The use of high halide-ion concentrations and automated phasing procedures for the structural analysis of BclA, the major component of the exosporium of Bacillus anthracis spores.

International audienceThe structure determination of the recombinant form of BclA, the major protein component of Bacillus anthracis exosporium, involved soaking in a high concentration of potassium iodide as the means of obtaining a good-quality heavy-atom derivative. The data to 2 angstroms resolution collected on a laboratory source were of sufficient quality to allow successful phasing and chain tracing by automated methods

Chromosomal inactivation of Bacillus subtilis exfusants: a prokaryotic model of epigenetic regulation.

International audienceEpigenetic mechanisms are not exclusively reserved to eukaryotic organisms. They are also observed in prokaryotes. As described first by Hotchkiss and Gabor, protoplast fusion between strains of Bacillus subtilis produces heterodiploid cells. Heterodiploidy is associated with the inactivation of one of the chromosomes. To study the physical structure of the fusion product and the molecular mechanisms of inactivation, we constructed heterodiploid clones containing two chromosomes labeled by a NotI restriction fragment length polymorphism. In the progeny, we identified haploid recombinant clones that contain a chromosome carrying large regions of inactivated DNA. Studies of both recombinants of the latter kind and heterodiploid cells indicated that chromosomal inactivation was not determined by alteration of the inactivated nucleotide sequence, but was probably due to a modification in the structure of the bacterial chromatin

Contribution of DNA Conformation and Topology in Right-handed DNA Wrapping by the Bacillus subtilis LrpC Protein

International audienceThe Bacillus subtilis LrpC protein belongs to the Lrp/AsnC family of transcriptional regulators. It binds the upstream region of the lrpC gene and autoregulates its expression. In this study, we have dissected the mechanisms that govern the interaction of LrpC with DNA by electrophoretic mobility shift assay, electron microscopy, and atomic force microscopy. LrpC is a structure-specific DNA binding protein that forms stable complexes with curved sequences containing phased A tracts and wraps DNA to form spherical, nucleosome-like structures. Formation of such wraps, initiated by cooperative binding of LrpC to DNA, results from optimal protein/protein interactions specified by the DNA conformation. In addition, we have demonstrated that LrpC constrains positive supercoils by wrapping the DNA in a right-handed superhelix, as visualized by electron microscopy

Metabolism of the lipophilic phycotoxin 13-Desmethylspirolide C using human and rat in vitro liver models

International audience13-Desmethylspirolide C (13-SPX-C) is a phycotoxin produced by dinoflagellates which can accumulate in shellfish. 13-SPX-C induces neurotoxic effects in rodents through blockade of nicotinic acetylcholine receptors. As no human intoxication has been to date attributed to the consumption of 13-SPX-C-contaminated seafood, this toxin is not regulated according to the Codex Alimentarius. Nevertheless, shellfish consumers can be exposed to 13-SPX-C via shellfish consumption. In order to follow the fate of the toxin after ingestion and to verify whether metabolic detoxification could explain the lack of human intoxications, we assessed the metabolism of 13-SPX-C using several in vitro liver systems. First, both phase I and II reactions occurring with rat and human liver S9 fractions were screened. Our results indicated that 13-SPX-C was almost completely metabolized with both rat and human liver S9. Using a receptor binding assay towards nicotinic acetylcholine receptors we demonstrated that the resulting metabolites showed less affinity towards nicotinic acetylcholine receptors than 13-SPX-C. Finally, we showed that 13-SPX-C induced a pronounced increase of gene expression of the drug-metabolizing enzyme cytochrome P450 (CYP) CYP1A2. The role of this CYP in 13-SPX-C metabolism was clarified using an innovative in vitro tool, CYP1A2-Silensomes™. In summary, this study highlights that liver first-pass metabolism can contribute to the detoxification of 13-SPX-C