Nelson L. Brock

The Sfp-Type 4′-Phosphopantetheinyl Transferase Ppt1 of Fusarium fujikuroi Controls Development, Secondary Metabolism and Pathogenicity

The heterothallic ascomycete Fusarium fujikuroi is a notorious rice pathogen causing super-elongation of plants due to the production of terpene-derived gibberellic acids (GAs) that function as natural plant hormones. Additionally, F. fujikuroi is able to produce a variety of polyketide- and non-ribosomal peptide-derived metabolites such as bikaverins, fusarubins and fusarins as well as metabolites from yet unidentified biosynthetic pathways, e.g. moniliformin. The key enzymes needed for their production belong to the family of polyketide synthases (PKSs) and non-ribosomal peptide synthases (NRPSs) that are generally known to be post-translationally modified by a Sfp-type 4′phosphopantetheinyl transferase (PPTase). In this study we provide evidence that the F. fujikuroi Sfp-type PPTase FfPpt1 is essentially involved in lysine biosynthesis and production of bikaverins, fusarubins and fusarins, but not moniliformin as shown by analytical methods. Concomitantly, targeted Ffppt1 deletion mutants reveal an enhancement of terpene-derived metabolites like GAs and volatile substances such as α-acorenol. Pathogenicity assays on rice roots using fluorescent labeled wild-type and Ffppt1 mutant strains indicate that lysine biosynthesis and iron acquisition but not PKS and NRPS metabolism is essential for establishment of primary infections of F. fujikuroi. Additionally, FfPpt1 is involved in conidiation and sexual mating recognition possibly by activating PKS- and/or NRPS-derived metabolites that could act as diffusible signals. Furthermore, the effect on iron acquisition of Ffppt1 mutants led us to identify a previously uncharacterized putative third reductive iron uptake system (FfFtr3/FfFet3) that is closely related to the FtrA/FetC system of A. fumigatus. Functional characterization provides evidence that both proteins are involved in iron acquisition and are liable to transcriptional repression of the homolog of the Aspergillus GATA-type transcription factor SreA under iron-replete conditions. Targeted deletion of the first Fusarium homolog of this GATA-type transcription factor-encoding gene, Ffsre1, strongly indicates its involvement in regulation of iron homeostasis and oxidative stress resistance.

Pathways and substrate specificity of DMSP catabolism in marine bacteria of the Roseobacter clade.

The volatiles released by Phaeobacter gallaeciensis, Oceanibulbus indolifex and Dinoroseobacter shibae have been investigated by GC‐MS, and several MeSH‐derived sulfur volatiles have been identified. An important sulfur source in the oceans is the algal metabolite dimethylsulfoniopropionate (DMSP). Labelled [2H6]DMSP was fed to the bacteria to investigate the production of volatiles from this compound through the lysis pathway to [2H6]dimethylsulfide or the demethylation pathway to [2H3]‐3‐(methylmercapto)propionic acid and lysis to [2H3]MeSH. [2H6]DMSP was efficiently converted to [2H3]MeSH by all three species. Several DMSP derivatives were synthesised and used in feeding experiments. Strong dealkylation activity was observed for the methylated ethyl methyl sulfoniopropionate and dimethylseleniopropionate, as indicated by the formation of EtSH‐ and MeSeH‐derived volatiles, whereas no volatiles were formed from dimethyltelluriopropionate. In contrast, the dealkylation activity for diethylsulfoniopropionate was strongly reduced, resulting in only small amounts of EtSH‐derived volatiles accompanied by diethyl sulfide in P. gallaeciensis and O. indolifex, while D. shibae produced the related oxidation product diethyl sulfone. The formation of diethyl sulfide and diethyl sulfone requires the lysis pathway, which is not active for [2H6]DMSP. These observations can be explained by a shifted distribution between the two competing pathways due to a blocked dealkylation of ethylated substrates.

Physiological diversity of Roseobacter clade bacteria co-occurring during a phytoplankton bloom in the North Sea.

Organisms of the Roseobacter clade are an important component in marine ecosystems, partially due to their metabolic variety. Not much is known, however, about the physiological diversity of different roseobacters present within one habitat. By using serial dilution cultures with low-nutrient media seven roseobacter strains, co-occurring during a phytoplankton bloom in the southern North Sea, were obtained in this study. Physiological characterization exhibited distinct substrate spectra of the isolates. Although no isolate showed growth on algal osmolyte dimethylsulfoniopropionate (DMSP), feeding experiments revealed that all new strains converted [²H₆]DMSP into a variety of volatile compounds. Six strains mainly decomposed DMSP via the demethylation pathway, but four strains were also capable of cleaving DMSP to DMS and acrylate. It is hypothesized that the great physiological diversity of the roseobacters reflects their ability to inhabit different ecological niches and enables the organisms to cope differently with changing substrate supplies during phytoplankton blooms. Denaturing gradient gel electrophoresis and sequencing of excised bands resulted in detection of five additional roseobacters. Three of these sequences showed affiliation with three of the four major clusters of the Roseobacter clade, consisting predominantly of uncultured organisms (i.e. the Roseobacter clade-affiliated (RCA)), the NAC11-7 and the CHAB-I-5 clusters.

Genetic Dissection of Sesquiterpene Biosynthesis by Fusarium fujikuroi

A treasure trove of terpenes: The products of two fungal sesquiterpene synthases from the rice pathogen Fusarium fujikuroi were identified by gene-knockout experiments, genetic engineering of the fungus for production optimization, isolation of the sesquiterpenes, and structure elucidation by spectroscopic methods.

Genetic Analysis of the Upper Phenylacetate Catabolic Pathway in the Production of Tropodithietic Acid by Phaeobacter gallaeciensis

ABSTRACT Production of the antibiotic tropodithietic acid (TDA) depends on the central phenylacetate catabolic pathway, specifically on the oxygenase PaaABCDE, which catalyzes epoxidation of phenylacetyl-coenzyme A (CoA). Our study was focused on genes of the upper part of this pathway leading to phenylacetyl-CoA as precursor for TDA. Phaeobacter gallaeciensis DSM 17395 encodes two genes with homology to phenylacetyl-CoA ligases (paaK1 and paaK2), which were shown to be essential for phenylacetate catabolism but not for TDA biosynthesis and phenylalanine degradation. Thus, in P. gallaeciensis another enzyme must produce phenylacetyl-CoA from phenylalanine. Using random transposon insertion mutagenesis of a paaK1-paaK2 double mutant we identified a gene (ior1) with similarity to iorA and iorB in archaea, encoding an indolepyruvate:ferredoxin oxidoreductase (IOR). The ior1 mutant was unable to grow on phenylalanine, and production of TDA was significantly reduced compared to the wild-type level (60%). Nuclear magnetic resonance (NMR) spectroscopic investigations using 13C-labeled phenylalanine isotopomers demonstrated that phenylalanine is transformed into phenylacetyl-CoA by Ior1. Using quantitative real-time PCR, we could show that expression of ior1 depends on the adjacent regulator IorR. Growth on phenylalanine promotes production of TDA, induces expression of ior1 (27-fold) and paaK1 (61-fold), and regulates the production of TDA. Phylogenetic analysis showed that the aerobic type of IOR as found in many roseobacters is common within a number of different phylogenetic groups of aerobic bacteria such as Burkholderia, Cupriavidis, and Rhizobia, where it may also contribute to the degradation of phenylalanine.

Microbial production of the drugs violacein and deoxyviolacein: analytical development and strain comparison.

Violacein and deoxyviolacein display a broad range of interesting biological properties but their production is rarely distinguished due to the lack of suitable analytical methods. An HPLC method has been developed for the separation and quantification of violacein and deoxyviolacein and can determine the content of both molecules in microbial cultures. A comparison of different production microorganisms, including recombinant Escherichia coli and the natural producer Janthinobacterium lividum, revealed that the formation of violacein and deoxyviolacein is strain-specific but showed significant variation during growth although the ratio between the two compounds remained constant.

Comparative "Omics" of the Fusarium fujikuroi Species Complex Highlights Differences in Genetic Potential and Metabolite Synthesis

Species of the Fusarium fujikuroi species complex (FFC) cause a wide spectrum of often devastating diseases on diverse agricultural crops, including coffee, fig, mango, maize, rice, and sugarcane. Although species within the FFC are difficult to distinguish by morphology, and their genes often share 90% sequence similarity, they can differ in host plant specificity and life style. FFC species can also produce structurally diverse secondary metabolites (SMs), including the mycotoxins fumonisins, fusarins, fusaric acid, and beauvericin, and the phytohormones gibberellins, auxins, and cytokinins. The spectrum of SMs produced can differ among closely related species, suggesting that SMs might be determinants of host specificity. To date, genomes of only a limited number of FFC species have been sequenced. Here, we provide draft genome sequences of three more members of the FFC: a single isolate of F. mangiferae, the cause of mango malformation, and two isolates of F. proliferatum, one a pathogen of maize and the other an orchid endophyte. We compared these genomes to publicly available genome sequences of three other FFC species. The comparisons revealed species-specific and isolate-specific differences in the composition and expression (in vitro and in planta) of genes involved in SM production including those for phytohormome biosynthesis. Such differences have the potential to impact host specificity and, as in the case of F. proliferatum, the pathogenic versus endophytic life style.

Biosynthesis of Sesquiterpenes by the Fungus Fusarium verticillioides

The volatiles of the fungus Fusarium verticillioides were analysed by GC‐MS. Sesquiterpenes dominated, with trichodiene as the principle component. Several other sesquiterpenes were detected in low amounts that were unambiguously identified from their mass spectra and retention indices. The absolute configurations of (R)‐β‐bisabolene, (R)‐cuparene, (+)‐β‐barbatene, (−)‐α‐cedrene, (+)‐β‐cedrene, and (+)‐α‐funebrene originating from different key cationic intermediates, were determined by chiral GC‐MS and proved to be related to the trichodiene stereostructure. The unusual compound (E)‐iso‐γ‐bisabolene was also found corroborating a previously suggested mechanism for the cyclisation of the bisabolyl to the cuprenyl cation that is based on quantum mechanical calculations (Y. J. Hong, D. J. Tantillo, Org. Lett. 2006, 8, 4601–4604). These analyses resulted in a revised biosynthesis scheme to trichodiene and the side products of the responsible terpene cyclase, trichodiene synthase, an enzyme that is well characterised from Fusarium sporotrichioides. Feeding studies with several deuterated mevalonolactone isotopomers unravelled stereochemical aspects of the late cyclisations towards trichodiene.

Biosynthesis of Sesqui- and Diterpenes by the Gibberellin Producer Fusarium fujikuroi

The fungus Fusarium fujikuroi IMI58289 emits a complex pattern of volatile terpenoids including two major compounds, the sesquiterpene alcohol α‐acorenol and the diterpene ent‐kaurene. ent‐Kaurene is the precursor for the phytohormone gibberellic acid (GA3) and is produced from geranylgeranyl diphosphate (GGPP) via ent‐copalyl diphosphate by the bifunctional ent‐copalyl diphosphate/ent‐kaurene synthase (CPS/KS). Several structurally related diterpenes were identified as side products of the CPS/KS. Deletion of the cps/ks gene or the whole GA3 biosynthetic gene cluster resulted in completely abolished diterpene production. Mutants with deletions of the cytochrome P450 monooxygenase gene P450‐4, which is responsible for the three oxidation steps from ent‐kaurene to ent‐kaurenoic acid en route to GA3, accumulate diterpene hydrocarbons. Feeding with [6,6,6‐2H3] mevalonolactone gave insights into the stereochemistry of the GGPP cyclisation, which operates with a chair–chair‐“antipodal” fold. A rational biosynthetic scheme for all identified sesquiterpenes demonstrated their formation from farnesyl diphosphate (FPP) via three alternative initial cyclisations. Genome sequencing revealed the presence of five putative sesquiterpene synthase genes in the F. fujikuroi genome. The structures of several trace compounds from other classes have been identified as new natural products; these were delineated from their mass spectra and unambiguously assigned by comparison to synthetic references.

Nucleobase Protection Strategy for Gene Cloning and Expression

Protecting group chemistry meets molecular biology: Chemically modified dATP carrying a bulky triethylsilylethynyl group was used in a PCR-based synthesis of a gene internally protected against cleavage by restriction endonucleases. The unmodified flanking regions were cleaved for cloning into a plasmid which was replicated by E. coli, and used for protein production.