-
Microbiological Research Jul 2022Pseudomonas chlororaphis G05 has the capability to repress the mycelial growth of many phytopathogenic fungi by producing and secreting certain antifungal compounds,...
Pseudomonas chlororaphis G05 has the capability to repress the mycelial growth of many phytopathogenic fungi by producing and secreting certain antifungal compounds, including phenazines and pyrrolnitrin. Although some regulatory genes have been identified to be involved in antifungal metabolite production, the regulatory mechanism and pathway of phenazine-1-carboxylic acid biosynthesis remain poorly defined. To identify more new regulatory genes, we applied transposon mutagenesis with the chromosomal lacZ fusion strain G05Δphz::lacZ as an acceptor. In the white conjugant colony G05W05, a novel transcriptional regulator gene, eppR, was verified to be interrupted by the transposon mini-Tn5Kan. To evaluate the specific function of eppR, we created a set of eppR-deletion mutants, including G05ΔeppR, G05Δphz::lacZΔeppR and G05Δprn::lacZΔeppR. By quantifying the production of antifungal compounds and β-galactosidase expression, we found that the expression of the phenazine biosynthetic gene cluster (phz) and the production of phenazine-1-carboxylic acid were markedly reduced in the absence of EppR. Moreover, the pathogen suppression test verified that the yield of phenazine-1-carboxylic acid was significantly decreased when eppR was deleted in frame. At the same time, no changes in the expression of the phzI/phzR quorum-sensing (QS) system and the production of N-acyl homoserine lactones (AHLs) and pyrrolnitrin were found in the EppR-deficient mutant. In addition, chromosomal fusion analyses and quantitative real-time polymerase chain reaction (qRT-PCR) results also showed that EppR could positively mediate the expression of the phz cluster at the posttranscriptional level. In summary, EppR is specifically essential for phenazine biosynthesis but not for pyrrolnitrin biosynthesis in P. chlororaphis.
Topics: Antifungal Agents; Bacterial Proteins; Gene Expression Regulation, Bacterial; Phenazines; Pseudomonas chlororaphis; Pyrrolnitrin
PubMed: 35504237
DOI: 10.1016/j.micres.2022.127050 -
Applied and Environmental Microbiology Jan 1994A soil isolate of Pseudomonas fluorescens (BL915) was shown to be an effective antagonist of Rhizoctonia solani-induced damping-off of cotton. Investigation of the...
A soil isolate of Pseudomonas fluorescens (BL915) was shown to be an effective antagonist of Rhizoctonia solani-induced damping-off of cotton. Investigation of the biological basis of this antagonism revealed that the strain produces pyrrolnitrin, a secondary metabolite known to inhibit R. solani and other fungi. Mutants of strain BL915 that did not produce pyrrolnitrin and did not suppress damping-off of cotton by R. solani were generated by exposure to N-methyl-N' -nitro-N-nitrosoguanidine. A gene region that was capable of restoring pyrrolnitrin production to the non-pyrrolnitrin-producing mutants and of conferring this ability upon two other P. fluorescens strains not otherwise known to produce this compound or to be capable of suppressing damping-off caused by R. solani was isolated from strain BL915. The non-pyrrolnitrin-producing strains (mutants of BL915 and the other two P. fluorescens strains) which synthesized pyrrolnitrin after the introduction of the gene region from strain BL915 were also shown to be equal to strain BL915 in their ability to suppress R. solani-induced damping-off of cotton. These results indicate that we have isolated from P. fluorescens BL915 a gene(s) that has a role in the synthesis of pyrrolnitrin and that the production of this compound has a role in the ability of this strain to control damping-off of cotton by R. solani.
PubMed: 16349167
DOI: 10.1128/aem.60.1.78-85.1994 -
Phytopathology May 2020A four-gene operon () from Pf-5 encoding the biosynthesis of the antibiotic pyrronitrin was introduced into (formerly ) 2-79, an aggressive root colonizer of both...
A four-gene operon () from Pf-5 encoding the biosynthesis of the antibiotic pyrronitrin was introduced into (formerly ) 2-79, an aggressive root colonizer of both dryland and irrigated wheat roots that naturally produces the antibiotic phenazine-1-carboxylic acid and suppresses both take-all and Rhizoctonia root rot of wheat. Recombinant strains ZHW15 and ZHW25 produced both antibiotics and maintained population sizes in the rhizosphere of wheat that were comparable to those of strain 2-79. The recombinant strains inhibited in vitro the wheat pathogens anastomosis group 8 (AG-8) and AG-2-1, var. , , , and significantly more than did strain 2-79. Both the wild-type and recombinant strains were equally inhibitory of . When applied as a seed treatment, the recombinant strains suppressed take-all, Rhizoctonia root rot of wheat, and Rhizoctonia root and stem rot of canola significantly better than did wild-type strain 2-79.
Topics: Plant Diseases; Pseudomonas; Pseudomonas fluorescens; Pyrrolnitrin
PubMed: 32065038
DOI: 10.1094/PHYTO-09-19-0367-R -
Biomedical Papers of the Medical... Sep 2014Screening of bacterial flora for strains producing metabolites with inhibitory effects on the human pathogenic oomycete Pythium insidiosum. Separation and...
AIMS
Screening of bacterial flora for strains producing metabolites with inhibitory effects on the human pathogenic oomycete Pythium insidiosum. Separation and characterization of extracts from Pseudomonas stutzeri with anti-Pythium inhibitory activity. Search for genes with anti-Pythium effect within the genome of P. stutzeri.
METHODS
A total of 88 bacterial strains were isolated from water resources in northeastern Thailand. Two screening methods were used to establish their inhibitory effects on P. insidiosum. One strain, P. stutzeri ST1302 was randomly chosen, and the extract with anti-P. insidiosum activity was fractionated and subfractionated using liquid column chromatography and purified by thin layer chromatography. The chemical structure of purified fractions was determined by Fourier transform infrared spectroscopy, nuclear magnetic resonance and mass spectrometry. Further, search for genes involved in the anti-Pythium activity (phenazine-1-carboxylic acid, 2,4-diacetylphloroglucinol, pyoluteorin and pyrrolnitrin) was undertaken in this P. stutzeri strain using primers described in the literature.
RESULTS
Anti-P. insidiosum activity was detected in 16 isolates (18.2%). In P. stutzeri ST1302, a subfraction labeled PYK7 exhibited strong activity against this oomycete. It was assigned to the diketopiperazines as cyclo(D-Pro-L-Val). In the search for genes, one gene region was successfully amplified. This corresponded to pyrrolnitrin. The results suggest the possibility of using the related metabolites against P. insidiosum. This is the first report on the inhibitory effects of P. stutzeri against this oomycete. The results may contribute to the development of antimicrobial drugs/probiotics against pythiosis.
Topics: Diketopiperazines; Genome, Bacterial; Microbial Sensitivity Tests; Pseudomonas stutzeri; Pyrrolnitrin; Pythiosis; Pythium; Thailand
PubMed: 23149469
DOI: 10.5507/bp.2012.090 -
Phytopathology Jun 1997ABSTRACT Pseudomonas fluorescens strain Pf-5, which produces several antifun-gal metabolites, including the antibiotics pyoluteorin, pyrrolnitrin, and...
ABSTRACT Pseudomonas fluorescens strain Pf-5, which produces several antifun-gal metabolites, including the antibiotics pyoluteorin, pyrrolnitrin, and 2,4-diacetylphloroglucinol, was tested for its ability to inhibit Sclerotinia homoeocarpa (causal agent of dollar spot) and Drechslerapoae (causal agent of 'melting-out') in vitro and in turfgrass; Tn5 mutants with altered antibiotic production also were tested. Inhibition in vitro differed with the medium used, but both fungi generally were inhibited by Pf-5. In most cases, a mutant deficient in pyoluteorin but not pyrrolnitrin or 2,4-di-acetylphloroglucinol was as inhibitory as Pf-5, whereas a pyrrolnitrin-deficient mutant was less inhibitory than Pf-5 in most fungus/medium combinations. High-performance liquid chromatography analysis of culture extracts showed that bacterial genotype and nutrition have an interactive effect on antibiotic production, such that conditions causing an increase in one antibiotic may increase or decrease another. The purported deficiencies for the pyrrolnitrin- and pyoluteorin-deficient mutants were confirmed. In S. homoeocarpa-infested grass clippings incubated in a moist chamber, Pf-5 reduced mycelial growth, whereas the pyrrolnitrin-deficient mutant did not and the pyoluteorin-deficient mutant was intermediate. In greenhouse experiments, Pf-5 reduced dollar spot disease incidence in bentgrass and bluegrass when sprayed over inoculated turf. In grass clippings infested with D. poae and incubated in a moist chamber under favorable conditions for spore production, Pf-5 did not reduce significantly the number of spores produced compared with the non-treated control. However, Pf-5 reduced melting-out disease incidence and severity in bluegrass inoculated with spores of D. poae under greenhouse conditions.
PubMed: 18945079
DOI: 10.1094/PHYTO.1997.87.6.614 -
Applied and Environmental Microbiology Jun 1997Pyrrolnitrin is a secondary metabolite of Pseudomonas and Burkholderia sp. strains with strong antifungal activity. Production of pyrrolnitrin has been correlated with... (Comparative Study)
Comparative Study
Pyrrolnitrin is a secondary metabolite of Pseudomonas and Burkholderia sp. strains with strong antifungal activity. Production of pyrrolnitrin has been correlated with the ability of some bacteria to control plant diseases caused by fungal pathogens, including the damping-off pathogen Rhizoctonia solani. Pseudomonas fluorescens BL915 has been reported to produce pyrrolnitrin and to be an effective biocontrol agent for this pathogen. We have isolated a 32-kb genomic DNA fragment from this strain that contains genes involved in the biosynthesis of pyrrolnitrin. Marker-exchange mutagenesis of this DNA with Tn5 revealed the presence of a 6.2-kb region that contains genes required for the synthesis of pyrrolnitrin. The nucleotide sequence of the 6.2-kb region was determined and found to contain a cluster of four genes that are required for the production of pyrrolnitrin. Deletion mutations in any of the four genes resulted in a pyrrolnitrin-nonproducing phenotype. The putative coding sequences of the four individual genes were cloned by PCR and fused to the tac promoter from Escherichia coli. In each case, the appropriate tac promoter-pyrrolnitrin gene fusion was shown to complement the pyrrolnitrin-negative phenotype of the corresponding deletion mutant. Transfer of the four gene cluster to E. coli resulted in the production of pyrrolnitrin by this organism, thereby demonstrating that the four genes are sufficient for the production of this metabolite and represent all of the genes required to encode the pathway for pyrrolnitrin biosynthesis.
Topics: Amino Acid Sequence; Antifungal Agents; Bacterial Proteins; Cloning, Molecular; DNA, Bacterial; Escherichia coli; Gene Deletion; Genes, Bacterial; Molecular Sequence Data; Multigene Family; Open Reading Frames; Phenotype; Plant Diseases; Plants; Polymerase Chain Reaction; Pseudomonas fluorescens; Pyrrolnitrin; Restriction Mapping; Sequence Homology, Amino Acid
PubMed: 9172332
DOI: 10.1128/aem.63.6.2147-2154.1997 -
Frontiers in Microbiology 2017Particular groups of plant-beneficial fluorescent pseudomonads are not only root colonizers that provide plant disease suppression, but in addition are able to infect...
Particular groups of plant-beneficial fluorescent pseudomonads are not only root colonizers that provide plant disease suppression, but in addition are able to infect and kill insect larvae. The mechanisms by which the bacteria manage to infest this alternative host, to overcome its immune system, and to ultimately kill the insect are still largely unknown. However, the investigation of the few virulence factors discovered so far, points to a highly multifactorial nature of insecticidal activity. Antimicrobial compounds produced by fluorescent pseudomonads are effective weapons against a vast diversity of organisms such as fungi, oomycetes, nematodes, and protozoa. Here, we investigated whether these compounds also contribute to insecticidal activity. We tested mutants of the highly insecticidal strains CHA0, PCL1391, and sp. CMR12a, defective for individual or multiple antimicrobial compounds, for injectable and oral activity against lepidopteran insect larvae. Moreover, we studied expression of biosynthesis genes for these antimicrobial compounds for the first time in insects. Our survey revealed that hydrogen cyanide and different types of cyclic lipopeptides contribute to insecticidal activity. Hydrogen cyanide was essential to full virulence of CHA0 and PCL1391 directly injected into the hemolymph. The cyclic lipopeptide orfamide produced by CHA0 and CMR12a was mainly important in oral infections. Mutants of CMR12a and PCL1391 impaired in the production of the cyclic lipopeptides sessilin and clp1391, respectively, showed reduced virulence in injection and feeding experiments. Although virulence of mutants lacking one or several of the other antimicrobial compounds, i.e., 2,4-diacetylphloroglucinol, phenazines, pyrrolnitrin, or pyoluteorin, was not reduced, these metabolites might still play a role in an insect background since all investigated biosynthetic genes for antimicrobial compounds of strain CHA0 were expressed at some point during insect infection. In summary, our study identified new factors contributing to insecticidal activity and extends the diverse functions of antimicrobial compounds produced by fluorescent pseudomonads from the plant environment to the insect host.
PubMed: 28217113
DOI: 10.3389/fmicb.2017.00100 -
Frontiers in Microbiology 2020For plants, the advantages of associating with beneficial bacteria include plant growth promotion, reduction of abiotic and biotic stresses and enhanced protection...
For plants, the advantages of associating with beneficial bacteria include plant growth promotion, reduction of abiotic and biotic stresses and enhanced protection against various pests and diseases. Beneficial bacteria rightly equipped for successful plant colonization and showing antagonistic activity toward plant pathogens seem to be actively recruited by plants. To gain more insights into the genetic determinants responsible for plant colonization and antagonistic activities, we first sequenced and assembled the complete genomes of nine strains that had exhibited varying antagonistic potential against the notorious oomycete , placed them into the phylogenomic context of known biocontrol strains and carried out a comparative genomic analysis to define core, accessory (i.e., genes found in two or more, but not all strains) and unique genes. Next, we assessed the colonizing abilities of these strains and used bioassays to characterize their inhibitory effects against different stages of ' lifecycle. The phenotype data were then correlated with genotype information, assessing over three hundred genes encoding known factors for plant colonization and antimicrobial activity as well as secondary metabolite biosynthesis clusters predicted by antiSMASH. All strains harbored genes required for successful plant colonization but also distinct arsenals of antimicrobial compounds. We identified genes coding for phenazine, hydrogen cyanide, 2-hexyl, 5-propyl resorcinol and pyrrolnitrin synthesis, as well as various siderophores, pyocins and type VI secretion systems. Additionally, the comparative genomic analysis revealed about a hundred accessory genes putatively involved in anti- activity, including a type II secretion system (T2SS), several peptidases and a toxin. Transcriptomic studies and mutagenesis are needed to further investigate the putative involvement of the novel candidate genes and to identify the various mechanisms involved in the inhibition of by different strains.
PubMed: 32425922
DOI: 10.3389/fmicb.2020.00857 -
Applied and Environmental Microbiology Jun 1999Understanding the environmental factors that regulate the biosynthesis of antimicrobial compounds by disease-suppressive strains of Pseudomonas fluorescens is an...
Understanding the environmental factors that regulate the biosynthesis of antimicrobial compounds by disease-suppressive strains of Pseudomonas fluorescens is an essential step toward improving the level and reliability of their biocontrol activity. We used liquid culture assays to identify several minerals and carbon sources which had a differential influence on the production of the antibiotics 2,4-diacetylphloroglucinol (PHL), pyoluteorin (PLT), and pyrrolnitrin and the siderophores salicylic acid and pyochelin by the model strain CHA0, which was isolated from a natural disease-suppressive soil in Switzerland. Production of PHL was stimulated by Zn2+, NH4Mo2+, and glucose; the precursor compound mono-acetylphloroglucinol was stimulated by the same factors as PHL. Production of PLT was stimulated by Zn2+, Co2+, and glycerol but was repressed by glucose. Pyrrolnitrin production was increased by fructose, mannitol, and a mixture of Zn2+ and NH4Mo2+. Pyochelin production was increased by Co2+, fructose, mannitol, and glucose. Interestingly, production of its precursor salicylic acid was increased by different factors, i.e., NH4Mo2+, glycerol, and glucose. The mixture of Zn2+ and NH4Mo2+ with fructose, mannitol, or glycerol further enhanced the production of PHL and PLT compared with either the minerals or the carbon sources used alone, but it did not improve siderophore production. Extending fermentation time from 2 to 5 days increased the accumulation of PLT, pyrrolnitrin, and pyochelin but not of PHL. When findings with CHA0 were extended to an ecologically and genetically diverse collection of 41 P. fluorescens biocontrol strains, the effect of certain factors was strain dependent, while others had a general effect. Stimulation of PHL by Zn2+ and glucose was strain dependent, whereas PLT production by all strains that can produce this compound was stimulated by Zn2+ and transiently repressed by glucose. Inorganic phosphate reduced PHL production by CHA0 and seven other strains tested but to various degrees. Production of PLT but not pyrrolnitrin by CHA0 was also reduced by 100 mM phosphate. The use of 1/10-strength nutrient broth-yeast extract, compared with standard nutrient broth-yeast extract, amended with glucose and/or glycerol resulted in dramatically increased accumulations of PHL (but not PLT), pyochelin, and salicylic acid, indicating that the ratio of carbon source to nutrient concentration played a key role in the metabolic flow. The results of this study (i) provide insight into the biosynthetic regulation of antimicrobial compounds, (ii) limit the number of factors for intensive study in situ, and (iii) indicate factors that can be manipulated to improve bacterial inoculants.
Topics: Anti-Bacterial Agents; Colony Count, Microbial; Culture Media; Pest Control, Biological; Phenols; Phloroglucinol; Pseudomonas fluorescens; Pyrroles; Pyrrolnitrin; Salicylic Acid; Siderophores; Soil Microbiology; Thiazoles
PubMed: 10347023
DOI: 10.1128/AEM.65.6.2429-2438.1999 -
Applied and Environmental Microbiology Jun 2021Within animal-associated microbiomes, the functional roles of specific microbial taxa are often uncharacterized. Here, we use the fungus-growing ant system, a model for...
Within animal-associated microbiomes, the functional roles of specific microbial taxa are often uncharacterized. Here, we use the fungus-growing ant system, a model for microbial symbiosis, to determine the potential defensive roles of key bacterial taxa present in the ants' fungus gardens. Fungus gardens serve as an external digestive system for the ants, with mutualistic fungi in the genus converting the plant substrate into energy for the ants. The fungus garden is host to specialized parasitic fungi in the genus . Here, we examine the potential role of spp. that occur within ant fungus gardens in inhibiting We isolated members of the bacterial genera and from 50% of the 52 colonies sampled, indicating that members of the family are common inhabitants in the fungus gardens of a diverse range of fungus-growing ant genera. Using antimicrobial inhibition bioassays, we found that 28 out of 32 isolates inhibited at least one strain with a zone of inhibition greater than 1 cm. Genomic assessment of fungus garden-associated indicated that isolates with strong inhibition all belonged to the genus and contained biosynthetic gene clusters that encoded the production of two antifungals: burkholdine1213 and pyrrolnitrin. Organic extracts of cultured isolates confirmed that these compounds are responsible for antifungal activities that inhibit but, at equivalent concentrations, not spp. Overall, these new findings, combined with previous evidence, suggest that members of the fungus garden microbiome play an important role in maintaining the health and function of fungus-growing ant colonies. Many organisms partner with microbes to defend themselves against parasites and pathogens. Fungus-growing ants must protect spp., the fungal mutualist that provides sustenance for the ants, from a specialized fungal parasite, . The ants take multiple approaches, including weeding their fungus gardens to remove spores, as well as harboring spp., bacteria that produce antifungals that inhibit In addition, a genus of bacteria commonly found in fungus gardens, , is known to produce secondary metabolites that inhibit spp. In this study, we isolated spp. from fungus-growing ants, assessed the isolates' ability to inhibit spp., and identified two compounds responsible for inhibition. Our findings suggest that spp. are often found in fungus gardens, adding another possible mechanism within the fungus-growing ant system to suppress the growth of the specialized parasite .
Topics: Animals; Antifungal Agents; Ants; Burkholderia; Hypocreales; Lipopeptides; Microbiota; Multigene Family; Parasites; Phylogeny; Pyrrolnitrin; Symbiosis
PubMed: 33962985
DOI: 10.1128/AEM.00178-21