We present the first complete genome sequence of a Staphylococcus aureus strain assigned to clonal complex 12. The strain was isolated in a food poisoning outbreak due to contaminated potato salad in Switzerland in 2009, and it produces staphylococcal enterotoxin B.
We describe the complete genome sequence of Burkholderia pseudomallei MSHR305, a clinical isolate taken from a fatal encephalomyelitis case, a rare form of melioidosis. This sequence will be used for comparisons to identify the genes that are involved in neurological cases.
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.
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.
Anaerobic gut fungi represent a distinct early-branching fungal phylum (Neocallimastigomycota) and reside in the rumen, hindgut, and feces of ruminant and nonruminant herbivores. The genome of an anaerobic fungal isolate, Orpinomyces sp. strain C1A, was sequenced using a combination of Illumina and PacBio single-molecule real-time (SMRT) technologies. The large genome (100.95 Mb, 16,347 genes) displayed extremely low G+C content (17.0%), large noncoding intergenic regions (73.1%), proliferation of microsatellite repeats (4.9%), and multiple gene duplications. Comparative genomic analysis identified multiple genes and pathways that are absent in Dikarya genomes but present in early-branching fungal lineages and/or nonfungal Opisthokonta. These included genes for posttranslational fucosylation, the production of specific intramembrane proteases and extracellular protease inhibitors, the formation of a complete axoneme and intraflagellar trafficking machinery, and a near-complete focal adhesion machinery. Analysis of the lignocellulolytic machinery in the C1A genome revealed an extremely rich repertoire, with evidence of horizontal gene acquisition from multiple bacterial lineages. Experimental analysis indicated that strain C1A is a remarkable biomass degrader, capable of simultaneous saccharification and fermentation of the cellulosic and hemicellulosic fractions in multiple untreated grasses and crop residues examined, with the process significantly enhanced by mild pretreatments. This capability, acquired during its separate evolutionary trajectory in the rumen, along with its resilience and invasiveness compared to prokaryotic anaerobes, renders anaerobic fungi promising agents for consolidated bioprocessing schemes in biofuels production.
Single-molecule sequencing allows simultaneous reading of DNA bases and their methylation state in bacterial genomes.
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.
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.
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.
New technologies are providing a more complete view of the functional dynamics of microorganisms such as E. coli and V. cholerae.
Genome assembly using high throughput data with short reads, arguably, remains an unresolvable task in repetitive genomes, since when the length of a repeat exceeds the read length, it becomes difficult to unambiguously connect the flanking regions. The emergence of third generation sequencing (Pacific Biosciences) with long reads enables the opportunity to resolve complicated repeats that could not be resolved by the short read data. However, these long reads have high error rate and it is an uphill task to assemble the genome without using additional high quality short reads. Recently, Koren et al. 2012 proposed an approach to use high quality short reads data to correct these long reads and, thus, make the assembly from long reads possible. However, due to the large size of both dataset (short and long reads), error-correction of these long reads requires excessively high computational resources, even on small bacterial genomes. In this work, instead of error correction of long reads, we first assemble the short reads and later map these long reads on the assembly graph to resolve repeats.
Haplogroup H dominates present-day Western European mitochondrial DNA variability (>40%), yet was less common (~19%) among Early Neolithic farmers (~5450 BC) and virtually absent in Mesolithic hunter-gatherers. Here we investigate this major component of the maternal population history of modern Europeans and sequence 39 complete haplogroup H mitochondrial genomes from ancient human remains. We then compare this ‘real-time’ genetic data with cultural changes taking place between the Early Neolithic (~5450 BC) and Bronze Age (~2200 BC) in Central Europe. Our results reveal that the current diversity and distribution of haplogroup H were largely established by the Mid Neolithic (~4000 BC), but with substantial genetic contributions from subsequent pan-European cultures such as the Bell Beakers expanding out of Iberia in the Late Neolithic (~2800 BC). Dated haplogroup H genomes allow us to reconstruct the recent evolutionary history of haplogroup H and reveal a mutation rate 45% higher than current estimates for human mitochondria.
We report here the complete genome sequence of Staphylococcus aureus Tager 104, originally isolated from a cutaneous abscess in 1947 by Morris Tager. Sequence typing of the strain revealed its membership in sequence type 49 (ST49), a previously unknown multilocus sequence type (MLST) in clinical samples.
Recent studies have found methyl-6-adenosine in thousands of mammalian genes, and this modification is most pronounced near the beginning of the 3′ UTR. We present a perspective on current work and new single-molecule sequencing methods for detecting RNA base modifications.
Exceptionally accurate genome reference sequences have proven to be of great value to microbial researchers. Thus, to date, about 1800 bacterial genome assemblies have been “finished” at great expense with the aid of manual laboratory and computational processes that typically iterate over a period of months or even years. By applying a new laboratory design and new assembly algorithm to 16 samples, we demonstrate that assemblies exceeding finished quality can be obtained from whole-genome shotgun data and automated computation. Cost and time requirements are thus dramatically reduced.
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