Study On The Interaction Between PprM Protien And PprI/recA/pprA Genes

Background and purpose:Deinococcus radiodurans(DR)is one of the most powerful forms of life in the world,which is known for its resistance to ionizing radiation.The cleavage of negative regulatory factor DdrO by PprI's metalloproteinase,activating the expression of multiple downstream genes,such as pprA,ddrD,gyrA,which participate in radiation resistance,thus having an efficient DNA damage response capability in Deinococcus radiodurans.PprM was found to be a novel regulatory protein involved in the PprI-dependent radiation response,however the mechanism is not clear for now.The aim of this study was to investigate the relationship between PprM protein and three genes of pprI,recA and ppr A,to further explore the PprM protein involved in the PprI-dependent signaling pathways,and to understand the role of PprM in the extreme resistance network of Deinococcus radiodurans.Methods:(1)The Ppr M protein may bind to the promoter region of three key genes pprI/recA/pprA,was screened by the method of chromosome immunoprecipitation(ChIP)combined with PCR.The recombinant plasmid pGEX-6p-1-pprM was expressed and purified to obtain GST-PprM fusion protein;(2)Design the pprI/recA/pprA promoter region upstream and downstream primers,synthetic biotin labeled probe.The interaction of pprI/recA/pprA promoter probe and GST-PprM fusion protein was verified by electrophoretic mobility shift assay(EMSA),further predicting their binding sequence;(3)The expression of pprI/recA/pprA mRNA in pprM mutant and DR wild-type bacteria was detected by qRT-PCR,to understand the effect of PprM on these three genes at transcription level;(4)The survival curves of pprM mutant strains and DR wild strains were tested at low temperature.Results: Successfully induced expression and purification into GST-PprM fusion protein;The PprM protein is capable to bindthe pprI/recA/pprA promoter region in vitro;The binding sequence of the pprI promoter is mainly located in the third pair of probe sequences,while the recA/pprA promoter binding sequence is mainly located in the respective second pair of probe sequences,the binding sequence may be GCCGTGAA or GACCTGAA;The expression level of pprI/pprA m RNA in pprM mutant strain was higher than that in DR wild-type strain,but the expression level of recA mRNA was not significantly different;The anti-cold ability of pprM mutant strain was higher than that of DR wild strain.Conclusions:1.EMSA confirms that the PprM protein interacts with the promoter region of the three genes of pprI/recA/pprA,and The binding sequence is located in the third sequence of the pprI promoter and the second sequence of the recA/pprA promoter,respectively.2.The PprM protein have a certain inhibitory effect on the expression of pprI/pprA at transcriptional level,but has no effect on recA.3.The deletion of the pprM gene leads to an increase in the cold resistance of Deinococcus radiodurans.