Cold Shock Response Of Deinococcus Radiodurans And Its Regulation Via Cold Shock Protein Csp

Low temperature is a major environmental stress factor for bacteria to survive. When bacteria are suddenly exposed to a cold environment, the cells will express a set of specific small molecule proteins, which called cold shock proteins(Csps). A large number of experiments show that Csp plays an important role either under normal growth conditions or during cold-shock adaptation. Radiation-resistant organism Deinococcus radiodurans can survival from extremely environment including low temperature, but the mechanism of cold shock response is still not clear, and only a few studies on regulatory mechanism of Csps were reported. In this dissertation, the regulatory network of Csp in Deinococcus radiodurans were studied by combined transcriptome and phenotype analysis of the cold shock response, real-time PCR confirmation of the transcriptome data for specific genes, and measurement of physiological and biochemical phenotype. The main results obtained are as follows:1. Western blot result demonstrated that quantity of Csp obviously increased after 4 h of incubation at 15°C compared to the amount observed when the cells were in the exponential phase at 30°C. To investigate the cold shock response of Deinococcus radiodurans, we performed comparative transcriptome analysis of Deinococcus radiodurans after a temperature downshift from 30°C to 15 for 4 h. In total, 199 genes were found to be signi?cantly induced upon cold shock challenge, and 642 genes displayed signi?cant repression under chill-stress growth conditions. These genes including the pathway of low temperature perception, signal transduction and response. This experiment was also used to con?rm the classic cold shock response in Deinococcus radiodurans. 1) We also observed that the transcriptions of gyr A(DNA gyrase) and Uvr ABC pathway related genes were upregulated after a temperature downshift; 2) Some catalase genes such as kat A is also upregulated, these ?ndings were of special interest as they suggest that catalase may be one of the contributors to the ability of Deinococcus radiodurans to grow at low temperature; 3) S-layer proteins Hpi and Slp A are upregulated, these genes are involved in the maintenance and integrity of the S layer under cold shock. Previous studies investigated the cold shock response of E. coli in DNA repair and transport of osmotic protective substance. Besides that, Deinococcus radiodurans can respond to oxidation resistance and membrane damage.2. There is only one predicted csp gene to play a role in the radiation response and cold shock response in Deinococcus radiodurans. Sequence alignment and protein structure homology-modelling suggested that Csp contain two nucleic acid binding motifs, and was presumed to act as activators of the transcription of cold adaptive genes. csp is highly expressed gene under normal growth conditions. Phenotypic analysis of mutant(△csp) and complementation(△csp C) strains show that: 1) △csp grows slower than wild-type and △csp C, and the absent of csp is affected the cells growth rate; 2) △csp is sensitive to low temperature(15°C) and H2O2, but wild-type and △csp C are not. These results showed that csp is necessary at low temperature, and also involved many pathways under normal condition.3. To further study regulatory mechanism of Csp during cold shock, we carried out a comparison of the transcriptome pro?les of the wild-type and △csp mutant cells to identify the target genes of the Csp. The genes with the following sequences in the promoter are supposed to be positive regulated by Csp, such as: 1) CCAAT of gyr A(DNA gyrase) promoter; 2) ATTGG of kat A(catalase) promoter; 3) TTTT of mal E(maltose ABC transporter) promoter. Real-time PCR data confirmed that kat A and kat E, were induced 2.2-fold and 3-fold respectively at low temperature. Compared to normal growth conditions, catalase activity increased 1.6 times under low temperature, but repressed in △csp mutant, implying that cold-induced Csp can improve catalase activity to protect the bacteria against the damaging effects of ROS generated during cold shock. Csp helps cell to survive under low temperature.This dissertation is the first report on cold shock response of Deinococcus radiodurans, our research revealed that Csp was involved in the regulation of gyr A、kat A、mal E, proposed the Csp regulation of cold shock response in Deinococcus radiodurans, and provided theoretical basis for the further work.