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In vitro characterization of the ParA family protein Soj from bacillus subtilis McLeod, Brett

Abstract

In a response to deteriorating environmental conditions and high population density the common soil bacterium Bacillus subtilis undergoes a developmental process characterized by an unusual asymmetric division which results in the formation of two different cell types (the forespore and the mother cell) and ultimately the release of a dormant endospore. This developmental process is under multiple levels of control. Mutation in stage zero genes (spoO) block all morphological changes. One stage zero gene spoOJ and a companion gene in the same operon (soj), have been implicated in the negative regulation of sporulation. Soj and SpoOJ are members of ParA and ParB families respectively, implicated in the stable inheritance of low copy number plasmids between daughter cells in bacteria. The role that Soj and SpoOJ play in the negative regulation of sporulation is not fully understood. This thesis characterizes the interaction of Soj with nucleotides, DNA, and SpoOJ in vitro to better understand the activities of Soj in the cell. I characterized the dimerization and DNA binding of wild type Soj and two Soj point mutants (S0jG12V and S0jD4OA). Wild type Soj was shown to bind adenosine nucleotides, form dimers, and hydrolyze ATP. Multiple lines of evidence demonstrated the nucleotide bound state does not alter the dimerization of Soj but does alter its ability to bind DNA. Multiple Soj-ATP proteins bound DNA to form Soj-DNA complexes as determined by DNaseI protection, electrophoretic mobility shift assays, and electron microscopy. Fixed angle light scattering was used to monitor the binding of Soj to DNA. Time resolved light scattering allowed characterization of multiple stages of Soj DNA binding. Soj was also shown to interact with SpoOJ, which resulted in a stimulation of Soj ATPase activity providing evidence that SpoOJ can alter Soj activity. The interaction of Soj and SpoOJ resulted in the formation of large protein complexes detected by light scattering and by electron microscopy. This data indicated that SpoOJ is able to form multimeric structures in response to a Soj interaction. The data were interpreted in a model for changes in the state of Soj and SpoOJ.

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