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July 1, 2019

Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon.

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The…

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July 1, 2019

Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi.

The fast-growing Gram-negative bacterium Vibrio natriegens is an attractive microbial system for molecular biology and biotechnology due to its remarkably short generation time1,2 and metabolic prowess3,4. However, efforts to uncover and utilize the mechanisms underlying its rapid growth are hampered by the scarcity of functional genomic data. Here, we develop a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) screen to identify a minimal set of genes required for rapid wild-type growth. Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered core genes comprising putative essential and growth-supporting genes that are enriched for respiratory pathways. We…

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June 3, 2019

Application Brochure: Explore metagenomes in high resolution

The ability to identify and understand the functions of the complex microbial populations living in, on, and around us requires comprehensive characterization of each community member. Highly accurate long reads– HiFi reads–with single-molecule resolution make Single Molecule, Real-Time (SMRT) Sequencing ideal for full-length 16S rRNA sequencing, long-read metagenomic profiling, and shotgun metagenomic assembly.

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June 1, 2019

Towards PacBio-based pan-eukaryote metabarcoding using full-length ITS sequences.

Development of high-throughput sequencing techniques have greatly benefited our understanding about microbial ecology; yet the methods producing short reads suffer from species-level resolution and uncertainty of identification. Here we optimize PacBio-based metabarcoding protocols covering the Internal Transcribed Spacer (ITS region) and partial Small Subunit (SSU) of the rRNA gene for species-level identification of all eukaryotes, with a specific focus on Fungi (including Glomeromycota) and Stramenopila (particularly Oomycota). Based on tests on composite soil samples and mock communities, we propose best suitable degenerate primers, ITS9munngs + ITS4ngsUni for eukaryotes and selected groups therein and discuss pros and cons of long read-based…

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June 1, 2019

The complete genome sequence of Thalassospira indica PB8BT insights into adaptation to the marine environment

Thalassospira indica PB8BT was isolated from the deep water of the Indian Ocean. Here we report the complete genome sequence of type strain PB8BT, which comprises 4,701,725?bp with a G?+?C content of 54.9?mol%. We found that numerous genes related to iron acquisition, resistance, motility and chemotaxis, nitrogen, phosphorus and sulfur metabolism, and stress response. These metabolic features and related genes revealed genetic basis for the adaptation to the marine environment. The genome of T. indica PB8BT will be helpful for further insights into its adaptive evolution and ecological role in marine environment.

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June 1, 2019

Cobaviruses – a new globally distributed phage group infecting Rhodobacteraceae in marine ecosystems.

Bacteriophages are widely considered to influence bacterial communities, however most phages are still unknown or not studied well enough to understand their ecological roles. We have isolated two phages infecting Lentibacter sp. SH36, affiliated with the marine Roseobacter group, and retrieved similar phage genomes from publicly available metagenomics databases. Phylogenetic analysis placed the new phages within the Cobavirus group, in the here newly proposed genus Siovirus and subfamily Riovirinae of the Podoviridae. Gene composition and presence of direct terminal repeats in cultivated cobaviruses point toward a genome replication and packaging strategy similar to the T7 phage. Investigation of the genomes…

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May 20, 2019

Case Study: Sequencing an historic bacterial collection for the future

The UK’s National Collection of Type Cultures (NCTC) is a unique collection of more than 5,000 expertly preserved and authenticated bacterial cultures, many of historical significance. Founded in 1920, NCTC is the longest established collection of its type anywhere in the world, with a history of its own that has reflected — and contributed to — the evolution of microbiology for more than 100 years.

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May 1, 2019

Genome Sequence and Methylation Patterns of Halorubrum sp. Strain BOL3-1, the First Haloarchaeon Isolated and Cultured from Salar de Uyuni, Bolivia.

Halorubrum sp. strain BOL3-1 was isolated from Salar de Uyuni, Bolivia, and sequenced using single-molecule real-time sequencing. Its 3.7-Mbp genome was analyzed for gene content and methylation patterns and incorporated into the Haloarchaeal Genomes Database (http://halo.umbc.edu). The polyextremophilic character and high-elevation environment make the microbe of interest for astrobiology. Copyright © 2019 DasSarma et al.

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April 25, 2019

Webinar: Long-read sequencing and infectious disease: New insights into longstanding challenges

One of the longstanding challenges in infectious disease has been the lack of high-quality reference genomes. However, developments in genome sequencing are helping researchers overcome this barrier. Recently, highly contiguous genome assemblies of Plasmodium falciparum, Aedes aegypti, and multiple trypanosomes have become available. The number of reference genomes for bacteria that cause infectious disease is similarly expanding rapidly. In this webinar Meredith Ashby discusses how these new resources are already yielding new biological insights into critical questions in infectious disease research, including how parasites evade the immune system add how pathogens are adapting to evolutionary pressures.

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April 1, 2019

A New Species of the ?-Proteobacterium Francisella, F. adeliensis Sp. Nov., Endocytobiont in an Antarctic Marine Ciliate and Potential Evolutionary Forerunner of Pathogenic Species.

The study of the draft genome of an Antarctic marine ciliate, Euplotes petzi, revealed foreign sequences of bacterial origin belonging to the ?-proteobacterium Francisella that includes pathogenic and environmental species. TEM and FISH analyses confirmed the presence of a Francisella endocytobiont in E. petzi. This endocytobiont was isolated and found to be a new species, named F. adeliensis sp. nov.. F. adeliensis grows well at wide ranges of temperature, salinity, and carbon dioxide concentrations implying that it may colonize new organisms living in deeply diversified habitats. The F. adeliensis genome includes the igl and pdp gene sets (pdpC and pdpE…

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April 1, 2019

Medusavirus, a Novel Large DNA Virus Discovered from Hot Spring Water.

Recent discoveries of new large DNA viruses reveal high diversity in their morphologies, genetic repertoires, and replication strategies. Here, we report the novel features of medusavirus, a large DNA virus newly isolated from hot spring water in Japan. Medusavirus, with a diameter of 260?nm, shows a T=277 icosahedral capsid with unique spherical-headed spikes on its surface. It has a 381-kb genome encoding 461 putative proteins, 86 of which have their closest homologs in Acanthamoeba, whereas 279 (61%) are orphan genes. The virus lacks the genes encoding DNA topoisomerase II and RNA polymerase, showing that DNA replication takes place in the…

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April 1, 2019

Light Modulates the Physiology of Nonphototrophic Actinobacteria.

Light is a source of energy and an environmental cue that is available in excess in most surface environments. In prokaryotic systems, conversion of light to energy by photoautotrophs and photoheterotrophs is well understood, but the conversion of light to information and the cellular response to that information have been characterized in only a few species. Our goal was to explore the response of freshwater Actinobacteria, which are ubiquitous in illuminated aquatic environments, to light. We found that Actinobacteria without functional photosystems grow faster in the light, likely because sugar transport and metabolism are upregulated in the light. Based on…

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March 1, 2019

A unique methylation pattern by a type I HsdM methyltransferase prepares for DpnI rare cutting sites in the Pseudomonas aeruginosa PAO1 genome.

A physical genome map of Pseudomonas aeruginosa PAO1 had been constructed in the late 1980s by combinatorial SpeI/DpnI long-range restriction mapping. Whereas SpeI was known to cleave within the infrequent 5'-ACTAGT hexanucleotide, the rare recognition site 5'-Gm6ATC for the methylation-sensitive 4-cutter DpnI in the PAO1 genome remained elusive. Resequencing the PAO1 genome with the methylation-sensitive Single Molecule Real-Time (SMRT) sequencing technology identified DpnI to cleave the PAO1 genome at 15 palindromic GAC(N)6Gm6ATC(N)6GTC recognition sites with the central Gm6ATC palindrome to be methylated on both strands by the clone PAO1-specific type I HsdM superfamily methyltransferase M.PaePAOI (PA2735).© FEMS 2019.

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