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Asset Tag: Third generation sequencing

September 22, 2019

Evolution of host support for two ancient bacterial symbionts with differentially degraded genomes in a leafhopper host.

Plant sap-feeding insects (Hemiptera) rely on bacterial symbionts for nutrition absent in their diets. These bacteria experience extreme genome reduction and require genetic resources from their hosts, particularly for basic cellular processes other than nutrition synthesis. The host-derived mechanisms that complete these processes have remained poorly understood. It is also unclear how hosts meet the distinct needs of multiple bacterial partners with differentially degraded genomes. To address these questions, we investigated the cell-specific gene-expression patterns in the symbiotic organs of the aster leafhopper (ALF), Macrosteles quadrilineatus (Cicadellidae). ALF harbors two intracellular symbionts that have two of the smallest known bacterial genomes: Nasuia (112 kb) and Sulcia (190 kb). Symbionts are segregated into distinct host cell types (bacteriocytes) and vary widely in their basic cellular capabilities. ALF differentially expresses thousands of genes between the bacteriocyte types to meet the functional needs of each symbiont, including the provisioning of metabolites and support of cellular processes. For example, the host highly expresses genes in the bacteriocytes that likely complement gene losses in nucleic acid synthesis, DNA repair mechanisms, transcription, and translation. Such genes are required to function in the bacterial cytosol. Many host genes comprising these support mechanisms are derived from the evolution of novel functional traits via horizontally transferred genes, reassigned mitochondrial support genes, and gene duplications with bacteriocyte-specific expression. Comparison across other hemipteran lineages reveals that hosts generally support the incomplete symbiont cellular processes, but the origins of these support mechanisms are generally specific to the host-symbiont system.Copyright © 2018 the Author(s). Published by PNAS.

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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

Genome wide characterization of enterotoxigenic Escherichia coli serogroup O6 isolates from multiple outbreaks and sporadic infections from 1975-2016.

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhea globally, particularly among children under the age of five in developing countries. ETEC O6 is the most common ETEC serogroup, yet the genome wide population structure of isolates of this serogroup is yet to be determined. In this study, we have characterized 40 ETEC O6 isolates collected between 1975-2016 by whole genome sequencing (WGS) and by phenotypic antimicrobial susceptibility testing. To determine the relatedness of isolates, we evaluated two methods-whole genome high-quality single nucleotide polymorphism (whole genome-hqSNP) and core genome SNP analyses using Lyve-SET and Parsnp respectively. All isolates were tested for antimicrobial susceptibility using a panel of 14 antibiotics. ResFinder 2.1 and a custom quinolone resistance determinants workflow were used for resistance determinant detection. VirulenceFinder 1.5 was used for prediction of the virulence genes. Thirty-seven isolates clustered into three major clades (I, II, III) by whole genome-hqSNP and core genome SNP analyses, while three isolates included in the whole genome-hqSNP analysis only did not cluster with clades I-III by both analyses and formed a distantly related outgroup, designated clade IV. Median number of pairwise whole genome-hqSNPs in clonal ETEC O6 outbreaks ranged from 0 to 5. Of the 40 isolates tested for antimicrobial susceptibility, 18 isolates were pansusceptible. Twenty-two isolates were resistant to at least one antibiotic, nine of which were multidrug resistant. Phenotypic antimicrobial resistance (AR) correlated with AR determinants in 22 isolates. Thirty-two isolates harbored both enterotoxin virulence genes while the remaining 8 isolates had only one of the two virulence genes. In summary, whole genome-hqSNP and core genome SNP analyses from this study revealed similar evolutionary relationships and an overall diversity of ETEC O6 isolates independent of time of isolation. Less than 5 pairwise hqSNPs between ETEC O6 isolates is circumstantially indicative of an outbreak cluster. Findings from this study will be a basis for quicker outbreak detection and control by efficient subtyping by WGS.

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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

Genomic and transcriptomic comparisons of closely related malaria parasites differing in virulence and sequestration pattern.

Background: Malaria parasite species differ greatly in the harm they do to humans. While P. falciparum kills hundreds of thousands per year, P. vivax kills much less often and P. malariae is relatively benign. Strains of the rodent malaria parasite Plasmodium chabaudi show phenotypic variation in virulence during infections of laboratory mice. This make it an excellent species to study genes which may be responsible for this trait. By understanding the mechanisms which underlie differences in virulence we can learn how parasites adapt to their hosts and how we might prevent disease. Methods: Here we present a complete reference genome sequence for a more virulent P. chabaudi strain, PcCB, and perform a detailed comparison with the genome of the less virulent PcAS strain. Results: We found the greatest variation in the subtelomeric regions, in particular amongst the sequences of the pir gene family, which has been associated with virulence and establishment of chronic infection. Despite substantial variation at the sequence level, the repertoire of these genes has been largely maintained, highlighting the requirement for functional conservation as well as diversification in host-parasite interactions. However, a subset of pir genes, previously associated with increased virulence, were more highly expressed in PcCB, suggesting a role for this gene family in virulence differences between strains. We found that core genes involved in red blood cell invasion have been under positive selection and that the more virulent strain has a greater preference for reticulocytes, which has elsewhere been associated with increased virulence. Conclusions: These results provide the basis for a mechanistic understanding of the phenotypic differences between Plasmodium chabaudi strains, which might ultimately be translated into a better understanding of malaria parasites affecting humans.

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September 22, 2019

Genomic characterization of a B chromosome in Lake Malawi cichlid fishes.

B chromosomes (Bs) were discovered a century ago, and since then, most studies have focused on describing their distribution and abundance using traditional cytogenetics. Only recently have attempts been made to understand their structure and evolution at the level of DNA sequence. Many questions regarding the origin, structure, function, and evolution of B chromosomes remain unanswered. Here, we identify B chromosome sequences from several species of cichlid fish from Lake Malawi by examining the ratios of DNA sequence coverage in individuals with or without B chromosomes. We examined the efficiency of this method, and compared results using both Illumina and PacBio sequence data. The B chromosome sequences detected in 13 individuals from 7 species were compared to assess the rates of sequence replacement. B-specific sequence common to at least 12 of the 13 datasets were identified as the “Core” B chromosome. The location of B sequence homologs throughout the genome provides further support for theories of B chromosome evolution. Finally, we identified genes and gene fragments located on the B chromosome, some of which may regulate the segregation and maintenance of the B chromosome.

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September 22, 2019

N6-methyladenine DNA methylation in Japonica and Indica rice genomes and its association with gene expression, plant development, and stress responses.

N6-Methyladenine (6mA) DNA methylation has recently been implicated as a potential new epigenetic marker in eukaryotes, including the dicot model Arabidopsis thaliana. However, the conservation and divergence of 6mA distribution patterns and functions in plants remain elusive. Here we report high-quality 6mA methylomes at single-nucleotide resolution in rice based on substantially improved genome sequences of two rice cultivars, Nipponbare (Nip; Japonica) and 93-11 (Indica). Analysis of 6mA genomic distribution and its association with transcription suggest that 6mA distribution and function is rather conserved between rice and Arabidopsis. We found that 6mA levels are positively correlated with the expression of key stress-related genes, which may be responsible for the difference in stress tolerance between Nip and 93-11. Moreover, we showed that mutations in DDM1 cause defects in plant growth and decreased 6mA level. Our results reveal that 6mA is a conserved DNA modification that is positively associated with gene expression and contributes to key agronomic traits in plants. Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.

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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

How resurrection plants survive being hung out to dry.

Resurrection plants have the unique ability to survive extreme dehydration (desiccation), lying dormant for months or sometimes years until rehydration is possible. This formidable survival strategy has independently evolved several times across the land plant phylogeny, and several phylogenetically diverse resurrection plant genomes have been sequenced and assembled in an attempt to understand the causal genetic mechanisms. Large-scale comparisons across each of these phylogenetically distant resurrection plant genomes reveals that some conserved molecular signatures may underlie desiccation tolerance (Illing et al., 2005; Zhang and Bartels, 2018), but overall the genes, networks, and regulatory factors that underlie desiccation tolerance remain largely unknown.

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September 22, 2019

Role of phage ?1 in two strains of Salmonella Rissen, sensitive and resistant to phage ?1.

The study describes the Salmonella Rissen phage ?1 isolated from the ?1-sensitive Salmonella Rissen strain RW. The same phage was then used to select the resistant strain RR?1+, which can harbour or not ?1.Following this approach, we found that ?1, upon excision from RW cells with mitomycin, behaves as a temperate phage: lyses host cells and generates phage particles; instead, upon spontaneous excision from RR?1+ cells, it does not generate phage particles; causes loss of phage resistance; switches the O-antigen from the smooth to the rough phenotype, and favors the transition of Salmonella Rissen from the planktonic to the biofilm growth. The RW and RR?1+ strains differ by 10 genes; of these, only two (phosphomannomutase_1 and phosphomannomutase_2; both involved in the mannose synthesis pathway) display significant differences at the expression levels. This result suggests that phage resistance is associated with these two genes.Phage ?1 displays the unusual property of behaving as template as well as lytic phage. This feature was used by the phage to modulate several phases of Salmonella Rissen lifestyle.

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September 22, 2019

Comparative analysis of Faecalibacterium prausnitzii genomes shows a high level of genome plasticity and warrants separation into new species-level taxa.

Faecalibacterium prausnitzii is a ubiquitous member of the human gut microbiome, constituting up to 15% of the total bacteria in the human gut. Substantial evidence connects decreased levels of F. prausnitzii with the onset and progression of certain forms of inflammatory bowel disease, which has been attributed to its anti-inflammatory potential. Two phylogroups of F. prausnitzii have been identified, with a decrease in phylogroup I being a more sensitive marker of intestinal inflammation. Much of the genomic and physiological data available to date was collected using phylogroup II strains. Little analysis of F. prausnitzii genomes has been performed so far and genetic differences between phylogroups I and II are poorly understood.In this study we sequenced 11 additional F. prausnitzii genomes and performed comparative genomics to investigate intraspecies diversity, functional gene complement and the mobilome of 31 high-quality draft and complete genomes. We reveal a very low level of average nucleotide identity among F. prausnitzii genomes and a high level of genome plasticity. Two genomogroups can be separated based on differences in functional gene complement, albeit that this division does not fully agree with separation based on conserved gene phylogeny, highlighting the importance of horizontal gene transfer in shaping F. prausnitzii genomes. The difference between the two genomogroups is mainly in the complement of genes associated with catabolism of carbohydrates (such as a predicted sialidase gene in genomogroup I) and amino acids, as well as defense mechanisms.Based on the combination of ANI of genomic sequences, phylogenetic analysis of core proteomes and functional differences we propose to separate the species F. prausnitzii into two new species level taxa: F. prausnitzii sensu stricto (neotype strain A2-165T?=?DSM 17677T?=?JCM 31915T) and F. moorei sp. nov. (type strain ATCC 27768T?=?NCIMB 13872T).

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September 22, 2019

Comparative genomics of 84 Pectobacterium genomes reveals the variations related to a pathogenic lifestyle.

Pectobacterium spp. are necrotrophic bacterial plant pathogens of the family Pectobacteriaceae, responsible for a wide spectrum of diseases of important crops and ornamental plants including soft rot, blackleg, and stem wilt. P. carotovorum is a genetically heterogeneous species consisting of three valid subspecies, P. carotovorum subsp. brasiliense (Pcb), P. carotovorum subsp. carotovorum (Pcc), and P. carotovorum subsp. odoriferum (Pco).Thirty-two P. carotovorum strains had their whole genomes sequenced, including the first complete genome of Pco and another circular genome of Pcb, as well as the high-coverage genome sequences for 30 additional strains covering Pcc, Pcb, and Pco. In combination with 52 other publicly available genome sequences, the comparative genomics study of P. carotovorum and other four closely related species P. polaris, P. parmentieri, P. atrosepticum, and Candidatus P. maceratum was conducted focusing on CRISPR-Cas defense systems and pathogenicity determinants. Our analysis identified two CRISPR-Cas types (I-F and I-E) in Pectobacterium, as well as another I-C type in Dickeya that is not found in Pectobacterium. The core pathogenicity factors (e.g., plant cell wall-degrading enzymes) were highly conserved, whereas some factors (e.g., flagellin, siderophores, polysaccharides, protein secretion systems, and regulatory factors) were varied among these species and/or subspecies. Notably, a novel type of T6SS as well as the sorbitol metabolizing srl operon was identified to be specific to Pco in Pectobacterium.This study not only advances the available knowledge about the genetic differentiation of individual subspecies of P. carotovorum, but also delineates the general genetic features of P. carotovorum by comparison with its four closely related species, thereby substantially enriching the extent of information now available for functional genomic investigations about Pectobacterium.

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