Metabolism and Virulence Strategies in Dickeya-Host Interactions.

Dickeya, a genus of the Enterobacteriaceae family, all cause plant diseases. They are aggressive necrotrophs that have both a wide geographic distribution and a wide host range. As a plant pathogen, Dickeya has had to adapt to a vegetarian diet. Plants constitute a large storage of carbohydrates; they contain substantial amounts of soluble sugars and the plant cell wall is composed of long polysaccharides. Metabolic functions used by Dickeya in order to multiply during infection are essential aspects of pathogenesis. Dickeya is able to catabolize a large range of oligosaccharides and glycosides of plant origin. Glucose, fructose, and sucrose are all efficiently metabolized by the bacteria. To avoid the formation of acidic products, their final catabolism involves the butanediol pathway, a nonacidifying fermentative pathway. The assimilation of plant polysaccharides necessitates their prior cleavage into oligomers. Notably, the Dickeya virulence strategy is based on its capacity to dissociate the plant cell wall and, for this, the bacteria secrete an extensive set of polysaccharide degrading enzymes, composed mostly of pectinases. Since pectic polymers have a major role in plant tissue cohesion, pectinase action results in plant rot. The pectate lyases secreted by Dickeya play a double role as virulence factors and as nutrient providers. This dual function implies that the pel gene expression is regulated by both metabolic and virulence regulators. The control of sugar assimilation by specific or global regulators enables Dickeya to link its nutritional status to virulence, a coupling that optimizes the different phases of infection.

Draft Genome Sequence of a Highly Virulent Strain of the Plant Pathogen Dickeya solani, IFB0099.

Dickeya solani is an important bacterial pathogen of potato cultivars in Europe. Here, we present the draft genome of D. solani strain IFB0099 isolated from potato in Poland that shows a high level of pectinolytic activity and a high virulence. This genome sequence is 5,094,121 bp and contains 4,365 protein-coding sequences.

Catabolism of Hexuronides, Hexuronates, Aldonates, and Aldarates.

Following elucidation of the regulation of the lactose operon in Escherichia coli, studies on the metabolism of many sugars were initiated in the early 1960s. The catabolic pathways of D-gluconate and of the two hexuronates, D-glucuronate and D-galacturonate, were investigated. The post genomic era has renewed interest in the study of these sugar acids and allowed the complete characterization of the D-gluconate pathway and the discovery of the catabolic pathways for L-idonate, D-glucarate, galactarate, and ketogluconates. Among the various sugar acids that are utilized as sole carbon and energy sources to support growth of E. coli, galacturonate, glucuronate, and gluconate were shown to play an important role in the colonization of the mammalian large intestine. In the case of sugar acid degradation, the regulators often mediate negative control and are inactivated by interaction with a specific inducer, which is either the substrate or an intermediate of the catabolism. These regulators coordinate the synthesis of all the proteins involved in the same pathway and, in some cases, exert crosspathway control between related catabolic pathways. This is particularly well illustrated in the case of hexuronide and hexuronate catabolism. The structural genes encoding the different steps of hexuronate catabolism were identified by analysis of numerous mutants affected for growth with galacturonate or glucuronate. E. coli is able to use the diacid sugars D-glucarate and galactarate (an achiral compound) as sole carbon source for growth. Pyruvate and 2-phosphoglycerate are the final products of the D-glucarate/galactarate catabolism.

Identification of TogMNAB, an ABC transporter which mediates the uptake of pectic oligomers in Erwinia chrysanthemi 3937.

The bacterium Erwinia chrysanthemi, which causes soft rot disease on various plants, is able to use pectin as a carbon source for growth. Knowledge of the critical step in pectin catabolism which allows the entry of pectic oligomers into the cells is scarce. We report here the first example of a transport system involved in the uptake of pectic oligomers. The TogMNAB transporter of E. chrysanthemi is a member of the ATP-binding cassette (ABC) superfamily. TogM and TogN are homologous to the inner membrane components, TogA exhibits the signature of ABC ATPases and TogB shows similarity with periplasmic ligand-binding proteins. The TogMNAB transporter is a new member of the carbohydrate uptake transporter-1 family (CUT1, TC no. 3.1.1), which is specialized in the transport of complex sugars. The four genes, togM, togN, togA and togB, are apparently co-transcribed in a large operon which also includes the pectate lyase gene pelW. The transcription of the tog operon is induced in the presence of pectic derivatives and is affected by catabolite repression. It is controlled by the KdgR repressor and the CRP activator. The TogMNAB system is able to provide Escherichia coli with the ability to transport oligogalacturonides. In E. chrysanthemi, the TogMNAB system seems to play a major role in switching on the induction of pectin catabolism. TogB also acts as a specific receptor for chemotaxis towards oligogalacturonides. The decreased capacity of maceration of a togM mutant indicates the importance of transport and/or attraction of oligogalacturonides for E. chrysanthemi pathogenicity.

Two transporters, TogT and TogMNAB, are responsible for oligogalacturonide uptake in Erwinia chrysanthemi 3937.

Erwinia chrysanthemi causes soft rot of plants by secreting pectinases which cleave pectin, a polysaccharide cementing the plant cell wall constituents. We demonstrated that two transporters mediate the uptake of the extracellularly formed oligomers in E. chrysanthemi. TogMNAB, a multicomponent transporter member of the ATP-binding cassette (ABC) superfamily, is only partially responsible for the uptake of pectic oligomers. Its action is completed by that of the second transporter, TogT, a member of the glycoside-pentoside-hexuronide (GPH) family (TC no. 2.2) which includes transporters involved in the uptake of complex sugars, mostly oligosaccharides and glycosides. Each transport system, TogMNAB and TogT, is able to independently mediate the transport of oligogalacturonides and the simultaneous inactivation of both is necessary to give a total absence of growth with pectin as the carbon source. The togT gene constitutes an independent transcriptional unit. Its expression is induced in the presence of pectic derivatives and it is subject to catabolite repression. In vitro, the repressor KdgR and the activator CRP both interact directly with the togT regulatory region. The decreased pathogenicity of single and double togT, togM mutants indicated that a deficiency in uptake of pectic oligomers leads to reduced bacterial multiplication which, in turn, limits plant maceration.

Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity.

Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the opgGH operon of E. chrysanthemi, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus mdoGH of Escherichia coli. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the opgG or opgH gene abolish OPG synthesis. The opg mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.

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.

Performance of selected microbial pectinases on synthetic monomethyl-esterified di- and trigalacturonates.

Two monomethyl esters of alpha-(1-4)-linked D-galacturonic dimers and three monomethyl esters of alpha-(1-4)-linked D-galacturonic acid trimers were synthesized chemically and further used as substrates in order to establish the substrate specificity of six different endopolygalacturonases from Aspergillus niger, one exopolygalacturonase from Aspergillus tubingensis, and four selected Erwinia chrysanthemi pectinases; exopolygalacturonan hydrolase X (PehX), exopolygalacturonate lyase X (PelX), exopectate lyase W (PelW), and oligogalacturonan lyase (Ogl). All A. niger endopolygalacturonases (PGs) were unable to hydrolyze the two monomethyldigalacturonates and 2-methyltrigalacturonate, whereas 1-methyltrigalacturonate was only cleaved by PGI, PGII, and PGB albeit at an extremely low rate. The hydrolysis of 3-methyltrigalacturonate into 2-methyldigalacturonate and galacturonate by all endopolygalacturonases demonstrates that these enzymes can accommodate a methylgalacturonate at subsite -2. The A. tubingensis exopolygalacturonase hydrolyzed the monomethyl-esterified digalacturonates and trigalacturonates although at lower rates than for the corresponding oligogalacturonates. 1-Methyltrigalacturonate was hydrolyzed at the same rate as trigalacturonate which demonstrates that the presence of a methyl ester at the third galacturonic acid from the nonreducing end does not have any effect on the performance of exopolygalacturonase. Of the four E. chrysanthemi pectinases, Ogl was the only enzyme able to cleave digalacturonate, whereas all four enzymes cleaved trigalacturonate. Ogl does not cleave monomethyl-esterified digalacturonate and trigalacturonate in case the second galacturonic acid residue from the reducing end is methyl-esterified. PehX did not hydrolyze any of the monomethyl-esterified trigalacturonates. The two lyases, PelX and PelW, were both only able to cleave 1-methyltrigalacturonate into Delta4,5-unsaturated 1-methyldigalacturonate and galacturonate.

Analysis of three clustered polygalacturonase genes in Erwinia chrysanthemi 3937 revealed an anti-repressor function for the PecS regulator.

Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes including several pectate lyases encoded by the pel genes. We characterized a novel cluster of pectinolytic genes consisting of the three adjacent genes pehV, pehW and pehX, whose products have polygalacturonase activity. The high similarity between the three genes suggests that they result from duplication of an ancestral gene. The transcription of pehV, pehW and pehX is dependent on several environmental conditions. They are induced by pectin catabolic products and this induction results from inactivation of the KdgR repressor which controls almost all the steps of pectin catabolism. The presence of calcium ions strongly reduced the transcription of the three peh genes. Their expression was also affected by growth phase, osmolarity, oxygen limitation and nitrogen starvation. In addition, the pehX transcription is affected by catabolite repression and controlled by the activator protein CRP. PecS, which was initially isolated as a repressor of virulence factors, acts as an activator of the peh transcription. We showed that the three regulators KdgR, PecS and CRP act by direct interaction with the promoter regions of the peh genes. Analysis of simultaneous binding of KdgR, PecS, CRP and RNA polymerase indicated that the activator effect of PecS results from a competition between PecS and KdgR for the occupation of overlapping binding sites. Thus, to activate peh transcription, PecS behaves as an anti-repressor against KdgR.

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

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

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.

Characterization of the exopolygalacturonate lyase PelX of Erwinia chrysanthemi 3937.

Erwinia chrysanthemi 3937 secretes several pectinolytic enzymes, among which eight isoenzymes of pectate lyases with an endo-cleaving mode (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ) have been identified. Two exo-cleaving enzymes, the exopolygalacturonate lyase, PelX, and an exo-poly-alpha-D-galacturonosidase, PehX, have been previously identified in other E. chrysanthemi strains. Using a genomic bank of a 3937 mutant with the major pel genes deleted, we cloned a pectinase gene identified as pelX, encoding the exopolygalacturonate lyase. The deduced amino acid sequence of the 3937 PelX is very similar to the PelX of another E. chrysanthemi strain, EC16, except in the 43 C-terminal amino acids. PelX also has homology to the endo-pectate lyase PelL of E. chrysanthemi but has a N-terminal extension of 324 residues. The transcription of pelX, analyzed by gene fusions, is dependent on several environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, nitrogen starvation, and catabolite repression. Regulation of pelX expression is dependent on the KdgR repressor, which controls almost all the steps of pectin catabolism, and on the global activator of sugar catabolism, cyclic AMP receptor protein. In contrast, PecS and PecT, two repressors of the transcription of most pectate lyase genes, are not involved in pelX expression. The pelX mutant displayed reduced pathogenicity on chicory leaves, but its virulence on potato tubers or Saintpaulia ionantha plants did not appear to be affected. The purified PelX protein has no maceration activity on plant tissues. Tetragalacturonate is the best substrate of PelX, but PelX also has good activity on longer oligomers. Therefore, the estimated number of binding subsites for PelX is 4, extending from subsites -2 to +2. PelX and PehX were shown to be localized in the periplasm of E. chrysanthemi 3937. PelX catalyzed the formation of unsaturated digalacturonates by attack from the reducing end of the substrate, while PehX released digalacturonates by attack from the nonreducing end of the substrate. Thus, the two types of exo-degrading enzymes appeared complementary in the degradation of pectic polymers, since they act on both extremities of the polymeric chain.

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 Ca2+ and Mn2+ as cofactors while the other Pels require only Ca2+. 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.

Pectate lyase PelI of Erwinia chrysanthemi 3937 belongs to a new family.

Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pel4, pelB, pelC, pelD, and pelE genes and a set of secondary pectate lyases, two of which, pelL and pelZ, have been already identified. We cloned the pelI gene, encoding a ninth pectate lyase of E. chrysanthemi 3937. The pelI reading frame is 1,035 bases long, corresponding to a protein of 344 amino acids including a typical amino-terminal signal sequence of 19 amino acids. The purified mature PelI protein has an isoelectric point of about 9 and an apparent molecular mass of 34 kDa. PelI has a preference for partially methyl esterified pectin and presents an endo-cleaving activity with an alkaline pH optimum and an absolute requirement for Ca2+ ions. PelI is an extracellular protein secreted by the Out secretory pathway of E. chrysanthemi. The PelI protein is very active in the maceration of plant tissues. A pelI mutant displayed reduced pathogenicity on chicory leaves, but its virulence did not appear to be affected on potato tubers or Saintpaulia ionantha plants. The pelI gene constitutes an independent transcriptional unit. As shown for the other pel genes, the transcription of pelI is dependent on various environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, temperature, nitrogen starvation, and catabolite repression. Regulation of pelI expression appeared to be dependent on the three repressors of pectinase synthesis, KdgR, PecS, and PecT, and on the global activator of sugar catabolism, cyclic AMP receptor protein. A functional KdgR binding site was identified close to the putative pelI promoter. Analysis of the amino acid sequence of PelI revealed high homology with a pectate lyase from Erwinia carotovora subsp. carotovora (65% identity) and low homology with pectate lyases of the phytopathogenic fungus Nectria haematococca (Fusarium solani). This finding indicates that PelI belongs to pectate lyase class III. Using immunoblotting experiments, we detected PelI homologs in various strains of E. chrysanthemi and E. carotovora subsp. carotovora but not in E. carotovora subsp. atroseptica.

Identification of a bacterial pectin acetyl esterase in Erwinia chrysanthemi 3937.

Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. The structural complexity of pectin requires the combined action of several pectinases for its efficient breakdown. Three types of pectinases have so far been identified in E. chrysanthemi: two pectin methyl esterases (PemA, PemB), a polygalacturonase (PehX), and eight pectate lyases (PelA, PelB, PelC, PelD, PelE, PelL, PelZ, PelX). We report in this paper the analysis of a novel enzyme, the pectin acetyl esterase encoded by the paeY gene. No bacterial form of pectin acetyl esterases has been described previously, while plant tissues and some pectinolytic fungi were found to produce similar enzymes. The paeY gene is present in a cluster of five pectinase-encoding genes, pelA-pelE-pelD-paeY-pemA. The paeY open reading frame is 1650 bases long and encodes a 551-residue precursor protein of 60704Da, including a 25-amino-acid signal peptide. PaeY shares one region of homology with a rhamnogalacturonan acetyl esterase of Aspergillus aculeatus. To characterize the enzyme, the paeY gene was overexpressed and its protein product was purified. PaeY releases acetate from sugar-beet pectin and from various synthetic substrates. Moreover, the enzyme was shown to act in synergy with other pectinases. The de-esterification rate by PaeY increased after previous demethylation of the pectins by PemA and after depolymerization of the pectin by pectate lyases. In addition, the degradation of sugar-beet pectin by pectate lyases is favoured after the removal of methyl and acetyl groups by PemA and PaeY, respectively. The paeY gene was first identified on the basis of its regulation, which shares several characteristics with that of other pectinases. Analysis of the paeY transcription, using gene fusions, revealed that it is induced by pectic catabolic products and is affected by growth phase, oxygen limitation and catabolite repression. Regulation of paeY expression appears to be dependent on the KdgR repressor, which controls all the steps of pectin catabolism, and on the catabolite regulatory protein (CRP), the global activator of sugar catabolism. The contiguous pelD, paeY and pemA genes are transcribed as an operon from a promoter proximal to pelD which allows the regulation by KdgR and CRP. However, transcription can be interrupted at the intra-operon Rho-independent terminator situated between pelD and paeY. The paeY mutant inoculated into Saintpaulia plants was less invasive than the wild-type E. chrysanthemi strain 3937, demonstrating the important role of PaeY in the soft-rot disease.

Comparative analysis of the five major Erwinia chrysanthemi pectate lyases: enzyme characteristics and potential inhibitors.

In Erwinia chrysanthemi 3937, pectate lyase activity mainly results from the cumulative action of five major isoenzymes, PelA to PelE. Comparison of their amino acid sequences revealed two families, PelB-C and PelA-D-E. Molecular cloning permitted expression of the different pel genes in Escherichia coli and the isolation of each Pel independently from the other isoenzymes. We used similar experimental conditions to overproduce and purify the five Pels in a one-step chromatography method. We analyzed some of the basic enzymatic properties of these five isoenzymes. PelA has a low specific activity compared to the other four enzymes. PelB and PelC have a high affinity for their substrate: about 10-fold higher than the enzymes of the PelA-D-E group. The optimum pH is more alkaline for PelB and PelC (about 9.2) than for PelA, PelD, and PelE (from 8 to 8.8). Below pH 7, activity was negligible for PelB and PelC, while PelA, PelD, and PelE retained 25 to 30% of their activities. The temperature optima were determined to be 50 degrees C for PelD and PelE, 55 degrees C for PelA, and 60 degrees C for PelB and PelC. Enzymes of the PelB-C group are more stable than those of the PelA-D-E group. Use of substrates presenting various degrees of methylation revealed that PelA, PelD, and PelE are active only for very low levels of methylation, while PelB and PelC are more active on partially methylated pectins (up to 22% for PelC and up to 45% for PelB). Pectate lyases have an absolute requirement for Ca2+ ions. For the five isoenzymes, maximal activity was obtained at a Ca2+ concentration of 0.1 mM. None of the tested cations (Ba2+, Co2+, Cu2+, Mg2+, Mn2+, Sr2+, Zn2+) can substitute for Ca2+. At a high concentration (1 mM), most of the divalent cations inhibited pectate lyase activity. In addition, we demonstrated that two compounds present in plant tissues, epicatechin and salicylic acid, inhibit the pectate lyases at a concentration of 0.2 mM.

Characterization of a periplasmic peptidyl-prolyl cis-trans isomerase in Erwinia chrysanthemi.

The main determinant of the plant pathogen Erwinia chrysanthemi virulence is the production of extracellular enzymes, mainly pectate lyases. Adjacent to a pectate lyase encoding locus, we identified the gene rotA supposed to encode a folding catalyst. Overproduction of the protein and assay of activity using a synthetic substrate, confirmed that rotA encodes a periplasmic peptidyl-prolyl cis-trans isomerase. rotA disruption provokes no change in cell morphology, cell viability, growth rate or stability of the extracellular and periplasmic proteins. In addition, this mutation does not alter the activity of the pectate lyases, their stability in the periplasm during the transitory step of secretion or their recognition by the Out secretory system. rotA expression was followed using a rotA::uidA transcriptional fusion. Some environmental conditions, such as temperature variations and nitrogen starvation, modulate rotA expression. In contrast to the E. coli rotA gene, the E. chrysanthemi rotA possesses only one promoter and is not controlled by the CRP global regulator.

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.

Regulatory systems modulating the transcription of the pectinase genes of Erwinia chrysanthemi are conserved in Escherichia coli.

To depolymerize plant pectin, the phytopathogenic enterobacterium Erwinia chrysanthemi produces five isoenzymes of pectate lyases encoded by the five genes pelA, pelB, pelC, pelD and pelE. In Er. chrysanthemi, all genes involved in pectin degradation are specifically controlled by the KdgR repressor and are induced in the presence of a pectin catabolic product, 2-keto-3-deoxygluconate (KDG). transcription of the pectinase genes is dependent on many environmental conditions. Transcriptional fusions present on low-copy-number plasmids were used to study the regulation of the pel genes in a heterologous host, Escherichia coli. Some physiological regulations that take place in Er. chrysanthemi are conserved in E. coli. The five pel fusions in E. coli are affected by growth phase, catabolite repression and anaerobic growth conditions and are induced in the presence of galacturonate, a sugar whose catabolism leads to the formation of KDG, the inducer of pel transcription in Er. chrysanthemi. Expression of pelE increased with the osmolarity of the culture medium. In contrast, the regulation of pel expression by temperature or nitrogen starvation, observed in Er. chrysanthemi, was not conserved in E. coli, suggesting that the mechanisms responsible for these regulations are specific to Er. chrysanthemi. Analysis of different E. coli mutants allowed some regulators affecting the transcription of the pel genes to be identified. In E. coli, the growth-phase regulation of the pel genes is not dependent on the RpoS sigma factor and the fnr gene is not involved in the increase of pel expression in oxygen-limited conditions. The gene hns, involved in the regulation of numerous genes, appears to affect pel expression but the effects of E. coli hns mutations are not related to osmoregulation. In contrast, this analysis clearly demonstrates the interchangeability of two regulatory systems of E. coli and Er. chrysanthemi: the global control exerted by the catabolite activator protein CAP and the specific regulation mediated by the KdgR repressor.