Regulation of iron-sulfur cluster assemby in a facultative phototrophic alpha- proteobacterium

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Proteins with iron-sulfur (Fe-S) clusters fulfill many essential biological functions such as electron transfer, redox sensing, catalysis, or DNA replication and repair. Oxygen and reactive oxygen species (ROS) destabilize Fe-S clusters leading to the release of Fe2+ ions that in turn potentiate oxygen toxicity by the production of hydroxyl radicals in the Fenton reaction. Thus organisms had to develop systems that promote biogenesis of Fe-S proteins while protecting the cellular surrounding from the deleterious effects of free iron. Genes for such systems are therefore regulated by iron availability and oxidative stress. Escherichia coli and related species harbor two systems for Fe-S cluster assembly, Isc and Suf and the regulation of the corresponding operons was intensively studied. However, little is known about the regulation of Fe-S assembly in other bacteria.Rhodobacter sphaeroides is an alpha-proteobacterium, which forms photosynthetic complexes at low oxygen tension increasing the demand for Fe-S clusters and at the same time the risk for the formation of ROS. R. sphaeroides has the iscRS and suf genes arranged together and we have evidence for individual promoters for isc and suf genes, yet also for co-transcription. Moreover, differential RNAseq analyses also revealed transcripts, which are partially antisense to the iscR transcript. Our preliminary data demonstrated that iron availability and oxygen concentrations have a major impact on isc-suf operon expression and that several protein regulators are involved in its control, some of which harbour Fe-S, heme or iron cofactors. As in E. coli, IscR itself is a regulator for isc-suf expression and binds an Fe-S center. R. sphaeroides IscR lacks however the conserved residues involved in Fe S binding in other bacteria. Furthermore, the iron-dependently expressed sRNA SurS is controlled by IscR and affects genes of the sulfur metabolism and for iron storage. Hence regulation of Fe-S assembly in this phototroph appears to differ significantly from that in E. coli.We will elucidate the mechanisms that control expression of the isc-suf genes and coordinate Fe-S assembly and formation of photosynthetic complexes in R. sphaeroides. The effect of different growth conditions, protein and RNA regulators on the individual isc-suf promoters will be analyzed. Special emphasis will be given to the question of whether formation of photosynthetic complexes or rather photosynthesis affects isc-suf expression and what the underlying mechanisms are and how the Suf machinery affects formation of photosynthetic complexes. We will also attempt to define the new type of Fe S binding site and test whether this ligation type is of advantage for regulation in R. sphaeroides. We expect to elucidate new strategies for the regulation of Fe-S cluster assembly in the special surrounding of a phototrophic organism.