Identification And Charaterization Of Superoxide Dismutase From Marine Synechococcus

Marine Synechococcus species are single-celled picophytoplankton, and their distribution is so ubiquitous that can be found in vast tracts of the world’s oceans. They contribute significantly to chlorophyll （Chl） biomass and primary production underlying their ecological importance as significant contributors to photosynthesis, carbon fixation and food chain of marine ecosystem. Various kinds of environmental stress, such as high light irradiation, high/low temperature, salt stress or certain nutrient limitation may lead to oxidative stress which often take palce in their natural habitats. SODs are the first line of defence to alleviate oxidative stress in living organisms that survive in oxic environments, which can be classified into FeSOD、MnSOD、Cu/ZnSOD and NiSOD according to the metal they incorporated. To our knowledge, FeSOD and MnSOD have been intensively studied in the past decades, while little information is available about the expression, location and regulation of Cu/ZnSOD and NiSOD in cyanobacteria. Genetic manipulation technology on marine Synechococcus has not been widely applicated, and metalloprotein SODs function after incorporating corresponding metal. So metal clean technology was adopted to study the roles of the two kinds of SODs. Synechocystis sp. PCC 6803 is convenient to genetic manipulation, and it was used as a host to study the roles of Cu/ZnSOD and NiSOD. The detailed content and results of this study is as follows, There are two putative sod genes in coastal Synechococcus sp. CC9311, while there is only one sodN in open-ocean Synechococcus sp. WH8102. The effects of copper （Cu） or/and nickel （Ni） limitation on the growth and photosynthetic characteristics were studied in two marine Synechococcus strains. Cu and Ni colimited the growth of Synechococcus, and their maximal growth rates were achieved when both nutrients were added simultaneously to cultures. Coupled to the knowledge of Cu and Ni utilization in cyanobacteria, it was assumed that superoxide dismutase （SOD） could play key roles in growth of the two strains. Synechococcus sp. CC9311 possesses two proposed genes for SODs:sync₀755 and sync₁771, which were confirmed to have SOD activity in this study. According to the existence of conserved Ni or Cu binding sites and signal sequences in their protein sequences, it could conclude that sync₀755 and sync₁771 encoded NiSOD and Cu/ZnSOD, respectively. Subcellular localization results indicated that NiSOD was cytosolic, whereas Cu/ZnSOD was localized both in the cytosol and thylakoid membrane. Cu/ZnSOD has a predicted N-terminal signal peptide, so it is probably a lumen protein. No obvious change of NiSOD amount was observed under Cu limitation condition, implying that NiSOD can not be completely complementary to Cu/ZnSOD in marine Synechococcus. This was maybe related to their different subcellular localization. Previous study reported that when PSI activity in Synechococcus sp. WH8102 was decreased, plastoquinone terminal oxidase （PTOX） could extract electron from electron transport chain to avoid excess excitation of PSII. The electron transport rates of whole chain were significantly increased in both strains under Cu limitation condition, which suggested that a PTOX-like protein could be involved in extracting electron from the intersystem electron transport chain of Synechococcus sp. CC9311. Meanwhile, water-water cycle intermediated by thylakoid membrane-attached Cu/ZnSOD may be an alternative explanation for the accelerated electron transport rates of the whole chain in Synechococcus sp. CC9311. Knockout sodN in Synechococcus sp. CC9311 was lethal for the strain, so we choosed to heterogeneous expression this gene in Synechocystis sp. PCC 6803. Unfortunately, no NiSOD protein was detectable in transformed Synechocystis sp. PCC 6803 with immunoblotting or in gel activity staining after multiple attempts. It revealed that the maturation process of nickel-protein was complex. Maybe more auxiliary factors are needed to produce a functional and stable NiSOD by heterogeneous expression.Synechocystis sp. PCC 6803 possesses only one sod gene sodB encoding iron superoxide dismutase （FeSOD）. It could not be completely knocked out by direct insertion of the kanamycin resistance cassette. When the promoter of sodB in wild type Synechocystis was replaced with the Cu-regulated promoter PpetE, completely segregated PpetE-sodB strain could be obtained. When this strain was cultured in Cu-starved BG11 medium, Chi a content was greatly reduced, the growth was seriously inhibited, and the strain was nearly dead during the 8 days’growth, while wild type grew well under the same growth condition. These results indicated that sodB was essential for the photoautotrophic growth of Synechocystis. The reduction of sodB gene copies in Synechocystis genome rendered the cells more sensitive to oxidative stress produced by methyl viologen and norflurazon which generate superoxide anion and singlet oxygen, respectively. SodB still could not be completely knocked out after actively expression of sodC （encoding Cu/ZnSOD） from Synechococcus sp. CC9311 in the neutral site slr0168 under the control of psbAII promoter, which means the function of FeSOD could not be completely complemented by Cu/ZnSOD. Heterogenously expressed sodC increased the oxidation tolerance of Synechocystis sodB knockdown mutant. The expression of Cu/ZnSOD enhanced the repair of photodamaged PSII, but did not protect PSII from photodamage. Cu/ZnSOD played a role in repair of photodamaged PSII while FeSOD did not invovle in repair of photodamaged PSII. Membrane fractionation followed by immunoblotting revealed that FeSOD was localized in the cytoplasm, and Cu/ZnSOD was localized in the soluble and thylakoid membrane fractions of the transformed Synechocystis. Cu/ZnSOD has a predicted N-terminal signal peptide, so it is probably a lumen protein. The different subcellular localization of these two SODs may result in the failure substitution of sodC for sodB.