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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 -
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 -
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 -
Scientific Reports Dec 2017Tryptophan 7-halogenase catalyzes chlorination of free tryptophan to 7-chlorotryptophan, which is the first step in the antibiotic pyrrolnitrin biosynthesis. Many...
Tryptophan 7-halogenase catalyzes chlorination of free tryptophan to 7-chlorotryptophan, which is the first step in the antibiotic pyrrolnitrin biosynthesis. Many biologically and pharmaceutically active natural products contain chlorine and thus, an understanding of the mechanism of its introduction into organic molecules is important. Whilst enzyme-catalyzed chlorination is accomplished with ease, it remains a difficult task for the chemists. Therefore, utilizing enzymes in the synthesis of chlorinated organic compounds is important, and providing atomistic mechanistic insights about the reaction mechanism of tryptophan 7-halogenase is vital and timely. In this work, we examined a mechanism for the reaction of tryptophan chlorination, performed by tryptophan 7-halogenase, by calculating potential energy and free energy surfaces using two different Combined Quantum Mechanical/Molecular Mechanical (QM/MM) methods both employing Density Functional Theory (DFT) for the QM region. Both computational strategies agree on the nature of the rate-limiting step and provided close results for the reaction barriers of the two reaction steps. The calculations for both the potential energy and the free energy profiles showed very similar geometric features and hydrogen bonding interactions for the characterized stationary points.
Topics: Biocatalysis; Flavin-Adenine Dinucleotide; Halogenation; Hydrogen Bonding; Kinetics; Molecular Dynamics Simulation; Oxidoreductases; Pseudomonas fluorescens; Thermodynamics; Tryptophan
PubMed: 29234124
DOI: 10.1038/s41598-017-17789-x -
Molecular Plant Pathology May 2018The Gac/Rsm network regulates, at the transcriptional level, many beneficial traits in biocontrol-active pseudomonads. In this study, we used Phenotype MicroArrays,...
The Gac/Rsm network regulates, at the transcriptional level, many beneficial traits in biocontrol-active pseudomonads. In this study, we used Phenotype MicroArrays, followed by specific growth studies and mutational analysis, to understand how catabolism is regulated by this sensor kinase system in the biocontrol isolate Pseudomonas chlororaphis O6. The growth of a gacS mutant was decreased significantly relative to that of the wild-type on ornithine and arginine, and on the precursor of these amino acids, N-acetyl-l-glutamic acid. The gacS mutant also showed reduced production of polyamines. Expression of the genes encoding arginine decarboxylase (speA) and ornithine decarboxylases (speC) was controlled at the transcriptional level by the GacS sensor of P. chlororaphis O6. Polyamine production was reduced in the speC mutant, and was eliminated in the speAspeC mutant. The addition of exogenous polyamines to the speAspeC mutant restored the in vitro growth inhibition of two fungal pathogens, as well as the secretion of three biological control-related factors: pyrrolnitrin, protease and siderophore. These results extend our knowledge of the regulation by the Gac/Rsm network in a biocontrol pseudomonad to include polyamine synthesis. Collectively, our studies demonstrate that bacterial polyamines act as important regulators of bacterial cell growth and biocontrol potential.
Topics: Bacterial Proteins; Biosynthetic Pathways; Gene Expression Regulation, Bacterial; Mutation; Polyamines; Pseudomonas chlororaphis; RNA, Messenger; Substrate Specificity; Transcription, Genetic
PubMed: 28862813
DOI: 10.1111/mpp.12610 -
Research in Microbiology Oct 2017Fluorescent pseudomonads from bean root and rhizosphere in Iran were investigated for biocontrol of the fungal pathogen Rhizoctonia solani. Phylogenetic analysis of...
Fluorescent pseudomonads from bean root and rhizosphere in Iran were investigated for biocontrol of the fungal pathogen Rhizoctonia solani. Phylogenetic analysis of concatenated 16S rRNA, gyrB and rpoD sequences for 33 Pseudomonas isolates showed that 15 belonged to four clusters within the 'P. fluorescens' group, i.e. one corresponding to P. thivervalensis, two others including P. moraviensis or P. baetica, and the last one without closely-related established species. The 18 other isolates belonged to five clusters within the 'P. putida' group, one including P. mosselii and P. entomophila, another including strains currently described as P. putida, and three without closely-related species described. Ten isolates were selected based on in vitro inhibition of R. solani. Cellulase activity was identified in three pseudomonads, chitinase activity in two pseudomonads, extracellular protease activity in nine pseudomonads and hydrogen cyanide production in two pseudomonads. Genes coding for production of phenazine, pyoluteorin, pyrrolnitrin and 2,4-diacetylphloroglucinol were not found, whereas the 1-aminocyclopropane-1-carboxylate deamination gene acdS was present in three pseudomonads. The antagonistic acdS strain VKh13 from the 'P. putida' group effectively protected soil-grown bean from R. solani AG 4-HGI. Results show that pseudomonads from uncharacterized taxa were readily obtained from Iranian soils and displayed biocontrol potential against R. solani.
Topics: Antibiosis; Fabaceae; Iran; Phylogeny; Plant Diseases; Plant Roots; Pseudomonas; Rhizoctonia; Rhizosphere; Soil Microbiology
PubMed: 28851671
DOI: 10.1016/j.resmic.2017.08.002 -
Journal of Bacteriology Jul 2017ATCC 31433 is a Gram-negative bacterium, first isolated from Japanese soil samples, that produces the monobactam isosulfazecin and the β-lactam-potentiating bulgecins....
ATCC 31433 is a Gram-negative bacterium, first isolated from Japanese soil samples, that produces the monobactam isosulfazecin and the β-lactam-potentiating bulgecins. To characterize the biosynthetic potential of ATCC 31433, its complete genome was determined using single-molecule real-time DNA sequence analysis. The 7.8-Mb genome comprised four replicons, three chromosomal (each encoding rRNA) and one plasmid. Phylogenetic analysis demonstrated that ATCC 31433 was misclassified at the time of its deposition and is a member of the complex, most closely related to The sequenced genome shows considerable additional biosynthetic potential; known gene clusters for malleilactone, ornibactin, isosulfazecin, alkylhydroxyquinoline, and pyrrolnitrin biosynthesis and several uncharacterized biosynthetic gene clusters for polyketides, nonribosomal peptides, and other metabolites were identified. Furthermore, ATCC 31433 harbors many genes associated with environmental resilience and antibiotic resistance and was resistant to a range of antibiotics and metal ions. In summary, this bioactive strain should be designated complex strain ATCC 31433, pending further detailed taxonomic characterization. This work reports the complete genome sequence of ATCC 31433, a known producer of bioactive compounds. Large numbers of both known and novel biosynthetic gene clusters were identified, indicating that ATCC 31433 is an untapped resource for discovery of novel bioactive compounds. Phylogenetic analysis demonstrated that ATCC 31433 is in fact a member of the complex, most closely related to the species Further investigation of the classification and biosynthetic potential of ATCC 31433 is warranted.
Topics: Anti-Bacterial Agents; Burkholderia cepacia complex; DNA, Bacterial; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Genome, Bacterial; Phylogeny; Pseudomonas
PubMed: 28439036
DOI: 10.1128/JB.00125-17 -
Frontiers in Plant Science 2017Strains of that produce antimicrobial metabolites and control soilborne plant diseases have often been isolated from soils defined as disease-suppressive, i.e., soils,...
Strains of that produce antimicrobial metabolites and control soilborne plant diseases have often been isolated from soils defined as disease-suppressive, i.e., soils, in which specific plant pathogens are present, but plants show no or reduced disease symptoms. Moreover, it is assumed that pseudomonads producing antimicrobial compounds such as 2,4-diacetylphloroglucinol (DAPG) or phenazines (PHZ) contribute to the specific disease resistance of suppressive soils. However, pseudomonads producing antimicrobial metabolites are also present in soils that are conducive to disease. Currently, it is still unknown whether and to which extent the abundance of antimicrobials-producing pseudomonads is related to the general disease resistance of common agricultural soils. Moreover, virtually nothing is known about the conditions under which pseudomonads express antimicrobial genes in agricultural field soils. We present here results of the first side-by-side comparison of 10 representative Swiss agricultural soils with a cereal-oriented cropping history for (i) the resistance against two soilborne pathogens, (ii) the abundance of bacteria harboring genes involved in the biosynthesis of the antimicrobials DAPG, PHZ, and pyrrolnitrin on roots of wheat, and (iii) the ability to support the expression of these genes on the roots. Our study revealed that the level of soil disease resistance strongly depends on the type of pathogen, e.g., soils that are highly resistant to often are highly susceptible to and vice versa. There was no significant correlation between the disease resistance of the soils, the abundance of bacteria carrying DAPG, PHZ, and pyrrolnitrin biosynthetic genes, and the ability of the soils to support the expression of the antimicrobial genes. Correlation analyses indicated that certain soil factors such as silt, clay, and some macro- and micronutrients influence both the abundance and the expression of the antimicrobial genes. Taken together, the results of this study suggests that pseudomonads producing DAPG, PHZ, or pyrrolnitrin are present and abundant in Swiss agricultural soils and that the soils support the expression of the respective biosynthetic genes in these bacteria to various degrees. The precise role that these pseudomonads play in the general disease resistance of the investigated agricultural soils remains elusive.
PubMed: 28424714
DOI: 10.3389/fpls.2017.00427