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Abstract

Perception of environmental changes and subsequent signal transduction are key aspects for the survival of an organism. In bacteria, two component systems (TCSs) consisting of a homodimeric receptor histidine kinase (HK) and a response regulator (RR) are the prevalent mechanism of stimulus sensing and cell signalling. They regulate motility, metabolism and development, and in addition, they are essential for virulence in a number of pathogenic species. The investigation of the signal transduction in TCSs is therefore essential to deepen our understanding of cell signalling and to develop a new class of antimicrobial drugs. The Geobacillus thermodenitri cans citrate receptor A (CitA) is used in this study as a model system for HKs. CitA is responsible for the activation of the citrate metabolism together with its cognate regulate receptor CitB. The periplasmic PAS (Per-Arnt-Sim) receptor is able to recognise the citrate molecule. The signal is then transferred across the membrane to a second, cytosolic PAS domain before reaching the conserved kinase core and triggering the auto-phosphorylation reaction. Although both citrate-binding and phosphate transfer has been described thoroughly, the signal relay from one domain to another remain poorly understood. Therefore, a combination of liquid- and solid-state NMR is adopted in this PhD project together with other biophysical techniques, such as X-ray crystallography and uorescence microscopy, to unveil details of the signalling mechanism of CitA. Our results show that an helical element is formed at the periplasm-membrane interface upon the citrate binding event both in the isolated periplasmic PAS domain and in a liposome-embedded CitA construct. The formation of the C-terminal - helix extends the results previously published on CitA and con rm the piston model proposed for the transmembrane helix motion. In addition, the existence of di erent dimer arrangements of the cytosolic PAS domain previously observed only in crystal structures is con rmed also in solution. These alternative dimeric conformations are then associated for the rst time with the relaying mechanism of the cytosolic PAS, shedding light on the role of the additional signalling domains interposed between the membrane and the kinase core.