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Microbial Ecology May 2019This work reports the comparison of the genome sequence and the ability to inhibit fungal growth of two Pseudomonas protegens related strains that were isolated from the...
This work reports the comparison of the genome sequence and the ability to inhibit fungal growth of two Pseudomonas protegens related strains that were isolated from the same hydroponic culture of lamb's lettuce. The two strains were very similar in their core genome but one strain, Pf4, contained three gene clusters for the production of secondary metabolites, i.e., pyoluteorin (plt), pyrrolnitrin (prn), and rhizoxin (rzx), that were missing in the other strain, Pf11. The difference between the two strains was not due to simple insertion events, but to a relatively complex differentiation focused on the accessory genomes. In dual culture assays, both strains inhibited nearly all tested fungal strains, yet Pf4 exerted a significantly stronger fungal growth inhibition than Pf11. In addition to the differences in the secondary metabolite production associated genes abundance, the genome of Pf4 was more stable, smaller in size and with a lower number of transposons. The preservation of a dynamic equilibrium within natural populations of different strains comprised in the same species but differing in their secondary metabolite repertoire and in their genome stability may be functional to the adaptation to environmental changes.
Topics: Antifungal Agents; Genome, Bacterial; Hydroponics; Pseudomonas; Pythium; Rhizoctonia
PubMed: 30088023
DOI: 10.1007/s00248-018-1238-5 -
Angewandte Chemie (International Ed. in... Aug 2018Ketosynthase (KS) domains of modular type I polyketide synthases (PKSs) typically catalyze the Claisen condensation of acyl and malonyl units to form linear chains. In...
Ketosynthase (KS) domains of modular type I polyketide synthases (PKSs) typically catalyze the Claisen condensation of acyl and malonyl units to form linear chains. In stark contrast, the KS of the rhizoxin PKS branching module mediates a Michael addition, which sets the basis for a pharmacophoric δ-lactone moiety. The precise role of the KS was evaluated by site-directed mutagenesis, chemical probes, and biotransformations. Biochemical and kinetic analyses helped to dissect branching and lactonization reactions and unequivocally assign the entire sequence to the KS. Probing the range of accepted substrates with diverse synthetic surrogates in vitro, we found that the KS tolerates defined acyl chain lengths to produce five- to seven-membered lactones. These results show that the KS is multifunctional, as it catalyzes β-branching and lactonization. Information on the increased product portfolio of the unusual, TE-independent on-line cyclization is relevant for synthetic biology approaches.
Topics: Bacillus amyloliquefaciens; Burkholderia; Cyclization; Lactones; Macrolides; Models, Molecular; Mutagenesis, Site-Directed; Polyketide Synthases; Protein Domains; Substrate Specificity
PubMed: 29897642
DOI: 10.1002/anie.201804991 -
Environmental Microbiology Oct 2016Pseudomonas protegens strain Pf-5 is a soil bacterium that was first described for its capacity to suppress plant diseases and has since been shown to be lethal to...
Pseudomonas protegens strain Pf-5 is a soil bacterium that was first described for its capacity to suppress plant diseases and has since been shown to be lethal to certain insects. Among these is the common fruit fly Drosophila melanogaster, a well-established model organism for studies evaluating the molecular and cellular basis of the immune response to bacterial challenge. Pf-5 produces the insect toxin FitD, but a ΔfitD mutant of Pf-5 retained full toxicity against D. melanogaster in a noninvasive feeding assay, indicating that FitD is not a major determinant of Pf-5's oral toxicity against this insect. Pf-5 also produces a broad spectrum of exoenzymes and natural products with antibiotic activity, whereas a mutant with a deletion in the global regulatory gene gacA produces none of these exoproducts and also lacks toxicity to D. melanogaster. In this study, we made use of a panel of Pf-5 mutants having single or multiple mutations in the biosynthetic gene clusters for seven natural products and two exoenzymes that are produced by the bacterium under the control of gacA. Our results demonstrate that the production of rhizoxin analogs, orfamide A, and chitinase are required for full oral toxicity of Pf-5 against D. melanogaster, with rhizoxins being the primary determinant.
Topics: Animals; Bacterial Proteins; Chitinases; Drosophila melanogaster; Genes, Regulator; Lipopeptides; Mutation; Peptides, Cyclic; Pseudomonas; Virulence
PubMed: 27130686
DOI: 10.1111/1462-2920.13369 -
Fungal Biology Mar 2016Mucorales have been used for production of fermented food in Asia and Africa since time immemorial. Particularly Rhizopus species are rapidly growing, active producers...
Mucorales have been used for production of fermented food in Asia and Africa since time immemorial. Particularly Rhizopus species are rapidly growing, active producers of lipases and proteases and occur naturally during the first stages of soybean fermentation. Two biosafety issues have been raised in recent literature: (1) pathogenicity, Rhizopus species being prevalent opportunists causing erosive infections in severely compromised patients, and (2) toxicity, strains harbouring endosymbiotic Burkholderia producing toxic secondary metabolites. At the molecular level, based on different gene markers, species identity was found between strains used for food processing and clinical strains. In this study, we screened for bacterial symbionts in 64 Rhizopus strains by light microscopy, 16S rRNA sequencing, and HPLC. Seven strains (11 %) carried bacteria identified as Burkholderia rhizoxinica and Burkholderia endofungorum, and an unknown Burkholderia species. The Burkholderia isolates proved to be able to produce toxic rhizoxins. Strains with endosymbionts originated from food, soil, and a clinical source, and thus their presence could not be linked to particular habitats. The presence of Burkholderia in Rhizopus producing toxins could not be excluded as a potential risk for human health. In contrast, given the type of diseases caused by Rhizopus species, we regard the practical risk of infection via the food industry as negligible.
Topics: Burkholderiaceae; Chromatography, High Pressure Liquid; DNA, Bacterial; DNA, Ribosomal; Food Handling; Food Microbiology; Food Safety; Microscopy; Mucorales; Mucormycosis; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Soil Microbiology; Symbiosis
PubMed: 26895868
DOI: 10.1016/j.funbio.2015.12.001 -
Applied and Environmental Microbiology Dec 2015Pseudomonas protegens strain Pf-5 is a rhizosphere bacterium that suppresses soilborne plant diseases and produces at least seven different secondary metabolites with...
An Interspecies Signaling System Mediated by Fusaric Acid Has Parallel Effects on Antifungal Metabolite Production by Pseudomonas protegens Strain Pf-5 and Antibiosis of Fusarium spp.
Pseudomonas protegens strain Pf-5 is a rhizosphere bacterium that suppresses soilborne plant diseases and produces at least seven different secondary metabolites with antifungal properties. We derived mutants of Pf-5 with single and multiple mutations in biosynthesis genes for seven antifungal metabolites: 2,4-diacetylphoroglucinol (DAPG), pyrrolnitrin, pyoluteorin, hydrogen cyanide, rhizoxin, orfamide A, and toxoflavin. These mutants were tested for inhibition of the pathogens Fusarium verticillioides and Fusarium oxysporum f. sp. pisi. Rhizoxin, pyrrolnitrin, and DAPG were found to be primarily responsible for fungal antagonism by Pf-5. Previously, other workers showed that the mycotoxin fusaric acid, which is produced by many Fusarium species, including F. verticillioides, inhibited the production of DAPG by Pseudomonas spp. In this study, amendment of culture media with fusaric acid decreased DAPG production, increased pyoluteorin production, and had no consistent influence on pyrrolnitrin or orfamide A production by Pf-5. Fusaric acid also altered the transcription of biosynthetic genes, indicating that the mycotoxin influenced antibiotic production by Pf-5 at the transcriptional level. Addition of fusaric acid to the culture medium reduced antibiosis of F. verticillioides by Pf-5 and derivative strains that produce DAPG but had no effect on antibiosis by Pf-5 derivatives that suppressed F. verticillioides due to pyrrolnitrin or rhizoxin production. Our results demonstrated the importance of three compounds, rhizoxin, pyrrolnitrin, and DAPG, in suppression of Fusarium spp. by Pf-5 and confirmed that an interspecies signaling system mediated by fusaric acid had parallel effects on antifungal metabolite production and antibiosis by the bacterial biological control organism.
Topics: Antibiosis; Antifungal Agents; Culture Media; Fusaric Acid; Fusarium; Metabolic Networks and Pathways; Microbial Interactions; Pseudomonas; Signal Transduction; Transcription, Genetic
PubMed: 26655755
DOI: 10.1128/AEM.02574-15 -
Nature Chemical Biology Dec 2015Biosynthesis of rhizoxin in Burkholderia rhizoxinica affords an unusual polyketide synthase module with ketosynthase and branching domains that install the δ-lactone,...
Biosynthesis of rhizoxin in Burkholderia rhizoxinica affords an unusual polyketide synthase module with ketosynthase and branching domains that install the δ-lactone, conferring antimitotic activity. To investigate their functions in chain branching, we designed chimeric modules with structurally similar domains from a glutarimide-forming module and a dehydratase. Biochemical, kinetic and mutational analyses reveal a structural role of the accessory domains and multifarious catalytic actions of the ketosynthase.
Topics: Acyltransferases; Burkholderia; Macrolides; Molecular Conformation; Polyketide Synthases; Polyketides
PubMed: 26479442
DOI: 10.1038/nchembio.1932 -
Chemical Communications (Cambridge,... Jun 2015The versatility of the branching module of the rhizoxin polyketide synthase was tested in an in vitro enzyme assay with a polyketide mimic and branched...
The versatility of the branching module of the rhizoxin polyketide synthase was tested in an in vitro enzyme assay with a polyketide mimic and branched (di)methylmalonyl-CoA extender units. Comparison of the products with synthetic reference compounds revealed that the module is able to stereoselectively introduce two branches in one step by a Michael addition-lactonisation sequence, thus expanding the scope of previously studied PKS systems.
Topics: Acyl Coenzyme A; Antibiotics, Antineoplastic; Macrolides; Polyketide Synthases
PubMed: 25994388
DOI: 10.1039/c5cc03085d -
Medicinal Research Reviews Sep 2015Although fungi produce highly structurally diverse metabolites, many of which have served as excellent sources of pharmaceuticals, no fungi-derived agent has been... (Review)
Review
Although fungi produce highly structurally diverse metabolites, many of which have served as excellent sources of pharmaceuticals, no fungi-derived agent has been approved as a cancer drug so far. This is despite a tremendous amount of research being aimed at the identification of fungal metabolites with promising anticancer activities. This review discusses the results of clinical testing of fungal metabolites and their synthetic derivatives, with the goal to evaluate how far we are from an approved cancer drug of fungal origin. Also, because in vivo studies in animal models are predictive of the efficacy and toxicity of a given compound in a clinical situation, literature describing animal cancer testing of compounds of fungal origin is reviewed as well. Agents showing the potential to advance to clinical trials are also identified. Finally, the technological challenges involved in the exploitation of fungal biodiversity and procurement of sufficient quantities of clinical candidates are discussed, and potential solutions that could be pursued by researchers are highlighted.
Topics: Androstadienes; Animals; Antineoplastic Agents; Aphidicolin; Biological Products; Clinical Trials as Topic; Cyclohexanes; Diketopiperazines; Disease Models, Animal; Drug Design; Drug Resistance, Neoplasm; Fatty Acids, Unsaturated; Female; Fungi; Humans; Macrolides; Male; Mice; Neoplasms; Polycyclic Sesquiterpenes; Sesquiterpenes; Trichothecenes; Wortmannin
PubMed: 25850821
DOI: 10.1002/med.21348 -
Molecular Plant-microbe Interactions :... Mar 2015Two strains of Pseudomonas sp., Os17 and St29, were newly isolated from the rhizosphere of rice and potato, respectively, by screening for 2,4-diacetylphloroglucinol...
Two strains of Pseudomonas sp., Os17 and St29, were newly isolated from the rhizosphere of rice and potato, respectively, by screening for 2,4-diacetylphloroglucinol producers. These strains were found to be the same species and were the closest to but different from Pseudomonas protegens among the sequenced pseudomonads, based on 16S ribosomal RNA gene and whole-genome analyses. Strain Os17 was as effective a biocontrol agent as reported for P. protegens Cab57, whereas strain St29 was less effective. The whole-genome sequences of these strains were obtained: the genomes are organized into a single circular chromosome with 6,885,464 bp, 63.5% G+C content, and 6,195 coding sequences for strain Os17; and with 6,833,117 bp, 63.3% G+C content, and 6,217 coding sequences for strain St29. Comparative genome analysis of these strains revealed that the complete rhizoxin analog biosynthesis gene cluster (approximately 79 kb) found in the Os17 genome was absent from the St29 genome. In an rzxB mutant, which lacks the polyketide synthase essential for the production of rhizoxin analogs, the growth inhibition activity against fungal and oomycete pathogens and the plant protection efficacy were attenuated compared with those of wild-type Os17. These findings suggest that rhizoxin analogs are important biocontrol factors of this strain.
Topics: Anti-Bacterial Agents; Antibiosis; Bacillus; Bacterial Proteins; Base Sequence; Biological Control Agents; Cucumis sativus; Fusarium; Genes, Reporter; Genome, Bacterial; Macrolides; Molecular Sequence Data; Multigene Family; Phloroglucinol; Plant Diseases; Plant Roots; Polyketide Synthases; Pseudomonas; Pythium; Recombinant Fusion Proteins; Rhizosphere; Sequence Analysis, DNA; Species Specificity
PubMed: 25496595
DOI: 10.1094/MPMI-09-14-0294-FI -
ELife Sep 2014Some of the proteins and enzymes that allow bacteria to enter living fungal cells and cause rice seedling blight have been identified.
Some of the proteins and enzymes that allow bacteria to enter living fungal cells and cause rice seedling blight have been identified.
Topics: Burkholderia; Macrolides; Rhizopus; Symbiosis
PubMed: 25268072
DOI: 10.7554/eLife.04603