Biochemical, structural and genetic studies on the cytoskeletal proteins Fibril and MreBs from Spiroplasma

Abstract

Spiroplasmas are cell wall-deficient, helical bacteria with characteristic kinking motility. In absence of any external appendages, motility in Spiroplasma is achieved by dynamic changes in helicity. These changes are brought about by filaments formed by Fibril, a cytoskeletal protein of novel fold, and MreBs, the bacterial actins. The repeating unit in Fibril forms very stable, nucleotide-independent, constitutive filaments. The presence of five MreB homologues in Spiroplasma, an organism lacking cell wall, makes it an interesting system to explore novel functions of MreBs. In the absence of convenient molecular genetic tools, limited information is available on shape determinaion and motility in spiroplasmas. My work was aimed at understanding the molecular mechanism of shape determination and motility by Fibril and MreBs, through a combination of biochemical, structural and genetic studies. To perform structural characterization, Fibril filaments were heterologously expressed in E. coli and successfully purified, as observed using electron microscopy. Domain-wise dissection of Fibril based on sequence alignments and secondary structure prediction provided insights for designing non-polymerizing soluble constructs and a polymerizing construct of minimal length. To understand shape determination and motility in Spiroplasma, comparative studies between the wildtype and a naturally occurring mutant (non-helical, non-motile) strain were performed. These studies revealed that the mutant cells are deficient in one of the MreBs and the MreBs have non-overlapping functions in Spiroplasma. The comparative analysis and complementation studies provided the first evidence for a new role of MreB in rod-to-helical shape transition. To decipher the role of MreBs and Fibril, the process of obtaining Spiroplasma mutants with each of the mreB and fibril gene disrupted has been initiated. Results from these experiments will help us delineate the functional significance of each MreB and Fibril.