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April 21, 2020  |  

The Complete Genome of the Atypical Enteropathogenic Escherichia coli Archetype Isolate E110019 Highlights a Role for Plasmids in Dissemination of the Type III Secreted Effector EspT.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of moderate to severe diarrhea among young children in developing countries, and EPEC isolates can be subdivided into two groups. Typical EPEC (tEPEC) bacteria are characterized by the presence of both the locus of enterocyte effacement (LEE) and the plasmid-encoded bundle-forming pilus (BFP), which are involved in adherence and translocation of type III effectors into the host cells. Atypical EPEC (aEPEC) bacteria also contain the LEE but lack the BFP. In the current report, we describe the complete genome of outbreak-associated aEPEC isolate E110019, which carries four plasmids. Comparative genomic analysis demonstrated that the type III secreted effector EspT gene, an autotransporter gene, a hemolysin gene, and putative fimbrial genes are all carried on plasmids. Further investigation of 65 espT-containing E. coli genomes demonstrated that different espT alleles are associated with multiple plasmids that differ in their overall gene content from the E110019 espT-containing plasmid. EspT has been previously described with respect to its role in the ability of E110019 to invade host cells. While other type III secreted effectors of E. coli have been identified on insertion elements and prophages of the chromosome, we demonstrated in the current study that the espT gene is located on multiple unique plasmids. These findings highlight a role of plasmids in dissemination of a unique E. coli type III secreted effector that is involved in host invasion and severe diarrheal illness.Copyright © 2019 American Society for Microbiology.

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April 21, 2020  |  

Advantage of the F2:A1:B- IncF Pandemic Plasmid over IncC Plasmids in In Vitro Acquisition and Evolution of blaCTX-M Gene-Bearing Plasmids in Escherichia coli.

Despite a fitness cost imposed on bacterial hosts, large conjugative plasmids play a key role in the diffusion of resistance determinants, such as CTX-M extended-spectrum ß-lactamases. Among the large conjugative plasmids, IncF plasmids are the most predominant group, and an F2:A1:B- IncF-type plasmid encoding a CTX-M-15 variant was recently described as being strongly associated with the emerging worldwide Escherichia coli sequence type 131 (ST131)-O25b:H4 H30Rx/C2 sublineage. In this context, we investigated the fitness cost of narrow-range F-type plasmids, including the F2:A1:B- IncF-type CTX-M-15 plasmid, and of broad-range C-type plasmids in the K-12-like J53-2 E. coli strain. Although all plasmids imposed a significant fitness cost to the bacterial host immediately after conjugation, we show, using an experimental-evolution approach, that a negative impact on the fitness of the host strain was maintained throughout 1,120 generations with the IncC-IncR plasmid, regardless of the presence or absence of cefotaxime, in contrast to the F2:A1:B- IncF plasmid, whose cost was alleviated. Many chromosomal and plasmid rearrangements were detected after conjugation in transconjugants carrying the IncC plasmids but not in transconjugants carrying the F2:A1:B- IncF plasmid, except for insertion sequence (IS) mobilization from the fliM gene leading to the restoration of motility of the recipient strains. Only a few mutations occurred on the chromosome of each transconjugant throughout the experimental-evolution assay. Our findings indicate that the F2:A1:B- IncF CTX-M-15 plasmid is well adapted to the E. coli strain studied, contrary to the IncC-IncR CTX-M-15 plasmid, and that such plasmid-host adaptation could participate in the evolutionary success of the CTX-M-15-producing pandemic E. coli ST131-O25b:H4 lineage.Copyright © 2019 Mahérault et al.

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April 21, 2020  |  

A Highly Unusual V1 Region of Env in an Elite Controller of HIV Infection.

HIV elite controllers represent a remarkable minority of patients who maintain normal CD4+ T-cell counts and low or undetectable viral loads for decades in the absence of antiretroviral therapy. To examine the possible contribution of virus attenuation to elite control, we obtained a primary HIV-1 isolate from an elite controller who had been infected for 19?years, the last 10 of which were in the absence of antiretroviral therapy. Full-length sequencing of this isolate revealed a highly unusual V1 domain in Envelope (Env). The V1 domain in this HIV-1 strain was 49 amino acids, placing it in the top 1% of lengths among the 6,112 Env sequences in the Los Alamos National Laboratory online database. Furthermore, it included two additional N-glycosylation sites and a pair of cysteines suggestive of an extra disulfide loop. Virus with this Env retained good infectivity and replicative capacity; however, analysis of recombinant viruses suggested that other sequences in Env were adapted to accommodate the unusual V1 domain. While the long V1 domain did not confer resistance to neutralization by monoclonal antibodies of the V1/V2-glycan-dependent class, it did confer resistance to neutralization by monoclonal antibodies of the V3-glycan-dependent class. Our findings support results in the literature that suggest a role for long V1 regions in shielding HIV-1 from recognition by V3-directed broadly neutralizing antibodies. In the case of the elite controller described here, it seems likely that selective pressures from the humoral immune system were responsible for driving the highly unusual polymorphisms present in this HIV-1 Envelope.IMPORTANCE Elite controllers have long provided an avenue for researchers to reveal mechanisms underlying control of HIV-1. While the role of host genetic factors in facilitating elite control is well known, the possibility of infection by attenuated strains of HIV-1 has been much less studied. Here we describe an unusual viral feature found in an elite controller of HIV-1 infection and demonstrate its role in conferring escape from monoclonal antibodies of the V3-glycan class. Our results suggest that extreme variation may be needed by HIV-1 to escape neutralization by some antibody specificities. Copyright © 2019 Silver et al.

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April 21, 2020  |  

Hybrid de novo genome assembly of Chinese chestnut (Castanea mollissima).

The Chinese chestnut (Castanea mollissima) is widely cultivated in China for nut production. This plant also plays an important ecological role in afforestation and ecosystem services. To facilitate and expand the use of C. mollissima for breeding and its genetic improvement, we report here the whole-genome sequence of C. mollissima.We produced a high-quality assembly of the C. mollissima genome using Pacific Biosciences single-molecule sequencing. The final draft genome is ~785.53 Mb long, with a contig N50 size of 944 kb, and we further annotated 36,479 protein-coding genes in the genome. Phylogenetic analysis showed that C. mollissima diverged from Quercus robur, a member of the Fagaceae family, ~13.62 million years ago.The high-quality whole-genome assembly of C. mollissima will be a valuable resource for further genetic improvement and breeding for disease resistance and nut quality. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020  |  

Genome sequence of the Chinese white wax scale insect Ericerus pela: the first draft genome for the Coccidae family of scale insects.

The Chinese white wax scale insect, Ericerus pela, is best known for producing wax, which has been widely used in candle production, casting, Chinese medicine, and wax printing products for thousands of years. The secretion of wax, and other unusual features of scale insects, is thought to be an adaptation to their change from an ancestral ground-dwelling lifestyle to a sedentary lifestyle on the higher parts of plants. As well as helping to improve its economic value, studies of E. pela might also help to explain the adaptation of scale insects. However, no genomic data are currently available for E. pela.To assemble the E. pela genome, 303.92 Gb of data were generated using Illumina and Pacific Biosciences sequencing, producing 277.22 Gb of clean data for assembly. The assembled genome size was 0.66 Gb, with 1,979 scaffolds and a scaffold N50 of 735 kb. The guanine + cytosine content was 33.80%. A total of 12,022 protein-coding genes were predicted, with a mean coding sequence length of 1,370 bp. Twenty-six fatty acyl-CoA reductase genes and 35 acyltransferase genes were identified. Evolutionary analysis revealed that E. pela and aphids formed a sister group and split ~241.1 million years ago. There were 214 expanded gene families and 2,219 contracted gene families in E. pela.We present the first genome sequence from the Coccidae family. These results will help to increase our understanding of the evolution of unique features in scale insects, and provide important genetic information for further research. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020  |  

Salmonella Genomic Island 3 Is an Integrative and Conjugative Element and Contributes to Copper and Arsenic Tolerance of Salmonella enterica.

Salmonella genomic island 3 (SGI3) was first described as a chromosomal island in Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium. The SGI3 DNA sequence detected from Salmonella 4,[5],12:i:- isolated in Japan was identical to that of a previously reported one across entire length of 81?kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using the S. enterica serovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3′ end of the tRNA genes pheV or pheR The length of the target site was 52 or 55?bp, and a 55-bp attI sequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4 compared to the recipient strains under anaerobic conditions. Tolerance was defined by the cus gene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4 compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance of S. enterica.Copyright © 2019 American Society for Microbiology.

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April 21, 2020  |  

Outcomes and characterization of chromosomal self-targeting by native CRISPR-Cas systems in Streptococcus thermophilus.

CRISPR-Cas systems provide adaptive immunity against phages in prokaryotes via DNA-encoded, RNA-mediated, nuclease-dependent targeting and cleavage. Due to inefficient and relatively limited DNA repair pathways in bacteria, CRISPR-Cas systems can be repurposed for lethal DNA targeting that selects for sequence variants. In this study, the relative killing efficiencies of endogenous Type I and Type II CRISPR-Cas systems in the model organism Streptococcus thermophilus DGCC7710 were assessed. Additionally, the genetic and phenotypic outcomes of chromosomal targeting by plasmid-programmed Type I-E or Type II-A systems were analyzed. Efficient killing was observed using both systems, in a dose-dependent manner when delivering 0.4-400 ng of plasmid DNA. Targeted PCR screening and genome sequencing were used to determine the genetic basis enabling survival, showing that evasion of Type I-E self-targeting was primarily the result of low-frequency defective plasmids that excised the targeting spacer. The most notable genotype recovered from Type II-A targeting of genomic locus, lacZ, was a 34 kb-deletion derived from homologous recombination (HR) between identical conserved sequences in two separate galE coding regions, resulting in 2% loss of the genome. Collectively, these results suggest that HR contributes to the plasticity and remodeling of bacterial genomes, leading to evasion of genome targeting by CRISPR-Cas systems. © FEMS 2019.

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April 21, 2020  |  

Medusavirus, a Novel Large DNA Virus Discovered from Hot Spring Water.

Recent discoveries of new large DNA viruses reveal high diversity in their morphologies, genetic repertoires, and replication strategies. Here, we report the novel features of medusavirus, a large DNA virus newly isolated from hot spring water in Japan. Medusavirus, with a diameter of 260?nm, shows a T=277 icosahedral capsid with unique spherical-headed spikes on its surface. It has a 381-kb genome encoding 461 putative proteins, 86 of which have their closest homologs in Acanthamoeba, whereas 279 (61%) are orphan genes. The virus lacks the genes encoding DNA topoisomerase II and RNA polymerase, showing that DNA replication takes place in the host nucleus, whereas the progeny virions are assembled in the cytoplasm. Furthermore, the medusavirus genome harbored genes for all five types of histones (H1, H2A, H2B, H3, and H4) and one DNA polymerase, which are phylogenetically placed at the root of the eukaryotic clades. In contrast, the host amoeba encoded many medusavirus homologs, including the major capsid protein. These facts strongly suggested that amoebae are indeed the most promising natural hosts of medusavirus, and that lateral gene transfers have taken place repeatedly and bidirectionally between the virus and its host since the early stage of their coevolution. Medusavirus reflects the traces of direct evolutionary interactions between the virus and eukaryotic hosts, which may be caused by sharing the DNA replication compartment and by evolutionarily long lasting virus-host relationships. Based on its unique morphological characteristics and phylogenomic relationships with other known large DNA viruses, we propose that medusavirus represents a new family, MedusaviridaeIMPORTANCE We have isolated a new nucleocytoplasmic large DNA virus (NCLDV) from hot spring water in Japan, named medusavirus. This new NCLDV is phylogenetically placed at the root of the eukaryotic clades based on the phylogenies of several key genes, including that encoding DNA polymerase, and its genome surprisingly encodes the full set of histone homologs. Furthermore, its laboratory host, Acanthamoeba castellanii, encodes many medusavirus homologs in its genome, including the major capsid protein, suggesting that the amoeba is the genuine natural host from ancient times of this newly described virus and that lateral gene transfers have repeatedly occurred between the virus and amoeba. These results suggest that medusavirus is a unique NCLDV preserving ancient footprints of evolutionary interactions with its hosts, thus providing clues to elucidate the evolution of NCLDVs, eukaryotes, and virus-host interaction. Based on the dissimilarities with other known NCLDVs, we propose that medusavirus represents a new viral family, Medusaviridae.Copyright © 2019 Yoshikawa et al.

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April 21, 2020  |  

Chromosomal-level assembly of the blolsod clam, Scapharca (Anadara) broughtonii, using long sequence reads and Hi-C.

The blood clam, Scapharca (Anadara) broughtonii, is an economically and ecologically important marine bivalve of the family Arcidae. Efforts to study their population genetics, breeding, cultivation, and stock enrichment have been somewhat hindered by the lack of a reference genome. Herein, we report the complete genome sequence of S. broughtonii, a first reference genome of the family Arcidae.A total of 75.79 Gb clean data were generated with the Pacific Biosciences and Oxford Nanopore platforms, which represented approximately 86× coverage of the S. broughtonii genome. De novo assembly of these long reads resulted in an 884.5-Mb genome, with a contig N50 of 1.80 Mb and scaffold N50 of 45.00 Mb. Genome Hi-C scaffolding resulted in 19 chromosomes containing 99.35% of bases in the assembled genome. Genome annotation revealed that nearly half of the genome (46.1%) is composed of repeated sequences, while 24,045 protein-coding genes were predicted and 84.7% of them were annotated.We report here a chromosomal-level assembly of the S. broughtonii genome based on long-read sequencing and Hi-C scaffolding. The genomic data can serve as a reference for the family Arcidae and will provide a valuable resource for the scientific community and aquaculture sector. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020  |  

De novo genome assembly of the endangered Acer yangbiense, a plant species with extremely small populations endemic to Yunnan Province, China.

Acer yangbiense is a newly described critically endangered endemic maple tree confined to Yangbi County in Yunnan Province in Southwest China. It was included in a programme for rescuing the most threatened species in China, focusing on “plant species with extremely small populations (PSESP)”.We generated 64, 94, and 110 Gb of raw DNA sequences and obtained a chromosome-level genome assembly of A. yangbiense through a combination of Pacific Biosciences Single-molecule Real-time, Illumina HiSeq X, and Hi-C mapping, respectively. The final genome assembly is ~666 Mb, with 13 chromosomes covering ~97% of the genome and scaffold N50 sizes of 45 Mb. Further, BUSCO analysis recovered 95.5% complete BUSCO genes. The total number of repetitive elements account for 68.0% of the A. yangbiense genome. Genome annotation generated 28,320 protein-coding genes, assisted by a combination of prediction and transcriptome sequencing. In addition, a nearly 1:1 orthology ratio of dot plots of longer syntenic blocks revealed a similar evolutionary history between A. yangbiense and grape, indicating that the genome has not undergone a whole-genome duplication event after the core eudicot common hexaploidization.Here, we report a high-quality de novo genome assembly of A. yangbiense, the first genome for the genus Acer and the family Aceraceae. This will provide fundamental conservation genomics resources, as well as representing a new high-quality reference genome for the economically important Acer lineage and the wider order of Sapindales. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020  |  

Survey of the Bradysia odoriphaga Transcriptome Using PacBio Single-Molecule Long-Read Sequencing.

The damage caused by Bradysia odoriphaga is the main factor threatening the production of vegetables in the Liliaceae family. However, few genetic studies of B. odoriphaga have been conducted because of a lack of genomic resources. Many long-read sequencing technologies have been developed in the last decade; therefore, in this study, the transcriptome including all development stages of B. odoriphaga was sequenced for the first time by Pacific single-molecule long-read sequencing. Here, 39,129 isoforms were generated, and 35,645 were found to have annotation results when checked against sequences available in different databases. Overall, 18,473 isoforms were distributed in 25 various Clusters of Orthologous Groups, and 11,880 isoforms were categorized into 60 functional groups that belonged to the three main Gene Ontology classifications. Moreover, 30,610 isoforms were assigned into 44 functional categories belonging to six main Kyoto Encyclopedia of Genes and Genomes functional categories. Coding DNA sequence (CDS) prediction showed that 36,419 out of 39,129 isoforms were predicted to have CDS, and 4319 simple sequence repeats were detected in total. Finally, 266 insecticide resistance and metabolism-related isoforms were identified as candidate genes for further investigation of insecticide resistance and metabolism in B. odoriphaga.

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April 21, 2020  |  

Pseudomolecule-level assembly of the Chinese oil tree yellowhorn (Xanthoceras sorbifolium) genome.

Yellowhorn (Xanthoceras sorbifolium) is a species of the Sapindaceae family native to China and is an oil tree that can withstand cold and drought conditions. A pseudomolecule-level genome assembly for this species will not only contribute to understanding the evolution of its genes and chromosomes but also bring yellowhorn breeding into the genomic era.Here, we generated 15 pseudomolecules of yellowhorn chromosomes, on which 97.04% of scaffolds were anchored, using the combined Illumina HiSeq, Pacific Biosciences Sequel, and Hi-C technologies. The length of the final yellowhorn genome assembly was 504.2 Mb with a contig N50 size of 1.04 Mb and a scaffold N50 size of 32.17 Mb. Genome annotation revealed that 68.67% of the yellowhorn genome was composed of repetitive elements. Gene modelling predicted 24,672 protein-coding genes. By comparing orthologous genes, the divergence time of yellowhorn and its close sister species longan (Dimocarpus longan) was estimated at ~33.07 million years ago. Gene cluster and chromosome synteny analysis demonstrated that the yellowhorn genome shared a conserved genome structure with its ancestor in some chromosomes.This genome assembly represents a high-quality reference genome for yellowhorn. Integrated genome annotations provide a valuable dataset for genetic and molecular research in this species. We did not detect whole-genome duplication in the genome. The yellowhorn genome carries syntenic blocks from ancient chromosomes. These data sources will enable this genome to serve as an initial platform for breeding better yellowhorn cultivars. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020  |  

Identification of Initial Colonizing Bacteria in Dental Plaques from Young Adults Using Full-Length 16S rRNA Gene Sequencing.

Development of dental plaque begins with the adhesion of salivary bacteria to the acquired pellicle covering the tooth surface. In this study, we collected in vivo dental plaque formed on hydroxyapatite disks for 6 h from 74 young adults and identified initial colonizing taxa based on full-length 16S rRNA gene sequences. A long-read, single-molecule sequencer, PacBio Sequel, provided 100,109 high-quality full-length 16S rRNA gene sequence reads from the early plaque microbiota, which were assigned to 90 oral bacterial taxa. The microbiota obtained from every individual mostly comprised the 21 predominant taxa with the maximum relative abundance of over 10% (95.8?±?6.2%, mean ± SD), which included Streptococcus species as well as nonstreptococcal species. A hierarchical cluster analysis of their relative abundance distribution suggested three major patterns of microbiota compositions: a Streptococcus mitis/Streptococcus sp. HMT-423-dominant profile, a Neisseria sicca/Neisseria flava/Neisseria mucosa-dominant profile, and a complex profile with high diversity. No notable variations in the community structures were associated with the dental caries status, although the total bacterial amounts were larger in the subjects with a high number of caries-experienced teeth (=8) than in those with no or a low number of caries-experienced teeth. Our results revealed the bacterial taxa primarily involved in early plaque formation on hydroxyapatite disks in young adults.IMPORTANCE Selective attachment of salivary bacteria to the tooth surface is an initial and repetitive phase in dental plaque development. We employed full-length 16S rRNA gene sequence analysis with a high taxonomic resolution using a third-generation sequencer, PacBio Sequel, to determine the bacterial composition during early plaque formation in 74 young adults accurately and in detail. The results revealed 21 bacterial taxa primarily involved in early plaque formation on hydroxyapatite disks in young adults, which include several streptococcal species as well as nonstreptococcal species, such as Neisseria sicca/Nflava/Nmucosa and Rothia dentocariosa Given that no notable variations in the microbiota composition were associated with the dental caries status, the maturation process, rather than the specific bacterial species that are the initial colonizers, is likely to play an important role in the development of dysbiotic microbiota associated with dental caries. Copyright © 2019 Ihara et al.

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April 21, 2020  |  

The genomes of pecan and Chinese hickory provide insights into Carya evolution and nut nutrition.

Pecan (Carya illinoinensis) and Chinese hickory (C. cathayensis) are important commercially cultivated nut trees in the genus Carya (Juglandaceae), with high nutritional value and substantial health benefits.We obtained >187.22 and 178.87 gigabases of sequence, and ~288× and 248× genome coverage, to a pecan cultivar (“Pawnee”) and a domesticated Chinese hickory landrace (ZAFU-1), respectively. The total assembly size is 651.31 megabases (Mb) for pecan and 706.43 Mb for Chinese hickory. Two genome duplication events before the divergence from walnut were found in these species. Gene family analysis highlighted key genes in biotic and abiotic tolerance, oil, polyphenols, essential amino acids, and B vitamins. Further analyses of reduced-coverage genome sequences of 16 Carya and 2 Juglans species provide additional phylogenetic perspective on crop wild relatives.Cooperative characterization of these valuable resources provides a window to their evolutionary development and a valuable foundation for future crop improvement. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020  |  

De novo genome assembly of the white-spotted flower chafer (Protaetia brevitarsis).

Protaetia brevitarsis, commonly known as the white-spotted flower chafer, is an important Scarabaeidae insect that is distributed in most Asian countries. Recently, research on the insect’s harmfulness to crops, usefulness in agricultural waste utilization, edibility, medicinal value, and usability in insect immunology has provided sufficient impetus to demonstrate the need for a detailed study of its biology. Herein, we sequenced the whole genome of this species to improve our understanding and study of P. brevitarsis.We developed a highly reliable genome resource for P. brevitarsis (Lewis, 1879; Coleoptera: Cetoniinae) using Illumina and PacBio sequencing platforms. A total of 135.75 gigabases (Gb) was generated, providing 150-fold coverage based on the 810-megabases (Mb) estimated genome size. The assembled P. brevitarsis genome was 751 Mb (including the scaffolds longer than 2 kilobases (kb)) with 327 scaffolds, and the N50 length of the assembly was 2.94 Mb. A total of 34,110 (22,229 in scaffolds and 11,881 located in alleles) genes were identified using Evidence Modeler, which was based on the gene prediction results obtained from 3 different methods (ab initio, RNA sequencing based, and known gene based).We assembled a high-quality P. brevitarsis genome, which will not only provide insight into the biology of the species but also provide a wealth of information that will inform researchers on the evolution, control, and utilization of P. brevitarsis. © The Author(s) 2019. Published by Oxford University Press.

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