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PeerJ 2021strain PA23 is a biocontrol agent that is able to protect canola against the pathogenic fungus . This bacterium secretes a number of metabolites that contribute to...
BACKGROUND
strain PA23 is a biocontrol agent that is able to protect canola against the pathogenic fungus . This bacterium secretes a number of metabolites that contribute to fungal antagonism, including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN) and degradative enzymes. In order to be successful, a biocontrol agent must be able to persist in the environment and avoid the threat of grazing predators. The focus of the current study was to investigate whether PA23 is able to resist grazing by the protozoan predator (Ac) and to define the role of bacterial metabolites in the PA23-Ac interaction.
METHODS
Ac was co-cultured with PA23 WT and a panel of derivative strains for a period of 15 days, and bacteria and amoebae were enumerated on days 1, 5, 10 and 15. Ac was subsequently incubated in the presence of purified PRN, PHZ, and KCN and viability was assessed at 24, 48 and 72 h. Chemotactic assays were conducted to assess whether PA23 compounds exhibit repellent or attractant properties towards Ac. Finally, PA23 grown in the presence and absence of amoebae was subject to phenotypic characterization and gene expression analyses.
RESULTS
PRN, PHZ and HCN were found to contribute to PA23 toxicity towards Ac trophozoites, either by killing or inducing cyst formation. This is the first report of PHZ-mediated toxicity towards amoebae. In chemotaxis assays, amoebae preferentially migrated towards regulatory mutants devoid of extracellular metabolite production as well as a PRN mutant, indicating this antibiotic has repellent properties. Co-culturing of bacteria with amoebae led to elevated expression of the PA23 / quorum-sensing (QS) genes and and , which are under QS control. PHZ and PRN levels were similarly increased in Ac co-cultures, suggesting that PA23 can respond to predator cues and upregulate expression of toxins accordingly.
CONCLUSIONS
PA23 compounds including PRN, PHZ and HCN exhibited both toxic and repellent effects on Ac. Co-culturing of bacteria and amoebae lead to changes in bacterial gene expression and secondary metabolite production, suggesting that PA23 can sense the presence of these would-be predators and adjust its physiology in response.
PubMed: 33552738
DOI: 10.7717/peerj.10756 -
Antibiotics (Basel, Switzerland) Feb 2021Antimicrobial resistance in Gram-negative pathogens represents a global threat to human health. This study determines the antimicrobial potential of a taxonomically and...
Antimicrobial resistance in Gram-negative pathogens represents a global threat to human health. This study determines the antimicrobial potential of a taxonomically and geographically diverse collection of 263 (sensu lato) isolates and applies natural product dereplication strategies to identify potentially novel molecules. Antimicrobial activity is almost exclusively present in sensu stricto bacteria and rarely observed in the novel genera , , , , and . Fourteen isolates show a unique spectrum of antimicrobial activity and inhibited carbapenem-resistant Gram-negative bacterial pathogens. Dereplication of the molecules present in crude spent agar extracts identifies 42 specialized metabolites, 19 of which represented potentially novel molecules. The known identified metabolites include toxoflavin, reumycin, pyrrolnitrin, enacyloxin, bactobolin, cepacidin, ditropolonyl sulfide, and antibiotics BN-227-F and SF 2420B, as well as the siderophores ornibactin, pyochelin, and cepabactin. Following semipreparative fractionation and activity testing, a total of five potentially novel molecules are detected in active fractions. Given the molecular formula and UV spectrum, two of those putative novel molecules are likely related to bactobolins, and another is likely related to enacyloxins. The results from this study confirm and extend the observation that bacteria present exciting opportunities for the discovery of potentially novel bioactive molecules.
PubMed: 33540653
DOI: 10.3390/antibiotics10020147 -
3 Biotech Feb 2021In this study, nine strains of and , and two isolates of sp: At1RP4 and RS-1, were characterized for the in-vitro production of secondary metabolites in LB, DMB, and...
UNLABELLED
In this study, nine strains of and , and two isolates of sp: At1RP4 and RS-1, were characterized for the in-vitro production of secondary metabolites in LB, DMB, and King's B media, and of the genes responsible for the production of antagonistic metabolites. Based on 16S rRNA gene sequence, isolates At1RP4 and RS-1 were identified as strains of and . Five phenazine derivatives comprising phenazine, phenazine-1-carboxylic acid (PCA), 2-hydroxyphenazine-1-carboxylic acid (2-OH-Phz-1-COOH), phenazine-1,6-dicarboxylic acid (Phz-1,6-di-COOH), and 2-hydroxyphenazine (2-OH-Phz) were produced by all strains in all three culture media including DMB, King's B and LB. However, 2,8-dihydroxyphenazine, 6-methylphenazine-1-carboxylic acid, pyrrolnitrin, and the -dialkylaromatic acids, were produced by the and strains. In addition, all strains produced 2-acetamidophenol, pyochelin, and diketopiperazine derivatives in variable amounts in all three culture media used. Highest levels of quorum-sensing signal molecules including PQS, 2-Octyl-3-hydroxy-4(1H)-quinolone, and hexahydro-quinoxaline-1,4-dioxide were recorded for At1RP4. Moreover, all strains produced volatile hydrogen cyanide (0.95-6.68 µg/L) and the phytohormone indole-3-acetic acid (0.42-13.9 µM). Production of extracellular lipase and protease was recorded in all pseudomonads, whereas, cellulase production and phosphate solubilization were variable. Genes for hydrogen cyanide and phenazine-1-carboxylic acid were detected in all eleven strains while the gene for pyrrolnitrin biosynthesis was amplified in and strains. Comparative metabolomic analysis provided detailed insights about the strain-specific metabolites in pseudomonads, and their pseudo-relative quantification in different bacterial growth media to be used as single-strain biofertilizer and biocontrol inoculums.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s13205-020-02585-8.
PubMed: 33489669
DOI: 10.1007/s13205-020-02585-8 -
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 -
Microorganisms Apr 2020Plant growth-promoting rhizobacteria (PGPR) can potentially be used as an alternative strategy to control plant diseases. In this study, strain ST-TJ4 isolated from the...
Plant growth-promoting rhizobacteria (PGPR) can potentially be used as an alternative strategy to control plant diseases. In this study, strain ST-TJ4 isolated from the rhizosphere soil of a healthy poplar was found to have a strong antifungal activity against 11 phytopathogenic fungi in agriculture and forestry. Strain ST-TJ4 was identified as sp. based on 16S rRNA-encoding gene sequences. The bacterium can produce siderophores, cellulase, and protease, and has genes involved in the synthesis of phenazine, 1-phenazinecarboxylic acid, pyrrolnitrin, and hydrogen cyanide. Additionally, the volatile compounds released by strain ST-TJ4 can inhibit the mycelial growth of plant pathogenic fungi more than diffusible substances can. Based on volatile compound profiles of strain ST-TJ4 obtained from headspace collection and GC-MS/MS analysis, 1-undecene was identified. In summary, the results suggested that sp. ST-TJ4 can be used as a biocontrol agent for various plant diseases caused by phytopathogenic fungi.
PubMed: 32325752
DOI: 10.3390/microorganisms8040590 -
Microbes and Environments 2020More than 3,000 isolates of fluorescent pseudomonads have been collected from plant roots in Japan and screened for the presence of antibiotic-synthesizing genes. In...
More than 3,000 isolates of fluorescent pseudomonads have been collected from plant roots in Japan and screened for the presence of antibiotic-synthesizing genes. In total, 927 hydrogen cyanide (HCN)-, 47 2,4-diacetylphloroglucinol (PHL)-, 6 pyoluteorin (PLT)-, 14 pyrrolnitrin (PRN)-, and 8 phenazine (PHZ)-producing isolates have been detected. A cluster analysis (≥99% identity) identified 10 operational taxonomic units (OTUs) in antibiotic biosynthesis gene-possessing pseudomonads. OTU HLR (PHL, PLT, and PRN) contained four antibiotics: HCN, PHL, PLT, and PRN, while OTU RZ (PRN and PHZ) contained three: HCN, PRN, and PHZ. OTU H1, H2, H3, H4, H5, H6, and H7 (PHL1-7) contained two antibiotics: HCN and PHL, while OTU H8 (PHL8) contained one: PHL. Isolates belonging to OTU HLR and RZ suppressed damping-off disease in cabbage seedlings caused by Rhizoctonia solani. Effective strains belonging to OTU HLR and RZ were related to Pseudomonas protegens and Pseudomonas chlororaphis, respectively. Antibiotic biosynthesis gene-possessing fluorescent pseudomonads are distributed among different geographical sites in Japan and plant species.
Topics: Anti-Bacterial Agents; Biological Control Agents; Fluorescence; Genes, Bacterial; Genetic Variation; Japan; Plant Roots; Pseudomonas; Pseudomonas fluorescens; RNA, Ribosomal, 16S; Rhizosphere; Soil Microbiology
PubMed: 32269203
DOI: 10.1264/jsme2.ME19155 -
PloS One 2020Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungal pathogen Sclerotinia sclerotiorum....
Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungal pathogen Sclerotinia sclerotiorum. PA23 produces several inhibitory compounds that are under control of a complex regulatory network. Included in this cascade is the PhzRI quorum sensing (QS) system, which plays an essential role in PA23 biocontrol, as well as CsaRI and AurRI, which have not yet been characterized in PA23. The focus of the current study was to employ RNA sequencing to explore the spectrum of PA23 genes under QS control. In this work, we investigated genes under the control of the main QS transcriptional regulator, PhzR, as well as those differentially expressed in an AHL-deficient strain, PA23-6863, which constitutively expresses an AiiA lactonase, rendering the strain QS defective. Transcriptomic profiling revealed 545 differentially expressed genes (365 downregulated; 180 upregulated) in the phzR mutant and 534 genes (382 downregulated; 152 upregulated) in the AHL-deficient PA23-6863. In both strains, decreased expression of phenazine, pyrrolnitrin, and exoprotease biosynthetic genes was observed. We have previously reported that QS activates expression of these genes and their encoded products. In addition, elevated siderophore and decreased chitinase gene expression was observed in the QS-deficient stains, which was confirmed by phenotypic analysis. Inspection of the promoter regions revealed the presence of "phz-box" sequences in only 58 of the 807 differentially expressed genes, suggesting that much of the QS regulon is indirectly regulated. Consistent with this notion, 41 transcriptional regulators displayed altered expression in one or both of the QS-deficient strains. Collectively, our findings indicate that QS governs expression of approximately 13% of the PA23 genome affecting diverse functions ranging from secondary metabolite production to general metabolism.
Topics: Bacterial Proteins; Carboxylic Ester Hydrolases; Cell Movement; Chitinases; Gene Expression Regulation, Bacterial; Mutant Proteins; Pest Control, Biological; Pseudomonas chlororaphis; Quorum Sensing; RNA-Seq; Regulon; Siderophores; Trans-Activators; Transcriptome
PubMed: 32109244
DOI: 10.1371/journal.pone.0226232 -
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 -
Organic Letters Feb 2020Pyonitrins A-D are recently isolated natural products from the insect-associated strain, which were isolated from complex fractions that exhibited antifungal activity...
Pyonitrins A-D are recently isolated natural products from the insect-associated strain, which were isolated from complex fractions that exhibited antifungal activity via an murine candidiasis assay. Genomic studies of suggested that pyonitrins A-D are formed via a spontaneous nonenzymatic reaction between biosynthetic intermediates of two well-known natural products pyochelin and pyrrolnitrin. Herein we have accomplished the first biomimetic total synthesis of pyonitrins A-D in three steps and studied the nonenzymatic formation of the pyonitrins using N NMR spectroscopy.
Topics: Antifungal Agents; Candida albicans; Magnetic Resonance Spectroscopy; Microbial Sensitivity Tests; Molecular Structure; Pseudomonas; Stereoisomerism; Thiazoles
PubMed: 32017580
DOI: 10.1021/acs.orglett.0c00098 -
Journal of the American Chemical Society Oct 2019Bacterial symbionts frequently provide chemical defenses for their hosts, and such systems can provide discovery pathways to new antifungals and structurally intriguing...
Bacterial symbionts frequently provide chemical defenses for their hosts, and such systems can provide discovery pathways to new antifungals and structurally intriguing metabolites. This report describes a small family of naturally occurring small molecules with chimeric structures and a mixed biosynthesis that features an unexpected but key nonenzymatic step. An insect-associated strain's activity in an murine candidiasis assay led to the discovery of a family of highly hydrogen-deficient metabolites. Bioactivity- and mass-guided fractionation led to the pyonitrins, highly complex aromatic metabolites in which 10 of the 20 carbons are quaternary, and 7 of them are contiguous. The genome revealed that the production of the pyonitrins is the result of a spontaneous reaction between biosynthetic intermediates of two well-studied metabolites, pyochelin and pyrrolnitrin. The combined discovery of the pyonitrins and identification of the responsible biosynthetic gene clusters revealed an unexpected biosynthetic route that would have prevented the discovery of these metabolites by bioinformatic analysis alone.
Topics: Animals; Antifungal Agents; Bacterial Proteins; Biological Products; Biosynthetic Pathways; Candida albicans; Candidiasis; Chromatography, High Pressure Liquid; Drug Evaluation, Preclinical; Magnetic Resonance Spectroscopy; Mice; Molecular Structure; Phenols; Pseudomonas; Pyrrolnitrin; Thiazoles
PubMed: 31600443
DOI: 10.1021/jacs.9b09739