A global phylogenomic analysis of the shiitake genus Lentinula.

Lentinula is a broadly distributed group of fungi that contains the cultivated shiitake mushroom, L. edodes. We sequenced 24 genomes representing eight described species and several unnamed lineages of Lentinula from 15 countries on four continents. Lentinula comprises four major clades that arose in the Oligocene, three in the Americas and one in Asia-Australasia. To expand sampling of shiitake mushrooms, we assembled 60 genomes of L. edodes from China that were previously published as raw Illumina reads and added them to our dataset. Lentinula edodes sensu lato (s. lat.) contains three lineages that may warrant recognition as species, one including a single isolate from Nepal that is the sister group to the rest of L. edodes s. lat., a second with 20 cultivars and 12 wild isolates from China, Japan, Korea, and the Russian Far East, and a third with 28 wild isolates from China, Thailand, and Vietnam. Two additional lineages in China have arisen by hybridization among the second and third groups. Genes encoding cysteine sulfoxide lyase (lecsl) and γ-glutamyl transpeptidase (leggt), which are implicated in biosynthesis of the organosulfur flavor compound lenthionine, have diversified in Lentinula. Paralogs of both genes that are unique to Lentinula (lecsl 3 and leggt 5b) are coordinately up-regulated in fruiting bodies of L. edodes. The pangenome of L. edodes s. lat. contains 20,308 groups of orthologous genes, but only 6,438 orthogroups (32%) are shared among all strains, whereas 3,444 orthogroups (17%) are found only in wild populations, which should be targeted for conservation.

Polar bear evolution is marked by rapid changes in gene copy number in response to dietary shift.

Polar bear (Ursus maritimus) and brown bear (Ursus arctos) are recently diverged species that inhabit vastly differing habitats. Thus, analysis of the polar bear and brown bear genomes represents a unique opportunity to investigate the evolutionary mechanisms and genetic underpinnings of rapid ecological adaptation in mammals. Copy number (CN) differences in genomic regions between closely related species can underlie adaptive phenotypes and this form of genetic variation has not been explored in the context of polar bear evolution. Here, we analyzed the CN profiles of 17 polar bears, 9 brown bears, and 2 black bears (Ursus americanus). We identified an average of 318 genes per individual that showed evidence of CN variation (CNV). Nearly 200 genes displayed species-specific CN differences between polar bear and brown bear species. Principal component analysis of gene CN provides strong evidence that CNV evolved rapidly in the polar bear lineage and mainly resulted in CN loss. Olfactory receptors composed 47% of CN differentiated genes, with the majority of these genes being at lower CN in the polar bear. Additionally, we found significantly fewer copies of several genes involved in fatty acid metabolism as well as AMY1B, the salivary amylase-encoding gene in the polar bear. These results suggest that natural selection shaped patterns of CNV in response to the transition from an omnivorous to primarily carnivorous diet during polar bear evolution. Our analyses of CNV shed light on the genomic underpinnings of ecological adaptation during polar bear evolution.

Concerted copy number variation balances ribosomal DNA dosage in human and mouse genomes.

Tandemly repeated ribosomal DNA (rDNA) arrays are among the most evolutionary dynamic loci of eukaryotic genomes. The loci code for essential cellular components, yet exhibit extensive copy number (CN) variation within and between species. CN might be partly determined by the requirement of dosage balance between the 5S and 45S rDNA arrays. The arrays are nonhomologous, physically unlinked in mammals, and encode functionally interdependent RNA components of the ribosome. Here we show that the 5S and 45S rDNA arrays exhibit concerted CN variation (cCNV). Despite 5S and 45S rDNA elements residing on different chromosomes and lacking sequence similarity, cCNV between these loci is strong, evolutionarily conserved in humans and mice, and manifested across individual genotypes in natural populations and pedigrees. Finally, we observe that bisphenol A induces rapid and parallel modulation of 5S and 45S rDNA CN. Our observations reveal a novel mode of genome variation, indicate that natural selection contributed to the evolution and conservation of cCNV, and support the hypothesis that 5S CN is partly determined by the requirement of dosage balance with the 45S rDNA array. We suggest that human disease variation might be traced to disrupted rDNA dosage balance in the genome.

Immunomodulators targeting MARCO expression improve resistance to postinfluenza bacterial pneumonia.

Downregulation of the alveolar macrophage (AM) receptor with collagenous structure (MARCO) leads to susceptibility to postinfluenza bacterial pneumonia, a major cause of morbidity and mortality. We sought to determine whether immunomodulation of MARCO could improve host defense and resistance to secondary bacterial pneumonia. RNAseq analysis identified a striking increase in MARCO expression between days 9 and 11 after influenza infection and indicated important roles for Akt and Nrf2 in MARCO recovery. In vitro, primary human AM-like monocyte-derived macrophages (AM-MDMs) and THP-1 macrophages were treated with IFNγ to model influenza effects. Activators of Nrf2 (sulforaphane) or Akt (SC79) caused increased MARCO expression and a MARCO-dependent improvement in phagocytosis in IFNγ-treated cells and improved survival in mice with postinfluenza pneumococcal pneumonia. Transcription factor analysis also indicated a role for transcription factor E-box (TFEB) in MARCO recovery. Overexpression of TFEB in THP-1 cells led to marked increases in MARCO. The ability of Akt activation to increase MARCO expression in IFNγ-treated AM-MDMs was abrogated in TFEB-knockdown cells, indicating Akt increases MARCO expression through TFEB. Increasing MARCO expression by targeting Nrf2 signaling or the Akt-TFEB-MARCO pathway are promising strategies to improve bacterial clearance and survival in postinfluenza bacterial pneumonia.

Copy number variation contributes to cryptic genetic variation in outbreak lineages of Cryptococcus gattii from the North American Pacific Northwest.

BACKGROUND: Copy number variants (CNVs) are a class of structural variants (SVs) and are defined as fragments of DNA that are present at variable copy number in comparison with a reference genome. Recent advances in bioinformatics methodologies and sequencing technologies have enabled the high-resolution quantification of genome-wide CNVs. In pathogenic fungi SVs have been shown to alter gene expression, influence host specificity, and drive fungicide resistance, but little attention has focused specifically on CNVs. Using publicly available sequencing data, we identified 90 isolates across 212 Cryptococcus gattii genomes that belong to the VGII subgroups responsible for the recent deadly outbreaks in the North American Pacific Northwest. We generated CNV profiles for each sample to investigate the prevalence and function of CNV in C. gattii. RESULTS: We identified eight genetic clusters among publicly available Illumina whole genome sequence data from 212 C. gattii isolates through population structure analysis. Three clusters represent the VGIIa, VGIIb, and VGIIc subgroups from the North American Pacific Northwest. CNV was bioinformatically predicted and affected ~300-400 Kilobases (Kb) of the C. gattii VGII subgroup genomes. Sixty-seven loci, encompassing 58 genes, showed highly divergent patterns of copy number variation between VGII subgroups. Analysis of PFam domains within divergent CN variable genes revealed enrichment of protein domains associated with transport, cell wall organization and external encapsulating structure. CONCLUSIONS: CNVs may contribute to pathological and phenotypic differences observed between the C. gattii VGIIa, VGIIb, and VGIIc subpopulations. Genes overlapping with population differentiated CNVs were enriched for several virulence related functional terms. These results uncover novel candidate genes to examine the genetic and functional underpinnings of C. gattii pathogenicity.

A bioinformatics approach for integrated transcriptomic and proteomic comparative analyses of model and non-sequenced anopheline vectors of human malaria parasites.

Malaria morbidity and mortality caused by both Plasmodium falciparum and Plasmodium vivax extend well beyond the African continent, and although P. vivax causes between 80 and 300 million severe cases each year, vivax transmission remains poorly understood. Plasmodium parasites are transmitted by Anopheles mosquitoes, and the critical site of interaction between parasite and host is at the mosquito's luminal midgut brush border. Although the genome of the "model" African P. falciparum vector, Anopheles gambiae, has been sequenced, evolutionary divergence limits its utility as a reference across anophelines, especially non-sequenced P. vivax vectors such as Anopheles albimanus. Clearly, technologies and platforms that bridge this substantial scientific gap are required in order to provide public health scientists with key transcriptomic and proteomic information that could spur the development of novel interventions to combat this disease. To our knowledge, no approaches have been published that address this issue. To bolster our understanding of P. vivax-An. albimanus midgut interactions, we developed an integrated bioinformatic-hybrid RNA-Seq-LC-MS/MS approach involving An. albimanus transcriptome (15,764 contigs) and luminal midgut subproteome (9,445 proteins) assembly, which, when used with our custom Diptera protein database (685,078 sequences), facilitated a comparative proteomic analysis of the midgut brush borders of two important malaria vectors, An. gambiae and An. albimanus.

Transcriptomic analysis using RNA sequencing and phenotypic analysis of Salmonella enterica after acid exposure for different time durations using adaptive laboratory evolution.

Introduction: This study is the final part of a two-part series that delves into the molecular mechanisms driving adaptive laboratory evolution (ALE) of Salmonella enterica in acid stress. The phenotypic and transcriptomic alterations in the acid-evolved lineages (EL) of Salmonella enterica serovar Enteritidis after 70 days of acid stress exposure were analyzed. Materials and methods: The stability of phenotypic changes observed after 70 days in acetic acid was explored after stress removal using a newly developed evolutionary lineage EL5. Additionally, the impact of short-term acid stress on the previously adapted lineage EL4 was also examined. Results: The results indicate that the elevated antibiotic minimum inhibitory concentration (MIC) observed after exposure to acetic acid for 70 days was lost when acid stress was removed. This phenomenon was observed against human antibiotics such as meropenem, ciprofloxacin, gentamicin, and streptomycin. The MIC of meropenem in EL4 on day 70 was 0.094 mM, which dropped to 0.032 mM when removed from acetic acid stress after day 70. However, after stress reintroduction, the MIC swiftly elevated, and within 4 days, it returned to 0.094 mM. After 20 more days of adaptation in acetic acid, the meropenem MIC increased to 0.125 mM. The other human antibiotics that were tested exhibited a similar trend. The MIC of acetic acid in EL4 on day 70 was observed to be 35 mM, which remained constant even after the removal of acetic acid stress. Readaptation of EL4 in acetic acid for 20 more days caused the acetic acid MIC to increase to 37 mM. Bacterial whole genome sequencing of EL5 revealed base substitutions in several genes involved in pathogenesis, such as the phoQ and wzc genes. Transcriptomic analysis of EL5 revealed upregulation of virulence, drug resistance, toxin-antitoxin, and iron metabolism genes. Unstable Salmonella small colony variants (SSCV) of S. Enteritidis were also observed in EL5 as compared to the wild-type unevolved S. Enteritidis. Discussion: This study presents a comprehensive understanding of the evolution of the phenotypic, genomic, and transcriptomic changes in S. Enteritidis due to prolonged acid exposure through ALE.

Phylogenomics reveals extensive misidentification of fungal strains from the genus Aspergillus.

Modern taxonomic classification is often based on phylogenetic analyses of a few molecular markers, although single-gene studies are still common. Here, we leverage genome-scale molecular phylogenetics (phylogenomics) of species and populations to reconstruct evolutionary relationships in a dense data set of 710 fungal genomes from the biomedically and technologically important genus Aspergillus. To do so, we generated a novel set of 1,362 high-quality molecular markers specific for Aspergillus and provided profile Hidden Markov Models for each, facilitating their use by others. Examining the resulting phylogeny helped resolve ongoing taxonomic controversies, identified new ones, and revealed extensive strain misidentification (7.59% of strains were previously misidentified), underscoring the importance of population-level sampling in species classification. These findings were corroborated using the current standard, taxonomically informative loci. These findings suggest that phylogenomics of species and populations can facilitate accurate taxonomic classifications and reconstructions of the Tree of Life.IMPORTANCEIdentification of fungal species relies on the use of molecular markers. Advances in genomic technologies have made it possible to sequence the genome of any fungal strain, making it possible to use genomic data for the accurate assignment of strains to fungal species (and for the discovery of new ones). We examined the usefulness and current limitations of genomic data using a large data set of 710 publicly available genomes from multiple strains and species of the biomedically, agriculturally, and industrially important genus Aspergillus. Our evolutionary genomic analyses revealed that nearly 8% of publicly available Aspergillus genomes are misidentified. Our work highlights the usefulness of genomic data for fungal systematic biology and suggests that systematic genome sequencing of multiple strains, including reference strains (e.g., type strains), of fungal species will be required to reduce misidentification errors in public databases.

Global transcriptome changes underlying colony growth in the opportunistic human pathogen Aspergillus fumigatus.

Aspergillus fumigatus is the most common and deadly pulmonary fungal infection worldwide. In the lung, the fungus usually forms a dense colony of filaments embedded in a polymeric extracellular matrix. To identify candidate genes involved in this biofilm (BF) growth, we used RNA-Seq to compare the transcriptomes of BF and liquid plankton (PL) growth. Sequencing and mapping of tens of millions sequence reads against the A. fumigatus transcriptome identified 3,728 differentially regulated genes in the two conditions. Although many of these genes, including the ones coding for transcription factors, stress response, the ribosome, and the translation machinery, likely reflect the different growth demands in the two conditions, our experiment also identified hundreds of candidate genes for the observed differences in morphology and pathobiology between BF and PL. We found an overrepresentation of upregulated genes in transport, secondary metabolism, and cell wall and surface functions. Furthermore, upregulated genes showed significant spatial structure across the A. fumigatus genome; they were more likely to occur in subtelomeric regions and colocalized in 27 genomic neighborhoods, many of which overlapped with known or candidate secondary metabolism gene clusters. We also identified 1,164 genes that were downregulated. This gene set was not spatially structured across the genome and was overrepresented in genes participating in primary metabolic functions, including carbon and amino acid metabolism. These results add valuable insight into the genetics of biofilm formation in A. fumigatus and other filamentous fungi and identify many relevant, in the context of biofilm biology, candidate genes for downstream functional experiments.

Adaptive laboratory evolution of Salmonella enterica in acid stress.

Introduction: Adaptive laboratory evolution (ALE) studies play a crucial role in understanding the adaptation and evolution of different bacterial species. In this study, we have investigated the adaptation and evolution of Salmonella enterica serovar Enteritidis to acetic acid using ALE. Materials and methods: Acetic acid concentrations below the minimum inhibitory concentration (sub-MIC) were used. Four evolutionary lineages (EL), namely, EL1, EL2, EL3, and EL4, of S. Enteritidis were developed, each demonstrating varying levels of resistance to acetic acid. Results: The acetic acid MIC of EL1 remained constant at 27 mM throughout 70 days, while the MIC of EL2, EL3, and EL4 increased throughout the 70 days. EL4 was adapted to the highest concentration of acetic acid (30 mM) and demonstrated the highest increase in its MIC against acetic acid throughout the study, reaching an MIC of 35 mM on day 70. The growth rates of the evolved lineages increased over time and were dependent on the concentration of acetic acid used during the evolutionary process. EL4 had the greatest increase in growth rate, reaching 0.33 (h-1) after 70 days in the presence of 30 mM acetic acid as compared to EL1, which had a growth rate of 0.2 (h-1) after 70 days with no exposure to acetic acid. Long-term exposure to acetic acid led to an increased MIC of human antibiotics such as ciprofloxacin and meropenem against the S. enterica evolutionary lineages. The MIC of ciprofloxacin for EL1 stayed constant at 0.016 throughout the 70 days while that of EL4 increased to 0.047. Bacterial whole genome sequencing revealed single-nucleotide polymorphisms in the ELs in various genes known to be involved in S. enterica virulence, pathogenesis, and stress response including phoP, phoQ, and fhuA. We also observed genome deletions in some of the ELs as compared to the wild-type S. Enteritidis which may have contributed to the bacterial acid adaptation. Discussion: This study highlights the potential for bacterial adaptation and evolution under environmental stress and underscores the importance of understanding the development of cross resistance to antibiotics in S. enterica populations. This study serves to enhance our understanding of the pathogenicity and survival strategies of S. enterica under acetic acid stress.

Transcriptome of the adult female malaria mosquito vector Anopheles albimanus.

BACKGROUND: Human Malaria is transmitted by mosquitoes of the genus Anopheles. Transmission is a complex phenomenon involving biological and environmental factors of humans, parasites and mosquitoes. Among more than 500 anopheline species, only a few species from different branches of the mosquito evolutionary tree transmit malaria, suggesting that their vectorial capacity has evolved independently. Anopheles albimanus (subgenus Nyssorhynchus) is an important malaria vector in the Americas. The divergence time between Anopheles gambiae, the main malaria vector in Africa, and the Neotropical vectors has been estimated to be 100 My. To better understand the biological basis of malaria transmission and to develop novel and effective means of vector control, there is a need to explore the mosquito biology beyond the An. gambiae complex. RESULTS: We sequenced the transcriptome of the An. albimanus adult female. By combining Sanger, 454 and Illumina sequences from cDNA libraries derived from the midgut, cuticular fat body, dorsal vessel, salivary gland and whole body, we generated a single, high-quality assembly containing 16,669 transcripts, 92% of which mapped to the An. darlingi genome and covered 90% of the core eukaryotic genome. Bidirectional comparisons between the An. gambiae, An. darlingi and An. albimanus predicted proteomes allowed the identification of 3,772 putative orthologs. More than half of the transcripts had a match to proteins in other insect vectors and had an InterPro annotation. We identified several protein families that may be relevant to the study of Plasmodium-mosquito interaction. An open source transcript annotation browser called GDAV (Genome-Delinked Annotation Viewer) was developed to facilitate public access to the data generated by this and future transcriptome projects. CONCLUSIONS: We have explored the adult female transcriptome of one important New World malaria vector, An. albimanus. We identified protein-coding transcripts involved in biological processes that may be relevant to the Plasmodium lifecycle and can serve as the starting point for searching targets for novel control strategies. Our data increase the available genomic information regarding An. albimanus several hundred-fold, and will facilitate molecular research in medical entomology, evolutionary biology, genomics and proteomics of anopheline mosquito vectors. The data reported in this manuscript is accessible to the community via the VectorBase website (http://www.vectorbase.org/Other/AdditionalOrganisms/).

Evidence for genetic differentiation and variable recombination rates among Dutch populations of the opportunistic human pathogen Aspergillus fumigatus.

As the frequency of antifungal drug resistance continues to increase, understanding the genetic structure of fungal populations, where resistant isolates have emerged and spread, is of major importance. Aspergillus fumigatus is an ubiquitously distributed fungus and the primary causative agent of invasive aspergillosis (IA), a potentially lethal infection in immunocompromised individuals. In the last few years, an increasing number of A. fumigatus isolates has evolved resistance to triazoles, the primary drugs for treating IA infections. In most isolates, this multiple-triazole-resistance (MTR) phenotype is caused by mutations in the cyp51A gene, which encodes the protein targeted by the triazoles. We investigated the genetic differentiation and reproductive mode of A. fumigatus in the Netherlands, the country where the MTR phenotype probably originated, to determine their role in facilitating the emergence and distribution of resistance genotypes. Using 20 genome-wide neutral markers, we genotyped 255 Dutch isolates including 25 isolates with the MTR phenotype. In contrast to previous reports, our results show that Dutch A. fumigatus genotypes are genetically differentiated into five distinct populations. Four of the five populations show significant linkage disequilibrium, indicative of an asexual reproductive mode, whereas the fifth population is in linkage equilibrium, indicative of a sexual reproductive mode. Notably, the observed genetic differentiation among Dutch isolates does not correlate with geography, although all isolates with the MTR phenotype nest within a single, predominantly asexual, population. These results suggest that both reproductive mode and genetic differentiation contribute to the structure of Dutch A. fumigatus populations and are probably shaping the evolutionary dynamics of drug resistance in this potentially deadly pathogen.

Characterization of 260 Isolates of Aspergillus Section Flavi Obtained from Sesame Seeds in Punjab, Pakistan.

Sesame Sesamum indicum L. is a major oil-based seed crop that has been widely cultivated and consumed in Pakistan. Unfortunately, sesame is highly prone to Aspergillus fungal growth in the field, and under inappropriate storage conditions can become contaminated with aflatoxins, the most potent carcinogen found in nature. Here, we have isolated a high number of Aspergillus isolates from sesame seeds in fresh and stored conditions obtained from rainfed and irrigated zones of Punjab, Pakistan, and characterized them for aflatoxigenic potentials. Using morphological identification techniques, 260 isolates were grouped as potential Aspergillus section Flavi, with 126 and 134 originating from the rainfed and irrigated zones, respectively. Out of 260 in total, 188 isolates were confirmed to produce aflatoxins. There were no significant differences in potential aflatoxigenic isolates with respect to the rainfed and irrigated zones. However, the number of potential aflatoxigenic isolates was significantly higher (p < 0.05) in stored samples than that of those from fresh sesame seeds in the rainfed and irrigated zone. Whole genome sequencing and comparative analyses of 12 select isolates have revealed that one of the A. flavus isolates, which produced very low aflatoxins (AFP10), has an elevated missense variant rate, numerous high impact mutations, and a 600 base pair deletion in the norB gene. In summary, our study provides insights into aflatoxigenic potential and the associated genetic diversity of indigenous Aspergillus section Flavi isolates and potential management strategies for reducing aflatoxin contamination levels in a major crop consumed in Punjab, Pakistan.

Genomic and Molecular Identification of Genes Contributing to the Caspofungin Paradoxical Effect in Aspergillus fumigatus.

Aspergillus fumigatus is a deadly opportunistic fungal pathogen responsible for ~100,000 annual deaths. Azoles are the first line antifungal agent used against A. fumigatus, but azole resistance has rapidly evolved making treatment challenging. Caspofungin is an important second-line therapy against invasive pulmonary aspergillosis, a severe A. fumigatus infection. Caspofungin functions by inhibiting ß-1,3-glucan synthesis, a primary and essential component of the fungal cell wall. A phenomenon termed the caspofungin paradoxical effect (CPE) has been observed in several fungal species where at higher concentrations of caspofungin, chitin replaces ß-1,3-glucan, morphology returns to normal, and growth rate increases. CPE appears to occur in vivo, and it is therefore clinically important to better understand the genetic contributors to CPE. We applied genomewide association (GWA) analysis and molecular genetics to identify and validate candidate genes involved in CPE. We quantified CPE across 67 clinical isolates and conducted three independent GWA analyses to identify genetic variants associated with CPE. We identified 48 single nucleotide polymorphisms (SNPs) associated with CPE. We used a CRISPR/Cas9 approach to generate gene deletion mutants for seven genes harboring candidate SNPs. Two null mutants, ΔAfu3g13230 and ΔAfu4g07080 (dscP), resulted in reduced basal growth rate and a loss of CPE. We further characterized the dscP phosphatase-null mutant and observed a significant reduction in conidia production and extremely high sensitivity to caspofungin at both low and high concentrations. Collectively, our work reveals the contribution of Afu3g13230 and dscP in CPE and sheds new light on the complex genetic interactions governing this phenotype. IMPORTANCE This is one of the first studies to apply genomewide association (GWA) analysis to identify genes involved in an Aspergillus fumigatus phenotype. A. fumigatus is an opportunistic fungal pathogen that causes hundreds of thousands of infections and ~100,000 deaths each year, and antifungal resistance has rapidly evolved in this species. A phenomenon called the caspofungin paradoxical effect (CPE) occurs in some isolates, where high concentrations of the drug lead to increased growth rate. There is clinical relevance in understanding the genetic basis of this phenotype, since caspofungin concentrations could lead to unintended adverse clinical outcomes in certain cases. Using GWA analysis, we identified several interesting candidate polymorphisms and genes and then generated gene deletion mutants to determine whether these genes were important for CPE. Two of these mutant strains (ΔAfu3g13230 and ΔAfu4g07080/ΔdscP) displayed a loss of the CPE. This study sheds light on the genes involved in clinically important phenotype CPE.

Comparative Genomics Reveals a Single Nucleotide Deletion in pksP That Results in White-Spore Phenotype in Natural Variants of Aspergillus fumigatus.

Aspergillus fumigatus is a potentially deadly opportunistic human pathogen. A. fumigatus has evolved a variety of mechanisms to evade detection by the immune system. For example, the conidium surface is covered in a layer of 1,8-dihydroxynaphthalene (DHN) melanin which masks the antigen macrophages use for recognition. DHN melanin also protects conidia from ultraviolet radiation and gives A. fumigatus conidia their characteristic green-grayish color. Here, we conducted genomic analysis of two closely related white-spore natural variants of A. fumigatus in comparison to two closely related green-spore isolates to identify a genetic basis of the white-spore phenotype. Illumina whole-genome resequencing data of the four isolates was used to identify variants that were shared in the white-spore isolates and different from both the green-spore isolates and the Af293 reference genome (which is also a green-spore isolate). We identified 4,279 single nucleotide variants and 1,785 insertion/deletions fitting this pattern. Among these, we identified 64 variants predicted to be high impact, loss-of-function mutations. One of these variants is a single nucleotide deletion that results in a frameshift in pksP (Afu2g17600), the core biosynthetic gene in the DHN melanin encoding gene cluster. The frameshift mutation in the white-spore isolates leads to a truncated protein in which a phosphopantetheine attachment site (PP-binding domain) is interrupted and an additional PP-binding domain and a thioesterase domain are omitted. Growth rate analysis of white-spore and green-spore isolates at 37°C and 48°C revealed that white-spore isolates are thermosensitive. Growth rate of A. fumigatus Af293 and a pksP null mutant in the Af293 background suggests pksP is not directly involved in the thermosensitivity phenotype. Further, our study identified a mutation in a gene (Afu4g04740) associated with thermal sensitivity in yeasts which could also be responsible for the thermosensitivity of the white-spore mutants. Overall, we used comparative genomics to identify the mutation and protein alterations responsible for the white-spore phenotype of environmental isolates of A. fumigatus.

Examination of Genome-Wide Ortholog Variation in Clinical and Environmental Isolates of the Fungal Pathogen Aspergillus fumigatus.

Aspergillus fumigatus is both an environmental saprobe and an opportunistic human fungal pathogen. Knowledge of genomic variation across A. fumigatus isolates is essential for understanding the evolution of pathogenicity, virulence, and resistance to antifungal drugs. Here, we investigated 206 A. fumigatus isolates (133 clinical and 73 environmental isolates), aiming to identify genes with variable presence across isolates and test whether this variation was related to the clinical or environmental origin of isolates. The PanOrtho genome of A. fumigatus consists of 13,085 ortholog groups, of which 7,773 (59.4%) are shared by all isolates (core groups) and 5,312 (40.6%) vary in their gene presence across isolates (accessory groups plus singletons). Despite differences in the distribution of orthologs across all isolates, no significant differences were observed among clinical versus environmental isolates when phylogeny was accounted for. Orthologs that differ in their distribution across isolates tend to occur at low frequency and/or be restricted to specific isolates; thus, the degree of genomic conservation between orthologs of A. fumigatus is high. These results suggest that differences in the distribution of orthologs within A. fumigatus cannot be associated with the clinical or environmental origin of isolates. IMPORTANCE Aspergillus fumigatus is a cosmopolitan species of fungus responsible for thousands of cases of invasive disease annually. Clinical and environmental isolates of A. fumigatus exhibit extensive phenotypic differences, including differences related to virulence and antifungal drug resistance. A comprehensive survey of the genomic diversity present in A. fumigatus and its relationship to the clinical or environmental origin of isolates can contribute to the prediction of the mechanisms of evolution and infection of the species. Our results suggest that there is no significant variation in ortholog distribution between clinical and environmental isolates when accounting for evolutionary history. The work supports the hypothesis that environmental and clinical isolates of A. fumigatus do not differ in their gene contents.

Microbial enzymes induce colitis by reactivating triclosan in the mouse gastrointestinal tract.

Emerging research supports that triclosan (TCS), an antimicrobial agent found in thousands of consumer products, exacerbates colitis and colitis-associated colorectal tumorigenesis in animal models. While the intestinal toxicities of TCS require the presence of gut microbiota, the molecular mechanisms involved have not been defined. Here we show that intestinal commensal microbes mediate metabolic activation of TCS in the colon and drive its gut toxicology. Using a range of in vitro, ex vivo, and in vivo approaches, we identify specific microbial ß-glucuronidase (GUS) enzymes involved and pinpoint molecular motifs required to metabolically activate TCS in the gut. Finally, we show that targeted inhibition of bacterial GUS enzymes abolishes the colitis-promoting effects of TCS, supporting an essential role of specific microbial proteins in TCS toxicity. Together, our results define a mechanism by which intestinal microbes contribute to the metabolic activation and gut toxicity of TCS, and highlight the importance of considering the contributions of the gut microbiota in evaluating the toxic potential of environmental chemicals.

Population Genomic Analysis of Listeria monocytogenes From Food Reveals Substrate-Specific Genome Variation.

Listeria monocytogenes is the major causative agent of the foodborne illness listeriosis. Listeriosis presents as flu-like symptoms in healthy individuals, and can be fatal for children, elderly, pregnant women, and immunocompromised individuals. Estimates suggest that L. monocytogenes results in ∼1,600 illnesses and ∼260 deaths annually in the United States. L. monocytogenes can survive and persist in a variety of harsh environments, including conditions encountered in production of fermented dairy products such as cheese. For instance, microbial growth is often limited in soft cheese fermentation because of harsh pH, water content, and salt concentrations. However, L. monocytogenes has caused a number of deadly listeriosis outbreaks through the contamination of cheese. The purpose of this study was to understand if genetically distinct populations of L. monocytogenes are associated with particular foods, including cheese and dairy. To address this goal, we analyzed the population genetic structure of 504 L. monocytogenes strains isolated from food with publicly available genome assemblies. We identified 10 genetically distinct populations spanning L. monocytogenes lineages 1, II, and III and serotypes 1/2a, 1/2b, 1/2c, 4b, and 4c. We observed an overrepresentation of isolates from specific populations with cheese (population 2), fruit/vegetable (population 2), seafood (populations 5, 8 and 9) and meat (population 10). We used the Large Scale Blast Score Ratio pipeline and Roary to identify genes unique to population 1 and population 2 in comparison with all other populations, and screened for the presence of antimicrobial resistance genes and virulence genes across all isolates. We identified > 40 genes that were present at high frequency in population 1 and population 2 and absent in most other isolates. Many of these genes encoded for transcription factors, and cell surface anchored proteins. Additionally, we found that the virulence genes aut and ami were entirely or partially deleted in population 2. These results indicate that some L. monocytogenes populations may exhibit associations with particular foods, including cheese, and that gene content may contribute to this pattern.

Genome Sequence of Aspergillus aculeatinus IC_8, Isolated from an Indoor Air Sample of an Urban Housing Complex in Abidjan, Ivory Coast.

Aspergillus aculeatinus is an industrially important species of Aspergillus section Nigri capable of producing bioactive, antibiotic, and antitumor compounds. We sequenced the genome of a strain of A. aculeatinus that was isolated from the interior of a housing complex in Abidjan, Ivory Coast.

Comparison of Two Aspergillus oryzae Genomes From Different Clades Reveals Independent Evolution of Alpha-Amylase Duplication, Variation in Secondary Metabolism Genes, and Differences in Primary Metabolism.

Microbes (bacteria, yeasts, molds), in addition to plants and animals, were domesticated for their roles in food preservation, nutrition and flavor. Aspergillus oryzae is a domesticated filamentous fungal species traditionally used during fermentation of Asian foods and beverage, such as sake, soy sauce, and miso. To date, little is known about the extent of genome and phenotypic variation of A. oryzae isolates from different clades. Here, we used long-read Oxford Nanopore and short-read Illumina sequencing to produce a highly accurate and contiguous genome assemble of A. oryzae 14160, an industrial strain from China. To understand the relationship of this isolate, we performed phylogenetic analysis with 90 A. oryzae isolates and 1 isolate of the A. oryzae progenitor, Aspergillus flavus. This analysis showed that A. oryzae 14160 is a member of clade A, in comparison to the RIB 40 type strain, which is a member of clade F. To explore genome variation between isolates from distinct A. oryzae clades, we compared the A. oryzae 14160 genome with the complete RIB 40 genome. Our results provide evidence of independent evolution of the alpha-amylase gene duplication, which is one of the major adaptive mutations resulting from domestication. Synteny analysis revealed that both genomes have three copies of the alpha-amylase gene, but only one copy on chromosome 2 was conserved. While the RIB 40 genome had additional copies of the alpha-amylase gene on chromosomes III, and V, 14160 had a second copy on chromosome II and an third copy on chromosome VI. Additionally, we identified hundreds of lineage specific genes, and putative high impact mutations in genes involved in secondary metabolism, including several of the core biosynthetic genes. Finally, to examine the functional effects of genome variation between strains, we measured amylase activity, proteolytic activity, and growth rate on several different substrates. RIB 40 produced significantly higher levels of amylase compared to 14160 when grown on rice and starch. Accordingly, RIB 40 grew faster on rice, while 14160 grew faster on soy. Taken together, our analyses reveal substantial genome and phenotypic variation within A. oryzae.