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Ben Lugtenberg - One of the best experts on this subject based on the ideXlab platform.

  • role of phenazine 1 carboxamide produced by pseudomonas chlororaphis pcl1391 in the control of tomato foot and root rot
    2013
    Co-Authors: Ben Lugtenberg, Geneviève Girard
    Abstract:

    Tomato foot and root rot (TFRR) is a tomato root disease caused by the fungus Fusarium oxysporum f. sp. radicis-lycopersici (Forl). No chemicals are available which efficiently suppress TFRR. In this chapter we show that the bacterium Pseudomonas chlororaphis strain PCL1391 is able to suppress the disease. To this end it uses antibiosis as its (major) disease-suppressing mechanism. The produced antibiotic was identified as Phenazine-1-Carboxamide (PCN). In contrast to the PCN-producing bacterium, pseudomonads which produce the PCN biosynthetic precursor phenazine-1-carboxylic acid (PCA) as their major phenazine were not active in disease suppression. However, when PCA was converted to PCN by complementing these strains with the phzH gene, which encodes an amidotransferase, the complemented strains produced PCN and controled TFRR. In order to be effective in disease control, strain PCL1391 should be able to produce PCN under a variety of environmental conditions. We therefore studied the regulation of PCN production under various environmental factors, by regulatory genes, by the plant, and by the pathogenic fungus. Special attention was paid to the secondary metabolite fusaric acid secreted by the fungus. Fusaric acid is detected by the bacterium as a chemo-attractant to reach the fungus, to colonize its surface and to finally use it as a food source. Conversely, fusaric acid is used by the fungus to inhibit the production of PCN and to reduce the bacterial growth rate. It is clear that during disease control the PCN-producing bacterium wins this battle. The result of the evaluation of the described studies is that we can understand in quite some detail how P. chlororaphis strain PCL1391 acts as a disease control agent and also why it is not active under all environmental conditions.

  • Journal of Bacteriology - Pip, a novel activator of phenazine biosynthesis in Pseudomonas chlororaphis PCL1391.
    Journal of bacteriology, 2006
    Co-Authors: Geneviève Girard, Ben Lugtenberg, Sébastien Rigali, Sharief Barends, E. Tjeerd Van Rij, Guido V Bloemberg
    Abstract:

    Secondary metabolites are important factors for interactions between bacteria and other organisms. Pseudomonas chlororaphis PCL1391 produces the antifungal secondary metabolite Phenazine-1-Carboxamide (PCN) that inhibits growth of Fusarium oxysporum f. sp. radius lycopersici the causative agent of tomato foot and root rot. Our previous work unraveled a cascade of genes regulating the PCN biosynthesis operon, phzABCDEFGH. Via a genetic screen, we identify in this study a novel TetR/AcrR regulator, named Pip (phenazine inducing protein), which is essential for PCN biosynthesis. A combination of a phenotypical characterization of a pip mutant, in trans complementation assays of various mutant strains, and electrophoretic mobility shift assays identified Pip as the fifth DNA-binding protein so far involved in regulation of PCN biosynthesis. In this regulatory pathway, Pip is positioned downstream of PsrA (Pseudomonas sigma factor regulator) and the stationary-phase sigma factor RpoS, while it is upstream of the quorum-sensing system PhzI/PhzR. These findings provide further evidence that the path leading to the expression of secondary metabolism gene clusters in Pseudomonas species is highly complex.

  • biocontrol of avocado dematophora root rot by antagonistic pseudomonas fluorescens pcl1606 correlates with the production of 2 hexyl 5 propyl resorcinol
    Molecular Plant-microbe Interactions, 2006
    Co-Authors: Francisco M Cazorla, Ben Lugtenberg, Simon B Duckett, Ed Bergstrom, Sadaf Noreen, Roeland Odijk, Jane Thomasoates, Guido V Bloemberg
    Abstract:

    A collection of 905 bacterial isolates from the rhizospheres of healthy avocado trees was obtained and screened for antagonistic activity against Dematophora necatrix, the cause of avocado Dematophora root rot (also called white root rot). A set of eight strains was selected on the basis of growth inhibitory activity against D. necatrix and several other important soilborne phytopathogenic fungi. After typing of these strains, they were classified as belonging to Pseudomonas chlororaphis, Pseudomonas fluorescens, and Pseudomonas putida. The eight antagonistic Pseudomonas spp. were analyzed for their secretion of hydrogen cyanide, hydrolytic enzymes, and antifungal metabolites. P. chlororaphis strains produced the antibiotic phenazine-1-carboxylic acid and Phenazine-1-Carboxamide. Upon testing the biocontrol ability of these strains in a newly developed avocado-D. necatrix test system and in a tomato-F. oxysporum test system, it became apparent that P. fluorescens PCL1606 exhibited the highest biocontrol a...

  • Regulatory roles of psrA and rpoS in Phenazine-1-Carboxamide synthesis by Pseudomonas chlororaphis PCL1391.
    Microbiology (Reading England), 2006
    Co-Authors: Geneviève Girard, Ben Lugtenberg, E. Tjeerd Van Rij, Guido V Bloemberg
    Abstract:

    Production of the secondary metabolite Phenazine-1-Carboxamide (PCN) by Pseudomonas chlororaphis PCL1391 is crucial for biocontrol activity against the phytopathogen Fusarium oxysporum f. sp. radicis lycopersici on tomato. Regulation of PCN production involves the two-component signalling system GacS/GacA, the quorum-sensing system PhzI/PhzR and the regulator PsrA. This paper reports that a functional rpoS is required for optimal PCN and N-hexanoyl-L-homoserine lactone (C(6)-HSL) production. Constitutive expression of rpoS is able to complement partially the defect of a psrA mutant for PCN and N-acylhomoserine lactone production. Western blotting shows that rpoS is regulated by gacS. Altogether, these results suggest the existence of a cascade consisting of gacS/gacA upstream of psrA and rpoS, which influence expression of phzI/phzR. Overproduction of phzR complements the effects on PCN and C(6)-HSL production of all mutations tested in the regulatory cascade, which shows that a functional quorum-sensing system is essential and sufficient for PCN synthesis. In addition, the relative amounts of PCN, phenazine-1-carboxylic acid and C(6)-HSL produced by rpoS and psrA mutants harbouring a constitutively expressed phzR indicate an even more complex network of interactions, probably involving other genes. Preliminary microarray analyses of the transcriptomics of the rpoS and psrA mutants support the model of regulation described in this study and allow identification of new genes that might be involved in secondary metabolism.

  • Biocontrol of avocado dematophora root rot by antagonistic Pseudomonas fluorescens PCL1606 correlates with the production of 2-hexyl 5-propyl resorcinol.
    Molecular plant-microbe interactions : MPMI, 2006
    Co-Authors: Francisco M Cazorla, Ben Lugtenberg, Jane Thomas-oates, Simon B Duckett, Ed Bergstrom, Sadaf Noreen, Roeland Odijk, Guido V Bloemberg
    Abstract:

    A collection of 905 bacterial isolates from the rhizospheres of healthy avocado trees was obtained and screened for antagonistic activity against Dematophora necatrix, the cause of avocado Dematophora root rot (also called white root rot). A set of eight strains was selected on the basis of growth inhibitory activity against D. necatrix and several other important soilborne phytopathogenic fungi. After typing of these strains, they were classified as belonging to Pseudomonas chlororaphis, Pseudomonas fluorescens, and Pseudomonas putida. The eight antagonistic Pseudomonas spp. were analyzed for their secretion of hydrogen cyanide, hydrolytic enzymes, and antifungal metabolites. P. chlororaphis strains produced the antibiotic phenazine-1-carboxylic acid and Phenazine-1-Carboxamide. Upon testing the biocontrol ability of these strains in a newly developed avocado-D. necatrix test system and in a tomato-F oxysporum test system, it became apparent that P. fluorescens PCL1606 exhibited the highest biocontrol ability. The major antifungal activity produced by strain P. fluorescens PCL1606 did not correspond to any of the major classes of antifungal antibiotics produced by Pseudomonas biocontrol strains. This compound was purified and subsequently identified as 2-hexyl 5-propyl resorcinol (HPR). To study the role of HPR in biocontrol activity, two Tn5 mutants of P. fluorescens PCL1606 impaired in antagonistic activity were selected. These mutants were shown to impair HRP production and showed a decrease in biocontrol activity. As far as we know, this is the first report of a Pseudomonas biocontrol strain that produces HPR in which the production of this compound correlates with its biocontrol activity.

Guido V Bloemberg - One of the best experts on this subject based on the ideXlab platform.

  • Journal of Bacteriology - Pip, a novel activator of phenazine biosynthesis in Pseudomonas chlororaphis PCL1391.
    Journal of bacteriology, 2006
    Co-Authors: Geneviève Girard, Ben Lugtenberg, Sébastien Rigali, Sharief Barends, E. Tjeerd Van Rij, Guido V Bloemberg
    Abstract:

    Secondary metabolites are important factors for interactions between bacteria and other organisms. Pseudomonas chlororaphis PCL1391 produces the antifungal secondary metabolite Phenazine-1-Carboxamide (PCN) that inhibits growth of Fusarium oxysporum f. sp. radius lycopersici the causative agent of tomato foot and root rot. Our previous work unraveled a cascade of genes regulating the PCN biosynthesis operon, phzABCDEFGH. Via a genetic screen, we identify in this study a novel TetR/AcrR regulator, named Pip (phenazine inducing protein), which is essential for PCN biosynthesis. A combination of a phenotypical characterization of a pip mutant, in trans complementation assays of various mutant strains, and electrophoretic mobility shift assays identified Pip as the fifth DNA-binding protein so far involved in regulation of PCN biosynthesis. In this regulatory pathway, Pip is positioned downstream of PsrA (Pseudomonas sigma factor regulator) and the stationary-phase sigma factor RpoS, while it is upstream of the quorum-sensing system PhzI/PhzR. These findings provide further evidence that the path leading to the expression of secondary metabolism gene clusters in Pseudomonas species is highly complex.

  • biocontrol of avocado dematophora root rot by antagonistic pseudomonas fluorescens pcl1606 correlates with the production of 2 hexyl 5 propyl resorcinol
    Molecular Plant-microbe Interactions, 2006
    Co-Authors: Francisco M Cazorla, Ben Lugtenberg, Simon B Duckett, Ed Bergstrom, Sadaf Noreen, Roeland Odijk, Jane Thomasoates, Guido V Bloemberg
    Abstract:

    A collection of 905 bacterial isolates from the rhizospheres of healthy avocado trees was obtained and screened for antagonistic activity against Dematophora necatrix, the cause of avocado Dematophora root rot (also called white root rot). A set of eight strains was selected on the basis of growth inhibitory activity against D. necatrix and several other important soilborne phytopathogenic fungi. After typing of these strains, they were classified as belonging to Pseudomonas chlororaphis, Pseudomonas fluorescens, and Pseudomonas putida. The eight antagonistic Pseudomonas spp. were analyzed for their secretion of hydrogen cyanide, hydrolytic enzymes, and antifungal metabolites. P. chlororaphis strains produced the antibiotic phenazine-1-carboxylic acid and Phenazine-1-Carboxamide. Upon testing the biocontrol ability of these strains in a newly developed avocado-D. necatrix test system and in a tomato-F. oxysporum test system, it became apparent that P. fluorescens PCL1606 exhibited the highest biocontrol a...

  • Regulatory roles of psrA and rpoS in Phenazine-1-Carboxamide synthesis by Pseudomonas chlororaphis PCL1391.
    Microbiology (Reading England), 2006
    Co-Authors: Geneviève Girard, Ben Lugtenberg, E. Tjeerd Van Rij, Guido V Bloemberg
    Abstract:

    Production of the secondary metabolite Phenazine-1-Carboxamide (PCN) by Pseudomonas chlororaphis PCL1391 is crucial for biocontrol activity against the phytopathogen Fusarium oxysporum f. sp. radicis lycopersici on tomato. Regulation of PCN production involves the two-component signalling system GacS/GacA, the quorum-sensing system PhzI/PhzR and the regulator PsrA. This paper reports that a functional rpoS is required for optimal PCN and N-hexanoyl-L-homoserine lactone (C(6)-HSL) production. Constitutive expression of rpoS is able to complement partially the defect of a psrA mutant for PCN and N-acylhomoserine lactone production. Western blotting shows that rpoS is regulated by gacS. Altogether, these results suggest the existence of a cascade consisting of gacS/gacA upstream of psrA and rpoS, which influence expression of phzI/phzR. Overproduction of phzR complements the effects on PCN and C(6)-HSL production of all mutations tested in the regulatory cascade, which shows that a functional quorum-sensing system is essential and sufficient for PCN synthesis. In addition, the relative amounts of PCN, phenazine-1-carboxylic acid and C(6)-HSL produced by rpoS and psrA mutants harbouring a constitutively expressed phzR indicate an even more complex network of interactions, probably involving other genes. Preliminary microarray analyses of the transcriptomics of the rpoS and psrA mutants support the model of regulation described in this study and allow identification of new genes that might be involved in secondary metabolism.

  • Biocontrol of avocado dematophora root rot by antagonistic Pseudomonas fluorescens PCL1606 correlates with the production of 2-hexyl 5-propyl resorcinol.
    Molecular plant-microbe interactions : MPMI, 2006
    Co-Authors: Francisco M Cazorla, Ben Lugtenberg, Jane Thomas-oates, Simon B Duckett, Ed Bergstrom, Sadaf Noreen, Roeland Odijk, Guido V Bloemberg
    Abstract:

    A collection of 905 bacterial isolates from the rhizospheres of healthy avocado trees was obtained and screened for antagonistic activity against Dematophora necatrix, the cause of avocado Dematophora root rot (also called white root rot). A set of eight strains was selected on the basis of growth inhibitory activity against D. necatrix and several other important soilborne phytopathogenic fungi. After typing of these strains, they were classified as belonging to Pseudomonas chlororaphis, Pseudomonas fluorescens, and Pseudomonas putida. The eight antagonistic Pseudomonas spp. were analyzed for their secretion of hydrogen cyanide, hydrolytic enzymes, and antifungal metabolites. P. chlororaphis strains produced the antibiotic phenazine-1-carboxylic acid and Phenazine-1-Carboxamide. Upon testing the biocontrol ability of these strains in a newly developed avocado-D. necatrix test system and in a tomato-F oxysporum test system, it became apparent that P. fluorescens PCL1606 exhibited the highest biocontrol ability. The major antifungal activity produced by strain P. fluorescens PCL1606 did not correspond to any of the major classes of antifungal antibiotics produced by Pseudomonas biocontrol strains. This compound was purified and subsequently identified as 2-hexyl 5-propyl resorcinol (HPR). To study the role of HPR in biocontrol activity, two Tn5 mutants of P. fluorescens PCL1606 impaired in antagonistic activity were selected. These mutants were shown to impair HRP production and showed a decrease in biocontrol activity. As far as we know, this is the first report of a Pseudomonas biocontrol strain that produces HPR in which the production of this compound correlates with its biocontrol activity.

  • influence of fusaric acid on phenazine 1 carboxamide synthesis and gene expression of pseudomonas chlororaphis strain pcl1391
    Microbiology, 2005
    Co-Authors: Tjeerd E Van Rij, Ben Lugtenberg, Geneviève Girard, Guido V Bloemberg
    Abstract:

    Production of the antifungal metabolite Phenazine-1-Carboxamide (PCN) by Pseudomonas chlororaphis strain PCL1391 is essential for the suppression of tomato foot and root rot caused by the soil-borne fungus F. oxysporum f. sp. radicis-lycopersici. The authors have shown previously that fusaric acid (FA), a phytotoxin produced by Fusarium oxysporum, represses the production of PCN and of the quorum-sensing signal N-hexanoyl-l-homoserine lactone (C6-HSL). Here they report that PCN repression by FA is maintained even during PCN-stimulating environmental conditions such as additional phenylalanine, additional ferric iron and a low Mg2+ concentration. Constitutive expression of phzI or phzR increases the production of C6-HSL and abolishes the repression of PCN production by FA. Transcriptome analysis using P. chlororaphis PCL1391 microarrays showed that FA represses expression of the phenazine biosynthetic operon (phzABCDEFGH) and of the quorum-sensing regulatory genes phzI and phzR. FA does not alter expression of the PCN regulators gacS, rpoS and psrA. In conclusion, reduction of PCN levels by FA is due to direct or indirect repression of phzR and phzI. Microarray analyses identified genes of which the expression is strongly influenced by FA. Genes highly upregulated by FA are also upregulated by iron starvation in Pseudomonas aeruginosa. This remarkable overlap in the expression profile suggests an overlapping stress response to FA and iron starvation.

Thomas F C Chinawoeng - One of the best experts on this subject based on the ideXlab platform.

  • root colonization by phenazine 1 carboxamide producing bacterium pseudomonas chlororaphis pcl1391 is essential for biocontrol of tomato foot and root rot
    Molecular Plant-microbe Interactions, 2000
    Co-Authors: Thomas F C Chinawoeng, Linda C Dekkers, Ine H M Mulders, Guido V Bloemberg, Ben Lugtenberg
    Abstract:

    The Phenazine-1-Carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. To test whether root colonization is required for biocontrol, mutants impaired in the known colonization traits motility, prototrophy for amino acids, or production of the site-specific recombinase, Sss/XerC were tested for their root tip colonization and biocontrol abilities. Upon tomato seedling inoculation, colonization mutants of strain PCL1391 were impaired in root tip colonization in a gnotobiotic sand system and in potting soil. In addition, all mutants were impaired in their ability to control tomato foot and root rot, despite the fact that they produce wild-type levels of Phenazine-1-Carboxamide, the antifungal metabolite previously shown to be required for biocontrol. These results show, for what we believe to be the first time, that root colonization plays a crucial role in biocontrol, presumably by providing a delivery syste...

  • root colonization by phenazine 1 carboxamide producing bacterium pseudomonas chlororaphis pcl1391 is essential for biocontrol of tomato foot and root rot
    Molecular Plant-microbe Interactions, 2000
    Co-Authors: Thomas F C Chinawoeng, Linda C Dekkers, Ine H M Mulders, Guido V Bloemberg, Ben Lugtenberg
    Abstract:

    The Phenazine-1-Carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicislycopersici. To test whether root colonization is required for biocontrol, mutants impaired in the known colonization traits motility, prototrophy for amino acids, or production of the site-specific recombinase, Sss/XerC were tested for their root tip colonization and biocontrol abilities. Upon tomato seedling inoculation, colonization mutants of strain PCL1391 were impaired in root tip colonization in a gnotobiotic sand system and in potting soil. In addition, all mutants were impaired in their ability to control tomato foot and root rot, despite the fact that they produce wild-type levels of Phenazine-1-Carboxamide, the antifungal metabolite previously shown to be required for biocontrol. These results show, for what we believe to be the first time, that root colonization plays a crucial role in biocontrol, presumably by providing a delivery system for antifungal metabolites. The ability to colonize and produce Phenazine-1-Carboxamide is essential for control of F. oxysporum f. sp. radicis-lycopersici. Furthermore, there is a notable overlap of traits identified as being important for colonization of the rhizosphere and animal tissues.

  • the sss colonization gene of the tomato fusarium oxysporum f sp radicis lycopersici biocontrol strain pseudomonas fluorescens wcs365 can improve root colonization of other wild type pseudomonas spp bacteria
    Molecular Plant-microbe Interactions, 2000
    Co-Authors: Linda C Dekkers, Ine H M Mulders, Claartje C Phoelich, Thomas F C Chinawoeng, Andre H M Wijfjes, Ben Lugtenberg
    Abstract:

    We show that the disease tomato foot and root rot caused by the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici can be controlled by inoculation of seeds with cells of the efficient root colonizer Pseudomonas fluorescens WCS365, indicating that strain WCS365 is a bio-control strain. The mechanism for disease suppression most likely is induced systemic resistance. P. fluorescens strain WCS365 and P. chlororaphis strain PCL1391, which acts through the production of the antibiotic Phenazine-1-Carboxamide, were differentially labeled using genes encoding autofluorescent proteins. Inoculation of seeds with a 1:1 mixture of these strains showed that, at the upper part of the root, the two cell types were present as microcolonies of either one or both cell types. Microcolonies at the lower root part were predominantly of one cell type. Mixed inoculation tended to improve biocontrol in comparison with single inoculations. In contrast to what was observed previously for strain PCL1391, mutations in...

  • the sss colonization gene of the tomato fusarium oxysporum f sp radicis lycopersici biocontrol strain pseudomonas fluorescens wcs365 can improve root colonization of other wild type pseudomonas spp bacteria
    Molecular Plant-microbe Interactions, 2000
    Co-Authors: Linda C Dekkers, Ine H M Mulders, Claartje C Phoelich, Thomas F C Chinawoeng, Andre H M Wijfjes, Ben Lugtenberg
    Abstract:

    We show that the disease tomato foot and root rot caused by the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici can be controlled by inoculation of seeds with cells of the efficient root colonizer Pseudomonas fluorescens WCS365, indicating that strain WCS365 is a biocontrol strain. The mechanism for disease suppression most likely is induced systemic resistance. P. fluorescens strain WCS365 and P. chlororaphis strain PCL1391, which acts through the production of the antibiotic Phenazine-1-Carboxamide, were differentially labeled using genes encoding autofluorescent proteins. Inoculation of seeds with a 1:1 mixture of these strains showed that, at the upper part of the root, the two cell types were present as microcolonies of either one or both cell types. Microcolonies at the lower root part were predominantly of one cell type. Mixed inoculation tended to improve biocontrol in comparison with single inoculations. In contrast to what was observed previously for strain PCL1391, mutations in various colonization genes, including sss, did not consistently decrease the biocontrol ability of strain WCS365. Multiple copies of the sss colonization gene in WCS365 improved neither colonization nor biocontrol by this strain. However, introduction of the sss-containing DNA fragment into the poor colonizer P. fluorescens WCS307 and into the good colonizer P. fluorescens F113 increased the competitive tomato root tip colonization ability of the latter strains 16- to 40-fold and 8- to 16-fold, respectively. These results show that improvement of the colonization ability of wild-type Pseudomonas strains by genetic engineering is a realistic goal.

  • biocontrol by phenazine 1 carboxamide producing pseudomonas chlororaphis pcl1391 of tomato root rot caused by fusarium oxysporum f sp radicis lycopersici
    Molecular Plant-microbe Interactions, 1998
    Co-Authors: Thomas F C Chinawoeng, Guido V Bloemberg, A J Van Der Bij, K M G M Van Der Drift, J Schripsema, Bart Kroon, R J Scheffer, Christoph Keel, Peter A H M Bakker, H V Tichy
    Abstract:

    Seventy bacterial isolates from the rhizosphere of tomato were screened for antagonistic activity against the tomato foot and root rot-causing fungal pathogen Fusarium oxysporum f. sp. radicis-lycopersici. One isolate, strain PCL1391, appeared to be an efficient colonizer of tomato roots and an excellent biocontrol strain in an F. oxysporum/tomato test system. Strain PCL1391 was identified as Pseudomonas chlororaphis and further characterization showed that it produces a broad spectrum of antifungal factors (AFFs), including a hydrophobic compound, hydrogen cyanide, chitinase(s), and protease(s). Through mass spectrometry and nuclear magnetic resonance, the hydrophobic compound was identified as Phenazine-1-Carboxamide (PCN). We have studied the production and action of this AFF both in vitro and in vivo. Using a PCL1391 transposon mutant, with a lux reporter gene inserted in the phenazine biosynthetic operon (phz), we showed that this phenazine biosynthetic mutant was substantially decreased in both in vitro antifungal activity and biocontrol activity. Moreover, with the same mutant it was shown that the phz biosynthetic operon is expressed in the tomato rhizosphere. Comparison of the biocontrol activity of the PCN-producing strain PCL1391 with those of phenazine-1-carboxylic acid (PCA)producing strains P. fluorescens 2-79 and P. aureofaciens 30-84 showed that the PCN-producing strain is able to suppress disease in the tomato/F. oxysporum system, whereas the PCA-producing strains are not. Comparison of in vitro antifungal activity of PCN and PCA showed that the antifungal activity of PCN was at least 10 times higher at neutral pH, suggesting that this may contribute to the superior biocontrol performance of strain PCL1391 in the tomato/F. oxysporum system.

Thomas F. C. Chin-a-woeng - One of the best experts on this subject based on the ideXlab platform.

  • The Pseudomonas chlororaphis PCL1391 Sigma Regulator psrA Represses the Production of the Antifungal Metabolite Phenazine-1-Carboxamide
    Molecular plant-microbe interactions : MPMI, 2005
    Co-Authors: Thomas F. C. Chin-a-woeng, Ben Lugtenberg, Daan Van Den Broek, Guido V Bloemberg
    Abstract:

    The rhizobacterium Pseudomonas chlororaphis PCL1391 produces the antifungal metabolite Phenazine-1-Carboxamide (PCN), which is a crucial trait in its competition with the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici in the rhizosphere. The expression of the PCN biosynthetic gene cluster in PCL1391 is population density–dependent and is regulated by the quorum-sensing genes phzI and phzR via synthesis of the autoinducer Nhexanoyl-L-homoserine lactone (C6-HSL). Here, we describe the identification of an additional regulatory gene of PCN biosynthesis in PCL1391. A mutation in the psrA gene (Pseudomonas sigma regulator), the gene product of which is a member of the TetR/AcrR family of transcriptional regulators, resulted in increased production of autoinducer molecules and PCN. Expression studies showed that inactivation of psrA resulted in increased expression of the phzI and phzR genes and the phz biosynthetic operon and that introduction of functional copies of psrA represses the expression of these genes, resulting in reduced production of autoinducer signal and PCN. Surprisingly, inactivation of psrA in the phzI or phzR quorum-sensing mutants, which do not produce detectable amounts of PCN and autoinducers by themselves, restored PCN biosynthesis. This phenomenon was accompanied by the appearance of compounds with autoinducer activities migrating at the positions of C4-HSL and C6-HSL on C18 reverse phase–thin-layer chromatography. These observations indicate that PsrA also represses at least one silent, yet unidentified, quorum-sensing system or autoinducer biosynthetic pathway in PCL1391. The expression of psrA declines at the onset of the stationary phase at the same moment at which quorum-sensing (-regulated) genes are activated. In addition, expression studies in a psrA – and a multicopy psrA background showed that psrA is autoregulated. Multiple copies of psrA repress its own expression. Mutation of gacS, encoding the sensor kinase member of a two-component global regulatory system significantly reduced production of autoinducers and PCN. We show a novel link between global regulation and quorum sensing via the PsrA regulator.

  • Influence of environmental conditions on the production of Phenazine-1-Carboxamide by Pseudomonas chlororaphis PCL1391.
    Molecular plant-microbe interactions : MPMI, 2004
    Co-Authors: E. Tjeerd Van Rij, Thomas F. C. Chin-a-woeng, Guido V Bloemberg, Monique Wesselink, Ben Lugtenberg
    Abstract:

    Pseudomonas chlororaphis PCL1391 produces the secondary metabolite Phenazine-1-Carboxamide (PCN), which is an antifungal metabolite required for biocontrol activity of the strain. Identification of conditions involved in PCN production showed that some carbon sources and all amino acids tested promote PCN levels. Decreasing the pH from 7 to 6 or decreasing the growth temperature from 21 to 16°C decreased PCN production dramatically. In contrast, growth at 1% oxygen as well as low magnesium concentrations increased PCN levels. Salt stress, low concentrations of ferric iron, phosphate, sulfate, and ammonium ions reduced PCN levels. Fusaric acid, a secondary metabolite produced by the soilborne Fusarium spp. fungi, also reduced PCN levels. Different nitrogen sources greatly influenced PCN levels. Analysis of autoinducer levels at conditions of high and low PCN production demonstrated that, under all tested conditions, PCN levels correlate with autoinducer levels, indicating that the regulation of PCN product...

  • Interactions in the tomato rhizosphere of two Pseudomonas biocontrol strains with the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici.
    Molecular plant-microbe interactions : MPMI, 2003
    Co-Authors: Annouschka Bolwerk, Andre H M Wijfjes, Ben Lugtenberg, Thomas F. C. Chin-a-woeng, Anastasia L. Lagopodi, Gerda E. M. Lamers, Guido V Bloemberg
    Abstract:

    The fungus Fusarium oxysporum f. sp. radicis-lycopersici causes foot and root rot of tomato plants, which can be controlled by the bacteria Pseudomonas fluorescens WCS365 and P. chlororaphis PCL1391. Induced systemic resistance is thought to be involved in biocontrol by P. fluorescens WCS365. The antifungal metabolite Phenazine-1-Carboxamide (PCN), as well as efficient root colonization, are essential in the mechanism of biocontrol by P. chlororaphis PCL1391. To understand the effects of bacterial strains WCS365 and PCL1391 on the fungus in the tomato rhizosphere, microscopic analyses were performed using different autofluorescent proteins as markers. Tomato seedlings were inoculated with biocontrol bacteria and planted in an F. oxysporum f. sp. radicis-lycopersici-infested gnotobiotic sand system. Confocal laser scanning microscope analyses of the interactions in the tomato rhizosphere revealed that i) the microbes effectively compete for the same niche, and presumably also for root exudate nutrients; ii) the presence of either of the two bacteria negatively affects infection of the tomato root by the fungus; iii) both biocontrol bacteria colonize the hyphae extensively, which may represent a new mechanism in biocontrol by these pseudomonads; and iv) the production of PCN by P. chlororaphis PCL1391 negatively affects hyphal growth and branching, which presumably affects the colonization and infecting ability of the fungus.

  • Introduction of the phzH gene of Pseudomonas chlororaphis PCL1391 extends the range of biocontrol ability of phenazine-1-carboxylic acid-producing Pseudomonas spp. strains.
    Molecular plant-microbe interactions : MPMI, 2001
    Co-Authors: Thomas F. C. Chin-a-woeng, Ben Lugtenberg, Jane Thomas-oates, Guido V Bloemberg
    Abstract:

    Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. Its biocontrol activity is mediated by the production of Phenazine-1-Carboxamide (PCN). In contrast, the take-all biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84, which produce phenazine-1-carboxylic acid (PCA), do not control this disease. To determine the role of the amide group in biocontrol, the PCN biosynthetic genes of strain PCL1391 were identified and characterized. Downstream of phzA through phzG, the novel phenazine biosynthetic gene phzH was identified and shown to be required for the presence of the 1-carboxamide group of PCN because a phzH mutant of strain PCL1391 accumulated PCA. The deduced PhzH protein shows homology with asparagine synthetases that belong to the class II glutamine amidotransferases, indicating that the conversion of PCA to PCN occurs via a transamidase reaction catalyzed by PhzH. Mutation of phzH caused loss of biocontrol activity, showing that the 1-carboxamide group of PCN is crucial for control of tomato foot and root rot. PCN production and biocontrol activity of the mutant were restored by complementing the phzH gene in trans. Moreover, transfer of phzH under control of the tac promoter to the PCA-producing biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84 enabled these strains to produce PCN instead of PCA and suppress tomato foot and root rot. Thus, we have shown, for what we believe is the first time, that the introduction of a single gene can efficiently extend the range of the biocontrol ability of bacterial strains.

  • Phenazine-1-Carboxamide Production in the Biocontrol Strain Pseudomonas chlororaphis PCL1391 Is Regulated by Multiple Factors Secreted into the Growth Medium
    Molecular plant-microbe interactions : MPMI, 2001
    Co-Authors: Thomas F. C. Chin-a-woeng, Ben Lugtenberg, Jane Thomas-oates, Daan Van Den Broek, Gert De Voer, Koen M. G. M. Van Der Drift, Sietske Tuinman, Guido V Bloemberg
    Abstract:

    Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicislycopersici. The production of Phenazine-1-Carboxamide (PCN) is crucial for this biocontrol activity. In vitro production of PCN is observed only at high-population densities, suggesting that production is under the regulation of quorum sensing. The main autoinducer molecule produced by PCL1391 was identified structurally as Nhexanoyl-L-homoserine lactone (C6-HSL). The two other autoinducers that were produced comigrate with Nbutanoyl-L-homoserine lactone (C4-HSL) and N-octanoylL-homoserine lactone (C8-HSL). Two PCL1391 mutants lacking production of PCN were defective in the genes phzI and phzR, respectively, the nucleotide sequences of which were determined completely. Production of PCN by the phzI mutant could be complemented by the addition of exogenous synthetic C6-HSL, but not by C4-HSL, C8-HSL, or any other HSL tested. Expression analyses of Tn5luxAB reporter strains of phzI, phzR, and the phz biosynthetic operon clearly showed that phzI expression and PCN production is regulated by C6-HSL in a population densitydependent manner. The introduction of multiple copies of the regulatory genes phzI and phzR on various plasmids resulted in an increase of the production of HSLs, expression of the PCN biosynthetic operon, and consequently, PCN production, up to a sixfold increase in a copydependent manner. Surprisingly, our expression studies show that an additional, yet unidentified factor(s), which are neither PCN nor C4-HSL or C8-HSL, secreted into the growth medium of the overnight cultures, is involved in the positive regulation of phzI, and is able to induce PCN

Xuehong Zhang - One of the best experts on this subject based on the ideXlab platform.

  • Metabolic Engineering of Pseudomonas chlororaphis Qlu-1 for the Enhanced Production of Phenazine-1-Carboxamide.
    Journal of agricultural and food chemistry, 2020
    Co-Authors: Wentao Yao, Xuehong Zhang, Ruiming Wang, Li Piwu, Kai Yang, Tengfei Wang, Kaiquan Liu
    Abstract:

    Phenazine-1-carboxylic acid (PCA), the primary active ingredient of Shenqinmycin, was awarded the China Pesticide Certificate in 2011 due to its excellent antibacterial action. Phenazine-1-Carboxamide (PCN) is a derivative of PCA, which is modified by the phzH gene, and its anti-bacterial effect is better than that of PCA. At present, PCN can be produced via Pseudomonas fermentation using an opportunistic pathogen, Pseudomonas aeruginosa. Qlu-1 is an environmentally friendly strain of Pseudomonas chlororaphis that can produce phenazine derivatives. We replaced the phzO gene with the phzH gene from P. aeruginosa to achieve PCN accumulation. Different strategies were used to enhance PCN production: knocking out of negative regulatory factors, enhancing the shikimate pathway by gene overexpression and gene knocking, and using fed-batch fermentation. Finally, an engineered strain of P. chlororaphis was produced, which produced 11.45 g/L PCN. This achievement indicates that Qlu-1 could be modified as a potential microbial cell factory for PCN production by metabolic engineering.

  • Identification of a Strong Quorum Sensing- and Thermo-Regulated Promoter for the Biosynthesis of a New Metabolite Pesticide Phenazine-1-Carboxamide in Pseudomonas strain PA1201.
    ACS synthetic biology, 2020
    Co-Authors: Zi-jing Jin, Lian Zhou, Shuang Sun, Ying Cui, Kai Song, Xuehong Zhang
    Abstract:

    Phenazine-1-Carboxamide (PCN) produced by multifarious Pseudomonas strains represents a promising candidate as a new metabolite pesticide due to its broad-spectrum antifungal activity and capacity to induce systemic resistance in plants. The rice rhizosphere Pseudomonas strain PA1201 contains two reiterated gene clusters, phz1 and phz2, for phenazine-1-carboxylic acid (PCA) biosynthesis; PCA is further converted into PCN by this strain using a functional phzH-encoding glutamine aminotransferase. However, PCN levels in PA1201 constitute approximately one-fifth of PCA levels and the optimal temperature for PCN synthesis is 28 °C. In this study, the phzH open reading frame (ORF) and promoter region were investigated and reannotated. phzH promoter PphzH was found to be a weak promoter, and PhzH levels were not sufficient to convert all of the native PCA into PCN. Following RNA Seq and promoter-lacZ fusion analyses, a strong quorum sensing (QS)- and thermo-regulated promoter PrhlI was identified and characterized. The activity of PphzH is approximately 1% of PrhlI in PA1201. After three rounds of promoter editing and swapping by PrhlI, a new PCN-overproducing strain UP46 was generated. The optimal fermentation temperature for PCN biosynthesis in UP46 was increased from 28 to 37 °C and the PCN fermentation titer increased 179.5-fold, reaching 14.1 g/L, the highest ever reported.

  • Enhanced biosynthesis of Phenazine-1-Carboxamide by Pseudomonas chlororaphis strains using statistical experimental designs.
    World journal of microbiology & biotechnology, 2018
    Co-Authors: Huasong Peng, Muhammad Bilal, Wei Wang, Tan Jian, Xuehong Zhang
    Abstract:

    Phenazine-1-Carboxamide (PCN) is one of the major biocontrol agents produced by plant growth-promoting rhizosphere (PGPR) pseudomonads including Pseudomonas chlororaphis. In this study, a combined strategy of genetic modification and statistical experimental designs was applied to obtain mutants of P. chlororaphis strains with high-yield PCN production. To achieve this, the lon gene was knocked out in wild-type P. chlororaphis HT66 and the breeding mutant P3 strain with a non-scar deletion strategy. The resulting HT66Δlon and P3Δlon mutants produced a significantly higher PCN production in shake-flask cultures which was 5- and  9-folds greater than their native counterparts. The potential ability of strain P3Δlon for PCN production was further optimized by statistical designs. A two-level Plackett-Burman (PB) experimental design with six variables was employed to scrutinize medium components that significantly influence PCN production. Notably, glycerol, tryptone, and soy peptone were identified to be the most significant factors (p 

  • Enhanced biosynthesis of Phenazine-1-Carboxamide by engineered Pseudomonas chlororaphis HT66.
    Microbial cell factories, 2018
    Co-Authors: Huasong Peng, Muhammad Bilal, Wei Wang, Zhang Pingyuan, Xuehong Zhang
    Abstract:

    Phenazine-1-Carboxamide (PCN), a phenazine derivative, is strongly antagonistic to fungal phytopathogens. The high PCN biocontrol activity fascinated researcher’s attention in isolating and identifying novel bacterial strains combined with engineering strategies to target PCN as a lead molecule. The chemical route for phenazines biosynthesis employs toxic chemicals and display low productivities, require harsh reaction conditions, and generate toxic by-products. Phenazine biosynthesis using some natural phenazine-producers represent remarkable advantages of non-toxicity and possibly high yield in environmentally-friendlier settings. A biocontrol bacterium with antagonistic activity towards fungal plant pathogens, designated as strain HT66, was isolated from the rice rhizosphere. The strain HT66 was identified as Pseudomonas chlororaphis based on the colony morphology, gas chromatography of cellular fatty acids and 16S rDNA sequence analysis. The secondary metabolite produced by HT66 strain was purified and identified as PCN through mass spectrometry, and 1H, 13C nuclear magnetic resonance spectrum. The yield of PCN by wild-type strain HT66 was 424.87 mg/L at 24 h. The inactivation of psrA and rpeA increased PCN production by 1.66- and 3.06-fold, respectively, which suggests that psrA and rpeA are PCN biosynthesis repressors. qRT-PCR analysis showed that the expression of phzI, phzR, and phzE was markedly increased in the psrA and rpeA double mutant than in psrA or rpeA mutant. However, the transcription level of rpeA and rpeB in strain HT66ΔpsrA increased by 3.52- and 11.58-folds, respectively. The reduced psrA expression in HT66ΔrpeA strain evidenced a complex regulation mechanism for PCN production in HT66. In conclusion, the results evidence that P. chlororaphis HT66 could be modified as a potential cell factory for industrial-scale biosynthesis of PCN and other phenazine derivatives by metabolic engineering strategies.

  • Engineering and systems-level analysis of Pseudomonas chlororaphis for production of Phenazine-1-Carboxamide using glycerol as the cost-effective carbon source.
    Biotechnology for biofuels, 2018
    Co-Authors: Ruilian Yao, Huasong Peng, Keli Pan, Lei Feng, Xuehong Zhang
    Abstract:

    Glycerol, an inevitable byproduct of biodiesel, has become an attractive feedstock for the production of value-added chemicals due to its availability and low price. Pseudomonas chlororaphis HT66 can use glycerol to synthesize Phenazine-1-Carboxamide (PCN), a phenazine derivative, which is strongly antagonistic to fungal phytopathogens. A systematic understanding of underlying mechanisms for the PCN overproduction will be important for the further improvement and industrialization. We constructed a PCN-overproducing strain (HT66LSP) through knocking out three negative regulatory genes, lon, parS, and prsA in HT66. The strain HT66LSP produced 4.10 g/L of PCN with a yield of 0.23 (g/g) from glycerol, which was of the highest titer and the yield obtained among PCN-producing strains. We studied gene expression, metabolomics, and dynamic 13C tracer in HT66 and HT66LSP. In response to the phenotype changes, the transcript levels of phz biosynthetic genes, which are responsible for PCN biosynthesis, were all upregulated in HT66LSP. Central carbon was rerouted to the shikimate pathway, which was shown by the modulation of specific genes involved in the lower glycolysis, the TCA cycle, and the shikimate pathway, as well as changes in abundances of intracellular metabolites and flux distribution to increase the precursor availability for PCN biosynthesis. Moreover, dynamic 13C-labeling experiments revealed that the presence of metabolite channeling of 3-phosphoglyceric acid to phosphoenolpyruvate and shikimate to trans-2,3-dihydro-3-hydroxyanthranilic acid in HT66LSP could enable high-yielding synthesis of PCN. The integrated analysis of gene expression, metabolomics, and dynamic 13C tracer enabled us to gain a more in-depth insight into complex mechanisms for the PCN overproduction. This study provides important basis for further engineering P. chlororaphis for high PCN production and efficient glycerol conversion.