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Asset Tag: SMRT Sequencing

July 7, 2019

Complete genome sequence of bacteriochlorophyll-synthesizing bacterium Porphyrobacter neustonensis DSM 9434.

The genus Porphyrobacter belongs to aerobic anoxygenic phototrophic bacteria cluster. Porphyrobacter neustonensis DSM 9434 was isolated from a eutrophic freshwater pond in Australia, and is able to synthesize Bacteriochlorophyll a as well as grow under aerobic conditions. It is the type species of the genus Porphyrobacter. Here we describe the characteristics of the strain DSM 9434, including the genome sequence and annotation, synthesis of BChl a, and metabolic pathways of the organism. The genome of strain DSM 9434 comprises 3,090,363 bp and contains 2,902 protein-coding genes, 47 tRNA genes and 6 rRNA genes. Strain DSM 9434 encodes 46 genes which participate in BChl a synthesis and this investigation shed light on the evolution and functional implications regarding bacteriochlorophyll synthesis.

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July 7, 2019

Sequencing a piece of history: complete genome sequence of the original Escherichia coli strain.

In 1885, Theodor Escherich first described the Bacillus coli commune, which was subsequently renamed Escherichia coli. We report the complete genome sequence of this original strain (NCTC 86). The 5?144?392?bp circular chromosome encodes the genes for 4805 proteins, which include antigens, virulence factors, antimicrobial-resistance factors and secretion systems, of a commensal organism from the pre-antibiotic era. It is located in the E. coli A subgroup and is closely related to E. coli K-12 MG1655. E. coli strain NCTC 86 and the non-pathogenic K-12, C, B and HS strains share a common backbone that is largely co-linear. The exception is a large 2?803?932?bp inversion that spans the replication terminus from gmhB to clpB. Comparison with E. coli K-12 reveals 41 regions of difference (577?351?bp) distributed across the chromosome. For example, and contrary to current dogma, E. coli NCTC 86 includes a nine gene sil locus that encodes a silver-resistance efflux pump acquired before the current widespread use of silver nanoparticles as an antibacterial agent, possibly resulting from the widespread use of silver utensils and currency in Germany in the 1800s. In summary, phylogenetic comparisons with other E. coli strains confirmed that the original strain isolated by Escherich is most closely related to the non-pathogenic commensal strains. It is more distant from the root than the pathogenic organisms E. coli 042 and O157?:?H7; therefore, it is not an ancestral state for the species.

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July 7, 2019

Complete genome sequence of the Campylobacter cuniculorum type strain LMG 24588.

Campylobacter cuniculorum is a thermotolerant species isolated from farmed rabbits (Oryctolagus cuniculus). Although C. cuniculorum is highly prevalent in rabbits farmed for human consumption, the pathogenicity of this organism in humans is still unknown. This study describes the whole-genome sequence of the C. cuniculorum type strain LMG 24588 (=CCUG 56289(T)). Copyright © 2017 Miller et al.

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July 7, 2019

Genome-wide identification of the mutation underlying fleece variation and discriminating ancestral hairy species from modern woolly sheep.

The composition and structure of fleece variation observed in mammals is a consequence of a strong selective pressure for fiber production after domestication. In sheep, fleece variation discriminates ancestral species carrying a long and hairy fleece from modern domestic sheep (Ovis aries) owning a short and woolly fleece. Here, we report that the “woolly” allele results from the insertion of an antisense EIF2S2 retrogene (called asEIF2S2) into the 3′ UTR of the IRF2BP2 gene leading to an abnormal IRF2BP2 transcript. We provide evidence that this chimeric IRF2BP2/asEIF2S2 messenger 1) targets the genuine sense EIF2S2 RNA and 2) creates a long endogenous double-stranded RNA which alters the expression of both EIF2S2 and IRF2BP2 mRNA. This represents a unique example of a phenotype arising via a RNA-RNA hybrid, itself generated through a retroposition mechanism. Our results bring new insights on the sheep population history thanks to the identification of the molecular origin of an evolutionary phenotypic variation.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019

Gas fermentation: cellular engineering possibilities and scale up.

Low carbon fuels and chemicals can be sourced from renewable materials such as biomass or from industrial and municipal waste streams. Gasification of these materials allows all of the carbon to become available for product generation, a clear advantage over partial biomass conversion into fermentable sugars. Gasification results into a synthesis stream (syngas) containing carbon monoxide (CO), carbon dioxide (CO2), hydrogen (H2) and nitrogen (N2). Autotrophy-the ability to fix carbon such as CO2 is present in all domains of life but photosynthesis alone is not keeping up with anthropogenic CO2 output. One strategy is to curtail the gaseous atmospheric release by developing waste and syngas conversion technologies. Historically microorganisms have contributed to major, albeit slow, atmospheric composition changes. The current status and future potential of anaerobic gas-fermenting bacteria with special focus on acetogens are the focus of this review.

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July 7, 2019

Nonomuraea sp. ATCC 55076 harbours the largest actinomycete chromosome to date and the kistamicin biosynthetic gene cluster.

Glycopeptide antibiotics (GPAs) have served as potent clinical drugs and as an inspiration to chemists in various disciplines. Among known GPAs, complestatin, chloropeptin, and kistamicin are unique in that they contain an unusual indole-phenol crosslink. The mechanism of formation of this linkage is unknown, and to date, the biosynthetic gene cluster of only one GPA with an indole-phenol crosslink, that of complestatin, has been identified. Here, we report the genome sequence of the kistamicin producer Nonomuraea sp. ATCC 55076. We find that this strain harbours the largest actinobacterial chromosome to date, consisting of a single linear chromosome of ~13.1 Mbp. AntiSMASH analysis shows that ~32 biosynthetic gene clusters and ~10% of the genome are devoted to production of secondary metabolites, which include 1,6-dihydroxyphenazine and nomuricin, a new anthraquinone-type pentacyclic compound that we report herein. The kistamicin gene cluster (kis) was identified bioinformatically. A unique feature of kis is that it contains two cytochrome P450 enzymes, which likely catalyze three crosslinking reactions. These findings set the stage for examining the biosynthesis of kistamicin and its unusual indole-phenol crosslink in the future.

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July 7, 2019

Evolutionary strata on young mating-type chromosomes despite the lack of sexual antagonism.

Sex chromosomes can display successive steps of recombination suppression known as “evolutionary strata,” which are thought to result from the successive linkage of sexually antagonistic genes to sex-determining genes. However, there is little evidence to support this explanation. Here we investigate whether evolutionary strata can evolve without sexual antagonism using fungi that display suppressed recombination extending beyond loci determining mating compatibility despite lack of male/female roles associated with their mating types. By comparing full-length chromosome assemblies from five anther-smut fungi with or without recombination suppression in their mating-type chromosomes, we inferred the ancestral gene order and derived chromosomal arrangements in this group. This approach shed light on the chromosomal fusion underlying the linkage of mating-type loci in fungi and provided evidence for multiple clearly resolved evolutionary strata over a range of ages (0.9-2.1 million years) in mating-type chromosomes. Several evolutionary strata did not include genes involved in mating-type determination. The existence of strata devoid of mating-type genes, despite the lack of sexual antagonism, calls for a unified theory of sex-related chromosome evolution, incorporating, for example, the influence of partially linked deleterious mutations and the maintenance of neutral rearrangement polymorphism due to balancing selection on sexes and mating types.

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July 7, 2019

Emergence of a new Neisseria meningitidis clonal complex 11 lineage 11.2 clade as an effective urogenital pathogen.

Neisseria meningitidis (Nm) clonal complex 11 (cc11) lineage is a hypervirulent pathogen responsible for outbreaks of invasive meningococcal disease, including among men who have sex with men, and is increasingly associated with urogenital infections. Recently, clusters of Nm urethritis have emerged primarily among heterosexual males in the United States. We determined that nonencapsulated meningococcal isolates from an ongoing Nm urethritis outbreak among epidemiologically unrelated men in Columbus, Ohio, are linked to increased Nm urethritis cases in multiple US cities, including Atlanta and Indianapolis, and that they form a unique clade (the US Nm urethritis clade, US_NmUC). The isolates belonged to the cc11 lineage 11.2/ET-15 with fine type of PorA P1.5-1, 10-8; FetA F3-6; PorB 2-2 and express a unique FHbp allele. A common molecular fingerprint of US_NmUC isolates was an IS1301 element in the intergenic region separating the capsule ctr-css operons and adjacent deletion of cssA/B/C and a part of csc, encoding the serogroup C capsule polymerase. This resulted in the loss of encapsulation and intrinsic lipooligosaccharide sialylation that may promote adherence to mucosal surfaces. Furthermore, we detected an IS1301-mediated inversion of an ~20-kb sequence near the cps locus. Surprisingly, these isolates had acquired by gene conversion the complete gonococcal denitrification norB-aniA gene cassette, and strains grow well anaerobically. The cc11 US_NmUC isolates causing urethritis clusters in the United States may have adapted to a urogenital environment by loss of capsule and gene conversion of the Neisseria gonorrheae norB-aniA cassette promoting anaerobic growth.

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July 7, 2019

Molecular and genomic features of Mycobacterium bovis strain 1595 isolated from Korean cattle.

The aim of this study was to investigate the molecular characteristics and to conduct a comparative genomic analysis of Mycobacterium (M.) bovis strain 1595 isolated from a native Korean cow. Molecular typing showed that M. bovis 1595 has spoligotype SB0140 with mycobacterial interspersed repetitive units-variable number of tandem repeats typing of 4-2-5-3-2-7-5-5-4-3-4-3-4-3, representing the most common type of M. bovis in Korea. The complete genome sequence of strain 1595 was determined by single-molecule real-time technology, which showed a genome of 4351712 bp in size with a 65.64% G + C content and 4358 protein-coding genes. Comparative genomic analysis with the genomes of Mycobacterium tuberculosis complex strains revealed that all genomes are similar in size and G + C content. Phylogenetic analysis revealed all strains were within a 0.1% average nucleotide identity value, and MUMmer analysis illustrated that all genomes showed positive collinearity with strain 1595. A sequence comparison based on BLASTP analysis showed that M. bovis AF2122/97 was the strain with the greatest number of completely matched proteins to M. bovis 1595. This genome sequence analysis will serve as a valuable reference for improving understanding of the virulence and epidemiologic traits among M. bovis isolates in Korea.

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July 7, 2019

Characterization of miR-122-independent propagation of HCV.

miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV122KO, possessing G28A substitution in the 5’UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocyte-derived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients.

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July 7, 2019

PipeCraft: Flexible open-source toolkit for bioinformatics analysis of custom high-throughput amplicon sequencing data.

High-throughput sequencing methods have become a routine analysis tool in environmental sciences as well as in public and private sector. These methods provide vast amount of data, which need to be analysed in several steps. Although the bioinformatics may be applied using several public tools, many analytical pipelines allow too few options for the optimal analysis for more complicated or customized designs. Here, we introduce PipeCraft, a flexible and handy bioinformatics pipeline with a user-friendly graphical interface that links several public tools for analysing amplicon sequencing data. Users are able to customize the pipeline by selecting the most suitable tools and options to process raw sequences from Illumina, Pacific Biosciences, Ion Torrent and Roche 454 sequencing platforms. We described the design and options of PipeCraft and evaluated its performance by analysing the data sets from three different sequencing platforms. We demonstrated that PipeCraft is able to process large data sets within 24 hr. The graphical user interface and the automated links between various bioinformatics tools enable easy customization of the workflow. All analytical steps and options are recorded in log files and are easily traceable.© 2017 John Wiley & Sons Ltd.

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July 7, 2019

Characterization of the polymyxin D synthetase biosynthetic cluster and product profile of Paenibacillus polymyxa ATCC 10401.

The increasing prevalence of polymyxin-resistant bacteria has stimulated the search for improved polymyxin lipopeptides. Here we describe the sequence and product profile for polymyxin D nonribosomal peptide synthetase from Paenibacillus polymyxa ATCC 10401. The polymyxin D synthase gene cluster comprised five genes that encoded ABC transporters (pmxC and pmxD) and enzymes responsible for the biosynthesis of polymyxin D (pmxA, pmxB, and pmxE). Unlike polymyxins B and E, polymyxin D contains d-Ser at position 3 as opposed to l-a,?-diaminobutyric acid and has an l-Thr at position 7 rather than l-Leu. Module 3 of pmxE harbored an auxiliary epimerization domain that catalyzes the conversion of l-Ser to the d-form. Structural modeling suggested that the adenylation domains of module 3 in PmxE and modules 6 and 7 in PmxA could bind amino acids with larger side chains than their preferred substrate. Feeding individual amino acids into the culture media not only affected production of polymyxins D1 and D2 but also led to the incorporation of different amino acids at positions 3, 6, and 7 of polymyxin D. Interestingly, the unnatural polymyxin analogues did not show antibiotic activity against a panel of Gram-negative clinical isolates, while the natural polymyxins D1 and D2 exhibited excellent in vitro antibacterial activity and were efficacious against Klebsiella pneumoniae and Acinetobacter baumannii in a mouse blood infection model. The results demonstrate the excellent antibacterial activity of these unusual d-Ser(3) polymxyins and underscore the possibility of incorporating alternate amino acids at positions 3, 6, and 7 of polymyxin D via manipulation of the polymyxin nonribosomal biosynthetic machinery.

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July 7, 2019

Phylogeography of Burkholderia pseudomallei isolates, Western Hemisphere.

The bacterium Burkholderia pseudomallei causes melioidosis, which is mainly associated with tropical areas. We analyzed single-nucleotide polymorphisms (SNPs) among genome sequences from isolates of B. pseudomallei that originated in the Western Hemisphere by comparing them with genome sequences of isolates that originated in the Eastern Hemisphere. Analysis indicated that isolates from the Western Hemisphere form a distinct clade, which supports the hypothesis that these isolates were derived from a constricted seeding event from Africa. Subclades have been resolved that are associated with specific regions within the Western Hemisphere and suggest that isolates might be correlated geographically with cases of melioidosis. One isolate associated with a former World War II prisoner of war was believed to represent illness 62 years after exposure in Southeast Asia. However, analysis suggested the isolate originated in Central or South America.

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July 7, 2019

Natural competence rates are variable among Xylella fastidiosa strains and homologous recombination occurs in vitro between subspecies fastidiosa and multiplex.

Xylella fastidiosa, an etiological agent of emerging crop diseases around the world, is naturally competent for the uptake of DNA from the environment that is incorporated into its genome by homologous recombination. Homologous recombination between subspecies of X. fastidiosa was inferred by in silico studies and was hypothesized to cause disease emergence. However, no experimental data are available on the degree to which X. fastidiosa strains are capable of competence and whether recombination can be experimentally demonstrated between subspecies. Here, using X. fastidiosa strains from different subspecies, natural competence in 11 of 13 strains was confirmed with plasmids containing antibiotic markers flanked by homologous regions and, in three of five strains, with dead bacterial cells used as source of donor DNA. Recombination frequency differed among strains and was correlated to growth rate and twitching motility. Moreover, intersubspecific recombination occurred readily between strains of subsp. fastidiosa and multiplex, as demonstrated by movement of antibiotic resistance and green fluorescent protein from donor to recipient cells and confirmed by DNA sequencing of the flanking arms of recombinant strains. Results demonstrate that natural competence is widespread among X. fastidiosa strains and could have an impact in pathogen adaptation and disease development.

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