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.     

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.     

Evidence that polygalacturonase is a virulence determinant in Erwinia carotovora.

Polygalacturonase (PG) was purified from Erwinia carotovora EC. A hybrid cosmid, pSH711, that encodes PG activity but not pectate lyase activity was identified from an E. carotovora genomic library by an immunological screening method. A cell extract of Escherichia coli cells containing pSH711 was able to produce plant tissue maceration when spotted on carrot, potato, or turnip slices. In addition, the E. coli strain containing this plasmid was able to macerate carrot, potato, and turnip slices. Our results suggest that PG plays an important role in soft-rot disease.     

Erwinia carotovora subsp. carotovora and Erwinia-derived elicitors HrpN and PehA trigger distinct but interacting defense responses and cell death in Arabidopsis

We have used an hrp-positive strain of the soft rot pathogen Erwinia carotovora subsp. carotovora to elucidate plant responses to this bacterial necrotroph. Purified virulence determinants, harpin (HrpN) and polygalacturonase (PehA), were used as tools to facilitate this analysis. We show that HrpN elicits lesion formation in Arabidopsis and tobacco and triggers systemic resistance in Arabidopsis. Establishment of resistance is accompanied by the expression of salicylic acid (SA)-dependent, but also jasmonate/ethylene (JA/ET)-dependent, marker genes PR1 and PDF1.2, respectively, suggesting that both SA-dependent and JA/ET-dependent defense pathways are activated. Use of pathway-specific mutants and transgenic NahG plants show that both pathways are required for the induction of resistance. Arabidopsis plants treated simultaneously with both elictors PehA, known to trigger only JA/ET-dependent defense signaling, and HrpN react with accelerated and enhanced induction of the marker genes PR1 and PDF1.2 both locally and systemically. This mutual amplification of defense gene expression involves both SA-dependent and JA/ET-dependent defense signaling. The two elicitors produced by E. carotovora subsp. carotovora also cooperate in triggering increased production of superoxide and lesion formation.     

A putative three-dimensional targeting motif of polygalacturonase (PehA), a protein secreted through the type II (GSP) pathway in Erwinia carotovora

Intramolecular information specifying protein secretion through the type II (GSP) pathway of Gram-negative bacteria was investigated. Two regions of the polygalacturonase (PehA) of Erwinia carotovora containing residues proposed to be included in a targeting motif were located, one close to the C-terminus between residues 342 and 369 and another between residues 84 and 135 in the large central loops. The regions were required together to promote secretion. Further residues in the middle of the protein were required for proper positioning of the regions, suggesting that they were both involved in interaction with the GSP. To our knowledge, this is the first time that a possible three-dimensional targeting motif has been defined. At least one of the motifs comprises a cluster on the surface of the protein. The two motifs are structurally dissimilar, suggesting that there are two distinct recognition regions in the GSP apparatus. Finally, we propose that the targeting motifs are of a complex conformational nature with some variability accommodated, as illustrated by the observation that many mutations exhibited no clear phenotype individually but, in combination, severely compromised secretion.     

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.     

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.     

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.     

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.     

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.     

胡萝卜软腐欧文氏菌甜菜亚种Ecb菌株表达的一种Harpin蛋白

1986年,hrp基因被首次报道.由于Wei等(1992)从梨火疫病菌(Brwinia amylovora)中分离出了能激发过敏反应的HarpinEa蛋白,细菌所产生的Harpin分子受到了同行的重视.基于对Harpin蛋白的深入研究,国外已研制出了相关新型、高效、安全的生物农药.近年来,国内涉及Harpin蛋白结构与功能的研究课题组     

Genomic Diversity of Erwinia carotovora subsp. carotovora and Its Correlation with Virulence

We used genetic and biochemical methods to examine the genomic diversity of the enterobacterial plant pathogen Erwinia carotovora subsp. carotovora. The results obtained with each method showed that E. carotovora subsp. carotovora strains isolated from one ecological niche, potato plants, are surprisingly diverse compared to related pathogens. A comparison of 23 partial mdh sequences revealed a maximum pairwise difference of 10.49% and an average pairwise difference of 2.13%, values which are much greater than the maximum variation (1.81%) and average variation (0.75%) previously reported for Escherichia coli. Pulsed-field gel electrophoresis analysis of I-CeuI-digested genomic DNA revealed seven rrn operons in all E. carotovora subsp. carotovora strains examined except strain WPP17, which had only six copies. We identified 26 I-CeuI restriction fragment length polymorphism patterns and observed significant polymorphism in fragment sizes ranging from 100 to 450 kb for all strains. We detected large plasmids in two strains, including the model strain E. carotovora subsp. carotovora 71. The two least virulent strains had an unusual chromosomal structure, suggesting that a particular pulsotype is correlated with virulence. To compare chromosomal organization of multiple enterobacterial genomes, several genes were mapped onto I-CeuI fragments. We identified portions of the genome that appear to be conserved across enterobacteria and portions that have undergone genome rearrangements. We found that the least virulent strain, WPP17, failed to oxidize cellobiose and was missing several hrp and hrc genes. The unexpected variability among isolates obtained from clonal hosts in one region and in one season suggests that factors other than the host plant, potato, drive the evolution of this common environmental bacterium and key plant pathogen.     

Natural variability in the Arabidopsis response to infection with Erwinia carotovora subsp. carotovora

The natural variation in the response of Arabidopsis thaliana (L.) Heynh. to Erwinia carotovora subsp. carotovora has been studied in seven ecotypes and two mutants. The susceptibility of all the plant types was investigated by (i) macroscopic symptoms, (ii) fluorescence microscopy using green fluorescent protein (GFP) and (iii) bacterial growth in planta. Although all the plants were susceptible to the bacterium, there was no correlation in the degree of infection as ascertained by the three methods. The induction, upon infection, of several genes known to be involved in defense was analyzed by RNA blot hybridization. The patterns of expression of these genes differed according to the genotype. These results suggest that both salicylic and jasmonic acid play a role in the response of Arabidopsis to this bacterium.     

The ineffectuality of potato protease inhibitor on the extracellular protease from Erwinia carotovora subsp. carotovora

Growth conditions are described for optimum production of extracellular protease in batch cultures of the soft rot pathogen Erwinia carotovora subsp. carotovora . This protease was inhibited by approximately 96% by 1 mmol/1 EDTA and by 55–6% by 10 mmol/l cysteine thereby classifying it as a metalloprotease. It was not inhibited by a chymotrypsin inhibitor extracted from potato tubers. This evidence suggests that the potato chymotrypsin inhibitor is not associated with resistance of potatoes to E. carotovora subsp. carotovora .     

珠芽魔芋对细菌性软腐病的抗性鉴定研究

采用魔芋Amorphophallus spp.块茎点种、注射、灌根接种及田间调查等方法,对国内普遍栽种的珠芽红魔芋A. bulbifer、珠芽金魔芋A. muelleri、花魔芋A. konjac和白魔芋A. albus等12个种质材料进行抗软腐病鉴定、比较和评价,以分析珠芽魔芋对抗细菌性软腐病的抗病水平。结果表明,供试材料对软腐病抗性差异较大,珠芽金魔芋种质对细菌性软腐病均有免疫性(I),德宏及临沧珠芽红魔芋种质为高抗病品种(HR);缅甸珠芽红魔芋为抗病品种(R);富源花魔芋、楚雄花魔芋、日本农林2号、鄂魔芋1号、秦魔1号、昭通白魔芋、丽江白魔芋均属易感品种(S),与田间抗性调查情况基本相符。