Genetic regulation of pathogenicity and virulence factors in bacteria Erwinia carotovora subsp. atroseptica: phenotypic characteristic of bacteria with the mutant kduD gene

In contrast to the closely related bacteria Erwinia chrysanthemi, bacteria Erwinia carotovora subsp. atroseptica produce lower levels of main pathogenicity and virulence factors (pectate lyases, cellulases, and proteases) in the presence of pectins. This effect was shown to be connected with the accumulation of the intermediate product of intracellular degradation of these substances, 2,5-diketo-3-deoxygluconate (DK2). The presence of DK2 in the culture broth of mutant bacteria, connected to its export in the environment, was established. The production of pectate lyases, cellulases, and proteases is repressed by DK2 only at its high concentrations in the cultivation medium, whereas low concentrations of DK2 induce the production of virulence factors. Genes involved in the intracellular catabolism of pectin substances and induced by both low and high DK2 concentrations in the cultivation medium are not repressed by this metabolite.     

Genetic Regulation of Pathogenicity and Virulence Factors in Bacteria Erwinia carotovora subsp. atroseptica: Identification of Gene kduD

A mutant that cannot utilize pectin substances of plant cell walls was obtained via insertion of mini-Tn5xylE transposon into the chromosome of phytopathogenic bacteria Erwinia carotovora subsp. atroseptica. the inability of mutant cells to utilize these substrates was caused by a failure to accomplish the catabolism of unsaturated digalacturonic acid (UDA). Study of enzymatic activities has established that mutant bacteria lost the ability to produce 2,5-diketo-3-deoxygluconate dehydrogenase, an enzyme of intracellular UDA utilization. Molecular cloning of the mutant gene was conducted, and its nucleotide sequence was determined. It was shown that the nucleotide sequence of this gene had an 82% homology with the sequence of Erwinia chrysanthemi EC3937 kduD gene encoding 2,5-diketo-3-deoxygluconate dehydrogenase. The intergene kduI–kduD region in bacteria Erwinia carotovora subsp. atroseptica is shorter in length by 98 nucleotides than the corresponding region of Erwinia chrysanthemi and does not contain promoter sequences. The kduD gene was located at 126.8 min of the Erwinia carotovora subsp. atroseptica genetic map.     

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.     

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)     

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.     

Genetic Regulation of Pathogenicity and Virulence Factors in Bacteria Erwinia carotovora subsp. atroseptica: Phenotypic Characteristic of Bacteria Mutant for the kduD Gene

In contrast to the closely related bacteria Erwinia chrysanthemi, the kDu mutant of Erwinia carotovora subsp. atroseptica produce lower levels of main pathogenicity and virulence factors (pectate lyases, cellulases, and proteases) in the presence of pectins. This effect was shown to be connected with the accumulation of the intermediate product of intracellular degradation of these substances, 2,5-diketo-3-deoxygluconate (DK2). The presence of DK2 in the culture broth of mutant bacteria, connected to its export in the environment, was established. The production of pectate lyases, cellulases, and proteases is repressed by DK2 only at its high concentrations in the cultivation medium, whereas low concentrations of DK2 induce the production of virulence factors. Genes involved in the intracellular catabolism of pectin substances and induced by both low and high DK2 concentrations in the cultivation medium are not repressed by this metabolite.     

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.     

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.     

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.     

Mutation in a gene required for lipopolysaccharide and enterobacterial common antigen biosynthesis affects virulence in the plant pathogen Erwinia carotovora subsp. atroseptica.

Spontaneous bacteriophage-resistant mutants of the phytopathogen Erwinia carotovora subsp. atroseptica (Eca) SCRI1043 were isolated and, out of 40, two were found to exhibit reduced virulence in planta. One of these mutants, A5/22, showed multiple cell surface defects including alterations in synthesis of outer membrane proteins, lipopolysaccharide (LPS), enterobacterial common antigen (ECA), and flagella. Mutant A5/22 also showed reduced synthesis of the exoenzymes pectate lyase (Pel) and cellulase (Cel), major virulence factors for this pathogen. Genetic analysis revealed the pronounced pleiotropic mutant phenotype to be due to a defect in a single gene (rffG) that, in Escherichia coli, is involved in the production of ECA. We also show that while other enteric bacteria possess duplicate homologues of this gene dedicated separately to synthesis of LPS and ECA, Eca has a single gene.     

Rapid detection and identification of Erwinia carotovora subsp. atroseptica by a conjugated Staphylococcus aureus slide agglutination test

A. MCLEOD AND M.C.M. PEROMBELON. 1992. A conjugated Staphylococcus aureus slide agglutination test was used to detect and identify the potato blackleg pathogen, Erwinia carotovora subsp. atroseptica. Agglutination was obtained with > 10⁸ cfu/ml of the homologous strain with a polyclonal antiserum (171) against E.c. atroseptica serogroup I which is the predominant E.c. atroseptica serogroup on potatoes in Scotland. The titre of antiserum 171 against live cells of E.c. atroseptica groups I and XXII was 2000 whereas that of other serogroups was considerably less; only 1 and 4 out of 22 serogroups of E. carotovora subsp. carotovora reacted at 1:1500 and 1:1000 antiserum dilutions, respectively and one of the three less common other E.c. atroseptica serogroups reacted at 1:1000. When tested against 24 different bacterial species including E. chrysanthemi and saprophytic bacteria present in potato tuber rots, negative results were obtained with 1:1000 antiserum dilution. The titre against heat-treated (1 h, 70°C) cells of E.c. atroseptica serogroups I and XXII was1700–2000 whereas it was < 10 against other bacteria including E.c. carotovora. Detection of E.c. atroseptica serogroups I and XXII in diseased potato tissues was achieved directly by the slide agglutination test, but lower antiserum dilutions (1:700–1000) were needed. Still lower antiserum dilutions were needed with heat-treated test material for E.c. atroseptica identification.     

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.     

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.