The in situ physiology of "Nostocoida limicola" II, a filamentous bacterial morphotype in bulking activated sludge, using fluorescence in situ hybridization and microautoradiography

The in situ physiology of the actinobacterial bulking and foaming filamentous bacterium "Nostocoida limicola" II was studied by fluorescence in situ hybridization/microautoradiography. Substrate assimilation patterns of pure cultures of this bacterium were different to those seen in activated sludge biomass samples. There was no evidence to suggest that "N. limicola" II preferred hydrophobic substrates, but evidence was produced to support the view that it is metabolically active under anaerobic conditions in activated sludge.

Lupin kernel fiber consumption modifies fecal microbiota in healthy men as determined by rRNA gene flourescent in situ by hybridization

Background Changes in the composition of gastrointestinal microbiota by dietary interventions using pro- and prebiotics provide opportunity for improving health and preventing disease. However, the capacity of lupin kernel fiber (LKFibre), a novel legume-derived food ingredient, to act as a prebiotic and modulate the colonic microbiota in humans needed investigation.

Aim of the study The present study aimed to determine the effect of LKFibre on human intestinal microbiota by quantitative fluorescent in situ hybridization (FISH) analysis.

Design A total of 18 free-living healthy males between the ages of 24 and 64 years consumed a control diet and a LKFibre diet (containing an additional 17–30 g/day fiber beyond that of the control—incorporated into daily food items) for 28 days with a 28-day washout period in a single-blind, randomized, crossover dietary intervention design.
Methods Fecal samples were collected for 3 days towards the end of each diet and microbial populations analyzed by FISH analysis using 16S rRNA gene-based oligonucleotide probes targeting total and predominant microbial populations.

Results Significantly higher levels of Bifidobacterium spp. (P = 0.001) and significantly lower levels of the clostridia group of C. ramosum, C. spiroforme and C. cocleatum (P = 0.039) were observed on the LKFibre diet compared with the control. No significant differences between the LKFibre and the control diet were observed for total bacteria, Lactobacillus spp., the Eubacterium spp., the C. histolyticum/C. lituseburense group and the Bacteroides–Prevotella group.
Conclusions Ingestion of LKFibre stimulated colonic bifidobacteria growth, which suggests that this dietary fiber may be considered as a prebiotic and may beneficially contribute to colon health.

RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae

We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 3′-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 3′-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 3′-end cleavage and polyadenylation, that is, cotranscriptionally.