Function of c-di-AMP in archaea

Very little knowledge is available about the occurrence and function of cyclic nucleotides as second messengers in Archaea. We have demonstrated that the extremely halophilic archaeon Haloferax volcanii produces c-di-AMP and now want to understand its function in this organism. First experiments hint at a function in osmoregulation as an H. volcanii strain producing less c-di-AMP swells up under hyposalt conditions (Braun et al., 2019). Moreover, the euryarchaeal diadenylate cyclase exhibits an additional N-terminal domain otherwise found in pyruvate kinases. We want to study the influence of this domain on the function of the diadenylate cyclase.

Molecular basis for the control of K+ uptake via KtrAB and KimA by cyclic di-AMP

Potassium homeostasis is essential for bacterial survival and controlled by the orchestrated function of various K+ importers and exporters. Recently, cyclic di-AMP has been identified as overarching regulatory signaling molecule for K+ homeostasis in different Gram-positive bacteria. It regulates the uptake and release of potassium by controlling gene expression and protein activity. In Bacillus subtilis, cyclic di-AMP directly binds to K+ importers KtrAB and KimA leading to their inhibition.

We aim to shed light on the underlying mechanisms of protein deactivation elucidating the molecular principles of cyclic di-AMP binding and transport inhibition.

Structural biochemistry of c-di-AMP degrading enzymes and their regulation

In our group, we are working on the biophysical and structural analysis of proteins in the c-di-AMP framework. Within this project we are focussing on the characterization of DHH/DHHA1-type phosphodiesterases that degrade c-di-AMP in two steps, i.e. c-di-AMP → 5’-pApA (step I) carried out by GdpP-type multidomain membrane proteins and 5’-pApA → 2AMP (step II) carried out by soluble phosphodiesterases.

Besides of the lacking structural information of the multidomain protein and the role of the GGDEF and PAS domains, we aim to solve the question why GdpP-type and soluble PDEs have different substrate specificities despite their highly homologous DHH/DHHA1-domains.

Cyclic di-AMP metabolism and functions in Streptomyces

The Gram-positive soil bacteria streptomycetes are an important source of diverse antibiotics and other natural products. The production of these secondary metabolites is temporally and genetically coordinated with a complex developmental life cycle involving growth as vegetative mycelium and dispersion via spores.

The aim of the proposed work is to analyse and characterise in molecular detail the roles of c-di-AMP in S. venezuelae. We will determine how c-di-AMP contributes to differentiation and stress survival strategies employed by Streptomyces spp. and plan a detailed analysis of an up to now uncharacterised cyclic di-nucleotide in these bacteria.

Signal transduction by the essential signal molecule
c-di-AMP in Bacillus subtilis

Bacteria use specific signaling nucleotides as second messengers to adapt their cellular activities to changing environmental conditions. Among all known second messengers, cyclic di-AMP is unique as it is essential in the Gram-positive model organism Bacillus subtilis and in related pathogenic bacteria. Several protein and RNA targets for c-di-AMP have been identified. We have demonstrated that the control of potassium homeostasis is the essential function of c-di-AMP.

In this project, we want to unravel the mechanistic details of c-di-AMP action as well as the so far unknown functions of the target proteins that are not directly involved in potassium homeostasis.

Osmolarity-dependent control of cellular c-di-AMP levels and characterization of osmolyte transporters in Listeria monocytogenes

Bacteria rely on complex signal transduction systems to cope with environmental changes and respond appropriately. One important environmental parameter is osmolarity. The second messenger c-di-AMP controls the uptake of osmolytes in the Gram-positive bacterium Listeria monocytogenes as well as in phylogenetically related bacteria.

The project aims at elucidating the molecular mechanism of how the c-di-AMP-synthesizing and -degrading enzymes sense osmolarity to adjust the cellular osmolyte concentration accordingly. Genetic, biochemical and structural methodologies are applied to unravel how c-di-AMP affects the activity of osmolyte transport systems in L. monocytogenes.

Multiplicity and functional diversity of cyclic mononucleotide signaling in Sinorhizobium meliloti

Many members of the alphaproteobacterial order Rhizobiales are able to establish a nitrogen fixing symbiosis with leguminous host plants. These bacteria possess an exceptional high number of mononucleotide cyclases. The genome of Sinorhizobium meliloti, the microsymbiont of Medicago, Melilotus and Trigonella plants, encodes 28 putative class III adenylate- and guanylate cyclases (AC/GC), seven potential cNMP phosphodiesterases and ten CRP-like proteins potentially binding cNMPs. This high number of cAMP-/cGMP-related proteins suggests their functional diversification.

In the project, we aim at exploring the catalytic activities of mononucleotide cyclases, as well as sensory inputs and biological roles of cNMP-signaling in S. meliloti.