PCR and real-time PCR primers developed for detection and identification of <Emphasis Type="Italic">Bifidobacterium thermophilum</Emphasis>in faeces

Background  

Culture-independent methods based on the 16S ribosomal RNA molecule are nowadays widely used for assessment of the composition of the intestinal microbiota, in relation to host health or probiotic efficacy. Because Bifidobacterium thermophilum was only recently isolated from human faeces until now, no specific real-time PCR (qPCR) assay has been developed for detection of this species as component of the bifidobacterial community of the human intestinal flora.     

Developments toward large-scale bacterial bioprocesses in the presence of bulk amounts of organic solvents

Many pseudomonads and other bacteria can grow on aliphatic and aromatic hydrocarbons that occur in the environment. We are examining the potential of such organisms as biocatalysts for the oxidation of a variety of substituted aliphatic and aromatic compounds. To attain a high production rate of oxidation products via such biotransformations, we have focused on two-liquid phase culture systems. In these systems, cells are grown in liquid media consisting of an aqueous phase containing water-soluble growth substrates and droplets of a water-immiscible organic solvent containing bioconversion substrates and products. For industrial applications of such two-liquid phase processes, several questions remain. What are the maximum rates at which apolar compounds can be transferred from the apolar phase to cells growing in the aqueous phase, i.e., what are the maximum space-time yields attainable in two-liquid phase fermentations under practical conditions? What does an efficient downstream processing of two-liquid phase medium involve? What safety regimes should be considered in working with flammable organic solvents? Can elevated pressure be used to increase oxygen transfer? Based on answers to these questions, we have recently developed a high-pressure, explosion-proof bioreactor system with Bioengineering AG (Wald, Switzerland), which will be installed in our pilot plant and used to explore two-liquid phase bioconversions at a pilot scale.     

Abscisic acid and proline improve desiccation tolerance and increase fatty acid content of celery somatic embryos

Desiccation tolerance of celery (Apium graveolens L.) somatic embryos was increased by supplementation of embryo-production medium with 1 M abscisic acid (ABA) or 1 mM proline, with highest survival obtained with a combination of 1 M ABA and 1 mM proline. Addition of ABA and proline increased fatty acid accumulation by somatic embryos; the effect on fatty acid composition was inconsistent. Somatic embryos capable of germination differed from mature zygotic embryos by greater size, lower fatty acid level, and substantially lower proportion of oleic acid (18:1) as compared to linoleic acid (18:2).     

M?ssbauer spectroscopic studies of Escherichia coli sulfite reductase. Evidence for coupling between the siroheme and Fe4S4 cluster prosthetic groups

Escherichia coli NADPH-sulfite reductase is a complex hemoflavoprotein with an alpha 8 beta 4 subunit structure. The beta-subunits each contain one siroheme and a tetranuclear iron-sulfur center (Fe4S4). Isolated beta-monomers can catalyze the 6-electron reduction of sulfite to sulfide. We have studied the beta-monomers with M?ssbauer and EPR spectroscopy. The data show conclusively that the siroheme and the Fe4S4 cluster are strongly exchange-coupled. This is proven by the observations that (a) the two chromophores share a single electronic spin and (b) the addition of 1 electron to oxidized sulfite reductase changes the environments of 5 iron atoms. Spin-sharing is demonstrated in oxidized and 2-electron-reduced sulfite reductase and strongly implicated in 1-electron-reduced material. Thus, sulfite reductase provides the first example of an active site where a heme and an iron-sulfur cluster are closely linked as a functional unit, probably via a common bridging ligand.     

High-performance liquid chromatographic assays for bufuralol 1'-hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan O-demethylase in microsomes and purified cytochrome P-450 isozymes of human liver

Bufuralol, debrisoquine, and dextromethorphan are three prototype substrates of the common genetic deficiency of oxidative drug metabolism in man known as debrisoquine/sparteine-type polymorphism. We describe assays for the in vitro metabolism of (+)- and (-)-bufuralol, debrisoquine, and dextromethorphan in human liver microsomes and reconstituted purified cytochrome P-450 isozymes. These assays combine nonextractive sample preparation by precipitation of protein with perchloric acid with reversed-phase inorganic ion-pair HPLC and fluorescence detection. The minimal detectable levels of the major metabolites formed are 1'-hydroxybufuralol, 0.1 ng/ml; 4-hydroxydebrisoquine, 0.8 ng/ml; and dextrorphan, 0.1 ng/ml. Formation of these metabolites is linear for at least 45 min and between 1 and 100 micrograms of microsomal protein. Comparative kinetic analysis of the three monooxygenase reactions in human liver microsomes revealed an apparent biphasicity of (+)- and (-)-bufuralol 1'-hydroxylation and dextromethorphan O-demethylation but monophasic formation of 4-hydroxydebrisoquine in the substrate concentration range (less than 1 mM) studied. These data, in combination with those obtained by purified human cytochrome P-450 isozymes indicate the involvement of the same enzyme in the metabolism of all three substrates investigated. However, additional and distinct activities contribute to the metabolism of (+)- and (-)-bufuralol and dextromethorphan.     

Annotation of the pRhico plasmid of Azospirillum brasilense reveals its role in determining the outer surface composition

The plant growth-promoting soil bacterium Azospirillum brasilense enhances growth of economically important crops, such as wheat, corn and rice. In order to improve plant growth, a close bacterial association with the plant roots is needed. Genes encoded on a 90-MDa plasmid, denoted pRhico plasmid, present in A. brasilense Sp7, play an important role in plant root interaction. Sequencing, annotation and in silico analysis of this 90-MDa plasmid revealed the presence of a large collection of genes encoding enzymes involved in surface polysaccharide biosynthesis. Analysis of the 90-MDa plasmid genome provided evidence for its essential role in the viability of the bacterial cell.