The Study Of Intracellular Manganeseion Regulation Protein In Deinococcus Radiodurans

Homeostasis of metal ions is closely related to the physiological processes of growth in bacterial cells. Some transition metal ions, such as Fe(â…¡), Mn(â…¡), Zn(â…¡), Ca(â…¡), could not only consider as the protein cofactors joining in the reaction of oxidizing reduction, but also set as the signal factors involving in the regulation of gene transcription and affecting the cytotoxicity and extreme resistance. Therefore, it is essential in regulating metal ions as to maintain normal physiology activity in bacterial cells. Moreover, the study of the regulation of metal ions in bacterial cells has always been a hotspot in the field of microbiology.Deinococcus radiodurans, an important member of extreme micro-organisms family, has high resistances to ionizing radiation, ultraviolet (UV) radiation, chemical mutation and desiccation. However, more and more studies show that adaptability of D. radiodurans is benefit from its anti-oxidative ability. Furthermore, the high concentration of intracellular manganese ions is always considered to be one of the most important antioxidation mechanisms. Therefore, D. radiodurans is a great kind of model organisms to study in regulating manganese ions metabolism. By studying the regulatory metabolism of manganese ions, we could not only to delve more deeply into the extremely resistant mechanism of D. radiodurans, but it is also significant to widen the regulatory mechanisms of metal ions in bacterial cells. In this study, by researching the manganese acquisition and efflux mechanism, we investigated the regulation of intracellular manganese homeostasis of D. radiodurans. The results of the studies are listed as follows:1. First of all, we identified and evaluated a manganese efflux protein (Drl236) in D. radiodurans. Dr1236 is the second manganese efflux protein that indentified from bacterial and is also first verified as the third type of efflux protein. By constructing the Dr1236 deletion mutant, we found the resistance of Dr1236 to manganese ions was decreased. whereas the level of intracellular manganese concentration is markedly increased. This indicated that Dr1236 play an critical role in the manganese resistance mechanism and in regulating intracellular manganese levels. Further, compared with the wild-type strain, the bacteria has extremely higher resistance to ionizing radiation, ultraviolet (UV) radiation and H2O2 after intracellular manganese content in mutant increased. The results indicated that the manganese ions are indispensable involved in the resistance of D. radiodurans cells, and then the protective effects may be realized by reducing the challenge of free radical to protein. Though the mutant cells accumulated higher intracellular concentration of manganese ions and extreme resistance was also increased differently, the cell lifespan of mutant cells is far shorter than the wild-type strain. The result showed that manganese ions at high concentrations are toxic to cells. Moreover, manganese efflux protein plays the key role in D. radiodurans.2. Dr0865, which encodes the only Fur homologue, may be involved in the regulation of intracellular manganese levels in D. radiodurans. E. coli expression system was used to express the dr0865 and its biochemical characteristics were also investigated. Additionally, bioinformatics study showed that Dr0865 probably has three metal-binding sites and add up to a distinct advantage of biological function. DNA binding activity assay showed that Dr0865 could bind with the promoters of dr2283,dr2284,dr2523, the binding activities are positive proportional to the concentration of metal ions. On the other hand, Dr0865 could not bind with the promoter of dr 1709. (3-galactosidase activity assay showed that Dr0865 binds with promoters of dr2283,dr2284,dr2523 could promote gene transcription. Moreover, it should be considered that the DNA binding activity of Dr0865 is dependent to metal ions concentration and the mutant resistance of Dr0865 is enhanced to H2O2. We supposed that dr2283,dr2284,dr2523 probably encode a new kind of Fe2+efflux protein. Furthermore, the regulatory mechanism of Fe ions is still unclear and needs further study.3. Dr2539 belongs to the MntR/DtxR family, and this family plays important role in intracellular Mn- or Fe- homeostasis regulation. A solubility-enhancing partner of translation initiation factor (IF) was fused to dr2539, and Dr2539 was successfully expressed in vitro. EMSA results indicated that Dr2539 can bind with the dr1709 promoter and the DNA binding activity of Dr2539 depended on the concentration of Mn and Fe. It is quite different with previous reported MntR because it is always Mn specifically binding protein. Bioinformatic analysis has shown that Dr2539 has DtxR characteristics, and further site-directed mutagenesis was carried out in order to explain why Dr2539 can response to both Mn and Fe. Different Dr2539 mutants were constructed and complemented to dr2539 null mutant. By examine the manganese resistance of complemented strains (C-D11M,C-H98Y,C-E101D,C-Tru), we found that the manganese resistance of C-D11M and C-Tru decreased compared with wild strain. Surprisingly, the resistance of C-H98Y decreased dramatically in 6-7mM Mn(II), while that recovered in>7mM. It is consistent with previous reported three step active model of DtxR, therefore, we suppose that the Dr2539 may be active in DtxR-like pattern, and it may also be involved in Mn/Fe homeostasis regulation.In sum, we investigated the manganese efflux ans acquisition system, and the results indicate that the manganese involved in the extreme resistance of D. radiodurans. Moreover, the biochemical characteristics of metalloregulator Dr0865 and Dr2539 suggested that the intracellular metal concentration regulated system in D. radiodurans may be different with that of presented reported bacteria, and our studies gave a good first step for further Dr0865 and Dr2539 structure analysis.