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July 7, 2019

Contiguous and accurate de novo assembly of metazoan genomes with modest long read coverage.

Genome assemblies that are accurate, complete and contiguous are essential for identifying important structural and functional elements of genomes and for identifying genetic variation. Nevertheless, most recent genome assemblies remain incomplete and fragmented. While long molecule sequencing promises to deliver more complete genome assemblies with fewer gaps, concerns about error rates, low yields, stringent DNA requirements and uncertainty about best practices may discourage many investigators from adopting this technology. Here, in conjunction with the platinum standard Drosophila melanogaster reference genome, we analyze recently published long molecule sequencing data to identify what governs completeness and contiguity of genome assemblies. We also present a hybrid meta-assembly approach that achieves remarkable assembly contiguity for both Drosophila and human assemblies with only modest long molecule sequencing coverage. Our results motivate a set of preliminary best practices for obtaining accurate and contiguous assemblies, a ‘missing manual’ that guides key decisions in building high quality de novo genome assemblies, from DNA isolation to polishing the assembly.© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 7, 2019

Genetic characterization of a blaVEB-2-carrying plasmid in Vibrio parahaemolyticus.

This study reports the first detection of blaVEB-2 gene in Vibrio parahaemolyticus strain isolated from a shrimp sample. The blaVEB-2 was carried on a novel Inc type plasmid, was likely to originate from aquatic organisms upon comparison with other known genetic elements in the GenBank. However, the plasmid contains resistance elements usually harbored by members of Enterobacteriaceae, suggesting that gene transfer events occurred and contributed to the formation of this multidrug resistance-encoding plasmid. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Comparative genomics and physiology of the butyrate-producing bacterium Intestinimonas butyriciproducens.

Intestinimonas is a newly described bacterial genus with representative strains present in the intestinal tract of human and other animals. Despite unique metabolic features including the production of butyrate from both sugars and amino acids, there is to date no data on their diversity, ecology, and physiology. Using a comprehensive phylogenetic approach, Intestinimomas was found to include at least three species that colonize primarily the human and mouse intestine. We focused on the most common and cultivable species of the genus, Intestinimonas butyriciproducens, and performed detailed genomic and physiological comparison of strains SRB521(T) and AF211, isolated from the mouse and human gut respectively. The complete 3.3-Mb genomic sequences of both strains were highly similar with 98.8% average nucleotide identity, testifying to their assignment to one single species. However, thorough analysis revealed significant genomic rearrangements, variations in phage-derived sequences, and the presence of new CRISPR sequences in both strains. Moreover, strain AF211 appeared to be more efficient than strain SRB521(T) in the conversion of the sugars arabinose and galactose. In conclusion, this study provides genomic and physiological insight into Intestinimonas butyriciproducens, a prevalent butyrate-producing species, differentiating strains that originate from the mouse and human gut.© 2016 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and JohnWiley & Sons Ltd.

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July 7, 2019

The complete genome sequences of sulfur-oxidizing Gammaproteobacteria Sulfurifustis variabilis skN76(T) and Sulfuricaulis limicola HA5(T).

Sulfurifustis variabilis and Sulfuricaulis limicola are autotrophic sulfur-oxidizing bacteria belonging to the family Acidiferrobacteraceae in the order Acidiferrobacterales. The type strains of these species, strain skN76(T) and strain HA5(T), were isolated from lakes in Japan. Here we describe the complete genome sequences of Sulfurifustis variabilis skN76(T) and Sulfuricaulis limicola HA5(T). The genome of Sulfurifustis variabilis skN76(T) consists of one circular chromosome with size of 4.0 Mbp including 3864 protein-coding sequences. The genome of Sulfuricaulis limicola HA5(T) is 2.9 Mbp chromosome with 2763 protein-coding sequences. In both genomes, 46 transfer RNA-coding genes and one ribosomal RNA operon were identified. In the genomes, redundancies of the genes involved in sulfur oxidation and inorganic carbon fixation pathways were observed. This is the first report to show the complete genome sequences of bacteria belonging to the order Acidiferrobacterales in the class Gammaproteobacteria.

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July 7, 2019

Genetic basis of priority effects: insights from nectar yeast.

Priority effects, in which the order of species arrival dictates community assembly, can have a major influence on species diversity, but the genetic basis of priority effects remains unknown. Here, we suggest that nitrogen scavenging genes previously considered responsible for starvation avoidance may drive priority effects by causing rapid resource depletion. Using single-molecule sequencing, we de novo assembled the genome of the nectar-colonizing yeast, Metschnikowia reukaufii, across eight scaffolds and complete mitochondrion, with gap-free coverage over gene spaces. We found a high rate of tandem gene duplication in this genome, enriched for nitrogen metabolism and transport. Both high-capacity amino acid importers, GAP1 and PUT4, present as tandem gene arrays, were highly expressed in synthetic nectar and regulated by the availability and quality of amino acids. In experiments with competitive nectar yeast, Candida rancensis, amino acid addition alleviated suppression of C. rancensis by early arrival of M. reukaufii, corroborating that amino acid scavenging may contribute to priority effects. Because niche pre-emption via rapid resource depletion may underlie priority effects in a broad range of microbial, plant and animal communities, nutrient scavenging genes like the ones we considered here may be broadly relevant to understanding priority effects.© 2016 The Author(s).

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July 7, 2019

Complete genome sequence of Halomonas sp. R5-57.

The marine Arctic isolate Halomonas sp. R5-57 was sequenced as part of a bioprospecting project which aims to discover novel enzymes and organisms from low-temperature environments, with potential uses in biotechnological applications. Phenotypically, Halomonas sp. R5-57 exhibits high salt tolerance over a wide range of temperatures and has extra-cellular hydrolytic activities with several substrates, indicating it secretes enzymes which may function in high salinity conditions. Genome sequencing identified the genes involved in the biosynthesis of the osmoprotectant ectoine, which has applications in food processing and pharmacy, as well as those involved in production of polyhydroxyalkanoates, which can serve as precursors to bioplastics. The percentage identity of these biosynthetic genes from Halomonas sp. R5-57 and current production strains varies between 99 % for some to 69 % for others, thus it is plausible that R5-57 may have a different production capacity to currently used strains, or that in the case of PHAs, the properties of the final product may vary. Here we present the finished genome sequence (LN813019) of Halomonas sp. R5-57 which will facilitate exploitation of this bacterium; either as a whole-cell production host, or by recombinant expression of its individual enzymes.

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July 7, 2019

Complete genome sequencing and comparative genomic analysis of the thermotolerant acetic acid bacterium, Acetobacter pasteurianus SKU1108, provide a new insight into thermotolerance.

Acetobacter pasteurianus SKU1108 is a typical thermotolerant acetic acid bacterium. In this study, the complete genome sequence of the SKU1108 strain was elucidated, and information on genomic modifications due to the thermal adaptation of SKU1108 was updated. In order to obtain a clearer understanding of the genetic background responsible for thermotolerance, the SKU1108 genome was compared with those of two closely related complete genome strains, thermotolerant A. pasteurianus 386B and mesophilic A. pasteurianus NBRC 3283. All 24 “thermotolerant genes” required for growth at higher temperatures in the thermotolerant Acetobacter tropicalis SKU1100 strain were conserved in all three strains. However, these thermotolerant genes accumulated amino acid mutations. Some biased mutations, particularly those that occurred in xanthine dehydrogenase XdhA, may be related to thermotolerance. By aligning whole genome sequences, we identified ten SKU1108 strain-specific regions, three of which were conserved in the genomes of the two thermotolerant A. pasteurianus strains. One of the regions contained a unique paralog of the thermotolerant gene xdhA, which may also be responsible for conferring thermotolerance. Thus, comparative genomics of complete genome sequences may provide novel insights into the phenotypes of these thermotolerant strains.

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July 7, 2019

Full-length nucleotide sequences of mcr-1-harboring plasmids isolated from extended- spectrum-ß-lactamase-producing Escherichia coli isolates of different origins.

Here, we present the full sequences of three mcr-1-carrying plasmids isolated from extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli The plasmids belong to three different replicon types and are 34,640 bp, 209,401 bp, and 247,885 bp in size. We describe for the first time a composite transposon containing mcr-1 localized on a multidrug-resistant (MDR) IncHI2 plasmid harboring additional determinants of resistance to six different classes of antibiotics, including the ESBL gene blaCTX-M-1, and heavy metal resistance. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

The complete genome sequence of the nicotine-degrading bacterium Shinella sp. HZN7.

Nicotine is a natural alkaloid that is very toxic to humans. To eliminate the harmful effects of nicotine in the environment, biological methods employing microbes to degrade nicotine are required (Brandsch, 2006; Liu et al., 2015). Shinella sp. HZN7 can degrade nicotine efficiently via the variant of a pyridine and pyrrolidine pathways (VPP; Ma et al., 2013; Qiu et al., 2014, 2015). The main intermediates in this pathway include 6-hydroxy-nicotine, 6-hydroxy-N-methylmyosmine, 6-hydroxypseudooxynicotine, 6-hydroxy-3-succinoyl-pyridine, and 2,5-dihydroxypyridine. This strain is the first nicotine-degrading bacterium to be isolated from the genus Shinella.

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July 7, 2019

Comparative genomics reveals Lysinibacillus sphaericus group comprises a novel species.

Early in the 1990s, it was recognized that Lysinibacillus sphaericus, one of the most popular and effective entomopathogenic bacteria, was a highly heterogeneous group. Many authors have even proposed it comprises more than one species, but the lack of phenotypic traits that guarantee an accurate differentiation has not allowed this issue to be clarified. Now that genomic technologies are rapidly advancing, it is possible to address the problem from a whole genome perspective, getting insights into the phylogeny, evolutive history and biology itself.The genome of the Colombian strain L. sphaericus OT4b.49 was sequenced, assembled and annotated, obtaining 3 chromosomal contigs and no evidence of plasmids. Using these sequences and the 13 other L. sphaericus genomes available on the NCBI database, we carried out comparative genomic analyses that included whole genome alignments, searching for mobile elements, phylogenomic metrics (TETRA, ANI and in-silico DDH) and pan-genome assessments. The results support the hypothesis about this species as a very heterogeneous group. The entomopathogenic lineage is actually a single and independent species with 3728 core genes and 2153 accessory genes, whereas each non-toxic strain seems to be a separate species, though without a clear circumscription. Toxin-encoding genes, binA, B and mtx1, 2, 3 could be acquired via horizontal gene transfer in a single evolutionary event. The non-toxic strain OT4b.31 is the most related with the type strain KCTC 3346.The current L. sphaericus is actually a sensu lato due to a sub-estimation of diversity accrued using traditional non-genomics based classification strategies. The toxic lineage is the most studied with regards to its larvicidal activity, which is a greatly conserved trait among these strains and thus, their differentiating feature. Further studies are needed in order to establish a univocal classification of the non-toxic strains that, according to our results, seem to be a paraphyletic group.

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July 7, 2019

Tracking inter-institutional spread of NDM and identification of a novel NDM-positive plasmid, pSg1-NDM, using next-generation sequencing approaches.

Owing to gene transposition and plasmid conjugation, New Delhi metallo-ß-lactamase (NDM) is typically identified among varied Enterobacteriaceae species and STs. We used WGS to characterize the chromosomal and plasmid molecular epidemiology of NDM transmission involving four institutions in Singapore.Thirty-three Enterobacteriaceae isolates (collection years 2010-14) were sequenced using short-read sequencing-by-synthesis and analysed. Long-read single molecule, real-time sequencing (SMRTS) was used to characterize genetically a novel plasmid pSg1-NDM carried on Klebsiella pneumoniae ST147.In 20 (61%) isolates, blaNDM was located on the pNDM-ECS01 plasmid in the background of multiple bacterial STs, including eight K. pneumoniae STs and five Escherichia coli STs. In six (18%) isolates, a novel blaNDM-positive plasmid, pSg1-NDM, was found only in K. pneumoniae ST147. The pSg1-NDM-K. pneumoniae ST147 clone (Sg1-NDM) was fully sequenced using SMRTS. pSg1-NDM, a 90?103 bp IncR plasmid, carried genes responsible for resistance to six classes of antimicrobials. A large portion of pSg1-NDM had no significant homology to any known plasmids in GenBank. pSg1-NDM had no conjugative transfer region. Combined chromosomal-plasmid phylogenetic analysis revealed five clusters of clonal bacterial NDM-positive plasmid transmission, of which two were inter-institution clusters. The largest inter-institution cluster involved six K. pneumoniae ST147-pSg1-NDM isolates. Fifteen patients were involved in transmission clusters, of which four had ward contact, six had hospital contact and five had an unknown transmission link.A combined sequencing-by-synthesis and SMRTS approach can determine effectively the transmission clusters of blaNDM and genetically characterize novel plasmids. Plasmid molecular epidemiology is important to understanding NDM spread as blaNDM-positive plasmids can conjugate extensively across species and STs.© The Author 2016. 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

Plasmids from Shiga toxin-producing Escherichia coli strains with rare enterohemolysin gene (ehxA) subtypes reveal pathogenicity potential and display a novel evolutionary path.

Most Shiga toxin-producing Escherichia coli (STEC) strains associated with severe disease, such as hemolytic-uremic syndrome (HUS), carry large enterohemolysin-encoding (ehxA) plasmids, e.g., pO157 and pO103, that contribute to STEC clinical manifestations. Six ehxA subtypes (A through F) exist that phylogenetically cluster into eae-positive (B, C, F), a mix of eae-positive (E) and eae-negative (A), and a third, more distantly related, cluster of eae-negative (D) STEC strains. While subtype B, C, and F plasmids share a number of virulence traits that are distinct from those of subtype A, sequence data have not been available for subtype D and E plasmids. Here, we determined and compared the genetic composition of four subtype D and two subtype E plasmids to establish their evolutionary relatedness among ehxA subtypes and define their potential role in pathogenicity. We found that subtype D strains carry one exceptionally large plasmid (>200 kbp) that carries a variety of virulence genes that are associated with enterotoxigenic and enterohemorrhagic E. coli, which, quite possibly, enables these strains to cause disease despite being food isolates. Our data offer further support for the hypothesis that this subtype D plasmid represents a novel virulence plasmid, sharing very few genetic features with other plasmids; we conclude that these plasmids have evolved from a different evolutionary lineage than the plasmids carrying the other ehxA subtypes. In contrast, the 50-kbp plasmids of subtype E (pO145), although isolated from HUS outbreak strains, carried only few virulence-associated determinants, suggesting that the clinical presentation of subtype E strains is largely a result of chromosomally encoded virulence factors.Bacterial plasmids are known to be key agents of change in microbial populations, promoting the dissemination of various traits, such as drug resistance and virulence. This study determined the genetic makeup of virulence plasmids from rare enterohemolysin subtype D and E Shiga toxin-producing E. coli strains. We demonstrated that ehxA subtype D plasmids represent a novel E. coli virulence plasmid, and although subtype D plasmids were derived from nonclinical isolates, they encoded a variety of virulence determinants that are associated with pathogenic E. coli In contrast, subtype E plasmids, isolated from strains recovered from severely ill patients, carry only a few virulence determinants. The results of this study reemphasize the plasticity and vast diversity among E. coli plasmids. This work demonstrates that, although E. coli strains of certain serogroups may not be frequently associated with disease, they should not be underestimated in protecting human health and food safety. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Conservation genetics of an endangered grassland butterfly (Oarisma poweshiek) reveals historically high gene flow despite recent and rapid range loss

1. In poorly dispersing species gene flow can be facilitated when suitable habitat is widespread, allowing for increased dispersal between neighbouring locations. The Poweshiek skipperling [Oarisma poweshiek (Parker)], a federally endangered butterfly, has undergone a rapid, recent demographic decline following the loss of tallgrass prairie and fen habitats range wide. The loss of habitat, now restricted geographic range, and poor dispersal ability have left O. poweshiek at increased risk of extinction. 2. We studied the population genetics of six remaining populations of O. poweshiek in order to test the hypothesis that gene flow was historically high despite limited long-distance dispersal capability. Utilising nine microsatellite loci developed by PacBio sequencing, we tested for patterns of isolation by distance, low population genetic structure and alternative gene flow models. 3. Populations from southern Manitoba, Canada to the Lower Peninsula of Michigan, USA are only weakly genetically differentiated despite having low diversity. We found no support for isolation by distance, and Bayesian estimates of historical gene flow support our hypothesis that high levels of gene flow previously connected populations from Michigan to Wisconsin. 4. Prairie grasslands have been reduced tremendously over the past century, but the low mobility of O. poweshiek suggests that rapid loss of populations over the past decade cannot be simply explained by fragmentation of habitat. 5. As a species at high risk of extinction, understanding historical processes of gene flow will allow for informed management decisions with respect to head-starting individuals for population reintroductions and for conserving networks of habitat that will allow for high levels of gene flow.

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July 7, 2019

Interchromosomal core duplicons drive both evolutionary instability and disease susceptibility of the Chromosome 8p23.1 region.

Recurrent rearrangements of Chromosome 8p23.1 are associated with congenital heart defects and developmental delay. The complexity of this region has led to inconsistencies in the current reference assembly, confounding studies of genetic variation. Using comparative sequence-based approaches, we generated a high-quality 6.3-Mbp alternate reference assembly of an inverted Chromosome 8p23.1 haplotype. Comparison with nonhuman primates reveals a 746-kbp duplicative transposition and two separate inversion events that arose in the last million years of human evolution. The breakpoints associated with these rearrangements map to an ape-specific interchromosomal core duplicon that clusters at sites of evolutionary inversion (P = 7.8 × 10(-5)). Refinement of microdeletion breakpoints identifies a subgroup of patients that map to the same interchromosomal core involved in the evolutionary formation of the duplication blocks. Our results define a higher-order genomic instability element that has shaped the structure of specific chromosomes during primate evolution contributing to rearrangements associated with inversion and disease.© 2016 Mohajeri et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019

The evolution of orphan regions in genomes of a fungal pathogen of wheat.

Fungal plant pathogens rapidly evolve virulence on resistant hosts through mutations in genes encoding proteins that modulate the host immune responses. The mutational spectrum likely includes chromosomal rearrangements responsible for gains or losses of entire genes. However, the mechanisms creating adaptive structural variation in fungal pathogen populations are poorly understood. We used complete genome assemblies to quantify structural variants segregating in the highly polymorphic fungal wheat pathogen Zymoseptoria tritici The genetic basis of virulence in Z. tritici is complex, and populations harbor significant genetic variation for virulence; hence, we aimed to identify whether structural variation led to functional differences. We combined single-molecule real-time sequencing, genetic maps, and transcriptomics data to generate a fully assembled and annotated genome of the highly virulent field isolate 3D7. Comparative genomics analyses against the complete reference genome IPO323 identified large chromosomal inversions and the complete gain or loss of transposable-element clusters, explaining the extensive chromosomal-length polymorphisms found in this species. Both the 3D7 and IPO323 genomes harbored long tracts of sequences exclusive to one of the two genomes. These orphan regions contained 296 genes unique to the 3D7 genome and not previously known for this species. These orphan genes tended to be organized in clusters and showed evidence of mutational decay. Moreover, the orphan genes were enriched in genes encoding putative effectors and included a gene that is one of the most upregulated putative effector genes during wheat infection. Our study showed that this pathogen species harbored extensive chromosomal structure polymorphism that may drive the evolution of virulence.Pathogen outbreak populations often harbor previously unknown genes conferring virulence. Hence, a key puzzle of rapid pathogen evolution is the origin of such evolutionary novelty in genomes. Chromosomal rearrangements and structural variation in pathogen populations likely play a key role. However, identifying such polymorphism is challenging, as most genome-sequencing approaches only yield information about point mutations. We combined long-read technology and genetic maps to assemble the complete genome of a strain of a highly polymorphic fungal pathogen of wheat. Comparisons against the reference genome of the species showed substantial variation in the chromosome structure and revealed large regions unique to each assembled genome. These regions were enriched in genes encoding likely effector proteins, which are important components of pathogenicity. Our study showed that pathogen populations harbor extensive polymorphism at the chromosome level and that this polymorphism can be a source of adaptive genetic variation in pathogen evolution. Copyright © 2016 Plissonneau et al.

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