Studies On The Mechanisms For Iron Transport And Iron Homeostasis Regulation In Shewanella Oneidensis

Iron is a necessary element for virtually all living organisms,and participates in an array of biological processes mainly as prosthetic group and cofactors in proteins,such as electron transfer in respiration and photosynthesis,oxygen transport,DNA biosynthesis,RNA modification,nitrogen fixation and so on.Therefore,the research on iron biology is important for understanding the fundamental metabolism and features of living organisms to survive or adapt to environment changes,which lays the foundation for engineering and utilizing them,especially microbes.In this study,the focused model is facultative anaerobic metal reduction microbe Shewanella oneidensis,which is famous for the respiration versatility.This characteristic is owing to a large repertoire of cytochrome c(cyt c)and iron-sulfur(Fe-S)proteins as electron carries and reductases.However,to date studies on iron transport and homeostasis remain rather limited,resulting in a lack of the basic understanding of these processes in S.oneidensis.On the basis of our previous findings,this dissertation focuses on mechanisms underpinning iron transport system and iron homeostasis regulation,and the main contents are: i)to uncover that the ferrous transport pathway Feo system and the putrebactin mediated siderophore transport for ferric are the two main iron uptake pathways in S.oneidensis;ii)to illustrate the interplay between the physiology impacts of the iron-responsive global regulator fur mutant and iron homeostasis in S.oneidensis;iii)to explore the function of small RNA Ryh B in iron homeostasis regulation.The loss of siderophore uptake pathway for ferric iron hardly affects the growth of S.oneidensis in regular LB medium,suggesting the presence of alternative iron transport systems.A genomic analysis reveals a putative Feo system(including A and B two subunits),which was subsequently confirmed to be a ferrous transporter and its loss resulted in a severe growth defect.This indicates that Feo system is the primary iron uptake pathway in S.oneidensis.Additionally,a strain without both of Feo and siderophore systems hardly grows,indicating that S.oneidensis almost entirely depends on these two routes for iron transport.S.oneidensis owns up to 9 siderophore receptors but only Put A is responsible for uptake of endogenous siderophore(putrebactin).But other siderophore receptors are not redundant;they can be used for uptake of siderophores released from other bacteria.Further work shows that some a-type hydroxy acids in rich medium or nature environments are able to facilitate iron import through the Feo system.Because of reactive oxygen species(ROS),induced by the overloaded intracellular iron is toxic to the cell,thus S.oneidensis should strictly maintain the iron homeostasis.It mainly depends on the global regulator Fur and the loss of Fur results in intracellular iron unbalance.Compared to the wild type,the free iron content significantly increases but the total iron and cyt c contents decrease in the fur mutant.Even with excessive iron in the medium,the fur mutant cannot recover from the defect.A proteomics analysis reveals that the Fur loss compromises activity of iron proteins,especially iron-sulfur proteins and hemoproteins.Interestingly,the expression of heme degradation enzyme Hmu Z is significantly higher in the fur mutant than that in WT.The physiological characterizations suggest that the biosynthesis defects of iron proteins and up-regulation of iron transport systems may account for increased levels of free iron.In parallel,the up-regulation of Hmu Z that augments heme degradation also promotes the content of free iron.Eventually,the low level of heme leads to the impairment of cyt c biosynthesis in the fur mutant.However,this impairment can be reversed not only by deletion of hmu AXZ but also by supplement of membrane permeable iron chelator,such as 2,2-dipyridy.All of these findings for the first time conclude that free iron is critically related to the Fur physiology in S.oneidensis.In the aspect of iron homeostasis regulation,a small regulatory RNA Ryh B also plays an important role.The function mechanism of Ryh B has been studied well in Escherichia coli.Interestingly,the sequence and predicted secondary structure of S.oneidensis Ryh B are much different from that in E.coli.A bioinformatic analysis predicts targets of Ryh B and some of them are verified by molecular biological approaches.Results reveal that the targets of Ryh B have species identity in S.oneidensis and some of them participate in iron homeostasis and regulation of heme levels.Overall,this dissertation reveals that S.oneidensis mainly owns two iron transport systems,including siderophore transport and Feo system.An explanation for the mechanism of a-type hydroxy acids facilitating iron transport is provided.The notion that Fur and Ryh B mediate the intracellular iron levels is established,and the role of multiple homologies of siderophore receptor Put A is illustrated.The findings presented here,on one hand,promote the understanding of iron regulation.On the other hand,it displays the complexity of biological iron homeostasis because some questions still remain unanswered.For example,the mechanisms of Fur loss strangely leading to the increasing level of free iron and the cooperation between Fur and Ryh B with its chaperone Hfq in regulation iron homeostasis have not been clear.