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Access Microbiology 2023Several species of soil-dwelling nematodes are used in the biocontrol of crop pests, due to their natural capacity to kill diverse lepidopteran species. Although this...
Several species of soil-dwelling nematodes are used in the biocontrol of crop pests, due to their natural capacity to kill diverse lepidopteran species. Although this insect-killing trait is known to be augmented by the nematodes' endosymbionts, the role of other steinernematid-associated bacterial genera in the nematode lifecycle remains unclear. This genomic study aimed to determine the potential of to contribute to the entomopathogenicity of its host. Insect larvae were infected with three separate cultures. From each of the three treatments, the prevalent bacteria in the haemocoel of cadavers, four days post-infection, were isolated. These three bacterial isolates were morphologically characterised. DNA was extracted from each of the three bacterial isolates and used for long-read genome sequencing and assembly. Assemblies were used to delineate species and identify genes that encode insect toxins, antimicrobials, and confer antibiotic resistance. We assembled three complete genomes. Through digital DNA-DNA hybridisation analyses, we ascertained that the haemocoels of insect cadavers previously infected with sp. Kalro, sp. 75, and sp. 97 were dominated by Kalro, 75, and 97, respectively. Kalro and 97 formed a subspecies with other symbionts of steinernematids from Kenya. 75 phylogenetically clustered with pseudomonads that are characterised by high insecticidal activity. The 75 genome encoded the production pathway of insect toxins such as orfamides and rhizoxins, antifungals such as pyrrolnitrin and pyoluteorin, and the broad-spectrum antimicrobial 2,4-diacetylphloroglucinol. The 75 genome encoded resistance to over ten classes of antibiotics, including cationic lipopeptides. Steinernematid-associated bacteria hence have the biosynthetic potential to contribute to nematode entomopathogenicity.
PubMed: 37970093
DOI: 10.1099/acmi.0.000659.v3 -
Microorganisms Sep 2021The bacterial pathogen causes the destructive fruit blotch (BFB) on cucurbit plants. YL-1 is a bacterial strain isolated from Mississippi soil and its genome harbors...
The bacterial pathogen causes the destructive fruit blotch (BFB) on cucurbit plants. YL-1 is a bacterial strain isolated from Mississippi soil and its genome harbors some antimicrobial-related gene clusters, such as phenazine, pyrrolnitrin, and pyoverdine. Here, we evaluated the antimicrobial activity of strain YL-1 as compared with its deficient mutants of antimicrobial-related genes, which were obtained using a based site-specific mutagenesis strategy. We found that only phenazine-deficient mutants Δ and Δ almost lost the inhibitory effects against in LB plates compared with the wild-type strain YL-1, and that the main antibacterial compound produced by strain YL-1 in LB medium was phenazine-1-carboxylic acid (PCA) based on the liquid chromatography-mass spectrometry (LC-MS) analysis. Gene expression analyses revealed that PCA enhanced the accumulation of reactive oxygen species (ROS) and increased the activity of catalase (CAT) in . The inhibition effect of PCA against was lowered by adding exogenous CAT. PCA significantly upregulated the transcript level of from 6 to 10 h, which encodes CAT that helps to protect the bacteria against oxidative stress. Collectively, the findings of this research suggest PCA is one of the key antimicrobial metabolites of bacterial strain YL-1, a promising biocontrol agent for disease management of BFB of cucurbit plants.
PubMed: 34683333
DOI: 10.3390/microorganisms9102012 -
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 -
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 -
Pathogens (Basel, Switzerland) Mar 2022(strain DSMZ 13134) is a biocontrol agent with promising antagonistic activity hinging on antibiosis against the fungal forest pathogens spp. Here, by using...
(strain DSMZ 13134) is a biocontrol agent with promising antagonistic activity hinging on antibiosis against the fungal forest pathogens spp. Here, by using High-Performance Liquid Chromatography coupled to Mass Spectrometry (HPLC-MS), we assessed whether monocultures of (strain DSMZ 13134) produce the three major determinants of biocontrol activity known for the genus : 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin (PLT), and pyrrolnitrin (PRN). At the tested culture conditions, we observed the production of PLT at concentrations ranging from 0.01 to 10.21 mg/L and 2,4-DAPG at a concentration not exceeding 0.5 mg/L. Variations of culture conditions involving culture medium, incubation temperature, and incubation period had no consistent influence on PLT production by the bacterium. Assays using culture medium amended with PLT at the same concentration of that present in cell-free filtrate of the bacterium, i.e., 3.77 mg/L, previously documented as effective against spp., showed a remarkable activity of PLT against genotypes of all the four species present in Europe, including the non-native invasive . However, such antifungal activity decreased over time, and this may be a constraint for using this molecule as a pesticide against spp. When the bacterium was co-cultured in liquid medium with genotypes of the different species, an increased production of PLT was observed at 4 °C, suggesting the bacterium may perform better as a PLT producer in field applications under similar environmental conditions, i.e., at low temperatures. Our results demonstrated the role of PLT in the inhibition of spp., although all lines of evidence suggest that antibiosis does not rely on a single constitutively produced metabolite, but rather on a plethora of secondary metabolites. Findings presented in this study will help to optimize treatments based on (strain DSMZ 13134) against spp.
PubMed: 35456066
DOI: 10.3390/pathogens11040391 -
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 -
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 -
The Plant Pathology Journal Aug 2019In our previous study, pyrrolnitrin produced in G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in...
In our previous study, pyrrolnitrin produced in G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant G05ΔΔ:: and S17-1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on , however, it was reported that the deletion of the caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the -knockout mutant G05Δ and G05ΔΔ::Δ. By quantifying β-galactosidase activities, we found that deletion of the decreased the operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant G05Δ, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in G05.
PubMed: 31481858
DOI: 10.5423/PPJ.OA.01.2019.0011 -
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 -
Frontiers in Microbiology 2022Nematicidal potential of the common plant pathogen has been recently identified against . The current study was designed to investigate the detailed genetic mechanism...
Nematicidal potential of the common plant pathogen has been recently identified against . The current study was designed to investigate the detailed genetic mechanism of the bacterial pathogenicity by applying comparative genomics, transcriptomics, mutant library screening, and protein expression. Results showed that strain MB03 could kill in the liquid assay by gut colonization. The genome of MB03 was sequenced and comparative analysis including multi locus sequence typing, and genome-to-genome distance placed MB03 in phylogroup II of . Furthermore, comparative genomics of MB03 with nematicidal strains of (PAO1 and PA14) predicted 115 potential virulence factors in MB03. However, genes for previously reported nematicidal metabolites, such as phenazine, pyochelin, and pyrrolnitrin, were found absent in the MB03 genome. Transcriptomics analysis showed that the growth phase of the pathogen considerably affected the expression of virulence factors, as genes for the flagellum, glutamate ABC transporter, /, /, type VI secretion system, and serralysin were highly up-regulated when stationary phase MB03 cells interacted with Additionally, screening of a transposon insertion mutant library led to the identification of other nematicidal genes such as , and . Finally, the nematicidal activity of selected proteins was confirmed by heterologous expression in .
PubMed: 35356513
DOI: 10.3389/fmicb.2022.826962