Efficient transformation of Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica.

We used a modified version of the method of Hanahan (D. Hanahan, J. Mol. Biol. 166:557-580, 1983) to transform Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica with the plasmids pBR322, pBR325, and pAT153. The transformation frequency ranged from 1 X 10(2) to 4 X 10(4) colonies per micrograms of plasmid DNA. The nature of these transformants was confirmed by plasmid analysis. ColE1-based plasmids make potentially useful cloning vectors for the study of genes involved in the pathogenesis of this species.     

Transposon Tn5 mutagenesis in Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica

In matings between Escherichia coli 2492(pJB4JI) and Erwinia carotovora subsp. carotovora Ecc71 and E. carotovora subsp. atroseptica Eca12, Kmr Gms transconjugants were obtained at high frequencies, indicating instability of the Mu-containing plasmid pJB4JI and transposition of Tn5 into the recipient genome. This was verified by Southern blot hybridization with pRZ102 DNA containing Tn5 as the 32P-labeled probe. Examination of Kmr Gms transconjugants of Ecc71 and Eca12 disclosed that a proportion (2 to 3%) were either auxotrophic or defective in catabolism of specific carbohydrates. Spontaneous prototrophic revertants were obtained for all markers with the exception of ilv, tyr, and suc. Genetic and physical data indicate that scattered insertions of Tn5 from pJb4JI into the chromosome of Ecc71 and Eca12 produced a variety of altered phenotypes due mostly to single insertions of Tn5 not accompanied by Mu DNA.     

Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, and Erwinia carotovora subsp. atroseptica

Erwinia spp. that cause soft-rot diseases in plants produce a variety of extracellular pectic enzymes. To assess the correlation between patterns of pectic enzyme production and taxonomic classification, we compared the enzymes from representative strains. Supernatants obtained from polygalacturonate-grown cultures of nine strains of Erwinia chrysanthemi, three strains of E. carotovora subsp. carotovora, and three strains of E. carotovora subsp. atroseptica were concentrated and subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. Pectate lyase, polygalacturonase, and exo-poly-alpha-D-galacturonosidase activities were visualized by staining diagnostically buffered pectate-agarose overlays with ruthenium red after incubation of the overlays with the isoelectric focusing gels. The isoelectric focusing profiles of pectate lyase and polygalacturonase were nearly identical for strains of E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica, showing three pectate lyase isozymes with isoelectric points higher than 8.7 and a polygalacturonase with pI of ca. 10.2. Isoelectric focusing profiles of the E. chrysanthemi pectic enzymes were substantially different. Although there was considerable intraspecific heterogeneity, all strains produced at least four isozymes of pectate lyase, which could be divided into three groups: basic (pI, ca. 9.0 to 10.0), slightly basic (pI, ca. 7.0 to 8.5), and acidic (pI, ca. 4.0 to 5.0). Several strains of E. chrysanthemi also produced a single form of exo-poly-alpha-D-galacturonosidase (pI, ca. 8.0).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation of Erwinia carotovora subsp. atroseptica and carotovora by RAPD-PCR

Six different 10-mer oligonucleotide primers were used to differentiate Erwinia carotovora subsp. atroseptica (Eca) and carotovora (Ecc) using RAPD-PCR. All primers gave different banding patterns for Eca and Ecc indicating their value for identification. UPGMA clustering analysis clearly showed two separate clusters, one for Eca and the other for the Ecc group. Similarity within Eca strains was very high, over 85% among most isolates but within the Ecc group extensive genetic diversity was found and many of the Ecc strains were no more than 50% similar. Similarity between the 10 Eca and 10 Ecc strains was generally only 10–25% based on the results from six primers. Three RAPD fragments from Eca group, which were amplified by three different RAPD primers, were isolated and used as probes for Southern hybridisation to test, if homologous fragments were amplified from Ecc strains. All these probes hybridised only with Eca isolates indicating that these fragments could be useful in order to develop a PCR-based detection system for Eca strains.     

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.     

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.     

Major outer membrane proteins in the phytopathogenic bacteria Erwinia carotovora subsp. carotovora and subsp. atroseptica

Abstract Immunological similarities of heat-labile Escherichia coli enterotoxins pathogenic for man (LTh) and piglets (LTp) and cholera enterotoxin (CT) were examined quantitatively by the reversed Mancini test. The following results were obtained by analysis of rabbit antisera against these toxins. (1) 86% and 61% of the immunoglobulins in anti-CT antisera were antibodies cross-reacting with LTh and LTp, respectively; (2) 77% and 66% of the immunoglobulins in anti-LTh antisera were antibodies cross-reacting with LTp and CT, respectively; (3) 75% and 59% of the immunoglobulins in anti-LTp antisera were antibodies cross-reacting with LTh and CT, respectively.     

The extent and survival of contamination of potato stocks in Scotland by Erwinia carotovora var. carotovora and E. carotovora var.atroseptica*

The following results were obtained when fifty-seven bulk and crate-stored commercial seed potato stocks from the East of Scotland were examined in 1966-8 for contamination by pectolytic Erwinia spp. (1) Most tubers of all the cultivars and stocks examined, irrespective of whether they were obtained from blackleg-infected or blackleg-free crops, were contaminated with E. carotovora; (2) some 80% of the Erwinia isolates obtained were identified as var. atroseptica, the rest being var. carotovora; (3) the organisms survived in and on tubers for 6–7 months of bulk storage over the winter and up to planting time the following spring; (4) contrary to what is generally thought, the high incidence of contamination of all stocks, while suggesting that the seed itself is the major source of E. carotovora for the growing crop, emphasizes that other factors affect manifestation of blackleg in the field and soft rot in store.     

Survival of the phytopathogen Erwinia carotovora subsp. carotovora in sterile and non-sterile soil, sand and their admixture

Survival of phytopathogens in irrigation water and irrigated soil is of major concern to the agricultural community. In the present study, an Erwinia carotovora subsp. carotovora strain was tested for survival capability in three non-sterile and heat-sterilized soil matrices (soil, sand and soil + sand) over 35 d. In all non-sterile soil matrices, Erw. carotovora subsp. carotovora numbers declined below the detection limit over 35 d, the main variance being the decline rate related to nutrients available in the different matrices (soil, soil + sand and sand respectively). In heat-sterilized soil and soil + sand matrices Erw. carotovora subsp. carotovora revealed a regrowth, while in sterile sand matrix its decline was lower over the same time period. In previous published reports, when soil was sterilized by irradiation, such a regrowth was not observed. Application of an initial single load of sodium nitrate solution (70 mg l⁻¹) was found to extend bacteria survival rate in non-sterile and sterile soil columns. In sterile soil columns supplemented with sodium nitrate, Erw. carotovora subsp. carotovora did survive well for up to 60 d, with a major regrowth over the first 12 d and decline up to day 60, reaching initial loading numbers. The information on the potential survival of Erw. carotovora subsp. carotovora in soil for up to 35 d and regrowth in sterile soil should be of concern, especially when irrigation is performed with poor quality water.     

Serological characterization of potato isolates of Erwinia carotovora subsp. atroseptica and subsp. carotovora using polyclonal and monoclonal antibodies

Serological, biochemical and physiological characteristics of 81 strains of Erwinia carotovora subsp. atroseptica ( Eca ) and 67 strains of subsp. carotovora ( Ecc ) from potato, isolated in Spain and from several international collections, have been studied. Ouchterlony double diffusion (ODD), indirect immunofluorescence (IIF) and indirect enzyme-linked immunosorbent assay (ELISA) were the methods used. The antibodies were polyclonals from eight antisera prepared with Eca serogroup I and Ecc serogroup III and two monoclonal antibodies (MAbs), 4G4 from Spain and 4F6 from Canada, both prepared with Eca strains of serogroup I. Serogroup I for Eca and several serogroups for Ecc were the most commonly found in the collection studied. Serological relationships between Eca and Ecc independently of the serogroups were observed by IIF and ELISA using polyclonal antibodies. Common epitopes between all Eca and Ecc studied were detected. Both MAbs recognized epitopes in Eca strains of serogroups I and XXII in IIF and ELISA but they did not react with strains of other serogroups nor Ecc strains. The pattern of reaction against the strains assayed was rather similar but not identical indicating that they represent two different and well conserved epitopes. This study confirms the serological complexity of Ecc and Eca and gives information about the serological probes for detection of both subspecies.     

Specificity of an antiserum against Erwinia carotovora subsp. atroseptica in indirect ELISA

Attempts to differentiate Erwinia carotovora subsp. atroseptica (Eca) from Erwinia carotovora subsp. carotovora (Ecc) by indirect ELISA using polyclonal antisera against the former bacterium were unsuccessful. However, when bacterial cells were preincubated with an antiserum against Eca serogroup I and excess serum washed away prior to coating on micro-ELISA plates, specificity was improved. This modified indirect ELISA was able to separate Eca serogroups I, XVIII and XXII from all the Ecc serogroups tested. Cross adsorption of the antiserum with Ecc serogroup XXIX resulted in greatly reduced absorbance values for all strains/serogroups except Eca serogroups I and XXII. Cross adsorption with the homologous Eca strain reduced absorbance values for all strains/serogroups. It is suggested that the differentiation of Eca serogroups I and XXII obtained with the modified indirect ELISA could be attributed to the removal of antibodies cross reacting to soluble antigens and the retention of antibodies to specific cell surface antigens.     

Serological and biochemical variation among potato strains of Erwinia carotovora subsp. atroseptica and their taxonomic relationship to other E. carotovora strains

The serological and biochemical characteristics of 32 Erwinia carotovora subsp. atroseptica strains from potato were compared with 48 other pectolytic Erwinia strains. Biochemical characteristics were examined by the API 20E and API 50CHE systems. Numerical analysis using the Euclidean distance coefficients and clustering by the unweighted average pair group method indicated that these E. carotovora subsp. atroseptica strains formed a distinct cluster (subphenon A₁) that could be differentiated from other E. carotovora strains. Three non-potato strains also belonged to this group; two of these were from tomato and the other from Chinese cabbage. Named E. carotovora subsp. atroseptica strains from other hosts clustered into other phenons. Sixty-three per cent of subphenon A₁ strains tested in this study typed into serogroup I. One potato strain in another phenon also typed into this serogroup. The subphenon A₁ strains that did not type into serogroup I typed into serogroups XVIII, XX, or XXII. Many of these strains, however, expressed several different O antigens which were also expressed by E. carotovora strains in other phenons.     

Anaerobic Nitrate Respiration by Erwinia carotovora subsp. atroseptica during Potato Tuber Invasion

The in planta induction of anaerobic nitrate respiration by Erwinia carotovora subsp. atroseptica in relation to the in situ oxygen status in soft rotting potato tubers has been investigated. In vitro experiments have shown that nitrate was required for the induction of respiratory nitrate reductase activity in E. carotovora. In addition, oxygen was found to repress this activity. Expression of respiratory nitrate reductase was found in E. carotovora cells extracted from soft rotting potato tuber tissue. However, the rate of nitrite production in these cells was approximately 70-fold lower than the rate recorded in fully induced anaerobic cultures. Oxygen measurements in soft rotting potato tubers indicated that the invading bacteria encounter the lowest oxygen concentration at the interphase between healthy and macerated tissue. Consequently, growth of bacteria present in this specific zone will be stimulated by nitrate which is present in sufficient amounts in tuber tissue. A high nitrate content of the tuber will most likely facilitate the proliferation of E. carotovora in the tuber tissue.     

Reannotation of hypothetical ORFs in plant pathogen Erwinia carotovora subsp. atroseptica SCRI1043

Over-annotation of hypothetical ORFs is a common phenomenon in bacterial genomes, which necessitates confirming the coding reliability of hypothetical ORFs and then predicting their functions. The important plant pathogen Erwinia carotovora subsp. atroseptica SCRI1043 (Eca1043) is a typical case because more than a quarter of its annotated ORFs are hypothetical. Our analysis focuses on annotation of Eca1043 hypothetical ORFs, and comprises two efforts: (a) based on the Z-curve method, 49 originally annotated hypothetical ORFs are recognized as noncoding, this is further supported by principal components analysis and other evidence; and (b) using sequence-alignment tools and some functional resources, more than a half of the hypothetical genes were assigned functions. The potential functions of 427 hypothetical genes are summarized according to the cluster of orthologous groups functional category. Moreover, 114 and 86 hypothetical genes are recognized as putative 'membrane proteins' and 'exported proteins', respectively. Reannotation of Eca1043 hypothetical ORFs will benefit research into the lifestyle, metabolism and pathogenicity of the important plant pathogen. Also, our study proffers a model for the reannotation of hypothetical ORFs in microbial genomes.     

Bioconversion of amino acids into flavouring alcohols and esters by Erwinia carotovora subsp. atroseptica

Summary Erwinia carotovora subsp. atroseptica produced flavour compounds when infecting endives (Cichorium intybus). These compounds were identified as esters and branched-chain alcohols.They were produced from amino acids and some of them such as methionol, methionol acetate, isobutanol, isobutyl acetate, -phenyl ethanol and tryptophol were produced with good yields.     

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.     

Nonsense-suppressor mutants of Erwinia carotovora subsp. carotovora

Abstract A promiscuous plasmid (pLM2) carrying amber mutations in two antibiotic-resistance genes was transferred to a derivative of Erwinia carotovora subsp. carotovora strain SCRI193. Following mutagenesis, two putative amber-suppressing mutants of this strain were isolated. The genotype of these mutants was confirmed by use of rep _am plasmid-specific phage. This constitutes the first isolation of amber-suppressing mutants in Erwinia spp.