Alternative hosts for functional (meta)genome analysis

Microorganisms are ubiquitous on earth, often forming complex microbial communities in numerous different habitats. Most of these organisms cannot be readily cultivated in the laboratory using standard media and growth conditions. However, it is possible to gain access to the vast genetic, enzymatic, and metabolic diversity present in these microbial communities using cultivation-independent approaches such as sequence- or function-based metagenomics. Function-based analysis is dependent on heterologous expression of metagenomic libraries in a genetically amenable cloning and expression host. To date, Escherichia coli is used in most cases; however, this has the drawback that many genes from heterologous genomes and complex metagenomes are expressed in E. coli either at very low levels or not at all. This review emphasizes the importance of establishing alternative microbial expression systems consisting of different genera and species as well as customized strains and vectors optimized for heterologous expression of membrane proteins, multigene clusters encoding protein complexes or entire metabolic pathways. The use of alternative host-vector systems will complement current metagenomic screening efforts and expand the yield of novel biocatalysts, metabolic pathways, and useful metabolites to be identified from environmental samples.     

Virtual Screening as a Strategy for the Identification of Xenobiotics Disrupting Corticosteroid Action

Background

Impaired corticosteroid action caused by genetic and environmental influence, including exposure to hazardous xenobiotics, contributes to the development and progression of metabolic diseases, cardiovascular complications and immune disorders. Novel strategies are thus needed for identifying xenobiotics that interfere with corticosteroid homeostasis. 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) and mineralocorticoid receptors (MR) are major regulators of corticosteroid action. 11β-HSD2 converts the active glucocorticoid cortisol to the inactive cortisone and protects MR from activation by glucocorticoids. 11β-HSD2 has also an essential role in the placenta to protect the fetus from high maternal cortisol concentrations.

Methods and Principal Findings

We employed a previously constructed 3D-structural library of chemicals with proven and suspected endocrine disrupting effects for virtual screening using a chemical feature-based 11β-HSD pharmacophore. We tested several in silico predicted chemicals in a 11β-HSD2 bioassay. The identified antibiotic lasalocid and the silane-coupling agent {"type":"entrez-nucleotide","attrs":{"text":"AB110873","term_id":"44885399","term_text":"AB110873"}}AB110873 were found to concentration-dependently inhibit 11β-HSD2. Moreover, the silane {"type":"entrez-nucleotide","attrs":{"text":"AB110873","term_id":"44885399","term_text":"AB110873"}}AB110873 was shown to activate MR and stimulate mitochondrial ROS generation and the production of the proinflammatory cytokine interleukin-6 (IL-6). Finally, we constructed a MR pharmacophore, which successfully identified the silane {"type":"entrez-nucleotide","attrs":{"text":"AB110873","term_id":"44885399","term_text":"AB110873"}}AB110873.

Conclusions

Screening of virtual chemical structure libraries can facilitate the identification of xenobiotics inhibiting 11β-HSD2 and/or activating MR. Lasalocid and {"type":"entrez-nucleotide","attrs":{"text":"AB110873","term_id":"44885399","term_text":"AB110873"}}AB110873 belong to new classes of 11β-HSD2 inhibitors. The silane {"type":"entrez-nucleotide","attrs":{"text":"AB110873","term_id":"44885399","term_text":"AB110873"}}AB110873 represents to the best of our knowledge the first industrial chemical shown to activate MR. Furthermore, the MR pharmacophore can now be used for future screening purposes.     

Base excision repair of 8-oxoG in dinucleosomes

In this work we have studied the effect of chromatin structure on the base excision repair (BER) efficiency of 8-oxoG. As a model system we have used precisely positioned dinucleosomes assembled with linker histone H1. A single 8-oxoG was inserted either in the linker or the core particle DNA within the dinucleosomal template. We found that in the absence of histone H1 the glycosylase OGG1 removed 8-oxoG from the linker DNA and cleaved DNA with identical efficiency as in the naked DNA. In contrast, the presence of histone H1 resulted in close to 10-fold decrease in the efficiency of 8-oxoG initiation of repair in linker DNA independently of linker DNA length. The repair of 8-oxoG in nucleosomal DNA was very highly impeded in both absence and presence of histone H1. Chaperone-induced uptake of H1 restored the efficiency of the glycosylase induced removal of 8-oxoG from linker DNA, but not from the nucleosomal DNA. We show, however, that removal of histone H1 and nucleosome remodelling are both necessary and sufficient for an efficient removal of 8-oxoG in nucleosomal DNA. Finally, a model for BER of 8-oxoG in chromatin templates is suggested.     

Insights into extreme thermoacidophily based on genome analysis of Picrophilus torridus and other thermoacidophilic archaea

Thermoacidophiles are prokaryotic microorganisms with the stunning capability to survive and multiply at extremely low pH and simultaneously at high temperatures. The mechanisms by which these organisms, exclusively members of the Archaea, cope with their harsh surroundings are poorly understood. The genome sequences of several representatives of the thermoacidophilic genera Picrophilus, Thermoplasma and Sulfolobus have recently become available. Genome-wide comparison has revealed a number of features as possible facets of the overall acidophilic survival strategy of the most thermoacidophilic organisms known, such as a high ratio of secondary over primary transport systems, the composition of the respiratory chain, and the frequent genetic input via lateral gene transfer (LGT) during evolution.     

Bacteriocin production by strain <Emphasis Type="Italic">Lactobacillus delbruecki</Emphasis>i ssp. <Emphasis Type="Italic">bulgaricus</Emphasis> BB18 during continuous prefermentation of yogurt starter culture and subsequent batch coagulation of milk

By screening for bacteriocin-producing lactic acid bacteria of 1,428 strains isolated from authentic Bulgarian dairy products, Lb. bulgaricus BB18 strain obtained from kefir grain was selected. Out of 11 yogurt starters containing Lb. bulgaricus BB18 and S. thermophilus strains resistant to bacteriocin secreted by Lb. bulgaricus BB18 a yogurt culture (S. thermophilus 11A+Lb. bulgaricus BB18) with high growth and bacteriocinogenic activity in milk was selected. Continuous (pH-stat 5.7) prefermentation processes were carried out in milk at 37 degrees C in a 2l MBR bioreactor (MBR AG, Zurich, Switzerland) with an IMCS controller for agitation speed, temperature, dissolved oxygen, CO2 and pH. Prefermented milk with pH 5.7 coagulated in a thermostat at 37 degrees C until pH 4.8-4.9. S. thermophilus 11A and Lb. bulgaricus BB18 grew independently in a continuous mode at similar and sufficiently high-dilution rates (D=1.83 h(-1)-S. thermophilus 11A; D=1.80 h(-1)-Lb. bulgaricus BB18). The yogurt cultures developed in a stream at a high-dilution rate (D=2.03-2.28 h(-1)). The progress of both processes (growth and bacteriocin production) depended on the initial ratio between the two microorganisms. The continuous prefermentation process promoted conditions for efficient fermentation and bacteriocinogenesis of the starter culture during the batch process: strong reduction of the times for bacteriocin production and coagulation of milk (to 4.5-5.0 h); high cell productivity (lactobacilli-4x10(12) CFU ml(-1), streptococci-6x10(12) CFU ml(-1)); high productivity of bacteriocins (4,500 BU ml(-1))-1.7 times higher than the bacteriocinogenic activity of the batch starter culture.     

Molecular genetics of Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) epidemiology and pathogenesis.

Kaposi's sarcoma had been recognized as unique human cancer for a century before it manifested as an AIDS-defining illness with a suspected infectious etiology. The discovery of Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, in 1994 by using representational difference analysis, a subtractive method previously employed for cloning differences in human genomic DNA, was a fitting harbinger for the powerful bioinformatic approaches since employed to understand its pathogenesis in KS. Indeed, the discovery of KSHV was rapidly followed by publication of its complete sequence, which revealed that the virus had coopted a wide armamentarium of human genes; in the short time since then, the functions of many of these viral gene variants in cell growth control, signaling apoptosis, angiogenesis, and immunomodulation have been characterized. This critical literature review explores the pathogenic potential of these genes within the framework of current knowledge of the basic herpesvirology of KSHV, including the relationships between viral genotypic variation and the four clinicoepidemiologic forms of Kaposi's sarcoma, current viral detection methods and their utility, primary infection by KSHV, tissue culture and animal models of latent- and lytic-cycle gene expression and pathogenesis, and viral reactivation from latency. Recent advances in models of de novo endothelial infection, microarray analyses of the host response to infection, receptor identification, and cloning of full-length, infectious KSHV genomic DNA promise to reveal key molecular mechanisms of the candidate pathogeneic genes when expressed in the context of viral infection.     

Biosynthesis of invertase by Saccharomyces cerevisiae with sugarcane molasses as substrate

Biosynthesis of invertase by Saccharomyces cerevisiae 01K32 was inversely proportional to the concentration of sugarcane blackstrap molasses included in the medium. In a fermenter, an intracellular invertase activity of 440 U/g dry cells was obtained.     

Crosslinking proteins to nucleic acids by ultraviolet laser irradiation.

Ultraviolet (UV) irradiation can initiate complex formation between proteins and DNA or RNA and so can be used to study such interactions. However, crosslink formation by standard UV light sources can take up to several hours. More recently, a beam of monochromatic UV light from a laser has been used to initiate crosslinking in nano- and picosecond time intervals. As noted in an earlier TIBS article 'the advantages of short pulse times and high-energy fluxes should make this a valuable technique in the future'. In this review we characterize laser-induced crosslinking and explore the applications of this method.     

PhenomicDB: a multi-species genotype/phenotype database for comparative phenomics

SUMMARY: We have created PhenomicDB, a multi-species genotype/phenotype database by merging public genotype/phenotype data from a wide range of model organisms and Homo sapiens. Until now these data were available in distinct organism-specific databases (e.g. WormBase, OMIM, FlyBase and MGI). We compiled this wealth of data into a single integrated resource by coarse-grained semantic mapping of the phenotypic data fields, by including common gene indices (NCBI Gene), and by the use of associated orthology relationships. With its use-case-oriented user interface, PhenomicDB allows scientists to compare and browse known phenotypes for a given gene or a set of genes from different organisms simultaneously. AVAILABILITY: PhenomicDB has been implemented at Schering AG as described below. A PhenomicDB implementation differing in some technical details has been set up for the public at Metalife AG http://www.phenomicDB.de SUPPLEMENTARY INFORMATION: database model, semantic mapping table.     

A genetic-algorithm-based approach to optimization of bioprocesses described by fuzzy rules

A new approach to optimization of bioprocesses described by fuzzy rules is introduced in the paper. It is based on genetic algorithms (GA) and allows to determine optimal values or profiles of control variables and to optimize fuzzy rules (parameters of membership functions). The process can be described by linguistic variables and fuzzy rules. An algorithm and related software was developed. The approach was applied to an industrial antibiotic fermentation. The optimal profile of a physical variable of the preculture was determined which leads to an increasing output product concentration in the main culture of about 5%.