- University of Reading
- Start date
- End date
- 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/D001943/1.
- Funding Source
- Biotechnology and Biological Sciences Research Council
- Project number
- Natural Toxins