Heavy Metal Tolerance in Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is an aerobic, non-fermentative Gram-negative bacterium widespread in the environment. S. maltophilia Sm777 exhibits innate resistance to multiple antimicrobial agents. Furthermore, this bacterium tolerates high levels (0.1 to 50 mM) of various toxic metals, such as Cd, Pb, Co, Zn, Hg, Ag, selenite, tellurite and uranyl. S. maltophilia Sm777 was able to grow in the presence of 50 mM selenite and 25 mM tellurite and to reduce them to elemental selenium (Se0) and tellurium (Te0) respectively. Transmission electron microscopy and energy dispersive X-ray analysis showed cytoplasmic nanometer-sized electron-dense Se0 granules and Te0 crystals. Moreover, this bacterium can withstand up to 2 mM CdCl2 and accumulate this metal up to 4% of its biomass. The analysis of soluble thiols in response to ten different metals showed eightfold increase of the intracellular pool of cysteine only in response to cadmium. Measurements by Cd K-edge EXAFS spectroscopy indicated the formation of Cd-S clusters in strain Sm777. Cysteine is likely to be involved in Cd tolerance and in CdS-clusters formation. Our data suggest that besides high tolerance to antibiotics by efflux mechanisms, S. maltophilia Sm777 has developed at least two different mechanisms to overcome metal toxicity, reduction of oxyanions to non-toxic elemental ions and detoxification of Cd into CdS

The effect of zeaxanthin as the only xanthophyll on the structure and function of the photosynthetic apparatus in Arabidopsis thaliana.

In green plants, the xanthophyll carotenoid zeaxanthin is synthesized transiently under conditions of excess light energy and participates in photoprotection. In the Arabidopsis lut2 npq2 double mutant, all xanthophylls were replaced constitutively by zeaxanthin, the only xanthophyll whose synthesis was not impaired. The relative proportions of the different chlorophyll antenna proteins were strongly affected with respect to the wild-type strain. The major antenna, LHCII, did not form trimers, and its abundance was strongly reduced as was CP26, albeit to a lesser extent. In contrast, CP29, CP24, LHCI proteins, and the PSI and PSII core complexes did not undergo major changes. PSII-LHCII supercomplexes were not detectable while the PSI-LHCI supercomplex remained unaffected. The effect of zeaxanthin accumulation on the stability of the different Lhc proteins was uneven: the LHCII proteins from lut2 npq2 had a lower melting temperature as compared with the wild-type complex while LHCI showed increased resistance to heat denaturation. Consistent with the loss of LHCII, light-state 1 to state 2 transitions were suppressed, the photochemical efficiency in limiting light was reduced and photosynthesis was saturated at higher light intensities in lut2 npq2 leaves, resulting in a photosynthetic phenotype resembling that of high light-acclimated leaves. Zeaxanthin functioned in vivo as a light-harvesting accessory pigment in lut2 npq2 chlorophyll antennae. As a whole, the in vivo data are consistent with the results obtained by using recombinant Lhc proteins reconstituted in vitro with purified zeaxanthin. While PSII photoinhibition was similar in wild type and lut2 npq2 exposed to high light at low temperature, the double mutant was much more resistant to photooxidative stress and lipid peroxidation than the wild type. The latter observation is consistent with an antioxidant and lipid protective role of zeaxanthin in viv

The Green Microalga Chlamydomonas reinhardtii Has a Single -3 Fatty Acid Desaturase That Localizes to the Chloroplast and Impacts Both Plastidic and Extraplastidic Membrane Lipids

International audienceThe v-3 polyunsaturated fatty acids account for more than 50% of total fatty acids in the green microalga Chlamydomonas reinhardtii, where they are present in both plastidic and extraplastidic membranes. In an effort to elucidate the lipid desaturation pathways in this model alga, a mutant with more than 65% reduction in total v-3 fatty acids was isolated by screening an insertional mutant library using gas chromatography-based analysis of total fatty acids of cell pellets. Molecular genetics analyses revealed the insertion of a TOC1 transposon 113 bp upstream of the ATG start codon of a putative v-3 desaturase (CrFAD7; locus Cre01.g038600). Nuclear genetic complementation of crfad7 using genomic DNA containing CrFAD7 restored the wild-type fatty acid profile. Under standard growth conditions, the mutant is indistinguishable from the wild type except for the fatty acid difference, but when exposed to short-term heat stress, its photosynthesis activity is more thermotolerant than the wild type. A comparative lipidomic analysis of the crfad7 mutant and the wild type revealed reductions in all v-3 fatty acid-containing plastidic and extraplastidic glycerolipid molecular species. CrFAD7 was localized to the plastid by immunofluorescence in situ hybridization. Transformation of the crfad7 plastidial genome with a codon-optimized CrFAD7 restored the v-3 fatty acid content of both plastidic and extraplastidic lipids. These results show that CrFAD7 is the only v-3 fatty acid desaturase expressed in C. reinhardtii, and we discuss possible mechanisms of how a plastid-located desaturase may impact the v-3 fatty acid content of extraplastidic lipids

Genome-wide transcriptome profiling of the early cadmium response of Arabidopsis roots and shoots.

Transcriptional regulation in response to cadmium treatment was investigated in both roots and leaves of Arabidopsis, using the whole genome CATMA microarray containing at least 24,576 independent probe sets. Arabidopsis plants were hydroponically treated with low (5 muM) or high (50 muM) cadmium concentrations during 2, 6, and 30 hours. At each time point, Cd level was determined using ICP-AES showing that both plant tissues are able to accumulate the heavy metal. RT-PCR of eight randomly selected genes confirmed the reliability of our microarray results. Analyses of response profiles demonstrate the existence of a regulatory network that differentially modulates gene expression in a tissue- and kinetic-specific manner in response to cadmium. One of the main response observed in roots was the induction of genes involved in sulfur assimilation-reduction and glutathione (GSH) metabolism. In addition, HPLC analysis of GSH and phytochelatin (PC) content shows a transient decrease of GSH after 2 and 6 h of metal treatment in roots correlated with an increase of PC contents. Altogether, our results suggest that to cope with cadmium, plants activate the sulfur assimilation pathway by increasing transcription of related genes to provide an enhanced supply of GSH for PC biosynthesis. Interestingly, in leaves an early induction of several genes encoding enzymes involved in the biosynthesis of phenylpropanoids was observed. Finally, our results provide new insights to understand the molecular mechanisms involved in transcriptional regulation in response to cadmium exposure in plants

A type II NAD(P)H dehydrogenase mediates light-independent plastoquinone reduction in the chloroplast of Chlamydomonas

In photosynthetic eukaryotes, nonphotochemical plastoquinone (PQ) reduction is important for the regulation of photosynthetic electron flow. In green microalgae where this process has been demonstrated, the chloroplastic enzyme that catalyses nonphotochemical PQ reduction has not been identified yet. Here, we show by an RNA interference (RNAi) approach that the NDA2 gene, belonging to a type II NAD(P)H dehydrogenases family in the green microalga Chlamydomonas reinhardtii, encodes a chloroplastic dehydrogenase that functions to reduce PQ nonphotochemically in this alga. Using a specific antibody, we show that the Nda2 protein is localized in chloroplasts of wild-type cells and is absent in two Nda2-RNAi cell lines. In both mutant cell lines, nonphotochemical PQ reduction is severely affected, as indicated by altered chlorophyll fluorescence transients after saturating illumination. Compared with wild type, change in light excitation distribution between photosystems ('state transition') upon inhibition of mitochondrial electron transport is strongly impaired in transformed cells because of inefficient PQ reduction. Furthermore, the amount of hydrogen produced by Nda2-RNAi cells under sulfur deprivation is substantially decreased compared with wild type, which supports previous assumptions that endogenous substrates serve as source of electrons for hydrogen formation. These results demonstrate the importance of Nda2 for nonphotochemical PQ reduction and associated processes in C. reinhardtii.Micro-H

Mechanism and dynamics of fatty acid photodecarboxylase

International audienceFatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer