Research: Membrane-bound DNA transport machines involved in antibiotic resistance, chromosome segregation, and spore formation How do cells move one of the largest and most hydrophilic biological molecules across hydrophobic membrane barriers? This is the broad question our laboratory seeks to understand. In bacteria alone, this process is involved in the transfer of antibiotic resistance, spore formation, and proper chromosome segregation during growth. Yet, very little is known about the molecular mechanism of DNA translocation across membranes in any system, as tools to study the mechanism of DNA transporters in their biological context at the membrane have been lacking. We combine in vitro and in vivo biochemistry, microscopy, microbiology and molecular biology to study these DNA transport complexes. DNA transport during sporulation Proper chromosome segregation is essential for successful cell division in all organisms. Interestingly, bacterial cells often form a division septum prior to completion of DNA segregation. Sporulating Bacillus subtilis cells provide an extreme example of this phenomenon in that they must transport more than 3 Megabases of a chromosome across a division septum and into the small cellular compartment that will become the spore. SpoIIIE, a member of a large family of bacterial and archeal membrane-bound ATPases (FtsK/SpoIIIE), is required for active transport of the chromosomal DNA at the division septum during sporulation. Using quantitative fluorescence microscopy, and in vivo DNA transport assays to study the oligomeric state and transport properties of the SpoIIIE complex led to the very surprising and novel result that the DNA is transported across two membranes during sporulation. These data predict a new model for DNA transport in which the transmembrane segments of the transporter form linked DNA-conducting channels across the two lipid bilayers of the septum. This system provides a unique opportunity to tackle this interesting phenomenon using complimentary in vivo and in vitro approaches and to address issues which have significant implications for our understanding of how cells efficiently move of large nucleic acids across membranes.
Selected Publications: Marquis KA, Burton BM, Nollmann M, Ptacin JL, Bustamante C, Ben-Yehuda S, Rudner DZ. (2008). SpoIIIE strips proteins off the DNA during chromosome translocation. Genes and Development, 22: 1786-1795. Burton BM, Marquis KA, Sullivan NL, Rapoport TA, and Rudner DZ. (2007) The ATPase SpoIIIE transports DNA across fused septal membranes during sporulation in Bacillus subtilis. Cell, 131: 1301-1312. Burton BM and Baker TA. (2005). Remodeling protein complexes: insights from the AAA+ unfoldase ClpX and Mu transposase. Protein Sci. 14:1945-1954. Review. Sauer RT, Bolon DN, Burton BM, et al. (2004). Sculpting the proteome with AAA(+) proteases and disassembly machines. Cell, 119:9-18. Review. Burton BM and Baker TA. (2003). Mu Transpososome Architecture Directs Unfolding by ClpX and Proteolysis by ClpXP to Remodel but Not Destroy the Complex. Chem Biol, 10; 463-472. Burton BM, Williams TL, and Baker TA. (2001). ClpX-Mediated Remodeling of Mu Transpososomes: Selective Unfolding of Subunits Destabilizes the Entire Complex. Molecular Cell, 8; 449-454. |
