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Asset Tag: PacBio RS II

July 7, 2019

Complete chloroplast genome sequences of Eucommia ulmoides: genome structure and evolution.

Eucommia ulmoides is an important traditional medicinal plant that is used for the production of locative Eucommia rubber. In this study, the complete chloroplast (cp) genome sequence of E. ulmoides was obtained by total DNA sequencing; this is the first cp genome sequence of the order Garryales. The cp genome of E. ulmoides was 163,341 bp long and included a pair of inverted repeat (IR) regions (31,300 bp), one large single copy (LSC) region (86,592 bp), and one small single copy (SSC) region (14,149 bp). The genome structure and GC content were similar to those of typical angiosperm cp genomes and contained 115 unique genes, including 80 protein-coding genes, 31 transfer RNA (tRNAs), and four ribosomal RNA (rRNAs). Compared with the entire cp genome sequence, three unique genome rearrangements were observed in the LSC region. Moreover, compared with the Sesamum and Nicotiana cp genomes, E. ulmoides contained no indels in the IR regions, and variable regions were identified in noncoding regions. The E. ulmoides cp genome showed extreme expansion at the IR/SSC boundary owing to the integration of an additional complete gene, ycf1. Twenty-nine simple sequence repeats (SSRs) were identified in the E. ulmoides cp genome. In addition, 36 protein-coding genes were used for phylogenetic inference, supporting a sister relationship between E. ulmoides and Aucuba, which belongs to Euasterids I. In summary, we described the complete cp genome sequence of E. ulmoides; this information will be useful for phylogenetic and evolutionary studies.

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July 7, 2019

Isolation and complete genome sequence of the thermophilic Geobacillus sp. 12AMOR1 from an Arctic deep-sea hydrothermal vent site.

Members of the genus Geobacillus have been isolated from a wide variety of habitats worldwide and are the subject for targeted enzyme utilization in various industrial applications. Here we report the isolation and complete genome sequence of the thermophilic starch-degrading Geobacillus sp. 12AMOR1. The strain 12AMOR1 was isolated from deep-sea hot sediment at the Jan Mayen hydrothermal Vent Site. Geobacillus sp. 12AMOR1 consists of a 3,410,035 bp circular chromosome and a 32,689 bp plasmid with a G?+?C content of 52 % and 47 %, respectively. The genome comprises 3323 protein-coding genes, 88 tRNA species and 10 rRNA operons. The isolate grows on a suite of sugars, complex polysaccharides and proteinous carbon sources. Accordingly, a versatility of genes encoding carbohydrate-active enzymes (CAZy) and peptidases were identified in the genome. Expression, purification and characterization of an enzyme of the glycoside hydrolase family 13 revealed a starch-degrading capacity and high thermal stability with a melting temperature of 76.4 °C. Altogether, the data obtained point to a new isolate from a marine hydrothermal vent with a large bioprospecting potential.

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July 7, 2019

Antibiotic resistance mechanisms of Myroides sp.

Bacteria of the genus Myroides (Myroides spp.) are rare opportunistic pathogens. Myroides sp. infections have been reported mainly in China. Myroides sp. is highly resistant to most available antibiotics, but the resistance mechanisms are not fully elucidated. Current strain identification methods based on biochemical traits are unable to identify strains accurately at the species level. While 16S ribosomal RNA (rRNA) gene sequencing can accurately achieve this, it fails to give information on the status and mechanisms of antibiotic resistance, because the 16S rRNA sequence contains no information on resistance genes, resistance islands or enzymes. We hypothesized that obtaining the whole genome sequence of Myroides sp., using next generation sequencing methods, would help to clarify the mechanisms of pathogenesis and antibiotic resistance, and guide antibiotic selection to treat Myroides sp. infections. As Myroides sp. can survive in hospitals and the environment, there is a risk of nosocomial infections and pandemics. For better management of Myroides sp. infections, it is imperative to apply next generation sequencing technologies to clarify the antibiotic resistance mechanisms in these bacteria.

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July 7, 2019

PEPR: pipelines for evaluating prokaryotic references.

The rapid adoption of microbial whole genome sequencing in public health, clinical testing, and forensic laboratories requires the use of validated measurement processes. Well-characterized, homogeneous, and stable microbial genomic reference materials can be used to evaluate measurement processes, improving confidence in microbial whole genome sequencing results. We have developed a reproducible and transparent bioinformatics tool, PEPR, Pipelines for Evaluating Prokaryotic References, for characterizing the reference genome of prokaryotic genomic materials. PEPR evaluates the quality, purity, and homogeneity of the reference material genome, and purity of the genomic material. The quality of the genome is evaluated using high coverage paired-end sequence data; coverage, paired-end read size and direction, as well as soft-clipping rates, are used to identify mis-assemblies. The homogeneity and purity of the material relative to the reference genome are characterized by comparing base calls from replicate datasets generated using multiple sequencing technologies. Genomic purity of the material is assessed by checking for DNA contaminants. We demonstrate the tool and its output using sequencing data while developing a Staphylococcus aureus candidate genomic reference material. PEPR is open source and available at https://github.com/usnistgov/pepr .

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July 7, 2019

Dynamics of mutations during development of resistance by Pseudomonas aeruginosa against five antibiotics.

Pseudomonas aeruginosa is an opportunistic pathogen that causes considerable morbidity and mortality, specifically in the intensive care. Antibiotic resistant variants of this organism are more difficult to treat and cause substantial extra costs compared to susceptible strains. In the laboratory, P. aeruginosa rapidly developed resistance against five medically relevant antibiotics upon exposure to step-wise increasing concentrations. At several time points during the acquisition of resistance samples were taken for whole genome sequencing. The increase of MIC for ciprofloxacin was linked to specific mutations in gyrA, parC and gyrB, appearing sequentially. In the case of tobramycin, mutations were induced in fusA, HP02880, rplB and capD The MIC for the beta-lactam compounds meropenem, ceftazidime and the combination piperacillin/tazobactam correlated linearly with the beta-lactamase activity, but not always with individual mutations. The genes that were mutated during development of beta-lactam resistance differed for each antibiotic. A quantitative relationship between the frequency of mutations and the increase in resistance could not be established for any of the antibiotics. When the adapted strains are grown in the absence of the antibiotic, some mutations remained and others were reverted, but this reversal did not necessarily lower the MIC. The increased MIC came at the cost of moderately reduced cellular functions, or somewhat lower growth rate. In all cases except ciprofloxacin, the increase of resistance seems to be the result of a complex interaction between several cellular systems, rather than individual mutations. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Genome sequence of Ustilaginoidea virens IPU010, a rice pathogenic fungus causing false smut.

Ustilaginoidea virens is a rice pathogenic fungus that causes false smut disease, a disease that seriously damages the yield and quality of the grain. Analysis of the U. virens IPU010 33.6-Mb genome sequence will aid in the understanding of the pathogenicity of the strain, particularly in regard to effector proteins and secondary metabolic genes. Copyright © 2016 Kumagai et al.

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July 7, 2019

Near-Complete Genome Sequence of Clostridium paradoxum Strain JW-YL-7.

Clostridium paradoxum strain JW-YL-7 is a moderately thermophilic anaerobic alkaliphile isolated from the municipal sewage treatment plant in Athens, GA. We report the near-complete genome sequence of C. paradoxum strain JW-YL-7 obtained by using PacBio DNA sequencing and Pilon for sequence assembly refinement with Illumina data. Copyright © 2016 Lancaster et al.

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July 7, 2019

Genomic organization of the zebrafish (Danio rerio) T cell receptor alpha/delta locus and analysis of expressed products.

In testing the hypothesis that all jawed vertebrate classes employ immunoglobulin heavy chain V (IgHV) gene segments in their T cell receptor (TCR)d encoding loci, we found that some basic characterization was required of zebrafish TCRd. We began by annotating and characterizing the TCRa/d locus of Danio rerio based on the most recent genome assembly, GRCz10. We identified a total of 141 theoretically functional V segments which we grouped into 41 families based upon 70 % nucleotide identity. This number represents the second greatest count of apparently functional V genes thus far described in an antigen receptor locus with the exception of cattle TCRa/d. Cloning, relative quantitative PCR, and deep sequencing results corroborate that zebrafish do express TCRd, but these data suggest only at extremely low levels and in limited diversity in the spleens of the adult fish. While we found no evidence for IgH-TCRd rearrangements in this fish, by determining the locus organization we were able to suggest how the evolution of the teleost a/d locus could have lost IgHVs that exist in sharks and frogs. We also found evidence of surprisingly low TCRd expression and repertoire diversity in this species.

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July 7, 2019

Elucidating the triplicated ancestral genome structure of radish based on chromosome-level comparison with the Brassica genomes.

This study presents a chromosome-scale draft genome sequence of radish that is assembled into nine chromosomal pseudomolecules. A comprehensive comparative genome analysis with the Brassica genomes provides genomic evidences on the evolution of the mesohexaploid radish genome. Radish (Raphanus sativus L.) is an agronomically important root vegetable crop and its origin and phylogenetic position in the tribe Brassiceae is controversial. Here we present a comprehensive analysis of the radish genome based on the chromosome sequences of R. sativus cv. WK10039. The radish genome was sequenced and assembled into 426.2 Mb spanning >98 % of the gene space, of which 344.0 Mb were integrated into nine chromosome pseudomolecules. Approximately 36 % of the genome was repetitive sequences and 46,514 protein-coding genes were predicted and annotated. Comparative mapping of the tPCK-like ancestral genome revealed that the radish genome has intermediate characteristics between the Brassica A/C and B genomes in the triplicated segments, suggesting an internal origin from the genus Brassica. The evolutionary characteristics shared between radish and other Brassica species provided genomic evidences that the current form of nine chromosomes in radish was rearranged from the chromosomes of hexaploid progenitor. Overall, this study provides a chromosome-scale draft genome sequence of radish as well as novel insight into evolution of the mesohexaploid genomes in the tribe Brassiceae.

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July 7, 2019

Complete genome sequence of a CTX-M-15-producing Escherichia coli strain from the H30Rx subclone of sequence type 131 from a patient with recurrent urinary tract infections, closely related to a lethal urosepsis isolate from the patient’s sister.

We report here the complete genome sequence, including five plasmid sequences, of Escherichia coli sequence type 131 (ST131) strain JJ1887. The strain was isolated in 2007 in the United States from a patient with recurrent cystitis, whose caregiver sister died from urosepsis caused by a nearly identical strain. Copyright © 2016 Johnson et al.

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July 7, 2019

Genetic diversity of O-antigens in Hafnia alvei and the development of a suspension array for serotype detection.

Hafnia alvei is a facultative and rod-shaped gram-negative bacterium that belongs to the Enterobacteriaceae family. Although it has been more than 50 years since the genus was identified, very little is known about variations among Hafnia species. Diversity in O-antigens (O-polysaccharide, OPS) is thought to be a major factor in bacterial adaptation to different hosts and situations and variability in the environment. Antigenic variation is also an important factor in pathogenicity that has been used to define clones within a number of species. The genes that are required to synthesize OPS are always clustered within the bacterial chromosome. A serotyping scheme including 39 O-serotypes has been proposed for H. alvei, but it has not been correlated with known OPS structures, and no previous report has described the genetic features of OPS. In this study, we obtained the genome sequences of 21 H. alvei strains (as defined by previous immunochemical studies) with different lipopolysaccharides. This is the first study to show that the O-antigen gene cluster in H. alvei is located between mpo and gnd in the chromosome. All 21 of the OPS gene clusters contain both the wzx gene and the wzy gene and display a large number of polymorphisms. We developed an O serotype-specific wzy-based suspension array to detect all 21 of the distinct OPS forms we identified in H. alvei. To the best of our knowledge, this is the first report to identify the genetic features of H. alvei antigenic variation and to develop a molecular technique to identify and classify different serotypes.

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July 7, 2019

Antibiotic failure mediated by a resistant subpopulation in Enterobacter cloacae.

Antibiotic resistance is a major public health threat, further complicated by unexplained treatment failures caused by bacteria that appear antibiotic susceptible. We describe an Enterobacter cloacae isolate harbouring a minor subpopulation that is highly resistant to the last-line antibiotic colistin. This subpopulation was distinct from persisters, became predominant in colistin, returned to baseline after colistin removal and was dependent on the histidine kinase PhoQ. During murine infection, but in the absence of colistin, innate immune defences led to an increased frequency of the resistant subpopulation, leading to inefficacy of subsequent colistin therapy. An isolate with a lower-frequency colistin-resistant subpopulation similarly caused treatment failure but was misclassified as susceptible by current diagnostics once cultured outside the host. These data demonstrate the ability of low-frequency bacterial subpopulations to contribute to clinically relevant antibiotic resistance, elucidating an enigmatic cause of antibiotic treatment failure and highlighting the critical need for more sensitive diagnostics.

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