Anti-stress activity of Propionibacterium freudenreichii: identification of a reactivative protein

Propionibacterium freudenreichii subsp. shermanii is known to prevent mutations caused by various agents such as N-methyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, 4-nitro-quinoline-1-oxide and by UV radiation in both prokaryotic and eukaryotic cells. It was also shown to prevent or repair damage caused by H(2)O(2) or UV radiation in Salmonella typhimurium and Escherichia coli, a characteristic previously designated as reactivative effect. In order to characterise this effect at the molecular level, we have purified the active component from a P. freudenreichii cell-free extract using a combination of ammonium sulfate precipitation, anion-exchange and size-exclusion chromatography. The isolated 35 kDa protein was then identified using both N-terminal and internal peptide sequencing as a cysteine synthase. The latter was localised in the P. freudenreichii proteomic map. It is constitutively expressed but also clearly induced during adaptation to detergent and heat, but not acid, stresses. The biological meaning of cysteine synthase in the context of adaptation to oxidative and non-oxidative stresses is discussed.     

Evidence for a composite phylogenetic origin of the plastid genome of the brown alga Pylaiella littoralis (L.) Kjellm

The nucleotide sequence and the 5 flanking region of the rbcL gene coding for the large subunit of ribulose bisphosphate-1,5-carboxylase/oxygenase of Pylaiella littoralis, a brown alga, has been determined and the deduced amino-acid sequence has been compared to those of various photosynthetic and chemoautotrophic Eubacteria, of a red alga and of green plastids (Euglena gracilis, green algae and higher plants). Unlike the rbcL genes of green plastids which are more closely related to those of cyanobacteria the P. littoralis rbcL gene is more closely related to that of a -purple bacterium, as was found for the rbcS gene of another chromophytic alga [Boczar et al., Proc Natl Acad Sci USA 86: 4996–4999, 1989]. Matrix data of homology between the rbcL gene of P. littoralis and the same gene of other organisms are presented. Based on our previous report, the gene coding for the 16S rRNA from P. littoralis is closely related to that of E. gracilis (Markowicz et al., Curr Genet 14: 599–608, 1988). We suggest that the large plastid DNA molecule of P. littoralis is a phylogenetically composite genome which probably resulted from mixed endosymbiosis events, or from a horizontal transfer of DNA.     

PHENOPSIS, an automated platform for reproducible phenotyping of plant responses to soil water deficit in Arabidopsis thaliana permitted the identification of an accession with low sensitivity to soil water deficit

The high-throughput phenotypic analysis of Arabidopsis thaliana collections requires methodological progress and automation. Methods to impose stable and reproducible soil water deficits are presented and were used to analyse plant responses to water stress. Several potential complications and methodological difficulties were identified, including the spatial and temporal variability of micrometeorological conditions within a growth chamber, the difference in soil water depletion rates between accessions and the differences in developmental stage of accessions the same time after sowing. Solutions were found. Nine accessions were grown in four experiments in a rigorously controlled growth-chamber equipped with an automated system to control soil water content and take pictures of individual plants. One accession, An1, was unaffected by water deficit in terms of leaf number, leaf area, root growth and transpiration rate per unit leaf area. Methods developed here will help identify quantitative trait loci and genes involved in plant tolerance to water deficit.     

Accumulation of phosphate and polyphosphate by Cryptococcus humicola and Saccharomyces cerevisiae in the absence of nitrogen

The search for new phosphate-accumulating microorganisms is of interest in connection with the problem of excess phosphate in environment. The ability of some yeast species belonging to ascomycetes and basidiomycetes for phosphate (P (i) ) accumulation in nitrogen-deficient medium was studied. The ascomycetous Saccharomyces cerevisiae and Kuraishia capsulata and basidiomycetous Cryptococcus humicola, Cryptococcus curvatus, and Pseudozyma fusiformata were the best in P (i) removal. The cells of Cryptococcus humicola and S.?cerevisiae took up 40% P (i) from the media containing P (i) and glucose (5 and 30?mM, respectively), and up to 80% upon addition of 5?mM MgSO(4) (.) The cells accumulated P (i) mostly in the form of polyphosphate (PolyP). In the presence of Mg(2+) , the content of PolyP with longer average chain length increased in both yeasts; they both had numerous inclusions fluorescing in the yellow region of the spectrum, typical of DAPI-PolyP complexes. Among the yeast species tested, Cryptococcus humicola is a new promising model organisms to study phosphorus removal from the media and biomineralization in microbial cells.     

Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes

During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers the caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors [Kagan, V. E., et al. (2005) Nat. Chem. Biol. 1, 223-232]. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of the CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp(59) fluorescence and absorbance at 695 nm (Fe-S(Met(80)) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H(2)O(2) concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H(2)O(2) can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.     

Mass mortality of macrobenthic communities in the lagoon of Hikueru atoll (French Polynesia)

Epibenthic macrofauna communities (corals, molluscs, echinoderms, and macroalgae) were investigated at Hikueru Atoll, Tuamotu Archipelago. The very low diversity and abundance that we observed (1 coral genus, and 2 mollusc, 1 echinoderm, and 17 macroalgal species) is likely to be the result of a mass mortality event that occurred 18 months earlier. Hydrological surveys suggest that this high mortality was the result of a major dystrophic crisis. Other atoll lagoons in the Tuamotu Archipelago have also been affected by high mortalities in the past, but Hikueru appears to have been the most frequently and intensely affected. The present study confirms that mass mortalities associated with phytoplankton blooms may be one of the major disturbances affecting coral reef ecosystems. Accepted: 14 May 2000