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.

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.

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.

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.

Cloning of genes encoding pectolytic enzymes from a genomic library of the phytopathogenic bacterium, Erwinia chrysanthemi.

Erwinia chrysanthemi are phytopathogenic enterobacteria causing soft-rot disease due to pectolytic enzymes degrading plant cell walls. We constructed a genomic library from Sau3A-digested E. chrysanthemi B374 DNA cloned in the BamHI site of the broad-host-range cosmid pMMB33 grown in Escherichia coli. Out of 1500 kanamycin-resistant (KmR) transductants of E. coli, nine pectolytic-enzyme-positive clones were identified. One of these contained the pEW325 cosmid with a 35-kb insert of Erwinia DNA. Cell extracts of E. coli harboring the cosmid pEW325 were fractionated on a polyacrylamide electrofocusing gel; bands with pectolytic activity were found to co-focus with pectolytic enzymes of E. chrysanthemi B374 strain. Cosmid pEW325 encodes three pectolytic enzymes PL10, PL20 and PL130 with isoelectric points of about 9.3, 9.2 and 4.6, respectively. These enzymes are lyases that cleave polygalacturonate by transelimination, and give rise to unsaturated products. A 15-kb HindIII fragment coding for polygalacturonate lyases was subcloned in pBR322, and a physical map of the resulting plasmid pPL01 was constructed. Starting from the pPL01, various endonuclease-generated fragments were subcloned into pBR322. Genes encoding pectate lyases were localized within an 8-kb fragment (pPL04) and then in a 2.7-kb fragment (pPL03). Polygalacturonate lyases are expressed at various levels; they accumulated in the periplasmic space of E. coli host, whereas E. chrysanthemi secreted these enzymes into the culture medium.

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.

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.

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.

Molecular characterization of the Erwinia chrysanthemi kdgK gene involved in pectin degradation.

The pathways of pectin and galacturonate catabolism in Erwinia chrysanthemi converge to form a common intermediate, 2-keto-3-deoxygluconate (KDG), which is phosphorylated by KDG kinase encoded by the kdgK gene. We cloned the kdgK gene of E. chrysanthemi 3937 by complementing an Escherichia coli kdgK mutation, using an RP4-derivative plasmid. One of the kdgK R-prime plasmids harbored a DNA insert of about 80 kb and carried the uxuA and uxuB genes involved in glucuronate catabolism and the celY gene coding for an E. chrysanthemi cellulase. The kdgK and celY genes were precisely located on this plasmid, and their respective transcriptional directions were determined. The nucleotide sequence of the kdgK region indicated that the kdgK reading frame is 981 bases long, corresponding to a protein of 329 amino acids with a molecular mass of 36,377 Da. Analysis of the deduced primary amino acid sequence showed that this enzyme is a new member of the PfkB family of carbohydrate kinases. Expression of kdgK is controlled by a negative regulatory gene, kdgR, which represses all the steps of pectin degradation. Near the putative promoter of the kdgK gene, we identified a putative KdgR-binding site and demonstrated that the KdgR protein specifically binds in vitro to this DNA region. The KdgR-KDG couple directly mediates the phenomenon of repression or induction. The KDG kinase, by limiting the intracellular inducer concentration, appears to be a key enzyme in induction of the whole catabolic pathway.

Expanded linkage map of Erwinia chrysanthemi strain 3937.

In this paper we describe the chromosomal location of various loci in Erwinia chrysanthemi strain 3937. Auxotrophic markers were obtained by chemical mutagenesis, antibiotic resistances were isolated spontaneously and mutations in sugar utilization were obtained by means of Mu insertions. These markers were located on the genetic linkage map of strain 3937 by using a conjugative system mediated by RP4::mini-Mu plasmids which permitted transfer of genetic material from any point of origin. The location of these markers was compared to that of previously located mutations. Many genes involved in pectinolysis were also located on the E. chrysanthemi 3937 map. These results permitted us to present a new genetic map containing 61 markers distributed over 34 widely scattered loci on the chromosome. Some pairs of markers giving high cotransfer frequencies were tested for cotransduction mediated by the generalized transducing phage phi-EC2; nine cotransducing pairs were found. It appears that the chromosomal locations of many of these loci are quite different to those of the well-known enterobacterium Escherichia coli but seem similar to those described for other E. chrysanthemi strains.

Isolation of Erwinia chrysanthemi kduD mutants altered in pectin degradation.

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

Environmental conditions affect transcription of the pectinase genes of Erwinia chrysanthemi 3937.

To depolymerize plant pectin, the phytopathogenic enterobacterium Erwinia chrysanthemi produces a series of enzymes which include a pectin-methyl-esterase encoded by the pem gene and five isoenzymes of pectate lyases encoded by the five genes pelA, pelB, pelC, pelD, and pelE. We have constructed transcriptional fusions between the pectinase gene promoters and the uidA gene, encoding beta-glucuronidase, to study the regulation of these E. chrysanthemi pectinase genes individually. The transcription of the pectinase genes is dependent on many environmental conditions. All the fusions were induced by pectic catabolic products and responded, to different degrees, to growth phase, catabolite repression, temperature, and nitrogen starvation. Transcription of pelA, pelD, and pelE was also increased in anaerobic growth conditions. High osmolarity of the culture medium increased expression of pelE but decreased that of pelD; the other pectinase genes were not affected. The level of expression of each gene was different. Transcription of pelA was very low under all growth conditions. The expression of the pelB, pelC, and pem genes was intermediate. The pelE gene had a high basal level of expression. Expression of pelD was generally the most affected by changes in culture conditions and showed a low basal level but very high induced levels. These differences in the expression of the pectinase genes of E. chrysanthemi 3937 presumably reflect their role during infection of plants, because the degradation of pectic polymers of the plant cell walls is the main determinant of tissue maceration caused by soft rot erwiniae.

Molecular cloning of the outJ gene involved in pectate lyase secretion by Erwinia chrysanthemi.

The phytopathogenic enterobacterium Erwinia chrysanthemi secretes a number of enzymes involved in plant-tissue degradation, notably the five isoenzymes of pectate lyase. We have cloned a region involved in pectate lyase and cellulase secretion by complementation of non-secretory outJ mutants of E. chrysanthemi strain 3937 using the RP4::miniMu plasmid pULB110. The cloned region maps near the ade-22 marker on the E. chrysanthemi 3937 chromosome. An R-prime containing a chromosomal DNA insert of about 30kb was first obtained; subcloning into pBR325 permitted the isolation of a 4kb ClaI/SspI fragment able to complement outJ mutations in E. chrysanthemi. The isolation of phoA fusions in this fragment allowed us to determine the direction of transcription of the encoding region, which extends over about 2.5kb, and demonstrate that this region encodes exported protein(s). When the TnphoA insertions were transferred back into E. chrysanthemi chromosome, the recombined strains no longer secreted pectate lyases or cellulases. Identification of the products encoded by the ClaI/SspI fragment demonstrated that outJ encodes an 83 kD polypeptide which is processed to an 81 kD polypeptide by cleavage of a signal sequence. The cloned DNA fragment did not endow Escherichia coli with the ability to secrete pectate lyases.

Analysis of the regulation of the pelBC genes in Erwinia chrysanthemi 3937.

Erwinia chrysanthemi secretes five major isoenzymes of pectate lyases encoded by the pelABCDE genes. The nucleotide sequence of the region surrounding the pelB gene of E. chrysanthemi 3937 was determined, including the regulatory regions involved in pelB and pelC expression. Analysis of the transcripts showed that transcription of pelB or pelC gave, in both cases, only one transcript. The transcription initiation sites of both pelB and pelC were precisely determined as well as the position of the transcription termination of pelB. The pelB and pelC promoters are very similar, showing a good homology with the -35 consensus region but low homology with the -10 consensus. In both cases a KdgR-box overlaps the -35 region. The pelC gene may have two KdgR operators. Moreover, the pelB and pelC genes are preceded by other sequences presenting the typical symmetry of operator sites that could be involved in more specific regulations. Comparison of E. chyrsanthemi pel regulatory regions revealed three classes of homology: pelA, pelB-pelC and pelD-pelE. The sole regulatory sequence conserved among the three classes corresponds to the KdgR-binding site. Moreover, all the pel regulatory regions are AT-rich in contrast to the coding regions which are GC-rich. Gel retardation experiments with fragments overlapping the pelB or pelC regulatory regions demonstrated that the KdgR protein specifically binds to these regions. Other proteins probably also interact with these DNA fragments. Transcription of pelB terminates in a region corresponding to a GC-rich inverted repeat followed by a run of T residues, typical of rho-independent transcription termination sites. Moreover, preliminary results imply that a region adjacent to pelC provoke, directly or indirectly, the repression of pelB and pelC expression.

Hexuronate catabolism in Erwinia chrysanthemi.

In the phytopathogenic enterobacterium Erwinia chrysanthemi, the catabolism of hexuronates is linked to the degradation of pectic polymers. We isolated Mu lac insertions in each gene of the hexuronate pathway and used genetic fusions with lacZ (the beta-galactosidase gene of Escherichia coli) to study the regulation of this pathway. Three independent regulatory genes (exuR, uxuR, and kdgR) were found. Galacturonate and glucuronate were converted into 2-keto-3-deoxygluconate (KDG) by separate three-step pathways encoded by the uxaC, uxaB, and uxaA genes and the uxaC, uxuB, and uxuA genes, respectively. The two aldohexuronates entered the cell by a specific transport system, encoded by exuT. Wild-type strain 3937 was unable to use glucuronate as a carbon source since glucuronate was unable to induce the exuT expression. Mutants able to use glucuronate possessed an inactivated exuR gene. The product of the regulatory gene exuR negatively controlled the expression of exuT, uxaC, uxaB, and uxaA, which was inducible in the presence of galacturonate. The two genes specifically involved in glucuronate catabolism, uxuA and uxuB, formed two independent transcriptional units regulated separately, uxuB expression was not inducible, whereas uxuA expression was induced in the presence of glucuronate and controlled by the uxuR product. KDG, the common end product of both pathways, is cleaved by the kdgK and kdgA gene products. KDG enters the cell by a specific transport system, encoded by kdgT. The regulatory gene kdgR controlled the expression of kdgT, kdgK, and kdgA and partially that of the pel genes encoding pectate-lyases. The real inducer of pectate-lyase synthesis, originating from catabolism of galacturonate or glucuronate, appeared to be KDG. The genes of E. chrysanthemi affecting hexuronate catabolism are separated into six independent transcriptional units exuT, uxaCBA, uxuA, uxuB, kdgK, and kdgA, but only three gene clusters were localized on the genetic map: exuT-uxaCBA, uxuA-uxuB-kdgK, and kdgA-exuR.

Isolation of Erwinia chrysanthemi mutants altered in pectinolytic enzyme production.

Various mutations in the pectin catabolic pathway of Erwinia chrysanthemi were isolated by selection of Mu-lac insertions, resulting in expression of the lac genes inducible by pectin degradation products. This approach allowed us to isolate lacZ fusions with the genes pelC, pelD, ogl and pem, encoding pectate lyases PLc and PLd, oligogalacturonate lyase and pectin methylesterase, respectively. Moreover, we obtained mutations affecting the regulation of pectinolytic enzymes; a locus called pecl appeared to be involved in induction of pectate lyases and pectin methylesterase. A second locus, called pecL, may encode an activator protein acting on pectate lyase production. Both pecl and pecL expression are induced in the presence of pectic polymers. The expression of the pem gene was studied in more detail by analysis of the pem-lacZ fusions. The expression of pem appears to be controlled by the negative regulatory gene kdgR, which controls all the genes involved in pectin degradation (pem, pel, ogl, kduD, kdul, kdgK, kdgA). This study confirmed that 2-keto-3-deoxygluconate is a key intermediate for the induction of the pectin catabolic pathway. The three genes pem, pelD and pecl were localized in the same region, near the ade-377 marker on the genetic map of the E. chrysanthemi strain 3937. The pem gene was located more precisely on an 18kb DNA fragment containing the pelADE cluster. However, this 18kb DNA fragment did not complement the pecl mutation. The pecL mutations were located near the ile-2 marker on the genetic map of E. chrysanthemi strain 3937.

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.

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.

Regulation of expression of the uxu operon and of the uxuR regulatory gene in Escherichia coli K12.

Gene fusions between the lac structural genes and various genes of the hexuronate system of Escherichia coli K12 were isolated by the technique of Casadaban. Mud(Aprlac) and lambda plac-Mu insertion mutants were constructed in which the lac genes were fused to the regulatory region of the uxu operon. In all the uxu-lac fusion strains, beta-galactosidase expression was shown to be inducible by the natural inducers of the uxu operon (glucuronate and fructuronate) and sensitive to catabolite repression by glucose. In addition we isolated a Mud(Aprlac) fusion where the lac genes were fused to the uxuR regulatory gene. In this fusion the synthesis of beta-galactosidase reflects the regulation of the uxuR gene. In the presence of a wild-type uxuR allele, partial repression of beta-galactosidase expression was found; the repression was removed when inducer was added. This result indicates that while the uxuR gene is subject to autogenous control, the uxuR repressor may have only a low affinity for its own operator.

Aldohexuronate transport system in Erwinia carotovora.

The biochemical and physiological aspects of hexuronate transport in Erwinia carotovora were studied to approach the genetic regulation of the hexuronate degradative pathway in this bacterial species. An active transport system for glucuronate and galacturonate uptake exists in E. carotovora. The glucuronate entry reaction displayed saturation kinetics with an apparent Km of 0.05 mM (at 25 degrees C; pH 7). Galacturonate appeared to be a competitive inhibitor of glucuronate uptake with a Ki of 0.1 mM. Glucuronate permeation was not induced by glucuronate itself in wild-type strains. Galacturonate induced the uptake of glucuronate (about fivefold). The induced synthesis of the transport system was sensitive to catabolite repression by glucose. Mutants able to grow on glucuronate as the sole carbon source showed constitutive synthesis of the hexuronate transport system.