In-depth analysis of the genome sequence of a clinical, extensively drug-resistant Mycobacterium bovis strain.

Although human-to-human transmission of Mycobacterium bovis strains and other members of the animal lineage of the tubercle bacilli is a rare event, an extensively drug resistant (XDR) strain, named M. bovis B strain, caused a lethal outbreak in the nineties in Spain. The genome of M. bovis B strain was re-sequenced by SOLiD platform and mapped to the reference M. bovis AF2122/97. The genetic polymorphisms detected have been analysed in depth. One hundred and fifty-eight specific non-synonymous SNPs were detected; ninety-two of these were non-conservative. In addition, one specific 3195-bp insertion could be identified as an ABC transporter gene by homology with tbd2 gene, which was found to be present in other clinical M. bovis strains. Its peculiar phenotype profile of resistance was explained by molecular characteristics, including a 5685-bp specific deletion that revealed a novel polymorphism associated with resistance to paraminosalicilic acid. From a phylogenetical point of view, according to the SNPs detected, M. bovis B could be included into the clonal complex M. bovis European 2. This is the first time that a deep analysis of the whole-genome sequencing of an extensively drug-resistant M. bovis strain is detailed. It offers the explanation for the resistance and found several data to be incorporated for future research.

Regulation of the chloroplastic copper chaperone (CCS) and cuprozinc superoxide dismutase (CSD2) by alternative splicing and copper excess in Glycine max.

Metal homeostasis is an important aspect of plant physiology, and the copper transport into the chloroplast and its fate after delivery is of special relevance for plants. In this work, the regulation of the chloroplastic copper chaperone for the cuprozinc superoxide dismutase (GmCCS) and its target, the cuprozinc superoxide dismutase (GmCSD2), was investigated in photosynthetic cell suspensions and entire plants from Glycine max (L.) Merr. Both genes were expressed in cell suspensions and in all plant tissues analysed, and their RNAs matured by alternative splicing with intron retention (IntronR). This mechanism generated a spliced and three non-spliced mRNAs in the case of GmCCS but only a spliced and a non-spliced mRNAs in GmCSD2. Copper excess strongly upregulated the expression of both fully spliced mRNAs but mostly unaffected the non-spliced forms. In entire plants, some tissue specificity was also observed depending on copper content status. At the protein level, the GmCCS was mostly unaffected but the GmCSD2 was strongly induced under copper excess in all subcellular fractions analysed, suggesting a post-transcriptional regulation for the former. This different protein regulation of the chaperone and its target may indicate some additional function for the CSD2 protein. In addition to its well-known superoxide dismutase (SOD) activity, it may also function as a metal sink in copper excess availability to avoid metal cell damage. Furthermore, the GmCCS seems to be present in the stroma only but the GmCSD2 was present in both stroma and thylakoids despite the general idea that the SOD enzymes are typically soluble stroma proteins. The presence of the SOD enzyme on the surface of the thylakoid membranes is reasonable considering that the superoxide radical (O2-) is preferentially formed at the acceptor side of the PSI.

Characterization of the recombinant copper chaperone (CCS) from the plant Glycine (G.) max.

The goal of the present work was to characterize the recombinant copper chaperone (CCS) from soybean. Very little is known about plant copper chaperones, which makes this study of current interest, and allows for a comparison with the better known homologues from yeast and humans. To obtain sizeable amounts of pure protein suitable for spectroscopic characterization, we cloned and overexpressed the G. max CCS chaperone in E. coli in the presence of 0.5 mM CuSO(4) and 0.5 mM ZnSO(4) in the broth. A pure protein preparation was obtained by using two IMAC steps and pH gradient chromatography. Most of the proteins were obtained as apo-form, devoid of copper atoms. The chaperone showed a high content (i.e., over 40%) of loops, turns and random coil as determined both by circular dichroism and homology modelling. The homology 3-D structural model suggests the protein might fold in three structural protein domains. The 3-D model along with the primary structure and spectroscopic data may suggest that copper atoms occupy the two metal binding sites, MKCEGC and CTC, within the N-terminal domain I and C-terminal domain III, respectively. But only one Zn-binding site was obtained spectroscopically.