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July 7, 2019

Sequencing of plasmids pAMBL1 and pAMBL2 from Pseudomonas aeruginosa reveals a blaVIM-1 amplification causing high-level carbapenem resistance.

Carbapenemases are a major concern for the treatment of infectious diseases caused by Gram-negative bacteria. Although plasmids are responsible for the spread of resistance genes among these pathogens, there is limited information on the nature of the mobile genetic elements carrying carbapenemases in Pseudomonas aeruginosa.We combined data from two different next-generation sequencing platforms, Illumina HiSeq2000 and PacBio RSII, to obtain the complete nucleotide sequences of two blaVIM-1-carrying plasmids (pAMBL1 and pAMBL2) isolated from P. aeruginosa clinical isolates.Plasmid pAMBL1 has 26?440 bp and carries a RepA_C family replication protein. pAMBL1 is similar to plasmids pNOR-2000 and pKLC102 from P. aeruginosa and pAX22 from Achromobacter xylosoxidans, which also carry VIM-type carbapenemases. pAMBL2 is a 24?133 bp plasmid with a replication protein that belongs to the Rep_3 family. It shows a high degree of homology with a fragment of the blaVIM-1-bearing plasmid pPC9 from Pseudomonas putida. Plasmid pAMBL2 carries three copies of the blaVIM-1 cassette in an In70 class 1 integron conferring, unlike pAMBL1, high-level resistance to carbapenems.We present two new plasmids coding for VIM-1 carbapenemase from P. aeruginosa and report that the presence of three copies of blaVIM-1 in pAMBL2 produces high-level resistance to carbapenems.© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

The genome of the Saprophytic fungus Verticillium tricorpus reveals a complex effector repertoire resembling that of its pathogenic relatives.

Vascular wilts caused by Verticillium spp. are destructive plant diseases affecting hundreds of hosts. Only a few Verticillium spp. are causal agents of vascular wilt diseases, of which V. dahliae is the most notorious pathogen, and several V. dahliae genomes are available. In contrast, V. tricorpus is mainly known as a saprophyte and causal agent of opportunistic infections. Based on a hybrid approach that combines second and third generation sequencing, a near-gapless V. tricorpus genome assembly was obtained. With comparative genomics, we sought to identify genomic features in V. dahliae that confer the ability to cause vascular wilt disease. Unexpectedly, both species encode similar effector repertoires and share a genomic structure with genes encoding secreted proteins clustered in genomic islands. Intriguingly, V. tricorpus contains significantly fewer repetitive elements and an extended spectrum of secreted carbohydrate- active enzymes when compared with V. dahliae. In conclusion, we highlight the technical advances of a hybrid sequencing and assembly approach and show that the saprophyte V. tricorpus shares many hallmark features with the pathogen V. dahliae.

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July 7, 2019

Retrohoming of a mobile group II intron in human cells suggests how eukaryotes limit group II intron proliferation.

Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a “ribozyme”) and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed “retrohoming”. Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the evolution of introns and gene expression mechanisms.

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July 7, 2019

Complete mitochondrial genome of the medicinal fungus Ophiocordyceps sinensis.

As part of a genome sequencing project for Ophiocordyceps sinensis, strain 1229, a complete mitochondrial (mt) genome was assembled as a single circular dsDNA of 157,510?bp, one of the largest reported for fungi. Conserved genes including the large and small rRNA subunits, 27 tRNA and 15 protein-coding genes, were identified. In addition, 58 non-conserved open reading frames (ncORFs) in the intergenic and intronic regions were also identified. Transcription analyses using RNA-Seq validated the expression of most conserved genes and ncORFs. Fifty-two introns (groups I and II) were found within conserved genes, accounting for 68.5% of the genome. Thirty-two homing endonucleases (HEs) with motif patterns LAGLIDADG (21) and GIY-YIG (11) were identified in group I introns. The ncORFs found in group II introns mostly encoded reverse transcriptases (RTs). As in other hypocrealean fungi, gene contents and order were found to be conserved in the mt genome of O. sinensis, but the genome size was enlarged by longer intergenic regions and numerous introns. Intergenic and intronic regions were composed of abundant repetitive sequences usually associated with mobile elements. It is likely that intronic ncORFs, which encode RTs and HEs, may have contributed to the enlarged mt genome of O. sinensis.

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July 7, 2019

The Lingula genome provides insights into brachiopod evolution and the origin of phosphate biomineralization.

The evolutionary origins of lingulid brachiopods and their calcium phosphate shells have been obscure. Here we decode the 425-Mb genome of Lingula anatina to gain insights into brachiopod evolution. Comprehensive phylogenomic analyses place Lingula close to molluscs, but distant from annelids. The Lingula gene number has increased to ~34,000 by extensive expansion of gene families. Although Lingula and vertebrates have superficially similar hard tissue components, our genomic, transcriptomic and proteomic analyses show that Lingula lacks genes involved in bone formation, indicating an independent origin of their phosphate biominerals. Several genes involved in Lingula shell formation are shared by molluscs. However, Lingula has independently undergone domain combinations to produce shell matrix collagens with EGF domains and carries lineage-specific shell matrix proteins. Gene family expansion, domain shuffling and co-option of genes appear to be the genomic background of Lingula’s unique biomineralization. This Lingula genome provides resources for further studies of lophotrochozoan evolution.

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July 7, 2019

Dual functions of Macpiwi1 in transposon silencing and stem cell maintenance in the flatworm Macrostomum lignano.

PIWI proteins and piRNA pathways are essential for transposon silencing and some aspects of gene regulation during animal germline development. In contrast to most animal species, some flatworms also express PIWIs and piRNAs in somatic stem cells, where they are required for tissue renewal and regeneration. Here, we have identified and characterized piRNAs and PIWI proteins in the emerging model flatworm Macrostomum lignano. We found that M. lignano encodes at least three PIWI proteins. One of these, Macpiwi1, acts as a key component of the canonical piRNA pathway in the germline and in somatic stem cells. Knockdown of Macpiwi1 dramatically reduces piRNA levels, derepresses transposons, and severely impacts stem cell maintenance. Knockdown of the piRNA biogenesis factor Macvasa caused an even greater reduction in piRNA levels with a corresponding increase in transposons. Yet, in Macvasa knockdown animals, we detected no major impact on stem cell self-renewal. These results may suggest stem cell maintenance functions of PIWI proteins in flatworms that are distinguishable from their impact on transposons and that might function independently of what are considered canonical piRNA populations.© 2015 Zhou et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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July 7, 2019

Gossypium barbadense genome sequence provides insight into the evolution of extra-long staple fiber and specialized metabolites.

Of the two cultivated species of allopolyploid cotton, Gossypium barbadense produces extra-long fibers for the production of superior textiles. We sequenced its genome (AD)2 and performed a comparative analysis. We identified three bursts of retrotransposons from 20 million years ago (Mya) and a genome-wide uneven pseudogenization peak at 11-20 Mya, which likely contributed to genomic divergences. Among the 2,483 genes preferentially expressed in fiber, a cell elongation regulator, PRE1, is strikingly At biased and fiber specific, echoing the A-genome origin of spinnable fiber. The expansion of the PRE members implies a genetic factor that underlies fiber elongation. Mature cotton fiber consists of nearly pure cellulose. G. barbadense and G. hirsutum contain 29 and 30 cellulose synthase (CesA) genes, respectively; whereas most of these genes (>25) are expressed in fiber, genes for secondary cell wall biosynthesis exhibited a delayed and higher degree of up-regulation in G. barbadense compared with G. hirsutum, conferring an extended elongation stage and highly active secondary wall deposition during extra-long fiber development. The rapid diversification of sesquiterpene synthase genes in the gossypol pathway exemplifies the chemical diversity of lineage-specific secondary metabolites. The G. barbadense genome advances our understanding of allopolyploidy, which will help improve cotton fiber quality.

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July 7, 2019

Genome and transcriptome of the regeneration-competent flatworm, Macrostomum lignano.

The free-living flatworm, Macrostomum lignano has an impressive regenerative capacity. Following injury, it can regenerate almost an entirely new organism because of the presence of an abundant somatic stem cell population, the neoblasts. This set of unique properties makes many flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell-fate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of M. lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ~75% of its sequence being comprised of simple repeats and transposon sequences. This has made high-quality assembly from Illumina reads alone impossible (N50 = 222 bp). We therefore generated 130× coverage by long sequencing reads from the Pacific Biosciences platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene-expression patterns during regeneration, examining pathways important to stem cell function.

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July 7, 2019

CHOgenome.org 2.0: Genome resources and website updates.

Chinese hamster ovary (CHO) cells are a major host cell line for the production of therapeutic proteins, and CHO cell and Chinese hamster (CH) genomes have recently been sequenced using next-generation sequencing methods. CHOgenome.org was launched in 2011 (version 1.0) to serve as a database repository and to provide bioinformatics tools for the CHO community. CHOgenome.org (version 1.0) maintained GenBank CHO-K1 genome data, identified CHO-omics literature, and provided a CHO-specific BLAST service. Recent major updates to CHOgenome.org (version 2.0) include new sequence and annotation databases for both CHO and CH genomes, a more user-friendly website, and new research tools, including a proteome browser and a genome viewer. CHO cell-line specific sequences and annotations facilitate cell line development opportunities, several of which are discussed. Moving forward, CHOgenome.org will host the increasing amount of CHO-omics data and continue to make useful bioinformatics tools available to the CHO community. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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July 7, 2019

A synteny-based draft genome sequence of the forage grass Lolium perenne.

Here we report the draft genome sequence of perennial ryegrass (Lolium perenne), an economically important forage and turf grass species that is widely cultivated in temperate regions worldwide. It is classified along with wheat, barley, oats and Brachypodium distachyon in the Pooideae sub-family of the grass family (Poaceae). Transcriptome data was used to identify 28 455 gene models, and we utilized macro-co-linearity between perennial ryegrass and barley, and synteny within the grass family, to establish a synteny-based linear gene order. The gametophytic self-incompatibility mechanism enables the pistil of a plant to reject self-pollen and therefore promote out-crossing. We have used the sequence assembly to characterize transcriptional changes in the stigma during pollination with both compatible and incompatible pollen. Characterization of the pollen transcriptome identified homologs to pollen allergens from a range of species, many of which were expressed to very high levels in mature pollen grains, and are potentially involved in the self-incompatibility mechanism. The genome sequence provides a valuable resource for future breeding efforts based on genomic prediction, and will accelerate the development of new varieties for more productive grasslands.© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

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July 7, 2019

The genome and methylome of a beetle with complex social behavior, Nicrophorus vespilloides (Coleoptera: Silphidae).

Testing for conserved and novel mechanisms underlying phenotypic evolution requires a diversity of genomes available for comparison spanning multiple independent lineages. For example, complex social behavior in insects has been investigated primarily with eusocial lineages, nearly all of which are Hymenoptera. If conserved genomic influences on sociality do exist, we need data from a wider range of taxa that also vary in their levels of sociality. Here, we present the assembled and annotated genome of the subsocial beetle Nicrophorus vespilloides, a species long used to investigate evolutionary questions of complex social behavior. We used this genome to address two questions. First, do aspects of life history, such as using a carcass to breed, predict overlap in gene models more strongly than phylogeny? We found that the overlap in gene models was similar between N. vespilloides and all other insect groups regardless of life history. Second, like other insects with highly developed social behavior but unlike other beetles, does N. vespilloides have DNA methylation? We found strong evidence for an active DNA methylation system. The distribution of methylation was similar to other insects with exons having the most methylated CpGs. Methylation status appears highly conserved; 85% of the methylated genes in N. vespilloides are also methylated in the hymentopteran Nasonia vitripennis. The addition of this genome adds a coleopteran resource to answer questions about the evolution and mechanistic basis of sociality and to address questions about the potential role of methylation in social behavior. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019

Enzymatic degradation of phenazines can generate energy and protect sensitive organisms from toxicity.

Diverse bacteria, including several Pseudomonas species, produce a class of redox-active metabolites called phenazines that impact different cell types in nature and disease. Phenazines can affect microbial communities in both positive and negative ways, where their presence is correlated with decreased species richness and diversity. However, little is known about how the concentration of phenazines is modulated in situ and what this may mean for the fitness of members of the community. Through culturing of phenazine-degrading mycobacteria, genome sequencing, comparative genomics, and molecular analysis, we identified several conserved genes that are important for the degradation of three Pseudomonas-derived phenazines: phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), and pyocyanin (PYO). PCA can be used as the sole carbon source for growth by these organisms. Deletion of several genes in Mycobacterium fortuitum abolishes the degradation phenotype, and expression of two genes in a heterologous host confers the ability to degrade PCN and PYO. In cocultures with phenazine producers, phenazine degraders alter the abundance of different phenazine types. Not only does degradation support mycobacterial catabolism, but also it provides protection to bacteria that would otherwise be inhibited by the toxicity of PYO. Collectively, these results serve as a reminder that microbial metabolites can be actively modified and degraded and that these turnover processes must be considered when the fate and impact of such compounds in any environment are being assessed.Phenazine production by Pseudomonas spp. can shape microbial communities in a variety of environments ranging from the cystic fibrosis lung to the rhizosphere of dryland crops. For example, in the rhizosphere, phenazines can protect plants from infection by pathogenic fungi. The redox activity of phenazines underpins their antibiotic activity, as well as providing pseudomonads with important physiological benefits. Our discovery that soil mycobacteria can catabolize phenazines and thereby protect other organisms against phenazine toxicity suggests that phenazine degradation may influence turnover in situ. The identification of genes involved in the degradation of phenazines opens the door to monitoring turnover in diverse environments, an essential process to consider when one is attempting to understand or control communities influenced by phenazines. Copyright © 2015 Costa et al.

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July 7, 2019

Chromosomal rearrangements as barriers to genetic homogenization between archaic and modern humans.

Chromosomal rearrangements, which shuffle DNA throughout the genome, are an important source of divergence across taxa. Using a paired-end read approach with Illumina sequence data for archaic humans, I identify changes in genome structure that occurred recently in human evolution. Hundreds of rearrangements indicate genomic trafficking between the sex chromosomes and autosomes, raising the possibility of sex-specific changes. Additionally, genes adjacent to genome structure changes in Neanderthals are associated with testis-specific expression, consistent with evolutionary theory that new genes commonly form with expression in the testes. I identify one case of new-gene creation through transposition from the Y chromosome to chromosome 10 that combines the 5′-end of the testis-specific gene Fank1 with previously untranscribed sequence. This new transcript experienced copy number expansion in archaic genomes, indicating rapid genomic change. Among rearrangements identified in Neanderthals, 13% are transposition of selfish genetic elements, whereas 32% appear to be ectopic exchange between repeats. In Denisovan, the pattern is similar but numbers are significantly higher with 18% of rearrangements reflecting transposition and 40% ectopic exchange between distantly related repeats. There is an excess of divergent rearrangements relative to polymorphism in Denisovan, which might result from nonuniform rates of mutation, possibly reflecting a burst of transposable element activity in the lineage that led to Denisovan. Finally, loci containing genome structure changes show diminished rates of introgression from Neanderthals into modern humans, consistent with the hypothesis that rearrangements serve as barriers to gene flow during hybridization. Together, these results suggest that this previously unidentified source of genomic variation has important biological consequences in human evolution. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Contiguity: Contig adjacency graph construction and visualisation

Contiguity is interactive software for the visualization and manipulation of de novo genome assemblies. 14 Contiguity creates and displays information on contig adjacency which is contextualized by the 15 simultaneous display of a comparison between assembled contigs and reference sequence. Where 16 scaffolders allow unambiguous connections between contigs to be resolved into a single scaffold, 17 Contiguity allows the user to create all potential scaffolds in ambiguous regions of the genome. This 18 enables the resolution of novel sequence or structural variants from the assembly. In addition, 19 Contiguity provides a sequencing and assembly agnostic approach for the creation of contig adjacency 20 graphs. To maximize the number of contig adjacencies determined, Contiguity combines information 21 from read pair mappings, sequence overlap and De Bruijn graph exploration. We demonstrate how 22 highly sensitive graphs can be achieved using this method. Contig adjacency graphs allow the user to 23 visualize potential arrangements of contigs in unresolvable areas of the genome. By combining 24 adjacency information with comparative genomics, Contiguity provides an intuitive approach for 25 exploring and improving sequence assemblies. It is also useful in guiding manual closure of long read 26 sequence assemblies. Contiguity is an open source application, implemented using Python and the 27 Tkinter GUI package that can run on any Unix, OSX and Windows operating system. It has been 28 designed and optimized for bacterial assemblies. Contiguity is available at 29 http://mjsull.github.io/Contiguity .

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