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July 7, 2019

Comparing the genomes of Helicobacter pylori clinical strain UM032 and mice-adapted derivatives.

Helicobacter pylori is a Gram-negative bacterium that persistently infects the human stomach inducing chronic inflammation. The exact mechanisms of pathogenesis are still not completely understood. Although not a natural host for H. pylori, mouse infection models play an important role in establishing the immunology and pathogenicity of H. pylori. In this study, for the first time, the genome sequences of clinical H. pylori strain UM032 and mice-adapted derivatives, 298 and 299, were sequenced using the PacBio Single Molecule, Real-Time (SMRT) technology.Here, we described the single contig which was achieved for UM032 (1,599,441 bp), 298 (1,604,216 bp) and 299 (1,601,149 bp). Preliminary analysis suggested that methylation of H. pylori genome through its restriction modification system may be determinative of its host specificity and adaptation.Availability of these genomic sequences will aid in enhancing our current level of understanding the host specificity of H. pylori.

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July 7, 2019

Genome sequence of the phage-gene rich marine Phaeobacter arcticus type strain DSM 23566(T.).

Phaeobacter arcticus Zhang et al. 2008 belongs to the marine Roseobacter clade whose members are phylogenetically and physiologically diverse. In contrast to the type species of this genus, Phaeobacter gallaeciensis, which is well characterized, relatively little is known about the characteristics of P. arcticus. Here, we describe the features of this organism including the annotated high-quality draft genome sequence and highlight some particular traits. The 5,049,232 bp long genome with its 4,828 protein-coding and 81 RNA genes consists of one chromosome and five extrachromosomal elements. Prophage sequences identified via PHAST constitute nearly 5% of the bacterial chromosome and included a potential Mu-like phage as well as a gene-transfer agent (GTA). In addition, the genome of strain DSM 23566(T) encodes all of the genes necessary for assimilatory nitrate reduction. Phylogenetic analysis and intergenomic distances indicate that the classification of the species might need to be reconsidered.

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July 7, 2019

Complete genome sequence of Bacillus subtilis strain PY79.

Bacillus subtilis is a Gram-positive soil-dwelling and endospore-forming bacterium in the phylum Firmicutes. B. subtilis strain PY79 is a prototrophic laboratory strain that has been highly used for studying a wide variety of cellular pathways. Here, we announce the complete whole-genome sequence of B. subtilis PY79.

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July 7, 2019

Single-molecule fluorescence imaging of processive myosin with enhanced background suppression using linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC).

Resolving single fluorescent molecules in the presence of high fluorophore concentrations remains a challenge in single-molecule biophysics that limits our understanding of weak molecular interactions. Total internal reflection fluorescence (TIRF) imaging, the workhorse of single-molecule fluorescence microscopy, enables experiments at concentrations up to about 100 nM, but many biological interactions have considerably weaker affinities, and thus require at least one species to be at micromolar or higher concentration. Current alternatives to TIRF often require three-dimensional confinement, and thus can be problematic for extended substrates, such as cytoskeletal filaments. To address this challenge, we have demonstrated and applied two new single-molecule fluorescence microscopy techniques, linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC), for imaging the processive motion of molecular motors myosin V and VI along actin filaments. Both technologies will allow imaging in the presence of higher fluorophore concentrations than TIRF microscopy. They will enable new biophysical measurements of a wide range of processive molecular motors that move along filamentous tracks, such as other myosins, dynein, and kinesin. A particularly salient application of these technologies will be to examine chemomechanical coupling by directly imaging fluorescent nucleotide molecules interacting with processive motors as they traverse their actin or microtubule tracks.

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July 7, 2019

StatsDB: platform-agnostic storage and understanding of next generation sequencing run metrics.

Modern sequencing platforms generate enormous quantities of data in ever-decreasing amounts of time. Additionally, techniques such as multiplex sequencing allow one run to contain hundreds of different samples. With such data comes a significant challenge to understand its quality and to understand how the quality and yield are changing across instruments and over time. As well as the desire to understand historical data, sequencing centres often have a duty to provide clear summaries of individual run performance to collaborators or customers. We present StatsDB, an open-source software package for storage and analysis of next generation sequencing run metrics. The system has been designed for incorporation into a primary analysis pipeline, either at the programmatic level or via integration into existing user interfaces. Statistics are stored in an SQL database and APIs provide the ability to store and access the data while abstracting the underlying database design. This abstraction allows simpler, wider querying across multiple fields than is possible by the manual steps and calculation required to dissect individual reports, e.g. “provide metrics about nucleotide bias in libraries using adaptor barcode X, across all runs on sequencer A, within the last month”. The software is supplied with modules for storage of statistics from FastQC, a commonly used tool for analysis of sequence reads, but the open nature of the database schema means it can be easily adapted to other tools. Currently at The Genome Analysis Centre (TGAC), reports are accessed through our LIMS system or through a standalone GUI tool, but the API and supplied examples make it easy to develop custom reports and to interface with other packages.

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July 7, 2019

Hammondia hammondi, an avirulent relative of Toxoplasma gondii, has functional orthologs of known T. gondii virulence genes.

Toxoplasma gondii is a ubiquitous protozoan parasite capable of infecting all warm-blooded animals, including humans. Its closest extant relative, Hammondia hammondi, has never been found to infect humans and, in contrast to T. gondii, is highly attenuated in mice. To better understand the genetic bases for these phenotypic differences, we sequenced the genome of a H. hammondi isolate (HhCatGer041) and found the genomic synteny between H. hammondi and T. gondii to be >95%. We used this genome to determine the H. hammondi primary sequence of two major T. gondii mouse virulence genes, TgROP5 and TgROP18. When we expressed these genes in T. gondii, we found that H. hammondi orthologs of TgROP5 and TgROP18 were functional. Similar to T. gondii, the HhROP5 locus is expanded, and two distinct HhROP5 paralogs increased the virulence of a T. gondii TgROP5 knockout strain. We also identified a 107 base pair promoter region, absent only in type III TgROP18, which is necessary for TgROP18 expression. This result indicates that the ROP18 promoter was active in the most recent common ancestor of these two species and that it was subsequently inactivated in progenitors of the type III lineage. Overall, these data suggest that the virulence differences between these species are not solely due to the functionality of these key virulence factors. This study provides evidence that other mechanisms, such as differences in gene expression or the lack of currently uncharacterized virulence factors, may underlie the phenotypic differences between these species.

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July 7, 2019

Coordinated conformational and compositional dynamics drive ribosome translocation.

During translation elongation, the ribosome compositional factors elongation factor G (EF-G; encoded by fusA) and tRNA alternately bind to the ribosome to direct protein synthesis and regulate the conformation of the ribosome. Here, we use single-molecule fluorescence with zero-mode waveguides to directly correlate ribosome conformation and composition during multiple rounds of elongation at high factor concentrations in Escherichia coli. Our results show that EF-G bound to GTP (EF-G-GTP) continuously samples both rotational states of the ribosome, binding with higher affinity to the rotated state. Upon successful accommodation into the rotated ribosome, the EF-G-ribosome complex evolves through several rate-limiting conformational changes and the hydrolysis of GTP, which results in a transition back to the nonrotated state and in turn drives translocation and facilitates release of both EF-G-GDP and E-site tRNA. These experiments highlight the power of tracking single-molecule conformation and composition simultaneously in real time.

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