Observing Bacterial Chromatin Protein-DNA Interactions by Combining DNA Flow-Stretching with Single-Molecule Imaging

Nucleoid-associated proteins bind to DNA specifically and nonspecifically to perform various roles in chromosome organization and segregation. In this chapter, we describe how the interaction between nucleoid-associated proteins and flow-stretched DNAs can be visualized on the single-molecule level. We describe three different experimental schemes that allow one to directly observe how these proteins that make up bacterial chromatin, associate with and act on DNAs. First, we describe how to visualize the diffusion of fluorescently labeled proteins on flow stretched DNAs. Second, we describe how the binding of bacterial chromatin proteins can be correlated with DNA condensation. Lastly, we describe the DNA motion capture assay, which allows one to probe the mechanism of DNA condensation by tracking how different segments of a flow stretched DNA are compacted by bacterial chromatin proteins.