Characterization of Spiroplasma citri MreB1

Abstract

For a living cell, maintenance of its shape is important to interact with its environment for numerous functional aspects like cell signalling, motility, biofilm formation, etc. The cytoskeletal proteins play a crucial role in the determination of cell shape. Complex eukaryotic cells, as well as prokaryotes, possess different cytoskeletal proteins. In most non-spherical, cell-walled bacteria, MreB, which is a homolog of eukaryotic actin, coordinates with the peptidoglycan synthesis machinery for maintaining cell shape. Surprisingly, Spiroplasma is able to maintain a helical cell shape even in the absence of a cell wall. It has a unique cytoskeletal protein, Fibril and five MreB paralogs, out of which MreB5 was recently shown to be involved in attaining motility and helicity in a plant pathogen, Spiroplasma citri, indicating that the function of MreB paralogs might be non-redundant in this organism. The aim of my MS thesis work was to characterize S.citri MreB1, which is one of the five MreB paralogs. SUMO tag fusion-based approach was utilized for standardizing the purification of MreB1. The ATPase activity was quantified for all the different constructs of MreB1. CD spectroscopy confirmed the presence of secondary structures. Filaments were visualized using Transmission electron microscopy. In future, the project will focus on understanding the protein interaction with cell membrane and its partner proteins - Fibril and MreB paralogs. Further, it will pitch into designing ATPase mutants and polymerization mutants to understand filament dynamics and the possible role of nucleotides in the same.