Iron mobilisation in the bacterial cell

Nearly all organisms depend on iron for their existence. Iron is a key element for life because of its abundance in the earths crust and its useful chemical and physical properties - e.g. it is very good at transferring electrons, and activating oxygen for reaction with a range of substrates. Both of these are key processes in respiration - the means by which energy stored in foods is converted into a useable form - thus illustrating why organisms are dependent on this metal. Despite its abundance and useful properties, iron presents organisms with two problems. Firstly, ever since the oxygen levels in the earth's atmosphere began to increase, iron has been present largely in an oxidised and highly insoluble mineral form that is not readily available for utilisation by organisms. As a consequence, iron is often a limiting nutrient for growth and its availability can, for example, determine whether or not a bacterial pathogen can successfully colonise its host. Secondly, the chemistry that makes iron so useful means that it can also be highly toxic to the cell. To counter these problems, a whole range of smart mechanisms have evolved that enable organisms to scavenge iron from their environment, to stock pile it when it is found in excess of immediate requirements, and to maintain it within the cell in a non-toxic form. The latter two are achieved by iron-storage molecules that are found in all cell types from the simplest to the most complex. Iron-storage proteins belong to the ferritin super-family and have unusual structures consisting of 24 subunits arranged to form a large, spherical protein shell surrounding a central cavity where up to 4,500 iron atoms can be stored. Bacteria, which are the simplest of organisms, often contain two different types of ferritin: a ferritin (Ftn) which resembles closely the archetypal ferritins found in mammals, and a bacterioferritin (BFR) which are more distantly related heme-containing proteins, so far found only in bacteria. We and others have studied these iron-storage proteins in the model bacterium E. coli, and so understand in some detail how their synthesis is controlled and how they are able to store large quantities of iron. In times of environmental iron deficiency, bacteria and other organisms mobilise their iron stores to compensate for the lack of external iron. However, very little is known about how iron is released from iron stores, either in bacteria or in more complex organisms such as mammals. We and others have identified a small electron transfer protein, Bfd, which we propose plays a key role in iron mobilisation from BFR. We now wish to test our hypothesis and to characterise, for the first time in any organism, the processes involved in iron mobilisation from iron-storage proteins. The research proposed here uniquely brings together our expertises in the physiology of iron metabolism (at Reading) and the biochemistry of iron-storage proteins (at UEA) in order to tackle this major remaining question of iron metabolism. Using a truly multi-disciplinary approach employing a wide range of genetic, biochemical and bioanalytical methods, we will study in detail iron mobilisation from BFR and Ftn in E. coli, and the role played by Bfd and other relevant factors in this process. We will also clarify the respective roles of BFR and Ftn in the iron-storage process (it is unclear why E. coli and other bacteria possess two such distinct iron-storage proteins) and seek to identify other cellular factors that interact with these proteins. This work will have a major impact on our understanding of how bacteria utilise previously stored iron for synthetic processes. Joint with BB/D002435/1.

Nearly all organisms have an absolute requirement for iron. However, its poor solubility and bio-reactivity can lead to problems of iron deficiency and toxicity. An important, and almost universal, strategy adopted involves the use of iron-storage molecules (mainly the 24meric ferritins/Ftn and bacterioferritins/BFR). These proteins can detoxify iron, and provide an intracellular iron source for use when external supplies are restricted. Although much is known about the processes of iron storage, little is known concerning mobilisation of intracellular iron reserves. This is one of the last unexplored aspects of iron homeostasis and is the major focus of the studies proposed here. We will exploit our expertise in bacterial iron storage and complementary skills in molecular genetics and inorganic biochemistry to investigate the release of iron from iron-storage proteins (BFR and FtnA) in E. coli (and Salmonella). The main objective is to determine whether Bfd, a ferredoxin associated with BFR, is involved in iron release from BFR. There is significant evidence in support of this possibility. Most bfr genes are associated with a bfd gene so it is likely that any mechanism defined in E. coli (or Salmonella) will be of general relevance to bacteria. We will measure iron release in vivo by monitoring loss of 55Fe from cellular BFR and FtnA through autoradiographic analysis of native gels containing 55Fe-labelled soluble cell extracts. The effect of bfd mutation and overexpression, and lack of the BFR heme group, will be determined. We will also assess the ability of bfr and bfd genes from other bacteria to complement iron-storage defects (to test for the specificity of the Bfd-BFR interaction). The release of iron from FtnA will also be characterised using similar approaches and we will test whether ferritins, bacterioferritins or Dps proteins from other organisms can complement an ftnA mutation in E. coli since this will help to determine whether the release of iron from ferritins requires a species-specific protein-protein interaction. We will also investigate whether iron release from FtnA and/or BFR involves degradation of the corresponding protein shells (lysosomal ferritin degradation is thought to be the major iron release mechanism in eukaryotes). The specific roles of BFR and FtnA in Salmonella will be tested by performing careful growth comparison experiments. The release of iron from BFR and FtnA will also be examined using in vitro approaches. We will monitor iron release spectroscopically using a range of colorimetric ferrous-iron traps with appropriate reductants. In the cell there may be a number of alternate electron donors and iron acceptors and, using this approach, we will investigate the effect on iron release of varying the thermodynamic driving force. For BFR, the ability of Bfd (from E. coli and other organisms) to mediate this process, the role of its Fe-S cluster and the effects of various mutations (eg in BFR ferroxidase centre and heme-binding site) will be considered. The physical interaction of Bfd and BFR and electron transfer between them will be studied by a range of biophysical techniques. These will enable us to obtain affinities, stoichiometry and rates of interaction. We will test the importance of the BFR assembly state, and its heme, ferroxidase centre and iron core for the interaction. These studies should allow us to locate the likely site of interaction of Bfd with BFR. Other proteins that may interact with BFR, Bfd and FtnA will be isolated by affinity chromatography and identified by proteomic approaches. Cystallisation trials with Bfd and Bfd-BFR complexes will be initiated in collaboration with Dr K Watsons group (Reading). Finally, the role of a second ferritin (FtnB) in E. coli (and Salmonella) will be examined through co-assembly studies, followed by analysis of the iron uptake properties of the resulting FtnAB heteropolymer. (Joint with BB/D002435/1).