Abundant positively-charged proteins underlie JCVI-Syn3A’s expanded nucleoid and ribosome distribution
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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.
Author summary
The chemical elements of DNA in cells — its genes — carry the instructions for it to grow, replicate, and divide but its physical organization keeps those instructions in order and easy to read. In bacterial cells, DNA occupies a specific region that determines where its mRNA transcripts are ultimately translated by ribosomes into the proteins that help the cell grow and adapt. This structure differs markedly between bacteria in two prominent genera: Escherichia and Mycoplasmas . Previous models and experiments explain why Escherichia ’s DNA tends to be compact and centralized, expelling ribosomes to the cell periphery, separating transcription from translation. But many Mycoplasmas , much simpler cells, have expanded DNA — and their ribosomes are co-located within the DNA’s structure. It’s been proposed that the same proteins that help cells adapt to stress may underlie this behavior, and they are far less abundant in Mycoplasmas , suggesting that expanded DNA may help improve low adaptability. To interrogate the interplay between mesoscale physical DNA architecture, stress proteins, and physical interactions in the cell, we built a computational model of the Mycoplasma -derived minimal cell JCVI-Syn3A, representing all of its DNA, ribosomes, and proteins with explicit physical resolution and interactions. By testing the conditions that drive these divergent “physiotypes”, we found an explanation in Syn3A’s atypically positively-charged proteins, which cloak ribosomes, letting them enter and expand the nucleoid.
