Genome concentration limits cell growth and modulates proteome composition in Escherichia coli

Defining the cellular factors that drive growth rate and proteome composition is essential for understanding and manipulating cellular systems. In bacteria, ribosome concentration is known to be a constraining factor of cell growth rate, while gene concentration is usually assumed not to be limiting. Here, using single-molecule tracking, quantitative single-cell microscopy, and modeling, we show that genome dilution in Escherichia coli cells arrested for DNA replication results in a decrease in the concentration of active RNA polymerases and ribosomes. The resulting sub-linear scaling of total active RNA polymerases and ribosomes with cell size leads to sub-exponential growth, even within physiological cell sizes. Cell growth rate scales proportionally with the total number of active ribosomes in a DNA concentration-dependent manner. Tandem-mass-tag mass spectrometry experiments further revealed that a decrease in DNA-to-cell-volume ratio also incrementally remodels proteome composition with cell size. Altogether, our findings indicate that genome concentration is an important driver of exponential cell growth and a global modulator of proteome composition in E. coli. Comparison with studies on eukaryotic cells suggests DNA concentration-dependent scaling principles of gene expression across domains of life. Overall design: RNA-seq libraries associated with the project; CRISPRi oriC strain (SJ_XTL676) was grown in M9glyCAAT at 37°C and cells were collected 60, 120, 200, and 240 min after addition of 0.2% L-arabinose. For each timepoint, the aliquot was spun down, the supernatant was removed, and the pellet was flash-frozen. E. coli pellets were resuspended in ice cold phosphate buffer saline solution (PBS) and mixed with C. crescentus cells in approximately a 1-to-1 ratio based on OD600, with the intent of using the latter as a spike-in reference.