Bacterial iron acquisition

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Iron is essential for most forms of life. It is a cofactor for cell sustaining reactions such as mediating single electron transfer reactions of the electron transport chain or the formation of deoxyribonucleotides. This special reactivity of metals such as iron necessitates tight control of uptake and handling by the cell to avoid toxic side effects. The research program is directed at understanding the cellular mechanisms of iron homeostasis in microbes. The Fet system is used by bacteria from many phyla to acquire iron under microaerophilic or low pH conditions. The best studied Fet proteins are from Escherichia coli and Campylobacter jejuni. The system is found broadly in nature in both animal associated and environmental bacteria. The core of the system is composed of FetM, an iron-transporter, and FetP, a periplasmic copper-containing protein. The genes encoding FetMP are part of a larger cluster that encodes 5 additional proteins (FetABCDE). In C. jejuni, the FetA-E are required for growth in cell culture under iron restriction. The copper-sites of FetP are required for iron-uptake function and the FetP dimer interacts with a periplasmic domain of FetM. We hypothesize that FetP is an oxidoreductase that delivers iron to FetM for transport into the cell. The functions of FetA-F are remain unknown. FetA is a predicted transmembrane protein with a large periplasmic region composed of a DUF2318 domain and YHS domain. The DUF2318 domain is predicted to harbour an iron sulfur cluster and we hypothesize the FetA-F is a redox system to control the oxidation state of iron through the action of FetP. Using E. coli as a model system, the specific aims are: 1.We will identify interaction interface between FetP and FetM and determine if the interaction between these proteins is metal dependent. We will use site-directed mutagenesis to probe the interface and structural biology to build a structural model of the complex to gain functional insight. Binding studies will be performed in the presence of transition metals with a focus on iron in the ferric and ferrous oxidation states. 2.We will characterize the role FetA in iron-uptake by FetMP. We have expressed a soluble construct containing two periplasmic domains of FetA (DUF2318/YHS). We will engineer the expression system to produce this domain with an iron sulfur cluster and characterize the redox and properties. We will test if the periplasmic localization of this domain is dependent on the TAT secretion system. Acidic conditions reduce the affinity of siderophores for iron and reduced oxygen favours the ferrous iron oxidation state. The results of this work will inform on how bacteria are able to acquire iron these conditions commonly found nature. This understanding is fundamental to broadening the conditions under which bacteria can be used for synthetic biology.