Department of Physics and Section of Molecular Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Proc Natl Acad Sci U S A. 2012 Oct 2;109(40):E2649-56. doi: 10.1073/pnas.1208689109. Epub 2012 Sep 14.
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.
复制的细菌染色体不断相互分离，并在细胞内称为核区的紧凑体积内进行分离。尽管已经鉴定出许多参与该过程的蛋白质，但由于对染色体的微观机械特性的了解有限，导致塑造和分离染色体的全局力的性质仍然不清楚。在这项工作中，我们通过实验证明了细菌染色体的基本柔软性质，以及在拥挤的细胞内环境中可以使其浓缩的熵力。我们开发了一种独特的“微活塞”，并测量了单个大肠杆菌染色体在受限空间中的力-压缩行为。我们的数据表明，约 100 pN 的力和约 10^5 kBT 的自由能足以将染色体压缩到其体内大小。相比之下，将染色体保持在这种大小所需的压力比细胞内周围的膨压小一千倍。此外，通过操纵分子拥挤条件（熵力），我们能够实时观察到单个染色体在受限空间中的快速（约 10 s）、突然、可逆和可重复的浓缩-去浓缩循环。相比之下，我们观察到组蛋白样蛋白 HU 从体内到体外转变过程中从整个染色体上解离的动力学要慢得多。这些结果首次为物理模型提供了定量的、实验性的支持，该模型表明细菌染色体在体内表现为负载的熵弹簧。
