Predicting antimicrobial mechanism-of-action from transcriptomes: A generalizable explainable artificial intelligence approach.

To better combat the expansion of antibiotic resistance in pathogens, new compounds, particularly those with novel mechanisms-of-action [MOA], represent a major research priority in biomedical science. However, rediscovery of known antibiotics demonstrates a need for approaches that accurately identify potential novelty with higher throughput and reduced labor. Here we describe an explainable artificial intelligence classification methodology that emphasizes prediction performance and human interpretability by using a Hierarchical Ensemble of Classifiers model optimized with a novel feature selection algorithm called Clairvoyance; collectively referred to as a CoHEC model. We evaluated our methods using whole transcriptome responses from Escherichia coli challenged with 41 known antibiotics and 9 crude extracts while depositing 122 transcriptomes unique to this study. Our CoHEC model can properly predict the primary MOA of previously unobserved compounds in both purified forms and crude extracts at an accuracy above 99%, while also correctly identifying darobactin, a newly discovered antibiotic, as having a novel MOA. In addition, we deploy our methods on a recent E. coli transcriptomics dataset from a different strain and a Mycobacterium smegmatis metabolomics timeseries dataset showcasing exceptionally high performance; improving upon the performance metrics of the original publications. We not only provide insight into the biological interpretation of our model but also that the concept of MOA is a non-discrete heuristic with diverse effects for different compounds within the same MOA, suggesting substantial antibiotic diversity awaiting discovery within existing MOA.

Mechanism-of-Action Classification of Antibiotics by Global Transcriptome Profiling.

Antimicrobial resistance (AMR) is an ever-growing public health problem worldwide. The low rate of antibiotic discovery coupled with the rapid spread of drug-resistant bacterial pathogens is causing a global health crisis. To facilitate the drug discovery processes, we present a large-scale study of reference antibiotic challenge bacterial transcriptome profiles, which included 37 antibiotics across 6 mechanisms of actions (MOAs) and provide an economical approach to aid in antimicrobial dereplication in the discovery process. We demonstrate that classical MOAs can be sorted based upon the magnitude of gene expression profiles despite some overlap in the secondary effects of antibiotic exposures across MOAs. Additionally, using gene subsets, we were able to subdivide broad MOA classes into subMOAs. Furthermore, we provide a biomarker gene set that can be used to classify most antimicrobial challenges according to their canonical MOA. We also demonstrate the ability of this rapid MOA diagnostic tool to predict and classify the expression profiles of pure compounds and crude extracts to their expression profile-associated MOA class.

Mutual independence of alkaline- and calcium-mediated signalling in Aspergillus fumigatus refutes the existence of a conserved druggable signalling nexus.

Functional coupling of calcium- and alkaline responsive signalling occurs in multiple fungi to afford efficient cation homeostasis. Host microenvironments exert alkaline stress and potentially toxic concentrations of Ca2+ , such that highly conserved regulators of both calcium- (Crz) and pH- (PacC/Rim101) responsive signalling are crucial for fungal pathogenicity. Drugs targeting calcineurin are potent antifungal agents but also perturb human immunity thereby negating their use as anti-infectives, abrogation of alkaline signalling has, therefore, been postulated as an adjunctive antifungal strategy. We examined the interdependency of pH- and calcium-mediated signalling in Aspergillus fumigatus and found that calcium chelation severely impedes hyphal growth indicating a critical requirement for this ion independently of ambient pH. Transcriptomic responses to alkaline pH or calcium excess exhibited minimal similarity. Mutants lacking calcineurin, or its client CrzA, displayed normal alkaline tolerance and nuclear translocation of CrzA was unaffected by ambient pH. Expression of a highly conserved, alkaline-regulated, sodium ATPase was tolerant of genetic or chemical perturbations of calcium-mediated signalling, but abolished in null mutants of the pH-responsive transcription factor PacC, and PacC proteolytic processing occurred normally during calcium excess. Taken together our data demonstrate that in A. fumigatus the regulatory hierarchy governing alkaline tolerance circumvents calcineurin signalling.

Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus's Role in Virulence.

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F9 MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence associated traits.

Reducing the Bottleneck in Discovery of Novel Antibiotics.

Most antibiotics were discovered by screening soil actinomycetes, but the efficiency of the discovery platform collapsed in the 1960s. By now, more than 3000 antibiotics have been described and most of the current discovery effort is focused on the rediscovery of known compounds, making the approach impractical. The last marketed broad-spectrum antibiotics discovered were daptomycin, linezolid, and fidaxomicin. The current state of the art in the development of new anti-infectives is a non-existent pipeline in the absence of a discovery platform. This is particularly troubling given the emergence of pan-resistant pathogens. The current practice in dealing with the problem of the background of known compounds is to use chemical dereplication of extracts to assess the relative novelty of a compound it contains. Dereplication typically requires scale-up, extraction, and often fractionation before an accurate mass and structure can be produced by MS analysis in combination with 2D NMR. Here, we describe a transcriptome analysis approach using RNA sequencing (RNASeq) to identify promising novel antimicrobial compounds from microbial extracts. Our pipeline permits identification of antimicrobial compounds that produce distinct transcription profiles using unfractionated cell extracts. This efficient pipeline will eliminate the requirement for purification and structure determination of compounds from extracts and will facilitate high-throughput screen of cell extracts for identification of novel compounds.

The tandem repeats enabling reversible switching between the two phases of ß-lactamase substrate spectrum.

Expansion or shrinkage of existing tandem repeats (TRs) associated with various biological processes has been actively studied in both prokaryotic and eukaryotic genomes, while their origin and biological implications remain mostly unknown. Here we describe various duplications (de novo TRs) that occurred in the coding region of a ß-lactamase gene, where a conserved structure called the omega loop is encoded. These duplications that occurred under selection using ceftazidime conferred substrate spectrum extension to include the antibiotic. Under selective pressure with one of the original substrates (amoxicillin), a high level of reversion occurred in the mutant ß-lactamase genes completing a cycle back to the original substrate spectrum. The de novo TRs coupled with reversion makes a genetic toggling mechanism enabling reversible switching between the two phases of the substrate spectrum of ß-lactamases. This toggle exemplifies the effective adaptation of de novo TRs for enhanced bacterial survival. We found pairs of direct repeats that mediated the DNA duplication (TR formation). In addition, we found different duos of sequences that mediated the DNA duplication. These novel elements-that we named SCSs (same-strand complementary sequences)-were also found associated with ß-lactamase TR mutations from clinical isolates. Both direct repeats and SCSs had a high correlation with TRs in diverse bacterial genomes throughout the major phylogenetic lineages, suggesting that they comprise a fundamental mechanism shaping the bacterial evolution.

The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary aspergillosis.

Destruction of the pulmonary epithelium is a major feature of lung diseases caused by the mould pathogen Aspergillus fumigatus. Although it is widely postulated that tissue invasion is governed by fungal proteases, A. fumigatus mutants lacking individual or multiple enzymes remain fully invasive, suggesting a concomitant requirement for other pathogenic activities during host invasion. In this study we discovered, and exploited, a novel, tissue non-invasive, phenotype in A. fumigatus mutants lacking the pH-responsive transcription factor PacC. Our study revealed a novel mode of epithelial entry, occurring in a cell wall-dependent manner prior to protease production, and via the Dectin-1 ß-glucan receptor. ΔpacC mutants are defective in both contact-mediated epithelial entry and protease expression, and significantly attenuated for pathogenicity in leukopenic mice. We combined murine infection modelling, in vivo transcriptomics, and in vitro infections of human alveolar epithelia, to delineate two major, and sequentially acting, PacC-dependent processes impacting epithelial integrity in vitro and tissue invasion in the whole animal. We demonstrate that A. fumigatus spores and germlings are internalised by epithelial cells in a contact-, actin-, cell wall- and Dectin-1 dependent manner and ΔpacC mutants, which aberrantly remodel the cell wall during germinative growth, are unable to gain entry into epithelial cells, both in vitro and in vivo. We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection. Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences. Infecting ΔpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy.

A systematic evaluation of high-dimensional, ensemble-based regression for exploring large model spaces in microbiome analyses.

BACKGROUND: Microbiome studies incorporate next-generation sequencing to obtain profiles of microbial communities. Data generated from these experiments are high-dimensional with a rich correlation structure but modest sample sizes. A statistical model that utilizes these microbiome profiles to explain a clinical or biological endpoint needs to tackle high-dimensionality resulting from the very large space of variable configurations. Ensemble models are a class of approaches that can address high-dimensionality by aggregating information across large model spaces. Although such models are popular in fields as diverse as economics and genetics, their performance on microbiome data has been largely unexplored. RESULTS: We developed a simulation framework that accurately captures the constraints of experimental microbiome data. Using this setup, we systematically evaluated a selection of both frequentist and Bayesian regression modeling ensembles. These are represented by variants of stability selection in conjunction with elastic net and spike-and-slab Bayesian model averaging (BMA), respectively. BMA ensembles that explore a larger space of models relative to stability selection variants performed better and had lower variability across simulations. However, stability selection ensembles were able to match the performance of BMA in scenarios of low sparsity where several variables had large regression coefficients. CONCLUSIONS: Given a microbiome dataset of interest, we present a methodology to generate simulated data that closely mimics its characteristics in a manner that enables meaningful evaluation of analytical strategies. Our evaluation demonstrates that the largest ensembles yield the strongest performance on microbiome data with modest sample sizes and high-dimensional measurements. We also demonstrate the ability of these ensembles to identify microbiome signatures that are associated with opportunistic Candida albicans colonization during antibiotic exposure. As the focus of microbiome research evolves from pilot to translational studies, we anticipate that our strategy will aid investigators in making evaluation-based decisions for selecting appropriate analytical methods.

Aspergillus galactosaminogalactan mediates adherence to host constituents and conceals hyphal ß-glucan from the immune system.

Aspergillus fumigatus is the most common cause of invasive mold disease in humans. The mechanisms underlying the adherence of this mold to host cells and macromolecules have remained elusive. Using mutants with different adhesive properties and comparative transcriptomics, we discovered that the gene uge3, encoding a fungal epimerase, is required for adherence through mediating the synthesis of galactosaminogalactan. Galactosaminogalactan functions as the dominant adhesin of A. fumigatus and mediates adherence to plastic, fibronectin, and epithelial cells. In addition, galactosaminogalactan suppresses host inflammatory responses in vitro and in vivo, in part through masking cell wall ß-glucans from recognition by dectin-1. Finally, galactosaminogalactan is essential for full virulence in two murine models of invasive aspergillosis. Collectively these data establish a role for galactosaminogalactan as a pivotal bifunctional virulence factor in the pathogenesis of invasive aspergillosis.

Polysome profiling reveals broad translatome remodeling during endoplasmic reticulum (ER) stress in the pathogenic fungus Aspergillus fumigatus.

BACKGROUND: The unfolded protein response (UPR) is a network of intracellular signaling pathways that supports the ability of the secretory pathway to maintain a balance between the load of proteins entering the endoplasmic reticulum (ER) and the protein folding capacity of the ER lumen. Current evidence indicates that several pathogenic fungi rely heavily on this pathway for virulence, but there is limited understanding of the mechanisms involved. The best known functional output of the UPR is transcriptional upregulation of mRNAs involved in ER homeostasis. However, this does not take into account mechanisms of translational regulation that involve differential loading of ribosomes onto mRNAs. In this study, a global analysis of transcript-specific translational regulation was performed in the pathogenic mold Aspergillus fumigatus to determine the nature and scope of the translational response to ER stress. RESULTS: ER stress was induced by treating the fungus with dithiothreitol, tunicamycin, or a thermal up-shift. The mRNAs were then fractionated on the basis of ribosome occupancy into an under-translated pool (U) and a well-translated pool (W). The mRNAs were used to interrogate microarrays and the ratio of the hybridization signal (W/U) was used as an indicator of the relative translational efficiency of a mRNA under each condition. The largest category of translationally upregulated mRNAs during ER stress encoded proteins involved in translation. Components of the ergosterol and GPI anchor biosynthetic pathways also showed increased polysome association, suggesting an important role for translational regulation in membrane and cell wall homeostasis. ER stress induced limited remodeling of the secretory pathway translatome. However, a select group of transcription factors was translationally upregulated, providing a link to subsequent modification of the transcriptome. Finally, we provide evidence that one component of the ER stress translatome is a novel mRNA isoform from the yvc1 gene that is induced by ER stress in a UPR-dependent manner. CONCLUSIONS: Together, these findings define a core set of mRNAs subject to translational control during the adaptive response to acute ER stress in A. fumigatus and reveal a remarkable breadth of functions that are needed to resolve ER stress in this organism.

Transcript amplification from single bacterium for transcriptome analysis.

Total transcript amplification (TTA) from single eukaryotic cells for transcriptome analysis is established, but TTA from a single prokaryotic cell presents additional challenges with much less starting material, the lack of poly(A)-tails, and the fact that the messages can be polycistronic. Here, we describe a novel method for single-bacterium TTA using a model organism, Burkholderia thailandensis, exposed to a subinhibitory concentration of the antibacterial agent, glyphosate. Utilizing a B. thailandensis microarray to assess the TTA method showed low fold-change bias (less than twofold difference and Pearson correlation coefficient R ≈ 0.87-0.89) and drop-outs (4%-6% of 2842 detectable genes), compared with data obtained from the larger-scale nonamplified RNA samples. Further analysis of the microarray data suggests that B. thailandensis, when exposed to the aromatic amino acid biosynthesis inhibitor glyphosate, induces (or represses) genes to possibly recuperate and balance the intracellular amino acid pool. We validated our single-cell microarray data at the multi-cell and single-cell levels with lacZ and gfp reporter-gene fusions, respectively. Sanger sequencing of 192 clones generated from the TTA product of a single cell, with and without enrichment by elimination of rRNA and tRNA, detected only B. thailandensis sequences with no contamination. These data indicate that RNA-seq of TTA from a single cell is possible using this novel method.

Clonality despite sex: the evolution of host-associated sexual neighborhoods in the pathogenic fungus Penicillium marneffei.

Molecular genetic approaches typically detect recombination in microbes regardless of assumed asexuality. However, genetic data have shown the AIDS-associated pathogen Penicillium marneffei to have extensive spatial genetic structure at local and regional scales, and although there has been some genetic evidence that a sexual cycle is possible, this haploid fungus is thought to be genetically, as well as morphologically, asexual in nature because of its highly clonal population structure. Here we use comparative genomics, experimental mixed-genotype infections, and population genetic data to elucidate the role of recombination in natural populations of P. marneffei. Genome wide comparisons reveal that all the genes required for meiosis are present in P. marneffei, mating type genes are arranged in a similar manner to that found in other heterothallic fungi, and there is evidence of a putatively meiosis-specific mutational process. Experiments suggest that recombination between isolates of compatible mating types may occur during mammal infection. Population genetic data from 34 isolates from bamboo rats in India, Thailand and Vietnam, and 273 isolates from humans in China, India, Thailand, and Vietnam show that recombination is most likely to occur across spatially and genetically limited distances in natural populations resulting in highly clonal population structure yet sexually reproducing populations. Predicted distributions of three different spatial genetic clusters within P. marneffei overlap with three different bamboo rat host distributions suggesting that recombination within hosts may act to maintain population barriers within P. marneffei.

The inhibitory effect of Bacillus megaterium on aflatoxin and cyclopiazonic acid biosynthetic pathway gene expression in Aspergillus flavus.

Aspergillus flavus is one of the major moulds that colonize peanut in the field and during storage. The impact to human and animal health, and to the economy in agriculture and commerce, is significant since this mold produces the most potent known natural toxins, aflatoxins, which are carcinogenic, mutagenic, immunosuppressive, and teratogenic. A strain of marine Bacillus megaterium isolated from the Yellow Sea of East China was evaluated for its effect in inhibiting aflatoxin formation in A. flavus through down-regulating aflatoxin pathway gene expression as demonstrated by gene chip analysis. Aflatoxin accumulation in potato dextrose broth liquid medium and liquid minimal medium was almost totally (more than 98 %) inhibited by co-cultivation with B. megaterium. Growth was also reduced. Using expression studies, we identified the fungal genes down-regulated by co-cultivation with B. megaterium across the entire fungal genome and specifically within the aflatoxin pathway gene cluster (aflF, aflT, aflS, aflJ, aflL, aflX). Modulating the expression of these genes could be used for controlling aflatoxin contamination in crops such as corn, cotton, and peanut. Importantly, the expression of the regulatory gene aflS was significantly down-regulated during co-cultivation. We present a model showing a hypothesis of the regulatory mechanism of aflatoxin production suppression by AflS and AflR through B. megaterium co-cultivation.

Large-scale transcriptional response to hypoxia in Aspergillus fumigatus observed using RNAseq identifies a novel hypoxia regulated ncRNA.

We utilized RNAseq analysis of the Aspergillus fumigatus response to early hypoxic condition exposure. The results show that more than 89% of the A. fumigatus genome is expressed under normoxic and hypoxic conditions. Replicate samples were highly reproducible; however, comparisons between normoxia and hypoxia revealed that >23 and 35% of genes were differentially expressed after 30 and 120 min of hypoxia exposure, respectively. Consistent with our previous report detailing transcriptomic and proteomic responses at later time points, the results here show major repression of ribosomal function and induction of ergosterol biosynthesis, as well as activation of alternate respiratory mechanisms at the later time point. RNAseq data were used to define 32 hypoxia-specific genes, which were not expressed under normoxic conditions. Transcripts of a C6 transcription factor and a histidine kinase-response regulator were found only in hypoxia. In addition, several genes involved in the phosphoenylpyruvate and D-glyceraldehyde-3-phosphate metabolism were only expressed in hypoxia. Interestingly, a 216-bp ncRNA Afu-182 in the 3' region of insA (AFUB_064770) was significantly repressed under hypoxia with a 40-fold reduction in expression. A detailed analysis of Afu-182 showed similarity with several genes in the genome, many of which were also repressed in hypoxia. The results from this study show that hypoxia induces very early and widely drastic genome-wide responses in A. fumigatus that include expression of protein-coding and ncRNA genes. The role of these ncRNA genes in regulating the fungal hypoxia response is an exciting future research direction.

HacA-independent functions of the ER stress sensor IreA synergize with the canonical UPR to influence virulence traits in Aspergillus fumigatus.

Endoplasmic reticulum (ER) stress is a condition in which the protein folding capacity of the ER becomes overwhelmed by an increased demand for secretion or by exposure to compounds that disrupt ER homeostasis. In yeast and other fungi, the accumulation of unfolded proteins is detected by the ER-transmembrane sensor IreA/Ire1, which responds by cleaving an intron from the downstream cytoplasmic mRNA HacA/Hac1, allowing for the translation of a transcription factor that coordinates a series of adaptive responses that are collectively known as the unfolded protein response (UPR). Here, we examined the contribution of IreA to growth and virulence in the human fungal pathogen Aspergillus fumigatus. Gene expression profiling revealed that A. fumigatus IreA signals predominantly through the canonical IreA-HacA pathway under conditions of severe ER stress. However, in the absence of ER stress IreA controls dual signaling circuits that are both HacA-dependent and HacA-independent. We found that a ΔireA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasts the partial virulence of a ΔhacA mutant, suggesting that IreA contributes to pathogenesis independently of HacA. In support of this conclusion, we found that the ΔireA mutant had more severe defects in the expression of multiple virulence-related traits relative to ΔhacA, including reduced thermotolerance, decreased nutritional versatility, impaired growth under hypoxia, altered cell wall and membrane composition, and increased susceptibility to azole antifungals. In addition, full or partial virulence could be restored to the ΔireA mutant by complementation with either the induced form of the hacA mRNA, hacA(i), or an ireA deletion mutant that was incapable of processing the hacA mRNA, ireA(Δ10). Together, these findings demonstrate that IreA has both HacA-dependent and HacA-independent functions that contribute to the expression of traits that are essential for virulence in A. fumigatus.

Succession and determinants of the early life nasopharyngeal microbiota in a South African birth cohort.

BACKGROUND: Bacteria colonizing the nasopharynx play a key role as gatekeepers of respiratory health. Yet, dynamics of early life nasopharyngeal (NP) bacterial profiles remain understudied in low- and middle-income countries (LMICs), where children have a high prevalence of risk factors for lower respiratory tract infection. We investigated longitudinal changes in NP bacterial profiles, and associated exposures, among healthy infants from low-income households in South Africa. METHODS: We used short fragment (V4 region) 16S rRNA gene amplicon sequencing to characterize NP bacterial profiles from 103 infants in a South African birth cohort, at monthly intervals from birth through the first 12 months of life and six monthly thereafter until 30 months. RESULTS: Corynebacterium and Staphylococcus were dominant colonizers at 1 month of life; however, these were rapidly replaced by Moraxella- or Haemophilus-dominated profiles by 4 months. This succession was almost universal and largely independent of a broad range of exposures. Warm weather (summer), lower gestational age, maternal smoking, no day-care attendance, antibiotic exposure, or low height-for-age z score at 12 months were associated with higher alpha and beta diversity. Summer was also associated with higher relative abundances of Staphylococcus, Streptococcus, Neisseria, or anaerobic gram-negative bacteria, whilst spring and winter were associated with higher relative abundances of Haemophilus or Corynebacterium, respectively. Maternal smoking was associated with higher relative abundances of Porphyromonas. Antibiotic therapy (or isoniazid prophylaxis for tuberculosis) was associated with higher relative abundance of anerobic taxa (Porphyromonas, Fusobacterium, and Prevotella) and with lower relative abundances of health associated-taxa Corynebacterium and Dolosigranulum. HIV-exposure was associated with higher relative abundances of Klebsiella or Veillonella and lower relative abundances of an unclassified genus within the family Lachnospiraceae. CONCLUSIONS: In this intensively sampled cohort, there was rapid and predictable replacement of early profiles dominated by health-associated Corynebacterium and Dolosigranulum with those dominated by Moraxella and Haemophilus, independent of exposures. Season and antibiotic exposure were key determinants of NP bacterial profiles. Understudied but highly prevalent exposures prevalent in LMICs, including maternal smoking and HIV-exposure, were associated with NP bacterial profiles. Video Abstract.

Draft genome sequence determination for cystic fibrosis and chronic granulomatous disease Burkholderia multivorans isolates.

Burkholderia multivorans is a Gram-negative bacterium and a member of the Burkholderia cepacia complex, which is frequently associated with respiratory infections in people with cystic fibrosis (CF) and chronic granulomatous disease (CGD). We are reporting the genome sequences of 4 B. multivorans strains, 2 from CF patients and 2 from CGD patients.

Sequencing of mitochondrial genomes of nine Aspergillus and Penicillium species identifies mobile introns and accessory genes as main sources of genome size variability.

BACKGROUND: The genera Aspergillus and Penicillium include some of the most beneficial as well as the most harmful fungal species such as the penicillin-producer Penicillium chrysogenum and the human pathogen Aspergillus fumigatus, respectively. Their mitochondrial genomic sequences may hold vital clues into the mechanisms of their evolution, population genetics, and biology, yet only a handful of these genomes have been fully sequenced and annotated. RESULTS: Here we report the complete sequence and annotation of the mitochondrial genomes of six Aspergillus and three Penicillium species: A. fumigatus, A. clavatus, A. oryzae, A. flavus, Neosartorya fischeri (A. fischerianus), A. terreus, P. chrysogenum, P. marneffei, and Talaromyces stipitatus (P. stipitatum). The accompanying comparative analysis of these and related publicly available mitochondrial genomes reveals wide variation in size (25-36 Kb) among these closely related fungi. The sources of genome expansion include group I introns and accessory genes encoding putative homing endonucleases, DNA and RNA polymerases (presumed to be of plasmid origin) and hypothetical proteins. The two smallest sequenced genomes (A. terreus and P. chrysogenum) do not contain introns in protein-coding genes, whereas the largest genome (T. stipitatus), contains a total of eleven introns. All of the sequenced genomes have a group I intron in the large ribosomal subunit RNA gene, suggesting that this intron is fixed in these species. Subsequent analysis of several A. fumigatus strains showed low intraspecies variation. This study also includes a phylogenetic analysis based on 14 concatenated core mitochondrial proteins. The phylogenetic tree has a different topology from published multilocus trees, highlighting the challenges still facing the Aspergillus systematics. CONCLUSIONS: The study expands the genomic resources available to fungal biologists by providing mitochondrial genomes with consistent annotations for future genetic, evolutionary and population studies. Despite the conservation of the core genes, the mitochondrial genomes of Aspergillus and Penicillium species examined here exhibit significant amount of interspecies variation. Most of this variation can be attributed to accessory genes and mobile introns, presumably acquired by horizontal gene transfer of mitochondrial plasmids and intron homing.

Comparative genomic analysis of fruiting body formation in Myxococcales.

Genetic programs underlying multicellular morphogenesis and cellular differentiation are most often associated with eukaryotic organisms, but examples also exist in bacteria such as the formation of multicellular, spore-filled fruiting bodies in the order Myxococcales. Most members of the Myxococcales undergo a multicellular developmental program culminating in the formation of spore-filled fruiting bodies in response to starvation. To gain insight into the evolutionary history of fruiting body formation in Myxococcales, we performed a comparative analysis of the genomes and transcriptomes of five Myxococcales species, four of these undergo fruiting body formation (Myxococcus xanthus, Stigmatella aurantiaca, Sorangium cellulosum, and Haliangium ochraceum) and one does not (Anaeromyxobacter dehalogenans). Our analyses show that a set of 95 known M. xanthus development-specific genes--although suffering from a sampling bias--are overrepresented and occur more frequently than an average M. xanthus gene in S. aurantiaca, whereas they occur at the same frequency as an average M. xanthus gene in S. cellulosum and in H. ochraceum and are underrepresented in A. dehalogenans. Moreover, genes for entire signal transduction pathways important for fruiting body formation in M. xanthus are conserved in S. aurantiaca, whereas only a minority of these genes are conserved in A. dehalogenans, S. cellulosum, and H. ochraceum. Likewise, global gene expression profiling of developmentally regulated genes showed that genes that upregulated during development in M. xanthus are overrepresented in S. aurantiaca and slightly underrepresented in A. dehalogenans, S. cellulosum, and H. ochraceum. These comparative analyses strongly indicate that the genetic programs for fruiting body formation in M. xanthus and S. aurantiaca are highly similar and significantly different from the genetic program directing fruiting body formation in S. cellulosum and H. ochraceum. Thus, our analyses reveal an unexpected level of plasticity in the genetic programs for fruiting body formation in the Myxococcales and strongly suggest that the genetic program underlying fruiting body formation in different Myxococcales is not conserved. The evolutionary implications of this finding are discussed.

The early stage of bacterial genome-reductive evolution in the host.

The equine-associated obligate pathogen Burkholderia mallei was developed by reductive evolution involving a substantial portion of the genome from Burkholderia pseudomallei, a free-living opportunistic pathogen. With its short history of divergence (approximately 3.5 myr), B. mallei provides an excellent resource to study the early steps in bacterial genome reductive evolution in the host. By examining 20 genomes of B. mallei and B. pseudomallei, we found that stepwise massive expansion of IS (insertion sequence) elements ISBma1, ISBma2, and IS407A occurred during the evolution of B. mallei. Each element proliferated through the sites where its target selection preference was met. Then, ISBma1 and ISBma2 contributed to the further spread of IS407A by providing secondary insertion sites. This spread increased genomic deletions and rearrangements, which were predominantly mediated by IS407A. There were also nucleotide-level disruptions in a large number of genes. However, no significant signs of erosion were yet noted in these genes. Intriguingly, all these genomic modifications did not seriously alter the gene expression patterns inherited from B. pseudomallei. This efficient and elaborate genomic transition was enabled largely through the formation of the highly flexible IS-blended genome and the guidance by selective forces in the host. The detailed IS intervention, unveiled for the first time in this study, may represent the key component of a general mechanism for early bacterial evolution in the host.