Griese lab

We use a combination of molecular biology, biochemical and structural biology methods, including X-ray crystallography and single-particle cryo-EM, to investigate metal homeostasis regulation in a bacterial model organism, the actinomycete Saccharopolyspora erythraea. We aim to obtain a complete understanding from the regulatory networks to the underlying molecular mechanisms.

Popular science presentation

Life is not possible without metals. Metal ions stabilize the structure of proteins and nucleic acids and catalyze chemical reactions that enzymes alone cannot accomplish. About half of all enzymes contain metal cofactors. Nature uses mainly the transition metals manganese, iron, cobalt, nickel, copper and zinc. These metals have different properties and fulfil different functions in the cell. Any imbalance in the cellular metal contents can lead to metals binding to the wrong enzymes, rendering these enzymes non-functional. Therefore, cells carefully balance metal uptake, storage and export so that their demands for each particular metal are exactly met. This process is called metal homeostasis.

In bacteria, intracellular metal concentrations are regulated by specialized transcription factors. These proteins recognize and bind to specific metals and specific DNA sequences, thereby either blocking or enhancing the expression of neighbouring genes. Thus, when manganese is bound to a sensor responsible for regulating manganese uptake, the sensor blocks transcription of genes encoding for manganese importers, so that no more importers are made when free manganese is already present in the cell.

We study the metal sensors from the bacterium Saccharopolyspora erythraea using different biochemical, biophysical and molecular biology methods. Presently we are investigating how they recognize their DNA target sites, aiming to understand the underlying molecular mechanism. Ultimately we want to unravel the complex regulatory networks these metal sensors control.

S. erythraea is best known for being the producer of erythromycin, an important antibiotic. S. erythraea and related species from the actinomycete family produce a large variety of compounds such as erythromycin that are called secondary metabolites and are often potent antibiotics. However, most strains do not produce much or any of the metabolites they can make under laboratory conditions. Many interesting compounds therefore remain to be discovered. Since the cellular metal status has a profound influence on metabolism, our research may help the continuing efforts to improve secondary metabolite production in S. erythraea and other actinomycetes.

Illustration of metal homeostasis in gram-positive bacteria such as S. erythraea, using the example of manganese. Different types of membrane importers pump manganese into the cell. When the intracellular manganese concentration reaches a certain level, manganese binds to the manganese-responsive repressor MntR and activates it, so that it can bind to the promoters of genes involved in manganese metabolism, such as those encoding manganese importers, and repress their transcription.

Research projects

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.

Crystal structure of the S. erythraea iron-dependent regulator IdeR in complex with iron and its consensus DNA recognition sequence (PDB ID 7B20; Marcos-Torres et al., NAR 2021). Each IdeR subunit binds two metal ions in adjacent binding sites. Two IdeR dimers bind to the palindromic DNA recognition sequence.

Group members

Research leader: Julia Griese

People

Julia Griese, Assistant Professor, Principal Investigator
email: julia.griese@icm.uu.se
phone: +46-18-471 4982

Oksana Koshla, PhD, Postdoc

Former members

  • Linda Juniar, PhD, Postdoc
  • Shruthi Krishnaswamy, Project student
  • Stijn Tieleman, Bachelor student
  • Deniz Biçer, Project student
  • Emmanuel Nji, PhD, Visiting Researcher
  • Carita Hammar Emas, Master student spring 2022
  • Yozlem Dilaver, Project student summer 2021
  • Francisco Javier Marcos-Torres, PhD, Postdoc 2018-2020
  • Dirk Maurer, PhD, Researcher 2018-2020
  • Mangirdas Kežys, Master student spring 2020
  • Malin Svensson, Master student spring 2020
  • Grim Elison Kalman, Master student spring 2019
  • Dilay Yılmaz, Project student summer 2018

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