Amy Jo Powell

Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris

Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanisms in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.

Altered patterns of gene duplication and differential gene gain and loss in fungal pathogens

BackgroundDuplication, followed by fixation or random loss of novel genes, contributes to genome evolution. Particular outcomes of duplication events are possibly associated with pathogenic life histories in fungi. To date, differential gene gain and loss have not been studied at genomic scales in fungal pathogens, despite this phenomenons known importance in virulence in bacteria and viruses.ResultsTo determine if patterns of gene duplication differed between pathogens and non-pathogens, we identified gene families across nine euascomycete and two basidiomycete species. Gene family size distributions were fit to power laws to compare gene duplication trends in pathogens versus non-pathogens. Fungal phytopathogens showed globally altered patterns of gene duplication, as indicated by differences in gene family size distribution. We also identified sixteen examples of gene family expansion and five instances of gene family contraction in pathogenic lineages. Expanded gene families included those predicted to be important in melanin biosynthesis, host cell wall degradation and transport functions. Contracted families included those encoding genes involved in toxin production, genes with oxidoreductase activity, as well as subunits of the vacuolar ATPase complex. Surveys of the functional distribution of gene duplicates indicated that pathogens show enrichment for gene duplicates associated with receptor and hydrolase activities, while euascomycete pathogens appeared to have not only these differences, but also significantly more duplicates associated with regulatory and carbohydrate binding functions.ConclusionDifferences in the overall levels of gene duplication in phytopathogenic species versus non-pathogenic relatives implicate gene inventory flux as an important virulence-associated process in fungi. We hypothesize that the observed patterns of gene duplicate enrichment, gene family expansion and contraction reflect adaptation within pathogenic life histories. These adaptations were likely shaped by ancient, as well as contemporary, intimate associations with monocot hosts.

Thermophilic fungi in an aridland ecosystem

We report a comprehensive multi-year study of thermophilic fungi at the Sevilleta National Wildlife Refuge in central New Mexico. Recovery of thermophilic fungi from soils showed seasonal fluctuations, with greater abundance correlating with spring and summer precipitation peaks. In addition to grassland soils, we obtained and characterized isolates from grassland and riparian litter, herbivore dung and biological soil crusts. All strains belonged to either the Eurotiales or Sordariales (Chaetomiaceae). No particular substrate or microhabitat associations were detected. Molecular typing of strains revealed substantial phylogenetic diversity, eight ad hoc phylogroups across the two orders were identified and genetic diversity was present within each phylogroup. Growth tests over a range of temperatures showed substantial variation in maximum growth rates among strains and across phylogroups but consistency within phylogroups. Results demonstrated that 45–50 C represents the optimal temperature for growth of most isolates, with a dramatic decline at 60 C. Most strains grew at 60 C, albeit slowly, whereas none grew at 65 C, providing empirical confirmation that 60 C presents an evolutionary threshold for fungal growth. Our results support the hypothesis that fungal thermophily is an adaptation to transient seasonal and diurnal high temperatures, rather than simply an adaptation to specialized high-temperature environments. We note that the diversity observed among strains and the frequently confused taxonomy within these groups highlight the need for comprehensive biosystematic revision of thermophilic taxa in both orders.

Mycothermus thermophilus gen. et comb. nov., a new home for the itinerant thermophile Scytalidium thermophilum (Torula thermophila)

Thermophilic fungi have received substantial attention in industry for their potential to produce thermostable enzymes and as production platforms tolerant of high temperature. Studies exploring the ecology and biosystematics of thermophilic fungi have lagged behind studies in applied biology. The species commonly known as Scytalidium thermophilum (Chaetomiaceae) is one of the most frequently encountered organisms in surveys of thermophilic fungi. There is evidence that it is ecologically and economically important, for example in the context of commercial mushroom growing. As described here, this species should not be placed in the genus Scytalidium or any other existing genus. We propose a new genus and combination, Mycothermus thermophilus.

Genetics of mating in members of the Chaetomiaceae as revealed by experimental and genomic characterization of reproduction in Myceliophthora heterothallica.

Members of the Chaetomiaceae are among the most studied fungi in industry and among the most reported in investigations of biomass degradation in both natural and laboratory settings. The family is recognized for production of carbohydrate-active enzymes and antibiotics. Thermophilic species are of special interest for their abilities to produce thermally stable enzymes and to be grown under conditions that are unsuitable for potential contaminant microorganisms. Such interests led to the recent acquisition of genome sequences from several members of the family, including thermophilic species, several of which are reported here for the first time. To date, however, thermophilic fungi in industry have served primarily as parts reservoirs and there has been no good genetic model for species in the family Chaetomiaceae or for thermophiles in general. We report here on the reproductive biology of the thermophile Myceliophthora heterothallica, which is heterothallic, unlike most described species in the family. We confirmed heterothallism genetically by following the segregation of mating type idiomorphs and other markers. We have expanded the number of known sexually-compatible individuals from the original isolates from Indiana and Germany to include several isolates from New Mexico. An interesting aspect of development in M. heterothallica is that ascocarp formation is optimal at approximately 30 °C, whereas vegetative growth is optimal at 45 °C. Genome sequences obtained from several strains, including isolates of each mating type, revealed mating-type regions whose genes are organized similarly to those of other members of the Sordariales, except for the presence of a truncated version of the mat A-1 (MAT1-1-1) gene in mating-type a (MAT1-2) strains. In M. heterothallica and other Chaetomiaceae, mating-type A (MAT1-1) strains have the full-length version of mat A-1 that is typical of mating-type A strains of diverse Ascomycota, whereas a strains have only the truncated version. This truncated mat A-1 has an intact open reading frame and a derived start codon that is not present in mat A-1 from A strains. The predicted protein contains a region that is conserved across diverse mat A-1 genes, but it lacks the major alpha1 domain, which characterizes proteins in this family and is known to be required for fertility in A strains from other Ascomycota. Finally, we have used genes from M. heterothallica to probe for mating genes in other homothallic and heterothallic members of the Chaetomiaceae. The majority of homothallic species examined have a typical mat A-1,2,3 (MAT1-1-1,2,3) region in addition to an unlinked mat a-1 (MAT1-2-1) gene, reflecting one type of homothallism commonly observed in diverse Ascomycota.

Lignin-modifying processes in the rhizosphere of arid land grasses

Genes associated with elevated oxidative enzyme activities in arid systems have not been well characterized. To link measured oxidative activities with specific enzymes, we assembled protein-coding reads from the rhizospheres (RHZ) of two arid land grasses. Targeted gene scans for open reading frames, encoding genes potentially involved in lignin modification, resulted in 127 distinct assembly products. The putative genes included those significantly similar to Class II secretory fungal peroxidases. These genes are expressed at sufficiently high levels for assembly, annotation and differentiation across experimental conditions, and they demonstrate the interplay of root systems, environment and plant microbiomes. The genes assembled also included copper-dependent lytic polysaccharide monooxygenases. We detail the enzymes in the host grass RHZs and present a preliminary taxonomic microhabitat characterization. Our findings provide support for biologically mediated Fenton chemistry in the root zones of desert grasses, and provide insight into arid land carbon flow. These results also demonstrate a hyperdiverse microbial community. Both ribosomal RNA and messenger RNA sequences were dominated by bacteria, followed by fungal sequence abundance. Among the notable fungal sequences were those from the members of the arbuscular mycorrhizal fungi (Glomeromycota), which though abundant in this study, we rarely observed in previous PCR-based surveys.

Omics Metadata Management Software (OMMS).

Next-generation sequencing projects have underappreciated information management tasks requiring detailed attention to specimen curation, nucleic acid sample preparation and sequence production methods required for downstream data processing, comparison, interpretation, sharing and reuse. The few existing metadata management tools for genome-based studies provide weak curatorial frameworks for experimentalists to store and manage idiosyncratic, project-specific information, typically offering no automation supporting unified naming and numbering conventions for sequencing production environments that routinely deal with hundreds, if not thousands of samples at a time. Moreover, existing tools are not readily interfaced with bioinformatics executables, (e.g., BLAST, Bowtie2, custom pipelines). Our application, the Omics Metadata Management Software (OMMS), answers both needs, empowering experimentalists to generate intuitive, consistent metadata, and perform analyses and information management tasks via an intuitive web-based interface. Several use cases with short-read sequence datasets are provided to validate installation and integrated function, and suggest possible methodological road maps for prospective users. Provided examples highlight possible OMMS workflows for metadata curation, multistep analyses, and results management and downloading. The OMMS can be implemented as a stand alone-package for individual laboratories, or can be configured for webbased deployment supporting geographically-dispersed projects. The OMMS was developed using an open-source software base, is flexible, extensible and easily installed and executed. The OMMS can be obtained at http://omms.sandia.gov. Availability The OMMS can be obtained at http://omms.sandia.gov

GT-Miner: a graph-theoretic data miner, viewer, and model processor.

Inexpensive computational power combined with high-throughput experimental platforms has created a wealth of biological information requiring analytical tools and techniques for interpretation. Graph-theoretic concepts and tools have provided an important foundation for information visualization, integration, and analysis of datasets, but they have often been relegated to background analysis tasks. GT-Miner is designed for visual data analysis and mining operations, interacts with other software, including databases, and works with diverse data types. It facilitates a discovery-oriented approach to data mining wherein exploration of alterations of the data and variations of the visualization is encouraged. The user is presented with a basic iterative process, consisting of loading, visualizing, transforming, and then storing the resultant information. Complex analyses are built-up through repeated iterations and user interactions. The iterative process is optimized by automatic layout following transformations and by maintaining a current selection set of interest for elements modified by the transformations. Multiple visualizations are supported including hierarchical, spring, and force-directed self-organizing layouts. Graphs can be transformed with an extensible set of algorithms or manually with an integral visual editor. GT-Miner is intended to allow easier access to visual data mining for the non-expert. Availability The GT-Miner program and supplemental materials, including example uses and a user guide, are freely available from http://www.cifr.ncsu.edu/bioinformatics/downloads/

Understanding and regulation of microbial lignolysis for renewable platform chemicals

Lignin is often overlooked in the valorization of lignocellulosic biomass, but lignin-based materials and chemicals represent potential value-added products for biorefineries that could significantly improve the economics of a biorefinery. Fluctuating crude oil prices and changing fuel specifications are some of the driving factors to develop new technologies that could be used to convert polymeric lignin into low molecular weight lignin and or monomeric aromatic feedstocks to assist in the displacement of the current products associated with the conversion of a whole barrel of oil. Our project of understanding microbial lignolysis for renewable platform chemicals aimed to understand microbial and enzymatic lignolysis processes to break down lignin for conversion into commercially viable drop-in fuels. We developed novel lignin analytics to interrogate enzymatic and microbial lignolysis of native polymeric lignin and established a detailed understanding of lignolysis as a function of fungal enzyme, microbes and endophytes. Bioinformatics pipeline was developed for metatranscryptomic analysis of aridland ecosystem for investigating the potential discovery of new lignolysis gene and gene products.