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July 7, 2019

Whole-genome analysis of Exserohilum rostratum from an outbreak of fungal meningitis and other infections.

Exserohilum rostratum was the cause of most cases of fungal meningitis and other infections associated with the injection of contaminated methylprednisolone acetate produced by the New England Compounding Center (NECC). Until this outbreak, very few human cases of Exserohilum infection had been reported, and very little was known about this dematiaceous fungus, which usually infects plants. Here, we report using whole-genome sequencing (WGS) for the detection of single nucleotide polymorphisms (SNPs) and phylogenetic analysis to investigate the molecular origin of the outbreak using 22 isolates of E. rostratum retrieved from 19 case patients with meningitis or epidural/spinal abscesses, 6 isolates from contaminated NECC vials, and 7 isolates unrelated to the outbreak. Our analysis indicates that all 28 isolates associated with the outbreak had nearly identical genomes of 33.8 Mb. A total of 8 SNPs were detected among the outbreak genomes, with no more than 2 SNPs separating any 2 of the 28 genomes. The outbreak genomes were separated from the next most closely related control strain by ~136,000 SNPs. We also observed significant genomic variability among strains unrelated to the outbreak, which may suggest the possibility of cryptic speciation in E. rostratum. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Complete sequence of a conjugative IncN plasmid harboring blakpc-2, blashv-12, and qnrS1 from an Escherichia coli sequence type 648 strain

We sequenced a novel conjugative blaKPC-2-harboring IncN plasmid, pYD626E, from an Escherichia coli sequence type 648 strain previously identified in Pittsburgh, Pennsylvania. pYD626E was 72,800 bp long and carried four ß-lactamase genes, blaKPC-2, blaSHV-12, blaLAP-1, and blaTEM-1. In addition, it harbored qnrS1 (fluoroquinolone resistance) and dfrA14 (trimethoprim resistance). The plasmid profile and clinical history supported the in vivo transfer of this plasmid between Klebsiella pneumoniae and Escherichia coli. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Genome analysis of a major urban malaria vector mosquito, Anopheles stephensi.

Background Anopheles stephensi is the key vector of malaria throughout the Indian subcontinent and Middle East and an emerging model for molecular and genetic studies of mosquito-parasite interactions. The type form of the species is responsible for the majority of urban malaria transmission across its range.ResultsHere, we report the genome sequence and annotation of the Indian strain of the type form of An. stephensi. The 221 Mb genome assembly represents more than 92% of the entire genome and was produced using a combination of 454, Illumina, and PacBio sequencing. Physical mapping assigned 62% of the genome onto chromosomes, enabling chromosome-based analysis. Comparisons between An. stephensi and An. gambiae reveal that the rate of gene order reshuffling on the X chromosome was three times higher than that on the autosomes. An. stephensi has more heterochromatin in pericentric regions but less repetitive DNA in chromosome arms than An. gambiae. We also identify a number of Y-chromosome contigs and BACs. Interspersed repeats constitute 7.1% of the assembled genome while LTR retrotransposons alone comprise more than 49% of the Y contigs. RNA-seq analyses provide new insights into mosquito innate immunity, development, and sexual dimorphism.ConclusionsThe genome analysis described in this manuscript provides a resource and platform for fundamental and translational research into a major urban malaria vector. Chromosome-based investigations provide unique perspectives on Anopheles chromosome evolution. RNA-seq analysis and studies of immunity genes offer new insights into mosquito biology and mosquito-parasite interactions.

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July 7, 2019

The genomic landscape of the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV.

Aerobic methanotrophs can grow in hostile volcanic environments and use methane as their sole source of energy. The discovery of three verrucomicrobial Methylacidiphilum strains has revealed diverse metabolic pathways used by these methanotrophs, including mechanisms through which methane is oxidized. The basis of a complete understanding of these processes and of how these bacteria evolved and are able to thrive in such extreme environments partially resides in the complete characterization of their genome and its architecture.In this study, we present the complete genome sequence of Methylacidiphilum fumariolicum SolV, obtained using Pacific Biosciences single-molecule real-time (SMRT) sequencing technology. The genome assembles to a single 2.5 Mbp chromosome with an average GC content of 41.5%. The genome contains 2,741 annotated genes and 314 functional subsystems including all key metabolic pathways that are associated with Methylacidiphilum strains, including the CBB pathway for CO2 fixation. However, it does not encode the serine cycle and ribulose monophosphate pathways for carbon fixation. Phylogenetic analysis of the particulate methane mono-oxygenase operon separates the Methylacidiphilum strains from other verrucomicrobial methanotrophs. RNA-Seq analysis of cell cultures growing in three different conditions revealed the deregulation of two out of three pmoCAB operons. In addition, genes involved in nitrogen fixation were upregulated in cell cultures growing in nitrogen fixing conditions, indicating the presence of active nitrogenase. Characterization of the global methylation state of M. fumariolicum SolV revealed methylation of adenines and cytosines mainly in the coding regions of the genome. Methylation of adenines was predominantly associated with 5′-m6ACN4GT-3′ and 5′-CCm6AN5CTC-3′ methyltransferase recognition motifs whereas methylated cytosines were not associated with any specific motif.Our findings provide novel insights into the global methylation state of verrucomicrobial methanotroph M. fumariolicum SolV. However, partial conservation of methyltransferases between M. fumariolicum SolV and M. infernorum V4 indicates potential differences in the global methylation state of Methylacidiphilum strains. Unravelling the M. fumariolicum SolV genome and its epigenetic regulation allow for robust characterization of biological processes that are involved in oxidizing methane. In turn, they offer a better understanding of the evolution, the underlying physiological and ecological properties of SolV and other Methylacidiphilum strains.

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July 7, 2019

Methyltransferases acquired by lactococcal 936-type phage provide protection against restriction endonuclease activity

BACKGROUND:So-called 936-type phages are among the most frequently isolated phages in dairy facilities utilising Lactococcus lactis starter cultures. Despite extensive efforts to control phage proliferation and decades of research, these phages continue to negatively impact cheese production in terms of the final product quality and consequently, monetary return.RESULTS:Whole genome sequencing and in silico analysis of three 936-type phage genomes identified several putative (orphan) methyltransferase (MTase)-encoding genes located within the packaging and replication regions of the genome. Utilising SMRT sequencing, methylome analysis was performed on all three phages, allowing the identification of adenine modifications consistent with N-6 methyladenine sequence methylation, which in some cases could be attributed to these phage-encoded MTases. Heterologous gene expression revealed that M.Phi145I/M.Phi93I and M.Phi93DAM, encoded by genes located within the packaging module, provide protection against the restriction enzymes HphI and DpnII, respectively, representing the first functional MTases identified in members of 936-type phages.CONCLUSIONS:SMRT sequencing technology enabled the identification of the target motifs of MTases encoded by the genomes of three lytic 936-type phages and these MTases represent the first functional MTases identified in this species of phage. The presence of these MTase-encoding genes on 936-type phage genomes is assumed to represent an adaptive response to circumvent host encoded restriction-modification systems thereby increasing the fitness of the phages in a dynamic dairy environment.

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July 7, 2019

An evaluation of alternative methods for constructing phylogenies from whole genome sequence data: a case study with Salmonella.

Comparative genomics based on whole genome sequencing (WGS) is increasingly being applied to investigate questions within evolutionary and molecular biology, as well as questions concerning public health (e.g., pathogen outbreaks). Given the impact that conclusions derived from such analyses may have, we have evaluated the robustness of clustering individuals based on WGS data to three key factors: (1) next-generation sequencing (NGS) platform (HiSeq, MiSeq, IonTorrent, 454, and SOLiD), (2) algorithms used to construct a SNP (single nucleotide polymorphism) matrix (reference-based and reference-free), and (3) phylogenetic inference method (FastTreeMP, GARLI, and RAxML). We carried out these analyses on 194 whole genome sequences representing 107 unique Salmonella enterica subsp. enterica ser. Montevideo strains. Reference-based approaches for identifying SNPs produced trees that were significantly more similar to one another than those produced under the reference-free approach. Topologies inferred using a core matrix (i.e., no missing data) were significantly more discordant than those inferred using a non-core matrix that allows for some missing data. However, allowing for too much missing data likely results in a high false discovery rate of SNPs. When analyzing the same SNP matrix, we observed that the more thorough inference methods implemented in GARLI and RAxML produced more similar topologies than FastTreeMP. Our results also confirm that reproducibility varies among NGS platforms where the MiSeq had the lowest number of pairwise differences among replicate runs. Our investigation into the robustness of clustering patterns illustrates the importance of carefully considering how data from different platforms are combined and analyzed. We found clear differences in the topologies inferred, and certain methods performed significantly better than others for discriminating between the highly clonal organisms investigated here. The methods supported by our results represent a preliminary set of guidelines and a step towards developing validated standards for clustering based on whole genome sequence data.

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July 7, 2019

Pseudoautosomal region 1 length polymorphism in the human population.

The human sex chromosomes differ in sequence, except for the pseudoautosomal regions (PAR) at the terminus of the short and the long arms, denoted as PAR1 and PAR2. The boundary between PAR1 and the unique X and Y sequences was established during the divergence of the great apes. During a copy number variation screen, we noted a paternally inherited chromosome X duplication in 15 independent families. Subsequent genomic analysis demonstrated that an insertional translocation of X chromosomal sequence into theMa Y chromosome generates an extended PAR. The insertion is generated by non-allelic homologous recombination between a 548 bp LTR6B repeat within the Y chromosome PAR1 and a second LTR6B repeat located 105 kb from the PAR boundary on the X chromosome. The identification of the reciprocal deletion on the X chromosome in one family and the occurrence of the variant in different chromosome Y haplogroups demonstrate this is a recurrent genomic rearrangement in the human population. This finding represents a novel mechanism shaping sex chromosomal evolution.

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July 7, 2019

Seeking the source of Pseudomonas aeruginosa infections in a recently opened hospital: an observational study using whole-genome sequencing.

Pseudomonas aeruginosa is a common nosocomial pathogen responsible for significant morbidity and mortality internationally. Patients may become colonised or infected with P. aeruginosa after exposure to contaminated sources within the hospital environment. The aim of this study was to determine whether whole-genome sequencing (WGS) can be used to determine the source in a cohort of burns patients at high risk of P. aeruginosa acquisition.An observational prospective cohort study.Burns care ward and critical care ward in the UK.Patients with >7% total burns by surface area were recruited into the study.All patients were screened for P. aeruginosa on admission and samples taken from their immediate environment, including water. Screening patients who subsequently developed a positive P. aeruginosa microbiology result were subject to enhanced environmental surveillance. All isolates of P. aeruginosa were genome sequenced. Sequence analysis looked at similarity and relatedness between isolates.WGS for 141 P. aeruginosa isolates were obtained from patients, hospital water and the ward environment. Phylogenetic analysis revealed eight distinct clades, with a single clade representing the majority of environmental isolates in the burns unit. Isolates from three patients had identical genotypes compared with water isolates from the same room. There was clear clustering of water isolates by room and outlet, allowing the source of acquisitions to be unambiguously identified. Whole-genome shotgun sequencing of biofilm DNA extracted from a thermostatic mixer valve revealed this was the source of a P. aeruginosa subpopulation previously detected in water. In the remaining two cases there was no clear link to the hospital environment.This study reveals that WGS can be used for source tracking of P. aeruginosa in a hospital setting, and that acquisitions can be traced to a specific source within a hospital ward. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

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July 7, 2019

Genomics of wood-degrading fungi.

Woody plants convert the energy of the sun into lignocellulosic biomass, which is an abundant substrate for bioenergy production. Fungi, especially wood decayers from the class Agaricomycetes, have evolved ways to degrade lignocellulose into its monomeric constituents, and understanding this process may facilitate the development of biofuels. Over the past decade genomics has become a powerful tool to study the Agaricomycetes. In 2004 the first sequenced genome of the white rot fungus Phanerochaete chrysosporium revealed a rich catalog of lignocellulolytic enzymes. In the decade that followed the number of genomes of Agaricomycetes grew to more than 75 and revealed a diversity of wood-decaying strategies. New technologies for high-throughput functional genomics are now needed to further study these organisms. Copyright © 2014 Elsevier Inc. All rights reserved.

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July 7, 2019

The odd one out: Bacillus ACT bacteriophage CP-51 exhibits unusual properties compared to related Spounavirinae W.Ph. and Bastille.

The Bacillus ACT group includes three important pathogenic species of Bacillus: anthracis, cereus and thuringiensis. We characterized three virulent bacteriophages, Bastille, W.Ph. and CP-51, that infect various strains of these three species. We have determined the complete genome sequences of CP-51, W.Ph. and Bastille, and their physical genome structures. The CP-51 genome sequence could only be obtained using a combination of conventional and second and third next generation sequencing technologies – illustrating the problems associated with sequencing highly modified DNA. We present evidence that the generalized transduction facilitated by CP-51 is independent of a specific genome structure, but likely due to sporadic packaging errors of the terminase. There is clear correlation of the genetic and morphological features of these phages validating their placement in the Spounavirinae subfamily (SPO1-related phages) of the Myoviridae. This study also provides tools for the development of phage-based diagnostics/therapeutics for this group of pathogens. Copyright © 2014 Elsevier Inc. All rights reserved.

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July 7, 2019

The characterization of goat genetic diversity: Towards a genomic approach

The investigation of genetic diversity at molecular level has been proposed as a valuable complement and sometimes proxy to phenotypic diversity of local breeds and is presently considered as one of the FAO priorities for breed characterization. By recommending a set of selected molecular markers for each of the main livestock species, FAO has promoted the meta-analysis of local datasets, to achieve a global view of molecular genetic diversity. Analysis within the EU Globaldiv project of two large goat microsatellite datasets produced by the Econogene Consortium and the IAEA CRP–Asia Consortium, respectively, has generated a picture of goat diversity across continents. This indicates a gradient of decreasing diversity from the domestication centre towards Europe and Asia, a clear phylogeographic structure at the continental and regional levels, and in Asia a limited genetic differentiation among local breeds. The development of SNP panels that assay thousands of markers and the whole genome sequencing of livestock permit an affordable use of genomic technologies in all livestock species, goats included. Preliminary data from the Italian Goat Consortium indicate that the SNP panel developed for this species is highly informative. The existing panel can be improved by integrating additional SNPs identified from the whole genome sequence alignment of goats adapted to extreme climates. Part of this effort is being achieved by international projects (e.g. EU FP7 NextGen and 3SR projects), but a fair representation of the global diversity in goats requires a large panel of samples (i.e. as in the recently launched 1000 cattle genomes initiative). Genomic technologies offer new strategies to investigate complex traits difficult to measure. For example, the comparison of patterns of diversity among the genomes in selected groups of animals (e.g. adapted to different environments) and the integration of genome-wide diversity with new GIScience-based methods are able to identify molecular markers associated with genomic regions of putative importance in adaptation and thus pave the way for the identification of causative genes. Goat breeds adapted to different production systems in extreme and harsh environments will play an important role in this process. The new sequencing technologies also permit the analysis of the entire mitochondrial genome at maximum resolution. The complete mtDNA sequence is now the common standard format for the investigation of human maternal lineages. A preliminary analysis of the complete goat mtDNA genome supports a single Neolithic origin of domestic goats rather than multiple domestication events in different geographic areas.

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July 7, 2019

Expansion of the genetic toolkit for metabolic engineering of Clostridium pasteurianum: chromosomal gene disruption of the endogenous CpaAI restriction enzyme.

Clostridium pasteurianum is one of the most promising biofuel producers within the genus Clostridium owing to its unique metabolic ability to ferment glycerol into butanol. Although an efficient means is available for introducing foreign DNA to C. pasteurianum, major genetic tools, such as gene knockout, knockdown, or genome editing, are lacking, preventing metabolic engineering of C. pasteurianum.Here we present a methodology for performing chromosomal gene disruption in C. pasteurianum using the programmable lactococcus Ll.ltrB group II intron. Gene disruption was initially found to be impeded by inefficient electrotransformation of Escherichia coli-C. pasteurianum shuttle vectors, presumably due to host restriction. By assessing the ability of various vector deletion derivatives to electrotransform C. pasteurianum and probing the microorganism’s methylome using next-generation sequence data, we identified a new C. pasteurianum Type I restriction-methylation system, CpaAII, with a predicted recognition sequence of 5′-AAGNNNNNCTCC-3′ (N?=?A, C, G, or T). Following rescue of high-level electrotransformation via mutation of the sole CpaAII site within the shuttle vectors, we retargeted the intron to the cpaAIR gene encoding the CpaAI Type II restriction endonuclease (recognition site of 5′-CGCG-3′). Intron insertion was potentially hindered by low retrohoming efficiency, yet this limitation could be overcome by a procedure for enrichment of the intron insertion. The resulting ?cpaAIR mutant strain was efficiently electrotransformed with M.FnuDII-unmethylated plasmid DNA.The markerless and plasmidless ?cpaAIR mutant strain of C. pasteurianum developed in this study can serve as a general host strain for future genetic and metabolic manipulation. Further, the associated gene disruption protocol should not only serve as a guide for chromosomal gene inactivation studies involving mobile group II introns, but also prove invaluable for applying metabolic engineering strategies to C. pasteurianum.

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July 7, 2019

A precise chloroplast genome of Nelumbo nucifera (Nelumbonaceae) evaluated with Sanger, Illumina MiSeq, and PacBio RS II sequencing platforms: insight into the plastid evolution of basal eudicots.

BackgroundThe chloroplast genome is important for plant development and plant evolution. Nelumbo nucifera is one member of relict plants surviving from the late Cretaceous. Recently, a new sequencing platform PacBio RS II, known as `SMRT (Single Molecule, Real-Time) sequencing¿, has been developed. Using the SMRT sequencing to investigate the chloroplast genome of N. nucifera will help to elucidate the plastid evolution of basal eudicots.ResultsThe sizes of the de novo assembled complete chloroplast genome of N. nucifera were 163,307 bp, 163,747 bp and 163,600 bp with average depths of coverage of 7×, 712× and 105× sequenced by Sanger, Illumina MiSeq and PacBio RS II, respectively. The precise chloroplast genome of N. nucifera was obtained from PacBio RS II data proofread by Illumina MiSeq reads, with a quadripartite structure containing a large single copy region (91,846 bp) and a small single copy region (19,626 bp) separated by two inverted repeat regions (26,064 bp). The genome contains 113 different genes, including four distinct rRNAs, 30 distinct tRNAs and 79 distinct peptide-coding genes. A phylogenetic analysis of 133 taxa from 56 orders indicated that Nelumbo with an age of 177 million years is a sister clade to Platanus, which belongs to the basal eudicots. Basal eudicots began to emerge during the early Jurassic with estimated divergence times at 197 million years using MCMCTree. IR expansions/contractions within the basal eudicots seem to have occurred independently.ConclusionsBecause of long reads and lack of bias in coverage of AT-rich regions, PacBio RS II showed a great promise for highly accurate `finished¿ genomes, especially for a de novo assembly of genomes. N. nucifera is one member of basal eudicots, however, evolutionary analyses of IR structural variations of N. nucifera and other basal eudicots suggested that IR expansions/contractions occurred independently in these basal eudicots or were caused by independent insertions and deletions. The precise chloroplast genome of N. nucifera will present new information for structural variation of chloroplast genomes and provide new insight into the evolution of basal eudicots at the primary sequence and structural level.

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July 7, 2019

Phylogenomically guided identification of industrially relevant GH1 ß-glucosidases through DNA synthesis and nanostructure-initiator mass spectrometry.

Harnessing the biotechnological potential of the large number of proteins available in sequence databases requires scalable methods for functional characterization. Here we propose a workflow to address this challenge by combining phylogenomic guided DNA synthesis with high-throughput mass spectrometry and apply it to the systematic characterization of GH1 ß-glucosidases, a family of enzymes necessary for biomass hydrolysis, an important step in the conversion of lignocellulosic feedstocks to fuels and chemicals. We synthesized and expressed 175 GH1s, selected from over 2000 candidate sequences to cover maximum sequence diversity. These enzymes were functionally characterized over a range of temperatures and pHs using nanostructure-initiator mass spectrometry (NIMS), generating over 10,000 data points. When combined with HPLC-based sugar profiling, we observed GH1 enzymes active over a broad temperature range and toward many different ß-linked disaccharides. For some GH1s we also observed activity toward laminarin, a more complex oligosaccharide present as a major component of macroalgae. An area of particular interest was the identification of GH1 enzymes compatible with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), a next-generation biomass pretreatment technology. We thus searched for GH1 enzymes active at 70 °C and 20% (v/v) [C2mim][OAc] over the course of a 24-h saccharification reaction. Using our unbiased approach, we identified multiple enzymes of different phylogentic origin with such activities. Our approach of characterizing sequence diversity through targeted gene synthesis coupled to high-throughput screening technologies is a broadly applicable paradigm for a wide range of biological problems.

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July 7, 2019

Co-option of Sox3 as the male-determining factor on the Y chromosome in the fish Oryzias dancena.

Sex chromosomes harbour a primary sex-determining signal that triggers sexual development of the organism. However, diverse sex chromosome systems have been evolved in vertebrates. Here we use positional cloning to identify the sex-determining locus of a medaka-related fish, Oryzias dancena, and find that the locus on the Y chromosome contains a cis-regulatory element that upregulates neighbouring Sox3 expression in developing gonad. Sex-reversed phenotypes in Sox3(Y) transgenic fish, and Sox3(Y) loss-of-function mutants all point to its critical role in sex determination. Furthermore, we demonstrate that Sox3 initiates testicular differentiation by upregulating expression of downstream Gsdf, which is highly conserved in fish sex differentiation pathways. Our results not only provide strong evidence for the independent recruitment of Sox3 to male determination in distantly related vertebrates, but also provide direct evidence that a novel sex determination pathway has evolved through co-option of a transcriptional regulator potentially interacted with a conserved downstream component.

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