The NreA/NreB sensors of Staphylococcus carnosus: A sensor complex for coordinated-sensing of O2 and nitrate.

• Applicant: Professor Dr. Gottfried Unden
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 260693735

Bacteria respond rapidly to environmental stimuli, and adapt cellular composition, metabolism and other properties to optimal growth and survival. Electron acceptors like O2 and nitrate represent major stimuli for many bacteria that control expression of many genes, including those for aerobic and anaerobic respiration, fermentation, but also virulence and biotechnologically relevant traits. In Staphylococci response to O2 and nitrate turned out to use new components and to follow new strategies. The oxygen sensor NreB is part of a cytoplasmic two-component system (NreB/NreC). The NreB sensor kinase consists of a sensory PAS domain that binds a labile [4Fe-4S]2+ cluster for O2-sensing which is degraded by O2 to a [2Fe-2S]2+ cluster, and then completely lost, forming apoNreB. The latter forms are inactive in transcriptional activation of the genes of nitrate respiration. The nitrate receptor NreA is a small cytoplasmic GAF domain protein that binds nitrate at the active site. The structure of this novel nitrate receptor was characterized in collaboration with T. Stehle (Tübingen). Interaction between O2 and nitrate sensing was continued in Mainz. NreA interacts directly with NreB, and the interaction is controlled by nitrate. Nitrate-free NreA binds to NreB which is then inhibited in autophosphorylation. Consequently, phosphorylation of NreC and expression of target genes is decreased. The NreA/NreB interaction and inhibition of NreB phosphorylation is relieved when NreA exists in the nitrate-bound form. Thus NreA and NreB cooperate in a NreA/NreB sensor complex, where the primary sensor NreB is modulated in activity by the nitrate-receptor NreA; NreA is not able of directly controlling target genes. This mode of O2/nitrate sensing is in fundamental contrast to that of other bacteria like E. coli, Bacillus, Pseudomonas.The novel NreA/NreB sensor complex will be characterized at the molecular level for its characteristic properties regarding NreA/NreB interaction and control of the O2-sensor NreB by NreA and nitrate. Thus (i) the physical interaction sites on NreA and NreB, and (ii) the sites in NreB for the intramolecular signal transfer (FeS cluster, PAS domain regions, kinase domain) that are modulated by NreA, shall be identified at the molecular level. In addition (iii), NreB contains an active phosphatase domain, which will be characterized in its role for NreB activity. Structural studies (iv) on NreB have been started and will be continued (collaboration with R. Lancaster, Homburg).

• DFG Programme: Research Grants