Identification And Function Of Iron Permease FTR1 In The Cyanobacterium Synechocystis Sp. PCC6803

Iron is necessary mineral element for all the living organisms. Iron serves as an important role in photosynthesis, respiration, and nitrogen metabolism. Iron content is rich in the earth’s crust, but most of iron exists in the form of precipitation and its bioavalablity is very low. Cyanobacteria as important primary product in the world live in iron-limiting marine environment. But the growth of cyanobacteria need more iron (10 fold than Escherichia coli) to meet their own needs. Therefore, cyanbacterica form an efficient iron uptake mechanism in the long-time evolution. Cyanobacteria have multiple iron transporters on the plasma membrane. It is very beneficial for the iron uptake and transport of cyanobacteria. Synechocystis sp. PCC6803 is a unicellular cyanobacteria, it can use glucose for heterotrophic growth and also have natural exogenous DNA transformation system. Thus it becomes one of the model organisms for cyanobacteria molecular genetics research. The current research about the specific mechanism of iron uptake is not very clear. In this study, a new iron transporter Slr0964 has been identified, which has the characteristics of iron permease. Slr0964 locates on the plasma membrane of Synechocystis sp. PCC6803. The slr0964 gene plays an important role in iron uptake and transfer process of the cyanobacterial speices. Besides Slr0964, other iron transporters in the plasmic membrane of Synechocystis sp. PCC68O3 have also been investigated in this research.The main research contents and results are as follows:Firstly, the iron uptake function of Slr0964 protein in Synechocystis sp. PCC6803 has been investigated.The slr0964 mutant showed seriously decline growth rate and chlorophyll content compared to the wild-type Synechocystis sp. PCC6803 (WT6803). In the transcriptional level, RT-PCR and qRT PCR results proved that the slr0964 gene was induced in iron deficient conditions. Complementary and overexpression assays showed that the slr0964 complementary strains can restore the iron deficient phenotype of the mutant, and the over-expression strains grew much better than WT6803 under iron deficient conditions. The iron toxicity experiments showed that the slr0964 overexpression strains grew more slowly than WT6803. Therefore, it can be concluded that the slr0964 gene participates in iron uptake process in the Synechocystis sp. cells. Furthermore, the iron content and iron uptake rate results conformed that the slr0964 mutant strain exhibited a remarkble decrease compared to WT6803 and the slr0964 gene plays important role in iron uptake. By bioinformatics analysis, it showed that Slr0964 is likely to be an iron permease and has mutil-transmembrane domains. The iron permases from Yeast (FTR1) and Escherichia coli (EfeU) could resorte the iron defienct phenotypes, suggesting the encoding product of slr0964 gene have iron permase-like function. It is a further evidence that slr0964 gene play an important role in iron uptake. Water two phase method and sucrose density gradient centrifugation proved that the Slr0964 protein locates on the plasma membrane of Synechocystis sp. PCC6803. Slr0964 may play a specificity role for iron uptake, from the results that the mutation of the slr0964 gene did not affect the uptake of other metal elements such as Zn2+ and Mn2+.Secondly, the functions of other iron transporters besides Slr0964 in the plamic membrane have been investigated in Synechocystis sp. PCC6803. It is known that there are four iron transporters on the plasma membrane of Synechocystis sp. PCC6803, including Slr0327 (FutB), Slr1392 (FeoB), S111263 (CDF) and Slr0964 (FTR1). In addtion, periplasmic space proteins such as Slr0513 (FutA2) and Slr1295 (FutAl) are also involved in the iron uptake and transport. By homologous recombination, many mutants were obtained with double or triple genes been knockout or knockdown. By analyzing the growth rate and the intracellular iron concent of the mutants, it could be found that the plasma membrane protein Slr0327 (FutB) play a vital role in Synechocystis sp. PCC6803, and the other transporters play supplementary role during the cells grow under iron defient conditions. Besides, mutation of the slr0513 gene resulted in a resistance to high iron concentration stress, suggesting that the periplasmic space protein FutA2 plays a role of transport iron from periplasmic space to cytoplasm.