BackgroundPseudomonas fluorescens is a genetically and physiologically diverse species of bacteria present in many habitats and in association with plants. This species of bacteria produces a large array of secondary metabolites with potential as natural products. P. fluorescens isolate WH6 produces Germination-Arrest Factor (GAF), a predicted small peptide or amino acid analog with herbicidal activity that specifically inhibits germination of seeds of graminaceous species.ResultsWe used a hybrid next-generation sequencing approach to develop a high-quality draft genome sequence for P. fluorescens WH6. We employed automated, manual, and experimental methods to further improve the draft genome sequence. From this assembly of 6.27 megabases, we predicted 5876 genes, of which 3115 were core to P. fluorescens and 1567 were unique to WH6. Comparative genomic studies of WH6 revealed high similarity in synteny and orthology of genes with P. fluorescens SBW25. A phylogenomic study also placed WH6 in the same lineage as SBW25. In a previous non-saturating mutagenesis screen we identified two genes necessary for GAF activity in WH6. Mapping of their flanking sequences revealed genes that encode a candidate anti-sigma factor and an aminotransferase. Finally, we discovered several candidate virulence and host-association mechanisms, one of which appears to be a complete type III secretion system.ConclusionsThe improved high-quality draft genome sequence of WH6 contributes towards resolving the P. fluorescens species, providing additional impetus for establishing two separate lineages in P. fluorescens. Despite the high levels of orthology and synteny to SBW25, WH6 still had a substantial number of unique genes and represents another source for the discovery of genes with implications in affecting plant growth and health. Two genes are demonstrably necessary for GAF and further characterization of their proteins is important for developing natural products as control measure against grassy weeds. Finally, WH6 is the first isolate of P. fluorescens reported to encode a complete T3SS. This gives us the opportunity to explore the role of what has traditionally been thought of as a virulence mechanism for non-pathogenic interactions with plants.
Transposon mutagenesis and a cosmid genomic library of DNA from the bean pathogen Pseudomonas syringae pathovar syringae were used to identify and isolate sequences essential for pathogenesis. Strain PS9021, derived by TnS mutagenesis, was determined to be nonpathogenic on Phaseolus vulgaris cultivar Red Mexican and incapable of inducing a hypersensitive response in Nicotiana tabacum. This mutant also produced fluidal rather than firm colonies on selected agar media. A TnS-containing EcoRI fragment from PS9021 was cloned and used to probe 1500 members of a genomic library constructed with DNA from the pathogenic parent strain and the wide host range cosmid pVK102. One member that hybridized to the probe contained a cosmid with a 30-kilobase-pair insert (pOSU3101) that complemented the mutant phenotypes when mobilized into PS9021. A restriction endonuclease cleavage site map of pOSU3101 was constructed and sequences essential for pathogenesis were determined by subcloning. Approximately 8.5 kilobase pairs of the insert were essential for restoration by complementation of pathogenesis and hypersensitive response and wild-type colony morphology in strain PS9021.Pathogenic strains of Pseudomonas syringae pathovar syringae (P. s. pv. syringae), which are worldwide in distribution, incite disease in a variety of agronomically important plant species (1,2). Investigations concerning the pathogenicity of this bacterium indicate that specific ecotypes exist in nature that generally exhibit host specificity (3)(4)(5). Strains pathogenic on bush beah (Phaseolus vulgaris) incite brown spot disease, which is characterized by the appearance of irregular, necrotic lesions on leaves and pods (6). Characteristic of many strains is the production of syringomycin (SR), a broad spectrum biocide that is toxic to plants, fungi, and bacteria (7). The occurrence of pathogenic strains that do not produce SR (8,9) indicates that factors other than SR production are essential for pathogenesis. Pathogenic isolates of P. s. pv. syringae also induce a hypersensitive response (HR) in non-host plant species, which is considered to be a defense mechanism (10). Presently, neither the genetics nor the molecular basis of pathogenicity and HR-inducing ability of this pathogen is known.We have recently isolated several TnS mutants from P. s. 406The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Aims: The germination‐arrest factor (GAF) produced by Pseudomonas fluorescens WH6, and identified as 4‐formylaminooxyvinylglycine, specifically inhibits the germination of a wide range of grassy weeds. This study was undertaken to determine whether GAF has antimicrobial activity in addition to its inhibitory effects on grass seed germination. Methods and Results: Culture filtrate from Ps. fluorescens WH6 had little or no effect on 17 species of bacteria grown in Petri dish lawns, but the in vitro growth of Erwinia amylovora, the causal agent of the disease of orchard crops known as fire blight, was strongly inhibited by the filtrate. The anti‐Erwinia activity of WH6 culture filtrate was shown to be due to its GAF content, and a commercially available oxyvinylglycine, 4‐aminoethoxyvinylglycine (AVG), exhibited anti‐Erwinia activity similar to that of GAF. The effects of GAF on Erwinia were reversed by particular amino acids. Conclusions: The biological properties of GAF include a rather specific antimicrobial activity against Erw. amylovora. This may be a general property of oxyvinylglycines as AVG exhibited similar activity. The ability of particular amino acids to reverse GAF inhibition is consistent with a potential effect of this compound on the activity of aminotransferases. Significance and Impact of the Study: The results presented here demonstrate a novel antimicrobial activity of oxyvinylglycines and suggest that GAF and/or GAF‐producing bacteria may have potential for the control of fire blight.
A new oxyvinylglycine has been identified as a naturally occurring herbicide that irreversibly arrests germination of the seeds of grassy weeds, such as annual bluegrass (Poa annua), without significantly affecting the growth of established grass seedlings and mature plants or germination of the seeds of broadleaf plant species (dicots). Previously, Pseudomonas fluorescens WH6 and over twenty other rhizosphere bacteria were isolated and selected for their ability to arrest germination of P. annua seeds. The Germination-Arrest Factor (GAF, 1) responsible for this developmentally specific herbicidal action has now been isolated from the culture filtrate of P. fluorescens WH6. Purification of this highly polar, low molecular weight natural product allowed its structure to be assigned as 4-formylaminooxy-l-vinylglycine based on NMR spectroscopic and mass spectrometric data, in combination with d/l-amino acid oxidase reactions to establish the absolute configuration. Assay results for P. annua inhibition by related compounds known to regulate plant growth are presented, and a cellular target for 1 is proposed. Furthermore, using bioassays, TLC, and capillary NMR spectroscopy, it has been shown that GAF (1) is secreted by all other herbicidally-active rhizosphere bacteria in our collection.
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