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September 22, 2019

Regulation of yeast-to-hyphae transition in Yarrowia lipolytica.

The yeast Yarrowia lipolytica undergoes a morphological transition from yeast-to-hyphal growth in response to environmental conditions. A forward genetic screen was used to identify mutants that reliably remain in the yeast phase, which were then assessed by whole-genome sequencing. All the smooth mutants identified, so named because of their colony morphology, exhibit independent loss of DNA at a repetitive locus made up of interspersed ribosomal DNA and short 10- to 40-mer telomere-like repeats. The loss of repetitive DNA is associated with downregulation of genes with stress response elements (5′-CCCCT-3′) and upregulation of genes with cell cycle box (5′-ACGCG-3′) motifs in their promoter region. The stress response element is bound by the transcription factor Msn2p in Saccharomyces cerevisiae We confirmed that the Y. lipolyticamsn2 (Ylmsn2) ortholog is required for hyphal growth and found that overexpression of Ylmsn2 enables hyphal growth in smooth strains. The cell cycle box is bound by the Mbp1p/Swi6p complex in S. cerevisiae to regulate G1-to-S phase progression. We found that overexpression of either the Ylmbp1 or Ylswi6 homologs decreased hyphal growth and that deletion of either Ylmbp1 or Ylswi6 promotes hyphal growth in smooth strains. A second forward genetic screen for reversion to hyphal growth was performed with the smooth-33 mutant to identify additional genetic factors regulating hyphal growth in Y. lipolytica Thirteen of the mutants sequenced from this screen had coding mutations in five kinases, including the histidine kinases Ylchk1 and Ylnik1 and kinases of the high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase cascade Ylssk2, Ylpbs2, and Ylhog1 Together, these results demonstrate that Y. lipolytica transitions to hyphal growth in response to stress through multiple signaling pathways.IMPORTANCE Many yeasts undergo a morphological transition from yeast-to-hyphal growth in response to environmental conditions. We used forward and reverse genetic techniques to identify genes regulating this transition in Yarrowia lipolytica We confirmed that the transcription factor Ylmsn2 is required for the transition to hyphal growth and found that signaling by the histidine kinases Ylchk1 and Ylnik1 as well as the MAP kinases of the HOG pathway (Ylssk2, Ylpbs2, and Ylhog1) regulates the transition to hyphal growth. These results suggest that Y. lipolytica transitions to hyphal growth in response to stress through multiple kinase pathways. Intriguingly, we found that a repetitive portion of the genome containing telomere-like and rDNA repeats may be involved in the transition to hyphal growth, suggesting a link between this region and the general stress response. Copyright © 2018 Pomraning et al.

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September 22, 2019

Genomic insights into multidrug-resistance, mating and virulence in Candida auris and related emerging species.

Candida auris is an emergent multidrug-resistant fungal pathogen causing increasing reports of outbreaks. While distantly related to C. albicans and C. glabrata, C. auris is closely related to rarely observed and often multidrug-resistant species from the C. haemulonii clade. Here, we analyze near complete genome assemblies for the four C. auris clades and three related species, and map intra- and inter-species rearrangements across the seven chromosomes. Using RNA-Seq-guided gene predictions, we find that most mating and meiosis genes are conserved and that clades contain either the MTLa or MTLa mating loci. Comparing the genomes of these emerging species to those of other Candida species identifies genes linked to drug resistance and virulence, including expanded families of transporters and lipases, as well as mutations and copy number variants in ERG11. Gene expression analysis identifies transporters and metabolic regulators specific to C. auris and those conserved with related species which may contribute to differences in drug response in this emerging fungal clade.

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September 22, 2019

Investigation of a cluster of Sphingomonas koreensis infections.

Plumbing systems are an infrequent but known reservoir for opportunistic microbial pathogens that can infect hospitalized patients. In 2016, a cluster of clinical sphingomonas infections prompted an investigation.We performed whole-genome DNA sequencing on clinical isolates of multidrug-resistant Sphingomonas koreensis identified from 2006 through 2016 at the National Institutes of Health (NIH) Clinical Center. We cultured S. koreensis from the sinks in patient rooms and performed both whole-genome and shotgun metagenomic sequencing to identify a reservoir within the infrastructure of the hospital. These isolates were compared with clinical and environmental S. koreensis isolates obtained from other institutions.The investigation showed that two isolates of S. koreensis obtained from the six patients identified in the 2016 cluster were unrelated, but four isolates shared more than 99.92% genetic similarity and were resistant to multiple antibiotic agents. Retrospective analysis of banked clinical isolates of sphingomonas from the NIH Clinical Center revealed the intermittent recovery of a clonal strain over the past decade. Unique single-nucleotide variants identified in strains of S. koreensis elucidated the existence of a reservoir in the hospital plumbing. Clinical S. koreensis isolates from other facilities were genetically distinct from the NIH isolates. Hospital remediation strategies were guided by results of microbiologic culturing and fine-scale genomic analyses.This genomic and epidemiologic investigation suggests that S. koreensis is an opportunistic human pathogen that both persisted in the NIH Clinical Center infrastructure across time and space and caused health care-associated infections. (Funded by the NIH Intramural Research Programs.).

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September 22, 2019

Genomic characterization of ß-glucuronidase-positive Escherichia coli O157:H7 producing Stx2a.

Among Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 strains, those producing Stx2a cause more severe diseases. Atypical STEC O157:H7 strains showing a ß-glucuronidase-positive phenotype (GP STEC O157:H7) have rarely been isolated from humans, mostly from persons with asymptomatic or mild infections; Stx2a-producing strains have not been reported. We isolated, from a patient with bloody diarrhea, a GP STEC O157:H7 strain (PV15-279) that produces Stx2a in addition to Stx1a and Stx2c. Genomic comparison with other STEC O157 strains revealed that PV15-279 recently emerged from the stx1a/stx2c-positive GP STEC O157:H7 clone circulating in Japan. Major virulence genes are shared between typical (ß-glucuronidase-negative) and GP STEC O157:H7 strains, and the Stx2-producing ability of PV15-279 is comparable to that of typical STEC O157:H7 strains; therefore, PV15-279 presents a virulence potential similar to that of typical STEC O157:H7. This study reveals the importance of GP O157:H7 as a source of highly pathogenic STEC clones.

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September 22, 2019

Detection and visualization of complex structural variants from long reads.

With applications in cancer, drug metabolism, and disease etiology, understanding structural variation in the human genome is critical in advancing the thrusts of individualized medicine. However, structural variants (SVs) remain challenging to detect with high sensitivity using short read sequencing technologies. This problem is exacerbated when considering complex SVs comprised of multiple overlapping or nested rearrangements. Longer reads, such as those from Pacific Biosciences platforms, often span multiple breakpoints of such events, and thus provide a way to unravel small-scale complexities in SVs with higher confidence.We present CORGi (COmplex Rearrangement detection with Graph-search), a method for the detection and visualization of complex local genomic rearrangements. This method leverages the ability of long reads to span multiple breakpoints to untangle SVs that appear very complicated with respect to a reference genome. We validated our approach against both simulated long reads, and real data from two long read sequencing technologies. We demonstrate the ability of our method to identify breakpoints inserted in synthetic data with high accuracy, and the ability to detect and plot SVs from NA12878 germline, achieving 88.4% concordance between the two sets of sequence data. The patterns of complexity we find in many NA12878 SVs match known mechanisms associated with DNA replication and structural variant formation, and highlight the ability of our method to automatically label complex SVs with an intuitive combination of adjacent or overlapping reference transformations.CORGi is a method for interrogating genomic regions suspected to contain local rearrangements using long reads. Using pairwise alignments and graph search CORGi produces labels and visualizations for local SVs of arbitrary complexity.

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September 22, 2019

Complete and de novo assembly of the Leishmania braziliensis (M2904) genome.

Leishmania braziliensis is the etiological agent of American mucosal leishmaniasis, one of the most severe clinical forms of leishmaniasis. Here, we report the assembly of the L. braziliensis (M2904) genome into 35 continuous chromosomes. Also, the annotation of 8395 genes is provided. The public availability of this information will contribute to a better knowledge of this pathogen and help in the search for vaccines and novel drug targets aimed to control the disease caused by this Leishmania species.

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September 22, 2019

Trophoblast organoids as a model for maternal-fetal interactions during human placentation.

The placenta is the extraembryonic organ that supports the fetus during intrauterine life. Although placental dysfunction results in major disorders of pregnancy with immediate and lifelong consequences for the mother and child, our knowledge of the human placenta is limited owing to a lack of functional experimental models1. After implantation, the trophectoderm of the blastocyst rapidly proliferates and generates the trophoblast, the unique cell type of the placenta. In vivo, proliferative villous cytotrophoblast cells differentiate into two main sub-populations: syncytiotrophoblast, the multinucleated epithelium of the villi responsible for nutrient exchange and hormone production, and extravillous trophoblast cells, which anchor the placenta to the maternal decidua and transform the maternal spiral arteries2. Here we describe the generation of long-term, genetically stable organoid cultures of trophoblast that can differentiate into both syncytiotrophoblast and extravillous trophoblast. We used human leukocyte antigen (HLA) typing to confirm that the organoids were derived from the fetus, and verified their identities against four trophoblast-specific criteria3. The cultures organize into villous-like structures, and we detected the secretion of placental-specific peptides and hormones, including human chorionic gonadotropin (hCG), growth differentiation factor 15 (GDF15) and pregnancy-specific glycoprotein (PSG) by mass spectrometry. The organoids also differentiate into HLA-G+ extravillous trophoblast cells, which vigorously invade in three-dimensional cultures. Analysis of the methylome reveals that the organoids closely resemble normal first trimester placentas. This organoid model will be transformative for studying human placental development and for investigating trophoblast interactions with the local and systemic maternal environment.

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September 22, 2019

Genetics and genomics of an unusual selfish sex ratio distortion in an insect.

Diverse selfish genetic elements have evolved the ability to manipulate reproduction to increase their transmission, and this can result in highly distorted sex ratios [1]. Indeed, one of the major explanations for why sex determination systems are so dynamic is because they are shaped by ongoing coevolutionary arms races between sex-ratio-distorting elements and the rest of the genome [2]. Here, we use genetic crosses and genome analysis to describe an unusual sex ratio distortion with striking consequences on genome organization in a booklouse species, Liposcelis sp. (Insecta: Psocodea), in which two types of females coexist. Distorter females never produce sons but must mate with males (the sons of nondistorting females) to reproduce [3]. Although they are diploid and express the genes inherited from their fathers in somatic tissues, distorter females only ever transmit genes inherited from their mothers. As a result, distorter females have unusual chimeric genomes, with distorter-restricted chromosomes diverging from their nondistorting counterparts and exhibiting features of a giant non-recombining sex chromosome. The distorter-restricted genome has also acquired a gene from the bacterium Wolbachia, a well-known insect reproductive manipulator; we found that this gene has independently colonized the genomes of two other insect species with unusual reproductive systems, suggesting possible roles in sex ratio distortion in this remarkable genetic system. Copyright © 2018 Elsevier Ltd. All rights reserved.

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September 22, 2019

Chemical Synergy between Ionophore PBT2 and Zinc Reverses Antibiotic Resistance.

The World Health Organization reports that antibiotic-resistant pathogens represent an imminent global health disaster for the 21st century. Gram-positive superbugs threaten to breach last-line antibiotic treatment, and the pharmaceutical industry antibiotic development pipeline is waning. Here we report the synergy between ionophore-induced physiological stress in Gram-positive bacteria and antibiotic treatment. PBT2 is a safe-for-human-use zinc ionophore that has progressed to phase 2 clinical trials for Alzheimer’s and Huntington’s disease treatment. In combination with zinc, PBT2 exhibits antibacterial activity and disrupts cellular homeostasis in erythromycin-resistant group A Streptococcus (GAS), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE). We were unable to select for mutants resistant to PBT2-zinc treatment. While ineffective alone against resistant bacteria, several clinically relevant antibiotics act synergistically with PBT2-zinc to enhance killing of these Gram-positive pathogens. These data represent a new paradigm whereby disruption of bacterial metal homeostasis reverses antibiotic-resistant phenotypes in a number of priority human bacterial pathogens.IMPORTANCE The rise of bacterial antibiotic resistance coupled with a reduction in new antibiotic development has placed significant burdens on global health care. Resistant bacterial pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus are leading causes of community- and hospital-acquired infection and present a significant clinical challenge. These pathogens have acquired resistance to broad classes of antimicrobials. Furthermore, Streptococcus pyogenes, a significant disease agent among Indigenous Australians, has now acquired resistance to several antibiotic classes. With a rise in antibiotic resistance and reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. As stated by the WHO Director-General, “On current trends, common diseases may become untreatable. Doctors facing patients will have to say, Sorry, there is nothing I can do for you.” Copyright © 2018 Bohlmann et al.

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September 22, 2019

Description of Schaedlerella arabinophila gen. nov., sp. nov., a D-arabinose utilizing bacterium isolated from feces of C57BL/6J mice and a close relative of Clostridium sp. ASF 502

The use of gnotobiotics has gained large interest in recent years due to technological advances that have revealed the importance of host-associated microbiomes for host physiology and health. One of the oldest and most important gnotobiotics mouse model, the Altered Schaedler Flora (ASF) has been used for several decades. ASF comprises eight different bacterial species, which have been characterized to different extent, but only few are available through public strain collections. Here, the isolation of a close relative to one of the less studied ASF strains, Clostridium sp. ASF 502, is reported. Isolate TLL-A1, which shares 99.6% 16S rRNA gene sequence identity with Clostridium sp. ASF 502, was obtained from feces of C57BL/6J mice where is was detectable at a relative abundance of less than one percent. D-arabinose was used as sole carbon source in the anaerobic cultivation medium. Growth experiments with TLL-A1 on different carbon sources and analysis of its ~6.5 gigabase genome indicate that TLL-A1 harbors a large gene repertoire to utilize different carbohydrates for growth. Comparative genome analyses of TLL-A1 and Clostridium sp. ASF 502 reveal differences in genome content between the two strains, in particular with regards to carbohydrate activating enzymes. Based on physiology and genomic analysis it is proposed to name TLL-A1 to gen. nov. sp. nov Schaedlerella arabinophila TLL-A1 (DSMZ 106076T; KCTC 15657T). The closely related Clostridium sp. ASF 502 is proposed to be renamed to Schaedlerella arabinophila to reflect its taxonomic standing and to keep textquoterightASF 502textquoteright as strain designation.

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September 22, 2019

Phototaxis in a wild isolate of the cyanobacterium Synechococcus elongatus.

Many cyanobacteria, which use light as an energy source via photosynthesis, have evolved the ability to guide their movement toward or away from a light source. This process, termed “phototaxis,” enables organisms to localize in optimal light environments for improved growth and fitness. Mechanisms of phototaxis have been studied in the coccoid cyanobacterium Synechocystis sp. strain PCC 6803, but the rod-shaped Synechococcus elongatus PCC 7942, studied for circadian rhythms and metabolic engineering, has no phototactic motility. In this study we report a recent environmental isolate of S. elongatus, the strain UTEX 3055, whose genome is 98.5% identical to that of PCC 7942 but which is motile and phototactic. A six-gene operon encoding chemotaxis-like proteins was confirmed to be involved in phototaxis. Environmental light signals are perceived by a cyanobacteriochrome, PixJSe (Synpcc7942_0858), which carries five GAF domains that are responsive to blue/green light and resemble those of PixJ from Synechocystis Plate-based phototaxis assays indicate that UTEX 3055 uses PixJSe to sense blue and green light. Mutation of conserved functional cysteine residues in different GAF domains indicates that PixJSe controls both positive and negative phototaxis, in contrast to the multiple proteins that are employed for implementing bidirectional phototaxis in Synechocystis.

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September 22, 2019

CompStor Novos: a low cost yet fast assembly-based variant calling for personal genomes

Application of assembly methods for personal genome analysis from next generation sequencing data has been limited by the requirement for an expensive supercomputer hardware or long computation times when using ordinary resources. We describe CompStor Novos, achieving supercomputer-class performance in de novo assembly computation time on standard server hardware, based on a tiered-memory algorithm. Run on commercial off-the-shelf servers, Novos assembly is more precise and 10-20 times faster than that of existing assembly algorithms. Furthermore, we integrated Novos into a variant calling pipeline and demonstrate that both compute times and precision of calling point variants and indels compare well with standard alignment-based pipelines. Additionally, assembly eliminates bias in the estimation of allele frequency for indels and naturally enables discovery of breakpoints for structural variants with base pair resolution. Thus, Novos bridges the gap between alignment-based and assembly-based genome analyses. Extension and adaption of its underlying algorithm will help quickly and fully harvest information in sequencing reads for personal genome reconstruction.

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September 21, 2019

Toward complete bacterial genome sequencing through the combined use of multiple next-generation sequencing platforms.

PacBio’s long-read sequencing technologies can be successfully used for a complete bacterial genome assembly using recently developed non-hybrid assemblers in the absence of secondgeneration, high-quality short reads. However, standardized procedures that take into account multiple pre-existing second-generation sequencing platforms are scarce. In addition to Illumina HiSeq and Ion Torrent PGM-based genome sequencing results derived from previous studies, we generated further sequencing data, including from the PacBio RS II platform, and applied various bioinformatics tools to obtain complete genome assemblies for five bacterial strains. Our approach revealed that the hierarchical genome assembly process (HGAP) non-hybrid assembler resulted in nearly complete assemblies at a moderate coverage of ~75x, but that different versions produced non-compatible results requiring post processing. The other two platforms further improved the PacBio assembly through scaffolding and a final error correction.

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September 21, 2019

in silico Whole Genome Sequencer & Analyzer (iWGS): a computational pipeline to guide the design and analysis of de novo genome sequencing studies.

The availability of genomes across the tree of life is highly biased toward vertebrates, pathogens, human disease models, and organisms with relatively small and simple genomes. Recent progress in genomics has enabled the de novo decoding of the genome of virtually any organism, greatly expanding its potential for understanding the biology and evolution of the full spectrum of biodiversity. The increasing diversity of sequencing technologies, assays, and de novo assembly algorithms have augmented the complexity of de novo genome sequencing projects in non-model organisms. To reduce the costs and challenges in de novo genome sequencing projects and streamline their experimental design and analysis, we developed iWGS (in silico Whole Genome Sequencer and Analyzer), an automated pipeline for guiding the choice of appropriate sequencing strategy and assembly protocols. iWGS seamlessly integrates the four key steps of a de novo genome sequencing project: data generation (through simulation), data quality control, de novo assembly, and assembly evaluation and validation. The last three steps can also be applied to the analysis of real data. iWGS is designed to enable the user to have great flexibility in testing the range of experimental designs available for genome sequencing projects, and supports all major sequencing technologies and popular assembly tools. Three case studies illustrate how iWGS can guide the design of de novo genome sequencing projects and evaluate the performance of a wide variety of user-specified sequencing strategies and assembly protocols on genomes of differing architectures. iWGS, along with a detailed documentation, is freely available at https://github.com/zhouxiaofan1983/iWGS. Copyright © 2016 Author et al.

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September 21, 2019

Mistranslation drives the evolution of robustness in TEM-1 ß-lactamase.

How biological systems such as proteins achieve robustness to ubiquitous perturbations is a fundamental biological question. Such perturbations include errors that introduce phenotypic mutations into nascent proteins during the translation of mRNA. These errors are remarkably frequent. They are also costly, because they reduce protein stability and help create toxic misfolded proteins. Adaptive evolution might reduce these costs of protein mistranslation by two principal mechanisms. The first increases the accuracy of translation via synonymous “high fidelity” codons at especially sensitive sites. The second increases the robustness of proteins to phenotypic errors via amino acids that increase protein stability. To study how these mechanisms are exploited by populations evolving in the laboratory, we evolved the antibiotic resistance gene TEM-1 in Escherichia coli hosts with either normal or high rates of mistranslation. We analyzed TEM-1 populations that evolved under relaxed and stringent selection for antibiotic resistance by single molecule real-time sequencing. Under relaxed selection, mistranslating populations reduce mistranslation costs by reducing TEM-1 expression. Under stringent selection, they efficiently purge destabilizing amino acid changes. More importantly, they accumulate stabilizing amino acid changes rather than synonymous changes that increase translational accuracy. In the large populations we study, and on short evolutionary timescales, the path of least resistance in TEM-1 evolution consists of reducing the consequences of translation errors rather than the errors themselves.

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