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The Journal of General and Applied... Jan 2019In our recent work, we found that pyrrolnitrin, and not phenazines, contributed to the suppression of the mycelia growth of Fusarium graminearum that causes heavy...
Construction of a β-galactosidase-gene-based fusion is convenient for screening candidate genes involved in regulation of pyrrolnitrin biosynthesis in Pseudomonas chlororaphis G05.
In our recent work, we found that pyrrolnitrin, and not phenazines, contributed to the suppression of the mycelia growth of Fusarium graminearum that causes heavy Fusarium head blight (FHB) disease in cereal crops. However, pyrrolnitrin production of Pseudomonas chlororaphis G05 in King's B medium was very low. Although a few regulatory genes mediating the prnABCD (the prn operon, pyrrolnitrin biosynthetic locus) expression have been identified, it is not enough for us to enhance pyrrolnitrin production by systematically constructing a genetically-engineered strain. To obtain new candidate genes involved in the regulation of the prn operon expression, we successfully constructed a fusion mutant G05ΔphzΔprn::lacZ, in which most of the coding regions of the prn operon and the phzABCDEFG (the phz operon, phenazine biosynthetic locus) were deleted, and the promoter region plus the first thirty condons of the prnA was in-frame fused with the truncated lacZ gene on its chromosome. The expression of the fused lacZ reporter gene driven by the promoter of the prn operon made it easy for us to detect the level of the prn expression in terms of the color variation of colonies on LB agar plates supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). With this fusion mutant as a recipient strain, mini-Tn5-based random insertional mutagenesis was then conducted. By picking up colonies with color change, it is possible for us to screen and identify new candidate genes involved in the regulation of the prn expression. Identification of additional regulatory genes in further work could reasonably be expected to increase pyrrolnitrin production in G05 and to improve its biological control function.
Topics: Antifungal Agents; Bacterial Proteins; DNA Transposable Elements; Fusarium; Gene Deletion; Gene Expression Regulation, Bacterial; Mutagenesis, Insertional; Operon; Pest Control, Biological; Phenazines; Promoter Regions, Genetic; Pseudomonas chlororaphis; Pyrrolnitrin; beta-Galactosidase
PubMed: 29806629
DOI: 10.2323/jgam.2018.01.003 -
Canadian Journal of Microbiology Nov 2018Inoculation with antagonistic soil microorganisms has shown potential to suppress replant disease of apple in orchard soils. Pseudomonas spp. may have the potential to...
Inoculation with antagonistic soil microorganisms has shown potential to suppress replant disease of apple in orchard soils. Pseudomonas spp. may have the potential to reduce Pratylenchus penetrans populations on apple. Pseudomonas spp. were isolated from the rhizosphere of sweet cherry and screened for antagonistic characteristics. Two highly antagonistic Pseudomonas isolates, P10-32 and P10-42, were evaluated for growth promotion of apple seedlings, suppression of P. penetrans populations, and root colonization in soil from three orchards. During the isolate screening, Pseudomonas fluorescens P10-32 reduced in vitro growth of fungal pathogens, had protease activity, had capacity to produce pyrrolnitrin, suppressed P. penetrans populations, and increased plant biomass. Pseudomonas fluorescens P10-42 reduced in vitro growth of fungal pathogens, had protease activity, suppressed P. penetrans populations, and increased plant biomass. In potted orchard soil, inoculating apple with P. fluorescens P10-32 suppressed P. penetrans populations in one of the three soils examined. Inoculation with P. fluorescens P10-42 improved plant growth in two of the soils and suppressed P. penetrans abundance in one soil. In one of the soils, P. fluorescens P10-42 was detected on the roots 56 days postinoculation. Overall, we conclude that Pseudomonas spp. play a role in suppressing P. penetrans on apple in orchard soil.
Topics: Animals; Malus; Nematoda; Nematode Infections; Pest Control; Plant Diseases; Pseudomonas fluorescens; Rhizosphere; Soil Microbiology
PubMed: 29791808
DOI: 10.1139/cjm-2018-0040 -
Frontiers in Microbiology 2018The soilborne fungus anastomosis group (AG) 8 is a major pathogen of grain crops resulting in substantial production losses. In the absence of resistant cultivars of...
The soilborne fungus anastomosis group (AG) 8 is a major pathogen of grain crops resulting in substantial production losses. In the absence of resistant cultivars of wheat or barley, a sustainable and enduring method for disease control may lie in the enhancement of biological disease suppression. Evidence of effective biological control of AG8 through disease suppression has been well documented at our study site in Avon, South Australia. A comparative metatranscriptomic approach was applied to assess the taxonomic and functional characteristics of the rhizosphere microbiome of wheat plants grown in adjacent fields which are suppressive and non-suppressive to the plant pathogen AG8. Analysis of 12 rhizosphere metatranscriptomes (six per field) was undertaken using two bioinformatic approaches involving unassembled and assembled reads. Differential expression analysis showed the dominant taxa in the rhizosphere based on mRNA annotation were spp. and spp. for non-suppressive samples and spp. and spp. for the suppressive samples. The assembled metatranscriptome analysis identified more differentially expressed genes than the unassembled analysis in the comparison of suppressive and non-suppressive samples. Suppressive samples showed greater expression of a polyketide cyclase, a terpenoid biosynthesis backbone gene () and many cold shock proteins (). Non-suppressive samples were characterised by greater expression of antibiotic genes such as non-heme chloroperoxidase () which is involved in pyrrolnitrin synthesis, and phenazine biosynthesis family protein F ( and its transcriptional activator protein (). A large number of genes involved in detoxifying reactive oxygen species (ROS) and superoxide radicals () were also expressed in the non-suppressive rhizosphere samples most likely in response to the infection of wheat roots by AG8. Together these results provide new insight into microbial gene expression in the rhizosphere of wheat in soils suppressive and non-suppressive to AG8. The approach taken and the genes involved in these functions provide direction for future studies to determine more precisely the molecular interplay of plant-microbe-pathogen interactions with the ultimate goal of the development of management options that promote beneficial rhizosphere microflora to reduce AG8 infection of crops.
PubMed: 29780371
DOI: 10.3389/fmicb.2018.00859 -
ACS Omega May 2018Many natural organic compounds with pharmaceutical applications, including antibiotics (chlortetracycline and vancomycin), antifungal compounds (pyrrolnitrin), and...
Many natural organic compounds with pharmaceutical applications, including antibiotics (chlortetracycline and vancomycin), antifungal compounds (pyrrolnitrin), and chemotherapeutics (salinosporamide A and rebeccamycin) are chlorinated. Halogenating enzymes like tryptophan 7-halogenase (PrnA) and tryptophan 5-halogenase (PyrH) perform regioselective halogenation of tryptophan. In this study, the conformational dynamics of two flavin-dependent tryptophan halogenases-PrnA and PyrH-was investigated through molecular dynamics simulations, which are in agreement with the crystallographic and kinetic experimental studies of both enzymes and provide further explanation of the experimental data at an atomistic level of accuracy. They show that the binding sites of the cofactor-flavin adenine dinucleotide and the substrate do not come into close proximity during the simulations, thus supporting an enzymatic mechanism without a direct contact between them. Two catalytically important active site residues, glutamate (E346/E354) and lysine (K79/K75) in PrnA and PyrH, respectively, were found to play a key role in positioning the proposed chlorinating agent, hypochlorous acid. The changes in the regioselectivity between PrnA and PyrH arise as a consequence of differences in the orientation of substrate in its binding site.
PubMed: 31458701
DOI: 10.1021/acsomega.8b00385 -
Natural Product Reports Jul 2018Covering: up to the end of 2017 The roles played by Rieske non-heme iron-dependent oxygenases in natural product biosynthesis are reviewed, with particular focus on... (Review)
Review
Covering: up to the end of 2017 The roles played by Rieske non-heme iron-dependent oxygenases in natural product biosynthesis are reviewed, with particular focus on experimentally characterised examples. Enzymes belonging to this class are known to catalyse a range of transformations, including oxidative carbocyclisation, N-oxygenation, C-hydroxylation and C-C desaturation. Examples of such enzymes that have yet to be experimentally investigated are also briefly described and their likely functions are discussed.
Topics: Biological Products; Cyclization; Electron Transport Complex III; Heme; Heterocyclic Compounds, 3-Ring; Hydroxylation; Oxygenases; Prodigiosin; Pyrroles; Pyrrolnitrin; Spiro Compounds
PubMed: 29651484
DOI: 10.1039/c8np00004b -
BioMed Research International 2018The use of microbial technologies in agriculture is currently expanding quite rapidly with the identification of new bacterial strains, which are more effective in...
The use of microbial technologies in agriculture is currently expanding quite rapidly with the identification of new bacterial strains, which are more effective in promoting plant growth. In the present study 18 strains of were isolated from soil sample of Balochistan coastline. Among isolated strains four designated as SP19, SP22, PS24, and SP25 exhibited biocontrol activities against phytopathogenic fungi, that is, and ; PS24 identified as by 16srRNA gene bank accession number EU081518 was selected on the basis of its antifungal activity to explore its potential as plant growth promotion. PS24 showed multiple plant growth promoting attributes such as phosphate solubilization activity, indole acetic acid (IAA), siderophore, and HCN production. In order to determine the basis for antifungal properties, antibiotics were extracted from King B broth of PS24 and analyzed by TLC. Pyrrolnitrin antibiotic was detected in the culture of strain PS24. PS24 exhibited antifungal activities found to be positive for hydrogen cyanide synthase gene. Sequencing of gene of gene of strain PS24 revealed 99% homology with the . The sequence of PS24 had been submitted in gene bank accession number KR605499. PS24 with its multifunctional biocontrol possessions can be used to bioprotect the crop plants from phytopathogens.
Topics: Anti-Bacterial Agents; Antifungal Agents; Plant Development; Plant Diseases; Plant Growth Regulators; Pseudomonas aeruginosa; Soil Microbiology
PubMed: 29568759
DOI: 10.1155/2018/6147380 -
Applied Microbiology and Biotechnology Apr 2018The antibiotic pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite that plays an important role in the biocontrol of plant diseases due to its broad-spectrum...
The antibiotic pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite that plays an important role in the biocontrol of plant diseases due to its broad-spectrum of antimicrobial activities. The PRN biosynthetic gene cluster remains to be characterised in Serratia plymuthica, though it is highly conserved in PRN-producing bacteria. To better understand PRN biosynthesis and its regulation in Serratia, the prnABCD operon from S. plymuthica G3 was cloned, sequenced and expressed in Escherichia coli DH5α. Furthermore, an engineered strain prnind which is a conditional mutant of G3 prnABCD under the control of the Ptac promoter was constructed. This mutant was able to overproduce PRN with isopropylthiogalactoside (IPTG) induction by overexpressing prnABCD, whilst behaving as a conditional mutant of G3 prnABCD in the absence of IPTG. These results confirmed that prnABCD is responsible for PRN biosynthesis in strain G3. Further experiments involving lux-/dsRed-based promoter fusions, combined with site-directed mutagenesis of the putative σ extended -10 region in the prnA promoter, and liquid chromatography-mass spectrometry (LC-MS) analysis extended our previous knowledge about G3, revealing that quorum sensing (QS) regulates PRN biosynthesis through cross talk with RpoS, which may directly activated prnABCD transcription. These findings suggest that PRN in S. plymuthica G3 is produced in a tightly controlled manner, and has diverse functions, such as modulation of cell motility, in addition to antimicrobial activities. Meanwhile, the construction of inducible mutants could be a powerful tool to improve PRN production, beyond its potential use for the investigation of the biological function of PRN.
Topics: Gene Expression Regulation, Bacterial; Mutation; Operon; Pyrrolnitrin; Quorum Sensing; Serratia
PubMed: 29511844
DOI: 10.1007/s00253-018-8857-0 -
A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology.Journal of the American Chemical Society Mar 2018Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry,...
Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.
Topics: Aminoglycosides; Carbazoles; Computational Biology; Cyclohexane Monoterpenes; Enediynes; Escherichia coli; Fatty Alcohols; Furans; Genetic Engineering; Lactones; Molecular Structure; Monoterpenes; Peptides; Pressure; Pyrimidine Nucleosides; Pyrrolnitrin; Saccharomyces cerevisiae; Streptomyces; Thiazoles; Time Factors; Vincristine
PubMed: 29480720
DOI: 10.1021/jacs.7b13292 -
PloS One 2018The aim of the present study is to evaluate plant growth promoting and biocontrol efficacy of a Serratia marcescens strain ETR17 isolated from tea rhizosphere for the...
The aim of the present study is to evaluate plant growth promoting and biocontrol efficacy of a Serratia marcescens strain ETR17 isolated from tea rhizosphere for the effective management of root rot disease in tea. Isolated bacterial culture ETR17 showed significant level of in vitro antagonism against nine different foliar and root pathogens of tea. The phenotypic and molecular characterization of ETR17 revealed the identity of the bacterium as Serratia marcescens. The bacterium was found to produce several hydrolytic enzymes like chitinase, protease, lipase, cellulase and plant growth promoting metabolites like IAA and siderophore. Scanning electron microscopic studies on the interaction zone between pathogen and antagonistic bacterial isolate revealed severe deformities in the fungal mycelia. Spectral analyses (LC-ESI-MS, UV-VIS spectrophotometry and HPLC) and TLC indicated the presence of the antibiotics pyrrolnitrin and prodigiosin in the extracellular bacterial culture extracts. Biofilm formation by ETR17 on polystyrene surface was also observed. In vivo application of talc-based formulations prepared with the isolate ETR17 in tea plantlets under green house conditions revealed effective reduction of root-rot disease as well as plant growth promotion to a considerable extent. Viability studies with the ETR17 talc formulation showed the survivability of the isolate up to six months at room temperature. The sustenance of ETR17 (concentration of 8-9x108 cfu g-1) in the soil after the application of talc formulation was recorded by ELISA. Safety studies revealed that ETR17 did not produce hemolysin as observed in pathogenic Serratia strains. The biocontrol strain reported in this study can be used for field application in order to minimize the use of chemical fungicides for disease control in tea gardens.
Topics: Plant Diseases; Plant Roots; Rhizosphere; Serratia marcescens; Tea
PubMed: 29466418
DOI: 10.1371/journal.pone.0191761 -
The Science of the Total Environment Jun 2018Nanoparticles are widely used as antimicrobial compounds. At sub-lethal concentrations, they may also stress microbes, potentially inducing antibiosis. Here we assess...
Nanoparticles are widely used as antimicrobial compounds. At sub-lethal concentrations, they may also stress microbes, potentially inducing antibiosis. Here we assess whether nanoparticles can serve as an enhancer of antibiosis in beneficial microbes. Several host-associated bacteria can suppress pathogens, providing therefore a first line of defense against diseases. In the present study, we assessed whether nanoparticles stimulate the antifungal activity of Pseudomonas protegens CHA0, a model plant-associated bacterium, against the ascomycete yeast Candida albicans. We synthesized and characterized four of the most common nanoparticles, namely Ag, SiO, TiO and ZnO, with an average size of 25, 11, 25 and 35 nm, respectively. The dose-dependent effect of these nanoparticles on the growth of Pseudomonas protegens CHA0 was assessed. Ag, SiO, TiO and ZnO nanoparticles inhibited the growth of Pseudomonas protegens by 100, 22, 15 and 15%, respectively at a concentration of 250 μg/mL. We then selected sub-lethal dose (500 ng/mL) and assessed whether the same nanoparticles stimulated the production of antifungal compounds inhibiting C. albicans. Incubating the bacteria in the presence of nanoparticles led to a four-fold increase in antifungal activity. We finally show that nanoparticles induce the expression of the prn operon, responsible for the production of antifungal compound pyrrolnitrin, within hours after nanoparticle exposure. This study shows that nanoparticle application may be a valuable tool to stimulate the antifungal activity of fluorescent pseudomonads, potentially assisting the development of future sustainable disease control strategies.
Topics: Antibiosis; Antifungal Agents; Nanoparticles; Pseudomonas; Silicon Dioxide; Toxicity Tests
PubMed: 29426189
DOI: 10.1016/j.scitotenv.2018.01.257