Executive Summary : | Myxococcus xanthus, a soil bacterium, undergoes frequent reversals of cell polarity during directed motility. Frz pathway, homologous to the Che pathway of E. coli, is the machinery that helps the bacterium to detect environmental cues and communicate to effector molecules for driving polarity reversals. Frz pathway possesses a cytoplasmic methyl accepting chemosensory protein (MCP), FrzCD, which has a novel DNA binding property, instead of a trans-membrane domain as is typical in majority of the characterized bacterial chemoreceptors. Hence, this is an interesting bacterial chemoreceptor assembly that is technologically amenable to reconstitution as a soluble complex, in order to obtain mechanistic insights into the function and assembly of bacterial chemoreceptors. SCIENTIFIC OBJECTIVES: The main objective of the project is to reconstitute the soluble bacterial chemoreceptor formed by Frz proteins (FrzCD, FrzE, FrzA and FrzB) that assembles on a DNA scaffold, owing to the DNA-binding MCP, namely FrzCD. EXPERIMENTAL APPROACH: The protein components of the chemoreceptor array mainly, a histidine kinase FrzE, and the CheW equivalents FrzA and FrzB will be cloned, expressed and purified. These components will be characterized for their function by monitoring the enzymatic activity of FrzE, oligomerization status of FrzA and FrzB, and their structure determination using X-ray crystallography. The assembly mechanism of the chemosensory complex will be elucidated through pairwise interaction studies, electrophoretic mobility shift assays (EMSA), and reconstitution of the complex using fluorescently labeled components to understand the dynamics of assembly. The complex will be visualized using electron microscopy to confirm the formation of higher order assemblies. HYPOTHESES: The membrane acts as a scaffold to form hexagonal arrays for most MCPs, which is essential for amplifying signals that enable downstream reactions. In FrzCD, DNA-binding might perform an analogous function to membrane-binding. The proposed reconstitution approach of FrzE, FrzA and FrzB with FrzCD, will demonstrate the stabilization of the FrzCD trimer of dimer complex on DNA, leading to the formation of hexagonal array. Hence, it provides an excellent platform for studying the allostery and cooperativity of kinase activity, an intriguing concept in all bacterial chemosensory arrays. SIGNIFICANCE: The proposed work on reconstitution of a soluble bacterial chemoreceptor complex provides a unique opportunity to i) understand the assembly dynamics ii) obtain a high-resolution structure iii) provide insights on the mechanistic basis of kinase activation and allosteric effects on kinase activity, for a bacterial chemosensory array. A successful assembly of the complex thus provides a powerful handle for answering many open questions in the field of bacterial chemotaxis. |