The secret to cellular growth has been hiding in plain sight — within the concentration of DNA itself.
For decades, scientists have understood that ribosomes—the cellular protein factories—serve as the primary engine limiting bacterial growth rates. The more ribosomes a cell has, the faster it can grow, or so the thinking went. Meanwhile, the concentration of DNA in cells was largely considered a bystander in this process, with little direct role in determining growth rates.
Recent groundbreaking research reveals this assumption has been profoundly mistaken. Scientists have discovered that genome concentration itself directly limits cell growth and serves as a global modulator of cellular composition in Escherichia coli (E. coli), fundamentally reshaping our understanding of the central dogma of molecular biology 1 .
For years, microbiology textbooks have taught that bacterial growth follows exponential patterns during the cell cycle. As cells double in mass and volume between divisions, their biosynthetic capacity increases proportionally, resulting in the characteristic exponential growth curve observed in bacterial populations.
The prevailing "growth laws" of microbiology have primarily emphasized translation as the rate-governing process for cellular growth, with ribosome concentration and kinetics serving as the principal constraint. Most other cellular components essential for growth were thought to exist in considerable excess—at least an order of magnitude above required levels for proper enzymatic function.
While the importance of DNA concentration had been explored in eukaryotic systems, where changes in DNA-to-cell-volume ratio have been linked to cellular senescence and proteome remodeling, its potential role in bacterial growth had received scant attention 2 . An early population study on E. coli even suggested that global transcription was limited not by genome concentration but by RNA polymerase availability 3 .
The groundbreaking research from Mäkelä and colleagues fundamentally challenges these long-held assumptions through a sophisticated series of experiments demonstrating that chromosome concentration directly limits transcription and consequently modulates proteome composition in E. coli 1 .
The implications extend far beyond bacterial systems, as comparative experiments in Caulobacter crescentus and evaluation of eukaryotic studies reveal broadly conserved DNA concentration-dependent scaling principles of gene expression across the tree of life.
The research team hypothesized that as bacterial cells grow without replicating their DNA, the resulting dilution of genome concentration could create a physical limitation on the total transcriptional capacity of the cell. This would represent a previously unrecognized constraint on cellular growth processes—a bottleneck at the level of gene availability rather than just translational capacity.
To rigorously test this hypothesis, researchers designed an elegant experimental system that allowed them to precisely control DNA replication while monitoring growth at the single-cell level.
The team utilized two CRISPR interference strains with arabinose-inducible expression of dCas9. The first strain expressed a single-guide RNA against the origin of replication, preventing initiation of DNA replication and producing cells with only a single chromosome. The second strain targeted the cell division protein FtsZ, blocking division while allowing DNA replication to continue normally.
Using time-lapse microscopy at 37°C in controlled nutrient conditions, researchers tracked individual cells over time. Cell area was automatically detected from phase-contrast images using a deep convolutional network, enabling precise measurement of growth dynamics.
To confirm the single-chromosome state, researchers used a fluorescent label (mCherry fusion to the nucleoid-binding protein HupA) to visualize DNA. In validation experiments, 96% of cells with a single nucleoid contained no more than one origin of replication focus, confirming the successful creation of 1N cells.
As an additional control, the team used a temperature-sensitive dnaC2 mutant with a deficient DNA helicase loader, providing an independent method to block DNA replication at restrictive temperatures.
The researchers complemented microscopic analysis with RNA sequencing, tandem-mass-tag mass spectrometry, and single-molecule tracking to comprehensively assess transcriptional and proteomic changes.
| Strain Type | DNA Replication | Cell Division | Chromosome Number | Growth Pattern |
|---|---|---|---|---|
| CRISPRi oriC | Blocked | Blocked | Single (1N) | Sub-exponential |
| CRISPRi FtsZ | Proceeds | Blocked | Multiple (multi-N) | Near-exponential |
| dnaC2 mutant | Blocked at 37°C | Blocked | Single (1N) | Sub-exponential |
| Wild-type E. coli | Normal | Normal | Multiple | Exponential |
The experimental results revealed striking differences between the growth patterns of 1N and multi-N cells:
| Growth Parameter | 1N Cells | Multi-N Cells | Wild-type Cells |
|---|---|---|---|
| Absolute Growth Rate | Moderate increase with size, plateauing at large sizes | Rapid increase with size | Similar to multi-N cells |
| Relative Growth Rate | Decreases with cell size | Relatively constant | Relatively constant |
| DNA Concentration | Decreases with growth | Maintained with growth | Maintained with growth |
| Transcription Capacity | Limited | Scalable | Scalable |
Perhaps most remarkably, when researchers examined subpopulations of dnaC2 cells with varying numbers of nucleoids due to "leaky" phenotypic expression, they observed clear gradations in growth rate—cells with more nucleoids (and thus higher DNA concentration) grew faster than those with fewer nucleoids, providing compelling evidence for a dose-dependent relationship between genome concentration and growth capacity 1 .
This groundbreaking research was made possible through sophisticated experimental tools that allowed precise manipulation and measurement of cellular processes:
Arabinose-inducible dCas9 with sgRNAs provided targeted control of DNA replication initiation or cell division.
Allowed single-cell growth monitoring under controlled environmental conditions.
Provided comprehensive transcriptome profiling across different growth conditions.
Enabled multiplexed proteomic analysis of thousands of proteins simultaneously.
Automated cell segmentation and size quantification from phase-contrast images.
Allowed observation of individual RNA polymerase molecules and their activity.
The implications of this research extend far beyond E. coli. Follow-up experiments in Caulobacter crescentus demonstrated similar DNA concentration-dependent effects, suggesting a broadly conserved principle across bacterial species.
Single chromosome, limited growth
Multiple chromosomes, exponential growth
Normal replication, optimal growth
Even more remarkably, comparison with eukaryotic systems reveals parallel mechanisms—changes in DNA-to-cell-volume ratio have been shown to remodel the proteome and promote cellular senescence in yeast and human cells. This suggests that the relationship between DNA concentration and cellular physiology represents a fundamental organizing principle across the evolutionary spectrum.
The discovery also helps explain previously puzzling observations in other bacterial species. Bacillus subtilis has been shown to display small but reproducible deviations from exponential growth during the division cycle, with growth rates increasing after initiation of DNA replication—exactly what would be predicted if DNA concentration influences growth capacity 4 .
This research fundamentally reshapes our understanding of what controls cellular growth, adding genome concentration as a key limiting factor alongside the well-established role of ribosomes. The discovery that DNA dilution limits total RNA polymerase activity, leading to sub-linear scaling of active ribosomes and sub-exponential growth, represents a paradigm shift in microbial physiology.
The implications span multiple fields—from basic microbiology to biotechnology and medicine. Understanding how DNA concentration modulates global gene expression patterns could inform new approaches to antibiotic development, optimize microbial factories for biotechnology, and enhance our comprehension of cellular aging processes across species.
As with all transformative science, this discovery raises as many questions as it answers. How do cells precisely sense and respond to DNA concentration changes? What molecular mechanisms coordinate DNA concentration with global transcriptional output? And how do these principles operate in more complex eukaryotic systems?
What remains clear is that even in well-studied model organisms like E. coli, fundamental principles of cellular operation remain to be discovered—a humbling and exciting realization for scientists exploring the intricate workings of life at the molecular level.
This article summarizes research findings from "Genome concentration limits cell growth and modulates proteome composition in Escherichia coli" by Mäkelä et al., published in eLife (2024). For complete experimental details and data, refer to the original study.