Function And Regulation Of Alanine Metabolism Genes In Response To Oxidative Stress In Deinococcus Radiodurans

Deinococcus radiodurans is a robust bacterium best known for its resistance to oxidative stress. Oxidative stress is incurred by reactive oxygen species（ROS）, which can be produced metabolically or can form upon exposure to physical and chemical agents such as, ionizing radiation, UV radiation, desiccation or hydrogen peroxide. Until now the underlying mechanisms for oxidative stress resistance are unclear of D. radiodurans, especially the role of amino acids for the extremly stress resistance. In this study, firstly we used genome-wide transcriptomics to investigate the effects of the global regulator Irr E on the changes in gene expression in D.radiodurans under normal growth and UV radiation, and focused on the changes of genes about amino acids metabolism. Second, we studied the effects of the Lrp famliy protein Ald R（DR₁894） on abiotic stress resistance of D. radiodurans.1. Irr E, served as a global regulator, is important for extremly abiotic stress resistance of D. radiodurans. The transcriptome data showed that 86 genes were up-regulated at least 2-fold（log2 ratio ≥ 1 and p ≤ 0.05） in Δ irr E mutant, whereas 134 genes were down-regulated more than 2-fold（log2 ratio≤1 and p≤0.05） under normal growth and UV radiation conditions. We classified the differentially expressed genes into 3 major groups（Information storage and processing, Metabolism and Poorly characterized） according to the COG database. Among the Irr E-dependent genes, these down-regulated genes（ppr A, ddr A, ddr B, ddr D, mut S, mut L, ruv B） appeard to be critical to this specie ability to survive UV radiation and were involved in DNA break repair. And we also found some Irr E-regulated genes about amino acids metabolism changed, such as dr₀958 and dr₀959（peptide ABC transporter permease）, dr₀965 and dra0127（GMC oxidoreductase）, and dr₁895（alanine dehydrogenase, ald）. We selected some genes（ppr A, uvr B, rec A and ald） and examined their expression levels using real-time PCR（RT-PCR） to verify the transcriptome data, and found ald（alanine dehydrogenase） was IrrE-denpendent.2. Amino acids, basic components of proteins, are essential for growth of cells. We analysed the intracellular content of free amino acids in the Δirr E mutant and wild type D. radiodurans strains after abiotic stress as determined by HPLC. Under normal growth conditions, the amount of free amino acids in the Δirr E mutant was 39.1 ± 3.9 mg / g dry cells and that of wild type was 40.6 ±1.7 mg / g dry cells. After 48℃ heat shock, the free amino acids level in Δirr E mutant was 36.8 ± 0.3 mg / g dry cells and that of wild type was 40.2 ± 4.0 mg / g dry cells. After UV treat and H2O2, the free amino acids level in Δirr E mutant was 22.2 ± 1.4 and 38.5 ±1.0 mg / g dry cells, which of wild type was 19.5 ± 0.3 and 38.7 ± 2.9 mg / g dry cells respectively. Among of free amino acids, glutamic acid, alanine, leucine and lysine changed at different levels with stress shock change. For example, the content of alanine in wild type and Δirr E mutant was 6.4 ± 0.2 mg / g dry cells and 5.8 ± 0.7 mg / g dry cells under normal growth conditions, was 3.3 ± 0.1 mg / g dry cells and 4.3 ± 0.3 mg / g dry cells after UV shock. These results showed that Irr E influenced amino acids metabolism and involved in alanine metabolism.Our results dr₁894 may directly regulated the expression of ald（dr₁895）. To sutdy the funtion of DR₁894, we first analysed the the nucleic acid sequences and amino acid sequences, locution, organisition, 2D and 3D structure, function of dr₁894 by the tools of bioinformatics. Our results showed DR₁894 belonged the Lrp family transcriptional regulator, dr₁894 and dr₁895（ald） may be a gene cluster, and named as Ald R. In further study,we found that the expression of ald R was induced by abiotic stress, such as p H, heavy metal stress, H2O2, cold and UV radiation. We consturcted the mutant of ald R and found the ald R mutant have same ablity to UV radiation, dessciation and cold shock compared to wild type, whereas was sensitive to hyperoxide（cumene hydroperoxide and hydrogen peroxide）. RT-PCR results showed some genes related oxidative resistance were down-regulated, such as oxy R, sod C, kat E, osm C and grx.The carbon source utilization of aldR mutant was determined using Biolog system and growth in alterd 1% TGY medium adding different concentration alanine, the results showed that the alanine utilization of ald R mutant was lower than wild type. The cells dry weight of ald R mutant was lower than wild type when cultured to stationary phase in alterd 1% TGY medium adding alanine. After promoter analysis of ald gene by bioinformatic tools, we found a consensus region（CGCAACCGCGTTGCG） which Lrp famliy protein（Ald R） may bind to. The possible role of Ald R in the regulation of ald expression was investigated by determining the activity of β-galactosidase in the wild-type and ald R mutant strains with p RADZ3 grown in the presence or absence of L-alanine. And theβ-galactosidase activity of ald R mutant was lower 3-fold than wild type. In addtion, we found ald R mutant was sensitive to Na Cl and sorbitol shock than wild type, which may indicated alanine may decreased as osmotic protectant. At last, we found the content of alanine was changed in ald R mutant compared to wild type at different conditions by HPLC method.Cumulatively,ther free amino acids influenced resistance to oxidative stress of D.radiodurans, whereas the underlying mechanisms of free amino acids should be further study.