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.
