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April 21, 2020

Liriodendron genome sheds light on angiosperm phylogeny and species-pair differentiation.

The genus Liriodendron belongs to the family Magnoliaceae, which resides within the magnoliids, an early diverging lineage of the Mesangiospermae. However, the phylogenetic relationship of magnoliids with eudicots and monocots has not been conclusively resolved and thus remains to be determined1-6. Liriodendron is a relict lineage from the Tertiary with two distinct species-one East Asian (L. chinense (Hemsley) Sargent) and one eastern North American (L. tulipifera Linn)-identified as a vicariad species pair. However, the genetic divergence and evolutionary trajectories of these species remain to be elucidated at the whole-genome level7. Here, we report the first de novo genome assembly of a plant in the Magnoliaceae, L. chinense. Phylogenetic analyses suggest that magnoliids are sister to the clade consisting of eudicots and monocots, with rapid diversification occurring in the common ancestor of these three lineages. Analyses of population genetic structure indicate that L. chinense has diverged into two lineages-the eastern and western groups-in China. While L. tulipifera in North America is genetically positioned between the two L. chinense groups, it is closer to the eastern group. This result is consistent with phenotypic observations that suggest that the eastern and western groups of China may have diverged long ago, possibly before the intercontinental differentiation between L. chinense and L. tulipifera. Genetic diversity analyses show that L. chinense has tenfold higher genetic diversity than L. tulipifera, suggesting that the complicated regions comprising east-west-orientated mountains and the Yangtze river basin (especially near 30°?N latitude) in East Asia offered more successful refugia than the south-north-orientated mountain valleys in eastern North America during the Quaternary glacial period.


April 21, 2020

Using Cre-recombinase-driven Polylox barcoding for in vivo fate mapping in mice.

Fate mapping is a powerful genetic tool for linking stem or progenitor cells with their progeny, and hence for defining cell lineages in vivo. The resolution of fate mapping depends on the numbers of distinct markers that are introduced in the beginning into stem or progenitor cells; ideally, numbers should be sufficiently large to allow the tracing of output from individual cells. Highly diverse genetic barcodes can serve this purpose. We recently developed an endogenous genetic barcoding system, termed Polylox. In Polylox, random DNA recombination can be induced by transient activity of Cre recombinase in a 2.1-kb-long artificial recombination substrate that has been introduced into a defined locus in mice (Rosa26Polylox reporter mice). Here, we provide a step-by-step protocol for the use of Polylox, including barcode induction and estimation of induction efficiency, barcode retrieval with single-molecule real-time (SMRT) DNA sequencing followed by computational barcode identification, and the calculation of barcode-generation probabilities, which is key for estimations of single-cell labeling for a given number of stem cells. Thus, Polylox barcoding enables high-resolution fate mapping in essentially all tissues in mice for which inducible Cre driver lines are available. Alternative methods include ex vivo cell barcoding, inducible transposon insertion and CRISPR-Cas9-based barcoding; Polylox currently allows combining non-invasive and cell-type-specific labeling with high label diversity. The execution time of this protocol is ~2-3 weeks for experimental data generation and typically <2 d for computational Polylox decoding and downstream analysis.


April 21, 2020

Characterization of a blaIMP-4-carrying plasmid from Enterobacter cloacae of swine origin.

To characterize an MDR blaIMP-4-harbouring plasmid from Enterobacter cloacae EC62 of swine origin in China.Plasmid pIMP-4-EC62 from E. cloacae EC62 was transferred by conjugation via filter mating into Escherichia coli J53. Plasmid DNA was extracted from an E. coli J53 transconjugant and sequenced using single-molecule real-time (SMRT) technology. MIC values for both the isolate EC62 and the transconjugant were determined using the broth microdilution and agar dilution methods. Plasmid stability in both the isolate EC62 and the transconjugant was assessed through a series of passages on antibiotic-free media.Plasmid pIMP-4-EC62 is 314351?bp in length, encodes 369 predicted proteins and harbours a novel class 1 integron carrying blaIMP-4 and a group II intron. The blaIMP-4-bearing plasmid belongs to the IncHI2/ST1 incompatibility group. Sequence analysis showed that pIMP-4-EC62 carries four MDR regions and several gene clusters encoding heavy metal resistance. Plasmid pIMP-4-EC62 was stably maintained in both the E. cloacae EC62 isolate and the transconjugant E. coli J53-pIMP-4-EC62 in the absence of selective pressure. Analysis of the evolutionary relatedness of selected IncHI2 plasmids indicates that ST1-type plasmids are key carriers of carbapenemase genes among IncHI2 plasmids.pIMP-4-EC62 represents the first fully sequenced IncHI2-type blaIMP-4-harbouring plasmid from E. cloacae in China. Co-location of blaIMP-4 with other resistance genes on an MDR plasmid is likely to further accelerate the dissemination of blaIMP-4 by co-selection among bacteria from humans, animals and the environment under the selective pressure of other antimicrobial agents, heavy metals and disinfectants. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.


April 21, 2020

Sequential evolution of virulence and resistance during clonal spread of community-acquired methicillin-resistant Staphylococcus aureus.

The past two decades have witnessed an alarming expansion of staphylococcal disease caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The factors underlying the epidemic expansion of CA-MRSA lineages such as USA300, the predominant CA-MRSA clone in the United States, are largely unknown. Previously described virulence and antimicrobial resistance genes that promote the dissemination of CA-MRSA are carried by mobile genetic elements, including phages and plasmids. Here, we used high-resolution genomics and experimental infections to characterize the evolution of a USA300 variant plaguing a patient population at increased risk of infection to understand the mechanisms underlying the emergence of genetic elements that facilitate clonal spread of the pathogen. Genetic analyses provided conclusive evidence that fitness (manifest as emergence of a dominant clone) changed coincidently with the stepwise emergence of (i) a unique prophage and mutation of the regulator of the pyrimidine nucleotide biosynthetic operon that promoted abscess formation and colonization, respectively, thereby priming the clone for success; and (ii) a unique plasmid that conferred resistance to two topical microbiocides, mupirocin and chlorhexidine, frequently used for decolonization and infection prevention. The resistance plasmid evolved through successive incorporation of DNA elements from non-S. aureus spp. into an indigenous cryptic plasmid, suggesting a mechanism for interspecies genetic exchange that promotes antimicrobial resistance. Collectively, the data suggest that clonal spread in a vulnerable population resulted from extensive clinical intervention and intense selection pressure toward a pathogen lifestyle that involved the evolution of consequential mutations and mobile genetic elements.


April 21, 2020

The complete mitochondrial genome of the tree frog, Polypedates braueri (Anura, Rhacophoridae)

We determined the complete mitochondrial genome of the tree frog, Polypedates braueri using next generation sequencing (NGS) and Sanger sequencing. The mitogenome of P. braueri was 19,904?bp in length, which contained 12 protein-coding genes, 22 tRNAs, two rRNAs, and two control regions (D-Loop). A noncoding sequence (NC) was discovered between tRNALys and ATP6 gene, as well as replaced the original position of ATP8 gene. The ND5 gene was found between the two control regions. More mitochondrial genomic information will contribute to revealing the phylogenetic relationships among species of the genus Polypedates.


April 21, 2020

High-Resolution Evolutionary Analysis of Within-Host Hepatitis C Virus Infection.

Despite recent breakthroughs in treatment of hepatitis C virus (HCV) infection, we have limited understanding of how virus diversity generated within individuals impacts the evolution and spread of HCV variants at the population scale. Addressing this gap is important for identifying the main sources of disease transmission and evaluating the risk of drug-resistance mutations emerging and disseminating in a population.We have undertaken a high-resolution analysis of HCV within-host evolution from 4 individuals coinfected with human immunodeficiency virus 1 (HIV-1). We used long-read, deep-sequenced data of full-length HCV envelope glycoprotein, longitudinally sampled from acute to chronic HCV infection to investigate the underlying viral population and evolutionary dynamics.We found statistical support for population structure maintaining the within-host HCV genetic diversity in 3 out of 4 individuals. We also report the first population genetic estimate of the within-host recombination rate for HCV (0.28 × 10-7 recombination/site/year), which is considerably lower than that estimated for HIV-1 and the overall nucleotide substitution rate estimated during HCV infection.Our findings indicate that population structure and strong genetic linkage shapes within-host HCV evolutionary dynamics. These results will guide the future investigation of potential HCV drug resistance adaptation during infection, and at the population scale. © The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.


April 21, 2020

Microbial community and fermentation characteristic of Italian ryegrass silage prepared with corn stover and lactic acid bacteria.

The bacterial community determined via PacBio single molecule, real-time sequencing technology (SMRT) and the fermentation characteristics of Italian ryegrass (IR, 82% moisture) silage prepared with corn stover (CS) were investigated. A selected strain of Lactobacillus plantarum (L694) and a commercial inoculant stain of Lactobacillus plantarum (LP) were used as additives. Lactic acid bacteria (LAB) effectively improved silage quality. After fermentation, Lactobacillus plantarum was the dominant species in IR?+?LP and IR?+?L694 treatments, which led to higher (P?


April 21, 2020

Deciphering bacterial epigenomes using modern sequencing technologies.

Prokaryotic DNA contains three types of methylation: N6-methyladenine, N4-methylcytosine and 5-methylcytosine. The lack of tools to analyse the frequency and distribution of methylated residues in bacterial genomes has prevented a full understanding of their functions. Now, advances in DNA sequencing technology, including single-molecule, real-time sequencing and nanopore-based sequencing, have provided new opportunities for systematic detection of all three forms of methylated DNA at a genome-wide scale and offer unprecedented opportunities for achieving a more complete understanding of bacterial epigenomes. Indeed, as the number of mapped bacterial methylomes approaches 2,000, increasing evidence supports roles for methylation in regulation of gene expression, virulence and pathogen-host interactions.


April 21, 2020

Development of Tigecycline Resistance in Carbapenemase-Producing Klebsiella pneumoniae Sequence Type 147 via AcrAB Overproduction Mediated by Replacement of the ramA Promoter.

Carbapenem-resistant K. pneumoniae 2297, isolated from a patient treated with tigecycline for pneumonia, developed tigecycline resistance, in contrast to carbapenem-resistant isolate 1215, which was collected four months prior to the 2297 isolate. Mechanisms underlying tigecycline resistance were elucidated for the clinical isolates.The tigecycline minimum inhibitory concentration (MIC) was determined using the broth microdilution method, with or without phenylalanine-arginine ß-naphthylamide (PABN), and whole-genome sequencing was carried out by single-molecule real-time sequencing. The expression levels of the genes acrA, oqxA, ramA, rarA, and rpoB were determined by reverse-transcription quantitative PCR.Both isolates presented identical antibiograms, except for tigecycline, which showed an MIC of 0.5 mg/L in 1215 and 2 mg/L in 2297. The addition of PABN to tigecycline-resistant 2297 caused a four-fold decrease in the tigecycline MIC to 0.5 mg/L, although acrA expression (encoding the AcrAB efflux pump) was upregulated by 2.5 fold and ramA expression (encoding the pump activator RamA) was upregulated by 1.4 fold. We identified a 6,096-bp fragment insertion flanking direct TATAT repeats that disrupted the romA gene located upstream of ramA in the chromosome of K. pneumoniae 2297; the insertion led the ramA gene promoter replacement resulting in stronger activation of the gene.The K. pneumoniae isolate developed tigecycline resistance during tigecycline treatment. It was related to the overexpression of the AcrAB resistance-nodulation-cell division efflux system due to promoter replacement. © The Korean Society for Laboratory Medicine.


April 21, 2020

Antimicrobial resistance-encoding plasmid clusters with heterogeneous MDR regions driven by IS26 in a single Escherichia coli isolate.

IS26-flanked transposons played an increasingly important part in the mobilization and development of resistance determinants. Heterogeneous resistance-encoding plasmid clusters with polymorphic MDR regions (MRRs) conferred by IS26 in an individual Escherichia coli isolate have not yet been detected.To characterize the complete sequence of a novel blaCTX-M-65- and fosA3-carrying IncZ-7 plasmid with dynamic MRRs from an E. coli isolate, and to depict the mechanism underlying the spread of resistance determinants and genetic polymorphisms.The molecular characterization of a strain carrying blaCTX-M-65 and fosA3 was analysed by antimicrobial susceptibility testing and MLST. The transferability of a plasmid bearing blaCTX-M-65 and fosA3 was determined by conjugation assays, and the complete structure of the plasmid was obtained by Illumina, PacBio and conventional PCR mapping, respectively. The circular forms derived from IS26-flanked transposons were detected by reverse PCR and sequencing.A novel IncZ-7 plasmid pEC013 (~118kb) harbouring the blaCTX-M-65 and fosA3 genes was recovered from E. coli isolate EC013 belonging to D-ST117. The plasmid was found to have heterogeneous and dynamic MRRs in an individual strain and the IS26-flanked composite transposon-derived circular intermediates were identified and characterized in pEC013.The heterogeneous MRRs suggested that a single plasmid may actually be a cluster of plasmids with the same backbone but varied MRRs, reflecting the plasmid’s heterogeneity and the survival benefits of having a response to antimicrobial-related threatening conditions in an individual strain. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.


April 21, 2020

Methylome and Metabolome Analyses Reveal Adaptive Mechanisms in Geobacter sulfurreducens Grown on Different Terminal Electron Acceptors.

The Geobacter species evolved respiratory versatility to utilize a wide range of terminal electron acceptors. To explore this adaptive mechanism, Fe(III) citrate, hydrous ferric oxide, and fumarate were selected as electron acceptors, and the methylome and metabolome of Geobacter sulfurreducens PCA grown on each electron acceptor were investigated via third-generation, single-molecule real-time DNA sequencing and gas chromatography/time-of-flight mass spectrometry-based metabolomics, respectively. Results showed that the patterns of 4-methylcytosine (m4C) and 6-methyladenine (m6A) modification, the concentrations of fatty acids (e.g., caprylic acid, capric acid, and squalene), and the activity of antioxidant enzymes (e.g., superoxide dismutase, catalase, and glutathione reductase) were all varied in different electron acceptor cultures. Moreover, genes (e.g., GSU0466 and GSU1467) with low expression levels generally had high methylation levels. These findings suggest that m4C and m6A modifications, fatty acids, and antioxidant enzymes all play a role in the adaptation of G. sulfurreducens to diverse electron acceptors, and DNA methylation may be involved in the adaptation mainly via gene expression regulation.


April 21, 2020

Genomic Plasticity Mediated by Transposable Elements in the Plant Pathogenic Fungus Colletotrichum higginsianum.

Phytopathogen genomes are under constant pressure to change, as pathogens are locked in an evolutionary arms race with their hosts, where pathogens evolve effector genes to manipulate their hosts, whereas the hosts evolve immune components to recognize the products of these genes. Colletotrichum higginsianum (Ch), a fungal pathogen with no known sexual morph, infects Brassicaceae plants including Arabidopsis thaliana. Previous studies revealed that Ch differs in its virulence toward various Arabidopsis thaliana ecotypes, indicating the existence of coevolutionary selective pressures. However, between-strain genomic variations in Ch have not been studied. Here, we sequenced and assembled the genome of a Ch strain, resulting in a highly contiguous genome assembly, which was compared with the chromosome-level genome assembly of another strain to identify genomic variations between strains. We found that the two closely related strains vary in terms of large-scale rearrangements, the existence of strain-specific regions, and effector candidate gene sets and that these variations are frequently associated with transposable elements (TEs). Ch has a compartmentalized genome consisting of gene-sparse, TE-dense regions with more effector candidate genes and gene-dense, TE-sparse regions harboring conserved genes. Additionally, analysis of the conservation patterns and syntenic regions of effector candidate genes indicated that the two strains vary in their effector candidate gene sets because of de novo evolution, horizontal gene transfer, or gene loss after divergence. Our results reveal mechanisms for generating genomic diversity in this asexual pathogen, which are important for understanding its adaption to hosts. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.


April 21, 2020

Full-length transcriptome sequences obtained by a combination of sequencing platforms applied to heat shock proteins and polyunsaturated fatty acids biosynthesis in Pyropia haitanensis

Pyropia haitanensis is a high-yield commercial seaweed in China. Pyropia haitanensis farms often suffer from problems such as severe germplasm degeneration, while the mechanisms underlying resistance to abiotic stresses remain unknown because of lacking genomic information. Although many previous studies focused on using next-generation sequencing (NGS) technologies, the short-read sequences generated by NGS generally prevent the assembly of full-length transcripts, and then limit screening functional genes. In the present study, which was based on hybrid sequencing (NGS and single-molecular real-time sequencing) of the P. haitanensis thallus transcriptome, we obtained high-quality full-length transcripts with a mean length of 2998 bp and an N50 value of 3366 bp. A total of 14,773 unigenes (93.52%) were annotated in at least one database, while approximately 60% of all unigenes were assembled by short Illumina reads. Moreover, we herein suggested that the genes involved in the biosynthesis of polyunsaturated fatty acids and heat shock proteins play an important role in the process of development and resistance to abiotic stresses in P. haitanensis. The present study, together with previously published ones, may facilitate seaweed transcriptome research.


April 21, 2020

A physical and genetic map of Cannabis sativa identifies extensive rearrangements at the THC/CBD acid synthase loci.

Cannabis sativa is widely cultivated for medicinal, food, industrial, and recreational use, but much remains unknown regarding its genetics, including the molecular determinants of cannabinoid content. Here, we describe a combined physical and genetic map derived from a cross between the drug-type strain Purple Kush and the hemp variety “Finola.” The map reveals that cannabinoid biosynthesis genes are generally unlinked but that aromatic prenyltransferase (AP), which produces the substrate for THCA and CBDA synthases (THCAS and CBDAS), is tightly linked to a known marker for total cannabinoid content. We further identify the gene encoding CBCA synthase (CBCAS) and characterize its catalytic activity, providing insight into how cannabinoid diversity arises in cannabis. THCAS and CBDAS (which determine the drug vs. hemp chemotype) are contained within large (>250 kb) retrotransposon-rich regions that are highly nonhomologous between drug- and hemp-type alleles and are furthermore embedded within ~40 Mb of minimally recombining repetitive DNA. The chromosome structures are similar to those in grains such as wheat, with recombination focused in gene-rich, repeat-depleted regions near chromosome ends. The physical and genetic map should facilitate further dissection of genetic and molecular mechanisms in this commercially and medically important plant. © 2019 Laverty et al.; Published by Cold Spring Harbor Laboratory Press.


April 21, 2020

Maleness-on-the-Y (MoY) orchestrates male sex determination in major agricultural fruit fly pests.

In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regulatory module controlling sexual differentiation. In the agricultural pest Ceratitis capitata (Mediterranean fruit fly, or Medfly), we identified a Y-linked gene, Maleness-on-the-Y (MoY), encoding a small protein that is necessary and sufficient for male development. Silencing or disruption of MoY in XY embryos causes feminization, whereas overexpression of MoY in XX embryos induces masculinization. Crosses between transformed XY females and XX males give rise to males and females, indicating that a Y chromosome can be transmitted by XY females. MoY is Y-linked and functionally conserved in other species of the Tephritidae family, highlighting its potential to serve as a tool for developing more effective control strategies against these major agricultural insect pests.Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.


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