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July 7, 2019

Key features of mcr-1-bearing plasmids from Escherichia coli isolated from humans and food.

Mcr-1-harboring Enterobacteriaceae are reported worldwide since their first discovery in 2015. However, a limited number of studies are available that compared full-length plasmid sequences of human and animal origins.In this study, mcr-1-bearing plasmids from seven Escherichia coli isolates recovered from patients (n = 3), poultry meat (n = 2) and turkey meat (n = 2) in Switzerland were further analyzed and compared. Isolates were characterized by multilocus sequence typing (MLST). The mcr-1-bearing plasmids were transferred by transformation into reference strain E. coli DH5a and MCR-1-producing transformants were selected on LB-agar supplemented with 2 mg/L colistin. Purified plasmids were then sequenced and compared.MLST revealed six distinct STs, illustrating the high clonal diversity among mcr-1-positive E. coli isolates of different origins. Two different mcr-1-positive plasmids were identified from a single E. coli ST48 human isolate. All other isolates possessed a single mcr-1 harboring plasmid. Transferable IncI2 (size ca. 60-61 kb) and IncX4 (size ca. 33-35 kb) type plasmids each bearing mcr-1 were found associated with human and food isolates. None of the mcr-1-positive IncI2 and IncX4 plasmids possessed any additional resistance determinants. Surprisingly, all but one of the sequenced mcr-1-positive plasmids lacked the ISApl1 element, which is a key element mediating acquisition of mcr-1 into various plasmid backbones.There is strong evidence that the food chain may be an important transmission route for mcr-1-bearing plasmids. Our data suggest that some “epidemic” plasmids rather than specific E. coli clones might be responsible for the spread of the mcr-1 gene along the food chain.

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July 7, 2019

Complete genome of Arthrobacter alpinus strain R3.8, bioremediation potential unraveled with genomic analysis.

Arthrobacter alpinus R3.8 is a psychrotolerant bacterial strain isolated from a soil sample obtained at Rothera Point, Adelaide Island, close to the Antarctic Peninsula. Strain R3.8 was sequenced in order to help discover potential cold active enzymes with biotechnological applications. Genome analysis identified various cold adaptation genes including some coding for anti-freeze proteins and cold-shock proteins, genes involved in bioremediation of xenobiotic compounds including naphthalene, and genes with chitinolytic and N-acetylglucosamine utilization properties and also plant-growth-influencing properties. In this genome report, we present a complete genome sequence of A. alpinus strain R3.8 and its annotation data, which will facilitate exploitation of potential novel cold-active enzymes.

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July 7, 2019

Complete genome sequence of a strain of Bifidobacterium pseudolongum isolated from mouse feces and associated with improved organ transplant outcome.

Here, we report the complete genome sequence of Bifidobacterium pseudolongum strain UMB-MBP-01, isolated from the feces of C57BL/6J mice. This strain was identified in microbiome profiling studies and associated with improved transplant outcome in a murine model of cardiac heterotypic transplantation. Copyright © 2017 Mongodin et al.

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July 7, 2019

Natural product diversity associated with the nematode symbionts Photorhabdus and Xenorhabdus.

Xenorhabdus and Photorhabdus species dedicate a large amount of resources to the production of specialized metabolites derived from non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS). Both bacteria undergo symbiosis with nematodes, which is followed by an insect pathogenic phase. So far, the molecular basis of this tripartite relationship and the exact roles that individual metabolites and metabolic pathways play have not been well understood. To close this gap, we have significantly expanded the database for comparative genomics studies in these bacteria. Clustering the genes encoded in the individual genomes into hierarchical orthologous groups reveals a high-resolution picture of functional evolution in this clade. It identifies groups of genes-many of which are involved in secondary metabolite production-that may account for the niche specificity of these bacteria. Photorhabdus and Xenorhabdus appear very similar at the DNA sequence level, which indicates their close evolutionary relationship. Yet, high-resolution mass spectrometry analyses reveal a huge chemical diversity in the two taxa. Molecular network reconstruction identified a large number of previously unidentified metabolite classes, including the xefoampeptides and tilivalline. Here, we apply genomic and metabolomic methods in a complementary manner to identify and elucidate additional classes of natural products. We also highlight the ability to rapidly and simultaneously identify potentially interesting bioactive products from NRPSs and PKSs, thereby augmenting the contribution of molecular biology techniques to the acceleration of natural product discovery.

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July 7, 2019

Building a locally diploid genome and transcriptome of the diatom Fragilariopsis cylindrus.

The genome of the cold-adapted diatom Fragilariopsis cylindrus is characterized by highly diverged haplotypes that intersperse its homozygous genome. Here, we describe how a combination of PacBio DNA and Illumina RNA sequencing can be used to resolve this complex genomic landscape locally into the highly diverged haplotypes, and how to map various environmentally controlled transcripts onto individual haplotypes. We assembled PacBio sequence data with the FALCON assembler and created a haplotype resolved annotation of the assembly using annotations of a Sanger sequenced F. cylindrus genome. RNA-seq datasets from six different growth conditions were used to resolve allele-specifc gene expression in F. cylindrus. This approach enables to study differential expression of alleles in a complex genomic landscape and provides a useful tool to study how diverged haplotypes in diploid organisms are used for adaptation and evolution to highly variable environments.

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July 7, 2019

Rapid and affordable size-selected PacBio single-molecule real-time sequencing template library construction using the bead-beating DNA extraction method

This study demonstrated that bead-beating method facilitates a simple and rapid protocol for genomic DNA isolation for Pacific BioSciences (PacBio) sequencing with library construction of sufficient length. The protocol may also be beneficial for inactivating pathogens by simultaneous and instant DNA fragmentation, with no special equipment required to obtain large DNA fragments. This protocol was comparable in terms of quality to the standard protocol suggested by PacBioand represents an alternative, rapid shortcut for performing accurate PacBio sequencing.

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July 7, 2019

The Tartary buckwheat genome provides insights into rutin biosynthesis and abiotic stress tolerance.

Tartary buckwheat (Fagopyrum tataricum) is an important pseudocereal crop that is strongly adapted to growth in adverse environments. Its gluten-free grain contains complete proteins with a well-balanced composition of essential amino acids and is a rich source of beneficial phytochemicals that provide significant health benefits. Here, we report a high-quality, chromosome-scale Tartary buckwheat genome sequence of 489.3 Mb that is assembled by combining whole-genome shotgun sequencing of both Illumina short reads and single-molecule real-time long reads, sequence tags of a large DNA insert fosmid library, Hi-C sequencing data, and BioNano genome maps. We annotated 33 366 high-confidence protein-coding genes based on expression evidence. Comparisons of the intra-genome with the sugar beet genome revealed an independent whole-genome duplication that occurred in the buckwheat lineage after they diverged from the common ancestor, which was not shared with rosids or asterids. The reference genome facilitated the identification of many new genes predicted to be involved in rutin biosynthesis and regulation, aluminum stress resistance, and in drought and cold stress responses. Our data suggest that Tartary buckwheat’s ability to tolerate high levels of abiotic stress is attributed to the expansion of several gene families involved in signal transduction, gene regulation, and membrane transport. The availability of these genomic resources will facilitate the discovery of agronomically and nutritionally important genes and genetic improvement of Tartary buckwheat. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

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July 7, 2019

Whole-genome sequencing identification of a multidrug-resistant Salmonella enterica serovar Typhimurium strain carrying blaNDM-5 from Guangdong, China.

A carbapenem-resistant Salmonella enterica serovar Typhimurium (sequence type 34 [ST34]) strain was isolated from a fecal specimen from a child with acute diarrhea. Whole-genome sequencing revealed that the 84.5-kb IncFII plasmid pST41-NDM carrying the NDM-5 carbapenemase gene possesses a structure identical to that of the IncFII-type plasmid backbone. However, the blaNDM-5 flanking sequence found in this plasmid is identical to the blaNDM-5-positive IncX3 plasmids carried by 10 strains of Enterobacteriaceae identified in the same hospital. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition.

CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

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July 7, 2019

Avoidance of APOBEC3B-induced mutation by error-free lesion bypass.

APOBEC cytidine deaminases mutate cancer genomes by converting cytidines into uridines within ssDNA during replication. Although uracil DNA glycosylases limit APOBEC-induced mutation, it is unknown if subsequent base excision repair (BER) steps function on replication-associated ssDNA. Hence, we measured APOBEC3B-induced CAN1 mutation frequencies in yeast deficient in BER endonucleases or DNA damage tolerance proteins. Strains lacking Apn1, Apn2, Ntg1, Ntg2 or Rev3 displayed wild-type frequencies of APOBEC3B-induced canavanine resistance (CanR). However, strains without error-free lesion bypass proteins Ubc13, Mms2 and Mph1 displayed respective 4.9-, 2.8- and 7.8-fold higher frequency of APOBEC3B-induced CanR. These results indicate that mutations resulting from APOBEC activity are avoided by deoxyuridine conversion to abasic sites ahead of nascent lagging strand DNA synthesis and subsequent bypass by error-free template switching. We found this mechanism also functions during telomere re-synthesis, but with a diminished requirement for Ubc13. Interestingly, reduction of G to C substitutions in Ubc13-deficient strains uncovered a previously unknown role of Ubc13 in controlling the activity of the translesion synthesis polymerase, Rev1. Our results highlight a novel mechanism for error-free bypass of deoxyuridines generated within ssDNA and suggest that the APOBEC mutation signature observed in cancer genomes may under-represent the genomic damage these enzymes induce.© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 7, 2019

Single-virion sequencing of lamivudine-treated HBV populations reveal population evolution dynamics and demographic history.

Viral populations are complex, dynamic, and fast evolving. The evolution of groups of closely related viruses in a competitive environment is termed quasispecies. To fully understand the role that quasispecies play in viral evolution, characterizing the trajectories of viral genotypes in an evolving population is the key. In particular, long-range haplotype information for thousands of individual viruses is critical; yet generating this information is non-trivial. Popular deep sequencing methods generate relatively short reads that do not preserve linkage information, while third generation sequencing methods have higher error rates that make detection of low frequency mutations a bioinformatics challenge. Here we applied BAsE-Seq, an Illumina-based single-virion sequencing technology, to eight samples from four chronic hepatitis B (CHB) patients – once before antiviral treatment and once after viral rebound due to resistance.With single-virion sequencing, we obtained 248-8796 single-virion sequences per sample, which allowed us to find evidence for both hard and soft selective sweeps. We were able to reconstruct population demographic history that was independently verified by clinically collected data. We further verified four of the samples independently through PacBio SMRT and Illumina Pooled deep sequencing.Overall, we showed that single-virion sequencing yields insight into viral evolution and population dynamics in an efficient and high throughput manner. We believe that single-virion sequencing is widely applicable to the study of viral evolution in the context of drug resistance and host adaptation, allows differentiation between soft or hard selective sweeps, and may be useful in the reconstruction of intra-host viral population demographic history.

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July 7, 2019

Large-scale suppression of recombination predates genomic rearrangements in Neurospora tetrasperma.

A common feature of eukaryote genomes is large chromosomal regions where recombination is absent or strongly reduced, but the factors that cause this reduction are not well understood. Genomic rearrangements have often been implicated, but they may also be a consequence of recombination suppression rather than a cause. In this study, we generate eight high-quality genomic data sets of the filamentous ascomycete Neurospora tetrasperma, a fungus that lacks recombination over most of its largest chromosome. The genomes surprisingly reveal collinearity of the non-recombining regions and although large inversions are enriched in these regions, we conclude these inversions to be derived and not the cause of the suppression. To our knowledge, this is the first time that non-recombining, genic regions as large as 86% of a full chromosome (or 8?Mbp), are shown to be collinear. These findings are of significant interest for our understanding of the evolution of sex chromosomes and other supergene complexes.

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July 7, 2019

The asparagus genome sheds light on the origin and evolution of a young Y chromosome.

Sex chromosomes evolved from autosomes many times across the eukaryote phylogeny. Several models have been proposed to explain this transition, some involving male and female sterility mutations linked in a region of suppressed recombination between X and Y (or Z/W, U/V) chromosomes. Comparative and experimental analysis of a reference genome assembly for a double haploid YY male garden asparagus (Asparagus officinalis L.) individual implicates separate but linked genes as responsible for sex determination. Dioecy has evolved recently within Asparagus and sex chromosomes are cytogenetically identical with the Y, harboring a megabase segment that is missing from the X. We show that deletion of this entire region results in a male-to-female conversion, whereas loss of a single suppressor of female development drives male-to-hermaphrodite conversion. A single copy anther-specific gene with a male sterile Arabidopsis knockout phenotype is also in the Y-specific region, supporting a two-gene model for sex chromosome evolution.

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