Membrane Biochemistry

In general, we focus on the molecular mechanisms of bacterial membrane transport systems and particularly on potassium-translocating systems. K⁺ homeostasis in bacteria is essential for the survival of an individual cell and of a bacterial community. Among others, K⁺ uptake and release are required for osmoadaptation upon host invasion and electrical signalling within a bacterial biofilm. Thus, several bacterial K⁺ translocation systems have been suggested as general pathogenicity factors and are potential targets for newly developed drugs. We are aiming at elucidating the proteins’ mechanisms of K⁺ translocation as well as identifying molecules that regulate their function. Currently, we are focusing on three major topics: The molecular mechanisms of bacterial osmoadaptation, the control of potassium homeostasis by the second messenger cyclic di-AMP, and the role of potassium channels in bacterial cell-cell communication.

Potassium transport by the chimeric pump KdpFABC

K⁺ uptake at very low extracellular concentrations is essential to the survival of microorganisms, and important to a wide variety of pathogens. Many bacteria and archaea rely on the KdpFABC complex to fulfill this task. KdpFABC is unique for combining an active P-type ATPase pump (KdpB) with a channel-like protein from the Superfamily of K⁺ Transporters (KdpA), alongside the auxiliary subunits KdpF and KdpC. Together, these subunits form a chimeric active uptake system that ensures cellular survival under K⁺ limitation.

Considering the subunits from two very different classes of membrane translocators, KdpFABC poses a number of interesting questions: How does K⁺ pass through the complex during transport? How exactly is translocation of K⁺ coupled to the ATP hydrolysis in KdpB, required for active transport? What is the role of each subunit in the complex? How does the regulation of KdpFABC work? We approach these questions with a combination of biochemical methods, structural work (cryo-EM), and MD simulations. In addition to illuminating a unique membrane transporter complex, answering these questions could help understand an important process in bacterial pathogenesis. (Contact: Jakob Silberberg)

Molecular mechanism of potassium uptake through the channel KtrAB

Regulation of potassium uptake by the proton/potassium symporter KimA

Adaption to environmental changes is key for survival. Especially single cell organisms are exposed to drastic changes in their surrounding conditions. To keep the potassium homeostasis is critical to overcome osmotic fluctuations as well as maintain the whole cellular metabolism. KimA is part of the KUP (K⁺ Uptake) protein family. As a secondary active potassium transporter is KimA exploiting the proton motif force to take up potassium against its gradient even at low extracellular concentrations. KimA builds a homodimer in the plasma membrane whereby each monomer consists of a transmembrane part forming a pore and a cytoplasmic domain involved in regulation. The potassium uptake is inhibited by the cyclic nucleotide cyclic-di-AMP, which acts a newly discovered secondary messenger in bacteria. Still, it remains elusive how the regulation takes place on a molecular level. Which structural changes are triggered upon ligand binding? What are crucial amino acids relevant to fulfil inhibition? What cellular conditions are required for ligand binding? To mention only few open questions. We are trying to find answers by using structural biology (Cryo-EM/X-ray crystallography) as well as biochemical in vivo assays and in vitro assays (Ligand binding, protein purification etc.) approaches. (Contact: Michael Fuss)

Potassium-mediated Signalling in Biofilms

Bacterial biofilms can be found ubiquitously in a variety of environments from deep see vents to pipes of the drinking and sewage water system or medical indwelling devices and open wounds. Especially in the latter, biofilms impose a great threat to humanity by contributing largely to the development of antibiotic resistance and virulence. Recent studies have revealed an involvement of the putative potassium channel YugO in the development and upkeep of biofilms formed by the gram-positive soil-dwelling bacterium Bacillus subtilis. By sending waves of potassium throughout the biofilm, the cells on the interior of the biofilms can communicate their need for nutrients and induce a periodic halting of growth of the peripheral cells which allows nutrients to diffuse deeper into the biofilm. This ensures the survival of the cells in the interior of the biofilm and increases overall viability. However, open questions remain as to how this signalling process is achieved on a molecular basis. How is the opening of the potassium channel YugO achieved? Which ligand controls the opening and closing in response to the metabolic status of the cells? What is the role of the membrane potential in the signalling process and how do changes of intracellular potassium content and membrane potential influence biofilm viability? We are aiming at answering these questions using a wide range of in vivo and in vitro approaches including microfluidic studies of growing biofilms, the introduction of rhodopsins for targeted manipulation of the membrane potential as well as biochemical assays such as DSF, ITC and electrophysiology. Unravelling the molecular mechanisms of the signalling process could open the opportunity to a new field of antibiotic targets and therapeutical applications in the long run. (Contact: Celina Thiel)