NOVEL MOLECULAR MECHANISMS OF SIGNAL SENSING AND TRANSDUCTION IN BACTERIAL CHEMOTAXIS

Abstract

Bacterial chemotaxis is a behavior bacteria exhibit to alter their motility in response to their chemical environment. The underlying sensory pathway responsible for this movement involves transmembrane receptors called methyl accepting chemotaxis proteins (MCPs) that couple with the intracellular histidine kinase CheA and adaptor protein CheW. Bacterial chemotaxis has long served as a model system for understanding the mechanisms of signal transduction. However, the exact mechanisms for the remarkable sensitivity and signal amplification of this system remains to be fully understood. In this dissertation, I present structural and activities studies of various proteins involved in bacterial chemotaxis. In particular, I focus on elucidating the structural changes that occur in the dimeric histidine kinase CheA when it switches between the kinase-on and kinase-off activity states. In this work I have utilized a newly developed in vitro chemotaxis system that consists of soluble receptor mimetics that couple to CheA and CheW to form ternary complexes suitable for single-particle experiments. Electron paramagnetic resonance (EPR) and mass spectrometry experiments demonstrate that the deactivated kinase forms a more compact and rigid structure. Additionally, crystallography and cross-linking experiments demonstrate that domains in the dimer self-associate when CheA is deactivated. In addition, I have determined the role of a previously uncharacterized protein whose gene is often found next to those for cytoplasmic receptors. We have named this new chemotaxis protein ODP1 for Oxygen Di-iron Protein and I have extensively characterized homologs of ODP1 from Treponema denticola and Thermotoga maritima using structural and biochemical methods. Analytical and spectrometric techniques demonstrate that iron and oxygen are ligands of ODP1. Furthermore, in vivo assays with T. denticola confirm that ODP1 acts as an iron and oxygen sensor for the chemotaxis system. Crystal structures of both Td and Tm ODP1 suggests a mechanism for iron sensing. Finally, I have developed a new method for spin-labeling proteins for EPR experiments. Specifically, I have synthesized and isolated an analog of ADP (ADP-β-S-SL) that is covalently attached to a nitroxide spin label and can hence be reconstituted into ATP-binding proteins, such as CheA. Indeed, Double Electron Electron Resonance spectroscopy experiments on the CheA kinase confirm that ADP-β-S-SL is an appropriate method for determining distance distributions from non-covalently bound spin probes. In summary, my work has provided new insights into the domain rearrangements that occur in CheA due to receptor mediated modulation. Additionally, I’ve characterized a new chemotaxis sensor protein that is responsible for monitoring intracellular levels of iron and oxygen.