Domain-wise biochemical characterization of Myxococcus xanthus FrzE

Abstract

A Gram-negative soil bacterium, Myxococcus xanthus, has a complex life cycle that is dependent on its motility. It is known to have two types of directed motility, social motility (S-motility) and gliding or adventurous motility (A-motility). This motility is controlled by the cell polarity reversal frequencies. The frequency of the reversal is modulated by the Frz pathway, a two-component signal transduction system present in M. xanthus, . This system consists of proteins homologous to those in the Che system - a chemoreceptor FrzCD, adapter proteins FrzA and FrzB, a fusion protein of a histidine kinase and a response regulator FrzE and a fusion protein of two response regulators FrzZ. FrzE plays a major role in regulating the reversal frequencies by autophosphorylation at the histidine kinase domain upon stimulation by FrzCD via FrzA and transfers the phosphoryl group to the downstream regulators. A histidine residue (His49) in the HPT domain of FrzE gets phosphorylated by the phosphoryl group released from the ATP after its hydrolysis by HATPase domain. Phosphoryl group is transferred from the FrzE_HPT domain to an aspartate residue (Asp709) of the FrzE_Rec domain and inhibits further phosphorylation of FrzE. Biochemical characterization of FrzE will give insights into the mechanism of the Frz pathway and directed motility in M. xanthus. In this thesis, domain wise biochemical characterization of FrzE is being carried out. FrzE_Dim_HATPase, FrzE_CheW and FrzE_Rec domains were cloned and purified. ATPase activity for the FrzE_Dim_HATPase was quantified using malachite green assay. The stability of FrzE_Rec was checked using circular dichroism spectroscopy and thermal shift assay. Co-expression for FrzE_Dim_HATPase and FrzE_HPT and FrzE_CheW_Rec and FrzA was carried out for better expression of FrzE_Dim_HATPase and FrzA.