CHIMERIC TRANSDUCERS TO MIMIC OXYGEN SENSING IN BACTERIA

Heme-based sensors are important mediators of cellular responses to metabolic and envi-ronmental stimuli such as NO, CO and O2. They are multidomain proteins that tend to function in homo- and hetero-multimeric states. Changes in intracellular gas concentra-tions are sensed by a heme moiety and result in either aerotaxis or some sort of gene regulation. Presently, there are six known types of heme sensors. The HemATs are the only heme sensors known to provoke aero-taxis, whereas the remaining heme sensors function in gene regulation. The globin-cou-pled sensor (GCS), HemAT-Bs, an aerotactic receptor from Bacillus subtilis, consists of an N-terminal globin domain connected to a C-terminal signaling domain. HemAT-Bs senses oxygen via a chromophore linked to residue Histidine 123. The goal of this project was to investigate how oxygen is sensed by the globin domain. In order to do this, we created a chimeric receptor and tested its function in Escherichia coli. We also mutated Histidine 123 to Alanine and observed its effects on 1) heme and oxygen binding and 2) the ability to trigger an aerotactic response in E. coli. We discovered that the chimeric receptor did sense oxygen while the E.coli cells without the expressed protein failed to show any reaction. Therefore the protein caused an aerotactic response in E.coli when it effectively bound to heme and oxygen. However, the mutated histidine 123 prompted a slight aero-tactic response in E.coli. These results indicate that non-specific heme-binding may cause an aerotactic response or HemAT-Bs has multiple functions to trigger aerotactic responses