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Auto Tag: De novo assembly

September 22, 2019

Prevalence and genomic structure of bacteriophage phi3 in human derived livestock-associated MRSA from 2000 to 2015.

Whereas the emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) clonal complex 398 (CC398) in animal husbandry and its transmission to humans are well documented, less is known about factors driving the epidemic spread of this zoonotic lineage within the human population. One factor could be the bacteriophage phi3, which is rarely detected in S. aureus isolates from animals but commonly found among isolates from humans, including those of the human-adapted methicillin-susceptible S. aureus (MSSA) CC398 clade. The proportion of phi3-carrying MRSA spa-CC011 isolates, which constitute presumptively LA-MRSA within the multilocus sequence type (MLST) clonal complex 398, was systematically assessed for a period of 16 years to investigate the role of phi3 in the adaptation process of LA-MRSA to the human host. For this purpose, 632 MRSA spa-CC011 isolates from patients of a university hospital located in a pig farming-dense area in Germany were analyzed. Livestock-associated acquisition of MRSA spa-CC011 was previously reported as having increased from 1.8% in 2000 to 29.4% in 2014 in MRSA-positive patients admitted to this hospital. However, in this study, the proportion of phi3-carrying isolates rose only from 1.1% (2000 to 2006) to 3.9% (2007 to 2015). Characterization of the phi3 genomes revealed 12 different phage types ranging in size from 40,712 kb up to 44,003 kb, with four hitherto unknown integration sites (genes or intergenic regions) and several modified bacterial attachment (attB) sites. In contrast to the MSSA CC398 clade, phi3 acquisition seems to be no major driver for the readaptation of MRSA spa-CC011 to the human host. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Characterization of a novel SXT/R391 Integrative and Conjugative Element carrying cfr, blaCTX-M-65, fosA3 and aac(6′)-Ib-cr in Proteus mirabilis.

A novel 139,487-bp SXT/R391 integrative and conjugative element, ICEPmiChnBCP11, was characterized in Proteus mirabilis of swine origin in China. ICEPmiChnBCP11 harbors 20 different antimicrobial resistance genes, including the clinically important rRNA methyltransferase gene cfr, the extended-spectrum ß-lactamase gene blaCTX-M-65, fosfomycin resistance gene fosA3, and fluoroquinolone resistance gene aac(6′)-Ib-cr An ISPpu12-mediated composite transposon containing various resistance genes and 10 copies of IS26 is inserted in hot spot 4. ICEPmiChnBCP11 was successfully transferred to Escherichia coli. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Koala genome insights.

A new study in Nature Genetics leverages long-read sequencing to generate a high-quality reference genome for the modern koala, Phascolarctos cinereus, and reports various inferences about adaptation and conservation of this species classified as ‘vulnerable’.

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September 22, 2019

Long-term colonization dynamics of Enterococcus faecalis in implanted devices in research macaques.

Enterococcus faecalis is a common opportunistic pathogen that colonizes cephalic recording chambers (CRCs) of macaques used in cognitive neuroscience research. We previously characterized 15 E. faecalis strains isolated from macaques at the Massachusetts Institute of Technology (MIT) in 2011. The goal of this study was to examine how a 2014 protocol change prohibiting the use of antimicrobials within CRCs affected colonizing E. faecalis strains. We collected 20 E. faecalis isolates from 10 macaques between 2013 and 2017 for comparison to 4 isolates previously characterized in 2011 with respect to the sequence type (ST) distribution, antimicrobial resistance, biofilm formation, and changes in genes that might confer a survival advantage. ST4 and ST55 were predominant among the isolates characterized in 2011, whereas the less antimicrobial-resistant lineage ST48 emerged to dominance after 2013. Two macaques remained colonized by ST4 and ST55 strains for 5 and 4 years, respectively. While the antimicrobial resistance and virulence factors identified in these ST4 and ST55 strains remained relatively stable, we detected an increase in biofilm formation ability over time in both isolates. We also found that ST48 strains were typically robust biofilm formers, which could explain why this ST increased in prevalence. Finally, we identified mutations in the DNA mismatch repair genes mutS and mutL in separate ST55 and ST4 strains and confirmed that strains bearing these mutations displayed a hypermutator phenotype. The presence of a hypermutator phenotype may complicate future antimicrobial treatment for clinically relevant E. faecalis infections in macaques.IMPORTANCEEnterococcus faecalis is a common cause of health care-associated infections in humans, largely due to its ability to persist in the hospital environment, colonize patients, acquire antimicrobial resistance, and form biofilms. Understanding how enterococci evolve in health care settings provides insight into factors affecting enterococcal survival and persistence. Macaques used in neuroscience research have long-term cranial implants that, despite best practices, often become colonized by E. faecalis This provides a unique opportunity to noninvasively examine the evolution of enterococci on a long-term indwelling device. We collected E. faecalis strains from cephalic implants over a 7-year period and characterized the sequence type, antimicrobial resistance, virulence factors, biofilm production, and hypermutator phenotypes. Improved antimicrobial stewardship allowed a less-antimicrobial-resistant E. faecalis strain to predominate at the implant interface, potentially improving antimicrobial treatment outcomes if future clinical infections occur. Biofilm formation appears to play an important role in the persistence of the E. faecalis strains associated with these implants. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

The chromosome-level genome assemblies of two rattans (Calamus simplicifolius and Daemonorops jenkinsiana).

Calamus simplicifolius and Daemonorops jenkinsiana are two representative rattans, the most significant material sources for the rattan industry. However, the lack of reference genome sequences is a major obstacle for basic and applied biology on rattan.We produced two chromosome-level genome assemblies of C. simplicifolius and D. jenkinsiana using Illumina, Pacific Biosciences, and Hi-C sequencing data. A total of ~730 Gb and ~682 Gb of raw data covered the predicted genome lengths (~1.98 Gb of C. simplicifolius and ~1.61 Gb of D. jenkinsiana) to ~372 × and ~426 × read depths, respectively. The two de novo genome assemblies, ~1.94 Gb and ~1.58 Gb, were generated with scaffold N50s of ~160 Mb and ~119 Mb in C. simplicifolius and D. jenkinsiana, respectively. The C. simplicifolius and D. jenkinsiana genomes were predicted to harbor ?51,235 and ?53,342 intact protein-coding gene models, respectively. Benchmarking Universal Single-Copy Orthologs evaluation demonstrated that genome completeness reached 96.4% and 91.3% in the C. simplicifolius and D. jenkinsiana genomes, respectively. Genome evolution showed that four Arecaceae plants clustered together, and the divergence time between the two rattans was ~19.3 million years ago. Additionally, we identified 193 and 172 genes involved in the lignin biosynthesis pathway in the C. simplicifolius and D. jenkinsiana genomes, respectively.We present the first de novo assemblies of two rattan genomes (C. simplicifolius and D. jenkinsiana). These data will not only provide a fundamental resource for functional genomics, particularly in promoting germplasm utilization for breeding, but also serve as reference genomes for comparative studies between and among different species.

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September 22, 2019

Draft genome assembly of the invasive cane toad, Rhinella marina.

The cane toad (Rhinella marina formerly Bufo marinus) is a species native to Central and South America that has spread across many regions of the globe. Cane toads are known for their rapid adaptation and deleterious impacts on native fauna in invaded regions. However, despite an iconic status, there are major gaps in our understanding of cane toad genetics. The availability of a genome would help to close these gaps and accelerate cane toad research.We report a draft genome assembly for R. marina, the first of its kind for the Bufonidae family. We used a combination of long-read Pacific Biosciences RS II and short-read Illumina HiSeq X sequencing to generate 359.5 Gb of raw sequence data. The final hybrid assembly of 31,392 scaffolds was 2.55 Gb in length with a scaffold N50 of 168 kb. BUSCO analysis revealed that the assembly included full length or partial fragments of 90.6% of tetrapod universal single-copy orthologs (n = 3950), illustrating that the gene-containing regions have been well assembled. Annotation predicted 25,846 protein coding genes with similarity to known proteins in Swiss-Prot. Repeat sequences were estimated to account for 63.9% of the assembly.The R. marina draft genome assembly will be an invaluable resource that can be used to further probe the biology of this invasive species. Future analysis of the genome will provide insights into cane toad evolution and enrich our understanding of their interplay with the ecosystem at large.

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September 22, 2019

Draft genome of Glyptosternon maculatum, an endemic fish from Tibet Plateau.

Mechanisms for high-altitude adaption have attracted widespread interest among evolutionary biologists. Several genome-wide studies have been carried out for endemic vertebrates in Tibet, including mammals, birds, and amphibians. However, little information is available about the adaptive evolution of highland fishes. Glyptosternon maculatum (Regan 1905), also known as Regan or barkley and endemic to the Tibetan Plateau, belongs to the Sisoridae family, order Siluriformes (catfishes). This species lives at an elevation ranging from roughly 2,800 m to 4,200 m. Hence, a high-quality reference genome of G. maculatum provides an opportunity to investigate high-altitude adaption mechanisms of fishes.To obtain a high-quality reference genome sequence of G. maculatum, we combined Pacific Bioscience single-molecule real-time sequencing, Illumina paired-end sequencing, 10X Genomics linked-reads, and BioNano optical map techniques. In total, 603.99 Gb sequencing data were generated. The assembled genome was about 662.34 Mb with scaffold and contig N50 sizes of 20.90 Mb and 993.67 kb, respectively, which captured 83% complete and 3.9% partial vertebrate Benchmarking Universal Single-Copy Orthologs. Repetitive elements account for 35.88% of the genome, and ?22,066 protein-coding genes were predicted from the genome, of which 91.7% have been functionally annotated.We present the first comprehensive de novo genome of G. maculatum. This genetic resource is fundamental for investigating the origin of G. maculatum and will improve our understanding of high-altitude adaption of fishes. The assembled genome can also be used as reference for future population genetic studies of G. maculatum.

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September 22, 2019

Isolation, development, and genomic analysis of Bacillus megaterium SR7 for growth and metabolite production under supercritical carbon dioxide

Supercritical carbon dioxide (scCO2) is an attractive substitute for conventional organic solvents due to its unique transport and thermodynamic properties, its renewability and labile nature, and its high solubility for compounds such as alcohols, ketones, and aldehydes. However, biological systems that use scCO2 are mainly limited to in vitro processes due to its strong inhibition of cell viability and growth. To solve this problem, we used a bioprospecting approach to isolate a microbial strain with the natural ability to grow while exposed to scCO2. Enrichment culture and serial passaging of deep subsurface fluids from the McElmo Dome scCO2 reservoir in aqueous media under scCO2 headspace enabled the isolation of spore-forming strain Bacillus megaterium SR7. Sequencing and analysis of the complete 5.51 Mbp genome and physiological characterization revealed the capacity for facultative anaerobic metabolism, including fermentative growth on a diverse range of organic substrates. Supplementation of growth medium with L-alanine for chemical induction of spore germination significantly improved growth frequencies and biomass accumulation under scCO2 headspace. Detection of endogenous fermentative compounds in cultures grown under scCO2 represents the first observation of bioproduct generation and accumulation under this condition. Culturing development and metabolic characterization of B. megaterium SR7 represent initial advancements in the effort toward enabling exploitation of scCO2 as a sustainable solvent for in vivo bioprocessing.

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September 22, 2019

PacBio-based mitochondrial genome assembly of Leucaena trichandra (Leguminosae) and an intrageneric assessment of mitochondrial RNA editing.

Reconstructions of vascular plant mitochondrial genomes (mt-genomes) are notoriously complicated by rampant recombination that has resulted in comparatively few plant mt-genomes being available. The dearth of plant mitochondrial resources has limited our understanding of mt-genome structural diversity, complex patterns of RNA editing, and the origins of novel mt-genome elements. Here, we use an efficient long read (PacBio) iterative assembly pipeline to generate mt-genome assemblies for Leucaena trichandra (Leguminosae: Caesalpinioideae: mimosoid clade), providing the first assessment of non-papilionoid legume mt-genome content and structure to date. The efficiency of the assembly approach facilitated the exploration of alternative structures that are common place among plant mitochondrial genomes. A compact version (729 kbp) of the recovered assemblies was used to investigate sources of mt-genome size variation among legumes and mt-genome sequence similarity to the legume associated root holoparasite Lophophytum. The genome and an associated suite of transcriptome data from select species of Leucaena permitted an in-depth exploration of RNA editing in a diverse clade of closely related species that includes hybrid lineages. RNA editing in the allotetraploid, Leucaena leucocephala, is consistent with co-option of nearly equal maternal and paternal C-to-U edit components, generating novel combinations of RNA edited sites. A preliminary investigation of L. leucocephala C-to-U edit frequencies identified the potential for a hybrid to generate unique pools of alleles from parental variation through edit frequencies shared with one parental lineage, those intermediate between parents, and transgressive patterns.

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September 22, 2019

The genome of Naegleria lovaniensis, the basis for a comparative approach to unravel pathogenicity factors of the human pathogenic amoeba N. fowleri.

Members of the genus Naegleria are free-living eukaryotes with the capability to transform from the amoeboid form into resting cysts or moving flagellates in response to environmental conditions. More than 40 species have been characterized, but only Naegleria fowleri (N. fowleri) is known as a human pathogen causing primary amoebic meningoencephalitis (PAM), a fast progressing and mostly fatal disease of the central nervous system. Several studies report an involvement of phospholipases and other molecular factors, but the mechanisms involved in pathogenesis are still poorly understood. To gain a better understanding of the relationships within the genus of Naegleria and to investigate pathogenicity factors of N. fowleri, we characterized the genome of its closest non-pathogenic relative N. lovaniensis.To gain insights into the taxonomy of Naegleria, we sequenced the genome of N. lovaniensis using long read sequencing technology. The assembly of the data resulted in a 30 Mb genome including the circular mitochondrial sequence. Unravelling the phylogenetic relationship using OrthoMCL protein clustering and maximum likelihood methods confirms the close relationship of N. lovaniensis and N. fowleri. To achieve an overview of the diversity of Naegleria proteins and to assess characteristics of the human pathogen N. fowleri, OrthoMCL protein clustering including data of N. fowleri, N. lovaniensis and N. gruberi was performed. GO enrichment analysis shows an association of N. fowleri specific proteins to the GO terms “Membrane” and “Protein Secretion.”In this study, we characterize the hitherto unknown genome of N. lovaniensis. With the description of the 30 Mb genome, a further piece is added to reveal the complex taxonomic relationship of Naegleria. Further, the whole genome sequencing data confirms the hypothesis of the close relationship between N. fowleri and N. lovaniensis. Therefore, the genome of N. lovaniensis provides the basis for further comparative approaches on the molecular and genomic level to unravel pathogenicity factors of its closest human pathogenic relative N. fowleri and possible treatment options for the rare but mostly fatal primary meningoencephalitis.

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September 22, 2019

A model for the evolution of prokaryotic DNA restriction-modification systems based upon the structural malleability of Type I restriction-modification enzymes.

Restriction Modification (RM) systems prevent the invasion of foreign genetic material into bacterial cells by restriction and protect the host’s genetic material by methylation. They are therefore important in maintaining the integrity of the host genome. RM systems are currently classified into four types (I to IV) on the basis of differences in composition, target recognition, cofactors and the manner in which they cleave DNA. Comparing the structures of the different types, similarities can be observed suggesting an evolutionary link between these different types. This work describes the ‘deconstruction’ of a large Type I RM enzyme into forms structurally similar to smaller Type II RM enzymes in an effort to elucidate the pathway taken by Nature to form these different RM enzymes. Based upon the ability to engineer new enzymes from the Type I ‘scaffold’, an evolutionary pathway and the evolutionary pressures required to move along the pathway from Type I RM systems to Type II RM systems are proposed. Experiments to test the evolutionary model are discussed.

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September 22, 2019

Evolutionary history of human Plasmodium vivax revealed by genome-wide analyses of related ape parasites.

Wild-living African apes are endemically infected with parasites that are closely related to human Plasmodium vivax, a leading cause of malaria outside Africa. This finding suggests that the origin of P. vivax was in Africa, even though the parasite is now rare in humans there. To elucidate the emergence of human P. vivax and its relationship to the ape parasites, we analyzed genome sequence data of P. vivax strains infecting six chimpanzees and one gorilla from Cameroon, Gabon, and Côte d’Ivoire. We found that ape and human parasites share nearly identical core genomes, differing by only 2% of coding sequences. However, compared with the ape parasites, human strains of P. vivax exhibit about 10-fold less diversity and have a relative excess of nonsynonymous nucleotide polymorphisms, with site-frequency spectra suggesting they are subject to greatly relaxed purifying selection. These data suggest that human P. vivax has undergone an extreme bottleneck, followed by rapid population expansion. Investigating potential host-specificity determinants, we found that ape P. vivax parasites encode intact orthologs of three reticulocyte-binding protein genes (rbp2d, rbp2e, and rbp3), which are pseudogenes in all human P. vivax strains. However, binding studies of recombinant RBP2e and RBP3 proteins to human, chimpanzee, and gorilla erythrocytes revealed no evidence of host-specific barriers to red blood cell invasion. These data suggest that, from an ancient stock of P. vivax parasites capable of infecting both humans and apes, a severely bottlenecked lineage emerged out of Africa and underwent rapid population growth as it spread globally. Copyright © 2018 the Author(s). Published by PNAS.

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September 22, 2019

Genome analyses of the microalga Picochlorum provide insights into the evolution of thermotolerance in the green lineage.

While the molecular events involved in cell responses to heat stress have been extensively studied, our understanding of the genetic basis of basal thermotolerance, and particularly its evolution within the green lineage, remains limited. Here, we present the 13.3-Mb haploid genome and transcriptomes of a halotolerant and thermotolerant unicellular green alga, Picochlorum costavermella (Trebouxiophyceae) to investigate the evolution of the genomic basis of thermotolerance. Differential gene expression at high and standard temperatures revealed that more of the gene families containing up-regulated genes at high temperature were recently evolved, and less originated at the ancestor of green plants. Inversely, there was an excess of ancient gene families containing transcriptionally repressed genes. Interestingly, there is a striking overlap between the thermotolerance and halotolerance transcriptional rewiring, as more than one-third of the gene families up-regulated at 35?°C were also up-regulated under variable salt concentrations in Picochlorum SE3. Moreover, phylogenetic analysis of the 9,304 protein coding genes revealed 26 genes of horizontally transferred origin in P. costavermella, of which five were differentially expressed at higher temperature. Altogether, these results provide new insights about how the genomic basis of adaptation to halo- and thermotolerance evolved in the green lineage.

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September 22, 2019

The structure of a conserved telomeric region associated with variant antigen loci in the blood parasite Trypanosoma congolense

African trypanosomiasis is a vector-borne disease of humans and livestock caused by African trypanosomes (Trypanosoma spp.). Survival in the vertebrate bloodstream depends on antigenic variation of Variant Surface Glycoproteins (VSGs) coating the parasite surface. In T. brucei, a model for antigenic variation, monoallelic VSG expression originates from dedicated VSG expression sites (VES). Trypanosoma brucei VES have a conserved structure consisting of a telomeric VSG locus downstream of unique, repeat sequences, and an independent promoter. Additional protein-coding sequences, known as “Expression Site Associated Genes (ESAGs)”, are also often present and are implicated in diverse, bloodstream-stage functions. Trypanosoma congolense is a related veterinary pathogen, also displaying VSG-mediated antigenic variation. A T. congolense VES has not been described, making it unclear if regulation of VSG expression is conserved between species. Here, we describe a conserved telomeric region associated with VSG loci from long-read DNA sequencing of two T. congolense strains, which consists of a distal repeat, conserved noncoding elements and other genes besides the VSG; although these are not orthologous to T. brucei ESAGs. Most conserved telomeric regions are associated with accessory minichromosomes, but the same structure may also be associated with megabase chromosomes. We propose that this region represents the T. congolense VES, and through comparison with T. brucei, we discuss the parallel evolution of antigenic switching mechanisms, and unique adaptation of the T. brucei VES for developmental regulation of bloodstream-stage genes. Hence, we provide a basis for understanding antigenic switching in T. congolense and the origins of the African trypanosome VES.

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September 22, 2019

Genomic insights into host adaptation between the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici) and the barley stripe rust pathogen (Puccinia striiformis f. sp. hordei).

Plant fungal pathogens can rapidly evolve and adapt to new environmental conditions in response to sudden changes of host populations in agro-ecosystems. However, the genomic basis of their host adaptation, especially at the forma specialis level, remains unclear.We sequenced two isolates each representing Puccinia striiformis f. sp. tritici (Pst) and P. striiformis f. sp. hordei (Psh), different formae speciales of the stripe rust fungus P. striiformis highly adapted to wheat and barley, respectively. The divergence of Pst and Psh, estimated to start 8.12 million years ago, has been driven by high nucleotide mutation rates. The high genomic variation within dikaryotic urediniospores of P. striiformis has provided raw genetic materials for genome evolution. No specific gene families have enriched in either isolate, but extensive gene loss events have occurred in both Pst and Psh after the divergence from their most recent common ancestor. A large number of isolate-specific genes were identified, with unique genomic features compared to the conserved genes, including 1) significantly shorter in length; 2) significantly less expressed; 3) significantly closer to transposable elements; and 4) redundant in pathways. The presence of specific genes in one isolate (or forma specialis) was resulted from the loss of the homologues in the other isolate (or forma specialis) by the replacements of transposable elements or losses of genomic fragments. In addition, different patterns and numbers of telomeric repeats were observed between the isolates.Host adaptation of P. striiformis at the forma specialis level is a complex pathogenic trait, involving not only virulence-related genes but also other genes. Gene loss, which might be adaptive and driven by transposable element activities, provides genomic basis for host adaptation of different formae speciales of P. striiformis.

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