Bacteria typically store their genetic information in a single circular chromosome that is several million DNA bases long. In order to maintain and duplicate this chromosome, called the nucleoid, bacteria must accomplish two major feats of structural engineering: First, a giant 1.5 millimeter-long DNA molecule must be packaged into a bacterial cell that is over a thousand times shorter. Second, newly replicated sister chromosomes must be disentangled and separated in preparation for cell division. Work over the last several decades has identified a number of nucleoid-associated proteins (NAPs) that play essential roles in these processes, yet it remains unclear how various NAP-DNA interactions collectively regulate nucleoid architecture. Using single-molecule biochemical assays, this project aims to investigate the molecular mechanisms by which nucleoid-associated proteins from the model bacterium Bacillus subtilis organize and segregate the bacterial chromosome. This study will focus on five key NAPs (Spo0J, HBsu, SASP, Rok and SMC) selected due to their essential functions throughout different parts of the B. subtilis life cycle. To develop a mechanistic understanding of how these NAPs function, a novel single-molecule assay will be employed that correlates NAP-DNA binding with changes in the elastic properties of individual DNA molecules. These techniques will be applied to address the following aims: (1) To determine how each of the individual NAPs associates with DNA and modulates its mechanical properties. (2) To establish how HBsu, the major non-specific NAP in B. subtilis, facilitates or antagonizes the interactions of Spo0J, SASP and Rok with DNA. (3) To characterize the SMC complex and establish if DNA compaction is driven by intra- or intermolecular SMC interactions. These studies will take the first steps toward reconstituting and ultimately dissecting the complexity of the chromosome by investigating cooperativity between NAPs, while the techniques developed will be broadly applicable to other studies of protein-DNA interactions.
Broader Impacts The single-molecule methods employed in this project provide a quantitative description of the dynamics of proteins and biological macromolecules, such as DNA and RNA, yet they have not gained popularity in the classroom because they are regarded as too difficult and expensive. In order to increase accessibility, an inexpensive single-molecule microscope will be designed that can be used in the undergraduate and high school teaching laboratory. A suite of experiments will be developed for this microscope to introduce biophysical concepts such as Brownian motion, DNA polymer dynamics and the principles of molecular motors. These experiments will be piloted in an undergraduate laboratory course and introduced to high school teachers and students through summer workshops in which the PI will participate.
