Functional Identification Of Sor1 And Sig2 Resistance To Radiation - Resistant Isosporine And Transcription Analysis Of Heat Shock Stress

a factor is a subunit of RNA polymerase of prokaryotes and is necessary for transcription initiation. It can activate initiated transcription by reversibly binding with the active catalytic site of the RNA polymerase core enzyme, affecting the correct identification of the transcription initiation site for RNA polymerase and the specific expression of initiated corresponding gene. With strong adaptability to extreme environmental stress and resistance to extreme radiation-induced oxidation, Deinococcus radiodurans consists of three a factors, they are, Sig1（DR₀180）, Sig2（DR₀804） and SigA（DR₀916）. It remains unknown about the functions of Sigl and Sig2, which are less reported.In this study, we constructed single mutant strains and double mutant strain of sigl and sig2, and intensively investigated the function of these two genes along with the regulation function based on stress impact experiment, Realtime PCR and transcription analysis. The main results obtained are as follows:1. Deinococcus radiodurans R1 Sigl is predicted to be a putative ECF a factor based on bioinformatic analysis. It belongs to a70 family 4 and is encoded by DR₀180 in chromosome I,690 bp,25.89 kDa with 229 aa. Sig2（DR₀804） also belongs to the σ70 family, whose physiological function is unknown. Sig2 is encoded by DR₀804 in chromosome I,849 bp, 29.91 kDa with 282 aa. By comparing the amino acid sequences, it demonstrated that Sig1 shows great similarity to the congeneric a factors of Deinococcus, exhibiting the over 64% in similarity, and is highly conserved in the domain of ECF family. Sig2 has the highest identity with sequence of a24 in Deinococcus sp. YIM 77859 of only 47%. In addition, Sig1 and Sig2 both have most conserved domain 2 of a-70 protein, which is the key domain for σ-factor to identify the core enzyme.2. In order to investigate the biological functions of Sig1 and Sig2, The sig1 deletion mutation strain （Δsig1）, the sig2 deletion mutation strain （Δsig2） and the double mutant strain of sigl and sig2 were constructed by means of the fusion PCR. The phenotypic analysis demonstrated that the growth of the strain is not affected by the sigl deletion mutation, sig2 deletion mutation and their double mutant of sigl and sig2. After 5M NaCl stress for 6h, the Δsigl showed decreased survival ability than the wild type, which indicated that sigl mutation resulted in the stress sensitive to acids and salts, and the stress sensitive to heat（48℃） and oxidation（10mM hydrogen peroxide） is caused by the single mutation of double mutation of sigl and sig2.3. To further study the regulation effect of Sigl and Sig2 on cellular metabolism, we carried out the transcriptome analysis of the sigl mutant and sig2 under normal growth conditions and heat shock at 48℃ for 2h. The results showed that 656 genes changed significantly in Deinococcus radiodurans, and 329 genes were up-regulated while 327 down-regulated.23% of differential expression genes are the genes for the genetic information storage and processing; 28% are for metabolism; 49% was genes of unknown function; The differential expression genes were related to several metabolic pathways. The sigl mutation resulted in 265 genes which altered significantly. Among them there were 59 genes up-regulated and 206 genes down-regulated. In addition,293 genes altered significantly after heat shock for 2h. There were 92 genes up-regulated and 201 genes down-regulated. Among them, there were 12 gene related to ribosomal protein structure, whose expression is enhanced, including rpsJ, rplC, rplW, rplB, rpsS, rplV, rplP, rplN, rplF, rp10, rpsM and rpsD. The sig2 mutation resulted in 317 genes which altered significantly. Among them,130 genes were up-regulated and 187 genes down-regulated. In addition,376 genes altered significantly after heat shock for 2h. There were 279 genes up-regulated and 97 genes down-regulated. The products of differential expression 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. The molecular chaperone DnaK, which was up-regulated in 1.3 times in Δsigl and down-regulated in 1.5 times in Δsig2 after heat shock at 48℃ for 2h. Real-time quantitative PCR results showed that the molecular chaperone groES, groEL, dnaK, dnaJ and heat shock genes DR 1314, DR₀972 and DR₀326 changed significantly after heat shock for 1h and 3h. Combining transcription data and real-time quantitative PCR results, we speculated that sigl, sig2 may be involved in these genes transcription. By comparing the transcription data of Sigl and Sig2 after heat shock, it was found that 28 genes are the same which changed significantly. DR₀179、DR₁783 and DR_A0248 were all up-regulated in the strains of single mutation. DR_A0248 regulated the transcription of MarR family proteins, which indicated that the transcription expression of MarR family proteins can be regulated directly under heat shock stress by Sigl and Sig2.This study demonstrated that the Deinococcus radiodurans R1 sigl and sig2 affects the reaction of several genetic physiological heat shock stress, including the protection from heat shock stress, protection of antioxidation and the synthesis of ribosomal protein. It can serve as a theoretical underpinning and assist in the reveal of the adaption mechanism for the extreme environment of Deinococcus radiodurans.