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Jessica M. Mann
Microbiology and Molecular Genetics Program
B.S. Biochemistry 2007
Juniata College, Huntingdon PA
Thesis Advisor: David Dubnau, Ph.D.
Department of Microbiology and Molecular Genetics
Wednesday, July 31, 2013
10:00 A.M., ICPH-Auditorium
At the onset of stationary phase, the Gram-positive soil bacterium Bacillus subtilis, can enter into several developmental states, including competence for natural transformation. Transformation is the process by which exogenous DNA is transported into the cytoplasm of a cell, through the coordinated actions of a multiprotein machine. To initiate the process, a DNA substrate must be bound to the outside of a cell. After the DNA is bound to the cell, it is transported through an aqueous channel into the cytoplasm. The movement of the DNA to this aqueous channel and the subsequent transport to the cytoplasm, is likely a dynamic process requiring many proteins. A subset of these transformation proteins shares homology with components of Type 4 pilus and Type 2 secretion systems. In all three systems, a protein complex (the pilus) is assembled from a major pilin. Three or four less abundant minor pilins, a membrane associated ATPase, a multipass membrane protein, and a pre-pilin peptidase are essential for pilus assembly and function. We show that the B. subtilis minor pilins form a complex, which is part of the transformation machine. We demonstrate that the minor pilins interact directly in the membrane, and engage in direct interactions with other components of the system, especially the cytoplasmic ATPase. We also show, unexpectedly, that one of the minor pilins requires other members of the complex to be processed by the peptidase. We propose a model whereby the minor pilins form multiple complexes that function in pilus initiation as well as elongation.
ComGA, the peripheral membrane ATPase, is the only known transformation protein essential for the first step of transformation, DNA-binding. Since ComGA is localized inside the cell, it cannot bind the DNA-directly. Presumably ComGA is required for binding by an unidentified receptor. We have adopted two approaches toward the identification this receptor. We have tested knockouts of proteins interacting with ComGA for transformation deficient phenotypes and we have developed a method for isolating proteins that have been cross-linked to transforming DNA. Although the identity of the receptor remains elusive, this work provides the foundation for future studies.