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.

Interaction of ammonium, glucose, and chitin regulates the expression of cell wall-degrading enzymes in Trichoderma atroviride strain P1.

Chitinolytic and glucanolytic fungal cell wall-degrading enzymes have been suggested to be primary determinants of biocontrol by Trichoderma spp. We examined the effects of ammonium, glucose, chitin, and chito-oligomers on transcription of specific genes and secretion of fungal cell wall-degrading enzymes. The genes ech42, nag1, and gluc78 were examined, as were the enzymes they encode (endochitinase CHIT42, N-acetylhexosaminidase CHIT73, and glucan exo-1,3-beta-glucanase GLUC78, respectively). gluc78 could be induced by nitrogen starvation alone, while both ech42 and nag1 required nitrogen starvation and the presence of chitin for induction. Starvation for both ammonium and glucose resulted in very early expression and secretion of all cell wall-degrading enzymes examined. In the presence of low levels of ammonium (10 mM), both chito-oligomers and chitin triggered CHIT42 and CHIT40 (chitobiosidase) production. CHIT73 secretion occurred in the presence of N-acetylglucosamine and chito-oligomers, while chitin was less effective. The presence of different chito-oligomers resulted in secretion of specific N-acetylhexosaminidases, of which CHIT73 is one. Our results indicate that the expression and secretion of cell wall-degrading enzymes is nitrogen repressed, that effects of carbon and nitrogen nutrition are interactive, and that especially for chitinolytic enzymes, the inductive effect of chitin is altered by the level of ammonium or glucose in the medium.

Cloning, sequence and structure of a gene encoding an antifungal glucan 1,3-beta-glucosidase from Trichoderma atroviride (T. harzianum).

A gene (gluc78) encoding an antifungal glucan 1,3-beta-glucosidase was cloned from strain P1 of the biocontrol fungus Trichoderma atroviride (formerly T. harzianum). A putative regulatory sequence upstream from the coding region was cloned using single-strand extension from a primer in the known portion of the gene, circularized with T4 ligase, and then reamplified with PCR to generate double-stranded DNA. The entire genomic DNA sequence consisted of 3440 bp, with 559 and 579 bp, respectively, in 5' and 3' untranslated regions. The transcription unit contains a single intron, positioned in the 5' untranslated region. The gene encodes for a protein of 770 aa, including a 40 aa signal peptide. Symmetry between the first and second halves of the mature protein was found. The gene is present as a single copy in T. atroviride and a similar gene also is present in T. harzianum and T. virens. The encoded protein has similarity to a small group of sequences from filamentous fungi and no significant similarity to 1,3-beta-glucanases or glucosidases from other organisms. Northern analysis indicates that the gene is repressed in the presence of 3% glucose and expressed in media containing 0.1% of the sugar. Laminarin (0.1%) enhances expression after 18 h and other polymers such as scleroglucan and pustulan may enhance expression after 40 h of growth.