Organization of the bacterial nucleoid by DNA-bridging proteins and globular crowders

Abstract

The genomic DNA of bacteria occupies only a fraction of the cell called the nucleoid, although it is not bounded by any membrane and would occupy a volume hundreds of times larger than the cell in the absence of constraints. The two most important contributions to the compaction of the DNA coil are the cross-linking of the DNA by nucleoid proteins (like H-NS and StpA) and the demixing of DNA and other abundant globular macromolecules which do not bind to the DNA (like ribosomes). The present work deals with the interplay of DNA-bridging proteins and globular macromolecular crowders, with the goal of determining the extent to which they collaborate in organizing the nucleoid. In order to answer this question, a coarse-grained model was developed and its properties were investigated through Brownian dynamics simulations. These simulations reveal that the radius of gyration of the DNA coil decreases linearly with the effective volume ratio of globular crowders and the number of DNA bridges formed by nucleoid proteins in the whole range of physiological values. Moreover, simulations highlight the fact that the number of DNA bridges formed by nucleoid proteins depends crucially on their ability to self-associate (oligomerize). An explanation for this result is proposed in terms of the mean distance between DNA segments and the capacity of proteins to maintain DNA–bridging in spite of the thermal fluctuations of the DNA network. Finally, simulations indicate that non-associating proteins preserve a high mobility inside the nucleoid while contributing to its compaction, leading to a DNA/protein complex which looks like a liquid droplet. In contrast, self-associating proteins form a little deformable network which cross-links the DNA chain, with the consequence that the DNA/protein complex looks more like a gel.

Macromolecular Crowding, Phase Separation, and Homeostasis in the Orchestration of Bacterial Cellular Functions

“…The bacterial nucleoid, with its several megabases of chromosomal DNA, is remarkably confined and compact despite the lack of a dedicated membrane to enclose it. 207,208 Initially described as a collection of loops emanating from a dense core organized by proteins and RNA, 209,210 the nucleoid has since been revealed to be a condensed phase (Figure 8) formed by LLPS through the interaction of multivalent cations and proteins in the presence of crowding agents. 211−213 Mobility within this dynamic structure allows organization of the chromosomal loci as required during the cell cycle.…”

“…The bacterial nucleoid, with its several megabases of chromosomal DNA, is remarkably confined and compact despite the lack of a dedicated membrane to enclose it. , Initially described as a collection of loops emanating from a dense core organized by proteins and RNA, , the nucleoid has since been revealed to be a condensed phase (Figure ) formed by LLPS through the interaction of multivalent cations and proteins in the presence of crowding agents. − Mobility within this dynamic structure allows organization of the chromosomal loci as required during the cell cycle . Interestingly, its size and positioning within the cell are regulated by crowding and cell geometry. , Atomic force microscopy and simulations with varying DNA concentrations show that self-crowding modifies nucleoid shape and properties depending on supercoiling density, which is essential for DNA replication .…”

Compaction and Segregation of DNA in Escherichia coli

“…At slow growth a newborn cell in B-(G1-) period is assumed to contain 1 chromosome equivalent. 4) Reestimated using instrumental magnification of 23850x as indicated on the original photographic print. 5) Calculated from average cell Vcell = 0.7 µm3; Vnewborn = 0.7/2ln2 = 0.5 µm3.…”

“…2 Why is the DNA not dispersed throughout the whole cell, but does occur in distinct regions as observed by phase-contrast microscopy? This question has been studied by many groups applying molecular dynamics simulations [3,4] and theoretical computations based on equilibrium statistical mechanics [5,6]. These involve formulations of the free energy of the system according to models for interactions between the supercoiled DNA and macromolecular crowders that cause the compaction of the nucleoid.…”

Compaction and Segregation of DNA in Escherichia coli

“…Why is the nucleoid’s DNA not dispersed throughout the whole cell, and how do the distinct low-density regions, as observed by phase-contrast microscopy, originate? These questions have been studied by many groups by applying molecular dynamics simulations [ 3 , 4 ], which will not be discussed here. These questions have also been addressed in theoretical studies based on equilibrium statistical mechanics and on formulations of the free energy of cell systems.…”