Endo-pectate Lyase Research Articles

A Novel PL9 Pectate Lyase from Paenibacillus polymyxa KF-1: Cloning, Expression, and Its Application in Pectin Degradation.

Pectate lyases play an important role in pectin degradation, and therefore are highly useful in the food and textile industries. Here, we report on the cloning of an alkaline pectate lyase gene (pppel9a) from Paenibacillus polymyxa KF-1. The full-length gene (1350 bp) encodes for a 449-residue protein that belongs to the polysaccharide lyase family 9 (PL9). Recombinant PpPel9a produced in Escherichia coli was purified to electrophoretic homogeneity in a single step using Ni2+-NTA affinity chromatography. The enzyme activity of PpPel9a (apparent molecular weight of 45.3 kDa) was found to be optimal at pH 10.0 and 40 °C, with substrate preference for homogalacturonan type (HG) pectins vis-à-vis rhamnogalacturonan-I (RG-I) type pectins. Using HG-type pectins as substrate, PpPel9a showed greater activity with de-esterified HGs. In addition, PpPel9a was active against water-soluble pectins isolated from different plants. Using this lyase, we degraded citrus pectin, purified fractions using Diethylaminoethyl (DEAE)-sepharose column chromatography, and characterized the main fraction MCP-0.3. High-performance gel permeation chromatography (HPGPC) analysis showed that the molecular mass of citrus pectin (~230.2 kDa) was reduced to ~24 kDa upon degradation. Ultra-performance liquid chromatography - tandem mass spectrometer (UPLC-MS) and monosaccharide composition analyses demonstrated that PpPel9a worked as an endo-pectate lyase, which acted primarily on the HG domain of citrus pectin. In vitro testing showed that the degradation product MCP-0.3 significantly promotes the growth of Lactobacillus plantarum and L. rhamnosus. In this regard, the enzyme has potential in the preparation of pharmacologically active pectin products.

Structural Modelling, Substrate Binding and Stability Studies of Endopectate Lyase (PL1B) of Family 1 Polysaccharide Lyase from Clostridium thermocellum.

An endo-pectate lyase (PL1B) of family 1 polysaccharide lyase from Clostridium thermocellum was structurally characterized and its stability under chaotropic agent was determined. The putative domain PL1B was identified from the protein sequence ABN53381.1 belonging to superfamily 3 of pectate lyase. Multiple sequence alignment of PL1B with other known pectate lyases revealed the conserved and semi-conserved residues. The secondary structure of PL1B predicted by PsiPred and confirmed by Circular Dichroism showed the presence of 2 α-helices (2.06%), 26 β-strands (40.54%) and 29 random coils (57.4%). The modelled protein represented right handed parallel β-helix structure, where three parallel β-sheets linked by loops coils around to form the β-helix core. Quality assessment of energy minimized structure by Ramachandran plot displayed 82.8% residues in favoured region. Superposition of PL1B structure with Bsp165-PelA from Bacillus sp. revealed the substrate binding cleft formed by the amino acid residues from the loops and β-sheet. Molecular dynamic simulation of modelled PL1B structure inferred that it is quite stable and compact. Docking studies identified Asp151, Arg209, Asn234, Arg236, Tyr271 and Ser272 as the key residues of PL1B involved during catalysis. Among them Arg209 is responsible for proton abstraction during β-elimination. Protein melting studies on PL1B showed that there was 12°C shift of peak from 74 to 86°C in presence of 0.6 mM Ca(2+) ions, showing that they provide stability to the structure. The unfolding of PL1B by GuHCl or Urea by fluorescence study showed that the protein structure is stable and disintegrates at their higher concentrations.

Role of pectinolytic enzymes identified in Clostridium thermocellum cellulosome.

The cloning, expression and characterization of three cellulosomal pectinolytic enzymes viz., two variants of PL1 (PL1A and PL1B) and PL9 from Clostridium thermocellum was carried out. The comparison of the primary sequences of PL1A, PL1B and PL9 revealed that these proteins displayed considerable sequence similarities with family 1 and 9 polysaccharide lyases, respectively. PL1A, PL1B and PL9 are the putative catalytic domains of protein sequence ABN54148.1 and ABN53381.1 respectively. These two protein sequences also contain putative carbohydrate binding module (CBM) and type-I dockerin. The associated putative CBM of PL1A showed strong homology with family 6 CBMs while those of PL1B and PL9 showed homology with family 35 CBMs. Recombinant derivatives of these three enzymes showed molecular masses of approximately 34 kDa, 40 kDa and 32 kDa for PL1A, PL1B and PL9, respectively. PL1A, PL1B and PL9 displayed high activity toward polygalacturonic acid and pectin (up to 55% methyl-esterified) from citrus fruits. However, PL1B showed relatively higher activity towards 55% and 85% methyl-esterified pectin (citrus). PL1A and PL9 showed higher activity on rhamnogalacturonan than PL1B. Both PL1A and PL9 displayed maximum activity at pH 8.5 with optimum temperature of 50°C and 60°C respectively. PL1B achieved highest activity at pH 9.8, under an optimum temperature of 50°C. PL1A, PL1B and PL9 all produced two or more unsaturated galacturonates from pectic substrates as displayed by TLC analysis confirming that they are endo-pectate lyase belonging to family 1 and 9, respectively. This report reveals that pectinolytic activity displayed by Clostridium thermocellum cellulosome is coordinated by a sub-set of at least three multi-modular enzymes.

Deciphering the cleavage mechanism of thermostable family 1 Polysaccharide lyase (PL1B) from Clostridium thermocellum ATCC 27405 (LB130)

Deciphering the cleavage mechanism of thermostable family 1 Polysaccharide lyase (PL1B) from Clostridium thermocellum ATCC 27405 Soumyadeep Chakraborty, Arun Goyal, Indian Institute of Technology, Guwahati, Assam, India A protein sequence with GeneBank accession number ABN53381.1 from Clostridium thermocellum ATCC 27405 belonging to family 1 polysacchaide lyase of CAZy database was scavenged out for this study. This sequence consisted of 352 aa N‐terminal catalytic domain (PL1B) and a 123 aa family 35 carbohydrate binding domain designated as (CBM35) at the C‐terminal. The catalytic domain PL1B was cloned in pET28a expression vector and expressed in E.coli as a soluble protein of molecular size 40 kDa. PL1B was active at alkaline pH on pectic substrates. Optimization of enzymatic reaction conditions revealed that PL1B shows 100% (3005 U/mg) activity at 50ºC in 50 mM Tris‐HCl buffer, pH 9.8. Highest activity was achieved on reaction with non‐methylated polygalacturonic acid (PGA) and almost 75% of the total activity was found with methylated citrus pectin. As this enzyme showed highest activity on non‐methylated PGA hence it was named as pectate lyase. The cleavage mechanism of PL1B towards pectic substrates was determined after analysis of the oligosaccharide produced. The reaction was setup with PGA and citrus pectin separately under optimized conditions of pH and temperature and samples was collected at different time interval at 0 min up to 60 min. Analysis of the reaction products by thin layer chromatography (TLC) revealed that unsaturated di and tri‐galacturonate was produced after enzymatic cleavage together with other oligosaccharides. This finding elucidated that PL1B cleaves within the polysaccharide chain and hence it is an endo cleaving enzyme; thus PL1B can be named as endo‐pectate lyase. Enzymatic degradation products were further analyzed by HPEAC and mass spectrometry.

PelN Is a New Pectate Lyase of Dickeya dadantii with Unusual Characteristics

The plant-pathogenic bacterium Dickeya dadantii produces several pectinolytic enzymes that play a major role in the soft-rot disease. Eight characterized endopectate lyases are secreted in the extracellular medium by the type II secretion system, Out. They cleave internal glycosidic bonds of pectin, leading to plant tissue maceration. The D. dadantii pectate lyases belong to different families, namely, PL1, PL2, PL3, and PL9. Analysis of the D. dadantii 3937 genome revealed a gene encoding a new protein of the PL9 family, which already includes the secreted endopectate lyase PelL and the periplasmic exopectate lyase PelX. We demonstrated that PelN is an additional extracellular protein secreted by the Out system. However, PelN has some unusual characteristics. Although most pectate lyases require a very alkaline pH and Ca²⁺ for their activity, the PelN activity is optimal at pH 7.4 and in the presence of Fe²⁺ as a cofactor. PelN is only weakly affected by the degree of pectin methyl esterification. The PelN structural model, constructed on the basis of the PelL structure, suggests that the PelL global topology and its catalytic amino acids are conserved in PelN. Notable differences concern the presence of additional loops at the PelN surface, and the replacement of PelL charged residues, involved in substrate binding, by aromatic residues in PelN. The pelN expression is affected by different environmental conditions, such as pH, osmolarity, and temperature. It is controlled by the repressors KdgR and PecS and by the activator GacA, three regulators of D. dadantii pectinase genes. Since a pelN mutant had reduced virulence on chicory leaves, the PelN enzyme plays a role in plant infection, despite its low specific activity and its unusual cofactor requirement.

Commensal effect of pectate lyases secreted from Dickeya dadantii on proliferation of Escherichia coli O157:H7 EDL933 on lettuce leaves.

The outbreaks caused by enterohemorrhagic Escherichia coli O157:H7 on leafy greens have raised serious and immediate food safety concerns. It has been suggested that several phytopathogens aid in the persistence and proliferation of the human enteropathogens in the phyllosphere. In this work, we examined the influence of virulence mechanisms of Dickeya dadantii 3937, a broad-host-range phytopathogen, on the proliferation of the human pathogen E. coli O157:H7 EDL933 (EDL933) on postharvest lettuce by coinoculation of EDL933 with D. dadantii 3937 derivatives that have mutations in virulence-related genes. A type II secretion system (T2SS)-deficient mutant of D. dadantii 3937, A1919 (ΔoutC), lost the capability to promote the multiplication of EDL933, whereas Ech159 (ΔrpoS), a stress-responsive σ factor RpoS-deficient mutant, increased EDL933 proliferation on lettuce leaves. A spectrophotometric enzyme activity assay revealed that A1919 (ΔoutC) was completely deficient in the secretion of pectate lyases (Pels), which play a major role in plant tissue maceration. In contrast to A1919 (ΔoutC), Ech159 (ΔrpoS) showed more than 2-fold-greater Pel activity than the wild-type D. dadantii 3937. Increased expression of pelD (encodes an endo-pectate lyase) was observed in Ech159 (ΔrpoS) in planta. These results suggest that the pectinolytic activity of D. dadantii 3937 is the dominant determinant of enhanced EDL933 proliferation on the lettuce leaves. In addition, RpoS, the general stress response σ factor involved in cell survival in suboptimal conditions, plays a role in EDL933 proliferation by controlling the production of pectate lyases in D. dadantii 3937.

Metal-tolerant moss Scopelophila cataractae accumulates copper in the cell wall pectin of the protonema

The growth kinetics in the presence of copper (Cu) of the protonema of the moss Scopelophila cataractae and the matrix polysaccharides of its cell walls have been analyzed in this study. Protonemal cells cultured in a medium containing 0.2 mM CuSO 4 showed a rapid accumulation of Cu, reaching a maximum between 30 and 60 d at approximately 65 μmol g −1 DW. Uronic acids were found in similar amounts in cell walls of both control and Cu-treated cells, whereas arabinose and galactose decreased to 61–67% in the presence of Cu. Cell wall polysaccharides were determined after successive extraction with 50 mM CDTA, 50 mM Na 2CO 3, 1 M KOH, and 4 M KOH. The pectic fractions (CDTA- and Na 2CO 3-soluble) decreased to 47% and the hemicellulosic fractions (1 M KOH- and 4 M KOH-soluble) to 86% under Cu application. Approximately 43% of the Cu taken into cell walls was released following endo-pectate lyase treatment, suggesting that two-fifths of the total Cu accumulation was tightly bound to the homogalacturonan of the cell wall pectin.

Global Effect of Indole-3-Acetic Acid Biosynthesis on Multiple Virulence Factors of Erwinia chrysanthemi 3937

Production of the plant hormone indole-3-acetic acid (IAA) is widespread among plant-associated microorganisms. The non-gall-forming phytopathogen Erwinia chrysanthemi 3937 (strain Ech3937) possesses iaaM (ASAP16562) and iaaH (ASAP16563) gene homologues. In this work, the null knockout iaaM mutant strain Ech138 was constructed. The IAA production by Ech138 was reduced in M9 minimal medium supplemented with l-tryptophan. Compared with wild-type Ech3937, Ech138 exhibited reduced ability to produce local maceration, but its multiplication in Saintpaulia ionantha was unaffected. The pectate lyase production of Ech138 was diminished. Compared with wild-type Ech3937, the expression levels of an oligogalacturonate lyase gene, ogl, and three endopectate lyase genes, pelD, pelI, and pelL, were reduced in Ech138 as determined by a green fluorescent protein-based fluorescence-activated cell sorting promoter activity assay. In addition, the transcription of type III secretion system (T3SS) genes, dspE (a putative T3SS effector) and hrpN (T3SS harpin), was found to be diminished in the iaaM mutant Ech138. Compared with Ech3937, reduced expression of hrpL (a T3SS alternative sigma factor) and gacA but increased expression of rsmA in Ech138 was also observed, suggesting that the regulation of T3SS and pectate lyase genes by IAA biosynthesis might be partially due to the posttranscriptional regulation of the Gac-Rsm regulatory pathway.

Structural analysis of the polysaccharides from Echinacea angustifolia radix

In this paper we report the characterization by monosaccharide and linkage analyses and by NMR spectroscopy and size exclusion chromatography of the carbohydrate fraction extracted from Echinacea angustifolia radix. In addition, the products of endo-pectin lyase, endo-pectate lyase, endo-polygalacturonase, endo-galactanase, and endo-arabinase digestion were characterized by MALDI-TOF mass spectrometry. The data obtained during this study showed that the carbohydrate fraction extracted from E. angustifolia radix is constituted by two polysaccharides with molecular weight of about 128,000 and 4500 Da. The low molecular weight polysaccharide corresponds to inulin while the high molecular weight component is a high metoxy pectin in which the backbone structure of the smooth region is constituted by α-(1–4)-polygalacturonan partially methyl esterified (60%) and acetylated (9%) and with the hairy regions containing 2- O- and 2,4- O-rhamnopyranose, 5- O- and 3,5- O-arabinofuranose, 3,6-galactopyranose, and terminal rhamnopyranose, arabinofuranose, arabinopyranose, galactopyranose, and galacturonopyranose. Mass spectrometry data of a galactanase treated sample showed evidence of a novel structure in the pectin hairy region, namely a galactose–galacturonic acid alternating sequence.

Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2

An alkalophilic bacterium was isolated based on the potential of extra-cellular enzymes for bioscouring. The bacterium was identified as a new strain of Bacillus pumilus BK2 producing an extra-cellular endo-pectate lyase PL (EC 4.2.2.2). PL was purified to homogeneity in three steps and has a molecular mass of 37.3 ± 4.8 kDa as determined by SDS-PAGE and an isoelectric point of pH 8.5. Peptide mass mapping by nano-LC–MS of PL revealed 15% homology with a pectate lyase from Bacillus sp. The pectate lyase exhibited optimum activity at pH 8.5 and around 70 °C in Tris/HCl buffer. It showed a half-life at 30 °C of more than 75 h. Stability decreased with increasing temperature, extremely over 60 °C. The enzyme did not require Ca 2+ ions for activity, and was strongly inhibited by EDTA and Co 2+. PL was active on polygalacturonic acid and esterified pectin, but the affinity showed a maximum for intermediate esterified pectins and decreased over a value of 50% of esterification. The best substrate was 29.5% methylated pectin. PL cleaved polygalacturonic acid via a β-elimination mechanism as shown by NMR analysis. PL released unsaturated tetragalacturonic acid from citrus pectin and polygalacturonic acid, but did not show any side activities on other hemicelluloses. On polygalacturonic acid PL showed a K m of 0.24 g l −1 and a v max of 0.72 g l −1 min −1. The applicability of pectate lyase for the bioscouring process was tested on a cotton fabric. Removal of up to 80% of pectin was proven by means of ruthenium red dyeing and HPAEC (65%). Structural contact angle measurements clearly indicated the increased hydrophilicity of enzyme treated fabrics.

The secretome of the plant pathogenic bacterium Erwinia chrysanthemi.

Erwinia chrysanthemi causes soft-rot diseases of many plants by secreting a battery of enzymes which degrade the plant cell walls. We initiated a proteomic analysis to create a reference map of the E. chrysanthemi secretome. Extracellular proteins were isolated from E. chrysanthemi culture supernatants and resolved by two-dimensional electrophoresis. By analysis of mutants, Western blotting, and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) 55 spots representing 25 unique proteins were identified. In uninduced conditions, we identified spots corresponding to the cellulase Cel5, the proteases PrtA, PrtB, and PrtC, the flagellin FliC, and some intracellular proteins whose presence probably resulted from spontaneous cell lysis. We identified another secreted protein, AvrL, homologous to an avirulence protein of Xanthomonas campestris. After culture in conditions inducing pectinase production, i.e., in the presence of galacturonate and plant extract, we identified spots corresponding to the endopectate lyases PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ, the pectin acetylesterases PaeX and PaeY, the pectin methylesterase PemA, and the polygalacturonase PehX. In the presence of other inducing compounds, we detected the rhamnogalacturonate lyase RhiE and the esterase FaeD. Analysis of mutants, altered for one type of secretion system, was performed to determine the targets of each system. The type I system Prt was necessary for the secretion of three proteases. No proteins secreted by the type III Hrp system could be detected in E. chrysanthemi supernatants. In addition to the already known substrates (eleven pectinases and one cellulase), this analysis revealed that the type II Out system mediates secretion of the esterase FaeD and of the Avr-like protein AvrL.

PehN, a polygalacturonase homologue with a low hydrolase activity, is coregulated with the other Erwinia chrysanthemi polygalacturonases.

Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes, including at least eight endo-pectate lyases encoded by pel genes, which play a major role in the soft-rot disease caused by this bacterium on various plants. E. chrysanthemi also produces some hydrolases that cleave pectin. Three adjacent hydrolase genes, pehV, pehW, and pehX, encoding exo-poly-alpha-D-galacturonosidases, have been characterized. These enzymes liberate digalacturonides from the nonreducing end of pectin. We report the identification of a novel gene, named pehN, encoding a protein homologous to the glycosyl hydrolases of family 28, which includes mainly polygalacturonases. PehN has a low hydrolase activity on polygalacturonate and on various pectins. PehN action favors the activity of the secreted endo-pectate lyases, mainly PelB and PelC, and that of the periplasmic exo-pectate lyase PelX. However, removal of the pehN gene does not significantly alter the virulence of E. chrysanthemi. Regulation of pehN transcription was analyzed by using gene fusions. Like other pectinase genes, pehN transcription is dependent on several environmental conditions. It is induced by pectic catabolic products and is affected by growth phase, catabolite repression, osmolarity, anaerobiosis, nitrogen starvation, and the presence of calcium ions. The transcription of pehN is modulated by the repressor KdgR, which controls almost all the steps of pectin catabolism, and by cyclic AMP receptor protein (CRP), the global activator of sugar catabolism. The regulator PecS, which represses the transcription of the pel genes but activates that of pehV, pehW, and pehX, also activates transcription of pehN. The three regulators KdgR, PecS, and CRP act by direct interaction with the pehN promoter region. The sequences involved in the binding of these three regulators and of RNA polymerase have been precisely defined. Analysis of the simultaneous binding of these proteins indicates that CRP and RNA polymerase bind cooperatively and that the binding of KdgR could prevent pehN transcription. In contrast, the activator effect of PecS is not linked to competition with KdgR or to cooperation with CRP or RNA polymerase. This effect probably results from competition between PecS and an unidentified repressor involved in peh regulation.

Enzyme Assay for Identification of Pectin and Pectin Derivatives, Based on Recombinant Pectate Lyase

A simple method was developed for fast identification of pectin, based on a recombinant endopectate lyase cloned from Aspergillus niger. When pectin was demethylated and treated with pectate lyase, beta-elimination occurred, resulting in a double bond between C-4 and C-5 in the galacturonic acid residue of the released nonreducing end. The formation of double bonds produced an increase in light absorption, which was detected at 235 nm. The assay was tested on pectin of different origins (apple, orange, sugar beet, sunflower, celery, lemon), pectin derivatives (amidated pectin), and speciality types such as low molecular weight and low %DE (degree of esterification, percentage of galacturonic acid groups esterified with methanol) pectin. The highest response was given by pectate (pectin with %DE< 5) and the lowest by pectin extracted from sugar beet. No other gums (carboxymethylcellulose, carrageenan, locust bean gum, tragacanth, gellan, tamarind, xanthan, amylogum, sodium alginate, or agar) gave any response. Members of IPPA (International Pectin Producers Association) have evaluated the validity of the assay in a ring test. All members of the Association were able to identify pectin from other gums in a blind test. The method can replace more laborious and ambiguous identification tests which exist today.

Modes of Action of Five Different Endopectate Lyases from Erwinia chrysanthemi 3937

Modes of Action of Five Different Endopectate Lyases from <i>Erwinia chrysanthemi</i> 3937

Modes of action of five different endopectate lyases from Erwinia chrysanthemi 3937.

Five endopectate lyases from the phytopathogenic bacterium Erwinia chrysanthemi, PelA, PelB, PelD, PelI, and PelL, were analyzed with respect to their modes of action on polymeric and oligomeric substrates (degree of polymerization, 2 to 8). On polygalacturonate, PelB showed higher reaction rates than PelD, PelI, and PelA, whereas the reaction rates for PelL were extremely low. The product progression during polygalacturonate cleavage showed a typical depolymerization profile for each enzyme and demonstrated their endolytic character. PelA, PelI, and PelL released oligogalacturonates of different sizes, whereas PelD and PelB released mostly unsaturated dimer and unsaturated trimer, respectively. Upon prolonged incubation, all enzymes degraded the primary products further, to unsaturated dimer and trimer, except for PelL, which degraded the primary products to unsaturated tetramer and pentamer in addition to unsaturated dimer and trimer. The bond cleavage frequencies on oligogalacturonates revealed differences in the modes of action of these enzymes that were commensurate with the product progression profiles. The preferential products formed from the oligogalacturonates were unsaturated dimer for PelD, unsaturated trimer for PelB, and unsaturated tetramer for PelI and PelL. For PelA, preferential products were dependent on the sizes of the oligogalacturonates. Whereas PelB and PelD displayed their highest activities on hexagalacturonate and tetragalacturonate, respectively, PelA, PelI, and PelL were most active on the octamer, the largest substrate used. The bond cleavage frequencies and reaction rates were used to estimate the number of subsites of each enzyme.

Degradation of pectic substances by two pectate lyases from a human intestinal bacterium, Clostridium butyricum-beijerinckii group

Clostridium butyricum-beijerinckii group, an anaerobic human intestinal bacterium, produced exo-pectate lyase, endo-pectate lyase, and pectin methylesterase, but not polygalacturonase. The production of the two pectate lyases was strongly influenced by the composition of the carbon sources added to the medium. The enzymes secreted by the bacterium into the human large intestine cooperatively digested pectic substances, producing mainly 4,5-unsaturated digalacturonic acid with the participation of the pectin methylesterase. The findings suggest that in the human intestine the bacterium contributes to the digestion of ingested pectic substances.

Processing of the pectate lyase PelI by extracellular proteases of Erwinia chrysanthemi 3937.

Erwinia chrysanthemi causes soft rot on various plants. The maceration of plant tissues is mainly due to the action of endopectate lyases. The E. chrysanthemi strain 3937 produces eight endopectate lyases (PelA, PelB, PelC, PelD, PelE, PelI, PelL and PelZ) that are secreted by the Out pathway. The necrotic response elicited by the wild-type E. chrysanthemi strain on tobacco leaves is due to an extracellular protein secreted by the Out machinery. Purification of the active factor revealed that it corresponds to a pectate lyase presenting immunological cross-reaction with PelI. Analysis of pelI and out mutants indicated that the necrosis-inducing pectate lyase results from a post-translational modification of PelI occurring extracellularly both in culture media and in planta. This modification consists of the cleavage of 97 N-terminal amino acids by the extracellular proteases of E. chrysanthemi. The enzymatic properties of the maturated form, PelI-3, are not, or only weakly, modified. However, this maturation gives rise to a small size and basic form that is active as a defence elicitor in plants.

Biochemical characterization of the pectate lyase PelZ of Erwinia chrysanthemi 3937

To degrade the plant pectin, the phytopathogenic bacterium Erwinia chrysanthemi produces a set of at least seven endo-pectate lyases (Pels). Five major (PelA, PelB, PelC, PelD and PelE) and two minor isoenzymes (PelL and PelZ) have been identified. PelZ is an extracellular enzyme secreted by the Out system. According to its amino acid sequence, the PelZ protein belongs to a new family. The PelZ protein was overproduced in E. coli and purified to compare its enzymatic properties to that of the other Pels of E. chrysanthemi. PelZ exhibits a low specific activity but good affinity for the substrates including partially methylated pectins (up to 45% methylation). The main characteristic of PelZ is the requirement for both Ca 2+ and Mn 2+ as cofactors while the other Pels require only Ca 2+. The cooperative effect of these two cations suggests the presence of distinct binding sites. The PelZ activity is sensitive to inhibition by excess of substrate, by oligogalacturonides, by the ionic strength and by different plant compounds. PelZ was shown to act in synergy with the major isoenzyme PelE, while competition was observed between PelZ and the minor pectate lyase PelL. No synergistic action was observed between PelZ and PelA, PelB, PelC or PelD.

Regulation of pelZ, a gene of the pelB-pelC cluster encoding a new pectate lyase of Erwinia chrysanthemi 3937.

The phytopathogenic enterobacterium Erwinia chrysanthemi 3937 produces five major and several secondary endo-pectate lyases encoded by the pel genes. Most of these genes are arranged in clusters on the bacterial chromosome. The genomic region surrounding the pelB-pelC cluster was supposed to be involved in the regulation of PelB and PelC synthesis. We demonstrated that the variation of pelB expression resulted from the titration of a regulatory protein by the gene adjacent to pelC. This gene was renamed pelZ since it encodes a protein of 420 amino acids with an endo-pectate lyase activity. Regulation of pelZ expression was investigated by using transcriptional fusions and a study of mRNA synthesis. Its transcription depends on different environmental conditions. It is induced in planta and in the presence of pectic catabolite products. This induction seems to be partially mediated by the KdgR protein but does not result from a direct interaction of KdgR with the pelZ 5' region. The transcription of pelZ leads to the synthesis of a monocistronic mRNA. However, the synthesis of a polycistronic mRNA from the pelC promoter, regulated by KdgR, is responsible for increased production of PelZ under inducing conditions. pelZ transcription is also controlled by pecT, which regulates some other pel genes, but it is independent of the pecS regulatory locus. The pelZ gene appears to be widespread in different strains of E. chrysanthemi. Moreover, a gene homologous to pelZ exists in Erwinia carotovora subsp. atroseptica adjacent to the cluster containing the pectate lyase-encoding genes pel1, pel2, and pel3. This conservation could reflect a significant role of PelZ in the pectinolytic system of Erwiniae. We showed pelZ is not a predominant virulence factor of E. chrysanthemi but is involved in host specificity.

Identification of a NovelpelDGene Expressed Uniquely in Planta byFusarium solanif. sp.pisi(Nectria haematococca,Mating Type VI) and Characterization of Its Protein Product as an Endo-Pectate Lyase

Antibodies prepared against a pectin-inducible pectate lyase (PLA) produced by a phytopathogenic fungusFusarium solanif. sp.pisi(Nectria haematococca,mating type VI) were previously found to protect the host from infection. The gene (pelA) and two of its homologs were cloned and sequenced. Here we report the isolation of a new pectate lyase gene,pelD,from a genomic library ofF. solani pisi.A 1.5-kb DNA fragment containingpelDand its flanking regions was sequenced. The nucleotide sequence ofpelDwould encode a protein of 24.5 kDa which shares 49, 44, and 65% amino acid sequence identity with PLA, PLB, and PLC, respectively, from the same fungus. Because the first 19 amino acid residues appeared to be a signal peptide, the mature enzyme could be a 22.7-kDa protein.pelDtranscripts and PLD protein could not be detected in fungus cultured in glucose, pectin, pea epicotyl extract, or a pea cell wall preparation. However,pelDtranscripts were readily found by RT–PCR with RNA isolated from infected pea tissues. The cDNA ofpelD,thus obtained, was expressed inPichia pastoriswith the putativepelDsignal sequence. The secreted PLD was purified and characterized to be an endopectate lyase, and its lyase activity could be inhibited by anti-PLA IgG. Thus, protection of the host observed with the anti-PLA antibodies could reflect inhibition of immunologically related pectate lyases including PLD which is expressed uniquely in planta.