Pantoea agglomerans Strain EH318 Produces Two Antibiotics That Inhibit Erwinia amylovora In Vitro

Pantoea agglomerans (synonym: Erwinia herbicola) strain Eh318 produces through antibiosis a complex zone of inhibited growth in an overlay seeded with Erwinia amylovora, the causal agent of fire blight. This zone is caused by two antibiotics, named pantocin A and B. Using a genomic library of Eh318, two cosmids, pCPP702 and pCPP704, were identified that conferred on Escherichia coli the ability to inhibit growth of E. amylovora. The two cosmids conferred different antibiotic activities on E. coli DH5α and had distinct restriction enzyme profiles. A smaller, antibiotic-conferring DNA segment from each cosmid was cloned. Each subclone was characterized and mutagenized with transposons to generate clones that were deficient in conferring pantocin A and B production, respectively. Mutated subclones were introduced into Eh318 to create three antibiotic-defective marker exchange mutants: strain Eh421 (pantocin A deficient); strain Eh439 (pantocin B deficient), and Eh440 (deficient in both pantocins). Cross-hybridization results, restriction maps, and spectrum-of-activity data using the subclones and marker exchange mutants, supported the presence of two distinct antibiotics, pantocin A and pantocin B, whose biosynthetic genes were present in pCPP702 and pCPP704, respectively. The structure of pantocin A is unknown, whereas that of pantocin B has been determined as (R)-N-[((S)-2-amino-propanoylamino)-methyl]-2-methanesulfonyl-succinamic acid. The two pantocins mainly affect other enteric bacteria, based on limited testing.     

Mechanisms of inhibition of Erwinia amylovora by Erw. herbicola in vitro and in vivo

The mechanisms by which Erwinia herbicola inhibits Erwinia amylovora , the fire blight pathogen, were investigated. The optimum pH for growth of Erw. amylovora strain Ea273 in nutrient-yeast extract-glucose broth (NYGB) was 7.0 and growth was markedly reduced at pH values below 6.0. In contrast, the growth rates of Erw. herbicola strains Eh141 and Eh112Y were only slightly reduced at pH levels as low as 4.5, relative to pH 6-8. When Ea273 was grown in NYGB in the presence of Eh141 or Eh112Y, the media became acidic and lower populations of Ea273 were recovered, compared with populations from buffered NYGB. Acidification of plant tissue as a consequence of growth of Erw. herbicola did not occur, however, and thus acid-based inhibition of growth in planta is unlikely. The growth rates of nine strains of Erw. herbicola and their abilities to reduce the pH of NYGB did not correlate with their different abilities to prevent development of fire blight incited by Ea273 in a research apple orchard. When grown in mixed culture, Eh114 and Eh112Y grew to higher populations than Ea273 due to depletion of a nitrogen source needed by Ea273. The ability of 12 strains of Erw. herbicola to produce antibiotics inhibitory to Ea273 on a glucose-asparagine medium correlated with the effectiveness of the strains in suppressing fire blight. A crude preparation of the Eh318 antibiotic delayed development of disease in immature pear fruits incited by Ea273 but not by strain Ea273R318, which is resistant in vitro to the Eh318 antibiotic. Cells of Eh318 protected immature pear fruits more effectively from infection by Ea273 than from the resistant strain Ea273R318.     

Role of antibiotic production by Erwinia herbicola Eh252 in biological control of Erwinia amylovora.

Erwinia herbicola Eh252 is a nonpathogenic epiphytic bacterium that reduces fire blight incidence when sprayed onto apple blossoms before inoculation with Erwinia amylovora, the causal agent of fire blight. Eh252 was found to produce on minimal medium an antibiotic that inhibited the growth of E. amylovora. This antibiotic was inactivated by histidine but not by Fe(II), was sensitive to proteolytic enzymes, and showed a narrow host range of activity. To determine the role of this antibiotic in the control of fire blight, two prototrophic Tn5-induced mutants, 10:12 and 17:12, that had lost their ability to inhibit E. amylovora on plates (Ant- mutants) were compared with the wild-type strain for their ability to suppress fire blight in immature pear fruits. The two mutants had single Tn5 insertions in the chromosome; although they grew in immature pear fruits at a rate similar to that of the wild-type strain, neither of these mutants suppressed fire blight as well as Eh252 did. The Tn5-containing fragment isolated from 10:12 was used to mutagenize Eh252 by marker exchange. Derivatives that acquired the Tn5-containing fragment by homologous recombination lost the ability to inhibit E. amylovora on minimal medium. Furthermore, the three Ant- derivatives tested were also affected in their ability to inhibit E. amylovora in immature pear fruits. The results obtained suggest that antibiotic production is a determinant of the biological control of E. amylovora by Eh252, but that another mechanism(s) is involved.     

Construction and use of a gene bank of Alcaligenes eutrophus in the analysis of ribulose bisphosphate carboxylase genes.

A gene bank of the DNA from the hydrogen bacterium Alcaligenes eutrophus ATCC 17707 was constructed in the broad host range cosmid vector pVK102 and established in Escherichia coli. A triparental replica plating procedure was developed to allow rapid screening of large numbers of isolated E. coli gene bank clones for complementation of A. eutrophus mutants. This procedure was used to identify hybrid cosmids that complemented CO2 fixation-negative (Cfx-), H2 uptake-negative (Hup-), and auxotrophic A. eutrophus mutants. The average insert DNA size in these hybrid cosmids was 22 kilobases. Nine hybrid cosmids that complemented ribulose bisphosphate carboxylase-negative (RuBPCase-) mutants were characterized. They fell into two distinct groups with respect to their restriction patterns. Complementing subclones from the two groups contained no common restriction fragments, but hybridization experiments indicated a high degree of sequence homology. Restriction fragments corresponding to one of the subclones were absent in total DNA from a cured strain that had lost plasmid pAE7, indigenous to the wild type. It is concluded that functional CO2 fixation genes in the A. eutrophus ATCC 17707 chromosome are reiterated on plasmid pAE7.     

Characterization of the rcsB gene from Erwinia amylovora and its influence on exoploysaccharide synthesis and virulence of the fire blight pathogen.

RcsB belongs to a family of positive regulators of exopolysaccharide synthesis in various enterobacteria. The rcsB gene of the fire blight pathogen Erwinia amylovora was cloned by PCR amplification with consensus primers, and its role in exopolysaccharide (EPS) synthesis was investigated. Its overexpression from high-copy-number plasmids stimulated the synthesis of the acidic EPS amylovoran and suppressed expression of the levan-forming enzyme levansucrase. Inactivation of rcsB by site-directed mutagenesis created mutants that were deficient in amylovoran synthesis and avirulent on host plants. In addition, a cosmid which complemented rcsB mutants was selected from a genomic library. The spontaneous E. amylovora mutant E8 has a similar phenotype and was complemented by the cloned rcsB gene. The rcsB region of strain E8 was also amplified by PCR, and the mutation was characterized as a nine-nucleotide deletion at the start of the rcsB gene. Nucleotide sequence analysis of the E. amylovora rcsB region and the predicted amino acid sequence of RcsB revealed extensive homology to rcsB and the encoded protein of other bacteria such as Escherichia coli and Erwinia stewartii. In all three organisms, rcsB is localized adjacent to the rcsC gene, which is transcribed in the opposite direction of rcsB. The E. amylovora rcsB gene has now been shown to strongly affect the formation of disease symptoms of a plant pathogen.     

Penicillin-binding proteins from Erwinia amylovora: mutants lacking PBP2 are avirulent.

Radiolabelled penicillin G was used to examine penicillin-binding proteins (PBPs) from Erwinia amylovora (OT1). This procedure identified seven PBPs with molecular masses ranging from 22 to 83 kDa. E. amylovora PBPs were compared with those from Escherichia coli (JM101) and from two spherical, avirulent TnphoA mutants derived from OT1. Radiolabelled penicillin G bound to only six proteins from the spherical mutants which lacked a 69-kDa PBP. The spherical mutants could be complemented by the cloned E. coli pbpA-rodA operon, which restored both cell shape and virulence to apple seedlings. This suggested that the E. amylovora 69-kDa PBP is probably the functional equivalent of the E. coli PBP2 protein. Southern blot analysis using the E. coli rodA and pbpA genes as radiolabelled probes showed that TnphoA had inserted into the E. amylovora equivalent of the E. coli rodA-pbpA operon. Southern blots to chromosomal DNAs of the two spherical mutants, using the cloned hrp and dsp genes from E. amylovora as radiolabelled probes, confirmed that the TnphoA insertions were not located in the region of the E. amylovora chromosome postulated to encode known virulence factors. Both of the spherical TnphoA mutants synthesized amounts of extracellular polysaccharide equivalent to those synthesized by the wild-type strain (OT1), were resistant to lysis in distilled water and to lysozyme, and elicited the hypersensitive response on nonhost plants. These results indicate a possible role for cell shape in the virulence of this plant pathogen.     

The influence of antibiotic production and pre-emptive colonization on the population dynamics of Pantoea agglomerans (Erwinia herbicola) Eh1087 and Erwinia amylovora in planta

Stigma colonization by Erwinia amylovora is the crucial first step in the development of most fire blight infections in apple and pear trees. Suppression at this point of the disease process by antagonists of E. amylovora, such as Pantoea agglomerans (Erwinia herbicola) strain Eh1087, is a rational approach to control fire blight. We tested the hypothesis that the ability of E. amylovora to compete with Eh1087 for colonization of a stigma is reduced by the potential for Eh1087 to produce the phenazine antibiotic, d-alanylgriseoluteic acid (AGA). In competition experiments on the stigmas of apple flowers, E. amylovora was significantly less successful against Eh1087 (AGA+) than against EhDeltaAGA (AGA-). Further experiments to test the importance of pre-emptive colonization of the stigma by either the pathogen or the antagonist suggested that AGA production significantly enhanced the competitiveness of Eh1087 when it was applied at the same time or 24 h before the pathogen. We also found that pre-emptive stigma colonization by either the pathogen or the antagonist resulted in a population that was resilient to subsequent invasion by a second species suggesting that niche exclusion has a dominant influence on the dynamics of bacterial populations on stigmas.     

Site-directed and transposon-mediated mutagenesis with pfd-plasmids by electroporation of Erwinia amylovora and Escherichia coli cells.

The suicide plasmid pfdA31-Tn5 was constructed to mutagenize Erwinia amylovora and Escherichia coli strains by electorporation. This vector carries the bacteriophage fd replication origin, a beta-lactamase gene and the transposon Tn5. For propagation the plasmid depends on host cells producing fd gene-2 protein. Electroporation of E.amylovora or E.coli cells with plasmid pfdA31-Tn5 yielded more than 10(4) transposition events per micrograms DNA. We have produced and characterized transposon mutants of E.amylovora affecting either galactose metabolism or the synthesis of the phytotoxin (L)-2,5-dihydrophenylalanine. A Tn5-insertion in a gene, involved in exopolysaccharide synthesis of E.amylovora strain Ea7/74, was subcloned into vector pfdA31 and used to mutagenize E.amylovora strain Ea1/79 by site-directed recombination.     

软腐欧氏杆菌的Ⅲ型分泌系统对harpin蛋白的识别与分泌

本文研究软腐欧氏杆菌分泌致病蛋白的Ⅲ型分泌系统的组分是否能识别梨火疫欧氏杆菌存在于mRNA上的分泌识别信号。用PCR的方法,将带有上游75bp可以在其mRNA的5′端形成一个典型的作为Ⅲ型分泌识别信号的茎环结构的梨火疫欧氏杆菌的诱导植物过敏反应的harpin的基因hrpN,从带有hrp基因簇的质粒pCPP430上克隆到pGEM\|T载体上,获得重组质粒pWGF1,经化学法转化到hrpN基因的转座突变体DH5α(pCPP430hrpN-)中,同时将pWGF1电击转化到胡萝卜软腐欧氏杆菌Se9R中,转化子的无细胞抽提物(CFEP)经Western\|blot检测到harpin蛋白已被表达;带有双重质粒的DH5α(pCPP430hrpN-/pWGF1)在番茄植物上可以引起过敏反应,Se9R(pWGF1)在大白菜上的致病力明显低于Se9R,初步表明一种植物病菌的harpin蛋白可被另外一种病菌的Ⅲ型分泌系统所识别、分泌并保持生物活性。     

Genetic Transfer of Episomic Elements Among Erwinia Species and Other Enterobacteria: F′lac+

The episomic element F'lac(+) was transferred, probably by conjugation, from Escherichia coli to Lac(-) strains of Erwinia herbicola, Erwinia amylovora, and Erwinia chrysanthemi (but not to several other Erwinia spp. In preliminary trials). The lac genes in the exconjugants of the Erwinia spp. showed varying degrees of stability depending on the strain (stable in E. herbicola strains Y46 and Y74 and E. amylovora strain EA178, but markedly unstable in E. chrysanthemi strain EC16). The lac genes and the sex factor (F) were eliminated from the exconjugants by treatment with acridine orange, thus suggesting that both lac and F are not integrated in the Erwinia exconjugants. All of the tested Lac(+) exconjugants of E. herbicola strains Y46 and Y74 and E. amylovora strain EA178, but not of E. chrysanthemi strain EC 16, were sensitive to the F-specific phage M13. The heterogenotes (which harbored F'lac(+)) of E. herbicola strains Y46 and Y74, E. amylovora strain EA178, and E. chrysanthemi strain EC16 were able to transfer lac genes by conjugation to strains of E. herbicola, E. amylovora, E. chrysanthemi, Escherichia coli, and Shigella dysenteriae. The frequency of such transfer from Lac(+) exconjugants of Erwinia spp. was comparable to that achieved by using E. coli F'lac(+) as donors, thus indicating the stability, expression, and restriction-and-modification properties of the sex factor (F) in Erwinia spp.     

Partial characterization of an inhibitory strain of Erwinia herbicola with potential as a biocontrol agent for Erwinia amylovora, the fire blight pathogen

Erwinia herbicola Eh1087 isolated from apple blossom inhibits development of Erwinia amylovora in immature pear fruit and produces a broad spectrum antibiotic activity in vitro that is bactericidal for Erw. amylovora. The antibiotic activity is present in cell-free culture supernatant fluids of late log-early stationary phase cultures of Eh1087. This antibiotic activity is not inhibited by proteases, excess ferric ions or essential amino acids. It is stable to acidic and basic pH and is inactivated at high temperature. The antibiotic activity is inactivated by β-lactamase digestion.     

The Rcs phosphorelay system is essential for pathogenicity in Erwinia amylovora

The Rcs phosphorelay system is a modified two-component signal transduction system found exclusively in Enterobacteriaceae . In this study, we characterized the roles of the Rcs system in Erwinia amylovora , a highly virulent and necrogenic enterobacterium causing fire blight disease on rosaceous plants. Our results showed that rcsB , rcsC , rcsD and rcsBD mutants were non-pathogenic on immature pear fruit. The bacterial growth of these mutants was also greatly reduced compared with that of the wild-type strain in immature pear fruit. In an in vitro amylovoran assay, rcsB and rcsD mutants were deficient in amylovoran production, whereas the rcsC mutant exhibited higher amylovoran production than that of the wild-type. Consistent with amylovoran production, expression of the amylovoran biosynthetic gene amsG , using green fluorescent protein as a reporter, was not detectable in rcsB , rcsD and rcsBD mutants both in vitro and in vivo . The expression of amsG in vitro was higher in the rcsC mutant than in the wild-type, whereas its expression in vivo was higher in the wild-type than in the rcsC mutant. In addition, rcs mutants were more susceptible to polymyxin B treatment than the wild-type, suggesting that the Rcs system conferred some level of resistance to polymyxin B. Furthermore, rcs mutants showed irregular and slightly reduced motility on swarming plates. Together, these results indicate that the Rcs system plays a major role in virulence and survival of E. amylovora in immature pear fruit.     

Cloning and heterologous expression of the phenazine biosynthetic locus from Pseudomonas aureofaciens 30-84.

Pseudomonas aureofaciens strain 30-84 suppresses take-all disease of wheat caused by Gaeumannomyces graminis var. tritici. Three antibiotics, phenazine-1-carboxylic acid, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine, were responsible for disease suppression. Tn5-induced mutants deficient in production of one or more of the antibiotics (Phz-) were significantly less suppressive than the parental strain. Cosmids pLSP259 and pLSP282 from a genomic library of strain 30-84 restored phenazine production and fungal inhibition to 10 different Phz- mutants. Sequences required for production of the phenazines were localized to a segment of approximately 2.8 kilobases that was present in both cosmids. Expression of this locus in Escherichia coli required the introduction of a functional promoter, was orientation-specific, and resulted in the production of all three phenazine antibiotics. These results strongly suggest that the cloned sequences encode a major portion of the phenazine biosynthetic pathway.     

Cloning of DNA fragments carrying hydrogenase genes of Rhodopseudomonas capsulata

A cosmid library of Rhodopseudomonas capsulata DNA was constructed in Escherichia coli HB101 using the broad-host-range cosmid vector pLAFR1. More than ninety per cent of the clones in the bank contained cosmids with DNA inserts averaging 20 kilobase pairs in length. Mutants deficient in uptake hydrogenase (Hup-) were obtained from R. capsulata strain B10 by ethylmethylsulfonate (EMS) mutagenesis. The content of hydrogenase protein in Hup- mutant cells was tested by rocket immunoelectrophoresis. Hup- mutants (Rifr) were complemented with the clone bank by conjugation and, from the transconjugants selected by rifampicin and tetracycline resistance, Hup+ transconjugants were screened for the ability to grow photoautotrophically and to reduce methylene blue in a colony assay. The recombinant plasmid pAC57 restored hydrogenase activity in the Hup- mutants RCC8, RCC10, RCC12 and ST410 whereas pAG202 restored that of IR4. The cloned R. capsulata DNA insert of pAC57 gave 5 restriction fragments by cleavage with EcoRI endonuclease. Fragment 1 (7 kb) restored hydrogenase activity in Hup- mutant strains RCC12 and ST410 and fragment 5 (1.3 kb) in strains RCC8 and RCC10. Since the 2 cosmids pAC57 and pAG202 are different cosmids, as indicated by restriction analyses and absence of cross hybridization, it is concluded that at least two hup genes are required for the expression of hydrogenase activity in R. capsulata.     

Characterization of a 56-kb plasmid of Erwinia amylovora Ea322: its noninvolvement in pathogenicity

A plasmid from Erwinia amylovora strain Ea322, pCPP60, was studied for its involvement in the phytopathogenicity of this strain. Eviction through incompatibility and curing with acridine orange did not affect the pathogenic capability of Ea322. The plasmid was characterized as self-transmissible with a narrow host range. Hybridization of its origin of replication with plasmids of different incompatibility groups revealed affiliation with IncF. The exact subgroup was not determined, although it does not belong to IncFI, IncFII, IncFIV, or IncFV. A sequence of 800 bp, required for conjugation in cis, was cloned in pUC9. A "miniplasmid" containing the origin of replication in a 1.2-kb sequence was constructed. Its high copy number was in contrast with the stringently controlled copy number of the native plasmid of one to three copies per chromosome equivalent.     

Cloning,sequencing and partial characterisation of sorbitol transporter (srlT) gene encoding phosphotransferase system,glucitol/sorbitol-specific IIBC components of Erwinia herbicola ATCC 21998 – Cloning,sequencing and partial characterisation of sorbitol specific transporter (srlT) gene of Erwinia herbicola ATCC 21998

A DNA fragment of approximately 1500 bp, harbouring the sorbitol transport gene (srlT), was amplified from the chromosomal DNA of Erwinia herbicola ATCC 21998 by PCR and cloned in Escherichia coli JM109. Degenerate oligonucleotide primers used were designed based on the conserved regions in the gene sequences within the gut operon of E. coli (Gene Bank accession no. J02708) and the srl operon of Erwinia amylovora (Gene Bank accession no. Y14603). The cloned DNA fragment was sequenced and found to contain an open reading frame of 1473 nucleotides coding for a protein of 491 amino acids, corresponding to a mass of 52410 Da. The nucleotide sequence of this ORF was highly homologous to that of the gutA gene of Escherichia coli gut operon, the srlE gene of Shigella flexrni and the sorbitol transporter gene sequence of Escherichia coli K12 (Gene Bank accession nos. J02708, AE016987 and D90892 respectively). The protein sequence showed significant homology to that of the phosphotransferase system i.e. the glucitol/sorbitol-specific IIBC components of Escherichia coli and Erwinia amylovora (P56580, O32522). The cloned DNA fragment was introduced into a pRA90 vector and the recombinant was used for developing srlT mutants of Erwinia herbicola, by homologous recombination. Mutants obtained were unable to grow on minimal medium with sorbitol. The insertion of the pRA90 vector inside the srlT gene sequence of the mutants was confirmed by DNA-DNA hybridisation.     

The rcsA gene from Erwinia amylovora: identification, nucleotide sequence, and regulation of exopolysaccharide biosynthesis

RcsA is a positive activator of extracellular polysaccharide synthesis in the Enterobacteriaceae. A cosmid clone containing the rcsA gene from Erwinia amylovora was identified by its ability to restore mucoidy to an E. stewartii rcsA mutant. The rcsA gene was subcloned on a 2.2-kilobase HindIII-PstI fragment that hybridized with an E. stewartii rcsA probe and complemented E. stewartii and Escherichia coli rcsA mutants. In addition, the cloned E. amylovora rcsA gene stimulated expression of cps::lac fusions in E. coli and E. stewartii. The rcsA region was sequenced, and one open reading frame of 211 amino acids was found. The predicted protein sequence specified by this open reading frame was 55% homologous with that of the Klebsiella pneumoniae RcsA protein. Highly conserved regions in the 3' and 5' ends of the two proteins were observed. An E. amylovora rcsA mutant was constructed by Tn5 mutagenesis of the cloned gene followed by recombination of the mutation into the chromosome of wild-type strain Ea1/79. The synthesis of both amylovorin and levan was reduced by more than 90% in this mutant, indicating common regulation of the two polysaccharides by rcsA. Virulence of the rcsA mutant on immature pear fruit was diminished but not completely abolished.     

Identification of the fire blight pathogen, Erwinia amylovora, by PCR assays with chromosomal DNA.

Erwinia amylovora, the causative agent of fire blight, was identified independently from the common plasmid pEA29 by three different PCR assays with chromosomal DNA. PCR with two primers was performed with isolated DNA and with whole cells, which were directly added to the assay mixture. The oligonucleotide primers were derived from the ams region, and the PCR product comprised the amsB gene, which is involved in exopolysaccharide synthesis. The amplified fragment of 1.6 kb was analyzed, and the sequence was found to be identical for two E. amylovora strains. The identity of the PCR products was further confirmed by restriction analysis. The 1.6-kb signal was also used for detection of the fire blight pathogen in the presence of other plant-associated bacteria and in infected plant tissue. For further identification of isolated strains, the 16S rRNA gene of E. amylovora and other plant-associated bacteria was amplified and the products were digested with the restriction enzyme HaeIII. The pattern obtained for E. amylovora was different from that of other bacteria. The sequence of the 16S rRNA gene was determined from a cloned fragment and was found to be closely related to the sequences of Escherichia coli and other Erwinia species. Finally, arbitrarily primed PCR with a 17-mer oligonucleotide derived from the sequence of transposon Tn5 produced a unique banding pattern for all E. amylovora strains investigated. These methods expand identification methods for E. amylovora, which include DNA hybridization and a PCR technique based on plasmid pEA29.     

Development and Properties of Streptomycin Resistant Cultures of Erwinia amylovora Derived from English Isolates

Resistance to 1000 p/m, streptomycin was developed in 3 out of 16 virulent strains of Erwinia amylovora by continuous subculturing on increasing concentrations of the drug. Resistance to various antibiotics, including streptomycin, was more readily developed in strains of Erwinia herbicola . Streptomycin resistance carried on an R factor was transferred by conjugation from Escherichia coli to E. amylovora and to E. herbicola . Resistance to streptomycin was associated with attenuation or, in one case, complete loss of virulence. Doubling times of resistant cultures were greater than those of the parent culture both in shaken broth culture and (with the two attenuated cultures) in apple seedlings. The avirulent culture appeared to persist longer in vivo in the presence of the virulent culture than alone.