Identification of a Pantoea biosynthetic cluster that directs the synthesis of an antimicrobial natural product.
ABSTRACT: Fire Blight is a destructive disease of apple and pear caused by the enteric bacterial pathogen, Erwinia amylovora. E. amylovora initiates infection by colonizing the stigmata of apple and pear trees, and entering the plants through natural openings. Epiphytic populations of the related enteric bacterium, Pantoea, reduce the incidence of disease through competition and antibiotic production. In this study, we identify an antibiotic from Pantoea ananatis BRT175, which is effective against E. amylovora and select species of Pantoea. We used transposon mutagenesis to create a mutant library, screened approximately 5,000 mutants for loss of antibiotic production, and recovered 29 mutants. Sequencing of the transposon insertion sites of these mutants revealed multiple independent disruptions of an 8.2 kb cluster consisting of seven genes, which appear to be coregulated. An analysis of the distribution of this cluster revealed that it was not present in any other of our 115 Pantoea isolates, or in any of the fully sequenced Pantoea genomes, and is most closely related to antibiotic biosynthetic clusters found in three different species of Pseudomonas. This identification of this biosynthetic cluster highlights the diversity of natural products produced by Pantoea.
Project description:Pantoea vagans C9-1 is a biocontrol strain that produces at least two antibiotics inhibiting the growth of Erwinia amylovora, the causal agent of fire blight disease of pear and apple. One antibiotic, herbicolin I, was purified from culture filtrates of P. vagans C9-1 and determined to be 2-amino-3-(oxirane-2,3-dicarboxamido)-propanoyl-valine, also known as N(ß)-epoxysuccinamoyl-DAP-valine. A plasposon library was screened for mutants that had lost the ability to produce herbicolin I. It was shown that mutants had reduced biocontrol efficacy in immature pear assays. The biosynthetic gene cluster in P. vagans C9-1 was identified by sequencing the flanking regions of the plasposon insertion sites. The herbicolin I biosynthetic gene cluster consists of 10 coding sequences (CDS) and is located on the 166-kb plasmid pPag2. Sequence comparisons identified orthologous gene clusters in Pantoea agglomerans CU0119 and Serratia proteamaculans 568. A low incidence of detection of the biosynthetic cluster in a collection of 45 Pantoea spp. from biocontrol, environmental, and clinical origins showed that this is a rare trait among the tested strains.
Project description:Erwinia amylovora is the causal agent of fire blight of apple and pear trees. Several bacteria have been shown to produce antibiotics that antagonize E. amylovora, including pantocins, herbicolins, dapdiamides, and the vinylglycines, 4-formylaminooxyvinylglycine (FVG) and 4-aminoethoxyvinylglycine (AVG). Pantoea ananatis BRT175 was previously shown to exhibit antibiotic activity against E. amylovora via the production of Pantoea natural product 1 (PNP-1), later shown to be FVG; however, exposure of E. amylovora to FVG results in spontaneously resistant mutants. To identify the mechanism of resistance, we used genome variant analysis on spontaneous FVG-resistant mutants of E. amylovora and identified null mutations in the l-asparagine permease gene ansP Heterologous expression of ansP in normally resistant Escherichia coli was sufficient to impart FVG susceptibility, suggesting that FVG is imported through this permease. Because FVG and AVG are structurally similar, we hypothesized that resistance to AVG would also be conferred through inactivation of ansP; however, ansP mutants were not resistant to AVG. We found that spontaneously resistant Ea321 mutants also arise in the presence of AVG, with whole-genome variant analysis revealing that resistance was due to inactivation of the arginine ABC transporter permease subunit gene artQ Heterologous expression of the predicted lysE-like transporter encoded within the Pantoea ananatis BRT175 FVG biosynthetic cluster, which is likely responsible for antibiotic export, was sufficient to confer resistance to both FVG and AVG. This work highlights the important roles of amino acid transporters in antibiotic import into bacteria and the potential utility of antimicrobial amino acid analogs as antibiotics.IMPORTANCE The related antibiotics formylaminooxyvinylglycine (FVG) and aminoethoxyvinylglycine (AVG) have been shown to have activity against the fire blight pathogen Erwinia amylovora; however, E. amylovora can develop spontaneous resistance to these antibiotics. By comparing the genomes of mutants to those of the wild type, we found that inactivation of the l-asparagine transporter conferred resistance to FVG, while inactivation of the l-arginine transporter conferred resistance to AVG. We also show that the transporter encoded by the FVG biosynthetic cluster can confer resistance to both FVG and AVG. Our work indicates the important role that amino acid transporters play in the import of antibiotics and highlights the possible utility in designer antibiotics that enter the bacterial cell through amino acid transporters.
Project description:Pantoea vagans is a Gram-negative enterobacterial plant epiphyte of a broad range of plants. Here we report the 4.89-Mb genome sequence of P. vagans strain C9-1 (formerly Pantoea agglomerans), which is commercially registered for biological control of fire blight, a disease of pear and apple trees caused by Erwinia amylovora.
Project description:The recently characterized strain Pseudomonas orientalis F9, an isolate from apple flowers in a Swiss orchard, exhibits antagonistic traits against phytopathogens. At high colonization densities, it exhibits phytotoxicity against apple flowers. P. orientalis F9 harbors biosynthesis genes for the siderophore pyoverdine as well as for the antibiotics safracin and phenazine. To elucidate the role of the three compounds in biocontrol, we screened a large random knockout library of P. orientalis F9 strains for lack of pyoverdine production or in vitro antagonism. Transposon mutants that lacked the ability for fluorescence carried transposons in pyoverdine production genes. Mutants unable to antagonize Erwinia amylovora in an in vitro double-layer assay carried transposon insertions in the safracin gene cluster. As no phenazine transposon mutant could be identified using the chosen selection criteria, we constructed a site-directed deletion mutant. Pyoverdine-, safracin-, and phenazine mutants were tested for their abilities to counteract the fire blight pathogen Erwinia amylovora ex vivo on apple flowers or the soilborne pathogen Pythium ultimum in vivo in a soil microcosm. In contrast to some in vitro assays, ex vivo and in vivo assays did not reveal significant differences between parental and mutant strains in their antagonistic activities. This suggests that, ex vivo and in vivo, other factors, such as competition for resources or space, are more important than the tested antibiotics or pyoverdine for successful antagonism of P. orientalis F9 against phytopathogens in the performed assays.IMPORTANCE Pseudomonas orientalis F9 is an antagonist of the economically important phytopathogen Erwinia amylovora, the causal agent of fire blight in pomme fruit. On King's B medium, P. orientalis F9 produces a pyoverdine siderophore and the antibiotic safracin. P. orientalis F9 transposon mutants lacking these factors fail to antagonize E. amylovora, depending on the in vitro assay. On isolated flowers and in soil microcosms, however, pyoverdine, safracin, and phenazine mutants control phytopathogens as clearly as their parental strains.
Project description:Antibiotics are used extensively to control animal, plant, and human pathogens. They are sprayed on apple and pear orchards to control the bacterium Erwinia amylovora, the causative agent of fire blight. This phytopathogen is developing antibiotic resistance and alternatives either have less efficacy, are phytotoxic, or more management intensive. The objective of our study was to develop an effective biological control agent colonizing the host plant and competing with Erwinia amylovora. It must not be phytotoxic, have a wide spectrum of activity, and be unlikely to induce resistance in the pathogen. To this end, several bacterial isolates from various environmental samples were screened to identify suitable candidates that are antagonistic to E. amylovora. We sampled bacteria from the flowers, leaves, and soil from apple and pear orchards from the springtime bloom period until the summer. The most effective bacteria, including isolates of Pseudomonas poae, Paenibacillus polymyxa, Bacillus amyloliquefaciens and Pantoea agglomerans, were tested in vitro and in vivo and formulated into products containing both the live strains and their metabolites that were stable for at least 9 months. Trees treated with the product based on P. agglomerans NY60 had significantly less fire blight than the untreated control and were statistically not different from streptomycin-treated control trees. With P. agglomerans NY60, fire blight never extended beyond the central vein of the inoculated leaf. The fire blight median disease severity score, 10 days after inoculation, was up to 70% less severe on trees treated with P. agglomerans NY60 as compared to untreated controls.
Project description:Construction of a genomic DNA library from Pantoea agglomerans strain CU0119 and screening against the plant pathogen Erwinia amylovora yielded a new family of antibiotics, dapdiamides A-E (1-5). The structures were established through 2D-NMR experiments and mass spectrometry, as well as the synthesis of dapdiamide A (1). Transposon mutagenesis of the active cosmid allowed identification of the biosynthetic gene cluster. The dapdiamide family's promiscuous biosynthetic pathway contains two unconventional amide ligases that are predicted to couple its constituent monomers.
Project description:For possible control of fire blight affecting apple and pear trees, we characterized Erwinia amylovora phages from North America and Germany. The genome size determined by electron microscopy (EM) was confirmed by sequence data and major coat proteins were identified from gel bands by mass spectroscopy. By their morphology from EM data, ?Ea1h and ?Ea100 were assigned to the Podoviridae and ?Ea104 and ?Ea116 to the Myoviridae. Host ranges were essentially confined to E. amylovora, strains of the species Erwinia pyrifoliae, E. billingiae and even Pantoea stewartii were partially sensitive. The phages ?Ea1h and ?Ea100 were dependent on the amylovoran capsule of E. amylovora, ?Ea104 and ?Ea116 were not. The Myoviridae efficiently lysed their hosts and protected apple flowers significantly better than the Podoviridae against E. amylovora and should be preferred in biocontrol experiments. We have also isolated and partially characterized E. amylovora phages from apple orchards in Germany. They belong to the Podoviridae or Myoviridae with a host range similar to the phages isolated in North America. In EM measurements, the genome sizes of the Podoviridae were smaller than the genomes of the Myoviridae from North America and from Germany, which differed from each other in corresponding nucleotide sequences.
Project description:Erwinia amylovora causes fire blight on several plant species such as apple and pear, which produce diverse phytoalexins as defence mechanisms. An evolutionary successful pathogen thus must develop resistance mechanisms towards these toxic compounds. The E. amylovora outer membrane protein, TolC, might mediate phytoalexin resistance through its interaction with the multidrug efflux pump, AcrAB. To prove this, a tolC mutant and an acrB/tolC double mutant were constructed. The minimal inhibitory concentrations of diverse antimicrobials and phytoalexins were determined for these mutants and compared with that of a previously generated acrB mutant. The tolC and arcB/tolC mutants were considerably more susceptible than the wild type but showed similar levels as the acrB mutant. The results clearly indicated that neither TolC nor AcrAB significantly interacted with other transport systems during the efflux of the tested toxic compounds. Survival and virulence assays on inoculated apple plants showed that pathogenicity and the ability of E. amylovora to colonize plant tissue were equally impaired by mutations of tolC and acrB/tolC. Our results allowed the conclusion that TolC plays an important role as a virulence and fitness factor of E. amylovora by mediating resistance towards phytoalexins through its exclusive interaction with AcrAB.
Project description:Bacteriophages, bacteria-infecting viruses, have been recently reconsidered as a biological control tool for preventing bacterial pathogens. Erwinia amylovora and E. pyrifoliae cause fire blight and black shoot blight disease in apple and pear, respectively. In this study, the bacteriophage phiEaP-8 was isolated from apple orchard soil and could efficiently and specifically kill both E. amylovora and E. pyrifoliae. This bacteriophage belongs to the Podoviridae family. Whole genome analysis revealed that phiEaP-8 carries a 75,929 bp genomic DNA with 78 coding sequences and 5 tRNA genes. Genome comparison showed that phiEaP-8 has only 85% identity to known bacteriophages at the DNA level. PhiEaP-8 retained lytic activity up to 50°C, within a pH range from 5 to 10, and under 365 nm UV light. Based on these characteristics, the bacteriophage phiEaP-8 is novel and carries potential to control both E. amylovora and E. pyrifoliae in apple and pear.
Project description:Poor survival on plants can limit the efficacy of Biological Control Agents (BCAs) in the field. Yet bacteria survive in the atmosphere, despite their exposure to high solar radiation and extreme temperatures. If conditions in the atmosphere are similar to, or more extreme than, the environmental conditions on the plant surface, then precipitation may serve as a reservoir of robust BCAs. To test this hypothesis, two hundred and fifty-four rain-borne isolates were screened for in vitro inhibition of Erwinia amylovora, the causal agent of fire blight, as well as of other plant pathogenic bacteria, fungi and oomycetes. Two isolates showed strong activity against E. amylovora and other plant pathogenic bacteria, while other isolates showed activity against fungal and oomycete pathogens. Survival assays suggested that the two isolates that inhibited E. amylovora were able to survive on apple blossoms and branches similarly to E. amylovora. Pathogen population size and associated fire blight symptoms were significantly reduced when detached apple blossoms were treated with the two isolates before pathogen inoculation, however, disease reduction on attached blossoms within an orchard was inconsistent. Using whole genome sequencing, the isolates were identified as Pantoea agglomerans and P. ananatis, respectively. A UV-mutagenesis screen pointed to a phenazine antibiotic D-alanylgriseoluteic acid synthesis gene cluster as being at the base of the antimicrobial activity of the P. agglomerans isolate. Our work reveals the potential of precipitation as an under-explored source of BCAs, whole genome sequencing as an effective approach to precisely identify BCAs, and UV-mutagenesis as a technically simple screen to investigate the genetic basis of BCAs. More field trials are needed to determine the efficacy of the identified BCAs in fire blight control.