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.     

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.     

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.     

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.     

Insertional specificity of transposon Tn5 in Acinetobacter sp.

Suicide plasmid pJB4JI, containing transposon Tn5 and phage Mu, was introduced from Escherichia coli 1830 into Acinetobacter sp. strain HO1-N and Acinetobacter calcoaceticus BD413. Kanamycin-resistant (Kmr) exconjugants of HO1-N and BD413, isolated on complex medium, were screened for auxotrophic requirements. Over 10,000 Kmr clones were examined, but no auxotrophs were detected. Several Kmr exconjugants of BD413 and HO1-N, obtained from independent matings, were chosen for further study. All Tn5-containing strains exhibited kanamycin phosphotransferase activity. Kmr strains lacked plasmid DNA as determined by three plasmid screening procedures, and the Kmr phenotype was not transferable by conjugal matings to kanamycin-sensitive BD413, HO1-N, or E. coli HB101. The chromosomal location of Tn5 insertions in independently isolated Kmr exconjugants of BD413 and HO1-N was characterized by restriction endonuclease mapping and hybridization studies. Results obtained from Southern hybridization studies were consistent with a single Tn5-specific insertion site in HO1-N and two such sites in BD413. Phage Mu sequences were not detected in Tn5-containing Acinetobacter sp.     

Cloning and expression in Escherichia coli of pectinase genes of Erwinia carotovora subsp. carotovora

Genes coding for an endo-pectate lyase, an exo-pectate lyase, and an endopolygalacturonase of Erwinia carotovora subsp. carotovora Ecc71 were cloned in Escherichia coli HB101, using the cosmid pHC79. The products of the cloned pectinase genes paralleled their counterparts in strain Ecc71 in isoelectric mobility, mode of substrate degradation, and ability to macerate potato tuber tissue.     

Enumeration of two competing Erwinia carotovora populations in potato tubers by a membrane filter-immunofluorescence procedure

Relative increases in cell populations of specific Erwinia carotovora strains injected into potato tubers either singly or in combination of two strains were determined by depositing tissue extracts on membrane filters and staining cells of individual strains by immunofluorescence. The population increase of an Erw. carotovora subsp. atroseptica (Eca) strain was, in general, not affected by the presence of an Erw. carotovora subsp. carotovora (Ecc) strain. However, the increase of an Ecc strain was inhibited by the presence of an Eca strain, especially at incubation temperatures of 5° and 15°C but not at 26°C. One Ecc strain consistently increased in population over another Ecc strain at a greater rate when they were inoculated together at the same loci in comparison to when they were inoculated separately at different loci.     

Cloning and expression in Escherichia coli of pectinase genes of Erwinia carotovora subsp. carotovora.

Genes coding for an endo-pectate lyase, an exo-pectate lyase, and an endopolygalacturonase of Erwinia carotovora subsp. carotovora Ecc71 were cloned in Escherichia coli HB101, using the cosmid pHC79. The products of the cloned pectinase genes paralleled their counterparts in strain Ecc71 in isoelectric mobility, mode of substrate degradation, and ability to macerate potato tuber tissue.     

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.     

Transposon mutagenesis in Caulobacter crescentus

Transposons Tn5 (Km) and Tn7 (Tp and Sm) were transferred to Caulobacter crescentus via P-type antibiotic resistance factors. Transposition was demonstrated by the isolation of chromosomal insertions of each transposon. With C. crescentus strains harboring RP4 aphA::Tn7, the introduction of a wild-type RP4 resulted in the loss of the resident plasmid. Simultaneous selection for Kmr and Smr yielded colonies with chromosomal insertions of Tn7. Examination of over 10,000 chromosomal insertions of Tn7 indicated no auxotrophic or motility mutants. Thus, Tn7 appears to have a high specificity of insertion in C. crescentus. The Mu-containing plasmid pJB4JI transferred Tn5 to C. crescentus, but the plasmid was not maintained. Control experiments showed that recovery of Mu-containing plasmids occurred at very low frequencies in C. crescentus and that the plasmids which were recovered had undergone extensive deletion of plasmid DNA. Presumably, some part of the Mu genome was not tolerated by C. crescentus. The instability of the Mu-containing plasmids makes them excellent vectors for the introduction of transposons, and we have used pJB4JI to isolated chromosomal insertions of Tn5. When several thousand of these insertion mutants were examined, we found auxotrophic and motility mutants at frequencies of 1 and 2%, respectively. These results indicate that Tn5 had a low specificity of insertion in C. crescentus and therefore would be a useful mutagen for obtaining a variety of mutant phenotypes.     

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.     

豌豆根瘤菌共生基因pJB5JI与华癸中生根瘤菌7653R共生质粒的相互关系

华癸中生根瘤菌(Mesorhizobium huakuii)7653R是分离自我国南方水稻田的一株根瘤菌,含有2个内源质粒:p7653Ra和p7653Rb,其中7653Rb是共生质粒.通过Tn5-sacB的插入方法来消除质粒,获得7653Rb消除的突变株7653RD.将豌豆根瘤菌T83K3的共生质粒pJB5JI导入7653R和7653RD中,盆栽结果表明含有pJB5JI的转移接合子7653R-197的竞争结瘤能力和共生固氮能力均高于7653R.pJB5JI不能恢复7653RD在紫云英上的结瘤能力.含有pJB5JI的7653RD可以在豌豆上结无效瘤,表明pJB5JI可以在7653R的染色体背景下表达其功能.对转移接合子中的质粒稳定性进行检测,结果表明pJB5JI在人工传代的情况下可以稳定存在,但经过共生之后发生了遗传分离,对转移接合子和出发菌株及分离菌株进行kan基因的PCR扩增,除了受体菌外其他菌株都可得到PCR产物,由此推测,pJB5JI可能部分或全部整合到了受体菌的染色体基因组中.     

Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions.

Rhizobium phaseoli CFN42 DNA was mutated by random insertion of Tn5 from suicide plasmid pJB4JI to obtain independently arising strains that were defective in symbiosis with Phaseolus vulgaris but grew normally outside the plant. When these mutants were incubated with the plant, one did not initiate visible nodule tissue (Nod-), seven led to slow nodule development (Ndv), and two led to superficially normal early nodule development but lacked symbiotic nitrogenase activity (Sna-). The Nod- mutant lacked the large transmissible indigenous plasmid pCFN42d that has homology to Klebsiella pneumoniae nitrogenase (nif) genes. The other mutants had normal plasmid content. In the two Sna- mutants and one Ndv mutant, Tn5 had inserted into plasmid pCFN42d outside the region of nif homology. The insertions of the other Ndv mutants were apparently in the chromosome. They were not in plasmids detected on agarose gels, and, in contrast to insertions on indigenous plasmids, they were transmitted in crosses to wild-type strain CFN42 at the same frequency as auxotrophic markers and with the same enhancement of transmission by conjugation plasmid R68.45. In these Ndv mutants the Tn5 insertions were the same as or very closely linked to mutations causing the Ndv phenotype. However, in two mutants with Tn5 insertions on plasmid pCFN42d, an additional mutation on the same plasmid, rather than Tn5, was responsible for the Sna- or Ndv phenotype. When plasmid pJB4JI was transferred to two other R. phaseoli strains, analysis of symbiotic mutants was complicated by Tn5-containing deleted forms of pJB4JI that were stably maintained.     

Cloning and expression of the Erwinia carotovora subsp. carotovora gene encoding the low-molecular-weight bacteriocin carocin S1

The purpose of this study was to clone the carocin S1 gene and express it in a non-carocin-producing strain of Erwinia carotovora. A mutant, TH22-10, which produced a high-molecular-weight bacteriocin but not a low-molecular-weight bacteriocin, was obtained by Tn5 insertional mutagenesis using H-rif-8-2 (a spontaneous rifampin-resistant mutant of Erwinia carotovora subsp. carotovora 89-H-4). Using thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of the contiguous 2,280-bp region were determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequence fragment. ORF2 and ORF3 were identified with the carocin S1 genes, caroS1K (ORF2) and caroS1I (ORF3), which, respectively, encode a killing protein (CaroS1K) and an immunity protein (CaroS1I). These genes were homologous to the pyocin S3 gene and the pyocin AP41 gene. Carocin S1 was expressed in E. carotovora subsp. carotovora Ea1068 and replicated in TH22-10 but could not be expressed in Escherichia coli (JM101) because a consensus sequence resembling an SOS box was absent. A putative sequence similar to the consensus sequence for the E. coli cyclic AMP receptor protein binding site (-312 bp) was found upstream of the start codon. Production of this bacteriocin was also induced by glucose and lactose. The homology search results indicated that the carocin S1 gene (between bp 1078 and bp 1704) was homologous to the pyocin S3 and pyocin AP41 genes in Pseudomonas aeruginosa. These genes encode proteins with nuclease activity (domain 4). This study found that carocin S1 also has nuclease activity.     

A competitive PCR-based method for the detection and quantification of Erwinia carotovora subsp. atroseptica On potato tubers

A PCR-based method was developed for the simultaneous detection and quantification of the potato pathogen Erwinia carotovora subsp. atroseptica (Eca) on potato tubers. The method incorporates a competitor PCR template cloned into Escherichia coli in vector pGEM-T (E. coli 4R l/l). Predetermined numbers of E. coli 4R were added to potato peel extract, either pre-inoculated with Eca or from naturally contaminated tubers, and Eca numbers estimated by comparing the ratio of products generated from Eca target DNA and competitor template DNA following PCR. Estimates of Eca numbers were consistent with counts obtained on crystal violet pectate medium and immunofluorescence colony staining. Unlike these methods, however, the PCR-based method is not affected by the presence of other erwinias and saprophytes and is able to detect all serogroups of Eca. Based on this method, a key was produced relating product ratios, obtained following PCR from contaminated tuber stocks, to the likelihood of blackleg disease incidence. This is the first quantitative PCR-based detection method described for Eca and is the first for any bacterial plant pathogen to incorporate a DNA extraction control.