Myxococcus xanthus is a long, rod-shaped bacterium that glides over surfaces using two different motors that operate simultaneously. The long term objective of this work is to identify the structural components of gliding and to understand how the direction of the two motors is coordinated during cell reversal. The immediate goals will focus on MgIA and MgIB, which appear to play a major role in motor switching, and AglZ, a protein that interacts with MgIA and which appears to be a structural component for one motility system. These proteins are counterparts of eukaryotic proteins, such as Ras, GNRF, and myosin, that govern essential regulatory processes in eukaryotic cells. Mutations in RAS that shift the balance toward the GTP-bound form result in transformation of eukaryotic cells which can culminate in a tumor. Similarly, mutations in MgIA, a Ras-like GTPase, affect critical, but non-essential process in M. xanthus. The similarities between these systems will be exploited to provide biologic and evolutionary information about GTPases and their effectors. The phenotype of an mgIA mutant suggests that MgIA relays information between the two motility systems so that engines reverse direction simultaneously. Hydrolysis is necessary for movement because mutations that affect residues of MgIA predicted to favor the ON (GTP-bound) state reduce or abolish gliding. Additional experiments to characterize MgIA biochemically are described. One type of mgIA mutation affects MgIA function in vivo without affecting activity in vitro and hints of the existence of a GAP protein. Experiments to identify a GAP homolog are described. Disruption of mgIB, a putative release factor for MgIA, reduces motility. AglZ is a myosin-like, filament-forming protein that is required for one of the motility systems. Experiments, such as FRAP analysis, to examine the dynamics and polarity of the AglZ structure in vivo and in vitro are described. Antibody and GFP-tagged proteins will be used to examine the interaction between MgIA and AglZ in vivo and in vitro.
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