Search

ABSTRACTThe entomopathogenic fungus Conoideocrella luteorostratahas recently been implicated in natural epizootics among exotic elongate hemlock scale (EHS) insects in Christmas tree farms in the eastern United States. Since 1913, C. luteorostratahas been reported from various plant feeding Hemiptera in the southeastern United States, but comprehensive morphological and phylogenetic studies of U.S. populations are lacking. The recovery of multiple strains of C. luteorostratafrom mycosed EHS in North Carolina provided an opportunity to conduct pathogenicity assays and morphological and phylogenetic studies to investigate genus- and species-level boundaries among the Clavicipitaceae. Pathogenicity assays confirmed that C. luteorostratacauses mortality of EHS crawlers, an essential first step in developing this fungus as a biocontrol. Morphological studies revealed that conidia aligned with previous measurements of the Paecilomyces-like asexual state of C. luteorostrata, with conidiophore morphology consistent with historical observations. Additionally, a Hirsutella-like synanamorph was observed in select C. luteorostratastrains. In both a four-locus, 54-taxon Clavicipitaceae-wide phylogenetic analysis including D1–D2 domains of the nuclear 28S rRNA region (28S), elongation factor 1 alpha (EF1-α), DNA-directed RNA polymerase II subunit 1 (RPB1), and DNA-directed RNA polymerase II subunit 2 (RPB2) and a two-locus, 38-taxon (28S and EF1-α) phylogenetic analysis, all three Conoideocrellaspecies were resolved as strongly supported monophyletic lineages across all loci and both methods (maximum likelihood and Bayesian inference) of phylogenetic inference except for 28S for C. tenuis. Despite the strong support for individual Conoideocrellaspecies, none of the analyses supported the monophyly of Conoideocrellawith the inclusion of Dussiella. Due to the paucity of RPB1and RPB2sequence data, EF1-αprovided superior delimitation of intraspecies groupings for Conoideocrellaand should be used in future studies. Further development of C. luteorostrataas a biocontrol against EHS will require additional surveys across diverse Hemiptera and expanded pathogenicity testing to clarify host range and efficacy of this fungus.

ABSTRACTSpecies of the phylum Blastocladiomycota, early-diverging zoosporic (flagellated) lineages of fungi, are vastly understudied. This phylum includes the genus Coelomomyces, which consists of more than 80 fungal species that are obligate parasites of arthropods. Known Coelomomycesspecies lack a complete asexual life cycle, instead surviving through an obligate heterecious alternation of generations life cycle. Despite their global distribution and interesting life cycle, little is known about the genomics of any Coelomomycesspecies. To address this, we generated three draft-level genomes and annotations for C. lativittatusrepresenting its haploid meiospore, orange gamete, and amber gamete life stages. These draft genome assemblies ranged in size from 5002 to 5799 contigs, with a total length of 19.8–22.8 Mb and a mean of 7416 protein-coding genes. We then demonstrated the utility of these genomes by combining the draft annotations as a reference for analysis of C. lativittatustranscriptomes. We analyzed transcriptomes from across host-associated life stages, including infected larvae and excised mature sporangia from the mosquito Anopheles quadrimaculatus. We identified differentially expressed genes and enriched GO terms both across and within life stages and used these to make hypotheses about C. lativittatusbiology. Generally, we found the C. lativittatustranscriptome to be a complex and dynamic expression landscape; GO terms related to metabolism and transport processes were enriched during infection and terms related to dispersal were enriched during sporulation. We further identified five high mobility group (HMG)-box genes in C. lativittatus, three belonging to clades with mating type (MAT) loci from other fungi, as well as four ortholog expansions in C. lativittatuscompared with other fungi. The C. lativittatusgenomes and transcriptomes reported here are a valuable resource and may be leveraged toward furthering understanding of the biology of these and other early-diverging fungal lineages.

ABSTRACTIn an era of rapid climate change and expansion of desertification, the extremely harsh conditions of drylands are a true challenge for microbial life. Under drought conditions, where most life forms cannot survive, rocks represent the main refuge for life. Indeed, the endolithic habitat provides thermal buffering, physical stability, and protection against incident ultraviolet (UV) radiation and solar radiation and, to some extent, ensures water retention to microorganisms. The study of these highly specialized extreme-tolerant and extremophiles may provide tools for understanding microbial interactions and processes that allow them to keep their metabolic machinery active under conditions of dryness and oligotrophy that are typically incompatible with active life, up to the dry limits for life. Despite lithobiontic communities being studied all over the world, a comprehensive understanding of their ecology, evolution, and adaptation is still nascent. Herein, we survey the fungal component of these microbial ecosystems. We first provide an overview of the main defined groups (i.e., lichen-forming fungi, black fungi, and yeasts) of the most known and studied Antarctic endolithic communities that are almost the only life forms ensuring ecosystem functionality in the ice-free areas of the continent. For each group, we discuss their main traits and their diversity. Then, we focus on the fungal taxonomy and ecology of other worldwide endolithic communities. Finally, we highlight the utmost importance of a global rock survey in order to have a comprehensive view of the diversity, distribution, and functionality of these fungi in drylands, to obtain tools in desert area management, and as early alarm systems to climate change.

ABSTRACTThe fungal genus Massospora(Zoopagomycota: Entomophthorales) includes more than a dozen obligate, sexually transmissible pathogenic species that infect cicadas (Hemiptera) worldwide. At least two species are known to produce psychoactive compounds during infection, which has garnered considerable interest for this enigmatic genus. As with many Entomophthorales, the evolutionary relationships and host associations of Massosporaspp. are not well understood. The acquisition of M. diceroproctaefrom Arizona, M. tettigatisfrom Chile, and M. platypediaefrom California and Colorado provided an opportunity to conduct molecular phylogenetic analyses and morphological studies to investigate whether these fungi represent a monophyletic group and delimit species boundaries. In a three-locus phylogenetic analysis including the D1–D2 domains of the nuclear 28S rRNA gene (28S), elongation factor 1 alpha-like (EFL), and beta-tubulin (BTUB), Massosporawas resolved in a strongly supported monophyletic group containing four well-supported genealogically exclusive lineages, based on two of three methods of phylogenetic inference. There was incongruence among the single-gene trees: two methods of phylogenetic inference recovered trees with either the same topology as the three-gene concatenated tree (EFL) or a basal polytomy (28S, BTUB). Massospora levisporaand M. platypediaeisolates formed a single lineage in all analyses and are synonymized here as M. levispora. Massospora diceroproctaewas sister to M. cicadinain all three single-gene trees and on an extremely long branch relative to the other Massospora, and even the outgroup taxa, which may reflect an accelerated rate of molecular evolution and/or incomplete taxon sampling. The results of the morphological study presented here indicate that spore measurements may not be phylogenetically or diagnostically informative. Despite recent advances in understanding the ecology of Massospora, much about its host range and diversity remains unexplored. The emerging phylogenetic framework can provide a foundation for exploring coevolutionary relationships with cicada hosts and the evolution of behavior-altering compounds.

During the construction and assembly of the Mars 2020 mission components at two different NASA cleanrooms, several fungal strains were isolated. Based on their colony morphology, two strains that showed yeast-like appearance were further characterized for their phylogenetic position. The species-level classification of these two novel strains, using traditional colony and cell morphology methods combined with the phylogenetic reconstructions using multi-locus sequence analysis (MLSA) based on several gene loci (ITS, LSU, SSU, RPB1, RPB2, CYTBand TEF1), and whole genome sequencing (WGS) was carried out. This polyphasic taxonomic approach supported the conclusion that the two basidiomycetous yeasts belong to hitherto undescribed species. The strain FJI-L2-BK-P3T, isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility, was placed in the Naganishia albidaclade(Filobasidiales, Tremellomycetes),but is genetically and physiologically different from other members of the clade. Another yeast strain FKI-L6-BK-PAB1T, isolated from the Kennedy Space Center Payload Hazardous and Servicing Facility, was placed in the genus Cystobasidium(Cystobasidiales, Cystobasidiomycetes) and is distantly related to C. benthicum.Here we propose two novel species with the type strains, Naganishia kalamiisp. nov. (FJI-L2-BK-P3T= NRRL 64466 = DSM 115730) and Cystobasidium onofriisp. nov. (FKI-L6-BK-PAB1T= NRRL 64426 = DSM 114625). The phylogenetic analyses revealed that single gene phylogenies (ITS or LSU) were not conclusive, and MLSA and WGS-based phylogenies were more advantageous for species discrimination in the two genera. The genomic analysis predicted proteins associated with dehydration and desiccation stress-response and the presence of genes that are directly related to osmotolerance and psychrotolerance in both novel yeasts described. Cells of these two newly-described yeasts were exposed to UV-C radiation and compared with N. onofrii,an extremophilic UV-C resistant cold-adapted Alpine yeast. Both novel species were UV resistant, emphasizing the need for collecting and characterizing extremotolerant microbes, including yeasts, to improve microbial reduction techniques used in NASA planetary protection programs.

Proteomic analysis of Ampulex compressavenom reveals a multifaceted attack on the host central nervous system. Rather than inducing paralysis or cytotoxicity, the venom appears to modify endogenous signaling, rendering the host lethargic and compliant. Some peptides and proteins in the venom are in precursor form, only to be processed into fully active form once injected into the pH-neutral host brain. This analysis deepens mechanistic understanding of venom action and points to signaling systems previously unrecognized in regulation of locomotion.

N6-methyldeoxyadenine (6mA) is a noncanonical DNA base modification present at low levels in plant and animal genomes, but its prevalence and association with genome function in other eukaryotic lineages remains poorly understood. Here we report that abundant 6mA is associated with transcriptionally active genes in early-diverging fungal lineages. Using single-molecule long-read sequencing of 16 diverse fungal genomes, we observed that up to 2.8% of all adenines were methylated in early-diverging fungi, far exceeding levels observed in other eukaryotes and more derived fungi. 6mA occurred symmetrically at ApT dinucleotides and was concentrated in dense methylated adenine clusters surrounding the transcriptional start sites of expressed genes; its distribution was inversely correlated with that of 5-methylcytosine. Our results show a striking contrast in the genomic distributions of 6mA and 5-methylcytosine and reinforce a distinct role for 6mA as a gene-expression-associated epigenomic mark in eukaryotes.

ABSTRACTIllumina amplicon sequencing of soil in a temperate pine forest in the southeastern United States detected an abundant, nitrogen (N)-responsive fungal genotype of unknown phylogenetic affiliation. Two isolates with ribosomal sequences consistent with that genotype were subsequently obtained. Examination of records in GenBank revealed that a genetically similar fungus had been isolated previously as an endophyte of moss in a pine forest in the southwestern United States. The three isolates were characterized using morphological, genomic, and multilocus molecular data (18S, internal transcribed spacer [ITS], and 28S rRNA sequences). Phylogenetic and maximum likelihood phylogenomic reconstructions revealed that the taxon represents a novel lineage in Mucoromycotina, only preceded by Calcarisporiella, the earliest diverging lineage in the subphylum. Sequences for the novel taxon are frequently detected in environmental sequencing studies, and it is currently part of UNITE’s dynamic list of most wanted fungi. The fungus is dimorphic, grows best at room temperature, and is associated with a wide variety of bacteria. Here, a new monotypic genus, Bifiguratus, is proposed, typified by Bifiguratus adelaidae.

Zygomycete fungi were classified as a single phylum, Zygomycota, based on sexual reproduction by zygospores, frequent asexual reproduction by sporangia, absence of multicellular sporocarps, and production of coenocytic hyphae, all with some exceptions. Molecular phylogenies based on one or a few genes did not support the monophyly of the phylum, however, and the phylum was subsequently abandoned. Here we present phylogenetic analyses of a genome-scale data set for 46 taxa, including 25 zygomycetes and 192 proteins, and we demonstrate that zygomycetes comprise two major clades that form a paraphyletic grade. A formal phylogenetic classification is proposed herein and includes two phyla, six subphyla, four classes and 16 orders. On the basis of these results, the phyla Mucoromycota and Zoopagomycota are circumscribed. Zoopagomycota comprises Entomophtoromycotina, Kickxellomycotina and Zoopagomycotina; it constitutes the earliest diverging lineage of zygomycetes and contains species that are primarily parasites and pathogens of small animals (e.g. amoeba, insects, etc.) and other fungi, i.e. mycoparasites. Mucoromycota comprises Glomeromycotina, Mortierellomycotina, and Mucoromycotina and is sister to Dikarya. It is the more derived clade of zygomycetes and mainly consists of mycorrhizal fungi, root endophytes, and decomposers of plant material. Evolution of trophic modes, morphology, and analysis of genome-scale data are discussed.

ABSTRACTCryptococcus, a major cause of disseminated infections in immunocompromised patients, kills over 600,000 people per year worldwide. Genes involved in the virulence of the meningitis-causing fungus are being characterized at an increasing rate, and to date, at least 648 Cryptococcusgene names have been published. However, these data are scattered throughout the literature and are challenging to find. Furthermore, conflicts in locus identification exist, so that named genes have been subsequently published under new names or names associated with one locus have been used for another locus. To avoid these conflicts and to provide a central source of Cryptococcusgene information, we have collected all published Cryptococcusgene names from the scientific literature and associated them with standard Cryptococcuslocus identifiers and have incorporated them into FungiDB (www.fungidb.org). FungiDB is a panfungal genome database that collects gene information and functional data and provides search tools for 61 species of fungi and oomycetes. We applied these published names to a manually curated ortholog set of all Cryptococcusspecies currently in FungiDB, including Cryptococcus neoformansvar. neoformansstrains JEC21 and B-3501A, C. neoformansvar. grubiistrain H99, and Cryptococcus gattiistrains R265 and WM276, and have written brief descriptions of their functions. We also compiled a protocol for gene naming that summarizes guidelines proposed by members of the Cryptococcusresearch community. The centralization of genomic and literature-based information for Cryptococcusat FungiDB will help researchers communicate about genes of interest, such as those related to virulence, and will further facilitate research on the pathogen.

Genome-enabled mycology is a rapidly expanding field that is characterized by the pervasive use of genome-scale data and associated computational tools in all aspects of fungal biology. Genome-enabled mycology is integrative and often requires teams of researchers with diverse skills in organismal mycology, bioinformatics and molecular biology. This issue of Mycologia presents the first complete fungal genomes in the history of the journal, reflecting the ongoing transformation of mycology into a genome-enabled science. Here, we consider the prospects for genome-enabled mycology and the technical and social challenges that will need to be overcome to grow the database of complete fungal genomes and enable all fungal biologists to make use of the new data.

Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.

Genome sequencing of filamentous fungi has demonstrated that most secondary metabolite biosynthetic gene clusters (BGCs) are silent under standard laboratory conditions. In this work, we have established an in vitro CRISPR-Cas9 system in Aspergillus wentii. To activate otherwise silent BGCs, we deleted the negative transcriptional regulator mcrA. Deletion of mcrA(mcrAΔ) resulted in differential production of 17 SMs in total when the strain was cultivated on potato dextrose media (PDA). Nine out of fifteen of these SMs were fully characterized, including emodin (1), physcion (2), sulochrin (3), physcion bianthrone (4), 14-O-demethylsulochrin (5), (trans/cis)-emodin bianthrone (6and 7), and (trans/cis)-emodin physcion bianthrone (8and 9). These compounds were all found to be produced by the same polyketide synthase (PKS) BGC. We then performed a secondary knockout targeting this PKS cluster in the mcrAΔ background. The metabolite profile of the dual-knockout strain revealed new metabolites that were not previously detected in the mcrAΔ parent strain. Two additional SMs were purified from the dual-knockout strain and were characterized as aspergillus acid B (16) and a structurally related but previously unidentified compound (17). For the first time, this work presents a facile genetic system capable of targeted gene editing in A. wentii.This work also illustrates the utility of performing a dual knockout to eliminate major metabolic products, enabling additional SM discovery.

ABSTRACTSexual reproduction of fungi is governed by the mating type (MAT) locus, a specialized region of the genome encoding key transcriptional regulators that direct regulatory networks to specify cell identity and fate. Knowledge of MATlocus structure and evolution has been considerably advanced in recent years as a result of genomic analyses that enable the definition of MATlocus sequences in many species as well as provide an understanding of the evolutionary plasticity of this unique region of the genome. Here, we extend this analysis to define the mating type locus of three dimorphic primary human fungal pathogens, Histoplasma capsulatum, Coccidioides immitis, and Coccidioides posadasii, using genomic analysis, direct sequencing, and bioinformatics. These studies provide evidence that all three species possess heterothallic bipolar mating type systems, with isolates encoding either a high-mobility-group (HMG) domain or an a-box transcriptional regulator. These genes are intact in all loci examined and have not been subject to loss or decay, providing evidence that the loss of fertility upon passage in H. capsulatumis not attributable to mutations at the MATlocus. These findings also suggest that an extant sexual cycle remains to be defined in both Coccidioidesspecies, in accord with population genetic evidence. Based on these MATsequences, a facile PCR test was developed that allows the mating type to be rapidly ascertained. Finally, these studies highlight the evolutionary forces shaping the MATlocus, revealing examples in which flanking genes have been inverted or subsumed and incorporated into an expanding MATlocus, allowing us to propose an expanded model for the evolution of the MATlocus in the phylum Ascomycota.

Sexual reproduction of fungi is governed by the mating type (MAT) locus, a specialized region of the genome encoding key transcriptional regulators that direct regulatory networks to specify cell identity and fate. Knowledge of MAT locus structure and evolution has been considerably advanced in recent years as a result of genomic analyses that enable the definition of MAT locus sequences in many species as well as provide an understanding of the evolutionary plasticity of this unique region of the genome. Here, we extend this analysis to define the mating type locus of three dimorphic primary human fungal pathogens, Histoplasma capsulatum, Coccidioides immitis, and Coccidioides posadasii, using genomic analysis, direct sequencing, and bioinformatics. These studies provide evidence that all three species possess heterothallic bipolar mating type systems, with isolates encoding either a high-mobility-group (HMG) domain or an -box transcriptional regulator. These genes are intact in all loci examined and have not been subject to loss or decay, providing evidence that the loss of fertility upon passage in H. capsulatum is not attributable to mutations at the MAT locus. These findings also suggest that an extant sexual cycle remains to be defined in both Coccidioides species, in accord with population genetic evidence. Based on these MAT sequences, a facile PCR test was developed that allows the mating type to be rapidly ascertained. Finally, these studies highlight the evolutionary forces shaping the MAT locus, revealing examples in which flanking genes have been inverted or subsumed and incorporated into an expanding MAT locus, allowing us to propose an expanded model for the evolution of the MAT locus in the phylum ASCOMYCOTA:

In the present study, we sought to elucidate the contribution of the Cryptococcus neoformans catalase gene family to antioxidant defense. We employed bioinformatics techniques to identify four members of the C. neoformans catalase gene family and created mutants lacking single or multiple catalase genes. Based on a phylogenetic analysis, CAT1 and CAT3 encode putative spore-specific catalases, CAT2 encodes a putative peroxisomal catalase, and CAT4 encodes a putative cytosolic catalase. Only Cat1 exhibited detectable biochemical activity in vitro, and Cat1 activity was constitutive in the yeast form of this organism. Although they were predicted to be important in spores, neither CAT1 nor CAT3 was essential for mating or spore viability. Consistent with previous studies of Saccharomyces cerevisiae, the single (cat1, cat2, cat3, and cat4) and quadruple (cat1 cat2 cat3 cat4) catalase mutant strains exhibited no oxidative-stress phenotypes under conditions in which either exogenous or endogenous levels of reactive oxygen species were elevated. In addition, there were no significant differences in the mean times to mortality between groups of mice infected with C. neoformans catalase mutant strains (the cat1 and cat1 cat2 cat3 cat4 mutants) and those infected with wild-type strain H99. We conclude from the results of this study that C. neoformans possesses a robust antioxidant system, composed of functionally overlapping and compensatory components that provide protection against endogenous and exogenous oxidative stresses.

ABSTRACTIn the present study, we sought to elucidate the contribution of the Cryptococcus neoformanscatalase gene family to antioxidant defense. We employed bioinformatics techniques to identify four members of the C. neoformanscatalase gene family and created mutants lacking single or multiple catalase genes. Based on a phylogenetic analysis, CAT1and CAT3encode putative spore-specific catalases, CAT2encodes a putative peroxisomal catalase, and CAT4encodes a putative cytosolic catalase. Only Cat1 exhibited detectable biochemical activity in vitro, and Cat1 activity was constitutive in the yeast form of this organism. Although they were predicted to be important in spores, neither CAT1nor CAT3was essential for mating or spore viability. Consistent with previous studies of Saccharomyces cerevisiae, the single (cat1, cat2, cat3, and cat4) and quadruple (cat1 cat2 cat3cat4) catalase mutant strains exhibited no oxidative-stress phenotypes under conditions in which either exogenous or endogenous levels of reactive oxygen species were elevated. In addition, there were no significant differences in the mean times to mortality between groups of mice infected with C. neoformanscatalase mutant strains (the cat1and cat1 cat2cat3 cat4mutants) and those infected with wild-type strain H99. We conclude from the results of this study that C. neoformanspossesses a robust antioxidant system, composed of functionally overlapping and compensatory components that provide protection against endogenous and exogenous oxidative stresses.

ABSTRACTIntrons are a defining feature of eukaryotic genomes, though the mechanism of intron gain or loss is not well understood. Reverse transcription of mRNA followed by homologous recombination with the genome has been posited as a mechanism of intron loss, though little direct evidence of recent loss events has been described to support this model. We find supporting evidence for an mRNA-mediated mechanism of loss through comparative genome analyses that revealed a recent loss of 10 adjacent introns in a 22-exon gene in the human-pathogenic fungus Cryptococcus neoformans. We surveyed the gene structures of the entire genomes of Cryptococcus gattii, which diverged from the C. neoformanslineage 37 million years ago (Mya), and C. neoformansvar. grubiiand var. neoformans, which diverged 18 Mya. Our comparison revealed greater than 99.9% intron conservation, with evidence from 20 genes showing evidence of intron loss, but no convincing evidence of intron gain. Our findings confirm that Cryptococcusintrons have been quite stable over recent evolutionary time, with occasional mRNA-mediated intron loss events.

Introns are a defining feature of eukaryotic genomes, though the mechanism of intron gain or loss is not well understood. Reverse transcription of mRNA followed by homologous recombination with the genome has been posited as a mechanism of intron loss, though little direct evidence of recent loss events has been described to support this model. We find supporting evidence for an mRNA-mediated mechanism of loss through comparative genome analyses that revealed a recent loss of 10 adjacent introns in a 22-exon gene in the human-pathogenic fungus Cryptococcus neoformans. We surveyed the gene structures of the entire genomes of Cryptococcus gattii, which diverged from the C. neoformans lineage 37 million years ago (Mya), and C. neoformans var. grubii and var. neoformans, which diverged 18 Mya. Our comparison revealed greater than 99.9% intron conservation, with evidence from 20 genes showing evidence of intron loss, but no convincing evidence of intron gain. Our findings confirm that Cryptococcus introns have been quite stable over recent evolutionary time, with occasional mRNA-mediated intron loss events.

ABSTRACTThe ability to survive and proliferate at 37°C is an essential virulence attribute of pathogenic microorganisms. A partial-genome microarray was used to profile gene expression in the human-pathogenic fungus Cryptococcus neoformansduring growth at 37°C. Genes with orthologs involved in stress responses were induced during growth at 37°C, suggesting that a conserved transcriptional program is used by C. neoformansto alter gene expression during stressful conditions. A gene encoding the transcription factor homolog Mga2 was induced at 37°C and found to be important for high-temperature growth. Genes encoding fatty acid biosynthetic enzymes were identified as potential targets of Mga2, suggesting that membrane remodeling is an important component of adaptation to high growth temperatures. mga2?mutants were extremely sensitive to the ergosterol synthesis inhibitor fluconazole, indicating a coordination of the synthesis of membrane component precursors. Unexpectedly, genes involved in amino acid and pyrimidine biosynthesis were repressed at 37°C, but components of these pathways were found to be required for high-temperature growth. Our findings demonstrate the utility of even partial-genome microarrays for delineating regulatory cascades that contribute to microbial pathogenesis.

The Bioperl project is an international open-source collaboration of biologists, bioinformaticians, and computer scientists that has evolved over the past 7 yr into the most comprehensive library of Perl modules available for managing and manipulating life-science information. Bioperl provides an easy-to-use, stable, and consistent programming interface for bioinformatics application programmers. The Bioperl modules have been successfully and repeatedly used to reduce otherwise complex tasks to only a few lines of code. The Bioperl object model has been proven to be flexible enough to support enterprise-level applications such as EnsEMBL, while maintaining an easy learning curve for novice Perl programmers. Bioperl is capable of executing analyses and processing results from programs such as BLAST, ClustalW, or the EMBOSS suite. Interoperation with modules written in Python and Java is supported through the evolving BioCORBA bridge. Bioperl provides access to data stores such as GenBank and SwissProt via a flexible series of sequence input/output modules, and to the emerging common sequence data storage format of the Open Bioinformatics Database Access project. This study describes the overall architecture of the toolkit, the problem domains that it addresses, and gives specific examples of how the toolkit can be used to solve common life-sciences problems. We conclude with a discussion of how the open-source nature of the project has contributed to the development effort.

The Generic Model Organism System Database Project (GMOD) seeks to develop reusable software components for model organism system databases. In this paper we describe the Generic Genome Browser (GBrowse), a Web-based application for displaying genomic annotations and other features. For the end user, features of the browser include the ability to scroll and zoom through arbitrary regions of a genome, to enter a region of the genome by searching for a landmark or performing a full text search of all features, and the ability to enable and disable tracks and change their relative order and appearance. The user can upload private annotations to view them in the context of the public ones, and publish those annotations to the community. For the data provider, features of the browser software include reliance on readily available open source components, simple installation, flexible configuration, and easy integration with other components of a model organism system Web site. GBrowse is freely available under an open source license. The software, its documentation, and support are available at http://www.gmod.org.

Aspergillus fumigatusinfection causes a spectrum of clinical manifestations. For individuals with cystic fibrosis (CF), allergic bronchopulmonary aspergillosis (ABPA) is an established complication, but there is a growing appreciation for A. fumigatusairway persistence in CF disease progression.

Understanding microbial evolution within patients is critical for managing chronic infections and understanding host-pathogen interactions. Here, our analysis of multiple MRR1alleles in isolates from a single Clavispora(Candida) lusitaniaeinfection revealed the selection for both high and low Mrr1 activity.

The manipulation of microbial biofilms in industrial and clinical applications remains a difficult task. The problem is particularly acute with regard to filamentous fungal biofilms for which molecular mechanisms of biofilm formation, maintenance, and function are only just being elucidated.

The fungal kingdom includes at least 6 million eukaryotic species and is remarkable with respect to its profound impact on global health, biodiversity, ecology, agriculture, manufacturing, and biomedical research. Approximately 625 fungal species have been reported to infect vertebrates, 200 of which can be human associated, either as commensals and members of our microbiome or as pathogens that cause infectious diseases. These organisms pose a growing threat to human health with the global increase in the incidence of invasive fungal infections, prevalence of fungal allergy, and the evolution of fungal pathogens resistant to some or all current classes of antifungals.

ABSTRACTModern genomics has shed light on many entomopathogenic fungi and expanded our knowledge widely; however, little is known about the genomic features of the insect-commensal fungi. Harpellales are obligate commensals living in the digestive tracts of disease-bearing insects (black flies, midges, and mosquitoes). In this study, we produced and annotated whole-genome sequences of nine Harpellales taxa and conducted the first comparative analyses to infer the genomic diversity within the members of the Harpellales. The genomes of the insect gut fungi feature low (26% to 37%) GC content and large genome size variations (25 to 102 Mb). Further comparisons with insect-pathogenic fungi (from both Ascomycota and Zoopagomycota), as well as with free-living relatives (as negative controls), helped to identify a gene toolbox that is essential to the fungus-insect symbiosis. The results not only narrow the genomic scope of fungus-insect interactions from several thousands to eight core players but also distinguish host invasion strategies employed by insect pathogens and commensals. The genomic content suggests that insect commensal fungi rely mostly on adhesion protein anchors that target digestive system, while entomopathogenic fungi have higher numbers of transmembrane helices, signal peptides, and pathogen-host interaction (PHI) genes across the whole genome and enrich genes as well as functional domains to inactivate the host inflammation system and suppress the host defense. Phylogenomic analyses have revealed that genome sizes of Harpellales fungi vary among lineages with an integer-multiple pattern, which implies that ancient genome duplications may have occurred within the gut of insects.IMPORTANCEInsect guts harbor various microbes that are important for host digestion, immune response, and disease dispersal in certain cases. Bacteria, which are among the primary endosymbionts, have been studied extensively. However, fungi, which are also frequently encountered, are poorly known with respect to their biology within the insect guts. To understand the genomic features and related biology, we produced the whole-genome sequences of nine gut commensal fungi from disease-bearing insects (black flies, midges, and mosquitoes). The results show that insect gut fungi tend to have low GC content across their genomes. By comparing these commensals with entomopathogenic and free-living fungi that have available genome sequences, we found a universal core gene toolbox that is unique and thus potentially important for the insect-fungus symbiosis. This comparative work also uncovered different host invasion strategies employed by insect pathogens and commensals, as well as a model system to study ancient fungal genome duplication within the gut of insects.

ABSTRACTPneumocystisspecies are opportunistic mammalian pathogens that cause severe pneumonia in immunocompromised individuals. These fungi are highly host specific and uncultivable in vitro. Human Pneumocystisinfections present major challenges because of a limited therapeutic arsenal and the rise of drug resistance. To investigate the diversity and demographic history of natural populations of Pneumocystisinfecting humans, rats, and mice, we performed whole-genome and large-scale multilocus sequencing of infected tissues collected in various geographic locations. Here, we detected reduced levels of recombination and variations in historical demography, which shape the global population structures. We report estimates of evolutionary rates, levels of genetic diversity, and population sizes. Molecular clock estimates indicate that Pneumocystisspecies diverged before their hosts, while the asynchronous timing of population declines suggests host shifts. Our results have uncovered complex patterns of genetic variation influenced by multiple factors that shaped the adaptation of Pneumocystispopulations during their spread across mammals.IMPORTANCEUnderstanding how natural pathogen populations evolve and identifying the determinants of genetic variation are central issues in evolutionary biology. Pneumocystis, a fungal pathogen which infects mammals exclusively, provides opportunities to explore these issues. In humans, Pneumocystiscan cause a life-threatening pneumonia in immunosuppressed individuals. In analysis of different Pneumocystisspecies infecting humans, rats, and mice, we found that there are high infection rates and that natural populations maintain a high level of genetic variation despite low levels of recombination. We found no evidence of population structuring by geography. Our comparisons of the times of divergence of these species to their respective hosts suggest that Pneumocystismay have undergone recent host shifts. The results demonstrate that Pneumocystisstrains are widely disseminated geographically and provide a new understanding of the evolution of these pathogens.

ABSTRACTThe mold Aspergillus fumigatuscauses invasive infection in immunocompromised patients. Recently, galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetylgalactosamine (GalNAc), was identified as a virulence factor required for biofilm formation. The molecular mechanisms underlying GAG biosynthesis and GAG-mediated biofilm formation were unknown. We identified a cluster of five coregulated genes that were dysregulated in GAG-deficient mutants and whose gene products share functional similarity with proteins that mediate the synthesis of the bacterial biofilm exopolysaccharide poly-(β1-6)-N-acetyl-d-glucosamine (PNAG). Bioinformatic analyses suggested that the GAG cluster gene agd3encodes a protein containing a deacetylase domain. Because deacetylation of N-acetylglucosamine residues is critical for the function of PNAG, we investigated the role of GAG deacetylation in fungal biofilm formation. Agd3 was found to mediate deacetylation of GalNAc residues within GAG and render the polysaccharide polycationic. As with PNAG, deacetylation is required for the adherence of GAG to hyphae and for biofilm formation. Growth of the Δagd3mutant in the presence of culture supernatants of the GAG-deficient Δuge3mutant rescued the biofilm defect of the Δagd3mutant and restored the adhesive properties of GAG, suggesting that deacetylation is an extracellular process. The GAG biosynthetic gene cluster is present in the genomes of members of the Pezizomycotinasubphylum of the Ascomycotaincluding a number of plant-pathogenic fungi and a single basidiomycete species, Trichosporon asahii, likely a result of recent horizontal gene transfer. The current study demonstrates that the production of cationic, deacetylated exopolysaccharides is a strategy used by both fungi and bacteria for biofilm formation.IMPORTANCEThis study sheds light on the biosynthetic pathways governing the synthesis of galactosaminogalactan (GAG), which plays a key role in A. fumigatusvirulence and biofilm formation. We find that bacteria and fungi use similar strategies to synthesize adhesive biofilm exopolysaccharides. The presence of orthologs of the GAG biosynthetic gene clusters in multiple fungi suggests that this exopolysaccharide may also be important in the virulence of other fungal pathogens. Further, these studies establish a molecular mechanism of adhesion in which GAG interacts via charge-charge interactions to bind to both fungal hyphae and other substrates. Finally, the importance of deacetylation in the synthesis of functional GAG and the extracellular localization of this process suggest that inhibition of deacetylation may be an attractive target for the development of novel antifungal therapies.

ABSTRACTMalasseziacommensal yeasts are associated with a number of skin disorders, such as atopic eczema/dermatitis and dandruff, and they also can cause systemic infections. Here we describe the 7.67-Mbp genome of Malassezia sympodialis, a species associated with atopic eczema, and contrast its genome repertoire with that of Malassezia globosa, associated with dandruff, as well as those of other closely related fungi. Ninety percent of the predicted M. sympodialisprotein coding genes were experimentally verified by mass spectrometry at the protein level. We identified a relatively limited number of genes related to lipid biosynthesis, and both species lack the fatty acid synthase gene, in line with the known requirement of these yeasts to assimilate lipids from the host. Malasseziaspecies do not appear to have many cell wall-localized glycosylphosphatidylinositol (GPI) proteins and lack other cell wall proteins previously identified in other fungi. This is surprising given that in other fungi these proteins have been shown to mediate interactions (e.g., adhesion and biofilm formation) with the host. The genome revealed a complex evolutionary history for an allergen of unknown function, Mala s 7, shown to be encoded by a member of an amplified gene family of secreted proteins. Based on genetic and biochemical studies with the basidiomycete human fungal pathogen Cryptococcus neoformans, we characterized the allergen Mala s 6 as the cytoplasmic cyclophilin A. We further present evidence that M. sympodialismay have the capacity to undergo sexual reproduction and present a model for a pseudobipolar mating system that allows limited recombination between two linked MATloci.IMPORTANCEMalasseziacommensal yeasts are associated with a number of skin disorders. The previously published genome of M. globosaprovided some of the first insights into Malasseziabiology and its involvement in dandruff. Here, we present the genome of M. sympodialis, frequently isolated from patients with atopic eczema and healthy individuals. We combined comparative genomics with sequencing and functional characterization of specific genes in a population of clinical isolates and in closely related model systems. Our analyses provide insights into the evolution of allergens related to atopic eczema and the evolutionary trajectory of the machinery for sexual reproduction and meiosis. We hypothesize that M. sympodialismay undergo sexual reproduction, which has important implications for the understanding of the life cycle and virulence potential of this medically important yeast. Our findings provide a foundation for the development of genetic and genomic tools to elucidate host-microbe interactions that occur on the skin and to identify potential therapeutic targets.

ABSTRACTBatrachochytrium dendrobatidisis the causative agent of chytridiomycosis, which is considered one of the driving forces behind the worldwide decline in populations of amphibians. As a member of the phylum Chytridiomycota, B. dendrobatidishas diverged significantly to emerge as the only pathogen of adult vertebrates. Such shifts in lifestyle are generally accompanied by various degrees of genomic modifications, yet neither its mode of pathogenicity nor any factors associated with it have ever been identified. Presented here is the identification and characterization of a unique expansion of the carbohydrate-binding module family 18 (CBM18), specific to B. dendrobatidis. CBM (chitin-binding module) expansions have been likened to the evolution of pathogenicity in a variety of fungus species, making this expanded group a prime candidate for the identification of potential pathogenicity factors. Furthermore, the CBM18 expansions are confined to three categories of genes, each having been previously implicated in host-pathogen interactions. These correlations highlight this specific domain expansion as a potential key player in the mode of pathogenicity in this unique fungus. The expansion of CBM18 in B. dendrobatidisis exceptional in its size and diversity compared to other pathogenic species of fungi, making this genomic feature unique in an evolutionary context as well as in pathogenicity.IMPORTANCEAmphibian populations are declining worldwide at an unprecedented rate. Although various factors are thought to contribute to this phenomenon, chytridiomycosis has been identified as one of the leading causes. This deadly fungal disease is cause by Batrachochytrium dendrobatidis, a chytrid fungus species unique in its pathogenicity and, furthermore, its specificity to amphibians. Despite more than two decades of research, the biology of this fungus species and its deadly interaction with amphibians had been notoriously difficult to unravel. Due to the alarming rate of worldwide spread and associated decline in amphibian populations, it is imperative to incorporate novel genomic and genetic techniques into the study of this species. In this study, we present the first reported potential pathogenicity factors in B. dendrobatidis. In silicostudies such as this allow us to identify putative targets for more specific molecular analyses, furthering our hope for the control of this pathogen.

Genome Browsers are software that allow the user to view genome annotations in the context of a reference sequence, such as a chromosome, contig, scaffold, etc. The Generic Genome Browser (GBrowse) is an open‐source genome browser package developed as part of the Generic Model Database Project (see UNIT; Stein et al., 2002). The increasing number of sequenced genomes has led to a corresponding growth in the field of comparative genomics, which requires methods to view and compare multiple genomes. Using the same software framework as GBrowse, the Generic Synteny Browser (GBrowse_syn) allows the comparison of colinear regions of multiple genomes using the familiar GBrowse‐style Web page. Like GBrowse, GBrowse_syn can be configured to display any organism, and is currently the synteny browser used for model organisms such as C. elegans(WormBase; http://www.wormbase.org; see UNIT) and Arabidopsis (TAIR; http://www.arabidopsis.org; see UNIT). GBrowse_syn is part of the GBrowse software package and can be downloaded from the Web and run on any Unix‐like operating system, such as Linux, Solaris, or MacOS X. GBrowse_syn is still under active development. This unit will cover installation and configuration as part of the current stable version of GBrowse (v. 1.71). Curr. Protoc. Bioinform. 31:9.12.1‐9.12.25. © 2010 by John Wiley & Sons, Inc.