Surviving salt fluctuations: stress and recovery in Halobacterium salinarum, an extreme halophilic Archaeon.

Halophilic proteins subjected to below about 15% salt in vitro denature through misfolding, aggregation and/or precipitation. Halobacteria, however, have been detected in environments of fluctuating salinity such as coastal salterns and even around fresh water springs in the depths of the Dead Sea. In order to identify the underlying mechanisms of low salt survival, we explored the reactivation capacity of Halobacterium (Hbt) salinarum sub-populations after incubation in low salt media and recovery in physiological salt. Respiratory oxygen consumption was assessed in stressed cells and cell viability was estimated by Live/Dead staining and flow cytometry. In vivo neutron scattering experiments showed that the recovery of Hbt salinarum sub-populations exposed to severe low salt conditions is related to a rapid retrieval of functional molecular dynamics in the proteome. In the hypothesis that the observations on Hbt salinarum have wider relevance, they could be of key ecological significance for the dispersion of extremophiles when environmental fluctuations become severe.

The gene encoding T protein of the glycine decarboxylase complex involved in the mitochondrial step of the photorespiratory pathway in plants exhibits features of light-induced genes.

We have isolated and characterized a genomic clone encoding the 41 kDa monomer T-protein. This gene called gdcT spans approximately 3 kbp and is composed of four exons interrupted by three introns (321, 691 and 114 bp). The splice sites for donor and acceptor are in agreement with the canonical GT/AG rule. Primer extension strongly suggests the presence of two major transcription start sites. The first transcription start site around 43 bases downstream of a putative TATA box was assigned the +1 position. The second (+31) is not correlated with a putative TATA box, but revealed a pyrimidine-rich region which is very similar to the initiator element. Sequence analysis of the 5'-upstream region of the gene reveals three consensus regions found in the nuclear genes encoding the chloroplastic proteins of ribulose-1,5-bisphosphate carboxylase (rbcS) and the chlorophyll a/b-binding protein (cab) such as an AT-rich sequence localized at -539 to -530, a box II core sequence GGTTAA (-123 to -118) and between -364 and -354 a tandem GATA motif. These elements are known to be involved respectively in the regulation of light-responsiveness and cell-type specificity expression of plant genes. Gel shift assays indicate that the box II core sequence could bind protein nuclear factors similar to the trans-acting factor which interact with corresponding promoter region of rbcS gene.

Regulation of the Expression of the Glycine Decarboxylase Complex during Pea Leaf Development.

The expression of the genes encoding the four proteins (P, H, T, and L) of glycine decarboxylase, a multienzymatic complex involved in the mitochondrial step of the photorespiration pathway, was examined during pea (Pisum sativum) leaf development in comparison with ribulose-1,5-bisphosphate carboxylase/oxygenase. Mitochondria from the primary leaf were isolated at several well-defined stages of development. Their capacity to oxidize glycine was negligible during the earlier stages but increased dramatically once the leaflet opened. This was correlated with the accumulation of the glycine decarboxylase complex (GDC) proteins, which was shown to occur in preexisting mitochondria, producing an increase in their density. The transcription of the GDC genes was coordinated and occurred early, with a peak at 7 d, a stage at which mitochondria are unable to oxidize glycine. This implies the existence of posttranscriptional control of gene expression. The comparison of the expression patterns of the genes encoding specific proteins of GDC with that of rbcS genes suggests a common regulation scheme that is related to light induction. However, ribulose-1,5-bisphosphate carboxylase/oxygenase is present in the chloroplast well before GDC fills the mitochondria, suggesting that the setup of photorespiration occurs in cells already engaged in active photosynthesis.

Glycine decarboxylase complex from higher plants. Molecular cloning, tissue distribution and mass spectrometry analyses of the T protein.

cDNA clones encoding the precursor of the T protein of the glycine decarboxylase complex have been isolated from a pea leaf cDNA library in lambda gt11. The longest cDNA insert of 1430 bp encodes a polypeptide of 408 amino acid residues of which 30 residues constitute an N-terminal cleavable presequence and 378 residues make up the mature protein. Several results confirmed the identity of the cDNA and the exactness of the predicted primary structure. Firstly, we purified the T protein to homogeneity and its mass was measured by mass spectrometry. The mass obtained (40966 +/- 5 Da) was the value predicted from the cDNA (40961 Da). Secondly, the purified T protein was chemically cleaved with cyanogen bromide and the peptide fragments were analysed by high-performance liquid chromatography/electrospray ionization mass spectrometry and/or fast-atom-bombardment mass spectrometry. The mass values of all the peptides generated by chemical cleavage and measured by these techniques were very close to the values calculated from the predicted primary structure. Thirdly, microsequencing of some of these peptides, which represent 35% of the total protein, fits perfectly with the primary structure deduced from the cDNA. In the present HPLC/electrospray ionization MS studies we never detected the presence of covalently bound tetrahydropteroylpolyglutamate (H4PteGlun), either in the native T protein or in the different peptide fragments generated by the chemical cleavage. The absence of H4PteGlun bound to the T protein in our experimental conditions demonstrates that H4PteGlun is not covalently linked to the T protein. Northern blot analysis showed that the steady-state level of the mRNA corresponding to the T protein was high in green leaves compared to the level in etiolated leaves (approximately 8-10-fold higher). Surprisingly, a non-negligible amount of mRNA corresponding to the T protein was present in roots whereas the mRNA encoding the H protein was not detectable. Western blot analysis showed that the P, L and T proteins of the glycine decarboxylase complex were present in roots whereas the H protein was not detectable. Southern hybridization to pea genomic DNA indicated the presence of a single gene encoding the T protein of the glycine decarboxylase complex in the haploid genome.