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July 7, 2019  |  

Closed genome sequence of Clostridium botulinum strain CFSAN064329 (62A).

Clostridium botulinum is a strictly anaerobic, Gram-positive, spore-forming bacterium that produces botulinum neurotoxin, a potent and deadly proteinaceous exotoxin. Clostridium botulinum strain CFSAN064329 (62A) produces an A1 serotype/subtype botulinum neurotoxin and is frequently utilized in food challenge and detection studies. We report here the closed genome sequence of Clostridium botulinum strain CFSAN064329 (62A).

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July 7, 2019  |  

Complete genome sequence of Streptomyces sp. strain BSE7F, a Bali mangrove sediment actinobacterium with antimicrobial activities.

The strain Streptomyces sp. BSE7F, a novel Streptomyces strain isolated from Indonesian mangrove sediment, displays antimicrobial activities against Gram-positive bacteria, Gram-negative bacteria, and yeast. Bioinformatic analysis of the genome sequence revealed the occurrence of 22 biosynthetic gene clusters disclosing the secondary metabolite capacity of strain BSE7F. Copyright © 2018 Handayani et al.

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July 7, 2019  |  

Interpreting whole-genome sequence analyses of foodborne bacteria for regulatory applications and outbreak investigations.

Whole-genome sequence (WGS) analysis has revolutionized the food safety industry by enabling high-resolution typing of foodborne bacteria. Higher resolving power allows investigators to identify origins of contamination during illness outbreaks and regulatory activities quickly and accurately. Government agencies and industry stakeholders worldwide are now analyzing WGS data routinely. Although researchers have published many studies that assess the efficacy of WGS data analysis for source attribution, guidance for interpreting WGS analyses is lacking. Here, we provide the framework for interpreting WGS analyses used by the Food and Drug Administration’s Center for Food Safety and Applied Nutrition (CFSAN). We based this framework on the experiences of CFSAN investigators, collaborations and interactions with government and industry partners, and evaluation of the published literature. A fundamental question for investigators is whether two or more bacteria arose from the same source of contamination. Analysts often count the numbers of nucleotide differences [single-nucleotide polymorphisms (SNPs)] between two or more genome sequences to measure genetic distances. However, using SNP thresholds alone to assess whether bacteria originated from the same source can be misleading. Bacteria that are isolated from food, environmental, or clinical samples are representatives of bacterial populations. These populations are subject to evolutionary forces that can change genome sequences. Therefore, interpreting WGS analyses of foodborne bacteria requires a more sophisticated approach. Here, we present a framework for interpreting WGS analyses that combines SNP counts with phylogenetic tree topologies and bootstrap support. We also clarify the roles of WGS, epidemiological, traceback, and other evidence in forming the conclusions of investigations. Finally, we present examples that illustrate the application of this framework to real-world situations.

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July 7, 2019  |  

Complete genome sequence of industrial biocontrol strain Paenibacillus polymyxa HY96-2 and further analysis of Its biocontrol mechanism.

Paenibacillus polymyxa (formerly known as Bacillus polymyxa) has been extensively studied for agricultural applications as a plant-growth-promoting rhizobacterium and is also an important biocontrol agent. Our team has developed the P. polymyxa strain HY96-2 from the tomato rhizosphere as the first microbial biopesticide based on P. polymyxa for controlling plant diseases around the world, leading to the commercialization of this microbial biopesticide in China. However, further research is essential for understanding its precise biocontrol mechanisms. In this paper, we report the complete genome sequence of HY96-2 and the results of a comparative genomic analysis between different P. polymyxa strains. The complete genome size of HY96-2 was found to be 5.75 Mb and 5207 coding sequences were predicted. HY96-2 was compared with seven other P. polymyxa strains for which complete genome sequences have been published, using phylogenetic tree, pan-genome, and nucleic acid co-linearity analysis. In addition, the genes and gene clusters involved in biofilm formation, antibiotic synthesis, and systemic resistance inducer production were compared between strain HY96-2 and two other strains, namely, SC2 and E681. The results revealed that all three of the P. polymyxa strains have the ability to control plant diseases via the mechanisms of colonization (biofilm formation), antagonism (antibiotic production), and induced resistance (systemic resistance inducer production). However, the variation of the corresponding genes or gene clusters between the three strains may lead to different antimicrobial spectra and biocontrol efficacies. Two possible pathways of biofilm formation in P. polymyxa were reported for the first time after searching the KEGG database. This study provides a scientific basis for the further optimization of the field applications and quality standards of industrial microbial biopesticides based on HY96-2. It may also serve as a reference for studying the differences in antimicrobial spectra and biocontrol capability between different biocontrol agents.

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July 7, 2019  |  

Characterization and genome analysis of a phthalate esters-degrading strain Sphingobium yanoikuyae SHJ.

A bacterium capable of utilizing dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP), and diisobuthyl phthalate (DIBP) as the sole carbon and energy source was isolated from shallow aquifer sediments. The strain was identified as Sphingobium yanoikuyae SHJ based on morphological characteristics, 16S rDNA gene phylogeny, and whole genome average nucleotide identity (ANI). The degradation half-life of DBP with substrate concentration of 8.5 and 50.0 mg/L by strain SHJ was 99.7 and 101.4 hours, respectively. The optimum degradation rate of DBP by SHJ was observed at 30°C and weak alkaline (pH 7.5). Genome sequence of the strain SHJ showed a circular chromosome and additional two circular plasmids with whole genome size of 5,669,383 bp and GC content of 64.23%. Functional annotation of SHJ revealed a total of 5,402 genes, with 5,183 protein-encoding genes, 143 pseudogenes, and 76 noncoding RNA genes. Based on genome annotation, 44 genes were identified to be involved in PAEs hydrolysis potentially. Besides, a region with size of about 6.9 kb comprised of seven ORFs, which is located on the smaller plasmid pSES189, was presumed to be responsible for the biodegradation of phthalate. These results provide insights into the genetic basis of DBP biodegradation in this strain.

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July 7, 2019  |  

sppIDer: a species identification tool to investigate hybrid genomes with high-throughput sequencing.

The genomics era has expanded our knowledge about the diversity of the living world, yet harnessing high-throughput sequencing data to investigate alternative evolutionary trajectories, such as hybridization, is still challenging. Here we present sppIDer, a pipeline for the characterization of interspecies hybrids and pure species, that illuminates the complete composition of genomes. sppIDer maps short-read sequencing data to a combination genome built from reference genomes of several species of interest and assesses the genomic contribution and relative ploidy of each parental species, producing a series of colorful graphical outputs ready for publication. As a proof-of-concept, we use the genus Saccharomyces to detect and visualize both interspecies hybrids and pure strains, even with missing parental reference genomes. Through simulation, we show that sppIDer is robust to variable reference genome qualities and performs well with low-coverage data. We further demonstrate the power of this approach in plants, animals, and other fungi. sppIDer is robust to many different inputs and provides visually intuitive insight into genome composition that enables the rapid identification of species and their interspecies hybrids. sppIDer exists as a Docker image, which is a reusable, reproducible, transparent, and simple-to-run package that automates the pipeline and installation of the required dependencies (https://github.com/GLBRC/sppIDer; last accessed September 6, 2018).

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July 7, 2019  |  

Clustering of circular consensus sequences: accurate error correction and assembly of single molecule real-time reads from multiplexed amplicon libraries.

Targeted resequencing with high-throughput sequencing (HTS) platforms can be used to efficiently interrogate the genomes of large numbers of individuals. A critical issue for research and applications using HTS data, especially from long-read platforms, is error in base calling arising from technological limits and bioinformatic algorithms. We found that the community standard long amplicon analysis (LAA) module from Pacific Biosciences is prone to substantial bioinformatic errors that raise concerns about findings based on this pipeline, prompting the need for a new method.A single molecule real-time (SMRT) sequencing-error correction and assembly pipeline, C3S-LAA, was developed for libraries of pooled amplicons. By uniquely leveraging the structure of SMRT sequence data (comprised of multiple low quality subreads from which higher quality circular consensus sequences are formed) to cluster raw reads, C3S-LAA produced accurate consensus sequences and assemblies of overlapping amplicons from single sample and multiplexed libraries. In contrast, despite read depths in excess of 100X per amplicon, the standard long amplicon analysis module from Pacific Biosciences generated unexpected numbers of amplicon sequences with substantial inaccuracies in the consensus sequences. A bootstrap analysis showed that the C3S-LAA pipeline per se was effective at removing bioinformatic sources of error, but in rare cases a read depth of nearly 400X was not sufficient to overcome minor but systematic errors inherent to amplification or sequencing.C3S-LAA uses a divide and conquer processing algorithm for SMRT amplicon-sequence data that generates accurate consensus sequences and local sequence assemblies. Solving the confounding bioinformatic source of error in LAA allowed for the identification of limited instances of errors due to DNA amplification or sequencing of homopolymeric nucleotide tracts. For research and development in genomics, C3S-LAA allows meaningful conclusions and biological inferences to be made from accurately polished sequence output.

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July 7, 2019  |  

Implementation of pharmacogenomics in everyday clinical settings.

Currently, germline pharmacogenomics (PGx) is successfully implemented within certain specialties in clinical care. With the integration of PGx in pharmacotherapy multiple stakeholders are involved, which are identified in this chapter. Clinically relevant pharmacogenes with their related PGx test are discussed, along with diagnostic test criteria to guide clinicians and policy makers in PGx test selection. The chapter further reviews the similarities and the differences between the guidelines of the Dutch Pharmacogenetics Working Group and the Clinical Pharmacogenetics Implementation Consortium which both support healthcare professionals in understanding PGx test results and help guiding pharmacotherapy by providing evidence-based dosing recommendations. Finally, clinical studies which provide scientific evidence and information on cost-effectiveness supporting clinical implementation of PGx in clinical care are discussed along with the remaining barriers for adoption of PGx testing by healthcare professionals.© 2018 Elsevier Inc. All rights reserved.

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July 7, 2019  |  

Complete genome sequence of the dissimilatory azo reducing thermophilic bacterium Novibacillus thermophiles SG-1.

With the isolation and identification of efficient azo-dye degradation bacteria, bioaugmentation with specific microbial strains has now become an effective strategy to promote the bioremediation of azo dye. However, Azo dye wastewater discharged at high temperature restricted the extensive application of the known mesophilic azoreducing microorganisms. Here we present the complete genome sequence of a bacterium capable of reducing azo dye under thermophilic condition, Novibacillus thermophiles SG-1 (=KCTC 33118T =CGMCC 1.12363T). The complete genome of strain SG-1 contains a circular chromosome of 3,629,225 bp with a G?+?C content of 50.44%. Genome analysis revealed that strain SG-1 possessed genes encoding riboflavin biosynthesis protein that would secrete riboflavin, which could act as electron shuttles to transport the electrons to extracellular azo dye in decolorization process. HPLC analysis showed that the concentration of riboflavin increased from 0.01?µM to 0.255?µM with the growth of strain SG-1 under azo dye reduction. Quantitative real-time PCR analysis further demonstrated that the gene encoding riboflavin biosynthesis protein would be involved in the azo dye decolorization. The results from this study would be beneficial to research the mechanism of anaerobic reduction of azo dye under thermophilic conditions. Copyright © 2018 Elsevier B.V. All rights reserved.

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July 7, 2019  |  

Nanoarrays on passivated aluminum surface for site-specific immobilization of biomolecules

The rapid development of biosensing platforms for highly sensitive and specific detection raises the desire of precise localization of biomolecules onto various material surfaces. Aluminum has been strategically employed in the biosensor system due to its compatibility with CMOS technology and its optical and electrical properties such as prominent propagation of surface plasmons. Herein, we present an adaptable method for preparation of carbon nanoarrays on aluminum surface passivated with poly(vinylphosphonic acid) (PVPA). The carbon nanoarrays were defined by means of electron beam induced deposition (EBID) and they were employed to realize site-specific immobilization of target biomolecules. To demonstrate the concept, selective streptavidin/neutravidin immobilization on the carbon nanoarrays was achieved through protein physisorption with a significantly high contrast of the carbon domains over the surrounding PVPA-modified aluminum surface. By adjusting the fabrication parameters, local protein densities could be varied on similarly sized nanodomains in a parallel process. Moreover, localization of single 40 nm biotinylated beads was achieved by loading them on the neutravidin-decorated nanoarrays. As a further demonstration, DNA polymerase with a streptavidin tag was bound to the biotin-beads that were immobilized on the nanoarrays and in situ rolling circle amplification (RCA) was subsequently performed. The observation of organized DNA arrays synthesized by RCA verified the nanoscale localization of the enzyme with retained biological activity. Hence, the presented approach could provide a flexible and universal avenue to precise localizing various biomolecules on aluminum surface for potential biosensor and bioelectronic applications.

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July 7, 2019  |  

Complete genome sequence of Bacillus sp. HBCD-sjtu, an efficient HBCD-degrading bacterium.

Environmental pollution caused by the release of industrial chemicals is currently one of the most important environmental harms. Manufacturing chemicals can be biodegraded, and valuable intermediates can be used as pharmacophores in drug targeting and have several other useful purposes. Hexabromocyclododecane (HBCD), a non-aromatic brominated flame retardant, is a toxic compound that consists of a cycloaliphatic ring of 12 carbon atoms to which six bromine atoms are attached. It is formed by bromination of cis-trans-trans-1,5,9-cyclododecatriene, but its use is now restricted in several countries, because it is an environmental pollutant. Little is known about whether bacteria can degrade HBCD. A bacterial strain that degrades HBCD was recently isolated using enrichment culture techniques. Based on morphological, biochemical and phylogenetic analysis this isolate was categorized as Bacillus cereus and named strain HBCD-sjtu. Maximum growth and HBCD-degrading activity were observed when this strain was grown at 30 °C, pH 7.0 and 200 RPM in mineral salt medium containing 0.5 mm HBCD. The genome of strain HBCD-sjtu, which consists of only one circular chromosome, was sequenced. This whole genome sequence will be crucial for illuminating the molecular mechanisms of HBCD degradation.

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July 7, 2019  |  

Genomic characterization of methylotrophy of Oharaeibacter diazotrophicus strain SM30T.

Oharaeibacter diazotrophicus strain SM30T, isolated from rice rhizosphere, is an aerobic, facultative lanthanide (Ln3+)-utilizing methylotroph and diazotroph that belongs to the Methylocystaceae family. In this research, the complete genome sequence of strain SM30T was determined, and its methylotrophy modules were characterized. The genome consists of one chromosome and two plasmids, comprising a total of 5,004,097 bp, and the GC content was 71.6 mol%. A total of 4497 CDSs, 67 tRNA, and 9 rRNA were encoded. Typical alpha-proteobacterial methylotrophy genes were found: pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) (mxaF and xoxF1-4), methylotrophy regulatory proteins (mxbDM and mxcQE), PQQ synthesis, H4F pathway, H4MPT pathway, formate oxidation, serine cycle, and ethylmalonyl-CoA pathway. SDS-PAGE and subsequent LC-MS analysis, and qPCR analysis revealed that MxaF and XoxF1 were the dominant MDH in the absence or presence of lanthanum (La3+), respectively. The growth of MDH gene-deletion mutants on alcohols and qPCR results indicated that mxaF and xoxF1 are also involved in ethanol and propanol oxidation, xoxF2 participates in methanol oxidation in the presence of La3+, while xoxF3 was associated with methanol and ethanol oxidation in the absence of La3+, implying that XoxF3 is a calcium (Ca2+)-binding XoxF. Four Ln3+ such as La3+, cerium (Ce3+), praseodymium (Pr3+), and neodymium (Nd3+) served as cofactors for XoxF1 by supporting ?mxaF growth on methanol. Some heavier lanthanides inhibited growth of SM30 on methanol. This study contributes to the understanding of the function of various XoxF-type MDHs and their roles in methylotrophs. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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July 7, 2019  |  

Bioaugmentated activated sludge degradation of progesterone: Kinetics and mechanism

Progesterone (PGT) is not completely removed in conventional treatment plants, and the processing results may have adverse effects on aquatic organisms. In this study, an effective PGT-degradation bacterium, Rhodococcus sp. HYW, was newly isolated from the pharmaceutical plant and was used to augment degradation of PGT. When grown in a mineral medium (MM) containing a trace amount of PGT (500?µg/L) as the sole carbon and energy source, the results show that 99% of PGT was degraded within 1?h and followed the first-order reaction kinetics. Bioaugmentation of PGT-contaminated activated sludge greatly enhanced the PGT degradation rate (~91%) and its derivatives degradation rate were also greatly improved (>83%). The process of PGT degradation in non-bioaugmented PGT-contaminated activated sludge (NBS) and bioaugmentation activated sludge with the bacterial consortium(BS) also conforms to the first-order kinetic model. Furthermore, 12 and 11 biodegradation products for PGT in the NBS and BS were identified using HPLC-LTQ-Orbitrap XL™, respectively. Based on these biodegradation products, two degradation pathways for PGT in NBS and BS were proposed, respectively. Comparing the degradation kinetics and metabolites, it was found that BS degrades PGT more rapidly and can further convert PGT to a small molecular acid. Finally, to reveal the probable cause for the differences in the PGT degradation efficiency and products in the NBS and BS.

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July 7, 2019  |  

The complete genome sequence of Bacillus halotolerans ZB201702 isolated from a drought- and salt-stressed rhizosphere soil.

Bacillus halotolerans is a rhizobacterium with the potential to promote plant growth and tolerance to drought and salinity stress. Here, we present the complete genome sequence of B. halotolerans ZB201702, which consists of 4,150,000 bp in a linear chromosome, including 3074 protein-coding sequences, 30 rRNAs, and 85 tRNAs. Genome analysis revealed many putative gene clusters involved in defense mechanisms. Activity analysis of the strain under salt and simulated drought stress suggests tolerance to abiotic stresses. The complete genome information of B. halotolerans ZB201702 could provide valuable insights into rhizobacteria-mediated plant salt and drought tolerance and rhizobacteria-based solutions for abiotic stress agriculture. Copyright © 2018 Elsevier Ltd. All rights reserved.

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