| Deinococcus radiodurans is unparalleled among all life in its capacity to survive oxidative stresses, and ionizing and ultraviolet (UV) radiations. IrrE is a global regulator, without ortholog in other genera, that greatly enhances the DNA repair capability and the radiation resistance of D. radiodurans. Our study demonstrated that heterologous expression of IrrE confers significantly enhanced salt tolerance in both E. coli and tobacco. However, the mechanisms and regulation of the enhanced stress resistance of IrrE-expressing cell is not fully understood. Furthermore, the heterologous expression of irrE increased tolerance to oxidative stress, heat shock and other stresses in E. coli. To better understand the global regulatory effects of IrrE we carried out a combined transcriptome and proteome analysis comparing of E. coli expressing irrE under normal and salt (NaCl) stress conditions.The main results obtained are as follows:1. In this work, we investigate that the IrrE-expressing strain was displayed better growth than the control strain with higher maximal cell density in LB medium. The carbon source utilization profile of E. coli expressing IrrE was determined using Biolog. IrrE greatly affected the types of substrates that were used and the efficiency with they were used. To better understand the global effects of IrrE on the regulatory networks, we carried out combined transcriptome and proteome analysis of E. coli expressing the IrrE protein. Our analysis showed that a large number of host genes with a significant change in expression, including those for trehalose biosynthesis, nucleotides biosynthesis, carbon source utilization, amino acid utilization, acid resistance, a hydrogenase and an oxidase. Also regulated were the EvgSA two-component system, the GadE, GadX and PurR master regulators, and 10 transcription factors (AppY, GadW, YhiF, AsnC, BetI, CynR, MhpR, PrpR, TdcA and KdgR). These results demonstrated that IrrE acts as global regulator and consequently improves abiotic stress tolerances in the heterologous host E. coli.2. We investigate that IrrE can be utilized to improve tolerance to various abiotic stresses. The results demonstrated that the heterologous expression of IrrE in E. coli increased tolerance to transient and continuous salt stress and heat shock, in addition to oxidative stress resistance. To investigate the regulatory mechanism of IrrE in response to salt stress, we performed comparative proteomics analyses on the IrrE-expressing strain and the control strain under salt stress condition. We detected significant changes for 124 proteins, with 66 proteins being upregulated in the IrrE-expressing strain. These up- or downregulated proteins can be grouped into 13 classes based on their common functional characteristics. Among them, a set of stress responsive proteins were upregulated by IrrE, including the molecular chaperone DnaK, the heat shock protein HslU, the osmotically inducible protein OsmY, catalase HPII KatE, general stress protein YhbO, the trigger factor Tig, the stress-inducible ATP-dependent protease Lon and stress response protein Dps. We found significant induction of general stress regulator RpoS, which exhibits a 3-fold-higher expression in the IrrE-expressing strain. After NaCl shock, the glycerol-degrading enzymes were down-regulated. The glycerol level in IrrE-expressing cells reached 37 nmol/mg dry weight and was approximately 2-fold higher than that in control cells.3. IrrE homologs from different Deinococcus species showed considerable N-terminal variation. Guided by the sequence alignments, four irrE deletions which removed 18, 26, 43 and 160 aa from the N-terminus of the 328 aa protein. Only the removal of 160 aa affected the UV and ionizing radiation resistance, abolishing it completely. |
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