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The microbial cofactor F 420 is crucial for processes like methanogenesis, antibiotics biosynthesis, drug resistance, and biocatalysis. Recently, a novel derivative of F 420 (3PG-F 420 ) was discovered, enabling the production and use of F 420 in heterologous hosts.

Upon injury, psychotropic psilocybin‐producing mushrooms instantly develop an intense blue color, the chemical basis and mode of formation of which has remained elusive. We report two enzymes from Psilocybe cubensis that carry out a two‐step cascade to prepare psilocybin for oxidative oligomerization that leads to blue products. The phosphatase PsiP removes the 4‐O‐phosphate group to yield psilocin, while PsiL oxidizes its 4‐hydroxy group. The PsiL reaction was monitored by in situ 13C NMR spectroscopy, which indicated that oxidative coupling of psilocyl residues occurs primarily via C‐5. MS and IR spectroscopy indicated the formation of a heterogeneous mixture of preferentially psilocyl 3‐ to 13‐mers and suggest multiple oligomerization routes, depending on oxidative power and substrate concentration. The results also imply that phosphate ester of psilocybin serves a reversible protective function., Magic mushroom blues: Hallucinogenic Psilocybe mushrooms turn blue when injured. We report that the phosphatase PsiP and the laccase PsiL degrade psilocybin and initiate blueing. MALDI‐MS and in situ NMR spectroscopy show that the blue color is due to a mixture of quinoid psilocyl oligomers, primarily coupled via carbon 5.

Coenzyme F420 is a microbial redox cofactor that is increasingly used for biocatalytic applications. Recently, diversified biosynthetic routes to F420 and the discovery of a derivative, 3PG-F420, were reported. 3PG-F420 is formed via activation of 3-phospho-D-glycerate (3-PG) by CofC, but the structural basis of substrate binding, its evolution, as well as the role of CofD in substrate selection remained elusive.Here, we present a crystal structure of the 3-PG-activating CofC from Mycetohabitans sp. B3 and define amino acids governing substrate specificity. Site-directed mutagenesis enabled bidirectional switching of specificity and thereby revealed the short evolutionary trajectory to 3PG-F420 formation. Furthermore, CofC stabilized its product, thus confirming the structure of the unstable molecule, revealing its binding mode and suggesting a substrate channeling mechanism to CofD. The latter enzyme was shown to significantly contribute to the selection of related intermediates to control the specificity of the combined biosynthetic CofC/D step. Taken together, this work closes important knowledge gaps and opens up perspectives for the discovery, enhanced biotechnological production, and engineering of coenzyme F420 derivatives in the future.ImportanceThe microbial cofactor F420 is crucial for processes like methanogenesis, antibiotics biosynthesis, drug resistance, and biocatalysis. Recently, a novel derivative of F420 (3PG-F420) was discovered, enabling the production and use of F420 in heterologous hosts.By analyzing the crystal structure of a CofC homolog whose substrate choice leads to formation of 3PG-F420, we defined amino acid residues governing the special substrate selectivity. A diagnostic residue enabled reprogramming of the substrate specificity, thus mimicking the evolution of the novel cofactor derivative and successfully guiding the identification of further 3-PG-activating enzymes.Furthermore, a labile reaction product of CofC was revealed that has not been directly detected so far and CofD was shown to provide as another layer of specificity of the combined CofC/D reaction, thus controlling the initial substrate choice of CofC. The latter finding resolves a current debate in the literature about the starting point of F420 biosynthesis in various organisms.

Morphotype switches frequently occur in Actinobacteria and are often associated with disparate natural product production. Here, we report on differences in the secondary metabolomes of two morphotypes of a

Fungi defend their ecological niche against antagonists by producing antibiosis molecules. Some of these molecules are only produced upon confrontation with the antagonist. The basidiomycete Coprinopsis cinerea induces the expression of the sesquiterpene synthase-encoding gene cop6 and its two neighboring genes coding for cytochrome P450 monooxygenases in response to bacteria. We further investigated this regulation of cop6 and examined if the gene product is involved in the production of antibacterials. Cell-free supernatants of axenic cultures of the Gram-positive bacterium Bacillus subtilis were sufficient to induce cop6 transcription assessed using a fluorescent reporter strain. Use of this strain in a microfluidic device revealed that the cop6 gene was induced in all hyphae directly exposed to the supernatant and that induction occurred within less than one hour. Targeted replacement of the cop6 gene demonstrated the requirement of the encoded synthase for the biosynthesis of the sesquiterpene lagopodin B, a previously reported antibacterial compound from related species. Accordingly, lagopodin B from C. cinerea inhibited the growth of several Gram-positive bacteria including B. subtilis but not Gram-negative bacteria. Our results demonstrate that the C. cinerea vegetative mycelium responds to soluble compounds of a bacterial culture supernatant by local production of an antibacterial secondary metabolite.

Modular biosynthetic machineries such as polyketide synthases (PKSs) or nonribosomal peptide synthetases (NRPSs) give rise to a vast structural diversity of bioactive metabolites indispensable in the treatment of cancer or infectious diseases. Here, we provide evidence for different evolutionary processes leading to the diversification of modular NRPSs and thus, their respective products. Discovery of a novel lipo-octapeptide family from Pseudomonas, the virginiafactins, and detailed structure elucidation of closely related peptides, the cichofactins and syringafactins, allowed retracing recombinational diversification of the respective NRPS genes. Bioinformatics analyses allowed us to spot an evolutionary snapshot of these processes, where recombination occurred both within the same and between different biosynthetic gene clusters. Our systems feature a recent diversification process, which may represent a typical paradigm to variations in modular biosynthetic machineries.

Morphotype switches frequently occur in Actinobacteria and are often associated with disparate natural product production. Here, we report on differences in the secondary metabolomes of two morphotypes of a Streptomyces species, including the discovery of a novel antimicrobial glycosylated macrolide, which we named termidomycin A. While exhibiting an unusual 46-member polyene backbone, termidomycin A (1) shares structural features with the clinically important antifungal agents amphotericin B and nystatin A1. Genomic analyses revealed a biosynthetic gene cluster encoding for a putative giant type I polyketide synthase (PKS), whose domain structure allowed us to propose the relative configuration of the 46-member macrolide. The architecture of the biosynthetic gene cluster was different in both morphotypes, thus leading to diversification of the product spectrum. Given the high frequency of genomic rearrangements in Streptomycetes, the metabolic analysis of distinct morphotypes as exemplified in this study is a promising approach for the discovery of bioactive natural products and pathways of diversification.

Coenzyme F420 is a redox cofactor involved in hydride transfer reactions in archaea and bacteria. Since F420-dependent enzymes are attracting increasing interest as tools in biocatalysis, F420 biosynthesis is being revisited. While it was commonly accepted for a long time that the 2-phospho-l-lactate (2-PL) moiety of F420 is formed from free 2-PL, it was recently shown that phosphoenolpyruvate is incorporated in Actinobacteria and that the C-terminal domain of the FbiB protein, a member of the nitroreductase (NTR) superfamily, converts dehydro-F420 into saturated F420 Outside the Actinobacteria, however, the situation is still unclear because FbiB is missing in these organisms and enzymes of the NTR family are highly diversified. Here, we show by heterologous expression and in vitro assays that stand-alone NTR enzymes from Thermomicrobia exhibit dehydro-F420 reductase activity. Metabolome analysis and proteomics studies confirmed the proposed biosynthetic pathway in Thermomicrobium roseum These results clarify the biosynthetic route of coenzyme F420 in a class of Gram-negative bacteria, redefine functional subgroups of the NTR superfamily, and offer an alternative for large-scale production of F420 in Escherichia coli in the future.IMPORTANCE Coenzyme F420 is a redox cofactor of Archaea and Actinobacteria, as well as some Gram-negative bacteria. Its involvement in processes such as the biosynthesis of antibiotics, the degradation of xenobiotics, and asymmetric enzymatic reductions renders F420 of great relevance for biotechnology. Recently, a new biosynthetic step during the formation of F420 in Actinobacteria was discovered, involving an enzyme domain belonging to the versatile nitroreductase (NTR) superfamily, while this process remained blurred in Gram-negative bacteria. Here, we show that a similar biosynthetic route exists in Thermomicrobia, although key biosynthetic enzymes show different domain architectures and are only distantly related. Our results shed light on the biosynthesis of F420 in Gram-negative bacteria and refine the knowledge about sequence-function relationships within the NTR superfamily of enzymes. Appreciably, these results offer an alternative route to produce F420 in Gram-negative model organisms and unveil yet another biochemical facet of this pathway to be explored by synthetic microbiologists.

Coenzyme F(420) is a redox cofactor involved in hydride transfer reactions in archaea and bacteria. Since F(420)-dependent enzymes are attracting increasing interest as tools in biocatalysis, F(420) biosynthesis is being revisited. While it was commonly accepted for a long time that the 2-phospho-l-lactate (2-PL) moiety of F(420) is formed from free 2-PL, it was recently shown that phosphoenolpyruvate is incorporated in Actinobacteria and that the C-terminal domain of the FbiB protein, a member of the nitroreductase (NTR) superfamily, converts dehydro-F(420) into saturated F(420). Outside the Actinobacteria, however, the situation is still unclear because FbiB is missing in these organisms and enzymes of the NTR family are highly diversified. Here, we show by heterologous expression and in vitro assays that stand-alone NTR enzymes from Thermomicrobia exhibit dehydro-F(420) reductase activity. Metabolome analysis and proteomics studies confirmed the proposed biosynthetic pathway in Thermomicrobium roseum. These results clarify the biosynthetic route of coenzyme F(420) in a class of Gram-negative bacteria, redefine functional subgroups of the NTR superfamily, and offer an alternative for large-scale production of F(420) in Escherichia coli in the future. IMPORTANCE Coenzyme F(420) is a redox cofactor of Archaea and Actinobacteria, as well as some Gram-negative bacteria. Its involvement in processes such as the biosynthesis of antibiotics, the degradation of xenobiotics, and asymmetric enzymatic reductions renders F(420) of great relevance for biotechnology. Recently, a new biosynthetic step during the formation of F(420) in Actinobacteria was discovered, involving an enzyme domain belonging to the versatile nitroreductase (NTR) superfamily, while this process remained blurred in Gram-negative bacteria. Here, we show that a similar biosynthetic route exists in Thermomicrobia, although key biosynthetic enzymes show different domain architectures and are only distantly related. Our results shed light on the biosynthesis of F(420) in Gram-negative bacteria and refine the knowledge about sequence-function relationships within the NTR superfamily of enzymes. Appreciably, these results offer an alternative route to produce F(420) in Gram-negative model organisms and unveil yet another biochemical facet of this pathway to be explored by synthetic microbiologists.

Among the invaluable benefits of basidiomycete genomics is the dramatically enhanced insight into the potential capacity to biosynthesize natural products. This study focuses on adenylate-forming reductases, which is a group of natural product biosynthesis enzymes that resembles non-ribosomal peptide synthetases, yet serves to modify one substrate, rather than to condense two or more building blocks. Phylogenetically, these reductases fall in four classes. The phylogeny of Heterobasidion annosum (Russulales) and Serpula lacrymans (Boletales) adenylate-forming reductases was investigated. We identified a previously unrecognized phylogenetic branch within class III adenylate-forming reductases. Three representatives were heterologously produced and their substrate preferences determined in vitro: NPS9 and NPS11 of S. lacrymans preferred l-threonine and benzoic acid, respectively, while NPS10 of H. annosum accepted phenylpyruvic acid best. We also investigated two class IV adenylate-forming reductases of Coprinopsis cinerea, which each were active with l-alanine, l-valine, and l-serine as substrates. Our results show that adenylate-forming reductases are functionally more diverse than previously recognized. As none of the natural products known from the species investigated in this study includes the identified substrates of their respective reductases, our findings may help further explore the diversity of these basidiomycete secondary metabolomes.

Mycofactocin (MFT) is a redox cofactor involved in alcohol metabolism of mycobacteria including Mycobacterium tuberculosis. In recent years, a preliminary biosynthetic model of MFT has been established by in-vitro studies, while the final structure of MFT remained elusive. Here, we report the discovery of MFT by metabolomics and establish a model of its biosynthesis in Mycolicibacterium smegmatis. Structure elucidation revealed that MFT is decorated with up to nine β-1,4-linked glucose residues. Dissection of biosynthetic genes demonstrated that the oligoglycosylation is catalyzed by the glycosyltransferase MftF. Furthermore, we confirm the cofactor function of MFT by activity-based metabolic profiling using the carveol dehydrogenase LimC and show that the MFT pool expands during cultivation on ethanol. Our results close an important gap of knowledge, will guide future studies into the physiological roles of MFT in bacteria and may inspire its utilization as a biomarker or potential drug target to combat mycobacterial diseases.

Coenzyme F

Ribosomally synthesized and posttranslationally modified peptide natural products (RiPPs) exhibit diverse structures and bioactivities and are classified into distinct biosynthetic families. A recently reported family is the proteusins, with the prototype members polytheonamides being generated by almost 50 maturation steps, including introduction of d-residues at multiple positions by an unusual radical SAM epimerase. A region in the protein-like N-terminal leader of proteusin precursors is identified that is crucial for epimerization. It resembles a precursor motif previously shown to mediate interaction in thioether bridge-formation in class I lanthipeptide biosynthesis. Beyond this region, similarities were identified between proteusin and further RiPP families, including class I lanthipeptides. The data suggest that common leader features guide distinct maturation types and that nitrile hydratase-like enzymes are ancestors of several RiPP classes.

Cyclic peptides containing non-proteinogenic amino acids often exhibit a broad bioactivity spectrum and many have entered clinical trials with good prospects for drug development. We recently reported the discovery of six cyclic tetrapeptides, pseudoxylallemycins A-F (1-6), from a termite-associated Pseudoxylaria sp. X802. These compounds contain a rare O-homoallenyl-l-tyrosine moiety and show promising antimicrobial activity against the Gram-negative pathogenic bacterium Pseudomonas aeruginosa. To perform more detailed structure-activity studies, we pursued a precursor-directed diversification strategy. Herein, we report the purification, identification, and testing of 21 new pseudoxylallemycin derivatives.

Chemical and biochemical analyses of one of the most basic nonribosomal peptide synthetases (NRPS) from a Pseudomonas fluorescens strain revealed its striking plasticity. Determination of the potential substrate scope enabled us to anticipate novel secondary metabolites that could subsequently be isolated and tested for their bioactivities. Detailed analyses of the monomodular pyreudione synthetase showed that the biosynthesis of the bacterial pyreudione alkaloids does not require additional biosynthetic enzymes. Heterologous expression of a similar and functional, yet cryptic, NRPS of Pseudomonas entomophila was successful and allowed us to perform a phylogenetic analysis of their thioesterase domains.

The Aspergillus fumigatus nonribosomal peptide synthetase FtpA is among the few of this species whose natural product has remained unknown. Both FtpA adenylation domains were characterized in vitro . Fumaric acid was identified as preferred substrate of the first and both l -tyrosine and l -phenylalanine as preferred substrates of the second adenylation domain. Genetically engineered A. fumigatus strains expressed either ftpA or the regulator gene ftpR , encoded in the same cluster of genes, under the control of the doxycycline-inducible tetracycline-induced transcriptional activation (tet-on) cassette. These strains produced fumaryl- l -tyrosine and fumaryl- l -phenylalanine which were identified by liquid chromatography and high-resolution mass spectrometry. Modeling of the first adenylation domain in silico provided insight into the structural requirements to bind fumaric acid as peptide synthetase substrate. This work adds aromatic fumaric acid amides to the secondary metabolome of the important human pathogen A. fumigatus which was previously not known as a producer of these compounds.

In this issue of Cell Chemical Biology, Li et al. (2017) report on the biosynthesis of the monobactam sulfazecin by Pseudomonas acidophila and hypothesize a novel mechanism of β-lactam ring formation. As monobactam antibiotics are unaffected by some emerging resistance mechanisms (particularly metallo-β-lactamases), this discovery opens prospects to engineer β-lactam antibiotics against multi-drug resistant pathogens.

The as-yet uncultured filamentous bacteria "Candidatus Entotheonella factor" and "Candidatus Entotheonella gemina" live associated with the marine sponge Theonella swinhoei Y, the source of numerous unusual bioactive natural products. Belonging to the proposed candidate phylum "Tectomicrobia," Candidatus Entotheonella members are only distantly related to any cultivated organism. The Ca E. factor has been identified as the source of almost all polyketide and modified peptides families reported from the sponge host, and both Ca Entotheonella phylotypes contain numerous additional genes for as-yet unknown metabolites. Here, we provide insights into the biology of these remarkable bacteria using genomic, (meta)proteomic, and chemical methods. The data suggest a metabolic model of Ca Entotheonella as facultative anaerobic, organotrophic organisms with the ability to use methanol as an energy source. The symbionts appear to be auxotrophic for some vitamins, but have the potential to produce most amino acids as well as rare cofactors like coenzyme F420 The latter likely accounts for the strong autofluorescence of Ca Entotheonella filaments. A large expansion of protein families involved in regulation and conversion of organic molecules indicates roles in host-bacterial interaction. In addition, a massive overrepresentation of members of the luciferase-like monooxygenase superfamily points toward an important role of these proteins in Ca Entotheonella. Furthermore, we performed mass spectrometric imaging combined with fluorescence in situ hybridization to localize Ca Entotheonella and some of the bioactive natural products in the sponge tissue. These metabolic insights into a new candidate phylum offer hints on the targeted cultivation of the chemically most prolific microorganisms known from microbial dark matter.

Aspirochlorine (1) is an epidithiodiketopiperazine (ETP) toxin produced from koji mold (Aspergillus oryzae), which has been used in the oriental cuisine for over two millennia. Considering its potential risk for food safety, we have elucidated the molecular basis of aspirochlorine biosynthesis. By a combination of genetic and chemical analyses we found the acl gene locus and identified the key role of AclH as a chlorinase. Stable isotope labeling, biotransformation, and mutational experiments, analysis of intermediates and an in vitro adenylation domain assay gave totally unexpected insights into the acl pathway: Instead of one Phe and one Gly, two Phe units are assembled by an iterative non-ribosomal peptide synthetase (NRPS, AclP), followed by halogenation and an unprecedented Phe to Gly amino acid conversion. Biological assays showed that both amino acid transformations are required to confer cytotoxicity and antifungal activity to the mycotoxin.

The filamentous mold Aspergillus fumigatus causes invasive aspergillosis, a potentially life-threatening infectious disease, in humans. The sidE gene encodes a bimodular peptide synthetase and was shown previously to be strongly upregulated during initiation of murine lung infection. In this study, we characterized the two adenylation domains of SidE with the ATP-[ 32 P]pyrophosphate exchange assay in vitro , which identified fumarate and l -alanine, respectively, as the preferred substrates. Using full-length holo -SidE, fumarylalanine (FA) formation was observed in vitro . Furthermore, FA was identified in A. fumigatus culture supernatants under inducing conditions, unless sidE was genetically inactivated. As FA is structurally related to established pharmaceutical products exerting immunomodulatory activity, this work may contribute to our understanding of the virulence of A. fumigatus .

SummaryLittle is known about polyketide biosynthesis in mushrooms (basidiomycota). In this study, we investigated the iterative type I polyketide synthase (PKS) ArmB of the tree pathogen Armillaria mellea, a producer of cytotoxic melleolides (i.e., polyketides esterified with various sesquiterpene alcohols). Heterologously produced ArmB showed orsellinic acid (OA) synthase activity in vitro. Further, we demonstrate cross-coupling activity of ArmB, which forms OA esters with various alcohols. Using a tricyclic Armillaria sesquiterpene alcohol, we reconstituted the biosynthesis of melledonol. Intermolecular transesterification reactions may represent a general mechanism of fungal PKSs to create structural diversity of small molecules. Phylogenetic network construction of thioesterase domains of both basidiomycetes and ascomycetes suggests that the fungal nonreducing PKS family has likely evolved from an ancient OA synthase and has gained versatility by adopting Claisen-like cyclase or transferase activity.

A considerable share of fungal small-molecule natural products is assembled via nonribosomal peptide synthetases (NRPSs). Here, we introduce the readers to two topical aspects which have emerged during work with enzymes of both asco- and basidiomycete origin. First, we highlight the function of non-canonical NRPS-like enzymes which lack the ability to make peptide bonds, and we summarize how they contribute to metabolic diversity. Second, we address the question to what extent substrates of fungal peptide synthetases and related enzymes can be predicted out of their primary sequence.

Numerous polyketides are known from bacteria, plants, and fungi. However, only a few have been isolated from basidiomycetes. Large scale genome sequencing projects now help anticipate the capacity of basidiomycetes to synthesize polyketides. In this study, we identified and annotated 111 type I and three type III polyketide synthase (PKS) genes from 35 sequenced basidiomycete genomes. Phylogenetic analysis of PKS genes suggests that all main types of fungal iterative PKS had already evolved before the Ascomycota and Basidiomycota diverged. A comparison of genomic and metabolomic data shows that the number of polyketide genes exceeds the number of known polyketide structures by far. Exploiting these results to design degenerate PCR primers, we amplified and cloned the complete sequence of armB, a PKS gene from the melleolide producer Armillaria mellea. We expect this study will serve as a guide for future genomic mining projects to discover structurally diverse mushroom-derived polyketides.

Caught in the act: imaging mass spectrometry of a button mushroom infected with the soft rot pathogen Janthinobacterium agaricidamnosum in conjunction with genome mining revealed jagaricin as a highly antifungal virulence factor that is not produced under standard cultivation conditions. The structure of jagaricin was rigorously elucidated by a combination of physicochemical analyses, chemical derivatization, and bioinformatics.

Ectomycorrhizal fungi are thought to have a key role in mobilizing organic nitrogen that is trapped in soil organic matter (SOM). However, the extent to which ectomycorrhizal fungi decompose SOM and the mechanism by which they do so remain unclear, considering that they have lost many genes encoding lignocellulose-degrading enzymes that are present in their saprotrophic ancestors. Spectroscopic analyses and transcriptome profiling were used to examine the mechanisms by which five species of ectomycorrhizal fungi, representing at least four origins of symbiosis, decompose SOM extracted from forest soils. In the presence of glucose and when acquiring nitrogen, all species converted the organic matter in the SOM extract using oxidative mechanisms. The transcriptome expressed during oxidative decomposition has diverged over evolutionary time. Each species expressed a different set of transcripts encoding proteins associated with oxidation of lignocellulose by saprotrophic fungi. The decomposition 'toolbox' has diverged through differences in the regulation of orthologous genes, the formation of new genes by gene duplications, and the recruitment of genes from diverse but functionally similar enzyme families. The capacity to oxidize SOM appears to be common among ectomycorrhizal fungi. We propose that the ancestral decay mechanisms used primarily to obtain carbon have been adapted in symbiosis to scavenge nutrients instead. This work was supported by grants from the Swedish Research Council (VR) (to A.T. and P.P.), the Knut and Alice Wallenberg Foundation (to A.T.), the strategic research programme Biodiversity and Ecosystem Services in a Changing Climate (BECC) (to A.T.) and the French National Research Agency through the Laboratory of Excellence ARBRE (ANR-11-LABX-0002-01) (to F.M.). Genome sequencing was conducted by the US Department of Energy Joint Genome Institute, supported by the Office of Science of the US Department of Energy. We also thank Annegret Kohler and Nicolas Cichocki for handling the fungal cultures, and principal investigators from the Mycorrhizal Genomics Initiative Consortium for providing the strain cultures and access to the genome sequences before publication.

Natural products play a key role in symbiotic interactions between microorganisms and higher organisms, covering all kingdoms of life. The function of these secondary metabolites may range from signaling compounds in mutualism to virulence factors and antibiotics in parasitic relationships. In many cases the interactions involve multiple partners and thus the biogenetic basis of chemical mediators can be quite complex. This complexity is well exemplified by the unparalleled tripartite relationship among the rice-seedling-blight fungus Rhizopus microsporus, its host plant Oryza sativa and endosymbiotic bacteria that reside in the fungal cytosol. The bacterial symbionts (Burkholderia species) produce a phytotoxin complex to assist the phytopathogenic fungus in colonizing rice seedlings. In turn the bacteria profit from a safe niche and access to nutrients released from the decaying plant. Initially, the macrolide rhizoxin (1, Figure 1) and various congeners such as WF-1360F (2) were isolated from cultures of R. microsporus van Tieghem var. chinensis and identified as the causative agent of rice seedling blight. Rhizoxin efficiently inhibits eukaryotic cell proliferation by binding to b-tubulin and thus blocking the formation of the mitotic spindle. Notably, the pure compound alone evokes the typical symptoms of seedling root swelling. Only recently, through detection, isolation, and cultivation of the endosymbionts we could unequivocally prove that actually associated bacteria are the true producers of the toxin complex. The importance of this metabolic capability has been underlined by the finding that fungal reproduction depends entirely on the presence of the bacterial symbionts. Survival of the toxinogenic symbiosis is warranted by the strict sporulation control and exclusive dispersal of spores harboring endosymbionts. Moreover, the unusual mutualism has been fine-tuned through symbiosis factors such as a type-III secretion system and a novel lipopolysaccharide O-antigen that sets the symbionts into a “stealth mode” by decorating the outer membrane of the endosymbionts. The host, on the other hand, acquired resistance towards rhizoxin by mutation of the b-tubulin. Because of its ecological and medicinal relevance as an antimitotic agent, the biosynthesis of rhizoxin has been studied. Cloning, sequencing, and molecular analyses of the rhizoxin (rhi) biosynthetic gene cluster in the genome of Burkholderia rhizoxinica revealed the molecular basis for a complex polyketide assembly line required for the biosynthesis of the virulence factor. Whereas the biosynthesis of the macrolide backbone has been decoded by mutational analyses, polyketide tailoring mechanisms and the biological role of the bis(epoxidation) have remained elusive. Herein we elucidate the dual epoxidation of rhizoxin and its impact on rice seedling blight and report an unprecedented case for symbiotic cooperation in the biosynthesis of an ecologically relevant natural product. In a broader survey on rhizoxin-positive Rhizopus species we discovered that the unusual bacterial–fungal association is not restricted to a single isolate but has spread worldwide. We have identified eight related Burkholderia–Rhizopus associations from geographically highly different regions on five continents; these findings underlign the ecological imporFigure 1. A) Structures of rhizoxin and congeners. B) Phylogenetic relationship of Rhizopus microsporus strains; structures of the corresponding metabolites indicate which strains can produce bisepoxides. The numbers on top of the branches indicate the clade probability values; the scale on the left site relates the length of a branch to the distance (number of changes that have taken place along a branch).

The gene greA was cloned from the genome of the basidiomycete Suillus grevillei. It encodes a monomodular natural product biosynthesis protein composed of three domains for adenylation, thiolation, and thioesterase and, hence, is reminiscent of a nonribosomal peptide synthetase (NRPS). GreA was biochemically characterized in vitro. It was identified as atromentin synthetase and therefore represents one of only a limited number of biochemically characterized NRPS-like enzymes which accept an aromatic α-keto acid. Specificity-conferring amino acid residues--collectively referred to as the nonribosomal code--were predicted for the primary sequence of the GreA adenylation domain and were an unprecedented combination for aromatic α-keto acids. Plausible support for this new code came from in silico simulation of the adenylation domain structure. According to the model, the predicted residues line the active site and, therefore, very likely contribute to substrate specificity.

Toxinogenic endobacteria were isolated from a collection of Rhizopus spp. representing highly diverse geographic origins and ecological niches. All endosymbionts belonged to the Burkholderia rhizoxinica complex according to matrix-assisted laser desorption ionization-time of flight biotyping and multilocus sequence typing, suggesting a common ancestor. Comparison of host and symbiont phylogenies provides insights into possible cospeciation and horizontal-transmission events.

Im Beitrag wird uber die Psychotherapie mit alteren Leuten berichtet, die als Kinder und Jugendliche Opfer des Naziterrors in Karnten geworden sind. Obwohl ihr Leid uber Jahrzehnte hinweg verleugnet und verdrangt wurde, erweist sich die traumatherapeutische Arbeit mit Mitteln des Psychodrama und der Psychodynamisch Imaginativen Traumatherapie als sinnvoll.

The benastatins, pradimicins, fredericamycins, and members of the griseorhodin/rubromycin family represent a structurally and functionally diverse group of long-chain polyphenols from actinomycetes. Comparison of their biosynthetic gene clusters (ben, prm, fdm, grh, rub) revealed that all loci harbor genes coding for a similar, yet uncharacterized, type of ketoreductases. In a phylogenetic survey of representative KRs involved in type II PKS systems, we found that it is generally possible to deduce the KR regiospecificity (C-9, C-15, C17) from the amino acid sequence and thus to predict the nature of the aromatic polyketide (e.g., angucycline, anthracycline, benzoisochromanequinones). We hypothezised that the new clade of KRs is characteristic for biosynthesis of polyphenols with an extended angular architecture we termed "pentangular". To test this hypothesis, we demonstrated the biogenetic relationship between benastatin and the structurally unrelated spiro ketal griseorhodin by generating a mutant producing collinone, a pentangular pathway intermediate. The benastatin pathway served as a model to characterize the KR. Gene inactivation of benL resulted in the formation of a series of 19-hydroxy benastatin and bequinostatin derivatives (e.g., benastatin K and benastatin L). These results clearly showed that BenL functions as a C-19 KR in pentangular pathways.

Janthinobacterium agaricidamnosum causes soft-rot disease of the cultured button mushroom Agaricus bisporus and is thus responsible for agricultural losses. Here, we present the genome sequence of J. agaricidamnosum DSM 9628. The 5.9-Mb genome harbors several secondary metabolite biosynthesis gene clusters, which renders this neglected bacterium a promising source for genome mining approaches.

L-α-Aminoadipic acid reductases catalyze the ATP- and NADPH-dependent reduction of L-α-aminoadipic acid to the corresponding 6-semialdehyde during fungal L-lysine biosynthesis. These reductases resemble peptide synthetases with regard to their multidomain composition but feature a unique domain of elusive function--now referred to as an adenylation activating (ADA) domain--that extends the reductase N-terminally. Truncated enzymes based on NPS3, the L-α-aminoadipic acid reductase of the basidiomycete Ceriporiopsis subvermispora, lacking the ADA domain either partially or entirely were tested for activity in vitro, together with an ADA-adenylation didomain and the ADA domainless adenylation domain. We provide evidence that the ADA domain is required for substrate adenylation: that is, the initial step of the catalytic turnover. Our biochemical data are supported by in silico modeling that identified the ADA domain as a partial peptide synthetase condensation domain.

International audience; Clonostachys rosea is a mycoparasitic fungus that can control several important plant diseases. Here, we report on the genome sequencing of C. rosea and a comparative genome analysis, in order to resolve the phylogenetic placement of C. rosea and to study the evolution of mycoparasitism as a fungal lifestyle. The genome of C. rosea is estimated to 58.3 Mb, and contains 14,268 predicted genes. A phylogenomic analysis shows that C. rosea clusters as sister taxon to plant pathogenic Fusarium species, with mycoparasitic/ saprotrophic Trichoderma species in an ancestral position. A comparative analysis of gene family evolution reveals several distinct differences between the included mycoparasites. Clonostachys rosea contains significantly more ATP-binding cassette (ABC) transporters , polyketide synthases, cytochrome P450 monooxygenases, pectin lyases, glucose-methanol-choline oxidoreductases, and lytic polysaccharide monooxygenases compared with other fungi in the Hypocreales. Interestingly, the increase of ABC transporter gene number in C. rosea is associated with phylogenetic subgroups B (multidrug resistance proteins) and G (pleiotropic drug resistance transporters), whereas an increase in subgroup C (multidrug resistance-associated proteins) is evident in Trichoderma virens. In contrast with mycoparasitic Trichoderma species, C. rosea contains very few chitinases. Expression of six group B and group G ABC transporter genes was induced in C. rosea during exposure to the Fusarium mycotoxin zearalenone, the fungicide Boscalid or metabolites from the biocontrol bacterium Pseudomonas chlororaphis. The data suggest that tolerance toward secondary metab-olites is a prominent feature in the biology of C. rosea.

The rice seedling blight fungus Rhizopus microsporus has an unusual symbiosis with a bacterium, Burkholderia rhizoxinica, which lives within the fungal cytosol and produces a potent phytotoxin that causes severe losses in agriculture. To gain insight into symbiosis factors we investigated the endosymbiont's exopolysaccharide (EPS), a secreted matrix that plays pivotal roles in mediating cell-environment interactions. By a combination of homo- and heteronuclear 2D NMR experiments, we elucidated a previously unknown EPS structure: a repeating tetrasaccharide unit bearing a nonstoichiometric acetyl group on a mannose residue. We also analyzed the EPS biosynthesis gene cluster and generated a targeted mutant to compare the phenotypes. Scanning electron microscope images revealed a reduced ability of the mutant to form extracellular polymers around cell aggregates. Phylogenetic analyses suggest that the symbiont's EPS genes are retained through evolutionary processes.

20 p.-3 tab.-9 fig., Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on freshcut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea’s extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes., The major portions of this work were performed under US Department of Agriculture Cooperative State, Research, Education, and Extension Service Grant 2007-35504-18257 (to DC and RAB). The US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy under Contract DE-AC02-05CH11231. This work was also supported by the HIPOP (BIO2011-26694) project of the Spanish Ministry of Economy and Competitiveness (MINECO) (to FJRD), the PEROXICATS (KBBE-2010-4-265397) and INDOX (KBBE-2013-.3.3-04-613549) European projects (to ATM), and the Chilean National Fund for Scientific and Technological Development Grant 1131030 (to LFL).

Mittels einer Falldarstellung beschreibe ich meine Arbeitsweise in der Psychotherapie mit traumatisierten Fluchtlingen. Zum Einsatz kommt die Psychodrama-Psychotherapie im Dolmetscherunterstutzten Einzelsetting. Groses Augenmerk wird auf die Stabilisierungsphase der Therapie gelegt, die sich aufgrund standig neu auftretender Umfeldbelastungen fur den Klienten bis zum Ende der Therapie durchzieht. Verschiedene psychodramatische Stabilisierungstechniken warden vorgestellt, wobei der "Sichere Ort" eine zentrale Stellung einnimmt. Nach Erlauterung einiger Grundzuge der Traumatherapie wird die Traumaexposition beschrieben, die mittels Distanzierungshilfen eine Annaherung an die traumatischen Erlebnisse ermoglicht.