The PDZ domain of OutC and the N-terminal region of OutD determine the secretion specificity of the type II out pathway of Erwinia chrysanthemi.

The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete multiple exoproteins by a type II pathway, the Out system. Secretion in Erwinia is species-specific: exoproteins of one species cannot be secreted by the other. We analysed the role of two components of the Out system, the bitopic inner membrane protein OutC and the secretin OutD, in the specific recognition of secreted proteins. We demonstrated that the PDZ domain of OutC determines its secretion specificity towards certain exoproteins. The secretin is the major determinant of specificity of the Out system: OutD of E. carotovora changes the secretion specificity of E. chrysanthemi and enables it to secrete heterologous exoproteins. Construction of chimeric OutD showed that the N-terminal region is the specificity domain of the secretin. Thus, both the PDZ domain of OutC and the N-terminal region of OutD are required for specific recognition of secreted proteins. Systematic analysis of the secretion of several exoproteins demonstrated that different exoproteins secreted by the Out machinery have different requirement for their presumed targeting signals on OutC and OutD. This strongly indicates that diverse exoproteins possess a variable number of targeting signals which are recognised by different regions of OutC and OutD.

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.

Crystallization and preliminary X-ray analysis of a member of a new family of pectate lyases, PeIL from Erwinia chrysanthemi.

PeIL, a pectate lyase (E.C. 4.2.2.9) from E. chrysanthemi 3937 that is not homologous to the lyases with known structures, has been purified and crystallized by the hanging-drop method using a variety of organic solvents as precipitant. Elongated lathes grown from poly(ethylene glycol) plus isopropanol belong to the space group P212121 with cell dimensions a = 55.5, b = 58.2, c = 16.4 A with a single molecule in the asymmetric unit. Although complete data sets have been collected to 2.3 A resolution, these crystals diffract to at least 1.9 A resolution and are suitable for structure determination. Chunky plates grown using other organic solvents as the precipitant diffracted to 3 A resolution and were partially characterized as a second orthorhombic crystal form with space group P21212 and cell dimensions a = 119.1, b = 140.5 and c = 105.4 A, suggesting four molecules in the asymmetric unit.

Synthesis of the two monomethyl esters of the disaccharide 4-O-alpha-D-galacturonosyl-D-galacturonic acid and of precursors for the preparation of higher oligomers methyl uronated in definite sequences.

Methyl (alpha-D-galactopyranosyluronic acid)-(1-->4)-D-galactopyranuronate and methyl alpha-D-galactopyranosyl-uronate-(1-->4)-D-galactopyranuronic acid have been synthesized by coupling methyl (benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate (3) or benzyl (benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate (4) with benzyl (phenyl 2,3,4-tri-O-benzyl-1-thio-beta-D-galactopyranosid)uronate and methyl (phenyl 2,3,4-tri-O-benzyl-1-thio-beta-D-galactopyranosid)uronate, respectively, using N-iodosuccinimide and trifluoromethanesulphonic acid as promoters, followed by removal of the benzyl groups. The 4'-OH unprotected dimers benzyl (methyl 2,3-di-O-benzyl-alpha-D-galactopyranosyluronate)-(1-->4)-(benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate and methyl (benzyl 2,3-di-O-benzyl-alpha-D-galactopyranosyluronate)-(1-->4)-(benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate were prepared from methyl (phenyl 2,3-di-O-benzyl-1-thio-4-O-trimethylsilyl-beta-D-galactopyranosid) uronate and benzyl (phenyl 2,3-di-O-benzyl-1-thio-4-O-trimethylsilyl-beta-D-galactopyranosid) uronate and acceptors 4 or 3, respectively. These compounds have been designed to serve as precursors for the preparation of higher-membered D-galacturonic acid oligomers methyl esterified in definite positions.

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.

Specific interaction between OutD, an Erwinia chrysanthemi outer membrane protein of the general secretory pathway, and secreted proteins.

OutD is an outer membrane component of the main terminal branch of the general secretory pathway (GSP) in Erwinia chrysanthemi. We analyzed the interactions of OutD with other components of the GSP (Out proteins) and with secreted proteins (PelB, EGZ and PemA). OutD is stabilized by its interaction with another GSP component, OutS. The 62 C-terminal amino acids of OutD are necessary for this interaction. In vivo formation of OutD multimers, up to tetramers, was proved after the dissociation in mild conditions of the OutD aggregates formed in the outer membrane. Thus, OutD could form a channel-like structure in the outer membrane. We showed that OutD is stabilized in vivo when co-expressed with Out-secreted proteins. This stabilization results from the formation of complexes that were detected in experiments of co-immunoprecipitation and co-sedimentation in sucrose density gradients. The presence of the N-terminal part of OutD is required for this interaction. The interaction between OutD and the secreted protein PelB was confirmed in vitro, suggesting that no other component of the GSP is required for this recognition. No interaction was observed between the E. carotovora PelC and the E. chrysanthemi OutD. Thus, the interaction between GspD and the secreted proteins present in the periplasm could be the key to the specificity of the secretion machinery and a trigger for that process.

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.

Characterization of pectin methylesterase B, an outer membrane lipoprotein of Erwinia chrysanthemi 3937.

The secretion of extracellular pectinases, among which there are least six isoenzymes of pectate lyase and one pectin methylesterase, allows the phytopathogenic bacterium Erwinia chrysanthemi to degrade pectin. A gene coding for a novel pectin methylesterase has been cloned from an E. chrysanthemi strain 3937 gene library. This gene, pemB, codes for a 433-amino-acid protein. The PemB N-terminal region has the characteristics of lipoprotein signal sequences. We have shown that the PemB precursor is processed and that palmitate is incorporated into the mature protein. The PemB lipoprotein is not released into the extracellular medium and is localized in the outer membrane. The PemB sequence presents homology with other pectin methylesterases from bacterial and plant origin. pemB-like proteins were detected in four other E. chrysanthemi strains but not in Erwinia carotovora strains. PemB was overproduced in Escherichia coli and purified to homogeneity. PemB activity is strongly increased by non-ionic detergents. The enzyme is more active on methylated oligogalacturonides than on pectin, and it is necessary for the growth of the bacteria on oligomeric substrates. PemB is more probably involved in the degradation of methylated oligogalacturonides present in the periplasm of the bacteria, rather than in a direct action on extracellular pectin. pemB expression is inducible in the presence of pectin and is controlled by the negative regulator KdgR.

Differential effect of dsbA and dsbC mutations on extracellular enzyme secretion in Erwinia chrysanthemi.

An Erwinia chrysanthemi gene able to complement an Escherichia coli dsbA mutation has been cloned and sequenced. This gene codes for a periplasmic protein with disulphide isomerase activity that has 69% identity and 94% similarity with the E. coli DsbA protein. An E. chrysanthemi dsbA-uidA fusion mutant has been constructed. dsbA expression seems to be constitutive. This mutant has multiple phenotypes resulting from the absence of disulphide bond formation in periplasmic and secreted proteins. Pectate lyases and the cellulase EGZ are rapidly degraded in the periplasm of the dsbA mutant. E. chrysanthemi synthesizes another periplasmic protein with disulphide isomerase activity, namely DsbC. The dsbC gene introduced on a multicopy plasmid in a dsbA mutant was only partially able to restore EGZ secretion, indicating that even if DsbA and DsbC possess disulphide oxydoreductase activity, they are not completely interchangeable. Moreover, pectate lyases expressed in an E. coli dsbA mutant were very instable but their stability was unaffected in a dsbC mutant. These results indicate that DsbA and DsbC could have different substrate specificities.

[Expression of pectate lyase genes of Erwinia carotovora subsp. carotovora 17A and Erwinia carotovora subsp. atroseptica 36A in Erwinia carotovor substp. atroseptica 36A cells]. / Ekspressiia genov pektatliaz Erwinia carotovora subsp. carotovora 17A i Erwinia carotovora subsp. atroseptica 36A v kletkakh Erwinia corotovora subsp. atroseptica 36A.

E.atroseptica 36A cells were transformed by the recombinant plasmids p27-1 and pEA364 (derivatives of the vector plasmid pUC19) containing pectate lyase genes of E.carotovora 17A and E.atroseptica 36A, respectively. The synthesis of pectate lyases determined by the cloned genes of bacteria of both subspecies, as well as the synthesis of the native enzymes, were induced by sodium poly pectate. Increase of the dose of pectate lyase genes did not result in alteration of pectate lyase secretion by E.atroseptica 36ApEA364 cells. At the same time, the efficiency of secretion of heterologous pectate lyases by E.atroseptica 36Ap27-1 cells was lower. The synthesis and secretion of the resident isoenzymes are as efficient as those of the parental cells. The results indicate a high specificity of the pectinase secretory system in Erwinia of different species and, moreover, subspecies.

Characterization of DsbC, a periplasmic protein of Erwinia chrysanthemi and Escherichia coli with disulfide isomerase activity.

We identified and characterized an Erwinia chrysanthemi gene able to complement an Escherichia coli dsbA mutation that prevents disulfide bond formation in periplasmic proteins. This gene, dsbC, codes for a 24 kDa periplasmic protein that contains a characteristic active site sequence of disulfide isomerases, Phe-X-X-X-X-Cys-X-X-Cys. Besides the active site, DsbC has no homology with DsbA, thioredoxin or eukaryotic protein disulfide isomerase and it could define a new subfamily of disulfide isomerases. Purified DsbC protein is able to catalyse insulin oxidation in a dithiothreitol dependent manner. The E.coli gene xprA codes for a protein functionally equivalent to DsbC. The in vivo function of DsbC seems to be the formation of disulfide bonds in proteins. The presence of XprA could explain the residual disulfide isomerase activity existing in dsbA mutants. Re-oxidation of XprA does not seem to occur through DsbB, the protein that probably re-oxidizes DsbA.

[Expression of pel genes of Erwinia chrysanthemi ENA49 in Erwinia carotovora var. atroseptica 36A cells]. / Ekspressiia pel-genov Erwinia chrysanthemi ENA49 v kletkakh Erwinia carotovora var. atroseptica 36A.

Erwinia atroseptica 36A cells were transformed by the recombinant plasmid pPL5-1 (a derivative of the vector plasmid pUC19) containing pelb and pelc genes which encode pectate lyases of Erwinia chrysanthemi ENA49. Synthesis of pectate lyases PLB and PLC determined by the cloned pel genes is constitutive in Erwinia atroseptica 36ApPL5-1 cells and not inducible by sodium polypectate. The major part of these enzymes was accumulated in the periplasmic fraction of Erwinia atroseptica and cells were unable to efficiently secrete the enzymes into the cultural medium. Synthesis and secretion of the native pectate lyases by Erwinia atroseptica harboring the plasmid were as efficient as by the parental cells. The obtained results suggest the high specificity of pectate lyase secretory systems of kindred Erwinias.

Production of pectolytic enzymes fromErwinia grown on different carbon sources.

A quantitative analysis of pectolytic enzymes (polygalacturonase (PG), pectin methyl esterase (PME) and six isoenzymes of pectate lyase (PL)) produced byErwinia bacteria in the presence of diverse carbon sources was made by preparative electrophoresis. Synthesis of each of these enzymes was regulated independently; different induction and repression ratios (about 10- to 1000-fold) were observed for diverse PL isoenzymes, PG and PME. The possibility of using specially constructed media for the production of pectinase complexes with a specific spectra of pectolytic enzymes has been demonstrated.

[Secretion of pectate lyase and protein composition of the outer membrane of Erwinia chrysanthemi ENA49 cells]. / Sekretsiia pektatliazy i sostav belkov naruzhnoi membrany kletok Erwinia chrysanthemi ENA49.

The combined changes (specters of isoenzymes of intracellular and extracellular pectatelyase, protein composition of periplasm and outer membrane) in the cells of E. chrysanthemi ENA49 from the periodical culture growing on the inducer containing medium have been studied. The beginning of active pectatelyase synthesis was established to be accompanied by the temporal intracellular accumulation of the enzyme. The cellular capability of pectatelyase secretion to the outer medium correlates with the presence of the definite protein in the outer membrane. The different pathways for secretion of pectatelyase isoenzymes PLb, PLc, PLd and PLe are suggested.

[Expression of the pectate lyase gene from Erwinia chrysanthemi ENA49 in cells of other Erwinia species]. / Ekspressiia gena pektatliazy Erwinia chrysanthemi ENA49 v kletkakh drugikh predstavitelei roda Erwinia.

The gene for a pectate lyase of E. chrysanthemi ENA49 cloned in a recombinant plasmid pPTL1 (a derivative of RSF1010) was transferred into E. carotovora. The pectate lyase determined by the cloned gene was secreted into the cultural medium from the cells of E. crysanthemi EC16. Partial secretion of the enzyme was registered for E. carotovora cells. The major part of EC1 E. chrysanthemi pectate lyase synthesized by E. carotovora cells is accumulated in periplasmic and cytoplasmic fractions. The obtained results suggest the different specificity or efficiency of pectate lyase secretion systems in the studied Erwinia strains.