The Negative Regulation Mechanism Of Deinococcus Radiodurans Global Regulator CarD In Response To Nutritional Stress

Nutritional stress is one of the most common environmental stress factors during bacterial physiological process.In order to adapt to the nutritional changes in the environment,bacteria quickly change the gene expression by mediating the string response,which regulated the r RNA synthesis and signal molecular(p)pp Gpp.It has been found that some bacteria have the global regulatory protein Car D which was similar to the eukaryotic HMGA(high-mobility group A),Car D showed importance in nutrition and abiotic stress.D.radiodurans is highly adaptable to the harsh extremely environmental condition.By analyzing the genome sequence of D.radiodurans,we found that it contains a Car D protein.However,no studies been published yet in regards to the role of the Car D in Deinococcus radiodurans.The current study established to investigate whether the Car D involves in cell growth and nutritional stress in D.radiodurans,in addition to studying the mechanism of Car D to control r RNA and(p)pp Gpp synthesis.The regulation of D.radiodurans' s Car D in nutritional stress was studied by constructing D.radiodurans Car D mutant,followed by phenotype analysis,?-galactosidaseactivity assay,q RT-PCR,CHIP,and comparative proteomic analysis.We achieved the following results:1.The Sequence of Deinococcus Car Ds has high similarity,which lead to the conclusion that is could be evolved from the same ancestor.On the other side Deinococcus radiodurans Car D also showed high similarity with the Car D of Thermus aquaticus,and less similarity with Myxobacteria,mycobacteria,concluding the different evolutionary branches.Myxobacteria Car D protein is 316 amino acids,with Car G form a global transcriptional regulatory complex.However,the Car G or Car G-similar proteins are absence in Mycobacteria and Deinococcus radiodurans,besides the length of the Car D protein is relatively shorter than the Myxobacteria(162 and 169 amino acids,respectively,).The Car D proteins in Mycobacteria and Deinococcus radiodurans showed very low similarity(26%),thus we inferred that the function of D.radiodurans Car D differ from Myxobacteria and Mycobacteria.2.Under nutritional stress,the expression of car D gene significantly increased(at least 2-fold),suggesting that car D gene was induced by nutrient-starved.In order to study the function of D.radiodurans Car D,we constructed car D deletion mutant,complement strain and overexpression strain.The results showed that the lack of car D gene resulted in 16 S r RNA gene was up-regulated more than 2-fold under the conditions of normal or nutrient stress,and rel A gene expression was significantly down-regulated by 2-fold under nutrient stress conditions.The promoter-activity assay of 16 S r RNA and rel A by reporter gene lac Z showed Car D negative impact on the expression of 16 S r RNA while it activating the rel A gene.Moreover,the results of CHIP confirmed that Car D protein interacting with 16 S r RNA and rel A gene promoter.For further study regards to the regulation of Car D we have constructed the overexpression strain,which been found that the overexpression of 16 S r RNA gene results in lower growth rate under several factors including nutritional stress,temperature sensitivity,enhanced the resistance to UV radiation,which consistent with car D deletion mutant strain.On the other hand,we constructed deletion mutant strains of(p)pp Gpp synthetase/hydrolase.D.radiodurans possessed two(p)pp Gpp synthetase/hydrolase genes(rel A,rel Q).Several growth assays have been applied to rel A,rel Q single,double mutant,and complement strains,showed that the deletion in rel A,rel Q resulting in bacteria sensitive to amino acid starvation,H2O2 and heat stress.The double mutant strain cannot grow in minimal medium.The overexpression synthase gene(rel Q)or Rel A synthetic conserved domain fragment(encoding Rel A N 'terminal HDc and RSD domain DNA sequence)resulted in a significant reduction in the ability of cell growth.Previous studies showed that(p)pp Gpp as a signal molecule regulating growth rate through string response,and(p)pp Gpp synthesis and hydrolysis activity of Rel A play a key role in control the intracellular(p)pp Gpp concentration.Under nutritional stress,D.radiodurans Car D control the content of intracellar(p)pp Gpp and inhibited cell growth by activating the expression of rel A gene.3.The results showed that the functions of D.radiodurans Car D share similarity with the functions of Car Ds in-depth study of Mycobacteria and Myxobacteria,while there are also some differences.Yeast two-hybrid showed that D.radiodurans Car D interacts with RNA polymerase ?' subunit,which is consistent with the findings of Mycobacteria.Myxobacteria Car D was induced by blue light and involved in carotenoids synthesis,while D.radiodurans Car D was induced by nutrition,oxidation and UV stresses and,Car D is not only response in nutritional stress,but also directly or indirectly involved in oxidative,UV radiation stresses and so on.The mechanism of global regulator Car D involved in multiple stresses is needed to be further studied.In summary,D.radiodurans Car D played negative regulation in stresses especially nutritional stress by negative regulating 16 S r RNA gene,or acting(p)pp Gpp synthase/hydrolase rel A gene,to control(p)pp Gpp contentant and growth rate.Gathering the Car D with stresses,especial-regard to the nutritional stress,have negative impact on the regulation of 16 S r RNA gene expression.On the other side,the later combination between Car D and stresses have positive impact to activate the expression of(p)pp Gpp synthesis/hydrolase rel A gene,which is controlling the cell content of(p)pp Gpp,sub-sequentially it will downlregulate cell growth rate and nutritional stress response.