The role of macromolecular crowders in the formation and compaction of the Escherichia coli nucleoid

The chromosomal DNA of Escherichia coli is approximately a thousand times longer than the linear dimensions of the cell it occupies. Nevertheless, it fills only about one-half of the cytosolic volume of the cell. The volume pervaded by the chromosomal DNA is known as nucleoid. The nucleoid is a ribosome-depleted region that behaves as a distinct liquid-like phase within the cytosol. In most bacteria, including E. coli , which lack membrane-enclosed organelles, the phase separation between the nucleoid and the ribosome-rich cytosolic fraction represents the most prominent organizational principle of the cell’s cytosolic interior. This review explores the mechanisms driving nucleoid phase separation, including the roles of DNA-binding proteins, supercoiling, and active DNA looping. Recent studies highlight macromolecular crowding as the dominant factor governing this spatial organization. The main focus of this review is on experimental and theoretical works—ranging from in vitro and in vivo studies to polymer physics-based models—that elucidate how macromolecular crowding drives nucleoid phase formation and regulates DNA compaction in E. coli .