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.     

Role of glutamine synthetase in phenazine antibiotic production by Pantoea agglomerans Eh1087

Pantoea agglomerans strain Eh1087 produces the phenazine antibiotic D-alanylgriseoluteic acid. A glutamine auxotroph harboring an insertion in a putative glnA gene was obtained by transposon-mutagenesis of Eh1087 that produced less D-alanylgriseoluteic acid than the parental strain (strain Eh7.1). Cosmids encoding the Eh1087 glnA were isolated by their ability to complement the mutant for prototrophy. The role of the Eh1087 glnA locus was functionally confirmed by complementation of an Escherichia coli glnA mutant. Analysis of the nucleotide and deduced amino acid sequences of the Eh1087 glnA gene indicated a high degree of similarity to the glnA genes and glutamine synthetase enzymes of other Enterobacteriaceae. Isotopic labelling experiments with 15N-labelled ammonium sulfate demonstrated that wild-type Eh1087 incorporated 15N into griseoluteic acid more readily than the glnA mutant Eh7.1. We conclude that the 2 nitrogens in the phenazine nucleus originate from glutamine and the intracellular glutamine synthesized by Eh1087 is a source of the phenazine nucleus nitrogens even in glutamine-rich environments.     

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.     

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.     

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 DH5alpha 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-s uccina mic acid. The two pantocins mainly affect other enteric bacteria, based on limited testing.     

Antibiotic Production by Erwinia herbicola Eh1087: Its Role in Inhibition of Erwinia amylovora and Partial Characterization of Antibiotic Biosynthesis Genes

Mutants of Erwinia herbicola Eh1087 (Ant⁻), which did not produce antibiotic activity against Erwinia amylovora, the fire blight pathogen, were selected after TnphoA mutagenesis. In immature pear fruit Ant⁻ mutants grew at the same rate as wild-type strain Eh1087 but did not suppress development of the disease caused by E. amylovora. These results indicated that antibiosis plays an important role in the suppression of disease by strain Eh1087. All of the Ant⁻ mutations obtained were located in a 2.2-kb region on a 200-kb indigenous plasmid. Sequence analysis of the mutated DNA region resulted in identification of six open reading frames, designated ORF1 through ORF6, four of which were essential to antibiotic expression. One gene was identified as a gene which encodes a translocase protein which is probably involved in antibiotic secretion. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of plasmid proteins produced in Escherichia coli minicells confirmed the presence of proteins whose sizes corresponded to the sizes of the predicted open reading frame products.     

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.     

The presence of diverse IS elements and an avrPphD homologue that acts as a virulence factor on the pathogenicity plasmid of Erwinia herbicola pv. gypsophilae

The pathogenicity of Erwinia herbicola pv. gypsophilae (Ehg) and Erwinia herbicola pv. betae (Ehb) is dependent on a native plasmid (pPATH(Ehg) or pPATH(Ehb)) that harbors the hrp gene cluster, genes encoding type III effectors, phytohormones, biosynthetic genes, and several copies of IS1327. Sequence analysis of the hrp-flanking region in pPATH(Ehg) (cosmid pLA150) revealed a cluster of four additional IS elements designated as ISEhel, ISEhe2, ISEhe3, and ISEhe4. Two copies of another IS element (ISEhe5) were identified on the upstream region of the indole-3-acetic acid operon located on the same cosmid. Based on homology of amino acids and genetic organization, ISEhe1 belongs to the IS630 family, ISEhe2 to the IS5 family, ISEhe3 and ISEhe4 to different groups of the IS3 family, and ISEhe5 to the IS1 family. With the exception of ISEhe4, one to three copies of all the other IS elements were identified only in pathogenic strains of Erwinia herbicola pv. gypsophilae and Erwinia herbicola pv. betae whereas ISEhe4 was present in both pathogenic and nonpathogenic strains. An open reading frame that exhibited high identity (89% in amino acids) to AvrPphD of Pseudomonas syringae pv. phaseolicola was present within the cluster of IS elements. An insertional mutation in the AvrPphDEh, reduced gall size in gypsophila by approximately 85%. In addition, remnants of known genes from four different bacteria were detected on the same cosmid.     

Analysis of bacterial carbapenem antibiotic production genes reveals a novel β-lactam biosynthesis pathway

Carbapenems are β-lactam antibiotics which have an increasing utility in chemotherapy, particularly for nosocomial, multidrug-resistant infections. Strain GS101 of the bacterial phytopathogen, Erwinia carotovora , makes the simple β-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid. We have mapped and sequenced the Erwinia genes encoding carbapenem production and have cloned these genes into Escherichia coli where we have reconstituted, for the first time, functional expression of the β-lactam in a heterologous host. The carbapenem synthesis gene products are unrelated to enzymes involved in the synthesis of the so-called sulphur-containing β-lactams, namely penicillins, cephamycins and cephalosporins. However, two of the carbapenem biosynthesis genes, carA and carC , encode proteins which show significant homology with proteins encoded by the Streptomyces clavuligerus gene cluster responsible for the production of the β-lactamase inhibitor, clavulanic acid. These homologies, and some similarities in genetic organization between the clusters, suggest an evolutionary relatedness between some of the genes encoding production of the antibiotic and the β-lactamase inhibitor. Our observations are consistent with the evolution of a second major biosynthetic route to the production of β-lactam-ring-containing antibiotics.     

Cloning and regulation of Erwinia herbicola pigment genes.

The genes coding for yellow pigment production in Erwinia herbicola Eho10 (ATCC 39368) were cloned and localized to a 12.4-kilobase (kb) chromosomal fragment. A 2.3-kb AvaI deletion in the cloned fragment resulted in the production of a pink-yellow pigment, a possible precursor of the yellow pigment. Production of yellow pigment in both E. herbicola Eho10 and pigmented Escherichia coli clones was inhibited by glucose. When the pigment genes were transformed into a cya (adenylate cyclase) E. coli mutant, no expression was observed unless exogenous cyclic AMP was provided, which suggests that cyclic AMP is involved in the regulation of pigment gene expression. In E. coli minicells, the 12.4-kb fragment specified the synthesis of at least seven polypeptides. The 2.3-kb AvaI deletion resulted in the loss of a 37K polypeptide and the appearance of a polypeptide of 40 kilodaltons (40K polypeptide). The synthesis of the 37K polypeptide, which appears to be required for yellow pigment production, was not repressed by the presence of glucose in the culture medium, as was the synthesis of other polypeptides specified by the 12.4-kb fragment, suggesting that there are at least two types of gene regulation involved in yellow pigment synthesis. DNA hybridization studies indicated that different yellow pigment genes exist among different E. herbicola strains. None of six pigmented plant pathogenic bacteria examined, Agrobacterium tumefaciens C58, Cornyebacterium flaccumfaciens 1D2, Erwinia rubrifaciens 6D364, Pseudomonas syringae ATCC 19310, Xanthomonas campestris 25D11, and "Xanthomonas oryzae" 17D54, exhibited homology with the cloned pigment genes.     

Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici.

Pseudomonas fluorescens 2-79 (NRRL B-15132) and its rifampin-resistant derivative 2-79RN10 are suppressive to take-all, a major root disease of wheat caused by Gaeumannomyces graminis var. tritici. Strain 2-79 produces the antibiotic phenazine-1-carboxylate, which is active in vitro against G. graminis var. tritici and other fungal root pathogens. Mutants defective in phenazine synthesis (Phz-) were generated by Tn5 insertion and then compared with the parental strain to determine the importance of the antibiotic in take-all suppression on wheat roots. Six independent, prototrophic Phz- mutants were noninhibitory to G. graminis var. tritici in vitro and provided significantly less control of take-all than strain 2-79 on wheat seedlings. Antibiotic synthesis, fungal inhibition in vitro, and suppression of take-all on wheat were coordinately restored in two mutants complemented with cloned DNA from a 2-79 genomic library. These mutants contained Tn5 insertions in adjacent EcoRI fragments in the 2-79 genome, and the restriction maps of the region flanking the insertions and the complementary DNA were colinear. These results indicate that sequences required for phenazine production were present in the cloned DNA and support the importance of the phenazine antibiotic in disease suppression in the rhizosphere.     

Identification of plant-inducible genes in Erwinia chrysanthemi 3937.

We present a method for identifying plant-inducible genes of Erwinia chrysanthemi 3937. Mutagenesis was done with the Mu dIIPR3 transposon, which carries a promoterless neomycin phosphotransferase gene (nptI), so upon insertion, the truncated gene can fuse to E. chrysanthemi promoters. Mutants containing insertions in plant-inducible genes were selected for their sensitivity to kanamycin on minimal plates and for their acquired resistance to this antibiotic when an S. ionantha plant extract was added to kanamycin minimal plates. The selection allowed the identification of E. chrysanthemi promoters inducible by host factors present in the S. ionantha plant extract. Using this method, we isolated 30 mutants and characterized 10 of them. Two mutants were defective in cation uptake, one was defective in the galacturonate degradation pathway, and another was altered in the production of the acidic pectate lyase. The functions of the other mutated genes are still unknown, but we show that most of them are involved in pathogenicity.     

Role of antibiotics and siderophores in biocontrol of take-all disease of wheat

Both antibiotics and siderophores have been implicated in the control of soilborne plant pathogens by fluorescent pseudomonads. In Pseudomonas fluorescens 2–79, which suppresses take-all of wheat, the importance of the antibiotic phenazine-1-carboxylic acid was established with mutants deficient or complemented for antiobiotic production and by isolation of the antibiotic from the roots of wheat colonized by the bacteria. Genetic and biochemical studies of phenazine synthesis have focused on two loci; the first is involved in production of both anthranilic acid and phenazine-1-carboxylic acid, and the second encodes genes involved directly in phenazine synthesis. Because the antibiotic does not account fully for the suppressiveness of strain 2-79, additional mutants were analyzed to evaluate the role of the fluorescent siderophore and of an antifungal factor (Aff, identified as anthranilic acid) that accumulates when iron is limiting. Whereas strains producing only the siderophore conferred little protection against take-all, Aff⁺ strains were suppressive, but much less so than phenazine-producing strains. Iron-regulated nonsiderophore antibiotics may be produced by fluorescent pseudomonads more frequently than previously recognized, and could be partly responsible for beneficial effects that were attributed in the past to fluorescent siderophores.     

Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola.

Erwinia herbicola 299R synthesizes indole-3-acetic acid (IAA) primarily by the indole-3-pyruvic acid pathway. A gene involved in the biosynthesis of IAA was cloned from strain 299R. This gene (ipdC) conferred the synthesis of indole-3-acetaldehyde and tryptophol upon Escherichia coli DH5 alpha in cultures supplemented with L-tryptophan. The deduced amino acid sequence of the gene product has high similarity to that of the indolepyruvate decarboxylase of Enterobacter cloacae. Regions within pyruvate decarboxylases of various fungal and plant species also exhibited considerable homology to portions of this gene. This gene therefore presumably encodes an indolepyruvate decarboxylase (IpdC) which catalyzes the conversion of indole-3-pyruvic acid to indole-3-acetaldehyde. Insertions of Tn3-spice within ipdC abolished the ability of strain 299R to synthesize indole-3-acetaldehyde and tryptophol and reduced its IAA production in tryptophan-supplemented minimal medium by approximately 10-fold, thus providing genetic evidence for the role of the indolepyruvate pathway in IAA synthesis in this strain. An ipdC probe hybridized strongly with the genomic DNA of all E. herbicola strains tested in Southern hybridization studies, suggesting that the indolepyruvate pathway is common in this species. Maximum parsimony analysis revealed that the ipdC gene is highly conserved within this group and that strains of diverse geographic origin were very similar with respect to ipdC.     

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.     

Cloning and partial characterization of zwittermicin A resistance gene cluster from Bacillus thuringiensis subsp. kurstaki strain HD1

AIM: The study seeks to shed light on the aminopolyol, broad-spectrum antibiotic zwittermicin A gene cluster of Bacillus thuringiensis subsp. kurstaki HD1 and to identify any new uncharacterized genes with an eventual goal to establish a better understanding of the resistance gene cluster. METHODS AND RESULTS: We screened 51 serovars of B. thuringiensis by PCR and identified 12 zmaR-positive strains. The zmaR-positive B. thuringiensis subsp. kurstaki HD1 strain displayed inhibition zones against indicator fungal strain Phytophthora meadii and bacterial strain Erwinia herbicola as well as against Rhizopus sp., Xanthomonas campestris and B. thuringiensis subsp. finitimus. The zmaR gene cluster of strain HD1 was partially cloned using a lambda library and was extensively characterized based on the information available from a study performed on a similar group of genes in Bacillus cereus. CONCLUSIONS: Three of the five genes in the zwittermicin gene cluster, including the zmaR gene, had counterparts in B. cereus, and the other two were new members of the B. thuringiensis zmaR gene cluster. SIGNIFICANCE AND IMPACT OF THE STUDY: The two new genes were extensively analysed and the data is presented. Understanding antifungal activity of B. thuringiensis may help us to design suitable Cry toxin delivery agents with antifungal activity as well as enhanced insecticidal activity.     

A gene cluster for amylovoran synthesis in Erwinia amylovora: characterization and relationship to cps genes in Erwinia stewartii

A large ams gene cluster required for production of the acidic extracellular polysaccharide (EPS) amylovoran by the fire blight pathogen Erwinia amylovora was cloned. Tn5 mutagenesis and gene replacement were used to construct chromosomal ams mutants. Five complementation groups, essential for amylovoran synthesis and virulence in E. amylovora, were identified and designated amsA-E. The ams gene cluster is about 7 kb in size and functionally equivalent to the cps gene cluster involved in EPS synthesis by the related pathogen Erwinia stewartii. Mucoidy and virulence were restored to E. stewartii mutants in four cps complementation groups by the cloned E. amylovora ams genes. Conversely, the E. stewartii cps gene cluster was able to complement mutations in E. amylovora ams genes. Correspondence was found between the amsA-E complementation groups and the cpsB-D region, but the arrangement of the genes appears to be different. EPS production and virulence were also restored to E. amylovora amsE and E. stewartii cpsD mutants by clones containing the Rhizobium meliloti exoA gene.     

A gene cluster for amylovoran synthesis in Erwinia amylovora: characterization and relationship to cps genes in Erwinia stewartii

A large ams gene cluster required for production of the acidic extracellular polysaccharide (EPS) amylovoran by the fire blight pathogen Erwinia amylovora was cloned. Tn5 mutagenesis and gene replacement were used to construct chromosomal ams mutants. Five complementation groups, essential for amylovoran synthesis and virulence in E. amylovora, were identified and designated amsA-E. The ams gene cluster is about 7 kb in size and functionally equivalent to the cps gene cluster involved in EPS synthesis by the related pathogen Erwinia stewartii. Mucoidy and virulence were restored to E. stewartii mutants in four cps complementation groups by the cloned E. amylovora ams genes. Conversely, the E. stewartii cps gene cluster was able to complement mutations in E. amylovora ams genes. Correspondence was found between the amsA-E complementation groups and the cpsB-D region, but the arrangement of the genes appears to be different. EPS production and virulence were also restored to E. amylovora amsE and E. stewartii cpsD mutants by clones containing the Rhizobium meliloti exoA gene.