Molecular Genetics of Secondary Chemistry in Metarhizium Fungi.

As with many microbes, entomopathogenic fungi from the genus Metarhizium produce a plethora of small molecule metabolites, often referred to as secondary metabolites. Although these intriguing compounds are a conspicuous feature of the biology of the producing fungi, their roles in pathogenicity and other interactions with their hosts and competing microbes are still not well understood. In this review, secondary metabolites that have been isolated from Metarhizium are cataloged along with the history of their discovery and structural elucidation and the salient biological activities attributed to them. Newly available genome sequences revealed an abundance of biosynthetic pathways and a capacity for producing SMs by Metarhizium species that far exceeds the known chemistry. Secondary metabolism genes identified in nine sequenced Metarhizium species are analyzed in detail and classified into distinct families based on orthology, phylogenetic analysis, and conservation of the gene organization around them. This analysis led to the identification of seven hybrid polyketide/nonribosomal peptide synthetases (M-HPNs), two inverted hybrid nonribosomal peptide/polyketide synthetases (M-IHs), 27 nonribosomal peptide synthetases (M-NRPSs), 14 nonribosomal peptide synthetase-like (M-NPL) pathways, 32 polyketide synthases, and 44 terpene biosynthetic genes having a nonuniform distribution and largely following established phylogenetic relationships within the genus Metarhzium. This systematization also identified candidate pathways for known Metarhizium chemistries and predicted the presence of unknown natural products for this genus by drawing connections between these pathways and natural products known to be produced by other fungi.

Involvement of a cytochrome P450 monooxygenase in thaxtomin A biosynthesis by Streptomyces acidiscabies.

The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification, N-methylation, and cyclization of two amino acid substrates are catalyzed by the txtAB-encoded thaxtomin synthetase. Nucleotide sequence analysis of the region 3' of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family. It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A. Comparisons of electrospray mass spectra as well as (1)H- and (13)C-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl), the didehydroxy analog of thaxtomin A. The ORF, designated txtC, was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts. TxtC produced in E. coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form. Based on these properties and the high similarity of TxtC to other well-characterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide.

Polyketide synthase genes in insect- and nematode-associated fungi.

Production of polyketides is accomplished through complex enzymes known as polyketide synthases (PKS); these enzymes have highly conserved domains that might be useful in screens for PKSs in diverse groups of organisms. A degenerate PCR-based approach was used to amplify PKS fragments of the ketosynthase domain from genomic DNA of a group of insect- and nematode-associated fungi. Of 157 isolates (representing 73 genera and 144 species) screened, 92 isolates generated PCR products of predicted size (approximately 300 bp). The ability to detect PKS domains was a function of the number of different primer pairs employed in the screen. Cloning and sequencing revealed that 66 isolates had at least one unique PKS sequence; ten members of this set contained multiple PKS fragments, for a total of 76 unique PKS fragments. Since PKS genes appear to be widespread among fungi, a PCR-based screening system appears to be an efficient, directed means to identify organisms having the potential to produce polyketides.

The txtAB genes of the plant pathogen Streptomyces acidiscabies encode a peptide synthetase required for phytotoxin thaxtomin A production and pathogenicity.

Four Streptomyces species have been described as the causal agents of scab disease, which affects economically important root and tuber crops worldwide. These species produce a family of cyclic dipeptides, the thaxtomins, which alone mimic disease symptomatology. Structural considerations suggest that thaxtomins are synthesized non-ribosomally. Degenerate oligonucleotide primers were used to amplify conserved portions of the acyladenylation module of peptide synthetase genes from genomic DNA of representatives of the four species. Pairwise Southern hybridizations identified a peptide synthetase acyladenylation module conserved among three species. The complete nucleotide sequences of two peptide synthetase genes (txtAB) were determined from S. acidiscabies 84.104 cosmid library clones. The organization of the deduced TxtA and TxtB peptide synthetase catalytic domains is consistent with the formation of N-methylated cyclic dipeptides such as thaxtomins. Based on high-performance liquid chromatography (HPLC) analysis, thaxtomin A production was abolished in txtA gene disruption mutants. Although the growth and morphological characteristics of the mutants were identical to those of the parent strain, txtA mutants were avirulent on potato tubers. Moreover, introduction of the thaxtomin synthetase cosmid into a txtA mutant restored both pathogenicity and thaxtomin A production, demonstrating a critical role for thaxtomins in pathogenesis.

Characterization of 6-epi-3-anhydroophiobolin B from Cochliobolus heterostrophus.

The new sesterterpenoid 6-epi-3-anhydroophiobolin B (1) and six known ophiobolins were isolated from the extracts of the fungus Cochliobolus heterostrophus race O. The structure of 6-epi-3-anhydroophiobolin B was deduced from analysis of spectral data and the structural characterization of dehydration and dimerization products. Ophiobolin A (2) showed potent activity in cytotoxicity assays and marginal activity in antimalarial assays.

Increased efficacy of Bacillus thuringiensis subsp. kurstaki in combination with tannic acid.

We identified tannic acid as an inexpensive additive that increased the efficacy of sublethal concentrations of Bacillus thuringiensis subsp. kurstaki (Berliner). Tannic acid mimicked the active constituents contained in an aqueous, tannin-rich extract of Taxus baccata (L.) bark that retarded development of Heliothis virescens (F.) larvae at 10,000 ppm; most larvae remained in first and second stage when treated with 250-10,000 ppm of tannic acid. Instar development of Trichoplusia ni (Hübner) larvae was affected in a concentration-dependent manner by 2.5-500 ppm of tannic acid. In subsequent bioassays, tannic acid at 25-500 ppm in combination with B. thuringiensis (1.63 micrograms [AI]/ml diet) yielded mean mortalities of 57-75%, whereas treatments with B. thuringiensis alone produced 10% mortality. Mean mortalities in the 3.0, 4.5, and 6.75 micrograms (AI) B. thuringiensis per milliliter of diet treatments (5.5; 8.0, and 30%, respectively) were significantly higher in the presence of 250 and 2,500 ppm tannic acid; in these treatments we observed 78-94% mortality. Addition of tannic acid increased the activity of concentrations of 3-4.5 micrograms (AI) B. thuringiensis per milliliter of diet to approximately that of a concentration of 13 micrograms (AI) B. thuringiensis per milliliter of diet alone (85-95% mortality). Although deaths caused by a formulation of B. thuringiensis + tannic acid occurred more slowly than with high rates of B. thuringiensis alone, such formulations would have the advantages of arresting development, minimizing foliar damage, and decreasing the concentration of B. thuringiensis used.

Identification of the antibiotic phomalactone from the entomopathogenic fungusHirsutella thompsonii var.synnematosa.

Dichloromethane extracts of culture broth from three strains of the entomopathogenic fungusHirsutella thompsonii var.synnematosa were toxic to two species of tephritid fruit fly and inhibited conidial germination in vitro in several other entomopathogenic fungi includingBeauveria bassiana, Tolypocladium spp., andMetarhizium anisopliae. A major metabolite, toxic to apple maggot,Rhagoletis pomonella, and inhibitory to conidial germination inB. bassiana, was isolated and identified as the antibiotic (+)-phomalactone, 6-(1-propenyl)-5,-6-dihydro-5-hydroxypyran-2-one. This is the first biologically active compound of low molecular weight isolated from the genusHirsutella.

Efrapeptin production byTolypocladium fungi (Deuteromycotina: Hyphomycetes): Intra- and interspecific variation.

Production of the mitochondrial ATPase inhibitory peptides efrapeptins was evaluated by HPLC analysis in 44 strains of nine species of the fungal genusTolypocladium (Deuteromycotina: Hyphomycetes). Efrapeptin identification was confirmed by mass spectral data for the first time in the speciesT. cylindrosporum. HPLC retention time data indicated thatT. nubicola andT. tundrense, two species not previously known to produce efrapeptins, also produce the peptides. No efrapeptins were detected (<0.3 mg/100 ml broth) in single strains each ofT. balanoides, T. extinguens, T. parasiticum, andT. microsporum. Five strains ofT. geodes produced detectable amounts of efrapeptins and had compound F, or F and G, as the major component(s) in the mixture with the order of abundance being F> or ∼G > H ∼ D ∼ E > C. Eleven strains ofT. niveum produced detectable amounts of efrapeptins and had efrapeptins D and E as the primary and secondary components, respectively, in the mixture with the order of abundance being D > E > F > C ∼ G. A singleT. niveum strain had an efrapeptin profile similar to that of theT. geodes strains. Ten strains ofT. cylindrosporum had detectable amounts of efrapeptins. Of these, nine had F and one had G as the major component.T. cylindrosporum had higher ratios of E to D than didT. geodes. Efrapeptins were detected in one of twoT. nubicola strains analyzed (F > G > H) and one of threeT. tundrense strains (F > G > H > E).T. niveum strains could, in most cases, be identified to species on the basis of their efrapeptin profiles.

Isolation of beauvericin as an insect toxin from Fusarium semitectum and Fusarium moniliforme var. subglutinans.

Nonpolar methylene chloride-soluble extracts from the mycelia of Fusarium semitectum and Fusarium moniliforme var. subglutinans were toxic to Colorado potato beetles. The major toxic metabolite was isolated and found to be the cyclodepsipeptide, beauvericin. This is the first report of the isolation of beauvericin from the genus Fusarium.

Identification and directed biosynthesis of efrapeptins in the fungusTolypocladium geodes gams (Deuteromycotina: Hyphomycetes).

HPLC analysis of crude dichloromethane extracts of shaken liquid cultures of the hyphomycetous fungusTolypocladium geodes Gams revealed the presence of efrapeptins. These peptides, which have mitochondrial ATPase inhibitory activity as well as antifungal and insecticidal properties, are previously known only from the congeneric species,T. niveum Rostrup. The identity of efrapeptins was confirmed by fast atom bombardment mass spectrometry and by amino acid analysis. HPLC analyses of efrapeptins extracted from single isolates of bothT. geodes andT. niveum indicated that both species produced the same efrapeptins but the profile of relative abundance of the compounds produced was diagnostic for the isolates examined. Efrapeptin F was the major component of the mixture fromT. geodes with the order of abundance of the six efrapeptins detected being F >G>D∼E>H>C. Efrapeptin D was the major component fromT. niveum with the order of abundance of the six efrapeptins detected being D >E>F>C∼G>H. Efrapeptin F diifers from efrapeptin D by a single amino acid residue, efrapeptin F having an alanine where efrapeptin D has a glycine. Addition of alanine to the culture medium increased the relative abundance of efrapeptin F in the profile of both species. Conversely, addition of glycine increased the relative abundance of efrapeptin D in the profile of both species.

Dihydropyrrolizine attractants for arctiid moths that visit plants containing pyrrolizidine alkaloids.

Adults of three species of arctiid moths (Cisseps fulvicollis, Ctenucha virginia, andHalysidota tessellaris) are attracted to plants that contain pyrrolizidine alkaloids (PAs). The moths use olfactory cues to locate these plants, then feed on leaves, flowers, and roots with the proboscis. To investigate the chemical basis of attraction, sticky traps were baited with roots of a PA-containing plant,Eupatorium maculatum, alkaloids ofE. maculatum, and several derivatives of these alkaloids. Volatile derivatives of the bicyclic pyrrolizidine skeleton attracted all three arctiid species. The dihydropyrrolizines, (S)-(+)-hydroxydanaidal and (R)-(-)-hydroxydanaidal, proved to be the most attractive compounds tested, accounting for over 70% of the moths captured. Different alkaloid derivatives attracted different proportions of male and femaleCisseps. Both (S)-(+)-hydroxydanaidal (52% male) and (R)-(-)-hydroxydanaidal (71% male) attracted a significantly lower percentage ofCisseps males thanE. maculatum roots (87% male).Cisseps males possess eversible scent organs (coremata) that are displayed during courtship. Analysis of corematal extracts revealed the presence of hydroxydanaidal.Cisseps moths thus resemble danaine and ithomiine butterflies, both in their attraction to PA sources and in the presence of PA derivatives in the male scent organs.

Quantitative and qualitative effects of larval diet on male scent secretions ofEstigmene acrea, Phragmatobia foliginosa, andPyrrharctia isabella (Lepidoptera: Arctiidae).

In feeding experiments with insects reared in the laboratory, the presence of the dihydropyrrolizines hydroxydanaidal and danaidal in the male scent organs (coremata) of the arctiids,Estigmene acrea (Drury),Phragmatobia fuliginosa (L.), andPyrrharctia isabella (J.E. Smith), was shown to depend on the presence of a source of pyrrolizidine alkaloids (PAs) in the larval diet.Phragmatobia males given an artificial diet supplemented with the powdered roots of the PA-containing plantSymphytum officinale L. (comfrey) produced more hydroxydanaidal than danaidal, whereas males given an artificial diet supplemented with dried whole plants of another PA-containing species,Senecio vulgaris L., produced more danaidal than hydroxydanaidal.Pyrrharctia males produced hydroxydanaidal with little if any danaidal, whether the source of PAs was comfrey orS. vulgaris. A behavioral bioassay showed that the coremata of PA-deniedPyrrharctia male progeny of PA-denied parents were pheromonally inactive, whereas those of PA-denied male progeny of PA-supplied parents (male and/or female) were often active. This indicates that a small amount of pheromone is made from PAs transferred from the female to her eggs and that males effect copulatory transfers of PAs that are, in turn, passed to the eggs by the mated female. Field observations ofPhragmatobia andPyrrharctia larvae feeding on sources of PAs were reported. The PA monocrotaline was shown to be a feeding stimulant forPyrrharctia larvae.

Quantitative and qualitative variation in male pheromones ofPhragmatobia fuliginosa andPyrrharctia isabella (Lepidoptera: Arctiidae).

The dihydropyrrolizine pheromones, hydroxydanaidal and danaidal, were identified from the scent organs of malePhragmatobia fuliginosa (L.) andPyrrharctia isabella (J.E. Smith). Qualitative and quantitative GLC analyses were conducted on ca. 80 field-collected males of each species. The total pheromone titer was distributed bimodally in each species with most males having either a small amount (< 10 ng) of pheromone or a large amount (1-10 µg inPyrrharctia and 0.3-3 µg inPhragmatobia).Pyrrharctia males in the 1- to 10-µg range had a predominance of hydroxydanaidal, with little if any danaidal. MostPhragmatobia males in the 0.3- to 3-µg range had danaidal with little if any hydroxydanaidal. These compounds elicited a courtship response in sexually receptive females of both species. A bioassay based on this response was used to measure the thresholds of female response to these compounds.Pyrrharctia females were more sensitive to (R)-(-)-hydroxydanaidal than to danaidal.Phragmatobia females were more sensitive to danaidal then to (R)-(-)-hydroxydanaidal.

Male wing-gland pheromone ofEphestia elutella.

Sex pheromone extracted from glands on the forewings of maleEphestia elutella (Hübner) elicits a stereotyped courtship response from conspecific females. A bioassay for this sex pheromone was developed based on this behavior. Maximum production and responsiveness for males and females, respectively, occurred in insects more than 24 hr old.E. elutella females were not responsive to extracts made fromE.figulilella Gregson,E. kuehniella Zeller,E. cautella (Walker), orPlodia interpunctella (Hübner) males.