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

## TL;DR

This study explains how a synthetic minimal cell, JCVI-Syn3A, maintains an expanded nucleoid with ribosomes distributed throughout the cell due to an abundance of positively charged proteins.

## Contribution

The study introduces a coarse-grained model linking genome-encoded proteome composition to cell-scale physical organization in a minimal cell.

## Key findings

- Positively charged proteins in JCVI-Syn3A partially shield ribosome-DNA repulsion, enabling ribosomes to enter the nucleoid.
- DNA stiffness and crowding favor nucleoid compaction, while electrostatic interactions and protein size diversity promote expansion.
- The model demonstrates how genome-encoded proteome composition shapes cell interior organization.

## Abstract

Nucleoid compaction in bacteria is commonly attributed to cytoplasmic crowding, DNA supercoiling, and nucleoid-associated proteins (NAPs). In most bacterial species, including E. coli, these effects condense the chromosome into a subcellular region and largely exclude ribosomes to the surrounding cytoplasm. In contrast, many Mycoplasma—including the Mycoplasma-derived synthetic cell JCVI-Syn3A—exhibit a cell-spanning nucleoid with ribosomes distributed throughout. Because Mycoplasma are evolutionarily distant from model bacteria like E. coli and have undergone extensive genome reduction, Syn3A is a natural testbed for genotype-to-‘physiotype’-to-phenotype, in which genome-encoded composition reshapes cell-scale organization. Here we show that this organization can arise from Syn3A’s unusually high abundance of positively charged proteins. We develop a coarse-grained model that explicitly and physically represents a sequence-accurate chromosome together with ribosomes and cytoplasmic proteins at physiological size, charge, and abundance. With DNA and ribosomes alone, the cell-spanning nucleoid relaxes toward a compacted state that sterically excludes ribosomes, indicating missing physics beyond polymer mechanics and excluded volume. When we include electrostatic interactions by assigning effective charges to each biomolecule, positively charged proteins dynamically enrich around ribosomes and DNA, partially screening ribosome–DNA repulsion. This charge shielding enables ribosomes to penetrate the nucleoid mesh and stabilizes a cell-spanning nucleoid consistent with experiment. This behavior is robust across parameter sweeps: DNA stiffness, heterogeneous mesh size, and crowding favor compaction, whereas electrostatics and size polydispersity promote expansion, with consequences for migration pathways within the nucleoid and thus transcription–translation dynamics. The framework is parameterized directly from genomic and proteomic composition and is transferable to other bacteria.

The chemical elements of DNA—its genes—carry the instructions for cells to grow and divide, but DNA is also a physical object packed into a crowded milieu, with organization that shapes how genetic information is accessed and used. In many bacteria such as Escherichia coli, the nucleoid is compacted, expelling ribosomes to the cell periphery, tending to separate transcription from translation. Yet in many Mycoplasma—including minimal cell JCVI-Syn3A—the nucleoid spans the entire cell, with ribosomes distributed throughout. How and why some genome-reduced bacteria adopt this expanded-nucleoid “physiotype” has remained unclear. To explore this, we built a whole-cell coarse-grained model that explicitly represents Syn3A DNA, ribosomes, and cytoplasmic proteins. The model confirms that DNA stiffness, DNA-binding proteins, and crowding all favor compaction. In contrast, we find that Syn3A’s unusually high abundance of positively charged proteins is a crucial ingredient for chromosome expansion. These proteins enrich around ribosomes and DNA, partially charge-shielding ribosomes, which enables them to enter the nucleoid mesh and stabilize a cell-spanning nucleoid. These results support a genotype-to-physiotype-to-phenotype link in which genome-encoded proteome composition reshapes the physical interactions that organize the cell interior.