INTRODUCTION

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The enterobacterium Erwinia chrysanthemi causes soft rot disease of various plants. Its pathogenicity is largely due to its ability to degrade pectin, a major constituent of plant cell walls and middle lamellae. The breakdown of pectin, a polysaccharide with a backbone consisting of partially esterified galacturonic acid, involves a battery of pectinases. The combination of different enzymatic activities allows E. chrysanthemi to efficiently degrade pectin and to subsequently use the liberated pectic oligomers as a carbon source for growth. The pectin esterases (pectin methylesterase and pectin acetylesterase) remove the ester groups of pectin and facilitate the action of depolymerases (pectate lyase and polygalacturonase) (12, 33). These depolymerases differ mainly in their reaction mechanisms (-elimination or hydrolysis) and in the random or terminal mode of attack of the polymer (endo or exo).

The majority of pectinases are secreted by E. chrysanthemi into the extracellular medium through the type II secretion machinery, the Out system (35). In E. chrysanthemi 3937, eight endo-pectate lyases (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ), the pectin methylesterase PemA, and the pectin acetylesterase PaeY are secreted by this system (12, 33). The endo-pectate lyases are the major pectinolytic enzymes produced by E. chrysanthemi, and they play an important role in the maceration of plant tissues. These enzymes randomly cleave, by -elimination, internal glycosidic linkages in pectic polymers, preferentially polygalacturonate or pectins with small numbers of methoxyl groups, and generate a series of oligogalacturonates with a 4,5-unsaturated residue at the nonreducing end.

Besides these extracellular enzymes, several cell-bound pectinases have been identified in E. chrysanthemi: the outer-membrane pectin methylesterase PemB (31), the periplasmic exopolygalacturonate lyase PelX and exo--D-polygalacturonosidase PehX (2, 8, 34), and the cytoplasmic oligogalacturonate lyase Ogl (3, 29). The two periplasmic exopectinases act on different extremities of the polymeric chain: PelX catalyzes the formation of unsaturated digalacturonates by attack from the reducing end, while PehX releases digalacturonates by attack from the nonreducing end (4, 34). The cytoplasmic oligogalacturonate lyase Ogl, like the pectate lyases, cleaves glycosidic linkages by -elimination, but in contrast to these enzymes, it is only active on short oligogalacturonates (22).

Pectate lyases are classified into five different families according to their primary amino acid sequences (15, 36). The E. chrysanthemi isoenzymes are distributed among these five families. Family 1 contains several bacterial pectate lyases, fungal pectin lyases, and plant proteins (9, 10, 36). It includes the E. chrysanthemi isoenzymes PelA to PelE, which are further separated into two subfamilies, PelADE and PelBC (39). Family 3 includes PelB/Pel-3 of Erwinia carotovora, PelI of E. chrysanthemi (36), and the four Pel proteins of the phytopathogenic fungus Nectria haematococca (Fusarium solani) (7). The endo-pectate lyase PelL (1, 18) and the exopolygalacturonate lyase PelX of E. chrysanthemi are the only members of family 4 (2, 34). PelZ of E. chrysanthemi is the sole characterized component of the fifth family (26). Family 2 contains the periplasmic pectate lyases PelB of Erwinia carotovora (11) and PelY of Yersinia pseudotuberculosis (20). Despite its homology with family 2 pectate lyases, no pectinolytic activity was observed for the product of the E. chrysanthemi kdgC gene (5).

In this study, we showed that the kdgC gene of E. chrysanthemi 3937 is longer than previously supposed (5). The corresponding protein was overproduced, and we demonstrated that it encodes an exopolygalacturonate lyase. Hence, the kdgC gene was renamed pelW. The biochemical properties of PelW and its cellular localization were analyzed. To clarify the role of this cytoplasmic enzyme in pectin degradation, we compared its enzymatic properties with those of the other E. chrysanthemi cytoplasmic pectinase, Ogl.

	MATERIALS AND METHODS

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Strains, media, and growth conditions. Bacterial strains and plasmids used in this study are listed in Table 1. Cells were grown in either Luria-Bertani (LB) or M63 medium (21). When required, the media were solidified with agar (15 g · liter¹). E. chrysanthemi cells were usually incubated at 30°C, and Escherichia coli cells were generally incubated at 37°C. Pectate agar medium was used to detect pectinolytic activity (14). Carbon sources were added at 2 g · liter¹ except for polygalacturonate (PGA) (grade II; Sigma), which was added at 4 g · liter¹. When required, antibiotics were added at the following concentrations: kanamycin, 20 µg · ml¹; ampicillin, 50 µg · ml¹; and chloramphenicol, 20 µg · ml¹.

Protein production and cell fractionation. The pelW gene was overexpressed by using the T7 promoter-T7 RNA polymerase system (38). The 2.9-kb EcoRV-ClaI fragment of pelW was inserted into the pT7-6 expression vector. The resulting plasmid, pT7-KC, was introduced into E. coli BL21(DE3). The BL21(DE3)/pT7-KC cells were grown at 30°C in LB medium supplemented with ampicillin (150 µg · ml¹). At an optical density at 600 nm (OD₆₀₀) of 0.8 to 1, 1 mM isopropyl--D-thiogalactopyranoside (IPTG) was added, and the cells were grown for an additional 2 to 3 h. The cells were harvested by centrifugation for 10 min at 5,000 × g and 4°C and were subjected to osmotic shock for extraction of the overproduced protein (6).

1

Protein labelling. Plasmid-encoded proteins were exclusively labelled with [³⁵S]cysteine-[³⁵S]methionine by using the T7 promoter-T7 polymerase system (38). For the cell fractionation experiments, cells were labelled for 10 min. Inhibition of the signal sequence processing was performed by adding 0.1 mM carbonylcyanide-m-chlorophenylhydrazone (CCCP) into an induced cell culture 2 min after addition of [³⁵S]cysteine-[³⁵S]methionine. The labelled amino acids were chased 3 min later, and cells were lysed in Laemmli sample buffer.

Analytical procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to the procedure of Laemmli (16a). Isoelectric focusing (IEF) was performed in a pH 3.5 to 10 gradient by using Pharmalytes. For differential detection of various pectate lyase activities after IEF, the gel was incubated in 50 mM Tris-HCl (pH 8.5) plus 5 g of PGA · liter¹ with either 1 mM CaCl₂ (for extracellular pectate lyase detection) or 1 mM CoCl₂ (for PelW detection). After 10 to 60 min of incubation at 25°C, the gel was rinsed with water and stained with 0.05% ruthenium red. PelW activity is sensitive to the position of the sample application papers on the IEF gel; it must be placed in the area of neutral or slightly alkaline pH.

Enzyme assays. Pectate lyase activity was determined by monitoring spectrophotometrically the formation of unsaturated products from substrate 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 PGA · liter¹. The appearance of products was monitored at 37°C over a period of 1 min (at 6-s intervals). The molar extinction coefficient of unsaturated oligogalacturonates used was 5,200 (23). One unit of activity was defined as the amount of enzyme required to produce 1 µmol of unsaturated product per minute. To assay the PelW activity, 0.1 mM MnCl₂ was substituted for the CaCl₂ and 0.1 mM trigalacturonic acid (Sigma) was substituted for the PGA.

Recombinant DNA techniques. Preparation of plasmid DNA, restriction digestions, ligations, DNA electrophoresis, and transformations were carried out as described by Sambrook et al. (30). Sequencing were performed by Genome Express SA (Grenoble, France).

Nucleotide sequence accession number. The nucleotide sequence of the pelW (kdgC) gene has been submitted to the EMBL, GenBank, and DDBJ nucleotide sequence databases under accession no. X62073.

	RESULTS

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Determination of the nucleotide sequence of the kdgC gene. The 2.3-kb EcoRV-PstI fragment containing the kdgC gene had been previously sequenced (5). However, no pectinolytic activity was found to be associated with the protein encoded by this fragment even after overproduction in different expression systems (data not shown). We decided to verify the sequence of this DNA region, mainly in the 5' and 3' ends of the potential open reading frame. This analysis revealed an error at the 3' end of the open reading frame which extends beyond the PstI site. The corrected gene is 1,659 nucleotides long and encodes a 553-amino-acid protein with a deduced molecular mass of 64,082 Da and a calculated pI of 7.7. Subcloning a 2.9-kb EcoRV-ClaI fragment downstream of the plac promoter of pBluescript (pBS-KC) allowed us to detect a moderate pectate agar pitting activity around the E. coli NM522 colonies carrying this plasmid. Assay of pectinolytic activities revealed a weak pectate lyase activity in the corresponding cell lysates. Thus, this pectate lyase gene was renamed pelW.

View larger version (44K): [in this window] [in a new window]   FIG. 1.   Comparison of the amino acid sequences of the family 2 pectate lyases. The PelY sequence of Y. pseudotuberculosis was determined by Manulis et al. (20), and PelB of E. carotovora was described by Hinton et al. (11). Vertical lines indicate identical residues. The putative signal sequence cleavage sites of PelY and PelB are indicated by triangles.

PelW identification and determination of its cellular localization. The PelW protein was overproduced in E. coli BL21(DE3)/pT7-KC cells (data not shown). A 65-kDa protein was detected by exclusive labelling of the plasmid-encoded proteins with [³⁵S]cysteine-[³⁵S]methionine (Fig. 2). Cell fractionation studies showed that this protein accumulated in the cytoplasm and that it was partially liberated by osmotic shock (Fig. 2).

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Cellular localization of PelW in E. coli. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gels were autoradiographed. E. coli BL21(DE3)/pT7-KC (PelW), E. coli BL21(DE3)/pT7-6 (pT7-6), E. coli BL21(DE3)/pT7-KCsp (PelW-SP), and E. coli K38/pGP1.2/pT-pelD (PelD) cells were fractionated and then labelled with [35S]cysteine-[35S]methionine in the absence () or in the presence (+) of CCCP. W, whole-cell lysate; P, periplasmic fraction; C, osmotically shocked cell fraction. Precursor (p) and mature (m) forms of PelW-SP and PelD are indicated by arrowheads.

View larger version (90K): [in this window] [in a new window]   FIG. 3.   Cellular localization of PelW in E. chrysanthemi. IEF was followed by specific detection of pectate lyase activity in the presence of either CaCl2 for 10 min (lanes 1 to 3) or CoCl2 for 40 min (lanes 4 to 10). The osmotic-shock-released fraction of BL21(DE3)/pT7-KC (PelW+) (lane 7) was used as a control. Subcellular fractionation of E. chrysanthemi A1077 (kdgR) (lanes 1 to 6) and E. chrysanthemi A3439 (kdgR pelW) (lanes 8 to 10) was performed. The positions of the five major E. chrysanthemi pectate lyases (PelA to PelE), PelX, and PelW are indicated. S, culture supernatant; P, periplasm; C, spheroplast lysate.

Ogl production and assay. Since PelW is a cytoplasmic pectate lyase, we decided to compare its properties with those of the other known cytoplasmic pectinase, the oligogalacturonate lyase Ogl. Overexpression of ogl in the E. coli recombinant strain BL21(DE3)/pT7-OGL yielded a high Ogl production level (about 0.4 mg · ml¹ of the culture), and the enzyme constituted about 90% of the cell protein content (data not shown). Therefore, the soluble cell fraction from BL21(DE3)/pT7-OGL was directly used for the enzyme analysis.

Substrate specificity of PelW and Ogl. To analyze the enzymatic properties of PelW, we used the fraction released by osmotic shock from E. coli BL 21(DE3)/pT7-KC. Despite the smaller amount of PelW in this fraction than in the total-cell lysate, the enzyme constituted about 20% of the total protein content of this fraction. The PelW enzymatic activity was first detected by using PGA as a substrate in the standard pectate lyase assay medium. Like the other E. chrysanthemi pectate lyases, PelW is active within a range of alkaline pH values, with an optimum at pH 8.5 in Tris-HCl buffer. In accordance with the cellular localization of this enzyme, it seemed unlikely that the polymer could be its natural substrate. Therefore, we analyzed the PelW activity on short oligogalacturonates, di-, tri-, and tetragalacturonate (G2, G3, and G4, respectively), which are more likely to be present in the E. chrysanthemi cytoplasm (Table 2). PelW did not cleave G2, while G3 and G4 were good substrates. The maximal activity was observed with G3. Determination of the K_m and V_max indicated that PelW exhibits the highest activity and affinity for G3 (Table 2). PGA was cleaved by this enzyme less effectively then G3 or G4. In comparison with the other E. chrysanthemi pectate lyases, PelW exhibits a low specific activity on PGA but a good affinity for this substrate. The higher activity observed with G3, compared with G4 or the polymer, suggested that the number of binding subsites for PelW was three, since the array of subsites is completely covered by G3. PelW shows almost the same initial reaction rate on PGA and on pectins with degrees of methylation up to 60% (data not shown). The enzyme retains about 50% of its maximal activity on 75% methylated pectin.

View larger version (38K): [in this window] [in a new window]   FIG. 4.   Identification of PelW and Ogl reaction products by thin-layer chromatography. The reaction mixtures contained 0.1 M Tris-HCl (pH 8), 0.1 mM MnCl2, 5 U of enzyme ml1, and 1.5 mg of the following substrates · ml1: digalacturonic acid (G2), trigalacturonic acid (G3), a mixture of unsaturated di- and trigalacturonic acids (uG2, uG3), a mixture of unsaturated oligogalacturonic acids (uG3 to uGn), and polygalacturonic acid (PGA). Incubations were performed at 30°C for 3 h after addition of PelW, Ogl, or PelX or without enzyme (). A 5-µl sample from each reaction was applied to a chromatogram sheet. The positions of the individual compounds are indicated by arrowheads. G1 and DKI are at the same position, but the staining method was not sensitive enough to detect the amounts of DKI applied to the chromatogram; 10 µg of DKI or G1 was applied to the lanes indicated by asterisks.

Cation requirements of PelW and Ogl. The characterized E. chrysanthemi pectate lyases have an absolute requirement for Ca²⁺. Addition of EDTA totally inhibits PelW, demonstrating its absolute cation requirement (Fig. 5A). PelW is weakly affected by the addition of Ca²⁺ but is strongly activated by Co²⁺, Mn²⁺, and Ni²⁺, with a maximum at concentrations of 0.1 to 1 mM (Fig. 5C and data not shown). Co²⁺ is the best cofactor for the enzyme. The effect of these cations is not additive, indicating that they have the same target on PelW and/or its substrate. It should be noted that the cation content of the commercially available G3 and PGA is high enough to give about 50% of the maximal PelW activity without addition of any cation (Fig. 5A and C).

View larger version (47K): [in this window] [in a new window]   FIG. 5.   Cation requirements of PelW and Ogl. The cation requirements of PelW (A) and Ogl (B) were tested by using 0.1 mM concentrations of their corresponding chloride forms, 25 µM EDTA, and 0.1 mM G3 in 0.1 M Tris-HCl (pH 8.5) as a substrate for PelW and 0.67 mM G2 in 0.1 M Tris-HCl (pH 7.0) as a substrate for Ogl. The dotted lines show the levels of enzyme activities in the absence of added EDTA or cations. , no cation added. (C) The influence of Mn2+ concentration on PelW activity was tested in 0.1 M Tris-HCl (pH 8.5)-0.1 mM G3, using various concentrations of EDTA or MnCl2. (D) The thermostability of PelW was monitored at 45°C after various incubation times in 0.1 M Tris-HCl (pH 8.0) containing 0.4 mM EDTA, either alone or with a 1 mM concentration of CaCl2, CoCl2, or MnCl2. The residual activity is given as a percentage of the initial activity.

	DISCUSSION

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This paper reports the characterization of PelW, the cytoplasmic exopolygalacturonate lyase of E. chrysanthemi 3937. It has been previously shown that the product of the kdgC gene is homologous to family 2 pectate lyases (5). However, an error in the gene sequence resulted in the failure of the authors to purify an active protein. We renamed this gene pelW after demonstrating that it encodes an exo-cleaving pectate lyase. It is not known whether the two other members of family 2, PelY of Y. pseudotuberculosis (20) and PelB of E. carotovora (11), are endo- or exo-cleaving enzymes. Their homology with PelW is not sufficient to infer a similar mode of substrate recognition. Indeed, pectate lyase family 3 includes both an exo- and an endo-cleaving enzyme, PelX and PelL, respectively (18, 34). Moreover, these enzymes are not situated in the same cellular compartment; PelL is extracellular, while PelX is located in the periplasm. Similarly, the cellular localization of the family 2 pectate lyases can differ. Both PelY and PelB possess a typical signal sequence and are periplasmic when expressed in E. coli (11, 20). We demonstrated that PelW has no signal sequence and is located in the cytoplasm. Thus, in addition to a set of at least eight extracellular endo-pectate lyases (36), E. chrysanthemi produces two intracellular exopolygalacturonate lyases, the periplasmic PelX and the cytoplasmic PelW. It is remarkable that these 10 pectate lyases are distributed among the five defined pectate lyase families, implying independent genetic acquisitions.

A cell-bound exo-pectate lyase activity was first reported in E. chrysanthemi CUCPB1237 (3). The pelX gene was then cloned from E. chrysanthemi EC16 (2) and E. chrysanthemi 3937 (34). Evidence that PelW, like PelX, is an exo-cleaving lyase arises from the fact that its action on a polymeric substrate generates a single product, unsaturated digalacturonate (Fig. 4). In contrast, an endo-cleaving enzyme would catalyze the formation of multiple products of various sizes (27). Despite the fact that the two E. chrysanthemi exopolygalacturonate lyases form the same product from the polymeric substrate PGA and show relatively good activity on this substrate, they exhibit some differences in activity on various oligomers. PelX shows a high level of activity on oligomers from G4 to G7, with a maximal activity on G4. G3 is a poor substrate for PelX (34). On the contrary, G3 is the best substrate of PelW, while this enzyme shows a lower level of activity on G4. The cellular localization of these two exopolygalacturonate lyases seems to be complementary. PelX could cleave oligomers when they pass through the periplasm, while PelW could cleave oligomers entering the cytoplasm. Thus, the two exopolygalacturonate lyases could ensure successive depolymerization of pectic substances, and their substrate preferences are linked to their cellular compartments. The data obtained with oligomers suggest that the number of subsites of PelW is only three (1 to +2); PelW seems to recognize only three residues at the reducing end of the polysaccharide. In contrast to the other pectate lyases, PelW is highly tolerant of the methylation of the substrate. It shows almost the same activity on PGA as it does on pectins with a degree of methylation up to 60%. Two E. chrysanthemi pectin methylesterases, the extracellular PemA and the outer-membrane-located PemB, are responsible for the demethylation of pectic substances in the extracellular medium and in the periplasm, respectively (17, 31). Nevertheless, the cytoplasmic lyase PelW might be able to cleave methylated oligomers escaping from the demethylation by these pectin methylesterases.

To better understand the role of the cytoplasmic pectinases in the final steps of pectin depolymerization in the E. chrysanthemi cytoplasm, we compared the properties of PelW with those of the oligogalacturonate lyase Ogl, which was the only known Erwinia cytoplasmic pectinase (3, 22). Ogl from E. carotovora was previously purified, and its catalytic properties were analyzed (22). Although the E. chrysanthemi ogl gene was cloned and sequenced (28, 29), enzymatic properties of the corresponding protein have never been studied. Nevertheless, this enzyme seems to play a key role in the pectin catabolic pathway, since E. chrysanthemi ogl mutants are unable to grow on PGA or digalacturonates as a carbon source (3, 29). This enzyme links the extracellular and intracellular steps of the pectin catabolic pathway because it cleaves the dimers produced by the combined action of all the other pectinases. However, the role of PelW in pectin catabolism is not negligible, since the E. chrysanthemi pelW (kdgC) mutant shows reduced growth on PGA (5). The role of the two enzymes PelW and Ogl is clearer when their substrate specificities are compared. Oligomers longer than dimers are very poor substrates for Ogl, while the trimer is the best substrate for PelW, which retains a good activity on the tetramer. Thus, these two enzymes seem to act in sequence during oligogalacturonate depolymerization in the cytoplasm. The substrate specificity of PelW implies that trimers, and probably tetramers, enter the E. chrysanthemi cytoplasm. The transport of pectic oligomers into the bacterial cells is the least-well-known step of pectin catabolism. Numerous mutants of E. chrysanthemi were isolated for their inability to grow on medium with PGA as the carbon source. None of them appeared to be affected with regard to transport of pectic oligomers. This result led us to theorize that several systems could be used for oligogalacturonate uptake.

The extracellular E. chrysanthemi endo-pectate lyases demonstrate an absolute Ca²⁺ requirement for their enzymatic activity (18, 25, 36, 39). These enzymes either are not affected or are inhibited in the presence of other divalent cations, with the exception of PelZ, which uses Mn²⁺ as a cofactor more efficiently than it uses Ca²⁺ (25, 39). The periplasmic exopolygalacturonate lyase PelX also requires cations for its activity and is weakly activated by Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺, or Cu²⁺ (34). It has been previously reported that the E. carotovora Ogl does not require calcium ions and is not inactivated by EDTA (22). Our results show that the activities of the two E. chrysanthemi cytoplasmic lyases, PelW and Ogl, are dependent on the presence of divalent cations, since both enzymes are inhibited by EDTA. However, Ca²⁺ is unable to restore their activity, while several other metal cations of similar size, such as Co²⁺, Mn²⁺, and Ni²⁺, can activate both PelW and Ogl with different efficiencies (Fig. 5A and B). The PelW thermoinactivation data indicate a direct binding of cation to the protein, resulting in the stabilization of the protein structure (Fig. 5D). It is remarkable that both cytoplasmic lyases are activated by almost the same spectrum of cations, which is very different from the cation requirement of the extracellular pectate lyases. Ca²⁺ binds to the extracellular pectate lyases and also interacts with the carboxyl groups of their substrate, PGA. These cations have been shown to be directly involved in catalysis (16). It is possible that the cation dependence of PelW and Ogl is related to specific features of the catalytic reactions carried out by these enzymes. However, the substrate specificities and the reaction products of PelW and Ogl are quite different. Moreover, except for the cation requirement, the enzymatic properties of PelW do not essentially differ from those of PelX or of the endo-pectate lyases. Another explanation for the similar cation requirement spectra of PelW and Ogl could be related to their cytoplasmic localization. The bacterial cells maintain an intracellular calcium level below that of the growth medium. For example, in E. coli, the intracellular calcium concentration is 0.1 µM, regardless of its extracellular concentration (37). On the other hand, several specific transport systems ensure the intracellular uptake of numerous other divalent metal cations (37). Thus, the cation content of the bacterial cytoplasm could probably constrain the bacteria to use other divalent cations in the place of Ca²⁺ for similar catalytic processes.