The genome of Anopheles darlingi, the main neotropical malaria vector.

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A.

Sequencing viral genomes from a single isolated plaque.

Background: Whole genome sequencing of viruses and bacteriophages is often hindered because of the need for large quantities of genomic material. A method is described that combines single plaque sequencing with an optimization of Sequence Independent Single Primer Amplification (SISPA). This method can be used for de novo whole genome next-generation sequencing of any cultivable virus without the need for large-scale production of viral stocks or viral purification using centrifugal techniques.

Isolation and genome analysis of single virions using 'single virus genomics'.

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation (1-3). Viruses, which are ubiquitous and the most numerous entities on our planet (4) and important in all environments (5), have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG).

The two-component response regulator LiaR regulates cell wall stress responses, pili expression and virulence in group B Streptococcus.

Group B Streptococcus (GBS) remains the leading cause of early onset sepsis among term infants. Evasion of innate immune defences is critical to neonatal GBS disease pathogenesis. Effectors of innate immunity, as well as numerous antibiotics, frequently target the peptidoglycan layer of the Gram-positive bacterial cell wall.

Sequencing of mitochondrial genomes of nine Aspergillus and Penicillium species identifies mobile introns and accessory genes as main sources of genome size variability.

Background: The genera Aspergillus and Penicillium include some of the most beneficial as well as the most harmful fungal species such as the penicillin-producer Penicillium chrysogenum and the human pathogen Aspergillus fumigatus, respectively. Their mitochondrial genomic sequences may hold vital clues into the mechanisms of their evolution, population genetics, and biology, yet only a handful of these genomes have been fully sequenced and annotated.

Results: Here we report the complete sequence and annotation of the mitochondrial genomes of six Aspergillus and three Penicillium species: A.

A Type III restriction-modification system in Mycoplasma mycoides subsp. capri.

The sequenced genome of Mycoplasma mycoides subsp. capri revealed the presence of a Type III restriction-modification system (MmyCI). The methyltransferase (modification) subunit of MmyCI (M.

Next generation sequencing to define prokaryotic and fungal diversity in the bovine rumen.

A combination of Sanger and 454 sequences of small subunit rRNA loci were used to interrogate microbial diversity in the bovine rumen of 12 cows consuming a forage diet. Observed bacterial species richness, based on the V1-V3 region of the 16S rRNA gene, was between 1,903 to 2,432 species-level operational taxonomic units (OTUs) when 5,520 reads were sampled per animal. Eighty percent of species-level OTUs were dominated by members of the order Clostridiales, Bacteroidales, Erysipelotrichales and unclassified TM7.

The role of synthetic biology for in situ resource utilization (ISRU).

A persistent presence in space can either be supported from Earth or generate the required resources for human survival from material already present in space, so called "in situ material." Likely, many of these resources such as water or oxygen can best be liberated from in situ material by conventional physical and chemical processes. However, there is one critical resource required for human life that can only be produced in quantity by biological processes: high-protein food.

Bioinformatics approaches and software for detection of secondary metabolic gene clusters.

The accelerating pace of microbial genomics is sparking a renaissance in the field of natural products research. Researchers can now get a preview of the organism's secondary metabolome by analyzing its genomic sequence. Combined with other -omics data, this approach may provide a cost-effective alternative to industrial high-throughput screening in drug discovery.

Predicting the functional effect of amino acid substitutions and indels.

As next-generation sequencing projects generate massive genome-wide sequence variation data, bioinformatics tools are being developed to provide computational predictions on the functional effects of sequence variations and narrow down the search of casual variants for disease phenotypes. Different classes of sequence variations at the nucleotide level are involved in human diseases, including substitutions, insertions, deletions, frameshifts, and non-sense mutations. Frameshifts and non-sense mutations are likely to cause a negative effect on protein function.

A case study for large-scale human microbiome analysis using JCVI's metagenomics reports (METAREP).

As metagenomic studies continue to increase in their number, sequence volume and complexity, the scalability of biological analysis frameworks has become a rate-limiting factor to meaningful data interpretation. To address this issue, we have developed JCVI Metagenomics Reports (METAREP) as an open source tool to query, browse, and compare extremely large volumes of metagenomic annotations. Here we present improvements to this software including the implementation of a dynamic weighting of taxonomic and functional annotation, support for distributed searches, advanced clustering routines, and integration of additional annotation input formats.

Development stage-specific proteomic profiling uncovers small, lineage specific proteins most abundant in the Aspergillus Fumigatus conidial proteome.

Background: The pathogenic mold Aspergillus fumigatus is the most frequent infectious cause of death in severely immunocompromised individuals such as leukemia and bone marrow transplant patients. Germination of inhaled conidia (asexual spores) in the host is critical for the initiation of infection, but little is known about the underlying mechanisms of this process.

Results: To gain insights into early germination events and facilitate the identification of potential stage-specific biomarkers and vaccine candidates, we have used quantitative shotgun proteomics to elucidate patterns of protein abundance changes during early fungal development.

Identifying low pH active and lactate-utilizing taxa within oral microbiome communities from healthy children using stable isotope probing techniques.

Background: Many human microbial infectious diseases including dental caries are polymicrobial in nature. How these complex multi-species communities evolve from a healthy to a diseased state is not well understood. Although many health- or disease-associated oral bacteria have been characterized in vitro, their physiology within the complex oral microbiome is difficult to determine with current approaches.

Dental caries pathogenicity: a genomic and metagenomic perspective.

In this review we address the subject of dental caries pathogenicity from a genomic and metagenomic perspective. The application of genomic technologies is certain to yield novel insights into the relationship between the bacterial flora, dental health and disease. Three primary attributes of bacterial species are thought to have direct impact on caries development, these include: adherence on tooth surfaces (biofilm formation), acid production and acid tolerance.

Separation of single-stranded DNA, double-stranded DNA and RNA from an environmental viral community using hydroxyapatite chromatography.

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments.

Genetic analysis of the Alteromonas macleodii [NiFe]-hydrogenase.

Alteromonas macleodii Deep ecotype is a marine, heterotrophic, gammaproteobacterium isolated in the Mediterranean Sea between depths of 1000 and 3500 m. The sequenced strain was previously reported to contain a [NiFe] hydrogenase. We verified the presence of this hydrogenase in other strains of A.

Comparative genomic analysis of Vibrio parahaemolyticus: serotype conversion and virulence.

Background: Vibrio parahaemolyticus is a common cause of foodborne disease. Beginning in 1996, a more virulent strain having serotype O3:K6 caused major outbreaks in India and other parts of the world, resulting in the emergence of a pandemic. Other serovariants of this strain emerged during its dissemination and together with the original O3:K6 were termed strains of the pandemic clone.

Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Synechococcus elongatus.

Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG.

Sim4db and Leaff: utilities for fast batch spliced alignment and sequence indexing.

Unlabelled: The large number of genomes that will be sequenced will need to be annotated with genes and other functional features. Aligning gene sequences from a related species to the target genome is an economical and highly reliable method to identify genes; unfortunately, existing tools have been lacking in sensitivity and speed. A program we reported, sim4cc, was shown to be highly accurate but is limited to comparing one cDNA with one genomic sequence.

Gene and genome construction in yeast.

The yeast Saccharomyces cerevisiae has the capacity to take up and assemble dozens of different overlapping DNA molecules in one transformation event. These DNA molecules can be single-stranded oligonucleotides, to produce gene-sized fragments, or double-stranded DNA fragments, to produce molecules up to hundreds of kilobases in length, including complete bacterial genomes. This unit presents protocols for designing the DNA molecules to be assembled, transforming them into yeast, and confirming their assembly.

Investigating the genome diversity of B. cereus and evolutionary aspects of B. anthracis emergence.

Here we report the use of a multi-genome DNA microarray to investigate the genome diversity of Bacillus cereus group members and elucidate the events associated with the emergence of Bacillus anthracis the causative agent of anthrax-a lethal zoonotic disease. We initially performed directed genome sequencing of seven diverse B. cereus strains to identify novel sequences encoded in those genomes.

Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Escherichia coli.

HynSL from Alteromonas macleodii 'deep ecotype' (AltDE) is an oxygen-tolerant and thermostable [NiFe] hydrogenase. Its two structural genes (hynSL), encoding small and large hydrogenase subunits, are surrounded by eight genes (hynD, hupH and hypCABDFE) predicted to encode accessory proteins involved in maturation of the hydrogenase. A 13 kb fragment containing the ten structural and accessory genes along with three additional adjacent genes (orf2, cyt and orf1) was cloned into an IPTG-inducible expression vector and transferred into an Escherichia coli mutant strain lacking its native hydrogenases.

Molecular characterization of a new species in the genus Alphacoronavirus associated with mink epizootic catarrhal gastroenteritis.

A coronavirus (CoV) previously shown to be associated with catarrhal gastroenteritis in mink (Mustela vison) was identified by electron microscopy in mink faeces from two fur farms in Wisconsin and Minnesota in 1998. A pan-coronavirus and a genus-specific RT-PCR assay were used initially to demonstrate that the newly discovered mink CoVs (MCoVs) were members of the genus Alphacoronavirus. Subsequently, using a random RT-PCR approach, full-genomic sequences were generated that further confirmed that, phylogenetically, the MCoVs belonged to the genus Alphacoronavirus, with closest relatedness to the recently identified but only partially sequenced (fragments of the polymerase, and full-length spike, 3c, envelope, nucleoprotein, membrane, 3x and 7b genes) ferret enteric coronavirus (FRECV) and ferret systemic coronavirus (FRSCV).

Isolation of circular yeast artificial chromosomes for synthetic biology and functional genomics studies.

Circular yeast artificial chromosomes (YACs) provide significant advantages for cloning and manipulating large segments of genomic DNA in Saccharomyces cerevisiae. However, it has been difficult to exploit these advantages, because circular YACs are difficult to isolate and purify. Here we describe a method for purification of large circular YACs that is more reliable compared with previously described protocols.

Chemical synthesis of the mouse mitochondrial genome.

We describe a one-step, isothermal assembly method for synthesizing DNA molecules from overlapping oligonucleotides. The method cycles between in vitro recombination and amplification until the desired length is reached. As a demonstration of its simplicity and robustness, we synthesized the entire 16.