The effect of wheat genotype on the microbiome is more evident in roots and varies through time.

Crop breeding has traditionally ignored the plant-associated microbial communities. Considering the interactions between plant genotype and associated microbiota is of value since different genotypes of the same crop often harbor distinct microbial communities which can influence the plant phenotype. However, recent studies have reported contrasting results, which led us to hypothesize that the effect of genotype is constrained by growth stages, sampling year and plant compartment.

Rhizosphere shotgun metagenomic analyses fail to show differences between ancestral and modern wheat genotypes grown under low fertilizer inputs.

It is thought that modern wheat genotypes have lost their capacity to associate with soil microbes that would help them acquire nutrients from the soil. To test this hypothesis, ten ancestral and modern wheat genotypes were seeded in a field experiment under low fertilization conditions. The rhizosphere soil was collected, its DNA extracted and submitted to shotgun metagenomic sequencing.

CaptureSeq: Hybridization-Based Enrichment of Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems.

Background: The molecular profiling of complex microbial communities has become the basis for examining the relationship between the microbiome composition, structure and metabolic functions of those communities. Microbial community structure can be partially assessed with "universal" PCR targeting taxonomic or functional gene markers. Increasingly, shotgun metagenomic DNA sequencing is providing more quantitative insight into microbiomes.

The Role of Localized Acidity Generation in Microbially Influenced Corrosion.

Microbially influenced corrosion is of great industrial concern. Microbial coupling of metal oxidation to sulfate-, nitrate-, nitrite-, or CO-reduction is proton-mediated, and some sulfate-reducing prokaryotes are capable of regulating extracellular pH. The analysis of the corrosive processes catalyzed by nitrate reducing bacteria and methanogenic archaea indicates that these microorganisms may be capable of regulating extracellular pH as well.

Quantitative molecular diagnostic assays of grain washes for Claviceps purpurea are correlated with visual determinations of ergot contamination.

We examined the epiphytic microbiome of cereal grain using the universal barcode chaperonin-60 (cpn60). Microbial community profiling of seed washes containing DNA extracts prepared from field-grown cereal grain detected sequences from a fungus identified only to Class Sordariomycetes. To identify the fungal sequence and to improve the reference database, we determined cpn60 sequences from field-collected and reference strains of the ergot fungus, Claviceps purpurea.

Enrichment and identification of biosurfactant-producing oil field microbiota utilizing electron acceptors other than oxygen and nitrate.

Microorganisms indigenous to an oil reservoir were grown in media containing either sucrose or proteins in four steel vessels under anoxic conditions at 30°C and 8.3MPa for 30days, to enrich biosurfactant producers. Fermentation of substrate was possible in the protein-containing medium and either fermentation or respiration through reduction of sulfate occurred in the sucrose-containing medium.

A Study of the Vaginal Microbiome in Healthy Canadian Women Utilizing cpn60-Based Molecular Profiling Reveals Distinct Gardnerella Subgroup Community State Types.

The vaginal microbiota is important in women's reproductive and overall health. However, the relationships between the structure, function and dynamics of this complex microbial community and health outcomes remain elusive. The objective of this study was to determine the phylogenetic range and abundance of prokaryotes in the vaginal microbiota of healthy, non-pregnant, ethnically diverse, reproductive-aged Canadian women.

Characterization of the vaginal microbiota of healthy Canadian women through the menstrual cycle.

Background: The vaginal microbial community plays a vital role in maintaining women's health. Understanding the precise bacterial composition is challenging because of the diverse and difficult-to-culture nature of many bacterial constituents, necessitating culture-independent methodology. During a natural menstrual cycle, physiological changes could have an impact on bacterial growth, colonization, and community structure.

mPUMA: a computational approach to microbiota analysis by de novo assembly of operational taxonomic units based on protein-coding barcode sequences.

Background: Formation of operational taxonomic units (OTU) is a common approach to data aggregation in microbial ecology studies based on amplification and sequencing of individual gene targets. The de novo assembly of OTU sequences has been recently demonstrated as an alternative to widely used clustering methods, providing robust information from experimental data alone, without any reliance on an external reference database.

Results: Here we introduce mPUMA (microbial Profiling Using Metagenomic Assembly, http://mpuma.

Simultaneous profiling of seed-associated bacteria and fungi reveals antagonistic interactions between microorganisms within a shared epiphytic microbiome on Triticum and Brassica seeds.

In order to address the hypothesis that seeds from ecologically and geographically diverse plants harbor characteristic epiphytic microbiota, we characterized the bacterial and fungal microbiota associated with Triticum and Brassica seed surfaces. The total microbial complement was determined by amplification and sequencing of a fragment of chaperonin 60 (cpn60). Specific microorganisms were quantified by qPCR.

The chaperonin-60 universal target is a barcode for bacteria that enables de novo assembly of metagenomic sequence data.

Barcoding with molecular sequences is widely used to catalogue eukaryotic biodiversity. Studies investigating the community dynamics of microbes have relied heavily on gene-centric metagenomic profiling using two genes (16S rRNA and cpn60) to identify and track Bacteria. While there have been criteria formalized for barcoding of eukaryotes, these criteria have not been used to evaluate gene targets for other domains of life.

Multiplex detection of bacteria in complex clinical and environmental samples using oligonucleotide-coupled fluorescent microspheres.

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others. This condition is associated with a range of negative health outcomes, including HIV acquisition, and it can be difficult to manage clinically. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria.

Pyrosequencing of chaperonin-60 (cpn60) amplicons as a means of determining microbial community composition.

The chaperonin-60 universal target (cpn60 UT) is generated from a set of PCR primers and provides a universally conserved, phylogenetically informative sequence signature for determining the composition of microbial communities by DNA sequencing. Pyrosequencing of cpn60 UT amplicons is emerging as a next-generation tool for providing unprecedented sequencing depth and resolution of microbial communities in individual samples. Owing to the increase in sequencing depth, the dynamic range across which the presence and abundance of individual species can be sampled experimentally also increases, significantly improving our ability to investigate microbial community richness and diversity.

Novel recombinant monoclonal antibodies for vitellogenin assays in cyprinid fish species.

Various polyclonal and monoclonal antibodies have been developed for vitellogenin (Vtg) bioassays in different aquatic species. Preparation of these reagents is time-consuming and expensive. In the present study, a phage-displayed, recombinant, single-chain variable fragment (scFv) format antibody library was constructed using splenic mRNA from non-immunized mice.

Essential role for Schizosaccharomyces pombe pik1 in septation.

Background: Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to bind Cdc4, but not Cdc4(G107S).

Principal Findings: Gene deletion revealed that pik1 is essential. In cells with pik1 deleted, ectopic expression of a loss-of-function allele, created by fusion to a temperature-sensitive dihydrofolate reductase, allowed normal cell proliferation at 25 degrees C.

A new fluorescent quantitative PCR-based in vitro neutralization assay for white spot syndrome virus.

A fluorescent quantitative PCR (FQ-PCR) assay utilizing SYBR green I dye is described for quantitation of white spot syndrome virus (WSSV) particles isolated from infected crayfish, Cambarus clarkii. For this assay, a primer set was designed which amplifies, with high efficiency and specificity, a 129bp target sequence within ORF167 of the WSSV genome. Conveniently, WSSV particles can be added into the FQ-PCR assay with a simple and convenient method to release its DNA.

Identification of a WSSV neutralizing scFv antibody by phage display technology and in vitro screening.

White spot syndrome virus (WSSV) is one of the most significant viral pathogens causing high mortality and economic damage in shrimp aquaculture. Although intensive efforts were undertaken to detect and characterize WSSV infection in shrimp during the last decade, we still lack methods either to prevent or cure white spot disease. Most of the studies on neutralizing antibodies from sera have been performed using in vivo assays.

Molecular characterization of microbial communities in Canadian pulp and paper activated sludge and quantification of a novel Thiothrix eikelboomii-like bulking filament.

We examined the microbial community structure and quantified the levels of the filamentous bulking organism Thiothrix eikelboomii in samples of activated sludge mixed liquor suspended solids (MLSS) from Canadian pulp and paper mills. Libraries of chaperonin 60 (cpn60) gene sequences were prepared from MLSS total microbial community DNA and each was compared with cpnDB, a reference database of cpn60 sequences (http://cpndb.cbr.

Biochemical and taxonomic characterization of bacteria associated with the crucifer root maggot (Delia radicum).

The crucifer root maggot, Delia radicum, is an important pest of cruciferous crops; however, little is known about its digestive biochemistry or resident gut microbiota. A culturing approach was used to survey the types of micro organisms associated with eggs, midgut, and faeces of larvae feeding on rutabaga. All bacteria isolated from the midgut and faecal materials were Gram-negative bacilli.

Characterization of intestinal microbiota and response to dietary virginiamycin supplementation in the broiler chicken.

The inclusion of antibiotic growth promoters, such as virginiamycin, at subtherapeutic levels in poultry feeds has a positive effect on health and growth characteristics, possibly due to beneficial effects on the host gastrointestinal microbiota. To improve our understanding of the chicken gastrointestinal microbiota and the effect of virginiamycin on its composition, we characterized the bacteria found in five different gastrointestinal tract locations (duodenal loop, mid-jejunum, proximal ileum, ileocecal junction, and cecum) in 47-day-old chickens that were fed diets excluding or including virginiamycin throughout the production cycle. Ten libraries (five gastrointestinal tract locations from two groups of birds) of approximately 555-bp chaperonin 60 PCR products were prepared, and 10,932 cloned sequences were analyzed.

Improved template representation in cpn60 polymerase chain reaction (PCR) product libraries generated from complex templates by application of a specific mixture of PCR primers.

Some classes of high G+C content organisms such as the Actinobacteria, which are known through culture-based studies to be present in large numbers in particular microbial communities, are under-represented or even absent from 16S rRNA or cpn60 polymerase chain reaction (PCR) product libraries derived from these templates. Using reference cpn60 sequence data from organisms with high G+C content genomes, a pair of PCR primers were designed which, when used in combination with the previously developed degenerate, universal cpn60 primers, improve the representation of templates with high G+C content. The primers were validated using a combination of traditional and quantitative real-time PCR on both manufactured template mixtures and biological samples.

Identification of Campylobacter spp. and discrimination from Helicobacter and Arcobacter spp. by direct sequencing of PCR-amplified cpn60 sequences and comparison to cpnDB, a chaperonin reference sequence database.

A robust method for the identification of Campylobacter spp. based on direct sequencing of PCR-amplified partial cpn60 sequences and comparison of these to a reference database of cpn60 sequences is reported. A total of 53 Campylobacter isolates, representing 15 species, were identified and distinguished from phenotypically similar Helicobacter and Arcobacter strains.

Identification of pathogenic Helicobacter species by chaperonin-60 differentiation on plastic DNA arrays.

A microarray method for bacterial species identification based on cpn60 and 16S rDNA hybridization was developed. Specific cpn60 or 16S rDNA oligonucleotides from various Helicobacter or Campylobacter species were printed and immobilized onto a proprietary plastic solid support. Using universal primers, fragments derived from either cpn60 or 16S rDNA genes from single isolates or from a complex human waste sludge DNA sample spiked with Helicobacter pylori were biotinylated and hybridized to the plastic slide.

Characterization of vaginal microflora of healthy, nonpregnant women by chaperonin-60 sequence-based methods.

Objective: The purpose of this study was to use a novel method that was based on the application of chaperonin-60 sequencing to describe the vaginal microflora of 16 healthy women.

Study Design: Asymptomatic women consented for vaginal swabs to be collected at the time of a clinical pelvic examination. Total genomic DNA was isolated from the vaginal swabs.

Dissecting the domain structure of Cdc4p, a myosin essential light chain involved in Schizosaccharomyces pombe cytokinesis.

Cytokinesis is the process by which one cell divides into two. Key in the cytokinetic mechanism of Schizosaccharomyces pombe is the contractile ring myosin, which consists of two heavy chains (Myo2p), two essential light chains (Cdc4p), and two regulatory light chains (Rlc1p). Cdc4p is a dumbbell-shaped EF-hand protein composed of N- and C-terminal domains separated by a flexible linker.