Modeling the Nucleoid and Cytoplasm Organization Inside a Minimal Synthetic Cell

The spatial arrangement of genetic material within cells can affect key processes such as transcription and translation. In bacteria such as E. coli, this genetic material is unbound but condensed into a nucleoid. This compact nucleoid partitions the cell into a porous region within, where transcription takes place, and a surrounding cytoplasm of mobile biomolecules where most of mRNA translation takes place at ribosomes. In a recent work, we observed that the E. coli nucleoid expands and contracts, changing its density and thus regulating protein expression by physical and electrostatic segregation of biomolecules in or out of the nucleoid. The compaction of the nucleoid is often attributed to a combined action of nucleoid-associated proteins (NAPs) and osmotic pressure from cytoplasmic biomolecules. In contrast to E. coli, some bacteria, such as many types of Mycoplasma species, possess a cell-spanning nucleoid. A notable example is the JCVI Syn3A synthetic cell, which is a model bacterium comprising the smallest known genome capable of life. In this work, we develop a colloidal-scale, coarse-grained model of the JCVI Syn3A minimal cell that captures the essential physics of DNA, native proteins, and ribosomes. Our model consists of a spherical enclosure, implicitly modeled cytosol, thousands of explicitly resolved biomolecules, and the chromosome, which is modeled as a semi-flexible bead chain. Through Monte Carlo simulations and Brownian Dynamics, we investigate the effects of DNA stiffness, native protein concentration, and charge distribution on nucleoid and cytoplasm organization. The effect of DNA-bending HU proteins is modeled as a random distribution of bending defects across the genome. The porous structure of the nucleoid is characterized by a Voronoi analysis, which suggests that a compact nucleoid exhibits a heterogeneous pore-size distribution that results in the separation and entrapment of larger particles. We find that an increased concentration of HU proteins may compact the chromosome and push ribosomes out into the surrounding cytoplasm, and that changes in crowder concentration and charge distribution can alter this effect. We propose how this may be connected to the experimental observation of a cell-spanning Syn3A nucleoid.