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Asset Tag: P4-C2 chemistry

July 19, 2019

The history of Bordetella pertussis genome evolution includes structural rearrangement.

Despite high pertussis vaccine coverage, reported cases of whooping cough (pertussis) have increased over the last decade in the United States and other developed countries. Although Bordetella pertussis is well known for its limited gene sequence variation, recent advances in long-read sequencing technology have begun to reveal genomic structural heterogeneity among otherwise indistinguishable isolates, even within geographically or temporally defined epidemics. We have compared rearrangements among complete genome assemblies from 257 B. pertussis isolates to examine the potential evolution of the chromosomal structure in a pathogen with minimal gene nucleotide sequence diversity. Discrete changes in gene order were identified that differentiated genomes from vaccine reference strains and clinical isolates of various genotypes, frequently along phylogenetic boundaries defined by single nucleotide polymorphisms. The observed rearrangements were primarily large inversions centered on the replication origin or terminus and flanked by IS481, a mobile genetic element with >240 copies per genome and previously suspected to mediate rearrangements and deletions by homologous recombination. These data illustrate that structural genome evolution in B. pertussis is not limited to reduction but also includes rearrangement. Therefore, although genomes of clinical isolates are structurally diverse, specific changes in gene order are conserved, perhaps due to positive selection, providing novel information for investigating disease resurgence and molecular epidemiology.IMPORTANCE Whooping cough, primarily caused by Bordetella pertussis, has resurged in the United States even though the coverage with pertussis-containing vaccines remains high. The rise in reported cases has included increased disease rates among all vaccinated age groups, provoking questions about the pathogen’s evolution. The chromosome of B. pertussis includes a large number of repetitive mobile genetic elements that obstruct genome analysis. However, these mobile elements facilitate large rearrangements that alter the order and orientation of essential protein-encoding genes, which otherwise exhibit little nucleotide sequence diversity. By comparing the complete genome assemblies from 257 isolates, we show that specific rearrangements have been conserved throughout recent evolutionary history, perhaps by eliciting changes in gene expression, which may also provide useful information for molecular epidemiology. Copyright © 2017 American Society for Microbiology.

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July 19, 2019

SMRT genome assembly corrects reference errors, resolving the genetic basis of virulence in Mycobacterium tuberculosis.

The genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication.Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. Further, one of the sequencing errors in H37Ra masks a true variant in common with the clinical strain CDC1551 which, when considered in the context of previous work, corresponds to a sequencing error in the H37Rv reference genome.Our results constrain the set of genomic differences possibly affecting virulence by more than half, which focuses laboratory investigation on pertinent targets and demonstrates the power of SMRT sequencing for producing high-quality reference genomes.

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July 19, 2019

Comparative genomics of two sequential Candida glabrata clinical isolates.

Candida glabrata is an important fungal pathogen which develops rapid antifungal resistance in treated patients. It is known that azole treatments lead to antifungal resistance in this fungal species and that multidrug efflux transporters are involved in this process. Specific mutations in the transcriptional regulator PDR1 result in upregulation of the transporters. In addition, we showed that the PDR1 mutations can contribute to enhance virulence in animal models. In this study, we were interested to compare genomes of two specific C. glabrata-related isolates, one of which was azole susceptible (DSY562) while the other was azole resistant (DSY565). DSY565 contained a PDR1 mutation (L280F) and was isolated after a time-lapse of 50 d of azole therapy. We expected that genome comparisons between both isolates could reveal additional mutations reflecting host adaptation or even additional resistance mechanisms. The PacBio technology used here yielded 14 major contigs (sizes 0.18-1.6 Mb) and mitochondrial genomes from both DSY562 and DSY565 isolates that were highly similar to each other. Comparisons of the clinical genomes with the published CBS138 genome indicated important genome rearrangements, but not between the clinical strains. Among the unique features, several retrotransposons were identified in the genomes of the investigated clinical isolates. DSY562 and DSY565 each contained a large set of adhesin-like genes (101 and 107, respectively), which exceed by far the number of reported adhesins (63) in the CBS138 genome. Comparison between DSY562 and DSY565 yielded 17 nonsynonymous SNPs (among which the was the expected PDR1 mutation) as well as small size indels in coding regions (11) but mainly in adhesin-like genes. The genomes contained a DNA mismatch repair allele of MSH2 known to be involved in the so-called hyper-mutator phenotype of this yeast species and the number of accumulated mutations between both clinical isolates is consistent with the presence of a MSH2 defect. In conclusion, this study is the first to compare genomes of C. glabrata sequential clinical isolates using the PacBio technology as an approach. The genomes of these isolates taken in the same patient at two different time points exhibited limited variations, even if submitted to the host pressure. Copyright © 2017 Vale-Silva et al.

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July 19, 2019

Contrasting evolutionary genome dynamics between domesticated and wild yeasts.

Structural rearrangements have long been recognized as an important source of genetic variation, with implications in phenotypic diversity and disease, yet their detailed evolutionary dynamics remain elusive. Here we use long-read sequencing to generate end-to-end genome assemblies for 12 strains representing major subpopulations of the partially domesticated yeast Saccharomyces cerevisiae and its wild relative Saccharomyces paradoxus. These population-level high-quality genomes with comprehensive annotation enable precise definition of chromosomal boundaries between cores and subtelomeres and a high-resolution view of evolutionary genome dynamics. In chromosomal cores, S. paradoxus shows faster accumulation of balanced rearrangements (inversions, reciprocal translocations and transpositions), whereas S. cerevisiae accumulates unbalanced rearrangements (novel insertions, deletions and duplications) more rapidly. In subtelomeres, both species show extensive interchromosomal reshuffling, with a higher tempo in S. cerevisiae. Such striking contrasts between wild and domesticated yeasts are likely to reflect the influence of human activities on structural genome evolution.

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July 19, 2019

Diversity of the TLR4 immunity receptor in Czech native cattle breeds revealed using the Pacific Biosciences sequencing platform.

The allelic variants of immunity genes in historical breeds likely reflect local infection pressure and therefore represent a reservoir for breeding. Screening to determine the diversity of the Toll-like receptor gene TLR4 was conducted in two conserved cattle breeds: Czech Red and Czech Red Pied. High-throughput sequencing of pooled PCR amplicons using the PacBio platform revealed polymorphisms, which were subsequently confirmed via genotyping techniques. Eight SNPs found in coding and adjacent regions were grouped into 18 haplotypes, representing a significant portion of the known diversity in the global breed panel and presumably exceeding diversity in production populations. Notably, the ancient Czech Red breed appeared to possess greater haplotype diversity than the Czech Red Pied breed, a Simmental variant, although the haplotype frequencies might have been distorted by significant crossbreeding and bottlenecks in the history of Czech Red cattle. The differences in haplotype frequencies validated the phenotypic distinctness of the local breeds. Due to the availability of Czech Red Pied production herds, the effect of intensive breeding on TLR diversity can be evaluated in this model. The advantages of the Pacific Biosciences technology for the resequencing of long PCR fragments with subsequent direct phasing were independently validated.

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July 19, 2019

Discovery and biosynthesis of gladiolin: A Burkholderia gladioli antibiotic with promising activity against Mycobacterium tuberculosis.

An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites.

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July 19, 2019

A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples.

VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, mediates the binding of P. falciparum-infected erythrocytes to chondroitin sulfate A, a surface-associated molecule expressed in placental cells, and plays a central role in the pathogenesis of placental malaria. VAR2CSA is a target of naturally acquired immunity and, as such, is a leading vaccine candidate against placental malaria. This protein is very polymorphic and technically challenging to sequence. Published var2csa sequences, mostly limited to specific domains, have been generated through the sequencing of cloned PCR amplicons using capillary electrophoresis, a method that is both time consuming and costly, and that performs poorly when applied to clinical samples that are commonly polyclonal. A next-generation sequencing platform, Pacific Biosciences (PacBio), offers an alternative approach to overcome these issues.PCR primers were designed that target a 5 kb segment in the 5′ end of var2csa and the resulting amplicons were sequenced using PacBio sequencing. The primers were optimized using two laboratory strains and were validated on DNA from 43 clinical samples, extracted from dried blood spots on filter paper or from cryopreserved P. falciparum-infected erythrocytes. Sequence reads were assembled using the SMRT-analysis ConsensusTools module.Here, a PacBio sequencing-based approach for recovering a segment encoding the majority of VAR2CSA’s extracellular region is described; this segment includes the totality of the first four domains in the 5′ end of var2csa (~5 kb), from clinical malaria samples. The feasibility of the method is demonstrated, showing a high success rate from cryopreserved samples and more limited success from dried blood spots stored at room temperature, and characterized the genetic variation of the var2csa locus.This method will facilitate a detailed analysis of var2csa genetic variation and can be adapted to sequence other hypervariable P. falciparum genes.

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July 19, 2019

Insight into the recent genome duplication of the halophilic yeast Hortaea werneckii: combining an improved genome with gene expression and chromatin structure.

Extremophilic organisms demonstrate the flexibility and adaptability of basic biological processes by highlighting how cell physiology adapts to environmental extremes. Few eukaryotic extremophiles have been well studied and only a small number are amenable to laboratory cultivation and manipulation. A detailed characterization of the genome architecture of such organisms is important to illuminate how they adapt to environmental stresses. One excellent example of a fungal extremophile is the halophile Hortaea werneckii (Pezizomycotina, Dothideomycetes, Capnodiales), a yeast-like fungus able to thrive at near-saturating concentrations of sodium chloride and which is also tolerant to both UV irradiation and desiccation. Given its unique lifestyle and its remarkably recent whole genome duplication, H. werneckii provides opportunities for testing the role of genome duplications and adaptability to extreme environments. We previously assembled the genome of H. werneckii using short-read sequencing technology and found a remarkable degree of gene duplication. Technology limitations, however, precluded high-confidence annotation of the entire genome. We therefore revisited the H. wernickii genome using long-read, single-molecule sequencing and provide an improved genome assembly which, combined with transcriptome and nucleosome analysis, provides a useful resource for fungal halophile genomics. Remarkably, the ~50 Mb H. wernickii genome contains 15,974 genes of which 95% (7608) are duplicates formed by a recent whole genome duplication (WGD), with an average of 5% protein sequence divergence between them. We found that the WGD is extraordinarily recent, and compared to Saccharomyces cerevisiae, the majority of the genome’s ohnologs have not diverged at the level of gene expression of chromatin structure. Copyright © 2017 Sinha et al.

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July 19, 2019

Genome and methylome variation in Helicobacter pylori with a cag pathogenicity island during early stages of human infection.

Helicobacter pylori is remarkable for its genetic variation. Yet little isknown about its genetic changes during early stages of human infection, as the bacteria adapt to their new environment. We analyzed genome and methylome variations in a fully virulent strain of H pylori strain during experimental infection.We performed a randomized Phase 1 and 2, observer-blind, placebo-controlled, study of 12 healthy, H pylori-negative adults in Germany from October 2008 through March 2010. The volunteers were given a prophylactic vaccine candidate (n=7) or placebo (n=5) and then challenged with H pylori strain BCM-300. Biopsy samples were collected and H pylori were isolated. Genomes of the challenge strain and 12 re-isolates, obtained 12 weeks after (or in 1 case, 62 weeks after) infection were sequenced by single-molecule, real-time technology, which, in parallel, permitted determination of genome-wide methylation patterns for all strains. Functional effects of genetic changes observed in H pylori strains during human infection were assessed by measuring release of interleukin 8 from AGS cells (to detect cag PAI function), neutral red uptake (to detect vacuolating cytotoxin activity), and adhesion assays.The observed mutation rate was in agreement with rates previously determined from patients with chronic H pylori infections, without evidence of a mutation burst. A loss; of cag PAI function was observed in 3 re-isolates. In addition, 3 re-isolates from the vaccine; group acquired mutations in the vacuolating cytotoxin gene vacA, resulting in loss of; vacuolization activity from gastric epithelial cells. We observed inter-strain variation in; methylomes due to phase variation in genes encoding methyltransferases.We analyzed adaptation of a fully virulent strain of H pylori to 12 differentvolunteers to obtain a robust estimate of the frequency of genetic and epigenetic changes inthe absence of inter-strain recombination. Our findings indicate that the large amount of; genetic variation in H pylori poses a challenge to vaccine development. ClinicalTrials.gov no: NCT00736476. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

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July 7, 2019

A novel Tn3-like composite transposon harboring blaVIM-1 in Klebsiella pneumoniae spp. pneumoniae isolated from river water.

We present a new plasmid (pOW16C2) with a novel Tn3-like transposon harboring blaVIM-1 from a Klebsiella pneumoniae strain isolated from river water in Switzerland.Complete nucleotide sequence of pOW16C2 was obtained using a Pacific Biosciences SMRT sequencing approach and coding sequences were predicted.The 59,228?bp sequence included a typical IncN-like backbone and a mosaic structure with blaVIM-1, aacA4, aphA15, aadA1, catB2, qnrS1, sul1, and dfrA14 conferring resistance to carbapenems and other ß-lactam antibiotics, aminoglycosides, chloramphenicol, quinolones, sulfonamides, and trimethoprim, respectively. Most of these resistance genes were inserted in a class 1 integron that was embedded in a novel Tn3-like composite transposon.IncN plasmids carrying carbapenemases are frequently isolated from K. pneumoniae strains in clinical settings. The dissemination of K. pneumoniae harboring blaVIM-1 in surface water is a cause for increased concern to public health.

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July 7, 2019

Emergence of scarlet fever Streptococcus pyogenes emm12 clones in Hong Kong is associated with toxin acquisition and multidrug resistance.

A scarlet fever outbreak began in mainland China and Hong Kong in 2011 (refs. 1-6). Macrolide- and tetracycline-resistant Streptococcus pyogenes emm12 isolates represent the majority of clinical cases. Recently, we identified two mobile genetic elements that were closely associated with emm12 outbreak isolates: the integrative and conjugative element ICE-emm12, encoding genes for tetracycline and macrolide resistance, and prophage FHKU.vir, encoding the superantigens SSA and SpeC, as well as the DNase Spd1 (ref. 4). Here we sequenced the genomes of 141 emm12 isolates, including 132 isolated in Hong Kong between 2005 and 2011. We found that the introduction of several ICE-emm12 variants, FHKU.vir and a new prophage, FHKU.ssa, occurred in three distinct emm12 lineages late in the twentieth century. Acquisition of ssa and transposable elements encoding multidrug resistance genes triggered the expansion of scarlet fever-associated emm12 lineages in Hong Kong. The occurrence of multidrug-resistant ssa-harboring scarlet fever strains should prompt heightened surveillance within China and abroad for the dissemination of these mobile genetic elements.

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July 7, 2019

Complete annotated genome sequence of Mycobacterium tuberculosis (Zopf) Lehmann and Neumann (ATCC35812) (Kurono).

We report the completely annotated genome sequence of Mycobacterium tuberculosis (Zopf) Lehmann and Neumann (ATCC35812) (Kurono), which is a used for virulence and/or immunization studies. The complete genome sequence of M. tuberculosis Kurono was determined with a length of 4,415,078 bp and a G+C content of 65.60%. The chromosome was shown to contain a total of 4,340 protein-coding genes, 53 tRNA genes, one transfer messenger RNA for all amino acids, and 1 rrn operon. Lineage analysis based on large sequence polymorphisms indicated that M. tuberculosis Kurono belongs to the Euro-American lineage (lineage 4). Phylogenetic analysis using whole genome sequences of M. tuberculosis Kurono in addition to 22 M. tuberculosis complex strains indicated that H37Rv is the closest relative of Kurono based on the results of phylogenetic analysis. These findings provide a basis for research using M. tuberculosis Kurono, especially in animal models. Copyright © 2014 Elsevier Ltd. All rights reserved.

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July 7, 2019

Complete genome sequence of Enterobacter cloacae GGT036: a furfural tolerant soil bacterium.

Enterobacter cloacae is a facultative anaerobic bacterium to be an important cause of nosocomial infection. However, the isolated E. cloacae GGT036 showed higher furfural-tolerant cellular growth, compared to industrial relevant strains such as Escherichia coli and Corynebacterium glutamicum. Here, we report the complete genome sequence of E. cloacae GGT036 isolated from Mt. Gwanak, Seoul, Republic of Korea. The genomic DNA sequence of E. cloacae GGT036 will provide valuable genetic resources for engineering of industrially relevant strains being tolerant to cellular inhibitors present in lignocellulosic hydrolysates. Copyright © 2014 Elsevier B.V. All rights reserved.

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July 7, 2019

Finished genome sequence of Collimonas arenae Cal35.

We announce the finished genome sequence of soil forest isolate Collimonas arenae Cal35, which comprises a 5.6-Mbp chromosome and 41-kb plasmid. The Cal35 genome is the second one published for the bacterial genus Collimonas and represents the first opportunity for high-resolution comparison of genome content and synteny among collimonads. Copyright © 2015 Wu et al.

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July 7, 2019

Chloroplast genome of Aconitum barbatum var. puberulum (Ranunculaceae) derived from CCS reads using the PacBio RS platform.

The chloroplast genome (cp genome) of Aconitum barbatum var. puberulum was sequenced using the third-generation sequencing platform based on the single-molecule real-time (SMRT) sequencing approach. To our knowledge, this is the first reported complete cp genome of Aconitum, and we anticipate that it will have great value for phylogenetic studies of the Ranunculaceae family. In total, 23,498 CCS reads and 20,685,462 base pairs were generated, the mean read length was 880 bp, and the longest read was 2,261 bp. Genome coverage of 100% was achieved with a mean coverage of 132× and no gaps. The accuracy of the assembled genome is 99.973%; the assembly was validated using Sanger sequencing of six selected genes from the cp genome. The complete cp genome of A. barbatum var. puberulum is 156,749 bp in length, including a large single-copy region of 87,630 bp and a small single-copy region of 16,941 bp separated by two inverted repeats of 26,089 bp. The cp genome contains 130 genes, including 84 protein-coding genes, 34 tRNA genes and eight rRNA genes. Four forward, five inverted and eight tandem repeats were identified. According to the SSR analysis, the longest poly structure is a 20-T repeat. Our results presented in this paper will facilitate the phylogenetic studies and molecular authentication on Aconitum.

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