Atomic Force Microscopy Imaging and Analysis of Prokaryotic Genome Organization

This protocol describes the application of atomic force microscopy for structural analysis of prokaryotic and organellar nucleoids. It is based on a simple cell manipulation procedure that enables stepwise dissection of the nucleoid. The procedure includes (i) on-substrate lysis of cells and (ii) enzyme treatment, followed by atomic force microscopy. This type of dissection analysis permits analysis of nucleoid structure ranging from the fundamental units assembled on DNA to higher-order levels of organization. The combination with molecular-genetic and biochemical techniques further permits analysis of the functions of key nucleoid factors relevant to signal-induced structural reorganization or building up of basic structures, as seen for Dps in Escherichia coli and TrmBL2 in Thermococcus kodakarensis. These systems are described here as examples of the successful application of AFM for this purpose. Moreover, we describe the procedures needed for quantitative analysis of the data.