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Molecular Plant Pathology Aug 2021The biocontrol rhizobacterium Pseudomonas protegens H78 can produce a large array of antimicrobial secondary metabolites, including pyoluteorin (Plt),...
The biocontrol rhizobacterium Pseudomonas protegens H78 can produce a large array of antimicrobial secondary metabolites, including pyoluteorin (Plt), 2,4-diacetylphloroglucinol (DAPG), and pyrrolnitrin (Prn). Our preliminary study showed that the biosynthesis of antibiotics including Plt is activated by the RNA chaperone Hfq in P. protegens H78. This prompted us to explore the global regulatory mechanism of Hfq, as well as the catabolite repression control (Crc) protein in H78. The antimicrobial capacity of H78 was positively controlled by Hfq while slightly down-regulated by knockout of crc. Similarly, cell growth of H78 was significantly impaired by deletion of hfq and slightly inhibited by knockout of crc. Transcriptomic profiling revealed that hfq mutation resulted in significant down-regulation of 688 genes and up-regulation of 683 genes. However, only 113 genes were significantly down-regulated and 105 genes up-regulated by the crc mutation in H78. Hfq positively regulated the expression of gene clusters involved in secondary metabolism (plt, prn, phl, hcn, and pvd), the type VI secretion system, and aromatic compound degradation. However, Crc only positively regulated the biosynthesis of Plt but not other antibiotics. Hfq also regulated expression of genes involved in oxidative phosphorylation and flagellar biogenesis. In addition, Hfq and Crc activated transcription of crcY/Z sRNAs by feedback. In summary, Hfq processes far more extensive and intensive regulatory capacity than Crc and shows small cross-regulation with Crc in H78. This study lays the foundation for clarifying the Hfq and/or Crc-dependent global regulatory network and improving antibiotic production by genetic engineering in P. protegens.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Gene Expression Regulation, Bacterial; Pseudomonas
PubMed: 33963656
DOI: 10.1111/mpp.13070 -
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 -
Scientific Reports Mar 2021Aspergillus fumigatus is an important fungal pathogen that represents a major threat for severely immunocompromised patients. Cases of invasive aspergillosis are...
Aspergillus fumigatus is an important fungal pathogen that represents a major threat for severely immunocompromised patients. Cases of invasive aspergillosis are associated with a high mortality rate, which reflects the limited treatment options that are currently available. The development of novel therapeutic approaches is therefore an urgent task. An interesting compound is fludioxonil, a derivative of the bacterial secondary metabolite pyrrolnitrin. Both agents possess potent antimicrobial activity against A. fumigatus and trigger a lethal activation of the group III hybrid histidine kinase TcsC, the major sensor kinase of the High Osmolarity Glycerol (HOG) pathway in A. fumigatus. In the current study, we have characterized proteins that operate downstream of TcsC and analyzed their roles in the antifungal activity of fludioxonil and in other stress situations. We found that the SskA-SakA axis of the HOG pathway and Skn7 can independently induce an increase of the internal glycerol concentration, but each of these individual responses amounts for only half of the level found in the wild type. The lethal fludioxonil-induced ballooning occurs in the sskA and the sakA mutant, but not in the skn7-deficient strain, although all three strains show comparable glycerol responses. This indicates that an elevated osmotic pressure is necessary, but not sufficient and that a second, decisive and Skn7-dependent mechanism mediates the antifungal activity. We assume that fludioxonil triggers a reorganization in the fungal cell wall that reduces its rigidity, which in combination with the elevated osmotic pressure executes the lethal expansion of the fungal cells. Two findings link Skn7 to the cell wall of A. fumigatus: (1) the fludioxonil-induced massive increase in the chitin content depends on Skn7 and (2) the skn7 mutant is more resistant to the cell wall stressor Calcofluor white. In conclusion, our data suggest that the antifungal activity of fludioxonil in A. fumigatus relies on two distinct and synergistic processes: A high internal osmotic pressure and a weakened cell wall. The involvement of Skn7 in both processes most likely accounts for its particular importance in the antifungal activity of fludioxonil.
Topics: Antifungal Agents; Aspergillus fumigatus; Dioxoles; Fungal Proteins; Pyrroles
PubMed: 33674651
DOI: 10.1038/s41598-021-84740-6 -
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 -
FEMS Microbiology Letters May 2020Pseudomonas putida and closely-related species such as Pseudomonas fluorescens and Pseudomonas brassicacearum have been reported as potential biocontrol agents and plant...
Pseudomonas putida and closely-related species such as Pseudomonas fluorescens and Pseudomonas brassicacearum have been reported as potential biocontrol agents and plant growth-promoters. Recently, we have described the biocontrol activity of P. putida B2017 against several phytopathogens of agricultural relevance. In this study, its ability to produce potential antibiotic / toxic metabolites was assessed by functional, chromatography-mass spectrometry and genomic analysis. Our results show that B2017 is not able to synthesize surfactants and common antibiotics produced by Pseudomonas spp., i.e. pyrrolnitrin, 2,4-diacetylphloroglucinol, pyoluteorin and pyocyanin, but it produces pyoverdine, a siderophore which is involved in its biocontrol activity. The non-production of other metabolites, such as cyanide, safracin, promysalin and lipopeptides between others, is also discussed. Our data suggest that the mode of action of B2017 is not mainly due to the production of antimicrobial / toxic metabolites. Moreover, these features make P. putida B2017 a promising biocontrol microorganism for plant protection without side effects on environment, non-target organisms and human health.
Topics: Anti-Bacterial Agents; Bacteria; Bacterial Proteins; Biological Control Agents; Fungi; Genome, Bacterial; Oligopeptides; Plant Diseases; Pseudomonas putida; Siderophores; Surface-Active Agents
PubMed: 32347922
DOI: 10.1093/femsle/fnaa075 -
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