Tight regulation of intracellular metal ion concentrations is crucial for any organism’s survival and plays a central role in the host-pathogen interaction. While metal homeostasis has been studied in different model organisms for a long time, evidence that different metals are not regulated independently from each other has only recently begun to emerge. Manganese and iron are particularly interesting because, although they have fairly similar chemical properties, they play opposing roles in the cell when it comes to oxidative stress: iron exacerbates oxidative stress, whereas manganese protects against it. Their intracellular concentrations are therefore cross-regulated.
The key players in prokaryotic metal homeostasis are metal-sensing transcription factors which regulate the expression of target genes such as metal transporters. We investigate the structure and function of the manganese, iron and peroxide sensors in an actinomycete model organism, Saccharopolyspora erythraea.
Currently we are addressing two main questions:
- How do metal sensors differentiate between different metal ions? Transition metals in general, and iron and manganese in particular, are biochemically difficult to tell apart, but they fulfill different cellular functions. Metal-sensing transcription factors must therefore be able to distinguish between them, but how they achieve selectivity is not fully understood.
- How do metal sensors recognize their DNA-binding sequences? Bacterial genomes generally encode several homologous metal sensors with different metal specificities. The DNA-binding domains and the DNA recognition sequences of different transcriptional regulators from the same protein family are usually very similar to each other, yet each regulator acts on a specific set of genes. Sequence specificity appears to be achieved by a combination of interactions with specific DNA bases and recognition of the DNA shape. It is unclear to which extent each of these factors contributes to specificity in metal-sensing transcription factors, and how shape recognition is accomplished.
Our methods include molecular biology, protein production in heterologous expression hosts, protein purification, X-ray crystallography and small-angle X-ray scattering, as well as different biochemical and biophysical techniques to study DNA and metal ion binding, such as electrophoretic mobility shift assays (EMSAs), fluorescence spectroscopy, microscale thermophoresis (MST) and isothermal titration calorimetry (ITC).
The overall goal of our research is to unravel the links between manganese and iron homeostasis and the oxidative stress response in actinomycetes. Metal homeostasis plays an important role in the production of medically interesting secondary metabolites by actinomycetes, such as the broad-spectrum antibiotic erythromycin by S. erythraea itself, as well as in virulence of pathogenic actinomycetes such as Mycobacterium tuberculosis. A better understanding of metal homeostasis in actinomycetes can therefore help in the development of new antibiotics in two ways, by enabling the production of novel secondary metabolites, as well as by identifying potential drug targets.