Phylogenomics reveals subfamilies of fungal nonribosomal peptide synthetases and their evolutionary relationships

<p><b>Abstract</b></p> <p>Background</p> <p>Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes, found in fungi and bacteria, which biosynthesize peptides without the aid of ribosomes. Although their metabolite products have been the subject of intense investigation due to their life-saving roles as medicinals and injurious roles as mycotoxins and virulence factors, little is known of the phylogenetic relationships of the corresponding NRPSs or whether they can be ranked into subgroups of common function. We identified genes (<it>NPS</it>) encoding NRPS and NRPS-like proteins in 38 fungal genomes and undertook phylogenomic analyses in order to identify fungal NRPS subfamilies, assess taxonomic distribution, evaluate levels of conservation across subfamilies, and address mechanisms of evolution of multimodular NRPSs. We also characterized relationships of fungal NRPSs, a representative sampling of bacterial NRPSs, and related adenylating enzymes, including α-aminoadipate reductases (AARs) involved in lysine biosynthesis in fungi.</p> <p>Results</p> <p>Phylogenomic analysis identified nine major subfamilies of fungal NRPSs which fell into two main groups: one corresponds to <it>NPS </it>genes encoding primarily mono/bi-modular enzymes which grouped with bacterial NRPSs and the other includes genes encoding primarily multimodular and exclusively fungal NRPSs. AARs shared a closer phylogenetic relationship to NRPSs than to other acyl-adenylating enzymes. Phylogenetic analyses and taxonomic distribution suggest that several mono/bi-modular subfamilies arose either prior to, or early in, the evolution of fungi, while two multimodular groups appear restricted to and expanded in fungi. The older mono/bi-modular subfamilies show conserved domain architectures suggestive of functional conservation, while multimodular NRPSs, particularly those unique to euascomycetes, show a diversity of architectures and of genetic mechanisms generating this diversity.</p> <p>Conclusions</p> <p>This work is the first to characterize subfamilies of fungal NRPSs. Our analyses suggest that mono/bi-modular NRPSs have more ancient origins and more conserved domain architectures than most multimodular NRPSs. It also demonstrates that the α-aminoadipate reductases involved in lysine biosynthesis in fungi are closely related to mono/bi-modular NRPSs. Several groups of mono/bi-modular NRPS metabolites are predicted to play more pivotal roles in cellular metabolism than products of multimodular NRPSs. In contrast, multimodular subfamilies of NRPSs are of more recent origin, are restricted to fungi, show less stable domain architectures, and biosynthesize metabolites which perform more niche-specific functions than mono/bi-modular NRPS products. The euascomycete-only NRPS subfamily, in particular, shows evidence for extensive gain and loss of domains suggestive of the contribution of domain duplication and loss in responding to niche-specific pressures.</p

Evolution Of Nonribosomal Peptide Synthetase Proteins Involved In Secondary Metabolism In Fungi

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes which biosynthesize peptides (NRPs) independently of ribosomes. Three core domains (adenylation (A), thiolation (T), condensation (C)) comprise a functional module for NRP biosynthesis. Although NRPSs produce a diversity of bioactive compounds, little is known about the evolutionary relationships of genes encoding NRPSs and the mechanisms by which they evolve. The objectives of this research were to perform phylogenomic analyses to identify major NRPS subclasses and determine evolutionary relationships and to elucidate fine-scale evolutionary mechanisms giving rise to the diverse NRPS domain structures in fungi. Chapter 2 is a published manuscript on ferrichrome synthetases tracking the evolution of domain architectures of these relatively conserved enzymes across fungi. Results supported the hypothesis that ferrichrome synthetases evolved by tandem duplication of complete modules (A-T-C) (single or double units) and loss of single A domains or complete A-T-C modules. A mechanism for evolution of iterative biosynthesis is proposed. Protein modeling of the A domain substrate binding pockets refined characterization of key residues involved in substrate specificity, by identifying novel sites. Chapter 3 reports a fungal kingdom-wide phylogenomic study of NRPSs, with the objective of identifying subclasses. Nine were identified which fell into two major groups. One consisted of primarily mono/bi-modular NRPSs with conserved domain architectures which group with bacterial NRPSs and whose products are associated with conserved metabolic roles. The other consisted of primarily multimodular and exclusively fungal NRPSs with variable domain architectures whose products perform niche-specific functions. All groups of NRPSs were much more common in Euascomycetes than in any other fungal taxonomic group. Although NRPSs are discontinuously distributed across fungal taxa, little evidence was found for horizontal gene transfer from bacteria to fungi. Overall, this study showed that both tandem duplication and loss, as well as recombination and rearrangement, of modular units (either complete A-T-C modules or single A domains) are mechanisms by which NRPSs and their chemical products evolve. Phylogenomic analysis identified subgroups of NRPSs possibly reflecting common function and suggested an older evolutionary origin of several mono/bimodular groups while multimodular fungal NRPSs are more recently derived and highly expanded in Euascomycetes

Seeing the Communities for the Carbon: Governance Challenges of Reducing Emissions from Deforestation and Forest Degradation in Nepal

oai:pubs.lib.umn.edu:article/563Reducing emissions from deforestation and forest degradation in developing countries (REDD), an emerging international climate change mitigation mechanism, would compensate developing countries with threatened forests for their conservation and reforestation efforts. The implications of this new scheme for governments, forests, communities and their development are still unclear. The preparation for REDD that is taking place in many countries includes little concern for this mechanism’s potential impacts on the rights and livelihoods of forest-dependent communities who have yet to be consulted. This paper analyzes both precedents and the current process of REDD readiness in Nepal, revealing discrepancies in forest governance that must be addressed before this carbon trading mechanism can successfully meet climate change mitigation, biodiversity conservation, or development goals. This paper finds that REDD readiness and policy formulation is being driven from the top down, with insufficient involvement and influence by local communities in planning and decision-making processes, and that a more adaptive, bottom-up approach is needed if REDD is to be economically beneficial, socially equitable, and environmentally sustainable in the long term

The genome of the truffle-parasite Tolypocladium ophioglossoides and the evolution of antifungal peptaibiotics

Abstract Background Two major mycoparasitic lineages, the family Hypocreaceae and the genus Tolypocladium, exist within the fungal order, Hypocreales. Peptaibiotics are a group of secondary metabolites almost exclusively described from Trichoderma species of Hypocreaceae. Peptaibiotics are produced by nonribosomal peptide synthetases (NRPSs) and have antibiotic and antifungal activities. Tolypocladium species are mainly truffle parasites, but a few species are insect pathogens. Results The draft genome sequence of the truffle parasite Tolypocladium ophioglossoides was generated and numerous secondary metabolite clusters were discovered, many of which have no known putative product. However, three large peptaibiotic gene clusters were identified using phylogenetic analyses. Peptaibiotic genes are absent from the predominantly plant and insect pathogenic lineages of Hypocreales, and are therefore exclusive to the largely mycoparasitic lineages. Using NRPS adenylation domain phylogenies and reconciliation of the domain tree with the organismal phylogeny, it is demonstrated that the distribution of these domains is likely not the product of horizontal gene transfer between mycoparasitic lineages, but represents independent losses in insect pathogenic lineages. Peptaibiotic genes are less conserved between species of Tolypocladium and are the product of complex patterns of lineage sorting and module duplication. In contrast, these genes are more conserved within the genus Trichoderma and consistent with diversification through speciation. Conclusions Peptaibiotic NRPS genes are restricted to mycoparasitic lineages of Hypocreales, based on current sampling. Phylogenomics and comparative genomics can provide insights into the evolution of secondary metabolite genes, their distribution across a broader range of taxa, and their possible function related to host specificity.http://deepblue.lib.umich.edu/bitstream/2027.42/112062/1/12864_2015_Article_1777.pd

Research, part of a Special Feature on REDD+ national policy networks: information flows, influence and coalitions for change REDD+ policy making in Nepal: toward state-centric, polycentric, or market-oriented governance?

ABSTRACT. Over the past 40 years, Nepal has become renowned for its community-based forestry policies, initiatives, and institutions, characterized by local autonomy in decisions about forest management and use and a gradual shift toward more inclusive national policy processes. In recent years, the government, international nongovernmental organizations (NGOs), donors, and some civil society organizations have instigated policy and piloting initiatives for an international climate change mitigation scheme known as &quot;reducing emissions from deforestation and forest degradation and enhancement of forest carbon stocks in developing countries&quot; (REDD+). Although many people see REDD+ as a means of bolstering forest conservation efforts and enhancing rural livelihoods, its broader implications for decentralized forest governance in Nepal and elsewhere remain uncertain and contested. Using policy network analysis and theories of polycentric and network governance, I examined influence, inclusiveness, and deliberation among actors involved in REDD+ policy making in Nepal. Data were collected between June and December 2011 through a survey of 34 organizations from government, civil society, educational and research institutions, international NGOs and donors, and the private sector. I investigated whether policy processes and the configurations of actors involved reflect state-centric, market-oriented, or polycentric governance, and I discuss the implications for decentralized forest governance in general and for the implementation of REDD+ in particular. Results indicate that REDD+ policy making is dominated by a &quot;development triangle&quot;, a tripartite coalition of key government actors, external organizations (international NGOs and donors), and select civil society organizations. As a result, the views and interests of other important stakeholders have been marginalized, threatening recentralized forest governance and hampering the effective implementation of REDD+ in Nepal. Key Words: climate change; conservation; decentralization; forest governance; policy networks; South Asia INTRODUCTION Reducing emissions from deforestation and forest degradation and enhancement of forest carbon stocks in developing countries (REDD+) has strong implications for forest governance worldwide. REDD+ is an emerging market-based mechanism designed to curb greenhouse gas emissions through performancebased payments for the protection and sustainable management of forests. Some stakeholders involved in REDD+ believe that it will inject renewed vigor, transparency, and accountability into forest governance and promote decentralized and inclusive modes of governance, considered necessary for forest conservation and reforestatio

The genome of the truffle-parasite Tolypocladium ophioglossoides and the evolution of antifungal peptaibiotics

Background Two major mycoparasitic lineages, the family Hypocreaceae and the genus Tolypocladium, exist within the fungal order, Hypocreales. Peptaibiotics are a group of secondary metabolites almost exclusively described from Trichoderma species of Hypocreaceae. Peptaibiotics are produced by nonribosomal peptide synthetases (NRPSs) and have antibiotic and antifungal activities. Tolypocladium species are mainly truffle parasites, but a few species are insect pathogens. Results The draft genome sequence of the truffle parasite Tolypocladium ophioglossoides was generated and numerous secondary metabolite clusters were discovered, many of which have no known putative product. However, three large peptaibiotic gene clusters were identified using phylogenetic analyses. Peptaibiotic genes are absent from the predominantly plant and insect pathogenic lineages of Hypocreales, and are therefore exclusive to the largely mycoparasitic lineages. Using NRPS adenylation domain phylogenies and reconciliation of the domain tree with the organismal phylogeny, it is demonstrated that the distribution of these domains is likely not the product of horizontal gene transfer between mycoparasitic lineages, but represents independent losses in insect pathogenic lineages. Peptaibiotic genes are less conserved between species of Tolypocladium and are the product of complex patterns of lineage sorting and module duplication. In contrast, these genes are more conserved within the genus Trichoderma and consistent with diversification through speciation. Conclusions Peptaibiotic NRPS genes are restricted to mycoparasitic lineages of Hypocreales, based on current sampling. Phylogenomics and comparative genomics can provide insights into the evolution of secondary metabolite genes, their distribution across a broader range of taxa, and their possible function related to host specificity.Keywords: Lineage sorting, Mycoparasites, Hypocreales, Secondary metabolis

Cyclosporine Biosynthesis in Tolypocladium inflatum Benefits Fungal Adaptation to the Environment

The cycloundecapeptide cyclosporin A (CsA) was first isolated from the insect-pathogenic fungus Tolypocladium inflatum for its antifungal activity and later developed as an immunosuppressant drug. However, the full biosynthetic mechanism of CsA remains unknown and has puzzled researchers for decades. In this study, the biosynthetic gene cluster is suggested to include 12 genes encoding enzymes, including the nonribosomal peptide synthetase (NRPS) (SimA) responsible for assembling the 11 amino acid substrates of cyclosporine and a polyketide synthase (PKS) (SimG) to mediate the production of the unusual amino acid (4R)-4-[(E)-2butenyl]-4-methyl-L-threonine (Bmt). Individual deletion of 10 genes, isolation of intermediates, and substrate feeding experiments show that Bmt is biosynthesized by three enzymes, including SimG, SimI, and SimJ. The substrate D-alanine is catalyzed from L-alanine by alanine racemase SimB. Gene cluster transcription is regulated by a putative basic leucine zipper (bZIP)-type protein encoded by the cluster gene SimL. We also found that the cluster cyclophilin (SimC) and transporter (SimD) genes contribute to the tolerance of CsA in the CsA-producing fungus. We also found that cyclosporine production could enable the fungus to outcompete other fungi during cocultivation tests. Deletion of the CsA biosynthetic genes also impaired fungal virulence against insect hosts. Taking all the data together, in addition to proposing a biosynthetic pathway of cyclosporines, the results of this study suggest that CsA produced by this fungus might play important ecological roles in fungal environment interactions. IMPORTANCE The cyclopeptide cyclosporin A was first isolated from the filamentous fungus Tolypocladium inflatum showing antifungal activity and was later developed as an immunosuppressant drug. We report the biosynthetic mechanism of cyclosporines that are mediated by a cluster of genes encoding NRPS and PKS controlled by a bZIP-type transcriptional regulator. The two unusual amino acids Bmt and D-Ala are produced by the PKS pathway and alanine racemase, respectively. The cyclophilin and transporter genes jointly contribute to fungal self-protection against cyclosporines. Cyclosporine confers on T. inflatum the abilities to outcompete other fungi in competitive interactions and to facilitate fungal infection of insect hosts, which therefore benefits fungal adaptations to different environments

Framing national REDD+ benefits, monitoring, governance and finance: A comparative analysis of seven countries

This article analyzes how and with what possible consequences REDD+ is framed in the national policy arena in Cameroon, Indonesia, Nepal, Papua New Guinea, Peru, Tanzania, and Vietnam. It analyzes the most prominent views and storylines around key REDD+ design features among policy actors and in policy documents. We focus on storylines related to four questions, namely: (1) What should REDD+ achieve: carbon or also non-carbon objectives? (2) Who should monitor REDD+ outcomes: only technical experts or also local communities? (3) At what level should REDD+ be governed: at national or sub-national level? and (4) How should REDD+ be financed: through market- or fund-based sources? The vast majority of policy actors and policy documents frame REDD+ as a mechanism that should also realize non-carbon benefits, yet non-carbon monitoring receives very little attention. In all but one country, policy documents contain plans to involve local communities in the design and/or execution of measuring, reporting and verifying REDD+ outcomes. With regard to the level at which REDD+ should be governed, while most policy documents contain elements of a nested approach to accounting, almost all countries envision a long-term transition to national accounting and benefit distribution. We found strikingly little discussion among policy actors and in policy documents of how to finance REDD+ and acquire results-based payments. In the conclusion we reflect on possible consequences of the prominence of REDD+ storylines in the seven countries, and argue that carbonization and centralization of forest governance are possible outcomes given the limited attention to non-carbon monitoring and the envisioned centralized approaches to REDD+