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April 21, 2020  |  

A chromosome-level sequence assembly reveals the structure of the Arabidopsis thaliana Nd-1 genome and its gene set.

In addition to the BAC-based reference sequence of the accession Columbia-0 from the year 2000, several short read assemblies of THE plant model organism Arabidopsis thaliana were published during the last years. Also, a SMRT-based assembly of Landsberg erecta has been generated that identified translocation and inversion polymorphisms between two genotypes of the species. Here we provide a chromosome-arm level assembly of the A. thaliana accession Niederzenz-1 (AthNd-1_v2c) based on SMRT sequencing data. The best assembly comprises 69 nucleome sequences and displays a contig length of up to 16 Mbp. Compared to an earlier Illumina short read-based NGS assembly (AthNd-1_v1), a 75 fold increase in contiguity was observed for AthNd-1_v2c. To assign contig locations independent from the Col-0 gold standard reference sequence, we used genetic anchoring to generate a de novo assembly. In addition, we assembled the chondrome and plastome sequences. Detailed analyses of AthNd-1_v2c allowed reliable identification of large genomic rearrangements between A. thaliana accessions contributing to differences in the gene sets that distinguish the genotypes. One of the differences detected identified a gene that is lacking from the Col-0 gold standard sequence. This de novo assembly extends the known proportion of the A. thaliana pan-genome.

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April 21, 2020  |  

Consensus and variations in cell line specificity among human metapneumovirus strains.

Human metapneumovirus (HMPV) has been a notable etiological agent of acute respiratory infection in humans, but it was not discovered until 2001, because HMPV replicates only in a limited number of cell lines and the cytopathic effect (CPE) is often mild. To promote the study of HMPV, several groups have generated green fluorescent protein (GFP)-expressing recombinant HMPV strains (HMPVGFP). However, the growing evidence has complicated the understanding of cell line specificity of HMPV, because it seems to vary notably among HMPV strains. In addition, unique A2b clade HMPV strains with a 180-nucleotide duplication in the G gene (HMPV A2b180nt-dup strains) have recently been detected. In this study, we re-evaluated and compared the cell line specificity of clinical isolates of HMPV strains, including the novel HMPV A2b180nt-dup strains, and six recombinant HMPVGFP strains, including the newly generated recombinant HMPV A2b180nt-dup strain, MG0256-EGFP. Our data demonstrate that VeroE6 and LLC-MK2 cells generally showed the highest infectivity with any clinical isolates and recombinant HMPVGFP strains. Other human-derived cell lines (BEAS-2B, A549, HEK293, MNT-1, and HeLa cells) showed certain levels of infectivity with HMPV, but these were significantly lower than those of VeroE6 and LLC-MK2 cells. Also, the infectivity in these suboptimal cell lines varied greatly among HMPV strains. The variations were not directly related to HMPV genotypes, cell lines used for isolation and propagation, specific genome mutations, or nucleotide duplications in the G gene. Thus, these variations in suboptimal cell lines are likely intrinsic to particular HMPV strains.

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April 21, 2020  |  

Double PIK3CA mutations in cis increase oncogenicity and sensitivity to PI3Ka inhibitors.

Activating mutations in PIK3CA are frequent in human breast cancer, and phosphoinositide 3-kinase alpha (PI3Ka) inhibitors have been approved for therapy. To characterize determinants of sensitivity to these agents, we analyzed PIK3CA-mutant cancer genomes and observed the presence of multiple PIK3CA mutations in 12 to 15% of breast cancers and other tumor types, most of which (95%) are double mutations. Double PIK3CA mutations are in cis on the same allele and result in increased PI3K activity, enhanced downstream signaling, increased cell proliferation, and tumor growth. The biochemical mechanisms of dual mutations include increased disruption of p110a binding to the inhibitory subunit p85a, which relieves its catalytic inhibition, and increased p110a membrane lipid binding. Double PIK3CA mutations predict increased sensitivity to PI3Ka inhibitors compared with single-hotspot mutations.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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April 21, 2020  |  

Complete Genome Sequence of the Wolbachia wAlbB Endosymbiont of Aedes albopictus.

Wolbachia, an alpha-proteobacterium closely related to Rickettsia, is a maternally transmitted, intracellular symbiont of arthropods and nematodes. Aedes albopictus mosquitoes are naturally infected with Wolbachia strains wAlbA and wAlbB. Cell line Aa23 established from Ae. albopictus embryos retains only wAlbB and is a key model to study host-endosymbiont interactions. We have assembled the complete circular genome of wAlbB from the Aa23 cell line using long-read PacBio sequencing at 500× median coverage. The assembled circular chromosome is 1.48 megabases in size, an increase of more than 300 kb over the published draft wAlbB genome. The annotation of the genome identified 1,205 protein coding genes, 34 tRNA, 3 rRNA, 1 tmRNA, and 3 other ncRNA loci. The long reads enabled sequencing over complex repeat regions which are difficult to resolve with short-read sequencing. Thirteen percent of the genome comprised insertion sequence elements distributed throughout the genome, some of which cause pseudogenization. Prophage WO genes encoding some essential components of phage particle assembly are missing, while the remainder are found in five prophage regions/WO-like islands or scattered around the genome. Orthology analysis identified a core proteome of 535 orthogroups across all completed Wolbachia genomes. The majority of proteins could be annotated using Pfam and eggNOG analyses, including ankyrins and components of the Type IV secretion system. KEGG analysis revealed the absence of five genes in wAlbB which are present in other Wolbachia. The availability of a complete circular chromosome from wAlbB will enable further biochemical, molecular, and genetic analyses on this strain and related Wolbachia. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020  |  

The whole genome insight on condition-specific redox activity and arsenopyrite interaction promoting As-mobilization by strain Lysinibacillus sp. B2A1.

A gram-positive spore former, Lysinibacillus sp. B2A1 was isolated from a high arsenic containing groundwater of Beimen2A well, Chianan Plain area, Southwestern Taiwan. Noteworthy, in the subsurface-mimicking anoxic incubation with a Na-lactate amendment system, this isolate could interact with arsenic-source mineral arsenopyrite and enhance arsenic mobilization. Further, the isolate showed elevated levels of arsenic resistance, 200?mM and 7.5?mM for arsenate and arsenite, respectively. Lysinibacillus sp. B2A1 demonstrated condition-specific redox activities including salient oxic oxidation of arsenite and anoxic reduction of arsenate. The elevated rate of As(III) oxidation (Vmax?=?0.13 µM min-1 per 106 cells, Km?=?15.3 µM) under oxic conditions was potent. Correlating with stout persistence in an arsenic-rich niche, remarkably, the lesser toxic effects of arsenic ions on bacterial sporulation frequency and germination highlight this strain’s ability to thrive under catastrophic conditions. Moreover, the whole genome analysis elucidated diverse metal redox/resistance genes that included a potential arsenite S-adenosylmethyltransferase capable of mitigating arsenite toxicity. Owing to its arsenic resistance, conditional redox activities and ability to interact with arsenic minerals leading to arsenic mobilization, the presence of such spore-forming strains could be a decisive indication towards arsenic mobilization in subsurface aquifers having a high concentration of soluble arsenic or its source minerals. Copyright © 2018 Elsevier B.V. All rights reserved.

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April 21, 2020  |  

eIF5B gates the transition from translation initiation to elongation.

Translation initiation determines both the quantity and identity of the protein that is encoded in an mRNA by establishing the reading frame for protein synthesis. In eukaryotic cells, numerous translation initiation factors prepare ribosomes for polypeptide synthesis; however, the underlying dynamics of this process remain unclear1,2. A central question is how eukaryotic ribosomes transition from translation initiation to elongation. Here we use in vitro single-molecule fluorescence microscopy approaches in a purified yeast Saccharomyces cerevisiae translation system to monitor directly, in real time, the pathways of late translation initiation and the transition to elongation. This transition was slower in our eukaryotic system than that reported for Escherichia coli3-5. The slow entry to elongation was defined by a long residence time of eukaryotic initiation factor 5B (eIF5B) on the 80S ribosome after the joining of individual ribosomal subunits-a process that is catalysed by this universally conserved initiation factor. Inhibition of the GTPase activity of eIF5B after the joining of ribosomal subunits prevented the dissociation of eIF5B from the 80S complex, thereby preventing elongation. Our findings illustrate how the dissociation of eIF5B serves as a kinetic checkpoint for the transition from initiation to elongation, and how its release may be governed by a change in the conformation of the ribosome complex that triggers GTP hydrolysis.

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April 21, 2020  |  

In-depth analysis of the genome of Trypanosoma evansi, an etiologic agent of surra.

Trypanosoma evansi is the causative agent of the animal trypanosomiasis surra, a disease with serious economic burden worldwide. The availability of the genome of its closely related parasite Trypanosoma brucei allows us to compare their genetic and evolutionarily shared and distinct biological features. The complete genomic sequence of the T. evansi YNB strain was obtained using a combination of genomic and transcriptomic sequencing, de novo assembly, and bioinformatic analysis. The genome size of the T. evansi YNB strain was 35.2 Mb, showing 96.59% similarity in sequence and 88.97% in scaffold alignment with T. brucei. A total of 8,617 protein-coding genes, accounting for 31% of the genome, were predicted. Approximately 1,641 alternative splicing events of 820 genes were identified, with a majority mediated by intron retention, which represented a major difference in post-transcriptional regulation between T. evansi and T. brucei. Disparities in gene copy number of the variant surface glycoprotein, expression site-associated genes, microRNAs, and RNA-binding protein were clearly observed between the two parasites. The results revealed the genomic determinants of T. evansi, which encoded specific biological characteristics that distinguished them from other related trypanosome species.

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April 21, 2020  |  

RADAR-seq: A RAre DAmage and Repair sequencing method for detecting DNA damage on a genome-wide scale.

RAre DAmage and Repair sequencing (RADAR-seq) is a highly adaptable sequencing method that enables the identification and detection of rare DNA damage events for a wide variety of DNA lesions at single-molecule resolution on a genome-wide scale. In RADAR-seq, DNA lesions are replaced with a patch of modified bases that can be directly detected by Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing. RADAR-seq enables dynamic detection over a wide range of DNA damage frequencies, including low physiological levels. Furthermore, without the need for DNA amplification and enrichment steps, RADAR-seq provides sequencing coverage of damaged and undamaged DNA across an entire genome. Here, we use RADAR-seq to measure the frequency and map the location of ribonucleotides in wild-type and RNaseH2-deficient E. coli and Thermococcus kodakarensis strains. Additionally, by tracking ribonucleotides incorporated during in vivo lagging strand DNA synthesis, we determined the replication initiation point in E. coli, and its relation to the origin of replication (oriC). RADAR-seq was also used to map cyclobutane pyrimidine dimers (CPDs) in Escherichia coli (E. coli) genomic DNA exposed to UV-radiation. On a broader scale, RADAR-seq can be applied to understand formation and repair of DNA damage, the correlation between DNA damage and disease initiation and progression, and complex biological pathways, including DNA replication.Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.

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April 21, 2020  |  

Determining stoichiometry and kinetics of two thermophilic nitrifying communities as a crucial step in the development of thermophilic nitrogen removal.

Nitrification and denitrification, the key biological processes for thermophilic nitrogen removal, have separately been established in bioreactors at 50?°C. A well-characterized set of kinetic parameters is essential to integrate these processes while safeguarding the autotrophs performing nitrification. Knowledge on thermophilic nitrifying kinetics is restricted to isolated or highly enriched batch cultures, which do not represent bioreactor conditions. This study characterized the stoichiometry and kinetics of two thermophilic (50?°C) nitrifying communities. The most abundant ammonia oxidizing archaea (AOA) were related to the Nitrososphaera genus, clustering relatively far from known species Nitrososphaera gargensis (95.5% 16S rRNA gene sequence identity). The most abundant nitrite oxidizing bacteria (NOB) were related to Nitrospira calida (97% 16S rRNA gene sequence identity). The nitrification biomass yield was 0.20-0.24?g VSS g-1 N, resulting mainly from a high AOA yield (0.16-0.20?g VSS g-1 N), which was reflected in a high AOA abundance in the community (57-76%) compared to NOB (5-11%). Batch-wise determination of decay rates (AOA: 0.23-0.29 d-1; NOB: 0.32-0.43 d-1) rendered an overestimation compared to in situ estimations of overall decay rate (0.026-0.078 d-1). Possibly, the inactivation rate rather than the actual decay rate was determined in batch experiments. Maximum growth rates of AOA and NOB were 0.12-0.15 d-1 and 0.13-0.33 d-1 respectively. NOB were susceptible to nitrite, opening up opportunities for shortcut nitrogen removal. However, NOB had a similar growth rate and oxygen affinity (0.15-0.55?mg O2 L-1) as AOA and were resilient towards free ammonia (IC50?>?16?mg NH3-N L-1). This might complicate NOB outselection using common practices to establish shortcut nitrogen removal (SRT control; aeration control; free ammonia shocks). Overall, the obtained insights can assist in integrating thermophilic conversions and facilitate single-sludge nitrification/denitrification. Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020  |  

Current advances in HIV vaccine preclinical studies using Macaque models.

The macaque simian or simian/human immunodeficiency virus (SIV/SHIV) challenge model has been widely used to inform and guide human vaccine trials. Substantial advances have been made recently in the application of repeated-low-dose challenge (RLD) approach to assess SIV/SHIV vaccine efficacies (VE). Some candidate HIV vaccines have shown protective effects in preclinical studies using the macaque SIV/SHIV model but the model’s true predictive value for screening potential HIV vaccine candidates needs to be evaluated further. Here, we review key parameters used in the RLD approach and discuss their relevance for evaluating VE to improve preclinical studies of candidate HIV vaccines.Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.

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April 21, 2020  |  

Function and Distribution of a Lantipeptide in Strawberry Fusarium Wilt Disease-Suppressive Soils.

Streptomyces griseus S4-7 is representative of strains responsible for the specific soil suppressiveness of Fusarium wilt of strawberry caused by Fusarium oxysporum f. sp. fragariae. Members of the genus Streptomyces secrete diverse secondary metabolites including lantipeptides, heat-stable lanthionine-containing compounds that can exhibit antibiotic activity. In this study, a class II lantipeptide provisionally named grisin, of previously unknown biological function, was shown to inhibit F. oxysporum. The inhibitory activity of grisin distinguishes it from other class II lantipeptides from Streptomyces spp. Results of quantitative reverse transcription-polymerase chain reaction with lanM-specific primers showed that the density of grisin-producing Streptomyces spp. in the rhizosphere of strawberry was positively correlated with the number of years of monoculture and a minimum of seven years was required for development of specific soil suppressiveness to Fusarium wilt disease. We suggest that lanM can be used as a diagnostic marker of whether a soil is conducive or suppressive to the disease.

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April 21, 2020  |  

A systematic review of the Trypanosoma cruzi genetic heterogeneity, host immune response and genetic factors as plausible drivers of chronic chagasic cardiomyopathy.

Chagas disease is a complex tropical pathology caused by the kinetoplastid Trypanosoma cruzi. This parasite displays massive genetic diversity and has been classified by international consensus in at least six Discrete Typing Units (DTUs) that are broadly distributed in the American continent. The main clinical manifestation of the disease is the chronic chagasic cardiomyopathy (CCC) that is lethal in the infected individuals. However, one intriguing feature is that only 30-40% of the infected individuals will develop CCC. Some authors have suggested that the immune response, host genetic factors, virulence factors and even the massive genetic heterogeneity of T. cruzi are responsible of this clinical pattern. To date, no conclusive data support the reason why a few percentages of the infected individuals will develop CCC. Therefore, we decided to conduct a systematic review analysing the host genetic factors, immune response, cytokine production, virulence factors and the plausible association of the parasite DTUs and CCC. The epidemiological and clinical implications are herein discussed.

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April 21, 2020  |  

Toxin and genome evolution in a Drosophila defensive symbiosis.

Defenses conferred by microbial symbionts play a vital role in the health and fitness of their animal hosts. An important outstanding question in the study of defensive symbiosis is what determines long term stability and effectiveness against diverse natural enemies. In this study, we combine genome and transcriptome sequencing, symbiont transfection and parasite protection experiments, and toxin activity assays to examine the evolution of the defensive symbiosis between Drosophila flies and their vertically transmitted Spiroplasma bacterial symbionts, focusing in particular on ribosome-inactivating proteins (RIPs), symbiont-encoded toxins that have been implicated in protection against both parasitic wasps and nematodes. Although many strains of Spiroplasma, including the male-killing symbiont (sMel) of Drosophila melanogaster, protect against parasitic wasps, only the strain (sNeo) that infects the mycophagous fly Drosophila neotestacea appears to protect against parasitic nematodes. We find that RIP repertoire is a major differentiating factor between strains that do and do not offer nematode protection, and that sMel RIPs do not show activity against nematode ribosomes in vivo. We also discovered a strain of Spiroplasma infecting a mycophagous phorid fly, Megaselia nigra. Although both the host and its Spiroplasma are distantly related to D. neotestacea and its symbiont, genome sequencing revealed that the M. nigra symbiont encodes abundant and diverse RIPs, including plasmid-encoded toxins that are closely related to the RIPs in sNeo. Our results suggest that distantly related Spiroplasma RIP toxins may perform specialized functions with regard to parasite specificity and suggest an important role for horizontal gene transfer in the emergence of novel defensive phenotypes.

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