Physical manipulation of the Escherichia coli chromosome reveals its soft nature

Abstract

Replicating bacterial chromosomes continuously demix from each other and segregate within a compact volume inside the cell called the nucleoid. Although many proteins involved in this process have been identified, the nature of the global forces that shape and segregate the chromosomes has remained unclear because of limited knowledge of the micromechanical properties of the chromosome. In this work, we demonstrate experimentally the fundamentally soft nature of the bacterial chromosome and the entropic forces that can compact it in a crowded intracellular environment. We developed a unique "micropiston" and measured the force-compression behavior of single Escherichia coli chromosomes in confinement. Our data show that forces on the order of 100 pN and free energies on the order of 10 5 k B T are sufficient to compress the chromosome to its in vivo size. For comparison, the pressure required to hold the chromosome at this size is a thousand-fold smaller than the surrounding turgor pressure inside the cell. Furthermore, by manipulation of molecular crowding conditions (entropic forces), we were able to observe in real time fast (approximately 10 s), abrupt, reversible, and repeatable compaction-decompaction cycles of individual chromosomes in confinement. In contrast, we observed much slower dissociation kinetics of a histone-like protein HU from the whole chromosome during its in vivo to in vitro transition. These results for the first time provide quantitative, experimental support for a physical model in which the bacterial chromosome behaves as a loaded entropic spring in vivo.chromosome segregation | depletion forces | polymer physics | mother machine | optical trap

Molecular simulations of cellular processes

“…1d). Although in line with some experimental observations (Pelletier et al 2012), this finding disagrees with evidence supporting the co-transcriptional translation, the mechanism by which ribosomes penetrate into the nucleoid and translate the mRNA transcripts while bound to the nucleoid.…”

“…Macromolecular crowders are unlikely to be the only determinants of chromosome compaction, as Shendruk et al (2015) concluded from observing a continuous rather than first-order transition as in experiments (Pelletier et al 2012). One such factor, i.e., DNA binding proteins (DBPs), has been studied by Brackley et al (2013b).…”

The effect of macromolecular crowding on mobility of biomolecules, association kinetics, and gene expression in living cells

“…Indeed, the fast real-time compaction of chromosomes to native volumes [126] is observed in systems crowded by polymers. Our predictions are obtained for exponential phase of growth of E. coli, for which nucleoid is in an expanded state.…”

Persistent super-diffusive motion of Escherichia coli chromosomal loci

“…Notably, the power law exponent B0.4 is not reproduced by standard polymer models 6 . The cytosol is a highly crowded environment, which promotes the chromosome to be (semi-)collapsed due to osmotic self-attraction 9,10 . However, in vivo the compacted chromosome (nucleoid) maintains a well-defined shape and tends to occupy a significantly smaller volume than the cell, likely a result of the activity of numerous DNA-associated proteins with varying functionality [11][12][13][14] .…”