dc.contributor.advisor | Lutkenhaus, Joe | |
dc.contributor.author | Hester, Christina Marie | |
dc.date.accessioned | 2008-08-05T02:47:39Z | |
dc.date.available | 2008-08-05T02:47:39Z | |
dc.date.issued | 2008-05-08 | |
dc.date.submitted | 2008 | |
dc.identifier.other | http://dissertations.umi.com/ku:2555 | |
dc.identifier.uri | http://hdl.handle.net/1808/4022 | |
dc.description.abstract | The ParA family of ATPases encompasses proteins with a wide variety of functions. The prototype of this family is the ParA ATPase from the P1 prophage plasmid, which, along with ParB and the parS binding site, is required for the faithful maintenance of this prophage in the episomal state. Many other low copy plasmids as well as bacterial chromosomes encode homologues of ParA. The function of these proteins in plasmid and chromosome segregation is still a mystery, yet their involvement in the process at some level is evident. Here, we have characterized a chromosomal ParA homologue, Soj from Bacillus subtilis, as it behaves and functions in plasmid partitioning in Escherichia coli and in the regulation of sporulation in B. subtilis. We have studied the localization and behavior of this protein in the heterologous host, E. coli. In B. subtilis, GFP-fusions to Soj localize to the nucleoids and poles and undergo movement from one end of the cell to the other on the DNA. We have observed this movement in E. coli in the absence of any other B. subtilis elements besides Spo0J (the B. subtilis ParB homologue) and the parS binding site, and we have determined that Spo0J and parS are required for this movement. These requirements are the same as those for the maintenance of a low copy plasmid by Soj and Spo0J in E. coli, supporting the possibility that Soj movement is required for its function in plasmid partitioning. We have also found that Soj binds non-specifically to DNA through conserved arginine residues that map to the surface of the structure of the Soj dimer. We identified these residues by alignment of Soj with other chromosomal ParA homologues and identification of conserved basic residues that mapped to the surface of the structure of Thermus thermophilus Soj. We were able to identify two key arginines that are important for the interaction of Soj with DNA. By mutating these residues, we were able to assess the importance of DNA binding for two of the known functions of Soj: for its role in plasmid maintenance in E. coli and for its role in regulation of sporulation in B. subtilis. We find that Soj can not function in either of these activities if it can not bind to DNA. Our findings allowed us to generate a model for how the Soj dimer is oriented on the DNA, enabling us to envision how the binding of dimers is propagated along the length of the DNA. We have extended the ParA/Soj DNA binding work by identifying conserved positively charged residues in ParA from the plasmid pB171 that may be important for its DNA binding. The mutation of these residues prevents ParA from binding to DNA in vivo or reduces its affinity for DNA in in vitro assays. This work has drawn attention to the importance of a characteristic of ParA proteins that has previously been overlooked: non-specific DNA binding. As we have determined that DNA binding is essential for Soj function, models for plasmid partitioning can now include the DNA binding property of ParA, and the role of DNA binding in the functions of these proteins can be further explored. | |
dc.format.extent | 164 pages | |
dc.language.iso | EN | |
dc.publisher | University of Kansas | |
dc.rights | This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author. | |
dc.subject | Biology | |
dc.subject | Microbiology | |
dc.title | Bacillus subtilis Soj (ParA): The involvement of DNA binding and dynamic localization in plasmid partitioning | |
dc.type | Dissertation | |
dc.contributor.cmtemember | Egan, Susan | |
dc.contributor.cmtemember | Swint-Kruse, Liskin | |
dc.contributor.cmtemember | Yankee, Thomas | |
dc.contributor.cmtemember | Zuckert, Wolfram R. | |
dc.thesis.degreeDiscipline | Microbiology, Molecular Genetics & Immunology | |
dc.thesis.degreeLevel | PH.D. | |
kusw.oastatus | na | |
kusw.oapolicy | This item does not meet KU Open Access policy criteria. | |
kusw.bibid | 6599435 | |
dc.rights.accessrights | openAccess | |