scholarly journals Agrobacterium-Mediated Disruption of a Nonribosomal Peptide Synthetase Gene in the Invertebrate Pathogen Metarhizium anisopliae Reveals a Peptide Spore Factor

2008 ◽  
Vol 74 (14) ◽  
pp. 4366-4380 ◽  
Author(s):  
Yong-Sun Moon ◽  
Bruno G. G. Donzelli ◽  
Stuart B. Krasnoff ◽  
Heather McLane ◽  
Mike H. Griggs ◽  
...  
Keyword(s):  
Metarhizium Anisopliae ◽  
Developmental Stages ◽  
Cyclic Peptide ◽  
Pathogenic Fungus ◽  
Nonribosomal Peptide Synthetase ◽  
Dna Polymorphisms ◽  
Beet Armyworm ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase

ABSTRACT Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium anisopliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption by Agrobacterium tumefaciens-mediated transformation a putative nonribosomal peptide synthetase (NPS) gene, MaNPS1. Four of six gene disruption mutants identified were examined further. Chemical analyses showed the presence of serinocyclins, cyclic heptapeptides, in the extracts of conidia of control strains, whereas the compounds were undetectable in ΔManps1 mutants treated identically or in other developmental stages, suggesting that MaNPS1 encodes a serinocyclin synthetase. Production of the cyclic depsipeptide destruxins, M. anisopliae metabolites also predicted to be synthesized by an NPS, was similar in ΔManps1 mutant and control strains, indicating that MaNPS1 does not contribute to destruxin biosynthesis. Surprisingly, a MaNPS1 fragment detected DNA polymorphisms that correlated with relative destruxin levels produced in vitro, and MaNPS1 was expressed concurrently with in vitro destruxin production. ΔManps1 mutants exhibited in vitro development and responses to external stresses comparable to control strains. No detectable differences in pathogenicity of the ΔManps1 mutants were observed in bioassays against beet armyworm and Colorado potato beetle in comparison to control strains. This is the first report of targeted disruption of a secondary metabolite gene in M. anisopliae, which revealed a novel cyclic peptide spore factor.

scholarly journals Characterization of the Ustilago maydis sid2 Gene, Encoding a Multidomain Peptide Synthetase in the Ferrichrome Biosynthetic Gene Cluster

2001 ◽  
Vol 183 (13) ◽  
pp. 4040-4051 ◽  
Author(s):  
Walter M. Yuan ◽  
Guillaume D. Gentil ◽  
Allen D. Budde ◽  
Sally A. Leong
Keyword(s):  
Amino Acids ◽  
Cyclic Peptide ◽  
Ustilago Maydis ◽  
Nonribosomal Peptide Synthetase ◽  
Hybridization Analysis ◽  
Northern Hybridization ◽  
Nucleotide Sequencing ◽  
Biosynthetic Gene ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase

ABSTRACT Ustilago maydis, the causal agent of corn smut disease, acquires and transports ferric ion by producing the extracellular, cyclic peptide, hydroxamate siderophores ferrichrome and ferrichrome A. Ferrichrome biosynthesis likely proceeds by hydroxylation and acetylation of l-ornithine, and later steps likely involve covalently bound thioester intermediates on a multimodular, nonribosomal peptide synthetase. sid1 encodesl-ornithine N 5-oxygenase, which catalyzes hydroxylation of l-ornithine, the first committed step of ferrichrome and ferrichrome A biosynthesis in U. maydis. In this report we characterize sid2, another biosynthetic gene in the pathway, by gene complementation, gene replacement, DNA sequence, and Northern hybridization analysis. Nucleotide sequencing has revealed that sid2 is located 3.7 kb upstream of sid1 and encodes an intronless polypeptide of 3,947 amino acids with three iterated modules of an approximate length of 1,000 amino acids each. Multiple motifs characteristic of the nonribosomal peptide synthetase protein family were identified in each module. A corresponding iron-regulated sid2 transcript of 11 kb was detected by Northern hybridization analysis. By contrast, constitutive accumulation of this large transcript was observed in a mutant carrying a disruption of urbs1, a zinc finger, GATA family transcription factor previously shown to regulate siderophore biosynthesis in Ustilago. Multiple GATA motifs are present in the intergenic region between sid1 and sid2, suggesting bidirectional transcription regulation by urbs1of this pathway. Indeed, mutation of two of these motifs, known to be important to regulation of sid1, altered the differential regulation of sid2 by iron.

scholarly journals A Highly Conserved Basidiomycete Peptide Synthetase Produces a Trimeric Hydroxamate Siderophore

2017 ◽  
Vol 83 (21) ◽  
Author(s):  
Eileen Brandenburger ◽  
Markus Gressler ◽  
Robin Leonhardt ◽  
Gerald Lackner ◽  
Andreas Habel ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Nonribosomal Peptide Synthetase ◽  
Full Length ◽  
Amide Bond ◽  
White Rot ◽  
Mass Spectrometry Analysis ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Peptide Synthetase

ABSTRACT The model white-rot basidiomycete, Ceriporiopsis (Gelatoporia) subvermispora B, encodes putative natural product biosynthesis genes. Among them is the gene for the seven-domain nonribosomal peptide synthetase CsNPS2. It is a member of the as-yet-uncharacterized fungal type VI siderophore synthetase family, which is highly conserved and widely distributed among the basidiomycetes. These enzymes include only one adenylation (A) domain, i.e., one complete peptide synthetase module, and two thiolation/condensation (T-C) didomain partial modules which together constitute an AT1C1T2C2T3C3 domain setup. The full-length CsNPS2 enzyme (274.5 kDa) was heterologously produced as a polyhistidine fusion in Aspergillus niger as a soluble and active protein. N 5-acetyl-N 5-hydroxy-l-ornithine (l-AHO) and N 5-cis-anhydromevalonyl-N 5 -hydroxy-l-ornithine (l-AMHO) were accepted as the substrates, based on results of an in vitro substrate-dependent [32P]ATP-pyrophosphate radioisotope exchange assay. Full-length holo-CsNPS2 catalyzed amide bond formation between three l-AHO molecules to release the linear l-AHO trimer, called basidioferrin, as the product in vitro, which was verified by liquid chromatography–high-resolution electrospray ionization–mass spectrometry analysis. Phylogenetic analyses suggested that type VI family siderophore synthetases are widespread in mushrooms and evolved in a common ancestor of basidiomycetes. IMPORTANCE The basidiomycete nonribosomal peptide synthetase CsNPS2 represents a member of a widely distributed but previously uninvestigated class (type VI) of fungal siderophore synthetases. Genes orthologous to CsNPS2 are highly conserved across various phylogenetic clades of the basidiomycetes. Hence, our work serves as a broadly applicable model for siderophore biosynthesis and iron metabolism in higher fungi. Also, our results on the amino acid substrate preference of CsNPS2 support a further understanding of the substrate selectivity of fungal adenylation domains. Methodologically, this report highlights the Aspergillus niger/SM-Xpress-based system as a suitable platform to heterologously express multimodular basidiomycete biosynthesis enzymes in the >250-kDa range in soluble and active form.

scholarly journals Characterization of the Saframycin A Gene Cluster from Streptomyces lavendulae NRRL 11002 Revealing a Nonribosomal Peptide Synthetase System for Assembling the Unusual Tetrapeptidyl Skeleton in an Iterative Manner

2007 ◽  
Vol 190 (1) ◽  
pp. 251-263 ◽  
Author(s):  
Lei Li ◽  
Wei Deng ◽  
Jie Song ◽  
Wei Ding ◽  
Qun-Fei Zhao ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Nonribosomal Peptide Synthetase ◽  
Combinatorial Biosynthesis ◽  
Common Mechanism ◽  
Nonribosomal Peptide ◽  
Streptomyces Lavendulae ◽  
Peptide Synthetase

ABSTRACT Saframycin A (SFM-A), produced by Streptomyces lavendulae NRRL 11002, belongs to the tetrahydroisoquinoline family of antibiotics, and its core is structurally similar to the core of ecteinascidin 743, which is a highly potent antitumor drug isolated from a marine tunicate. In this study, the biosynthetic gene cluster for SFM-A was cloned and localized to a 62-kb contiguous DNA region. Sequence analysis revealed 30 genes that constitute the SFM-A gene cluster, encoding an unusual nonribosomal peptide synthetase (NRPS) system and tailoring enzymes and regulatory and resistance proteins. The results of substrate prediction and in vitro characterization of the adenylation specificities of this NRPS system support the hypothesis that the last module acts in an iterative manner to form a tetrapeptidyl intermediate and that the colinearity rule does not apply. Although this mechanism is different from those proposed for the SFM-A analogs SFM-Mx1 and safracin B (SAC-B), based on the high similarity of these systems, it is likely they share a common mechanism of biosynthesis as we describe here. Construction of the biosynthetic pathway of SFM-Y3, an aminated SFM-A, was achieved in the SAC-B producer (Pseudomonas fluorescens). These findings not only shed new insight on tetrahydroisoquinoline biosynthesis but also demonstrate the feasibility of engineering microorganisms to generate structurally more complex and biologically more active analogs by combinatorial biosynthesis.

scholarly journals Deletion of the Nonribosomal Peptide Synthetase Gene nps1 in the Fungus Clonostachys rosea Attenuates Antagonism and Biocontrol of Plant Pathogenic Fusarium and Nematodes

2019 ◽  
Vol 109 (10) ◽  
pp. 1698-1709 ◽  
Author(s):  
Mudassir Iqbal ◽  
Mukesh Dubey ◽  
Anders Broberg ◽  
Maria Viketoft ◽  
Dan Funck Jensen ◽  
...  
Keyword(s):  
Pathogenic Fungus ◽  
Nonribosomal Peptide Synthetase ◽  
Parasitic Nematodes ◽  
Wild Type ◽  
Clonostachys Rosea ◽  
Wheat Seedlings ◽  
Nonribosomal Peptide ◽  
Plant Pathogenic Fungus ◽  
Culture Filtrates ◽  
Peptide Synthetase

Secondary metabolites produced by biological control agents may influence the outcome of their interactions with plant pathogenic microorganisms and plants. In the present study, we investigated the role of the nonribosomal peptide synthetase gene nps1 expressed by the biocontrol fungus Clonostachys rosea. A gene expression analysis showed that nps1 was induced during confrontations with the plant pathogenic fungus Botrytis cinerea. Gene deletion strains of nps1 displayed increased growth rates and conidiation. However, the nematicidal activity of culture filtrates from C. rosea Δnps1 strains was significantly weaker than that from wild-type filtrates (P ≤ 0.001); after 24 h of incubation with culture filtrates from nps1 deletion strains, only 13 to 33% of a mixed community of nematodes were dead compared with 42% of nematodes incubated with wild-type culture filtrates. The Δnps1 strains also showed reduced biocontrol efficacy during pot experiments, thus failing to protect wheat seedlings from foot rot disease caused by the plant pathogenic fungus Fusarium graminearum. Furthermore, C. rosea Δnps1 strains were not able to reduce populations of plant-parasitic nematodes in soil or in roots of wheat as efficiently as the wild-type strain. Both C. rosea wild-type and Δnps1 strains increased the dry shoot weight and shoot length of wheat by 20 and 13%, respectively. We showed that NPS1, a putative nonribosomal peptide synthetase encoded by nps1, is a biocontrol factor, presumably by producing a hitherto unknown nonribosomal peptide compound with antifungal and nematicidal properties that contributes to the biocontrol properties of C. rosea.

scholarly journals Comparison of Cyanopeptolin Genes in Planktothrix, Microcystis, and Anabaena Strains: Evidence for Independent Evolution within Each Genus

2007 ◽  
Vol 73 (22) ◽  
pp. 7322-7330 ◽  
Author(s):  
Trine B. Rounge ◽  
Thomas Rohrlack ◽  
Ave Tooming-Klunderud ◽  
Tom Kristensen ◽  
Kjetill S. Jakobsen
Keyword(s):  
Gene Cluster ◽  
Cyclic Peptide ◽  
Phylogenetic Analyses ◽  
Gene Clusters ◽  
Binding Pocket ◽  
Nonribosomal Peptide Synthetase ◽  
Glyceric Acid ◽  
Nonribosomal Peptide ◽  
Independent Evolution ◽  
Peptide Synthetase

ABSTRACT The major cyclic peptide cyanopeptolin 1138, produced by Planktothrix strain NIVA CYA 116, was characterized and shown to be structurally very close to the earlier-characterized oscillapeptin E. A cyanopeptolin gene cluster likely to encode the corresponding peptide synthetase was sequenced from the same strain. The 30-kb oci gene cluster contains two novel domains previously not detected in nonribosomal peptide synthetase gene clusters (a putative glyceric acid-activating domain and a sulfotransferase domain), in addition to seven nonribosomal peptide synthetase modules. Unlike in two previously described cyanopeptolin gene clusters from Anabaena and Microcystis, a halogenase gene is not present. The three cyanopeptolin gene clusters show similar gene and domain arrangements, while the binding pocket signatures deduced from the adenylation domain sequences and the additional tailoring domains vary. This suggests loss and gain of tailoring domains within each genus, after the diversification of the three clades, as major events leading to the present diversity. The ABC transporter genes associated with the cyanopeptolin gene clusters form a monophyletic clade and accordingly are likely to have evolved as part of the functional unit. Phylogenetic analyses of adenylation and condensation domains, including domains from cyanopeptolins and microcystins, show a closer similarity between the Planktothrix and Microcystis cyanopeptolin domains than between these and the Anabaena domain. No clear evidence of recombination between cyanopeptolins and microcystins could be detected. There were no strong indications of horizontal gene transfer of cyanopeptolin gene sequences across the three genera, supporting independent evolution within each genus.

scholarly journals Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

2017 ◽  
Vol 114 (27) ◽  
pp. 7025-7030 ◽  
Author(s):  
Nicholas C. Harris ◽  
Michio Sato ◽  
Nicolaus A. Herman ◽  
Frederick Twigg ◽  
Wenlong Cai ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Acyl Chain ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Nonribosomal Peptide Synthetase ◽  
Biosynthetic Gene ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase

A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a nonribosomal peptide synthetase. In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.

scholarly journals In Vitro Characterization of a Heterologously Expressed Nonribosomal Peptide Synthetase Involved in Phosphinothricin Tripeptide Biosynthesis

Biochemistry ◽  
2009 ◽  
Vol 48 (23) ◽  
pp. 5054-5056 ◽  
Author(s):  
Jin-Hee Lee ◽  
Bradley S. Evans ◽  
Gongyong Li ◽  
Neil L. Kelleher ◽  
Wilfred A. van der Donk
Keyword(s):  
Nonribosomal Peptide Synthetase ◽  
Nonribosomal Peptide ◽  
In Vitro Characterization ◽  
Peptide Synthetase
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