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July 7, 2019

From isolate to answer: how whole genome sequencing is helping us rapidly characterise nosocomial bacterial outbreaks

The occurrence of highly resistant bacterial pathogens has risen in recent years, causing immense strain on the healthcare industry. Hospital-acquired infections are arguably of most concern, as bacterial outbreaks in clinical settings provide an ideal environment for proliferation among vulnerable populations. Understanding these outbreaks beyond what can be determined with traditional clinical diagnostics and implementing these new techniques routinely in the hospital environment has now become a major focus. This brief review will discuss the three main whole genome sequence techniques available today, and how they are being used to further discriminate bacterial outbreaks in nosocomial settings.

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July 7, 2019

Pearl millet genome sequence provides a resource to improve agronomic traits in arid environments.

Pearl millet [Cenchrus americanus (L.) Morrone] is a staple food for more than 90 million farmers in arid and semi-arid regions of sub-Saharan Africa, India and South Asia. We report the ~1.79 Gb draft whole genome sequence of reference genotype Tift 23D2B1-P1-P5, which contains an estimated 38,579 genes. We highlight the substantial enrichment for wax biosynthesis genes, which may contribute to heat and drought tolerance in this crop. We resequenced and analyzed 994 pearl millet lines, enabling insights into population structure, genetic diversity and domestication. We use these resequencing data to establish marker trait associations for genomic selection, to define heterotic pools, and to predict hybrid performance. We believe that these resources should empower researchers and breeders to improve this important staple crop.

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July 7, 2019

Complete fusion of a transposon and herpesvirus created the Teratorn mobile element in medaka fish.

Mobile genetic elements (e.g., transposable elements and viruses) display significant diversity with various life cycles, but how novel elements emerge remains obscure. Here, we report a giant (180-kb long) transposon, Teratorn, originally identified in the genome of medaka, Oryzias latipes. Teratorn belongs to the piggyBac superfamily and retains the transposition activity. Remarkably, Teratorn is largely derived from a herpesvirus of the Alloherpesviridae family that could infect fish and amphibians. Genomic survey of Teratorn-like elements reveals that some of them exist as a fused form between piggyBac transposon and herpesvirus genome in teleosts, implying the generality of transposon-herpesvirus fusion. We propose that Teratorn was created by a unique fusion of DNA transposon and herpesvirus, leading to life cycle shift. Our study supports the idea that recombination is the key event in generation of novel mobile genetic elements. Teratorn is a large mobile genetic element originally identified in the small teleost fish medaka. Here, the authors show that Teratorn is derived from the fusion of a piggyBac superfamily DNA transposon and an alloherpesvirus and that it is widely found across teleost fish.

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July 7, 2019

Centrochromatin of fungi.

The centromere is an essential chromosomal locus that dictates the nucleation point for assembly of the kinetochore and subsequent attachment of spindle microtubules during chromosome segregation. Research over the last decades demonstrated that centromeres are defined by a combination of genetic and epigenetic factors. Recent work showed that centromeres are quite diverse and flexible and that many types of centromere sequences and centromeric chromatin (“centrochromatin”) have evolved. The kingdom of the fungi serves as an outstanding example of centromere plasticity, including organisms with centromeres as diverse as 0.15-300 kb in length, and with different types of chromatin states for most species examined thus far. Some of the species in the less familiar taxa provide excellent opportunities to help us better understand centromere biology in all eukaryotes, which may improve treatment options against fungal infection, and biotechnologies based on fungi. This review summarizes the current knowledge of fungal centromeres and centrochromatin, including an outlook for future research.

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July 7, 2019

Genome characterization of two bile-isolated Vibrio fluvialis strains: an insight into pathogenicity and bile salt adaption.

Vibrio fluvialis is recognized as an emerging pathogen. However, not much is known about the mechanism of its pathogenesis, and its adaptation to a special niche such as the gall bladder. Here we describe two V. fluvialis strains that cause acute cholecystitis. It is noteworthy that both strains were susceptible to all antibiotics tested, which is in contrast to previous studies, suggesting substantial genetic diversity among V. fluvialis isolates. In agreement with their survival and growth in the gall bladder, the genomes of strains 12605 and 3663 contain a considerable number of genes that confer resistance to bile, including toxR, omp U, tolC, cmeABC, rlpB, yrbK, rpoS, damX and gltK. Furthermore, integrative and conjugative elements (ICEs), virulence factors and prophage regions were also detected in strains 12605 and 3663, reflecting their flexibility in recombination during the evolution of pathogenicity. Comparative analysis of nine available genomes of V. fluvialis revealed a core genome consisting of 3,147 genes. Our results highlight the association of V. fluvialis with a rare disease profile and shed light on the evolution of pathogenesis and niche adaptation of V. fluvialis.

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July 7, 2019

Genomic structure and evolution of the mating type locus in the green seaweed Ulva partita.

The evolution of sex chromosomes and mating loci in organisms with UV systems of sex/mating type determination in haploid phases via genes on UV chromosomes is not well understood. We report the structure of the mating type (MT) locus and its evolutionary history in the green seaweed Ulva partita, which is a multicellular organism with an isomorphic haploid-diploid life cycle and mating type determination in the haploid phase. Comprehensive comparison of a total of 12.0 and 16.6?Gb of genomic next-generation sequencing data for mt(-) and mt(+) strains identified highly rearranged MT loci of 1.0 and 1.5?Mb in size and containing 46 and 67 genes, respectively, including 23 gametologs. Molecular evolutionary analyses suggested that the MT loci diverged over a prolonged period in the individual mating types after their establishment in an ancestor. A gene encoding an RWP-RK domain-containing protein was found in the mt(-) MT locus but was not an ortholog of the chlorophycean mating type determination gene MID. Taken together, our results suggest that the genomic structure and its evolutionary history in the U. partita MT locus are similar to those on other UV chromosomes and that the MT locus genes are quite different from those of Chlorophyceae.

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July 7, 2019

A step-by-step guide to assemble a reptilian genome.

Multiple technologies and software are now available facilitating the de novo sequencing and assembly of any vertebrate genome. Yet the quality of most available sequenced genomes is substantially poorer than that of the golden standard in the field: the human genome. Here, we present a step-by-step protocol for the successful sequencing and assembly of a high-quality snake genome that can be applied to any other reptilian or avian species. We combine the great sequencing depth and accuracy of short reads with the use of different insert size libraries for extended scaffolding followed by optical mapping. We show that this procedure improved the corn snake scaffold N50 from 3.7 kbp to 1.4 Mbp, currently making it one of the snake genomes with the longest scaffolds. Short guidelines are also given on the extraction of long DNA molecules from reptilian blood and the necessary modifications in DNA extraction protocols. This chapter is accompanied by a website ( www.reptilomics.org/stepbystep.html ), where we provide links to the suggested software, examples of input and output files, and running parameters.

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July 7, 2019

Draft genome sequences from a novel clade of Bacillus cereus sensu lato strains, isolated from the International Space Station.

The draft genome sequences of six Bacillus strains, isolated from the International Space Station and belonging to the Bacillus anthracis-B. cereus-B. thuringiensis group, are presented here. These strains were isolated from the Japanese Experiment Module (one strain), U.S. Harmony Node 2 (three strains), and Russian Segment Zvezda Module (two strains). Copyright © 2017 Venkateswaran et al.

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July 7, 2019

Identification and characterization of the novel colonization factor CS30 based on whole genome sequencing in enterotoxigenic Escherichia coli (ETEC).

The ability to colonize the small intestine is essential for enterotoxigenic Escherichia coli (ETEC) to cause diarrhea. Although 22 antigenically different colonization factors (CFs) have been identified and characterized in ETEC at least 30% of clinical ETEC isolates lack known CFs. Ninety-four whole genome sequenced “CF negative” isolates were searched for novel CFs using a reverse genetics approach followed by phenotypic analyses. We identified a novel CF, CS30, encoded by a set of seven genes, csmA-G, related to the human CF operon CS18 and the porcine CF operon 987P (F6). CS30 was shown to be thermo-regulated, expressed at 37?°C, but not at 20?°C, by SDS-page and mass spectrometry analyses as well as electron microscopy imaging. Bacteria expressing CS30 were also shown to bind to differentiated human intestinal Caco-2 cells. The genes encoding CS30 were located on a plasmid (E873p3) together with the genes encoding LT and STp. PCR screening of ETEC isolates revealed that 8.6% (n?=?13) of “CF negative” (n?=?152) and 19.4% (n?=?13) of “CF negative” LT?+?STp (n?=?67) expressing isolates analyzed harbored CS30. Hence, we conclude that CS30 is common among “CF negative” LT?+?STp isolates and is associated with ETEC that cause diarrhea.

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July 7, 2019

The Apostasia genome and the evolution of orchids.

Constituting approximately 10% of flowering plant species, orchids (Orchidaceae) display unique flower morphologies, possess an extraordinary diversity in lifestyle, and have successfully colonized almost every habitat on Earth. Here we report the draft genome sequence of Apostasia shenzhenica, a representative of one of two genera that form a sister lineage to the rest of the Orchidaceae, providing a reference for inferring the genome content and structure of the most recent common ancestor of all extant orchids and improving our understanding of their origins and evolution. In addition, we present transcriptome data for representatives of Vanilloideae, Cypripedioideae and Orchidoideae, and novel third-generation genome data for two species of Epidendroideae, covering all five orchid subfamilies. A. shenzhenica shows clear evidence of a whole-genome duplication, which is shared by all orchids and occurred shortly before their divergence. Comparisons between A. shenzhenica and other orchids and angiosperms also permitted the reconstruction of an ancestral orchid gene toolkit. We identify new gene families, gene family expansions and contractions, and changes within MADS-box gene classes, which control a diverse suite of developmental processes, during orchid evolution. This study sheds new light on the genetic mechanisms underpinning key orchid innovations, including the development of the labellum and gynostemium, pollinia, and seeds without endosperm, as well as the evolution of epiphytism; reveals relationships between the Orchidaceae subfamilies; and helps clarify the evolutionary history of orchids within the angiosperms.

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July 7, 2019

Single-molecule sequencing and Hi-C-based proximity-guided assembly of amaranth (Amaranthus hypochondriacus) chromosomes provide insights into genome evolution.

Amaranth (Amaranthus hypochondriacus) was a food staple among the ancient civilizations of Central and South America that has recently received increased attention due to the high nutritional value of the seeds, with the potential to help alleviate malnutrition and food security concerns, particularly in arid and semiarid regions of the developing world. Here, we present a reference-quality assembly of the amaranth genome which will assist the agronomic development of the species.Utilizing single-molecule, real-time sequencing (Pacific Biosciences) and chromatin interaction mapping (Hi-C) to close assembly gaps and scaffold contigs, respectively, we improved our previously reported Illumina-based assembly to produce a chromosome-scale assembly with a scaffold N50 of 24.4 Mb. The 16 largest scaffolds contain 98% of the assembly and likely represent the haploid chromosomes (n?=?16). To demonstrate the accuracy and utility of this approach, we produced physical and genetic maps and identified candidate genes for the betalain pigmentation pathway. The chromosome-scale assembly facilitated a genome-wide syntenic comparison of amaranth with other Amaranthaceae species, revealing chromosome loss and fusion events in amaranth that explain the reduction from the ancestral haploid chromosome number (n?=?18) for a tetraploid member of the Amaranthaceae.The assembly method reported here minimizes cost by relying primarily on short-read technology and is one of the first reported uses of in vivo Hi-C for assembly of a plant genome. Our analyses implicate chromosome loss and fusion as major evolutionary events in the 2n?=?32 amaranths and clearly establish the homoeologous relationship among most of the subgenome chromosomes, which will facilitate future investigations of intragenomic changes that occurred post polyploidization.

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July 7, 2019

Identification of novel conjugative plasmids with multiple copies of fosB that confer high-level fosfomycin resistance to vancomycin-resistant Enterococci.

To further characterize the fosB-carrying plasmids of 19 vancomycin-resistant enterococci, the complete sequences of the fosB- and vanA-containing plasmids of Enterococcus faecium (pEMA120) and E. avium (pEA19081) were obtained by single-molecule, real-time sequencing. We found that these two plasmids are essentially identical (99.99% nucleotide sequence identity), which proved the possibility of interspecies transmission. Comparative analysis of the plasmids revealed that the backbone of pEMA120 is 99% similar to a conjugative fosB-negative E. faecium plasmid, pZB18. There is a traE disrupted in the transfer region of pEMA120, in comparison to pZB18 with an intact traE. The difference of their transfer frequencies between pEMA120 and pZB18 suggests this interruption of traE might affect conjugative transfer. Two copies of the fosB gene linked to a tnpA gene, forming an ISL3-like transposon, were found at separate locations within pEMA120, which had not been reported previously. These two fosB-carrying transposons were confirmed to form circular intermediates by inverse PCR. The hybridization of plasmid DNA digested by BsaI, having restriction site within the fosB sequence, demonstrated that the presence of multiple copies of fosB per plasmid is common. The total copy number of the fosB gene as revealed by qRT-PCR did not correlate with fosfomycin MICs or growth rates at sub-MICs of fosfomycin in different transconjugants. From susceptibility tests, the fosB gene, regardless of the copy number, conferred high fosfomycin MICs that ranged from 16384 to 65536 µg/ml. This first complete nucleotide sequence of a plasmid carrying two copies of fosB in VRE suggests that the fosB gene can transfer to multiple loci of plasmids by the ISL3 family transposase TnpA, possibly in the form of circular intermediates, leading to the dissemination of high fosfomycin resistance in VRE.

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July 7, 2019

The mobilome; A major contributor to Escherichia coli stx2-positive O26:H11 strains intra-serotype diversity.

Shiga toxin-producing Escherichia coli of serotype O26:H11/H- constitute a diverse group of strains and several clones with distinct genetic characteristics have been identified and characterized. Whole genome sequencing was performed using Illumina and PacBio technologies on eight stx2-positive O26:H11 strains circulating in France. Comparative analyses of the whole genome of the stx2-positive O26:H11 strains indicate that several clones of EHEC O26:H11 are co-circulating in France. Phylogenetic analysis of the French strains together with stx2-positive and stx-negative E. coli O26:H11 genomes obtained from Genbank indicates the existence of four clonal complexes (SNP-CCs) separated in two distinct lineages, one of which comprises the “new French clone” (SNP-CC1) that appears genetically closely related to stx-negative attaching and effacing E. coli (AEEC) strains. Interestingly, the whole genome SNP (wgSNP) phylogeny is summarized in the cas gene phylogeny, and a simple qPCR assay targeting the CRISPR array specific to SNP-CC1 (SP_O26-E) can distinguish between the two main lineages. The PacBio sequencing allowed a detailed analysis of the mobile genetic elements (MGEs) of the strains. Numerous MGEs were identified in each strain, including a large number of prophages and up to four large plasmids, representing overall 8.7-19.8% of the total genome size. Analysis of the prophage pool of the strains shows a considerable diversity with a complex history of recombination. Each clonal complex (SNP-CC) is characterized by a unique set of plasmids and phages, including stx-prophages, suggesting evolution through separate acquisition events. Overall, the MGEs appear to play a major role in O26:H11 intra-serotype clonal diversification.

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July 7, 2019

The complete genome sequence of Streptomyces autolyticus CGMCC 0516, the producer of geldanamycin, autolytimycin, reblastatin and elaiophylin.

Streptomyces autolyticus CGMCC 0516 produces the anti-tumor benzoquinone ansamycins geldanamycin, autolytimycin, and reblastatin and the 16-membered macrodiolide elaiophylin. Here, we report the complete genome sequence of S. autolyticus CGMCC 0516, which consists of a 10,029,028bp linear chromosome and seven circular plasmids. Fifty-seven putative biosynthetic gene clusters for secondary metabolites were found. The geldanamycin, autolytimycin, and reblastatin biosynthetic gene clusters were located on the left arm (2.06-2.15Mb) of the chromosome, and the elaiophylin gene cluster was located on the right arm (9.45-9.53Mb). Twenty-one putative gene clusters with high or moderate similarity to important antibiotic biosynthetic gene clusters were found, including the antitumor agents echoside, bafilomycin, hygrocin, and toxoflavin; the antibacterial/antifungal agents nigericin, skyllamycin, kanamycin, naphthomycin, eco-02301, and bottromycin A2; the immunosuppressants meridamycin and brasilicardin A; the anti-inflammatory agent cyclooctatin; and the acute iron poisoning medication desferrioxamine B. The genome sequence reported here will enable us to study the biosynthetic mechanism of these important antibiotics and will facilitate the discovery of novel secondary metabolites with potential applications to human health. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Phase-variable methylation and epigenetic regulation by type I restriction-modification systems.

Epigenetic modifications in bacteria, such as DNA methylation, have been shown to affect gene regulation, thereby generating cells that are isogenic but with distinctly different phenotypes. Restriction-modification (RM) systems contain prototypic methylases that are responsible for much of bacterial DNA methylation. This review focuses on a distinctive group of type I RM loci that , through phase variation, can modify their methylation target specificity and can thereby switch bacteria between alternative patterns of DNA methylation. Phase variation occurs at the level of the target recognition domains of the hsdS (specificity) gene via reversible recombination processes acting upon multiple hsdS alleles. We describe the global distribution of such loci throughout the prokaryotic kingdom and highlight the differences in loci structure across the various bacterial species. Although RM systems are often considered simply as an evolutionary response to bacteriophages, these multi-hsdS type I systems have also shown the capacity to change bacterial phenotypes. The ability of these RM systems to allow bacteria to reversibly switch between different physiological states, combined with the existence of such loci across many species of medical and industrial importance, highlights the potential of phase-variable DNA methylation to act as a global regulatory mechanism in bacteria.© FEMS 2017.

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