INTRODUCTION

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The enterobacterium Erwinia chrysanthemi causes soft rot disease of various plants. The maceration process involves the depolymerization of the pectin of plant cell walls and the middle lamella. Pectin is a heteropolysaccharide with a backbone consisting of partially esterified galacturonic acid. Pectin degradation is accomplished by a variety of pectinases, which differ mainly in the reaction mechanism (-elimination or hydrolysis) and in the random or terminal mode of attack of the polymer (endo or exo).

Erwinia chrysanthemi is characterized by its ability to secrete multiple isoenzymes of endo-pectate lyases (endo-Pels), which play an important role in the soft rot disease (3). These enzymes randomly cleave, by -elimination, internal glycosidic linkages in pectic polymers, preferentially polygalacturonate or low-methoxylated pectin (up to 30%) (43). They generate a series of oligogalacturonates with a 4,5-unsaturated residue at the nonreducing end. Eight E. chrysanthemi 3937 endo-Pels have been characterized, PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ (16, 29, 39). The corresponding genes are organized in four clusters on the bacterial chromosome (pelA-pelE-pelD, pelB-pelC-pelZ, pelI, and pelL). Pectate lyases are classified in five different families according to their primary amino acid sequences (4a). PelA, PelB, PelC, PelD, and PelE belong to family 1, which contains several bacterial pectate lyases, fungal pectin lyases, and plant proteins (14, 15). PelI belongs to family 3 (39), which includes enzymes of Erwinia carotovora and of the phytopathogenic fungus Nectria haematococca. Family 4 contains PelL and PelX of E. chrysanthemi (1, 23). PelZ of E. chrysanthemi is the sole characterized component of family 5 (29). Two exo-cleaving depolymerases have also been identified in E. chrysanthemi. A cell-bound exopolygalacturonate lyase and an extracellular exo-poly--D-galacturonosidase were found in E. chrysanthemi CUCPB1237 (7, 8). The corresponding genes, pelX and pehX, respectively, were isolated from E. chrysanthemi EC16 (6, 12). PehX and PelX contribute to pectin catabolism but they have little direct involvement in plant maceration.

Transcription of the E. chrysanthemi pectinase genes is dependent on several environmental conditions (16). The genes are all induced by pectin catabolic products and by the late exponential growth phase. Most of them are repressed under conditions of catabolite repression, nitrogen starvation, and high temperature. Other conditions affect the transcription of a limited set of genes: pelA, pelD, and pelE expression is increased by oxygen limitation, while elevation of osmolarity activates only pelE expression (17). The regulation of pectinase gene transcription involves several regulatory systems. The KdgR repressor mediates the induction of the pectinolytic genes in the presence of pectin catabolites (28, 33). The PecS and PecT proteins, which are members of the MarR and LysR families of regulators, respectively, repress the transcription of most of the pectinase genes (34, 41). The signals triggering PecS and PecT controls have not been identified. The cyclic AMP receptor protein (CRP), which modulates the expression of many catabolite operons, is essential for activating the transcription of the pectinase genes (32).

In this paper, we present the identification of the pelX gene of E. chrysanthemi 3937 and the characterization of its product, the exopolygalacturonate lyase, PelX. pelX expression was monitored by using an uidA transcriptional fusion. We assayed the fusion under various environmental conditions and tested the involvement of KdgR, PecS, PecT, and CRP in pelX regulation. PelX was purified to analyze its biochemical properties. To clarify the role of PelX in pectin degradation, we determined its best substrate by using purified oligogalacturonates and its cellular localization in E. chrysanthemi 3937.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1.

Media and growth conditions. Cells were grown in either Luria-Bertani or M63 medium (25). When required, the media were solidified with agar (15 g · liter¹). E. chrysanthemi and Escherichia coli cells were usually incubated at 30 and 37°C, respectively. Carbon sources were added at 2 g · liter¹ except for polygalacturonate (grade II; Sigma Chemical Co.), which was used at 4 g · liter¹. Pectate agar medium was used to detect pectinolytic activity, which is visualized, after addition of 1 M CaCl₂, by pitting of the colony in the medium due to polygalacturonate cleavage (20). The media used to test the different physiological conditions have been described previously (17). When required, antibiotics were added at the following concentrations: kanamycin, 20 µg · ml¹; ampicillin, 50 µg · ml¹; chloramphenicol, 20 µg · ml¹; streptomycin, 100 µg · ml¹.

Matings and transductions. Plasmid pULB110, a kanamycin-sensitive RP4::mini Mu derivative (44), was used for chromosome mobilization as previously described (18). The pelX-Km insertion was transduced into various strains by using the phi-EC2 generalized transducing phage (31).

Cellular fractionation and analytical procedures. Periplasmic proteins were released from E. coli cells by osmotic shock (9) and from E. chrysanthemi cells by spheroplast formation (37). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on slab gels (4% stacking gel and 12% separating gel) with the mini-protean II system (Bio-Rad). Proteins were stained with Coomassie blue G-250. Isoelectrofocusing was performed in a 3.5 to 10 pH gradient with Pharmalytes. To perform the differential detection of pectin depolymerases after electrofocusing, the gel was incubated in 50 mM Tris-HCl (pH 8.5)-5 g of polygalacturonate liter¹ either with 1 mM CaCl₂ (to detect the major pectate lyases) or with 1 mM MnCl₂ (to detect PelX and PehX). After 10 to 60 min of incubation at 25°C, the gel was rinsed with water and stained with 0.05% ruthenium red.

Overproduction and purification of PelX. The pelX gene was overexpressed by using the T7 promoter-T7 RNA polymerase system (42). The 2.9-kb MunI-XbaI fragment from plasmid pPN1 was inserted into the pT7-5 expression vector. The resulting plasmid, pTaX, was introduced into E. coli K38/pGP1.2, which contains the T7 RNA polymerase gene under the control of the cI857 thermosensitive repressor. The plasmid-encoded proteins were labelled with [³⁵S]cysteine-[³⁵S]methionine after thermal induction of the T7 polymerase (42).

1

Enzyme assays. Pectate lyase activity was determined by spectrophotometrically monitoring the formation of unsaturated products from polygalacturonate at 230 nm. Unless otherwise specified, the standard assay mixture consisted of 50 mM Tris-HCl (pH 8.5), 0.1 mM CaCl₂, and 0.5 g of polygalacturonate per liter in a total volume of 1 ml. The appearance of products was monitored at 37°C for 2 min. The molar extinction coefficient of unsaturated oligogalacturonates was assumed to be 5,200 (26). One unit of activity was defined as the amount of enzyme required to produce 1 µmol of unsaturated product per min. The influence of Ca²⁺ was investigated by addition of CaCl₂ concentrations ranging from 0 to 1 mM. EDTA (0.5 and 1 mM) was added to verify the cation requirement. The optimum pH was determined by using Tris-HCl (pH 7 to 9), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)-NaOH (pH 5.5 to 7.5), and sodium phosphate (pH 6 to 8), each at 50 mM. The activity on substrates with various degrees of methylation was determined by substituting 7, 22, 45, 60, 75, and 90% esterified citrus pectins (Copenhagen Pectin) for polygalacturonate. Oligogalacturonates of defined length (G_n, with n = 2 to 7) were prepared as previously described (21).

Analysis of the depolymerase reaction products. Thin-layer chromatography was used to identify the products obtained after polygalacturonate degradation (23). The chromatogram was developed with a 5:3:2 mixture of n-butanol, water, and acetic acid, and the products were visualized by treatment with phosphomolybdic acid (23).

Recombinant DNA techniques. Preparation of plasmid or chromosomal DNA, restriction digestions, ligations, DNA electrophoresis, and transformations were carried out as described by Sambrook et al. (37). For the construction of the genomic library, E. chrysanthemi PMV4116 chromosomal DNA was partially digested with Sau3A and 3- to 6-kb fragments were isolated by electroelution. The sized fragments were ligated into pUC18 digested with BamHI and treated with alkaline phosphatase (Pharmacia). The deleted proteins PelXD1 and PelXD2 were obtained by in-frame deletions of the pelX gene present in plasmid pTaX. pTaXD1, encoding PelXD1, was obtained by deletion of a 0.9-kb NruI-SgrAI fragment (the SgrAI protruding end was filled in with Klenow enzyme before ligation). pTaXD2, encoding PelXD2, was obtained by deletion of a 0.6-kb MamI fragment.

Construction of the pelX::uidA fusion. Insertion of a uidA-Km cassette in the correct orientation generates transcriptional fusion (2). The uidA-Km cassette was liberated by SmaI digestion of pN416 and inserted into the NruI site of plasmid pPN1. In one of the recombinant plasmids, pPNa18, uidA is oriented in the same transcriptional direction as pelX, giving rise to a pelX::uidA fusion. The pPNa18 plasmid was introduced into E. chrysanthemi cells by electroporation. The pelX mutation was then integrated into the E. chrysanthemi chromosome by marker exchange recombination after successive cultures in low-phosphate medium in the presence of the appropriate antibiotic (36).

Maceration of plant tissue. Small cubes (3-mm sides) were cut from commercial Bindge potato tubers. They were placed into 0.1 M Tris-HCl (pH 8)-0.5 mM CaCl₂, and 0.1 U of PelX was added per ml. Samples were incubated at 30°C and examined after 1 to 24 h. The degree of tissue maceration was estimated by determining the ease with which the tissue could be pulled apart with a spatula. The PelD macerating enzyme was used as the positive control (23).

Pathogenicity test. Saintpaulia ionantha potted plants were infected as previously described (10). Plants were inoculated, after wounding of a leaf, with 50 µl of a bacterial suspension (10⁸ bacteria). Results of infections were scored daily for 10 days. For inoculation of potato tubers, sterile pipette tips containing 5 µl of bacterial suspension (10⁷ bacteria) were inserted into the tuber parenchyma (22). The tubers were inoculated and incubated in a dew chamber. After 1, 2, or 3 days, the tubers were sliced vertically through the inoculation point and the weight of decayed tissue was taken as the characteristic of disease severity. Chicory leaves were infected with 50 µl of a diluted bacterial suspension (10⁶ bacteria). After incubation in a dew chamber for 24 h, the length of rotted tissue was measured to estimate the disease severity. Pathogenicity tests were repeated in three independent experiments. In each experiment, both the wild-type strain and the pelX mutant were used and 30 plants were infected for each strain.

Nucleotide sequence accession number. The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank, and DDBJ nucleotide sequence databases under accession no. Y16797.

	RESULTS

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Isolation of the pelX gene and determination of its nucleotide sequence. A genomic library containing inserts of 3 to 6 kb was constructed in pUC18 by using DNA extracted from strain PMV4116, a 3937 derivative with the pelA-pelE-pelD and pelB-pelC-pelZ gene clusters deleted (4). About 3,500 clones were screened for pectinolytic activity on various media. Using a pectate semisolid agar medium, we could identify six transformants presenting a weak pectinolytic activity. The assay of enzymatic activity revealed that three clones encode a pectate lyase activity. Restriction analysis of the plasmids present in these three clones showed that pPN1, pPN15, and pPN9 contained overlapping fragments of 3.3, 4.4, and 4.5 kb, respectively (Fig. 1). Deletion analysis and subcloning experiments enabled us to reduce the size of the DNA fragment exhibiting the pectate lyase activity to a 2.8-kb MunI-PvuII fragment (Fig. 1).

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Physical map of the E. chrysanthemi 3937 pelX gene. The restriction map of the three PelX-encoding plasmids is indicated. The arrows below the MunI-PvuII fragment indicate the position and the transcription direction of the pelX gene and of adjacent genes. The flag shows the site of insertion of the uidA-Km cassette.

View larger version (69K): [in this window] [in a new window]   FIG. 2.   Comparison of the amino acid sequences of the PelX and PelL proteins. The PelX sequence of E. chrysanthemi EC16 is from reference 6, and PelL of strain 3937 was described by Lojkowska et al. (23). Colons indicates identical residues. The signal sequence of the proteins are underlined. D1 and D2 indicate the ends of the two deletions on 3937 PelX.

View larger version (8K): [in this window] [in a new window]   FIG. 3.   Alignment of the pelX promoters of E. chrysanthemi 3937 and EC16. The PelX sequence of E. chrysanthemi EC16 is from reference 6. Vertical lines indicate identical bases. The putative sequences corresponding to the translation start, Shine-Dalgarno (S.D.), 10 and 35 promoter, KdgR, and CRP-binding sites are underlined.

1

Expression of the pelX gene. A pelX::uidA transcriptional fusion was constructed by insertion of an uidA-Km cassette in the NruI site situated inside the pelX ORF (Fig. 1). After recombination into the E. chrysanthemi chromosome, the expression of the fusion was followed in various conditions (Table 2). Under noninducing conditions, pelX showed a low basal level of expression. In the presence of polygalacturonate, its transcription was stimulated about fourfold. By monitoring the expression of the fusion during the growth curve, we showed that pelX transcription increased about fourfold when the cells entered the late exponential growth phase and was coincident with production of the endo-Pels, as measured by pectate lyase activity (data not shown). In the presence of a readily utilizable carbon source, such as glucose, a fourfold decrease in pelX transcription was observed (Table 2). Oxygen limitation slightly increased pelX expression in the presence of polygalacturonate, while nitrogen starvation strongly inhibited pelX expression. Variations of the growth temperature (25, 30, or 37°C) or modification of the medium osmolarity had no significant effect on the pelX expression (Table 2).

Analysis of the pelX mutant. Growth of the pelX mutants A2524 and A2699 with polygalacturonate as the sole carbon source was compared with that of the parental strain, A350. The final growth yields were about 10⁹ bacteria per mg of polygalacturonate for the two strains. However, a clear difference was noticed in the lag phase and doubling times during exponential growth (about 110 min for A350 and 160 min for the pelX mutants) (Fig. 4). Thus, the pelX mutation affects the initiation of polygalacturonate catabolism.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Growth of E. chrysanthemi pelX mutants on polygalacturonate. The sole carbon source was polygalacturonate (4 g · liter1) in M63 medium. Growth was monitored by measuring the optical density of the culture at 600 nm (OD600). A350 is the parental strain of the two pelX mutants A2524 and A2699.

View larger version (14K): [in this window] [in a new window]   FIG. 5.   Location of the pelX gene on the genetic map of E. chrysanthemi 3937. Localization was performed by chromosomal mobilization with plasmid pULB110. The numbers indicate the percentage of cotransfer between two markers. Arrowheads point to the unselected marker.

Identification and purification of PelX. The PelX protein was overproduced in E. coli and purified to analyze its properties. Analysis by SDS-PAGE revealed the presence of a protein of about 76 kDa (Fig. 6A). Isoelectrofocusing followed by specific staining of pectinolytic activity indicated that the isoelectric point of PelX is about 9 (Fig. 6B). These data are in agreement with that deduced from the nucleotide sequence of the pelX gene. Indeed, the pelX gene of strain 3937 encodes a 734-amino-acid protein, including a typical amino-terminal signal sequence with a potential cleavage site between the two alanine residues at positions 26 and 27 (Fig. 2). A cleavage site was observed at the same position in EC16-PelX and confirmed by sequencing the N terminus of the mature protein (6). The mature 3937 PelX protein contains 708 amino acids and has a calculated molecular mass of 76,938 daltons and a calculated pI of 7.7. 

View larger version (53K): [in this window] [in a new window]   FIG. 6.   Identification of the PelX protein. (A) SDS-PAGE separation was followed by staining with Coomassie blue. Lanes: Whole-cell extract of BL21(DE3)/pTaX before (lane 1) and after (lane 2) IPTG induction; periplasmic fraction of BL21(DE3)/pTaX after IPTG induction (lane 3); pure PelX protein (lane 4). Molecular mass markers are indicated. (B) Electrofocusing was performed either at 2,500 V-h (lane 1) or at 1,500 V-h (lanes 2 to 11) and followed by specific revelation of pectate lyase activity in the presence of either CaCl2 for 10 min (lane 1) or MnCl2 for 40 min (lanes 2 to 11). Culture supernatants (lanes 1 to 5) and periplasmic fractions (lanes 6 to 9) of the E. chrysanthemi strains are shown: A837 (parental strain) (lanes 1, 2, and 6), A3497 (peh) (lanes 3 and 7), A3498 (peh pelX::Cm) (lanes 4 and 8), and A2737 (pelX::Km) (lanes 5 and 9). Lanes 10 and 11 contain purified PehX and PelX, respectively. The positions of the five major pectate lyases (PelA to PelE), PelX, and PehX are indicated. w.t., wild type. (C) Stability of the PelX deletion derivatives. E. coli K38/pGP1.2 cells carrying pTaX (lane 1), pTaXD2 (lane 2), or pTaXD1 (lane 3) were labelled with [35S]cysteine-[35S]methionine. An excess of "cold" cysteine-methionine was added 10 min later, and the cells were collected immediately (0 min) or incubated for additional 30 min at 37°C (30 min).

Biochemical characterization of PelX. The addition of EDTA totally inhibits PelX activity, but the enzyme is weakly activated by the addition of Ca²⁺ (Fig. 7A). Moreover, in contrast to the major E. chrysanthemi pectate lyases, PelX activity is not inhibited but weakly activated by other bivalent cations, such as Mn²⁺, Co²⁺, Ni²⁺, and Cu²⁺ (data not shown). Increasing the Tris-HCl concentration activated PelX, with a maximal activity at 100 mM (data not shown). This effect is linked to the ionic strength of the medium, since NaCl also affected PelX activity, mainly at low Tris-HCl concentrations. With 20 mM Tris-HCl, the addition of 50 mM NaCl provoked a twofold activation of PelX. With 100 mM Tris-HCl, the addition of 50 mM NaCl did not affect PelX activity. The optimum pH for the reaction is about 8.5, but the enzyme is active within a large range of pH (Fig. 7B). PelX retains more than 50% of its activity at neutral pH. Even at pH 7, Tris-HCl increased PelX activity in comparison with other tested buffers (Fig. 7B and data not shown). The stability of PelX (Fig. 7C) was studied by its incubation at 45°C under different conditions. In contrast to the extracellular E. chrysanthemi pectate lyases, addition of Ca²⁺ or EDTA to the protein did not modify its stability, since a decrease of 50% in PelX activity was observed after 5 min, either in the presence of one of these compounds or in their absence. In the presence of polygalacturonate, a decrease of only 25% in activity was observed after 30 min, indicating that the interaction of PelX with its substrate increased its thermostability. Pectins with up to 22% methylation were as good as polygalacturonate as substrates (Fig. 7D). The ability of PelX to macerate plant tissues was analyzed by incubating serial dilutions of purified enzymes with potato cubes. PelX was totally inactive in tissue maceration, since the addition of 0.1 U · ml¹ caused no tissue softening even after 24 h (data not shown).

View larger version (45K): [in this window] [in a new window]   FIG. 7.   Enzymatic properties of PelX. (A) The influence of Ca2+ was tested by using various concentrations of CaCl2 in 50 mM Tris-HCl (pH 8.5)-0.5 g of polygalacturonate per liter. The cation requirement was confirmed by addition of 0.5 and 1 mM EDTA. (B) The effect of pH was tested with 0.5 g of polygalacturonate per liter as substrate, in the presence of 0.1 mM CaCl2 in 50 mM ACES-NaOH buffer (pH 5.5 to 7.5 [open symbols]) or in 50 mM Tris-HCl buffer (pH 7 to 9 [solid symbols]). (C) The thermostability of PelX was monitored at 45°C after various incubation times in 50 mM Tris-HCl (pH 8.5) without (control) or with the addition of CaCl2 (0.5 mM), EDTA (0.5 mM), or polygalacturonate (0.5 g · liter1). The residual activity is given as a percentage of the initial activity. Activity toward pectins presenting various degrees of methylation (D) was determined in 50 mM Tris-HCl (pH 8.5)-0.1 mM CaCl2-0.5 g of substrate liter1.

Analysis of the PelX reaction products. To precisely determine the mode of action of PelX and PehX, the reaction products obtained on various substrates were characterized by thin-layer chromatography and high-performance anion-exchange chromatography. As previously observed (6, 8), with polygalacturonate as the substrate, both enzymes catalyze the formation of only one product. PehX liberates digalacturonate, while PelX liberates unsaturated digalacturonate (Fig. 8). PelX and PehX have no activity on saturated or unsaturated dimers. Both enzymes cleaved saturated and unsaturated trimers, but PehX activity was lower on unsaturated trimers (Fig. 8), indicating that modification of the residue situated at the nonreducing end influences the action of PehX. It was previously shown that PehX releases dimers from the nonreducing end of polygalacturonate (8). To clarify from which end of the substrate PelX releases dimers, we analyzed the products obtained after degradation of hexamers (G₆) and reduced hexamers (rG₆) by PelX and PehX (Fig. 9). PehX hydrolyzed G₆ into tetragalacturonate (G₄) and digalacturonate (G₂), while PelX cleaved G₆ into G₄ and unsaturated digalacturonate (uG₂) (Fig. 9A). The action of PehX on rG₆ demonstrated hydrolysis from the nonreducing end, since it led mainly to the appearance of G₂ and reduced tetramers (rG₄) (Fig. 9B). A small quantity of reduced dimers (rG₂) also appeared, due to further hydrolysis of rG₄ into G₂ and rG₂. PelX degraded rG₆ with very low efficiency, since a large decrease in reaction rate was observed by comparison with the action on G₆ (Fig. 9). Moreover, the weak cleavage of rG₆ by PelX did not lead to dimer formation but resulted in the formation of reduced unsaturated trigalacturonate (ruG₃) and trigalacturonate (G₃). Thus, modification of the reducing end inhibited PelX activity and provoked a shift in the PelX cleavage site (Fig. 9). These results indicate that the exopolygalacturonate lyase PelX attacks the reducing end of the substrate while the exo-poly--D-galacturonosidase PehX attacks its nonreducing end (Fig. 10).

View larger version (57K): [in this window] [in a new window]   FIG. 8.   Separation of the PelX and PehX reaction products by thin-layer chromatography. The reaction mixtures contained 0.1 M Tris-HCl (pH 8), 0.1 mM CaCl2, 5 U of enzymatic activity per ml, and 1.5 mg of the following substrates per ml: digalacturonic acid (G2), trigalacturonic acid (G3), a mixture of unsaturated di- and trigalacturonic acids (uG2, uG3), or polygalacturonic acid (PGA). Incubations were performed at 30°C for 3 h after addition of PelX or PehX or without enzyme (). A 5-µl sample from each reaction mixture was applied to chromatogram sheets. The positions of the individual compounds are indicated by arrows. It should be noted that the staining method used is not sensitive enough to detect the unsaturated monomer which appears by degradation of uG3 with PelX.

View larger version (30K): [in this window] [in a new window]   FIG. 9.   Analysis of PelX and PehX reaction products on hexagalacturonate (A) and reduced hexagalacturonate (B) by high-pressure liquid chromatography. For PelX, the reaction mixtures (0.5 ml) contained 0.5 mM substrate in 20 mM Tris-HCl (pH 8)-1 mM CaCl2. A 10- or 200-ng sample of PelX was used in a 30-min reaction for G6 (A) and rG6 (B), respectively. For PehX, the reaction mixtures (0.5 ml) contained 0.5 mM substrate in 20 mM Tris-HCl pH 7. 25 ng or 500 ng of PehX were used in a 5 min reaction for G6 (A) and rG6 (B), respectively. Reactions were carried out at 30°C. The blanks contained the substrate prior to enzyme addition. The reaction products were identified by using standard mixtures of uG2 and uG3 (curve 1), oligogalacturonates G1 to G6 (curve 3), and reduced oligogalacturonates rG1 to rG6 (curve 2).

View larger version (15K): [in this window] [in a new window]   FIG. 10.   Sites of attack of the E. chrysanthemi exo-enzymes PelX and PehX. The arrows indicate the site of cleavage of the polymer, polygalacturonate, for each type of enzyme produced by E. chrysanthemi, PehX, PelX, and the endo-Pels (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ). The sole product resulting from the action of the exo-polygalacturonase, PehX, or the exopolygalacturonate lyase, PelX, is indicated below the corresponding arrow.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

This paper reports the characterization of the pelX gene of E. chrysanthemi 3937 and of its product, the exopolygalacturonate lyase, PelX, which cleaves polygalacturonate by -elimination. E. chrysanthemi produces another type of exo-cleaving enzyme, the exo-poly--D-galacturonosidase, PehX, which hydrolyzes polygalacturonate (12). We showed that the two exo-cleaving pectinases, PelX and PehX, are located in the periplasm of E. chrysanthemi 3937. The exopolygalacturonate lyase activity was previously reported to be cell bound in E. chrysanthemi CUCPB1237 (7) and was supposed to be periplasmic in E. chrysanthemi EC16 (6). In contrast, PehX was found extracellularly in E. chrysanthemi CUCPB1237 and EC16 (8, 13). Analysis of various strains indicated that the polygalacturonase activity is detected in the culture supernatant of strains isolated from dicot but not from monocot hosts (35). These last strains could produce a periplasmic exo-poly--D-galacturonosidase, as shown for strain 3937.

PelX of strain 3937 was purified from recombinant E. coli cells. The activity of PelX on polygalacturonate and oligogalacturonates is inhibited by EDTA and requires low concentrations of Ca²⁺ or other bivalent cations, such as Mn²⁺, Co²⁺, Ni²⁺, or Cu²⁺. In contrast to the E. chrysanthemi endo-Pels (43), PelX is not highly dependent on pH, since good activity is observed within a wide range of pH, from 7 to 9.5. PelX can utilize polygalacturonate and partially methylated pectins as substrates. However, in contrast to EC16 PelX (6), 3937 PelX is not active on highly methylated pectins and its activity is only slightly stimulated in the presence of NaCl. The optimal conditions for 3937 PelX activity are also different from those observed for 3937 PelL, an endo-Pel having homology to the C-terminal amino acid sequence of PelX (23). This region contains a series of four to six identical residues that could be involved in the active site of these enzymes (Fig. 2). The E. chrysanthemi pectate lyases PelL and PelX belong to pectate lyase family 4 and have no homology to other described pectic enzymes. However, compared with PelL, PelX possesses an N-terminal extension of 324 residues, suggesting an organization of PelX in two domains. Exo-cleaving polysaccharidases, such as cellulases or xylanases, may present an N-terminal domain important for substrate recognition (11). To assess the role of the N-terminal extension of PelX, we constructed two derivatives containing important deletions in this region. The lack of pectinolytic activity of these deleted proteins indicated that the N-terminal region of PelX is necessary for its enzymatic activity.

Analysis of a pelX::uidA transcriptional fusion indicated that pelX expression increased in the presence of polygalacturonate, in the late exponential growth phase, and during oxygen limitation. In contrast, pelX expression was repressed in the presence of glucose and during nitrogen starvation. Therefore, like the other pel genes, the expression of pelX is controlled by several environmental stimuli. Inducibility of pectinases by pectic derivatives is mediated by the KdgR repressor, which binds to a specific DNA sequence present in the vicinity of the promoters of the controlled genes (28). The formation of pectin catabolic products, mainly KDG, provokes the dissociation of KdgR from its operators. The fact that expression of the pelX::uidA fusion is affected by kdgR or kdgK mutations indicates that pelX induction in the presence of polygalacturonate is dependent on the KdgR-KDG couple. Since pelX expression is very low in the crp mutant, CRP is necessary for activation of its expression. The presence of sequences highly homologous to the consensus determined for the KdgR-binding site, and for the CRP-binding site in the pelX regulatory region (Fig. 3), suggests that both KdgR and CRP probably control pelX transcription by direct binding. In contrast, pelX expression is not controlled by PecS or PecT, two other proteins involved in pectate lyase regulation in E. chrysanthemi (16).

While endo-enzymes catalyze the formation of multiple products of various sizes (30), the action of exo-cleaving enzymes on polymeric substrates generates a single product. The reaction products of PehX and PelX on polygalacturonate are in accordance with their exo-activity, since they generate only digalacturonate and unsaturated digalacturonate, respectively. To determine whether PehX and PelX attack from the nonreducing or the reducing end of the substrate, we used HPAEC to analyze the products obtained after degradation of hexamers, modified or not modified on the reducing end. The exopolygalacturonate lyase, PelX, degraded the hexagalacturonate to an unsaturated dimer and tetramer. Reduced hexagalacturonate was a very poor substrate of PelX, and its degradation resulted in a shift of the bond cleaved, with formation of a trimer and an unsaturated reduced trimer. The fact that modification of the reducing extremity interfered with PelX activity demonstrated that PelX attacks the oligomer from the reducing end. The exo-poly--D-galacturonosidase, PehX, degraded the hexagalacturonate into a dimer and a tetramer. The rate of hydrolysis of the reduced hexagalacturonate was similar (data not shown) and led to dimer and reduced-tetramer formation. As expected (8), the action of PehX is typical of an enzyme that attacks from the nonreducing end. It is remarkable that E. chrysanthemi, in addition to a set of at least eight endo-Pels (39), produces two exo-enzymes able to act on both ends of the substrate (Fig. 10). These two types of activity are complementary and may be necessary when one extremity of the polymer is blocked by modification of the terminal residue (by glycosylation with a neutral sugar or by esterification).

PelX plays some role in pectin catabolism since, during growth on polygalacturonate, the pelX mutant showed a longer lag phase and doubling time than did the wild-type strain. The virulence of the pelX mutant appeared not to be affected on Saintpaulia plants or potato tubers, but the maceration observed on chicory leaves slightly decreased, indicating that PelX action may be necessary for the total effect of E. chrysanthemi on some host plants. The periplasmic enzyme PelX is probably involved in the degradation of pectic oligomers that could enter the periplasm. Analysis of the PelX activity on oligomers of various length (2 to 7 residues) demonstrated that tetragalacturonate is its best substrate. While longer oligomers remain good substrates, trigalacturonate is a poor substrate of PelX. The data obtained with oligomers suggest that PelX recognizes only 4 residues at the reducing end of the polysaccharide. In E. coli, oligosaccharides as large as maltoheptaose can enter the periplasm by specific porines (45). The high PelX activity on tetra- to heptagalacturonates suggests that these pectic oligomers could enter the E. chrysanthemi periplasm. In the periplasm, the action of PelX enables further degradation of oligomers up to dimers and trimers that can probably enter the cytoplasm. Oligogalacturonates longer than trimers are poor substrates for the first enzyme of the cytoplasmic catabolic pathway, oligogalacturonate lyase (27). The role of the periplasmic exo-enzymes PelX and PehX is thus probably to be intermediate elements in pectin degradation, acting between the extracellular endo-Pels and the cytoplasmic oligogalacturonate lyase.