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.     

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.     

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.     

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)     

Temperature-dependent production of carotovoricin Er and pectin lyase in phytopathogenic Erwinia carotovora subsp. carotovora Er

The production of pectin lyase (Pnl) and carotovoricin (Ctv), as well as cell lysis in the plant-pathogenic bacterium Erwinia carotovora subsp. carotovora Er are induced by mitomycin C. Here, Pnl and Ctv production and cell lysis were found to be temperature-dependent. The optimal temperature for Pnl production was 30 degrees C. However, the optimal temperature for both Ctv production and cell lysis was 23 degrees C, at which Pnl production was reduced to 47% of the maximum. These data suggest the possible existence of novel regulation system(s) for the production of Pnl and Ctv, and cell lysis, in addition to the well-documented regulation system of recA, rdgA, and rdgB genes.     

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.     

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.     

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.     

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.     

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.     

Survival of soft rot coliforms, Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica in soil in Scotland

An enrichment method was used to monitor Erwinia carotovora in soil or the rhizosphere of different crops and weeds in 17 fields with different cropping histories on three farms. The bacteria were detected in all fields not cropped with potatoes, although not consistently, and the mean annual frequency of detection was generally low (< 10%). Fields in which potatoes were grown were extensively contaminated after harvest in September but contamination declined over the winter to very low levels by early summer in the following year. Contamination level tended to rise in some fields without potatoes regardless of their cropping history but for only a short time during autumn and winter. The bacteria were no more frequent in rhizosphere soil of any of the weeds or crops examined, with the exception of brassicas, than in bare soil. In fields where more than 16 months had elapsed since cropping with potatoes, 91% of erwinia isolates obtained were E. carotovora subsp. carotovora , the remainder being E. carotovora subsp. atroseptica. The bacteria were shortlived in soil and in the rhizospheres of inoculated field and pot grown crop and weed plants. Longevity was greater in dry (10% moisture) than in wet (21% moisture) soil and decreased as temperatures rose, particularly above 25°C. Survival was best in association with brassica plants, moderate on grasses and cereals, and least on potatoes and weeds. E.c. carotovora survived better than E.c. atroseptica. Because survival of the bacteria in soil is apparently restricted, their presence in fields could be attributed to recurrent introductions from different sources.     

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.     

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.     

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.     

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.