaga sp. in addition to short rods and long filaments were observed in two strains (ATCC 29583 and 29588) throughout the growth cycle and also in aged cultures of other strains. All strains examined were shown to degrade five natural or synthetic polymers (pectin, chitin, starch, protein, and carboxymethyl cellulose). Only six strains (including ATCC 17061, 29587, 29589, and 19366) were able to infect and macerate artificially wounded potato tubers and fruits of pepper, squash, and tomato. The pathogenic strains secreted more pectate lyase in broth medium than the nonpathogenic strains. C. johnsonae, generally known as a soil saprophyte, might occasionally act as an opportunistic pathogen, causing decay of fresh produce in storage or in transit.     

Properties of Cytophaga johnsonae strains causing spoilage of fresh produce at food markets

Two strains of gliding, orange-pigmented bacteria, isolated from fresh bell pepper and watermelon, respectively, showing soft-rot lesions, were identified as Cytophaga johnsonae. They differed from seven type strains of C. johnsonae deposited at the American Type Culture Collection (ATCC) in some properties, such as the ability to utilize glucose, xylose, trehalose, rhamnose, and sucrose. Spherical bodies resembling microcysts of Sporocytophaga sp. in addition to short rods and long filaments were observed in two strains (ATCC 29583 and 29588) throughout the growth cycle and also in aged cultures of other strains. All strains examined were shown to degrade five natural or synthetic polymers (pectin, chitin, starch, protein, and carboxymethyl cellulose). Only six strains (including ATCC 17061, 29587, 29589, and 19366) were able to infect and macerate artificially wounded potato tubers and fruits of pepper, squash, and tomato. The pathogenic strains secreted more pectate lyase in broth medium than the nonpathogenic strains. C. johnsonae, generally known as a soil saprophyte, might occasionally act as an opportunistic pathogen, causing decay of fresh produce in storage or in transit.     

Isolation of Erwinia chrysanthemi kduD mutants altered in pectin degradation.

Mutants of Erwinia chrysanthemi impaired in pectin degradation were isolated by chemical and Mu d(Ap lac) insertion mutagenesis. A mutation in the kduD gene coding for 2-keto-3-deoxygluconate oxidoreductase prevented the growth of the bacteria on polygalacturonate as the sole carbon source. Analysis of the kduD::Mu d(Ap lac) insertions indicated that kduD is either an isolated gene or the last gene of a polycistronic operon. Some of the Mu d(Ap lac) insertions were kduD-lac fusions in which beta-galactosidase synthesis reflected kduD gene expression. In all these fusions, beta-galactosidase activity was shown to be sensitive to catabolite repression by glucose and to be inducible by polygalacturonate, galacturonate, and other intermediates of polygalacturonate catabolism. Galacturonate-mediated induction was prevented by a mutation which blocked its metabolism to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate appeared to be the true inducer of kduD expression resulting from galacturonate degradation. 5-Keto-4-deoxyuronate or 2,5-diketo-3-deoxygluconate were the true inducers, originating from polygalacturonate cleavage. These three intermediates also appeared to induce pectate lyases, oligogalacturonate lyase, and 5-keto-4-deoxyuronate isomerase synthesis.     

Production of pectolytic enzymes fromErwinia grown on different carbon sources

A quantitative analysis of pectolytic enzymes (polygalacturonase (PG), pectin methyl esterase (PME) and six isoenzymes of pectate lyase (PL)) produced byErwinia bacteria in the presence of diverse carbon sources was made by preparative electrophoresis. Synthesis of each of these enzymes was regulated independently; different induction and repression ratios (about 10- to 1000-fold) were observed for diverse PL isoenzymes, PG and PME. The possibility of using specially constructed media for the production of pectinase complexes with a specific spectra of pectolytic enzymes has been demonstrated.     

Cellular and environmental factors affecting the synthesis of polygalacturonate lyase byBacillus subtilis

Summary Cellular and environmental factors affecting the synthesis of polygalacturonate lyase in batch and chemostat cultures ofBacillus subtilis were investigated. The lyase was produced constitutively during growth on a wide range of carbon sources in a defined minimal medium and in medium containing complex organic carbon and nitrogen sources. The highest activity was obtained during batch growth in minimal medium containing glucose and ammonium sulphate. Over 99% of the activity was present extracellularly in the supernatant medium at all stages of the batch growth cycle. Two distinct differential rates of synthesis were observed during exponential growth. The lyase was unable to attack pectin rapidly unless pectin methyl-esterase was also present. Pectin was a poor substrate for growth and polygalacturonate lyase induction because the organism did not produce pectin methyl-esterase. In continuous-flow chemostat cultures with glucose medium, polygalacturonate lyase activity declined to a very low level owing to the selection of non-productive mutant strains. Loss of activity did not occur when polypectate was the carbon source. Steady-state specific polygalacturonate lyase activity in polypectate medium was relatively independent of dilution rate in the range 0.04 to 0.36/h. When polypectate was supplied in excess of the growth requirement lyase activity was 5 times higher than during polypectate-limited growth.     

Pectinolytic Systems of Two Aerobic Sporogenous Bacterial Strains with High Activity on Pectin

Strains Paenibacillus sp. BP-23 and Bacillus sp. BP-7, previously isolated from soil from a rice field, secreted high levels of pectinase activity in media supplemented with pectin. Production of pectinases in strain Paenibacillus sp. BP-23 showed catabolite repression, while in Bacillus sp. BP-7 production of pectin degrading enzymes was not negatively affected by glucose. The two strains showed lyase activities as the predominant pectinases, while hydrolase activity was very low. Analysis of Paenibacillus sp. BP-23 in SDS–polyacrylamide gels and zymograms showed five pectinase activity bands. The strict requirement of Ca²⁺ for lyase activity of the strain indicates that correspond to pectate lyases. For Bacillus sp. BP-7, zymograms showed four bands of different size. The strain showed a Ca²⁺ requirement for lyase activity on pectate but not on pectin, indicating that the pectinolytic system of Bacillus sp. BP-7 is comprised of pectate lyases and pectin lyases. The results show differences in pectin degrading systems between the two aerobic sporogenous bacterial strains studied.     

Distribution of the 7S Proteins in Soybean Globulins by Gel Filtration with Sephadex G-200

The level of isocitrate lyase, an enzyme of glyoxylate cycle, in Candida tropicalis was enhanced at the later period of growth when the yeast was cultivated in a semisynthetic glucose medium. On the other hand, such increase in the enzyme activity was not observed in C. lipolytica grown under the same conditions. In the case of C. tropicalis, high concentrations of glucose remaining in the medium permitted the increase in the enzyme activity and the addition of ethanol, one of the major products from glucose, to the glucose medium did not stimulate the enzyme formation, indicating that the enhanced enzyme level in the yeast was not merely attributable to the release from the repression by glucose or to the induction by ethanol. Biotin, one of the growth-stimulating factors for C. tropicalis, affected markedly the level of isocitrate lyase. That is, the supplementation of biotin to the synthetic glucose medium inhibited completely the increase in the enzyme activity, and reversely the absence of biotin stimulated the enzyme formation in the glucose-assimilating cells. Thiamine, another growth-stimulating factor for C. tropicalis, did not show any effect on the level of isocitrate lyase in the yeast. The level of isocitrate lyase in C. lipolytica growing on glucose was not affected by biotin added exogenously.     

Silencing of SlPL,which encodes a pectate lyase in tomato,confers enhanced fruit firmness,prolonged shelf‐life and reduced susceptibility to grey mould

Pectate lyase genes have been documented as excellent candidates for improvement of fruit firmness. However, implementation of pectate lyase in regulating fruit postharvest deterioration has not been fully explored. In this report, 22 individual pectate lyase genes in tomato were identified, and one pectate lyase gene SlPL (Solyc03g111690) showed dominant expression during fruit maturation. RNA interference of SlPL resulted in enhanced fruit firmness and changes in pericarp cells. More importantly, the SlPL‐RNAi fruit demonstrated greater antirotting and pathogen‐resisting ability. Compared to wild‐type, SlPL‐RNAi fruit had higher levels of cellulose and hemicellulose, whereas the level of water‐soluble pectin was lower. Consistent with this, the activities of peroxidase, superoxide dismutase and catalase were higher in SlPL‐RNAi fruit, and the malondialdehyde concentration was lower. RNA‐Seq results showed large amounts of differentially expressed genes involved in hormone signalling, cell wall modification, oxidative stress and pathogen resistance. Collectively, these data demonstrate that pectate lyase plays an important role in both fruit softening and pathogen resistance. This may advance knowledge of postharvest fruit preservation in tomato and other fleshy fruit.     

Production of lyases byPhoma exigua associated with seed-rot ofVigna radiata

Phoma exigua associated with seed-rot ofVigna radiata produced lyases which varied with the media tested. The production of lyases was higher in pectin-supplemented media.Vigna seed meal medium was not suitable for induction of lyase production. The pectin lyase and pectate lyase was maximum after 11 d of incubation by which time the pH was shifted to alkaline side. Temperature of 25 °C and pH 9 was found to be optimum for the activity of pectin lyase and pectate lyase. Fungicides (antracol and panoctine), phenols (pyrocatechol and gallic acid) and growth substances (gibberellic acid and yeast extract) adversely affected the enzyme secretion.