Effects Of Ferritin On Iron Response,Iron Homeostasis,Antioxidation And Tumorigenicity Of Agrobacterium Tumefaciens

Agrobacterium tumefaciens is a gram-negative plant pathogenic bacterium that lives in the soil.It is a member of alphaproteobacteria and can cause crown gall tumor disease in dicotyledonous plants under natural conditions.In the process of infecting the host,A.tumefaciens can transfer and integrate the T-DNA of its Ti plasmid into the plant genome,so that the plants can undergo genetic transformation.Based on this characteristic,A tumefaciens is widely used in most dicotyledons and some monocotyledons for transgenic engineering.At present,the transformation mechanism of A.tumefaciens is clear,but there are still many unknown factors that affect its tumorigenic ability and transgenic efficiency.Among them,iron metabolism homeostasis and the ability of antioxidant stress of A.tumefaciens is closely related to its tumorigenic ability.Ferritin plays an important role in maintaining iron homeostasis and dealing with oxidative stress for bacterial cells.There are three types of ferritin in bacteria:ferritins(Ftn),bacterioferritins(Bfr)and the DNA-binding protein(Dps).However,there is only one Bfr and one Dps in A.tumefaciens C58.Compared with other bacteria,A.tumefaciens Dps lacks 11 amino acids at N-terminal.In vitro experiments show that A.tumefaciens Dps is unable to bind DNA but still play an important role in iron detoxification.A.tumefaciens Bfr has 8 more amino acids at N-terminal,and its specific function is still unknown.Based on the uniqueness of Agrobacterium tumefaciens ferritin and the influence of iron metabolism on the pathogenicity of A.tumefaciens,the study of the functions of A.tumefaciens Bfr and Dps has very important and potential application value.On the basis of previous research in the laboratory,the effects of Ferritin(Bfr and Dps)on iron response,intracellular iron balance,antioxidation and tumorigenicity of A tumefaciens were studied by reverse genetics.The following research results have been achieved.(1)By measuring the iron binding capacity and iron release rate of A.tumefaciens ferritin(Bfr and Dps)in vitro,we found that Bfr can bind approximately 3000 iron atoms,BfrM60L(a variant of Bfr)can bind approximately 2300 iron atoms and Dps can bind approximately 500 iron atoms.Bfr variant BfrM60L is the replacement of methionine Met from heme binding site of Bfr to leucine Leu,which can still bind iron,but its binding capacity has declined.The iron release rate of Bfr is significantly faster than that of BfrM60L In this study,we verified that A.tumefaciens Bfr and Dps are ferritin with iron storage function and A.tumefaciens Bfr is heme-Bfr.We also determined the iron storage capacity of A.tumefaciens ferritin.(2)The utilization of soluble iron in culture medium and growth curves of A.tumefaciens wild-type strain and ferritin deficient strains(?bfr,?dps and ?bfr-dps)were measured by AB-sucrose medium with different iron concentrations.We found that the deletion of Bfr would affect the growth of A.tumefaciens after depletion of soluble iron in the medium,and the deletion of Dps would not affect the growth of A.tumefaciens.In addition,the deletion of Bfr decreased the utilization rate of soluble iron by Agrobacterium tumefaciens.It is suggested that Bfr can regulate the intracellular free iron content of A.tumefaciens and the utilization rate and amount of environmental iron by A.tumefaciens through its iron storage and iron release function,and ultimately affect the growth of A.tumefaciens.(3)By comparing the iron content of A.tumefaciens wild-type strain and ferritin deficient strains,we found that under 9 ?M iron concentration,only the deletion of Bfr(?bfr and ?bfr-dps)seriously affected the iron storage capacity of cells.The deletion of Dps(?dps)had little effect on the iron storage capacity of cells.Under 20 ?M iron concentration,the deletion of Bfr and Dps(?bfr,?dps and ?bfr-dps)will obviously affect the iron storage capacity of cells.It is suggested that Bfr rather than Dps plays a major role in regulating iron homeostasis and iron storage in A.tumefaciens.(4)By comparing the antioxidation of A.tumefaciens wild-type strain and ferritin deficient strains,we found that the deletion of ferritin(especially the absence of Bfr)reduced the survival rate of A.tumefaciens under the short-term treatment of high concentration hydrogen peroxide.In the long-term treatment with low concentration of hydrogen peroxide,the deletion of ferritin will significantly affect the growth of A.tumefaciens.It is suggested that ferritin plays an important role in the response to oxidative stress.And Bfr has a more significant effect on the antioxidant capacity of A.tumefaciens.(5)By comparing the tumorigenic capacity of A.tumefaciens wild-type strain and its ferritin deficient strains,we found that the tumorigenic capacity of wild-type strain and ferritin deficient strains to potato tubers was basically the same in iron-free environment;the tumorigenic capacity of wild-type strain to potato tubers was significantly higher than that of ferritin deficient strains in the iron environment.It is suggested that ferritin affects the tumorigenicity of A.tumefaciens by regulating the iron homeostasis of A.tumefaciens.In addition,Bfr and Dps had the same effect on the tumorigenicity of A.tumefaciens in iron containing environment,but they had different effects on the iron storage capacity of A.tumefaciens.It is suggested that ferritin not only affects the tumorigenicity of A.tumefaciens by regulating the iron homeostasis of A.tumefaciens,but also affects the tumorigenicity of A.tumefaciens by other mechanisms.These results indicate that Bfr affects the iron response,Iron homeostasis,antioxidation and tumorigenicity of A.tumefacies.Dps only affects the antioxidation and tumorigenicity of A.tumefaciens,but has little effect on the iron storage capacity of A.tumefacies.Therefore,Bfr plays a more important role in A.tumefaciens than Dps.