An isoelectric focusing study of the effect of methyl-esterified pectic substances on the production of extracellular pectin isoenzymes by soft rot Erwinia spp.

The production of extracellular pectic isoenzymes by seven strains of soft rot bacteria, Erwinia carotovora subsp. carotovora, E.c. atroseptica and E. chrysanthemi , when grown in media containing four different pectic substances with different degrees of methylation or with potato tuber cell-wall extract was examined by isoelectric focusing activity staining. In addition to the isoenzymes of pectate lyase, polygalacturonase and pectin methyl esterase produced constitutively or following induction by polygalacturonic acid (PGA) and coded by known genes, between two and seven novel isoenzymes of the three enzymes with a wider pI range were apparently induced by the pectins and cell-wall extract. Pectin lyase, which is induced in vitro by DNA-damaging agents, was not produced in the absence of mitomycin C in a medium containing PGA but up to two isoenzymes were found with pectin or cell-wall extract. In contrast, cellulase isoenzyme production was not affected by pectin or cell-wall extract. A greater number of novel isoenzymes of all pectic enzymes except pectin lyase tended to be produced in media containing Link pectin, which is PGA methylated to 98%, than the other pectic substances and cell-wall extract. Pectate lyase and polygalacturonase were induced by pectin lyase-degraded products of highly methylated pectin but not by PGA in an E. chrysanthemi strain with all its known pei and peh genes mutated. The results suggest that the production of novel pectic isoenzymes could be related to the presence of CH⁺₃ groups and that their induction differs from that for isomers induced by PGA-degraded products and DNA-damaging agents or produced constitutively.     

pULB113, an RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of Erwinia carotovora.

The RP4::mini-Mu plasmid pULB113, transferred from Escherichia coli strain MXR, was stable and transfer proficient in Erwinia amylovora strain EA303, E. carotovora subsp. atroseptica strain ECA12, E. carotovora subsp. carotovora strain ECC193, and E. chrysanthemi strain EC183. The plasmid mobilized an array of Erwinia sp. chromosomal markers (E. amylovora: his+,ilv+,rbs+,ser+,thr+;E. chrysanthemi:arg+,his+,ilv+,leu+; E. carotovora subsp. atroseptica: arg+,gua+,leu+,lys+,pur+,trp+; E. carotovora subsp. carotovora: arg+,gua+,leu+,lys+,out+[export of enzymes],pur+,trp+), suggesting random interactions of the plasmid with the chromosomes. In E. carotovora subsp. carotovora, pULB113-mediated two-factor crosses revealed linkage between three auxotrophic markers and the out loci. The export of pectate lyase, polygalacturonase, and cellulase and the maceration of potato tuber tissue occurred with Out+, but not Out-, strains of E. carotovora subsp. carotovora, indicating the importance of enzyme export in plant tissue maceration. Erwinia sp. donors harboring pULB113 complemented mutations in various biosynthetic and catabolic genes (arg, gal, his, leu, met, pro, pur, thy) in Escherichia coli recA strains. Escherichia coli transconjugants harbored pULB113 primes as indicated by the cotransfer of Erwinia genes and pULB113 markers and a change in plasmid mass. Moreover, the PstI and SmaI cleavage patterns of selected pULB113 primes were different from those of pULB113. pULB113 primes carried DNA insertions ranging from 3 to about 160 kilobases. These findings indicate that pULB113 is useful for in vivo gene cloning and genetic analysis of various enterobacterial phytopathogens.     

Characterization of a monoclonal antibody against active pectate lyase from Erwinia carotovora

A monoclonal antibody (2E2) produced against pectate lyase from Erwinia carotovora ssp. carotovora reacted with a 41- and a 44-kilodaltion protein on Western blots of concentrated Erwinia culture supernatants resolved by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. It was unequivocally shown that monoclonal 2E2 reacted with an active form of pectate lyase by affinity purifying the antigen with the monoclonal. The affinity-purified antigen was enzymatically active and moved as a single protein band in a nonequilibrium isoelectric focusing gel. Monoclonal 2E2 reacted with the pectate lyases of a diverse range of E. carotovora ssp. carotovora, ssp. atroseptica, and ssp. betavasculorum strains, as well as with one of three strains of E. chrysanthemi. The electrophoretic mobility of the major protein (44 kilodaltons) that reacted with 2E2 was identical within a subspecies but differed among subspecies.     

pULB113, an RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of Erwinia carotovora

The RP4::mini-Mu plasmid pULB113, transferred from Escherichia coli strain MXR, was stable and transfer proficient in Erwinia amylovora strain EA303, E. carotovora subsp. atroseptica strain ECA12, E. carotovora subsp. carotovora strain ECC193, and E. chrysanthemi strain EC183. The plasmid mobilized an array of Erwinia sp. chromosomal markers (E. amylovora: his+,ilv+,rbs+,ser+,thr+;E. chrysanthemi:arg+,his+,ilv+,leu+; E. carotovora subsp. atroseptica: arg+,gua+,leu+,lys+,pur+,trp+; E. carotovora subsp. carotovora: arg+,gua+,leu+,lys+,out+[export of enzymes],pur+,trp+), suggesting random interactions of the plasmid with the chromosomes. In E. carotovora subsp. carotovora, pULB113-mediated two-factor crosses revealed linkage between three auxotrophic markers and the out loci. The export of pectate lyase, polygalacturonase, and cellulase and the maceration of potato tuber tissue occurred with Out+, but not Out-, strains of E. carotovora subsp. carotovora, indicating the importance of enzyme export in plant tissue maceration. Erwinia sp. donors harboring pULB113 complemented mutations in various biosynthetic and catabolic genes (arg, gal, his, leu, met, pro, pur, thy) in Escherichia coli recA strains. Escherichia coli transconjugants harbored pULB113 primes as indicated by the cotransfer of Erwinia genes and pULB113 markers and a change in plasmid mass. Moreover, the PstI and SmaI cleavage patterns of selected pULB113 primes were different from those of pULB113. pULB113 primes carried DNA insertions ranging from 3 to about 160 kilobases. These findings indicate that pULB113 is useful for in vivo gene cloning and genetic analysis of various enterobacterial phytopathogens.     

Characterization of transposon insertion out- mutants of Erwinia carotovora subsp. carotovora defective in enzyme export and of a DNA segment that complements out mutations in E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi.

Soft-rotting Erwinia spp. export degradative enzymes to the cell exterior (Out+), a process contributing to their ability to macerate plant tissues. Transposon (Tn5, Tn10, Tn10-lacZ) insertion Out- mutants were obtained in Erwinia carotovora subsp. carotovora 71 by using plasmid and bacteriophage lambda delivery systems. In these mutants, pectate lyases, polygalacturonase, and cellulase, which are normally excreted into the growth medium, accumulated in the periplasm. However, localization of the extracellular protease was not affected. The Out- mutants were impaired in their ability to macerate potato tuber tissue. Out+ clones were identified in a cosmid library of E. carotovora subsp. carotovora 71 by their ability to complement mutants. Localization of cyclic phosphodiesterase in the periplasm indicated that the Out+ plasmids did not cause lysis or a nonspecific protein release. The Out+ derivatives of the E. carotovora subsp. carotovora 71 mutants regained the ability to macerate potato tuber tissue. Our data indicate that a cluster of several genes is required for the Out+ phenotype. While one plasmid, pAKC260, restored the Out+ phenotype in each of the 31 mutants of E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi, it failed to render Escherichia coli export proficient. Homologs of E. carotovora subsp. carotovora 71 out DNA were detected by Southern hybridizations in subspecies of E. carotovora under high-stringency conditions. In contrast, E. chrysanthemi sequences bearing homology to the E. carotovora subsp. carotovora 71 out DNA were detectable only under low-stringency hybridization. Thus, although the out genes are functional in these two soft-rotting bacterial groups, the genes appear to have diverged.     

Comparative study of regulatory mechanisms for pectinase production by Erwinia carotovora subsp. carotovora and Erwinia chrysanthemi

The production of pectinase, the major virulence determinant of soft-rot Erwinia species, is controlled by many regulatory factors. We focused on the major regulatory proteins, KdgR, CRP, Pir, and PecS, characterized mainly in E. chrysanthemi, and tested for their presence and function in the control of pectate lyase (Pel) and polygalacturonase (Peh) production in E. carotovora subsp. carotovora. Homologues of kdgR and crp but not of pir and pecS were detected by Southern blot analyses in E. carotovora subsp. carotovora. In fact, KdgR and CRP homologues of E. carotovora subsp. carotovora had high amino acid identities to those of E. chrysanthemi, including a complete match of the hypothetical helix-turn-helix DNA-binding motif. However, in Western blot analyses using anti-Pir (E. chrysanthemi) antibodies, a cross-reacting protein was present in both Erwinia species, although Pel production in E. carotovora subsp. carotovora was not further stimulated by adding plant extract into the medium containing PGA (polygalacturonic acid) in which hyperinduction by Pir has been reported in E. chrysanthemi EC16. When plasmids that contained each of these regulatory genes from E. chrysanthemi were introduced into E. carotovora subsp. carotovora, Pel production was controlled as predicted from their roles in E. chrysanthemi, except for PecS. PecS exerted a positive control in E. carotovora subsp. carotovora, in contrast to a negative control in E. chrysanthemi. DNA-binding assays demonstrated that KdgR, CRP, Pir, and PecS of E. chrysanthemi and KdgR and CRP homologues of E. carotovora subsp. carotovora could bind to the promoter regions of pel-1, pel-3, and peh of E. carotovora subsp. carotovora. Taken together, KdgR and CRP homologues of E. carotovora subsp. carotovora may regulate Pel and Peh production as in E. chrysanthemi. However, the presence of Pir and PecS homologues in E. carotovora subsp. carotovora was not identified in this study, though these proteins of E. chrysanthemi were functional on the promoter regions of the pectinase genes of E. carotovora subsp. carotovora.     

Thermodependence of growth and enzymatic activities implicated in pathogenicity of two Erwinia carotovora subspecies (Pectobacterium spp.)

Erwinia carotovora subsp. atroseptica and Erwinia carotovora subsp. carotovora can cause substantial damage to economically important plant crops and stored products. The occurrence of the disease and the scale of the damage are temperature dependent. Disease development consists first of active multiplication of the bacteria in the infection area and then production of numerous extracellular enzymes. We investigated the effects of various temperatures on these two steps. We assayed the specific growth rate and the pectate lyase and protease activities for eight strains belonging to E. carotovora subsp. atroseptica and E. carotovora subsp. carotovora in vitro. The temperature effect on growth rate and on pectate lyase activity is different for the two subspecies, but protease activity appears to be similarly thermoregulated. Our results are in agreement with ecological data implicating E. carotovora subsp. atroseptica in disease when the temperature is below 20 degrees C. The optimal temperature for pathogenicity appears to be different from the optimal growth temperature but seems to be a compromise between this temperature and temperatures at which lytic activities are maximal.     

Purification to homogeneity of extracellular polygalacturonase and isoenzymes of pectate lyase of Erwinia carotovora subsp. atroseptica by column chromatography

Extracellular polygalacturonase (PG) and two pectate lyase isoenzymes (PLI and PLII) produced by a 48 h culture of Erwinia carotovora subsp. atroseptica in pectate-based medium were purified 2027, 2036 and 2374-fold respectively to homogeneity with corresponding 59%, 61% and 32% recovery. This was achieved first by ion exchange chromatography on a S-Sepharose fast flow column with 20 mmol/1 Tris at pH 8.0 followed by elution of bound proteins with a 1 mol/1 NaCl gradient which separated PG from PL. The two enzymes were then further purified to homogeneity (assessed by SDS-PAGE) by selective adsorption chromatography on a hydroxyapatite column equilibrated with distilled water; PG was eluted with a 3 mol/1 KCl gradient and PLI with a 3 mol/1 KCl gradient followed by a 1.2 mol/1 PO₄ buffer pH 6.5 gradient to elute PLII. The Mr of the three enzymes determined by SDS-PAGE was 39 kDa and the pI values for PG, PLI and PII were 10.3, 10.3 and 10.0 respectively as determined by isoelectric focusing (IEF)-gel electrophoresis followed by activity staining.     

Application of amplified fragment length polymorphism fingerprinting for taxonomy and identification of the soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi

The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment length polymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (clusters 1 to 4) resulted. Cluster 1 contained Erwinia carotovora subsp. carotovora (subclusters 1a and 1b) and Erwinia carotovora subsp. odorifera (subcluster 1c) strains, while cluster 2 contained Erwinia carotovora subsp. atroseptica (subcluster 2a) and Erwinia carotovora subsp. betavasculorum (subcluster 2b) strains. Clusters 3 and 4 contained Erwinia carotovora subsp. wasabiae and E. chrysanthemi strains, respectively. While E. carotovora subsp. carotovora and E. chrysanthemi showed a high level of molecular diversity (23 to 38% mean similarity), E. carotovora subsp. odorifera, E. carotovora subsp. betavasculorum, E. carotovora subsp. atroseptica, and E. carotovora subsp. wasabiae showed considerably less (56 to 76% mean similarity), which may reflect their limited geographical distributions and/or host ranges. The species- and subspecies-specific banding profiles generated from the AFLPs allowed rapid identification of unknown isolates and the potential for future development of diagnostics. AFLP fingerprinting was also found to be more differentiating than other techniques for typing the soft rot erwinias and was applicable to all strain types, including different serogroups.     

Differentiation of Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica isolated from potato by Western blot and subsequent indirect ELISA

Electrotransfer of protein bands from a polyacrylamide gel to a hydrophobic poly-vinylidene difluoride (PVDF) membrane (Western blot) and their serological determination by indirect ELISA (immunoblotting) were used to differentiate Erwinia carotovora subsp. carotovora (Ecc) from Erwinia carotovora subsp. atroseptica (Eca). Ninety strains: 69 Ecc, 19 Eca and two Erwinia chrysanthemi (Echr) were examined. Eight polyclonal antisera against whole cells, glutaraldehyde fixed cells, glycopro-teins, and somatic antigens were prepared. Antisera produced with glutaraldehyde fixed cells did not recognize any band of the protein pattern. The remaining antisera recognized a limited number of bands. Two protein bands allowed differentiation of the two subspecies by the antisera against glycoproteins. One band with an estimated molecular weight of 36000 Da was present in the 19 Eca strains tested and another band with an estimated molecular weight of 35 000 Da was present in the 69 Ecc strains, except for three cases. The strains of Echr showed a band with an estimated weight of 33 000 Da.     

Production of cell wall dissolving enzymes by Erwinia carotovora subsp. atroseptica in vitro at 27°C and 30°5C

Cultures of Erwinia carotovora subsp. atroseptica , grown at 27°C and 30·°C in different liquid media were assayed for activities of pectate lyase, polygalacturonase and cellulase. Total production of both pectate lyase and of polygalacturonase was 3–6 times less at 30·5°C than at 27°C; secretion of pectate lyase was similarly affected. Cellulase was cell bound and its production was not affected by the temperatures investigated. Growth, protein synthesis and protease activity were similar at the two temperatures and production of enzyme activity at 27°C and 30·5°C was independent of the growth medium.     

Isolation by genomic subtraction of DNA probes specific for Erwinia carotovora subsp. atroseptica.

Erwinia carotovora subsp. atroseptica is a pathogen of potatoes in Europe because of its ability to induce blackleg symptoms early in the growing season. However, E. carotovora subsp. carotovora is not able to produce such severe symptoms under the same conditions. On the basis of the technique described by Straus and Ausubel (Proc. Natl. Acad. Sci. USA 87:1889-1893, 1990), we isolated DNA sequences of E. carotovora subsp. atroseptica 86.20 that were absent from the genomic DNA of E. carotovora subsp. carotovora CH26. Six DNA fragments ranging from ca. 180 to 400 bp were isolated, cloned, and sequenced. Each fragment was further hybridized with 130 microorganisms including 87 E. carotovora strains. One probe was specific for typical E. carotovora subsp. atroseptica strains, two probes hybridized with all E. carotovora subsp. atroseptica strains and with a few E. carotovora subsp. carotovora strains, and two probes recognized only a subset of E. carotovora subsp. atroseptica strains. The last probe was absent from the genomic DNA of E. carotovora subsp. carotovora CH26 but was present in the genomes of many strains, including those of other species and genera. This probe is homologous to the putP gene of Escherichia coli, which encodes a proline carrier. Further use of the probes is discussed.     

PCR and restriction fragment length polymorphism of a pel gene as a tool to identify Erwinia carotovora in relation to potato diseases.

Using a sequenced pectate lyase-encoding gene (pel gene), we developed a PCR test for Erwinia carotovora. A set of primers allowed the amplification of a 434-bp fragment in E. carotovora strains. Among the 89 E. carotovora strains tested, only the Erwinia carotovora subsp. betavasculorum strains were not detected. A restriction fragment length polymorphism (RFLP) study was undertaken on the amplified fragment with seven endonucleases. The Sau3AI digestion pattern specifically identified the Erwinia carotovora subsp. atroseptica strains, and the whole set of data identified the Erwinia carotovora subsp. wasabiae strains. However, Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. odorifera could not be separated. Phenetic and phylogenic analyses of RFLP results showed E. carotovora subsp. atroseptica as a homogeneous group while E. carotovora subsp. carotovora and E. carotovora subsp. odorifera strains exhibited a genetic diversity that may result from a nonmonophyletic origin. The use of RFLP on amplified fragments in epidemiology and for diagnosis is discussed.     

Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil

AIMS: To determine the characteristics of bacteria associated with the blackleg disease of potato in Brazil and compare them with species and subspecies of pectolytic Erwinia. METHODS AND RESULTS: Biochemical and physiological characteristics of 16 strains from blackleg-infected potatoes in State of Rio Grande do Sul, Brazil, were determined and differentiated them from all the E. carotovora subspecies and E. chrysanthemi. Pathogenicity and maceration ability of the Brazilian strains were greater than those of E. carotovora subsp. atroseptica, the causal agent of potato blackleg in temperate zones. Analyses of serological reaction and fatty acid composition confirmed that the Brazilian strains differed from E. carotovora subsp. atroseptica, but the sequence of 16S rDNA gene and the 16S-23S intergenic spacer (IGS) region confirmed the Brazilian strains as pectolytic Erwinia. Restriction analysis of the IGS region differentiated the Brazilian strains from the subspecies of E. carotovora and from E. chrysanthemi. A unique SexAI restriction site in the IGS region was used as the basis for a primer to specifically amplify DNA from the Brazilian potato blackleg bacterium in PCR. CONCLUSIONS: The bacterium that causes the blackleg disease of potato in Brazil differs from E. carotovora subsp. atroseptica, the blackleg pathogen in temperate zones. It also differs from other subspecies of E. carotovora and from E. chrysanthemi and warrants status as a new subspecies, which would be appropriately named E. carotovora subsp. brasiliensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The blackleg disease of potato is caused by a different strain of pectolytic Erwinia in Brazil than in temperate potato-growing regions. The Brazilian strain is more virulent than E. carotovora subsp. atroseptica, the usual causal agent of potato blackleg.     

recA is required in the induction of pectin lyase and carotovoricin in Erwinia carotovora subsp. carotovora.

Pectin lyase (PNL) and the bacteriocin carotovoricin (CTV) were induced in Erwinia carotovora subsp. carotovora 71 by the DNA-damaging agents mitomycin C, nalidixic acid, and UV light. To determine whether the recA product was involved in the expression of these damage-inducible phenotypes, we cloned the E. carotovora subsp. carotovora recA+ gene, inactivated it by Tn5 insertion, and constructed an E. carotovora subsp. carotovora recA::Tn5 strain by gene replacement via homologous recombination. The RecA- strain was more sensitive to methyl methanesulfonate, nitroquinoline oxide, and UV light than its RecA+ parent. The recA mutation did not affect the production of pectate lyase, polygalacturonase, cellulase, and protease or the ability to cause soft rot of potato tubers. With this mutant, unlike with the RecA+ parent strain, PNL and CTV were not induced by mitomycin C or detected in potato tuber tissue. The RecA+ phenotype, including the inducibility of PNL and CTV, could, however, be restored in the mutant in trans by the recA+ gene from either E. carotovora subsp. carotovora or Escherichia coli. We conclude that, in E. carotovora subsp. carotovora, the recA product is required in the induction of PNL and CTV.     

Activity stain for rapid characterization of pectic enzymes in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels

A system was developed for the rapid characterization of microbial pectic enzyme complexes and then tested on Erwinia chrysanthemi and Sclerotium rolfsii. Pectic enzymes in minute samples of crude culture filtrates were resolved by ultrathin-layer polyacrylamide gel isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then assayed with an ultrathin pectate-agarose overlay stained with ruthenium red. The simple procedure can be completed within 30 min after isoelectric focusing, can detect extremely low levels of pectate lyase (6.4 x 10 mumol of product per min), and is sufficiently sensitive to determine the pectate lyase isozyme profile of a single bacterial colony with a diameter of 4 mm. Pectate lyases and polygalacturonases can be distinguished by altering buffer conditions in the overlays. The assay system revealed additional isozymes not resolved by classical techniques and generally corroborated the previously published isoelectric points and molecular weights of the pectate lyase isozymes and exo-poly-alpha-d-galacturonosidase produced by E. chrysanthemi and the endopolygalacturonase and exopolygalacturonase produced by S. rolfsii.     

Erwinia chrysanthemi EC16 Produces a Second Set of Plant-Inducible Pectate Lyase Isozymes

The enterobacterium Erwinia chrysanthemi causes soft-rot diseases involving extensive tissue maceration in a wide variety of plants and secretes multiple pectic enzymes that degrade plant cell walls and middle lamellae. An E. chrysanthemi mutant with directed deletions or insertions in genes pehX, pelX, pelA, pelB, pelC, and pelE, which encode exo-poly-alpha-d-galacturonosidase, exopolygalacturonate lyase, and four isozymes of pectate lyase, respectively, was constructed by the marker exchange of a cloned pehX::TnphoA fragment into E. chrysanthemi CUCPB5010, a Delta(pelA pelE) Delta(pelB pelC)::28bp Delta(pelX)Delta4bp derivative of strain EC16. This mutant, E. chrysanthemi CUCPB5012, no longer caused pitting in a standard pectate semisolid agar medium used to detect pectolytic activity in bacteria. Nevertheless, the mutant still macerated leaves of chrysanthemum (Chrysanthemum morifolium), although with reduced virulence. The mutant was found to produce significant pectate lyase activity in rotting chrysanthemum tissue and in minimal media containing chrysanthemum extracts or cell walls as the sole carbon source. Activity-stained, ultra-thin-layer isoelectric focusing gels revealed the presence in these preparations of several pectate lyase isozymes with pIs ranging from highly acidic to highly alkaline. Sterile culture fluids containing these isozymes were able to macerate chrysanthemum leaf tissue. Unlike the products of the pelA, pelB, pelC, and pelE genes in E. chrysanthemi EC16, these plant-inducible pectate lyase isozymes were not produced in minimal medium containing pectate. The results suggest that E. chrysanthemi produces two sets of independently regulated pectate lyase isozymes that are capable of macerating plant tissues.