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Asset Tag: PacBio RS II

July 7, 2019

Improve homology search sensitivity of PacBio data by correcting frameshifts.

Single-molecule, real-time sequencing (SMRT) developed by Pacific BioSciences produces longer reads than secondary generation sequencing technologies such as Illumina. The long read length enables PacBio sequencing to close gaps in genome assembly, reveal structural variations, and identify gene isoforms with higher accuracy in transcriptomic sequencing. However, PacBio data has high sequencing error rate and most of the errors are insertion or deletion errors. During alignment-based homology search, insertion or deletion errors in genes will cause frameshifts and may only lead to marginal alignment scores and short alignments. As a result, it is hard to distinguish true alignments from random alignments and the ambiguity will incur errors in structural and functional annotation. Existing frameshift correction tools are designed for data with much lower error rate and are not optimized for PacBio data. As an increasing number of groups are using SMRT, there is an urgent need for dedicated homology search tools for PacBio data.In this work, we introduce Frame-Pro, a profile homology search tool for PacBio reads. Our tool corrects sequencing errors and also outputs the profile alignments of the corrected sequences against characterized protein families. We applied our tool to both simulated and real PacBio data. The results showed that our method enables more sensitive homology search, especially for PacBio data sets of low sequencing coverage. In addition, we can correct more errors when comparing with a popular error correction tool that does not rely on hybrid sequencing.The source code is freely available at https://sourceforge.net/projects/frame-pro/yannisun@msu.edu. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Epigenetic mechanisms in microbial members of the human microbiota: current knowledge and perspectives.

The human microbiota and epigenetic processes have both been shown to play a crucial role in health and disease. However, there is extremely scarce information on epigenetic modulation of microbiota members except for a few pathogens. Mainly DNA adenine methylation has been described extensively in modulating the virulence of pathogenic bacteria in particular. It would thus appear likely that such mechanisms are widespread for most bacterial members of the microbiota. This review will present briefly the current knowledge on epigenetic processes in bacteria, give examples of known methylation processes in microbial members of the human microbiota and summarize the knowledge on regulation of host epigenetic processes by the human microbiota.

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July 7, 2019

Identification of a virulence determinant that is conserved in the Jawetz and Heyl biotypes of [Pasteurella] pneumotropica.

[Pasteurella] pneumotropica is a ubiquitous bacterium frequently isolated from laboratory rodents. Although this bacterium causes various diseases in immunosuppressed animals, little is known about major virulence factors and their roles in pathogenicity. To identify virulence factors, we sequenced the genome of [P.] pneumotropica biotype Heyl strain ATCC 12555, and compared the resulting non-contiguous draft genome sequence with the genome of biotype Jawetz strain ATCC 35149. Among a large number of genes encoding virulence-associated factors in both strains, four genes encoding for YadA-like proteins, which are known virulence factors that function in host cell adherence and invasion in many pathogens. In this study, we assessed YadA distribution and biological activity as an example of one of virulence-associated factor shared, with biotype Jawetz and Heyl. More than half of mouse isolates were found to have at least one of these genes; whereas, the majority of rat isolates did not. Autoagglutination activity, and ability to bind to mouse collagen type IV and mouse fibroblast cells, was significantly higher in YadA-positive than YadA-negative strains. To conclude, we identified a large number of candidate genes predicted to influence [P.] pneumotropica pathogenesis.© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Genomics-inspired discovery of three antibacterial active metabolites, aurantinins B, C, and D from compost-associated Bacillus subtilis fmb60.

Fmb60 is a wild-type Bacillus subtilis isolated from compost with significant broad-spectrum antimicrobial activities. Two novel PKS clusters were recognized in the genome sequence of fmb60, and then three polyene antibiotics, aurantinins B, C, and D, 1-3, were obtained by bioactivity-guided isolation from the fermentation of fmb60. The structures of aurantinins B-D were elucidated by LC-HRMS and NMR data analysis. Aurantinins C and D were identified as new antimicrobial compounds. The three aurantinins showed significant activity against multidrug-resistant Staphylococcus aureus and Clostridium sporogenes. However, aurantinins B-D did not exhibit any cytotoxicity (IC50 > 100 µg/mL) against LO2 and Caco2 cell lines by MTT assay. Furthermore, using S. aureus as a model bacterium to explore the antibacterial mechanism of aurantinins B-D, it was revealed that the bactericidal activity of aurantinins B-D was related to their ability to disrupt the cell membrane.

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July 7, 2019

Complete genome sequence and transcriptome regulation of the pentose utilizing yeast Sugiyamaella lignohabitans.

Efficient conversion of hexoses and pentoses into value-added chemicals represents one core step for establishing economically feasible biorefineries from lignocellulosic material. While extensive research efforts have recently provided advances in the overall process performance, the quest for new microbial cell factories and novel enzymes sources is still open. As demonstrated recently the yeast Sugiyamaella lignohabitans (formerly Candida lignohabitans) represents a promising microbial cell factory for the production of organic acids from lignocellulosic hydrolysates. We report here the de novo genome assembly of S. lignohabitans using the Single Molecule Real-Time platform, with gene prediction refined by using RNA-seq. The sequencing revealed a 15.98 Mb genome, subdivided into four chromosomes. By phylogenetic analysis, Blastobotrys (Arxula) adeninivorans and Yarrowia lipolytica were found to be close relatives of S. lignohabitans Differential gene expression was evaluated in typical growth conditions on glucose and xylose and allowed a first insight into the transcriptional response of S. lignohabitans to different carbon sources and different oxygenation conditions. Novel sequences for enzymes and transporters involved in the central carbon metabolism, and therefore of potential biotechnological interest, were identified. These data open the way for a better understanding of the metabolism of S. lignohabitans and provide resources for further metabolic engineering.© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Spontaneous chloroplast mutants mostly occur by replication slippage and show a biased pattern in the plastome of Oenothera.

Spontaneous plastome mutants have been used as a research tool since the beginning of genetics. However, technical restrictions have severely limited their contributions to research in physiology and molecular biology. Here, we used full plastome sequencing to systematically characterize a collection of 51 spontaneous chloroplast mutants in Oenothera (evening primrose). Most mutants carry only a single mutation. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms but are insertions/deletions originating from DNA replication slippage events. Only very few mutations appear to be caused by imprecise double-strand break repair, nucleotide misincorporation during replication, or incorrect nucleotide excision repair following oxidative damage. U-turn inversions were not detected. Replication slippage is induced at repetitive sequences that can be very small and tend to have high A/T content. Interestingly, the mutations are not distributed randomly in the genome. The underrepresentation of mutations caused by faulty double-strand break repair might explain the high structural conservation of seed plant plastomes throughout evolution. In addition to providing a fully characterized mutant collection for future research on plastid genetics, gene expression, and photosynthesis, our work identified the spectrum of spontaneous mutations in plastids and reveals that this spectrum is very different from that in the nucleus.© 2016 American Society of Plant Biologists. All rights reserved.

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July 7, 2019

Efficient, cost-effective, high-throughput, Multilocus Sequencing Typing (MLST) method, NGMLST, and the analytical software program MLSTEZ.

Multilocus sequence typing (MLST) has become the preferred method for genotyping many biological species. It can be used to identify major phylogenetic clades, molecular groups, or subpopulations of a species, as well as individual strains or clones. However, conventional MLST is costly and time consuming, which limits its power for genotyping large numbers of samples. Here, we describe a new MLST method that uses next-generation sequencing, a multiplexing protocol, and appropriate analytical software to provide accurate, rapid, and economical MLST genotyping of 96 or more isolates in a single assay.

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July 7, 2019

Transfer of the potato plant isolates of Pectobacterium wasabiae to Pectobacterium parmentieri sp. nov.

Pectobacterium wasabiae was originally isolated from Japanese horseradish (Eutrema wasabi), but recently some Pectobacterium isolates collected from potato plants and tubers displaying blackleg and soft rot symptoms were also assigned to P. wasabiae. Here, combining genomic and phenotypical data, we re-evaluated their taxonomic position. PacBio and Illumina technologies were used to complete the genome sequences of P. wasabiae CFBP 3304T and RNS 08-42-1A. Multi-locus sequence analysis showed that the P. wasabiae strains RNS 08-42-1A, SCC3193, CFIA1002 and WPP163, which were collected from potato plant environment, constituted a separate clade from the original Japanese horseradish P. wasabiae. The taxonomic position of these strains was also supported by calculation of the in-silico DNA-DNA hybridization, genome average nucleotide indentity, alignment fraction and average nucleotide indentity values. In addition, they were phenotypically distinguished from P. wasabiae strains by producing acids from (+)-raffinose, a-d(+)-a-lactose, d(+)-galactose and (+)-melibiose but not from methyl a-d-glycopyranoside, (+)-maltose or malonic acid. The name Pectobacterium parmentieri sp. nov. is proposed for this taxon; the type strain is RNS 08-42-1AT (=CFBP 8475T=LMG 29774T).

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July 7, 2019

Origins of the current seventh cholera pandemic.

Vibrio cholerae has caused seven cholera pandemics since 1817, imposing terror on much of the world, but bacterial strains are currently only available for the sixth and seventh pandemics. The El Tor biotype seventh pandemic began in 1961 in Indonesia, but did not originate directly from the classical biotype sixth-pandemic strain. Previous studies focused mainly on the spread of the seventh pandemic after 1970. Here, we analyze in unprecedented detail the origin, evolution, and transition to pandemicity of the seventh-pandemic strain. We used high-resolution comparative genomic analysis of strains collected from 1930 to 1964, covering the evolution from the first available El Tor biotype strain to the start of the seventh pandemic. We define six stages leading to the pandemic strain and reveal all key events. The seventh pandemic originated from a nonpathogenic strain in the Middle East, first observed in 1897. It subsequently underwent explosive diversification, including the spawning of the pandemic lineage. This rapid diversification suggests that, when first observed, the strain had only recently arrived in the Middle East, possibly from the Asian homeland of cholera. The lineage migrated to Makassar, Indonesia, where it gained the important virulence-associated elements Vibrio seventh pandemic island I (VSP-I), VSP-II, and El Tor type cholera toxin prophage by 1954, and it then became pandemic in 1961 after only 12 additional mutations. Our data indicate that specific niches in the Middle East and Makassar were important in generating the pandemic strain by providing gene sources and the driving forces for genetic events.

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July 7, 2019

Characterization of tet(Y)-carrying LowGC plasmids exogenously captured from cow manure at a conventional dairy farm.

Manure from dairy farms has been shown to contain diverse tetracycline resistance genes that are transferable to soil. Here, we focus on conjugative plasmids that may spread tetracycline resistance at a conventional dairy farm. We performed exogenous plasmid isolation from cattle feces using chlortetracycline for transconjugant selection. The transconjugants obtained harbored LowGC-type plasmids and tet(Y). A representative plasmid (pFK2-7) was fully sequenced and this was compared with previously described LowGC plasmids from piggery manure-treated soil and a GenBank record from Acinetobacter nosocomialis that we also identified as a LowGC plasmid. The pFK2-7 plasmid had the conservative backbone typical of LowGC plasmids, though this region was interrupted with an insert containing the tet(Y)-tet(R) tetracycline resistance genes and the strA-strB streptomycin resistance genes. Despite Acinetobacter populations being considered natural hosts of LowGC plasmids, these plasmids were not found in three Acinetobacter isolates from the study farm. The isolates harbored tet(Y)-tet(R) genes in identical genetic surroundings as pFK2-7, however, suggesting genetic exchange between Acinetobacter and LowGC plasmids. Abundance of LowGC plasmids and tet(Y) was correlated in manure and soil samples from the farm, indicating that LowGC plasmids may be involved in the spread of tet(Y) in the environment.© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Genomic insights into a sustained national outbreak of Yersinia pseudotuberculosis.

In 2014, a sustained outbreak of yersiniosis due to Yersinia pseudotuberculosis occurred across all major cities in New Zealand (NZ), with a total of 220 laboratory-confirmed cases, representing one of the largest ever reported outbreaks of Y. pseudotuberculosis. Here, we performed whole genome sequencing of outbreak-associated isolates to produce the largest population analysis to date of Y. pseudotuberculosis, giving us unprecedented capacity to understand the emergence and evolution of the outbreak clone. Multivariate analysis incorporating our genomic and clinical epidemiological data strongly suggested a single point-source contamination of the food chain, with subsequent nationwide distribution of contaminated produce. We additionally uncovered significant diversity in key determinants of virulence, which we speculate may help explain the high morbidity linked to this outbreak.

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July 7, 2019

Clonal dissemination of Pseudomonas aeruginosa sequence type 235 isolates carrying blaIMP-6 and emergence of blaGES-24 and blaIMP-10 on novel genomic islands PAGI-15 and -16 in South Korea.

A total of 431 Pseudomonas aeruginosa clinical isolates were collected from 29 general hospitals in South Korea in 2015. Antimicrobial susceptibility was tested by the disk diffusion method, and MICs of carbapenems were determined by the agar dilution method. Carbapenemase genes were amplified by PCR and sequenced, and the structures of class 1 integrons surrounding the carbapenemase gene cassettes were analyzed by PCR mapping. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed for strain typing. Whole-genome sequencing was carried out to analyze P. aeruginosa genomic islands (PAGIs) carrying the blaIMP-6, blaIMP-10, and blaGES-24 genes. The rates of carbapenem-nonsusceptible and carbapenemase-producing P. aeruginosa isolates were 34.3% (148/431) and 9.5% (41/431), respectively. IMP-6 was the most prevalent carbapenemase type, followed by VIM-2, IMP-10, and GES-24. All carbapenemase genes were located on class 1 integrons of 6 different types on the chromosome. All isolates harboring carbapenemase genes exhibited genetic relatedness by PFGE (similarity > 80%); moreover, all isolates were identified as sequence type 235 (ST235), with the exception of two ST244 isolates by MLST. The blaIMP-6, blaIMP-10, and blaGES-24 genes were found to be located on two novel PAGIs, designated PAGI-15 and PAGI-16. Our data support the clonal spread of an IMP-6-producing P. aeruginosa ST235 strain, and the emergence of IMP-10 and GES-24 demonstrates the diversification of carbapenemases in P. aeruginosa in Korea. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Complete genome sequence of a psychotrophic Pseudarthrobacter sulfonivorans strain Ar51 (CGMCC 4.7316), a novel crude oil and multi benzene compounds degradation strain.

Pseudarthrobacter sulfonivorans strain Ar51, a psychotrophic bacterium isolated from the Tibet permafrost of China, can degrade crude oil and multi benzene compounds efficiently in low temperature. Here we report the complete genome sequence of this bacterium. The complete genome sequence of Pseudarthrobacter sulfonivorans strain Ar51, consisting of a cycle chromosome with a size of 5.04Mbp and a cycle plasmid with a size of 12.39kbp. The availability of this genome sequence allows us to investigate the genetic basis of crude oil degradation and adaptation to growth in a nutrient-poor permafrost environment. Copyright © 2016 Elsevier B.V. All rights reserved.

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July 7, 2019

Complete genome sequence of Marivivens sp. JLT3646, a potential aromatic compound degrader

Marivivens sp. JLT3646 (CGMCC 1.15778), belonging to the phylum Alphaproteobacteria, was isolated from seawater, Kueishan Islet, offshore northeast of Taiwan. Here, we present the complete genome sequence of Marivivens sp. JLT3646, which contains a circular 2,978,145 bp chromosome with 56.2% G + C content, and one circular plasmid which is 169,066 bp in length. The genome data suggested that Marivivens sp. JLT3646 has the potential to degrade aromatic monomers, which might provide insight into biotechnological applications and facilitate the investigation of environmental bioremediation.

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July 7, 2019

Complete genome sequence of human pathogen Kosakonia cowanii type strain 888-76T.

Kosakonia cowanii type strain 888-76T is a human pathogen which was originally isolated from blood as NIH group 42. In this study, we report the complete genome sequence of K. cowanii 888-76T. 888-76T has 1 chromosome and 2 plasmids with a total genome size of 4,857,567bp and C+G 56.15%. This genome sequence will not only help us to understand the virulence features of K. cowanii 888-76T but also provide us the useful information for the study of evolution of Kosakonia genus. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

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