Functional Identification And Transcriptome Analysis Of Cold Shock Protein In Deinococcus Radiodurans

Cold shock proteins (Csps) are found in most animals, plants and eubacteria, but not in Archaea. Csps are small, 7-10 kd proteins with similar amino acid sequences and structures, and they contain a specific RNA and ssDNA binding cold shock domain. Csps are generally systhesised in large quantities in response to temperature downshifts and they function as RNA chaperones preventing secondary structure formation, and in some organisms also as transcriptional enhancers. D. radiodurans R1 contains a Csp-like protein which is presumably controlled by the global regulator PprI. A csp mutant was sensitive toγirradiation. So far there are few studies of the role of the D. radiodurans csp in the cold shock response. This work studies the function of Csp and its pleiotropic regulatory roles for cell metabolism.The csp mutant grew more slowly than the parent even at the optimal growth temparature of 30℃, and growth almost stopped at 15℃. Realtime PCR confirmed that csp was cold induced. Biolog assays revealed a change in the carbon source utilization of the csp mutant. Eight carbon sources, including glucose, were utilized less efficiently, and three more efficiently. Gel shift mobility assays showed that D. radiodurans Csp binded to ssDNA containing stretches of T or the ATTGG or the complementary CCAAT Y-box core sequence. The csp gene may increase its own expression by binding to the CCAAT sequence upstream of its promoter.To further study the effect of Csp on cellular metabolism, we carried out transcriptome analysis of the csp mutant under normal growth conditions. Compared to the parent, 466 genes were induced and 322 genes were repressed. The differently regulated genes were involved in most cell processes including cell wall/membrane/envelope biosynthesis, ribosomal structure and biogenesis, replication/recombination and repair, signal transduction, amino acid transport and metabolism, energy production and conversion, lipid transport and metabolism and secondary metabolites biosynthesis, transport and catabolism. Realtime PCR confirmed the transcriptome data for specific genes. In carotenoid synthesis, the key gene crtI was 2.8-fold induced, and both intracellular and extracellular carotenoid production increased rendering the cultures red. This suggested that csp participated in the regulation of carotenoid biosynthesis. DNA response and damage repair related genes such as recA, pprA, irrE, ddrB and ddrI were induced and antioxidant genes were repressed. The repression of genes involved in oxidative stress responses may be the predominant cause for the high sensitivity to UV radiation of the csp mutant strain. Genes involved in peptidoglycan synthesis and the cell envelope S-layer-like array-related protein genes were induced in the csp mutant. Transmission electron microscopy showed that the outermost layer of the cell wall had a loose appearance and featured extruded vesicles. Imaging mass spectroscopy revealed a change in the distribution of polypeptides or lipopeptides. This was consistent with observed changes in the expression of genes for ABC transporters. Those data indicated that Csp was important for the normal morphogenesis and compound transport in D. radiodurans. This study confirmed that the D. radiodurans csp gene was cold induced. Transcriptome, physiological and biochemical analysis revealed that Csp was involved in many cellular processes including cellular antioxidant protection, DNA damage repair and carotenoid synthesis. This enhances the understanding of the strain's adaptive response to extreme stress.