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Asset Tag: de novo assembly

April 21, 2020

Genome characterization of an extensively drug-resistant Streptococcus pneumoniae serotype 11A strain.

In this study, the whole genome sequences of two Streptococcus pneumoniae clinical isolates from South Korea were determined and compared. They were found to be the same serotype (11?A) and multilocus sequence typing analysis showed that they are single-locus variants (SLVs; ST8279 and ST166) of each other, differing at one allele (aroE). However, the ST8279 strain is extensively drug-resistant (XDR) whereas the ST166 strain is not. The genome of the XDR strain is very similar in structure to that of two previously reported genomes, AP200 (11?A:ST62) and 70585 (5:ST5803); however, some regions were inverted and there were some exogenous regions in the ST8279 strain. It was found that 6,502 single nucleotide polymorphisms are dispersed across the genome between the two serotype 11?A ST8279 and ST166 strains. Many of them are located in genes associated with antibiotic resistance. In addition, many amino acid differences were also identified in genes involved in DNA repair (mutL, uvrA and uvrC) and recombination (recU, recR and recA). On the basis of these results, it was inferred that the XDR strain did not evolve from its SLV via a single recombination event involving a large portion of the genome including the aroE gene. Rather, the strain likely evolved through many point mutations and recombination events involving small portions of the genome. © 2019 The Societies and John Wiley & Sons Australia, Ltd.

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April 21, 2020

The major histocompatibility complex of Old World camelids: Class I and class I-related genes.

The genomic structure of the Major Histocompatibility Complex (MHC) region and variation in selected MHC class I related genes in Old World camels, Camelus bactrianus and Camelus dromedaries were studied. The overall genomic organization of the camel MHC region follows a general pattern observed in other mammalian species and individual MHC loci appear to be well conserved. Selected MHC class I genes B-67 and BL3-7 exhibited unexpectedly low variability, even when compared to other camel MHC class I related genes MR1 and MICA. Interspecific SNP and allele sharing are relatively common, and frequencies of heterozygotes are usually low. Such a low variation in a genomic region generally considered as one of the most polymorphic in vertebrate genomes is unusual. Evolutionary relationships between MHC class I related genes and their counterparts from other species seem to be rather complex. Often, they do not follow the general evolutionary history of the species concerned. Close evolutionary relationships of individual MHC class I loci between camels, humans and dogs were observed. Based on the results of this study and on our data on MHC class II genes, the extent and the pattern of polymorphism of the MHC region of Old World camelids differed from most mammalian groups studied so far. Camels thus seem to be an important model for our understanding of the role of genetic diversity in immune functions, especially in the context of unique features of their immunoglobulin and T-cell receptor genes. © 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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April 21, 2020

Mate Selection in Self-Compatible Wild Tobacco Results from Coordinated Variation in Homologous Self-Incompatibility Genes.

In flowering plants, intraspecific mate preference is frequently related to mating systems: the rejection of self pollen in self-incompatible (SI) plants that prevents inbreeding is one of the best described examples. However, in other mating systems, more nuanced patterns of pollen rejection occur. In the self-compatible (SC) Nicotiana attenuata, in which SI is not found and all crosses are compatible, certain pollen genotypes are consistently selected in mixed pollinations. However, the molecular mechanisms of this polyandrous mate selection remain unknown. Style-expressed NaS-like-RNases and pollen-expressed NaSLF-like genes, homologous to SI factors in Solanaceae, were identified and examined for a role in N. attenuata’s mate selection. A comparison of two NaS-like-RNases and six NaSLF-like genes among 26 natural accessions revealed specific combinations of co-expression and direct protein-protein interactions. To evaluate their role in mate selection, we silenced the expression of specific NaS-like-RNases and NaSLF-like proteins and conducted diagnostic binary mixed pollinations and mixed pollinations with 14 different non-self pollen donors. Styles expressing particular combinations of NaS-like-RNases selected mates from plants with corresponding NaS-like-RNase expression patterns, while styles lacking NaS-like-RNase expression were non-selective in their fertilizations, which reflected the genotype ratios of pollen mixtures deposited on the stigmas. DNA methylation could account for some of the observed variation in stylar NaS-like-RNase patterns. We conclude that the S-RNase-SLF recognition mechanism plays a central role in polyandrous mate selection in this self-compatible species. These results suggest that after the SI-SC transition, natural variation of SI homologous genes was repurposed to mediate intraspecific mate selection. Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020

Genetic, structural, and functional diversity of low and high-affinity siderophores in strains of nitrogen fixing Azotobacter chroococcum.

To increase iron (Fe) bioavailability in surface soils, microbes secrete siderophores, chelators with widely varying Fe affinities. Strains of the soil bacterium Azotobacter chroococcum (AC), plant-growth promoting rhizobacteria used as agricultural inoculants, require high Fe concentrations for aerobic respiration and nitrogen fixation. Recently, A. chroococcum str. NCIMB 8003 was shown to synthesize three siderophore classes: (1) vibrioferrin, a low-affinity a-hydroxy carboxylate (pFe = 18.4), (2) amphibactins, high-affinity tris-hydroxamates, and (3) crochelin A, a high-affinity siderophore with mixed Fe-chelating groups (pFe = 23.9). The relevance and specific functions of these siderophores in AC strains remain unclear. We analyzed the genome and siderophores of a second AC strain, A. chroococcum str. B3, and found that it also produces vibrioferrin and amphibactins, but not crochelin A. Genome comparisons indicate that vibrioferrin production is a vertically inherited, conserved strategy for Fe uptake in A. chroococcum and other species of Azotobacter. Amphibactin and crochelin biosynthesis reflects a more complex evolutionary history, shaped by vertical gene transfer, gene gain and loss through recombination at a genomic hotspot. We found conserved patterns of low vs. high-affinity siderophore production across strains: the low-affinity vibrioferrin was produced by mildly Fe limited cultures. As cells became more severely Fe starved, vibrioferrin production decreased in favor of high-affinity amphibactins (str. B3, NCIMB 8003) and crochelin A (str. NCIMB 8003). Our results show the evolution of low and high-affinity siderophore families and conserved patterns for their production in response to Fe bioavailability in a common soil diazotroph.

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April 21, 2020

Development of a Molecular Marker Linked to the A4 Locus and the Structure of HD Genes in Pleurotus eryngii

Allelic differences in A and B mating-type loci are a prerequisite for the progression of mating in the genus Pleurotus eryngii; thus, the crossing is hampered by this biological barrier in inbreeding. Molecular markers linked to mating types of P. eryngii KNR2312 were investigated with randomly amplified polymorphic DNA to enhance crossing efficiency. An A4-linked sequence was identified and used to find the adjacent genomic region with the entire motif of the A locus from a contig sequenced by PacBio. The sequence-characterized amplified region marker 7-2299 distinguished A4 mating-type monokaryons from KNR2312 and other strains. A BLAST search of flanked sequences revealed that the A4 locus had a general feature consisting of the putative HD1 and HD2 genes. Both putative HD transcription factors contain a homeodomain sequence and a nuclear localization sequence; however, valid dimerization motifs were found only in the HD1 protein. The ACAAT motif, which was reported to have relevance to sex determination, was found in the intergenic region. The SCAR marker could be applicable in the classification of mating types in the P. eryngii breeding program, and the A4 locus could be the basis for a multi-allele detection marker.

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April 21, 2020

The complete mitochondrial genome of the tartar Sand Boa Eryx tataricus

Eryx is a genus of snakes belonging to the family Boidae. In this study, the mitochondrial genome sequence of Eryx tataricus was generated using a PacBio RSII DNA sequencer employing the single mol- ecule, real-time sequencing technology. A maximum-likelihood (ML) phylogenetic tree of 26 snakes was re-constructed based on the 13 protein-coding genes for convincing the mitochondrial DNA sequences.

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April 21, 2020

Ancestral Admixture Is the Main Determinant of Global Biodiversity in Fission Yeast.

Mutation and recombination are key evolutionary processes governing phenotypic variation and reproductive isolation. We here demonstrate that biodiversity within all globally known strains of Schizosaccharomyces pombe arose through admixture between two divergent ancestral lineages. Initial hybridization was inferred to have occurred ~20-60 sexual outcrossing generations ago consistent with recent, human-induced migration at the onset of intensified transcontinental trade. Species-wide heritable phenotypic variation was explained near-exclusively by strain-specific arrangements of alternating ancestry components with evidence for transgressive segregation. Reproductive compatibility between strains was likewise predicted by the degree of shared ancestry. To assess the genetic determinants of ancestry block distribution across the genome, we characterized the type, frequency, and position of structural genomic variation using nanopore and single-molecule real-time sequencing. Despite being associated with double-strand break initiation points, over 800 segregating structural variants exerted overall little influence on the introgression landscape or on reproductive compatibility between strains. In contrast, we found strong ancestry disequilibrium consistent with negative epistatic selection shaping genomic ancestry combinations during the course of hybridization. This study provides a detailed, experimentally tractable example that genomes of natural populations are mosaics reflecting different evolutionary histories. Exploiting genome-wide heterogeneity in the history of ancestral recombination and lineage-specific mutations sheds new light on the population history of S. pombe and highlights the importance of hybridization as a creative force in generating biodiversity. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020

The Genome Sequence of the Anthelmintic-Susceptible New Zealand Haemonchus contortus.

Internal parasitic nematodes are a global animal health issue causing drastic losses in livestock. Here, we report a H. contortus representative draft genome to serve as a genetic resource to the scientific community and support future experimental research of molecular mechanisms in related parasites. A de novo hybrid assembly was generated from PCR-free whole genome sequence data, resulting in a chromosome-level assembly that is 465 Mb in size encoding 22,341 genes. The genome sequence presented here is consistent with the genome architecture of the existing Haemonchus species and is a valuable resource for future studies regarding population genetic structures of parasitic nematodes. Additionally, comparative pan-genomics with other species of economically important parasitic nematodes have revealed highly open genomes and strong collinearities within the phylum Nematoda. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020

Evolution of Klebsiella pneumoniae with mucoid and non-mucoid type colonies within a single patient.

We obtained nine Klebsiella pneumoniae isolates successively isolated from a single patient. Four pairs (M1-M4 and NM1-NM4) obtained simultaneously from the same site showed different colony types, mucoid and non-mucoid, while the final isolate (M5) was isolated alone from the blood and showed a mucoid phenotype. The whole genome of isolate M5 was sequenced de novo using the PacBio RSII system, while the others were sequenced with an Illumina Hiseq4000 and mapped to the genome sequences of M5. To identify insertions or deletions in the cps locus, we amplified and sequenced cps locus genes. We identified insertion sequence (IS) elements in several genes of the cps locus or one amino acid substitution in WcaJ in all non-mucoid isolates. Five additional amino acid alterations in RpsJ, LolE, Lon-2, PpsE, and a hypothetical protein were detected in some mucoid and non-mucoid isolates. Based on the genome data and cps locus sequences, the mucoid phenotype may have been lost or converted into the non-mucoid phenotype because of the insertion of IS elements or amino acid alterations at this locus. We inferred a within-host evolutionary scenario, in which non-mucoid variants emerged repeatedly from mucoid isolates, but may be short-lived because of their low fitness.Copyright © 2019 Elsevier GmbH. All rights reserved.

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April 21, 2020

Human Migration and the Spread of the Nematode Parasite Wuchereria bancrofti.

The human disease lymphatic filariasis causes the debilitating effects of elephantiasis and hydrocele. Lymphatic filariasis currently affects the lives of 90 million people in 52 countries. There are three nematodes that cause lymphatic filariasis, Brugia malayi, Brugia timori, and Wuchereria bancrofti, but 90% of all cases of lymphatic filariasis are caused solely by W. bancrofti (Wb). Here we use population genomics to reconstruct the probable route and timing of migration of Wb strains that currently infect Africa, Haiti, and Papua New Guinea (PNG). We used selective whole genome amplification to sequence 42 whole genomes of single Wb worms from populations in Haiti, Mali, Kenya, and PNG. Our results are consistent with a hypothesis of an Island Southeast Asia or East Asian origin of Wb. Our demographic models support divergence times that correlate with the migration of human populations. We hypothesize that PNG was infected at two separate times, first by the Melanesians and later by the migrating Austronesians. The migrating Austronesians also likely introduced Wb to Madagascar where later migrations spread it to continental Africa. From Africa, Wb spread to the New World during the transatlantic slave trade. Genome scans identified 17 genes that were highly differentiated among Wb populations. Among these are genes associated with human immune suppression, insecticide sensitivity, and proposed drug targets. Identifying the distribution of genetic diversity in Wb populations and selection forces acting on the genome will build a foundation to test future hypotheses and help predict response to current eradication efforts. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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April 21, 2020

Structural and functional characterization of an intradiol ring-cleavage dioxygenase from the polyphagous spider mite herbivore Tetranychus urticae Koch.

Genome analyses of the polyphagous spider mite herbivore Tetranychus urticae (two-spotted spider mite) revealed the presence of a set of 17 genes that code for secreted proteins belonging to the “intradiol dioxygenase-like” subgroup. Phylogenetic analyses indicate that this novel enzyme family has been acquired by horizontal gene transfer. In order to better understand the role of these proteins in T. urticae, we have structurally and functionally characterized one paralog (tetur07g02040). It was demonstrated that this protein is indeed an intradiol ring-cleavage dioxygenase, as the enzyme is able to cleave catechol between two hydroxyl-groups using atmospheric dioxygen. The enzyme was characterized functionally and structurally. The active site of the T. urticae enzyme contains an Fe3+ cofactor that is coordinated by two histidine and two tyrosine residues, an arrangement that is similar to those observed in bacterial homologs. However, the active site is significantly more solvent exposed than in bacterial proteins. Moreover, the mite enzyme is monomeric, while almost all structurally characterized bacterial homologs form oligomeric assemblies. Tetur07g02040 is not only the first spider mite dioxygenase that has been characterized at the molecular level, but is also the first structurally characterized intradiol ring-cleavage dioxygenase originating from a eukaryote.Copyright © 2018 Elsevier Ltd. All rights reserved.

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April 21, 2020

Detecting a long insertion variant in SAMD12 by SMRT sequencing: implications of long-read whole-genome sequencing for repeat expansion diseases.

Long-read sequencing technology is now capable of reading single-molecule DNA with an average read length of more than 10?kb, fully enabling the coverage of large structural variations (SVs). This advantage may pave the way for the detection of unprecedented SVs as well as repeat expansions. Pathogenic SVs of only known genes used to be selectively analyzed based on prior knowledge of target DNA sequence. The unbiased application of long-read whole-genome sequencing (WGS) for the detection of pathogenic SVs has just begun. Here, we apply PacBio SMRT sequencing in a Japanese family with benign adult familial myoclonus epilepsy (BAFME). Our SV selection of low-coverage WGS data (7×) narrowed down the candidates to only six SVs in a 7.16-Mb region of the BAFME1 locus and correctly determined an approximately 4.6-kb SAMD12 intronic repeat insertion, which is causal of BAFME1. These results indicate that long-read WGS is potentially useful for evaluating all of the known SVs in a genome and identifying new disease-causing SVs in combination with other genetic methods to resolve the genetic causes of currently unexplained diseases.

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April 21, 2020

Tengunoibacter tsumagoiensis gen. nov., sp. nov., Dictyobacter kobayashii sp. nov., Dictyobacter alpinus sp. nov., and description of Dictyobacteraceae fam. nov. within the order Ktedonobacterales isolated from Tengu-no-mugimeshi, a soil-like granular mass of micro-organisms, and emended descriptions of the genera Ktedonobacter and Dictyobacter.

Three mesophilic, Gram-stain-positive, aerobic bacterial strains, designated Uno3T, Uno11T and Uno16T, were isolated from a soil-like granular micro-organism mass (termed Tengu-no-mugimeshi) collected from Tsumagoi, Gunma, Japan. They grow at 11-37?°C?and pH 4.0-8.0, form branched mycelia, and have a G+C?content between 49.4-50.3?mol%. The major menaquinone and fatty acid of Uno3T are MK-9 and iso-C16?:?0, respectively, whereas Uno11T and Uno16T share MK-9 (H2) and C16?:?1-2OH. The major cell-wall sugars are mannose (Uno3T and Uno11T) and glucose (Uno16T). Phylogenetic analysis based on 16S rRNA gene sequences indicated that these three strains belong to the order Ktedonobacterales and are most closely related to Dictyobacter aurantiacus S-27T (sequence similarity of 91.3, 96.4 and 95.5?%). Average nucleotide identity values were <79.9?% among Uno11T, Uno16T and D. aurantiacus S-27T, well below the 95-96?%?species circumscription threshold. Based on phenotypic features and phylogenetic positions, we propose that Uno3T represents a novel genus and species, Tengunoibacter tsumagoiensis gen. nov., sp. nov. (type strain Uno3T=NBRC 113152T=LMG 30471T=BCRC 81113T) within the new family Dictyobacteraceae fam. nov. Strains Uno11T and Uno16T are also considered to represent novel species: Dictyobacterkobayashii sp. nov. (type strain Uno11T=NBRC 113153T=LMG 30472T=BCRC 81114T) and Dictyobacteralpinus sp. nov. (type strain Uno16T=NBRC 113154T=BCRC 81115T). We also propose an emended description of the genus Dictyobacter, classifying it within family Dictyobacteraceae, and provide emended descriptions of the genera Dictyobacter and Ktedonobacter.

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April 21, 2020

Dissemination of multiple carbapenem resistance genes in an in vitro gut model simulating the human colon.

Carbapenemase-producing Enterobacteriaceae (CPE) pose a major global health risk. Mobile genetic elements account for much of the increasing CPE burden.To investigate CPE colonization and the impact of antibiotic exposure on subsequent resistance gene dissemination within the gut microbiota using a model to simulate the human colon.Gut models seeded with CPE-negative human faeces [screened with BioMérieux chromID® CARBA-SMART (Carba-Smart), Cepheid Xpert® Carba-R assay (XCR)] were inoculated with distinct carbapenemase-producing Klebsiella pneumoniae strains (KPC, NDM) and challenged with imipenem or piperacillin/tazobactam then meropenem. Resistant populations were enumerated daily on selective agars (Carba-Smart); CPE genes were confirmed by PCR (XCR, Check-Direct CPE Screen for BD MAX™). CPE gene dissemination was tracked using PacBio long-read sequencing.CPE populations increased during inoculation, plateauing at ~105?log10?cfu/mL in both models and persisting throughout the experiments (>65?days), with no evidence of CPE ‘washout’. After antibiotic administration, there was evidence of interspecies plasmid transfer of blaKPC-2 (111742?bp IncFII/IncR plasmid, 99% identity to pKpQIL-D2) and blaNDM-1 (~170?kb IncFIB/IncFII plasmid), and CPE populations rose from <0.01% to >45% of the total lactose-fermenting populations in the KPC model. Isolation of a blaNDM-1K. pneumoniae with one chromosomal single-nucleotide variant compared with the inoculated strain indicated clonal expansion within the model. Antibiotic administration exposed a previously undetected K. pneumoniae encoding blaOXA-232 (KPC model).CPE exposure can lead to colonization, clonal expansion and resistance gene transfer within intact human colonic microbiota. Furthermore, under antibiotic selective pressure, new resistant populations emerge, emphasizing the need to control exposure to antimicrobials. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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April 21, 2020

Lignin catabolic pathways reveal unique characteristics of dye-decolorizing peroxidases in Pseudomonas putida.

Lignin is one of the largest carbon reservoirs in the environment, playing an important role in the global carbon cycle. However, lignin degradation in bacteria, especially non-model organisms, has not been well characterized either enzymatically or genetically. Here, a lignin-degrading bacterial strain, Pseudomonas putida A514, was used as the research model. Genomic and proteomic analyses suggested that two B subfamily dye-decolorizing peroxidases (DypBs) were prominent in lignin depolymerization, while the classic O2 -dependent ring cleavage strategy was utilized in central pathways to catabolize lignin-derived aromatic compounds that were funnelled by peripheral pathways. These enzymes, together with a range of transporters, sequential and expression-dose dependent regulation and stress response systems coordinated for lignin metabolism. Catalytic assays indicated these DypBs show unique Mn2+ independent lignin depolymerization activity, while Mn2+ oxidation activity is absent. Furthermore, a high synergy between DypB enzymes and A514 cells was observed to promote cell growth (5 × 1012 cfus/ml) and lignin degradation (27%). This suggested DypBs are competitive lignin biocatalysts and pinpointed limited extracellular secretion capacity as the rate-limiting factor in bacterial lignin degradation. DypB production was, therefore, optimized in recombinant strains and a 14,141-fold increase in DypB activity (56,565?U/l) was achieved, providing novel insights for lignin bioconversion. © 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.

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