The overall goal of our research is to establish the regulatory networks controlling metal homeostasis in Saccharopolyspora erythraea, an actinomycete best known for being the producer of the macrolide antibiotic erythromycin. To obtain a comprehensive understanding, we aim to map out the regulatory networks as well as dissect the underlying molecular mechanisms.

Tight regulation of intracellular metal ion concentrations is crucial for any organism’s survival. The key players in prokaryotic metal homeostasis are metal-responsive transcriptional regulators. These metal sensors control the expression of genes encoding proteins responsible for metal uptake, efflux and storage, as well as metalloenzymes, to adjust metabolism in accordance with the cellular metal status.

We are currently investigating the metal sensors from S. erythraea, addressing two main questions:

1. How do the metal sensors recognize their target sites in DNA?

The fundamental mechanisms underlying protein–DNA recognition are incompletely understood. Although it is well-established that eukaryotic transcription factors recognize their target sites in the genome using a combination of interactions with specific DNA bases (base readout) and recognition of the DNA shape (shape readout), it remains unclear how transcription factors specifically recognize subtly different sequence-dependent DNA shapes. Moreover, the contribution of shape readout to target recognition has so far largely been overlooked when it comes to prokaryotic transcription factors. However, it is beginning to emerge that shape readout plays an important role even in these less complex systems (Marcos-Torres et al., NAR 2021). A central aim of our research is to fill in the gaps in our understanding of this fundamental molecular recognition mechanism. The relatively simple bacterial model systems that the metal sensors constitute allow us to dissect their protein–DNA recognition mechanism in detail and obtain a deeper molecular mechanistic understanding that will help us to understand the overarching principles of DNA shape readout.

2. Which genes do the metal sensors regulate?

Metal homeostasis has been intensively studied for actinomycete pathogens, in particular of the genera Mycobacterium and Corynebacterium, where the iron sensor IdeR/DtxR is required not only to control iron homeostasis, but also to maintain virulence. In contrast, metal homeostasis networks are not well described for antibiotic-producing actinomycetes such as S. erythraea. These networks can be expected to influence the production of medically interesting secondary metabolites such as erythromycin. Although much effort has been made to improve production of this secondary metabolite, the complex regulation of this process is still not completely understood. Our research will contribute to a better understanding of how primary and secondary metabolism are regulated in S. erythraea, ultimately enabling an improvement of secondary metabolite production and potentially the discovery of new medically interesting metabolites.

To address these questions, we use molecular and microbiology methods, recombinant protein production and purification, X-ray crystallography, small-angle X-ray scattering (SAXS), single-particle cryogenic electron microscopy (cryo-EM) and nuclear magnetic resonance spectroscopy (NMR), as well as different biochemical and biophysical techniques to study DNA binding, such as electrophoretic mobility shift assays (EMSAs) and fluorescence spectroscopy.