Abundant positively-charged proteins underlie JCVI-Syn3A's expanded nucleoid and ribosome distribution

Nucleoid compaction in bacterial cells has been attributed to cytoplasmic crowding, supercoiling effects, and the action of nucleoid-associated proteins (NAPs). In most bacteria, including E. coli , these mechanisms condense the nucleoid to a smaller volume within the cell, excluding most ribosomes to the surrounding cytoplasm. In contrast, the nucleoid in many Mycoplasma s, including the Mycoplasma -derived synthetic cell JCVI-Syn3A, spans the entire cell, with ribosomes distributed throughout. Recent models of Syn3A representing only DNA and ribosomes (both charge neutral) instantiated the experimentally-observed expanded nucleoid and ribosome distribution. However, we found that this configuration becomes dynamically unstable, giving way to a compacted nucleoid that expels ribosomes to the periphery, suggesting the need for a more detailed model. Speculation emerging from recent studies of Syn3A suggests that its lower concentration of NAPs underlie its expanded nucleoid. We are interested in this genotype-to-`physiotype'-to-phenotype implication: that coupled transcription, translation, and nucleoid remodeling lead to different phenotypical outcomes. We developed a coarse-grained computational model of Syn3A, physically and explicitly representing ribosomes, cytoplasmic proteins, and a sequence-accurate chromosome with physiological distributions of size, charge, and relative molecular abundance. An interplay between Brownian dynamics, DNA stiffness (both inherent and NAP-enhanced), and electrostatic charge led naturally to a stable molecular distribution. We find that an interplay between inherent and induced DNA stiffness, heterogeneous mesh size, and crowding enhances nucleoid compaction and ribosome expulsion via a competition between entropic and enthalpic forces. In contrast, electrostatic interactions and size-polydispersity counteract these effects and expand the nucleoid. In particular, Syn3A's atypically high abundance of positively-charged proteins shields ribosomes' negative charge, allowing them to interpenetrate the nucleoid. Finally, we observe condensate formation arising from electrostatic interactions with potential implications on transcription and translation rates.