of E. coli are physically together in 3-D space within the chromosome even though they are far apart by genetic distance. Spatial proximity of functionally-related genes not only make the biological functions more compartmentalized and efficient but would also contribute to the folding and spatial organization of the nucleoid. A recent study using fluorescent markers for detection of specific DNA loci examined pairwise physical distances between the seven rRNA operons that are genetically separated from each other (by as much as two million bp). It reported that all of the operons, except rrnC, were in physical proximity. Surprisingly, 3C-seq studies did not reveal the physical clustering of rrn operons, contradicting the results of the fluorescence-based study. Therefore, further investigation is required to resolve these contradicting observations. In another example, GalR, forms an interaction network of GalR binding sites that are scattered across the chromosome. GalR is a transcriptional regulator of the galactose regulon composed of genes encoding enzymes for transport and metabolism of the sugar D-galactose. GalR exists in only one to two foci in cells and can self-assemble into large ordered structures. Therefore, it appears that DNA-bound GalR multimerizes to form long-distance interactions. Global shape and structure Conventional transmission electron microscopy (TEM) of chemically fixed E. coli cells portrayed the nucleoid as an irregularly shaped organelle. However, wide-field fluorescence imaging of live nucleoids in 3D revealed a discrete, ellipsoid shape. The overlay of a phase-contrast image of the cell and the fluorescent image of the nucleoid showed a close juxtaposition only in the radial dimension along its entire length of the nucleoid to the cell periphery. This finding indicates radial confinement of the nucleoid. A detailed examination of the 3D fluorescence image after cross-sectioning perpendicular to its long axis further revealed two global features of the nucleoid: curvature and longitudinal, high-density regions. Examining the chirality of the centerline of the nucleoid by connecting the center of intensity of each cross-section showed that the overall nucleoid shape is curved. The fluorescence intensity distribution in the cross-sections revealed a density substructure, consisting of curved, high-density regions or bundles at the central core, and low-density regions at the periphery. One implication of the radial confinement is that it determines the curved shape of the nucleoid. According to one model, the nucleoid is forced to bend because it is confined into a cylindrical E. coli cell whose radius is smaller than its bendable