Characterising the Canine Oral Microbiome by Direct Sequencing of Reverse-Transcribed rRNA Molecules.

PCR amplification and sequencing of phylogenetic markers, primarily Small Sub-Unit ribosomal RNA (SSU rRNA) genes, has been the paradigm for defining the taxonomic composition of microbiomes. However, 'universal' SSU rRNA gene PCR primer sets are likely to miss much of the diversity therein. We sequenced a library comprising purified and reverse-transcribed SSU rRNA (RT-SSU rRNA) molecules from the canine oral microbiome and compared it to a general bacterial 16S rRNA gene PCR amplicon library generated from the same biological sample.

Development and application of a method for the purification of free shigatoxigenic bacteriophage from environmental samples.

Shiga toxin producing Escherichia coli (STEC) strains are foodborne pathogens whose ability to produce Shiga toxin (Stx) is due to the integration of Stx-encoding lambdoid bacteriophage (Stx phage). Circulating, infective Stx phages are very difficult to isolate, purify and propagate such that there is no information on their genetic composition and properties. Here we describe a novel approach that exploits the phage's ability to infect their host and form a lysogen, thus enabling purification of Stx phages by a series of sequential lysogen isolation and induction steps.

Comparative genomics of Shiga toxin encoding bacteriophages.

Background: Stx bacteriophages are responsible for driving the dissemination of Stx toxin genes (stx) across their bacterial host range. Lysogens carrying Stx phages can cause severe, life-threatening disease and Stx toxin is an integral virulence factor. The Stx-bacteriophage vB_EcoP-24B, commonly referred to as Ф24B, is capable of multiply infecting a single bacterial host cell at a high frequency, with secondary infection increasing the rate at which subsequent bacteriophage infections can occur.

Metagenomic approaches to the discovery of cellulases.

Most of the microorganisms responsible for nutrient cycling in the environment have yet to be cultivated, and this could include those species responsible for the degradation of cellulose. Known cellulases are well defined at the protein sequence level, but gene variants are difficult to amplify from environmental DNA. The identification of novel cellulase genes independent of DNA amplification is made possible by adopting a direct metagenome sequencing approach to provide genes that can be cloned, expressed, and characterized prior to potential exploitation, all in the absence of any information on the species from which they originated.

Methods for the isolation of cellulose-degrading microorganisms.

The biodegradation of lignocellulose, the most abundant organic material in the biosphere, is a feature of many aerobic, facultatively anaerobic and obligately anaerobic bacteria and fungi. Despite widely recognized difficulties in the isolation and cultivation of individual microbial species from complex microbial populations and environments, significant progress has been made in recovering cellulolytic taxa from a range of ecological niches including the human, herbivore, and termite gut, and terrestrial, aquatic, and managed environments. Knowledge of cellulose-degrading microbial taxa is of significant importance with respect to nutrition, biodegradation, biotechnology, and the carbon-cycle, providing insights into the metabolism, physiology, and functional enzyme systems of the cellulolytic bacteria and fungi that are responsible for the largest flow of carbon in the biosphere.

The microbial ecology of anaerobic cellulose degradation in municipal waste landfill sites: evidence of a role for fibrobacters.

Cellulose is reputedly the most abundant organic polymer in the biosphere, yet despite the fundamental role of cellulolytic microorganisms in global carbon cycling and as potential sources of novel enzymes for biotechnology, their identity and ecology is not well established. Cellulose is a major component of landfill waste and its degradation is therefore a key feature of the anaerobic microbial decomposition process. Here, we targeted a number of taxa containing known cellulolytic anaerobes (members of the bacterial genus Fibrobacter, lineages of Clostridium clusters I, III, IV and XIV, and anaerobic fungi of the Neocallimastigales) in landfill leachate and colonized cellulose 'baits' via PCR and quantitative PCR (qPCR).

Development and validation of a qPCR-based method for quantifying Shiga toxin-encoding and other lambdoid bacteriophages.

To address whether seasonal variability exists among Shiga toxin-encoding bacteriophage (Stx phage) numbers on a cattle farm, conventional plaque assay was performed on water samples collected over a 17 month period. Distinct seasonal variation in bacteriophage numbers was evident, peaking between June and August. Removal of cattle from the pasture precipitated a reduction in bacteriophage numbers, and during the winter months, no bacteriophage infecting Escherichia coli were detected, a surprising occurrence considering that 10(31) tailed-bacteriophages are estimated to populate the globe.

454-pyrosequencing: a molecular battiscope for freshwater viral ecology.

Viruses, the most abundant biological entities on the planet, are capable of infecting organisms from all three branches of life, although the majority infect bacteria where the greatest degree of cellular diversity lies. However, the characterization and assessment of viral diversity in natural environments is only beginning to become a possibility. Through the development of a novel technique for the harvest of viral DNA and the application of 454 pyrosequencing, a snapshot of the diversity of the DNA viruses harvested from a standing pond on a cattle farm has been obtained.