Due to the high concentration of proteins, nucleic acids, and other macromolecules, the bacterial cytoplasm is typically described as a crowded environment. However, the extent to which each of these macromolecules individually affects the mobility of macromolecular complexes, and how this depends on growth conditions, is presently unclear. In this study, we sought to quantify the crowding experienced by an exogenous 40 nm fluorescent particle in the cytoplasm of under different growth conditions. By performing single-particle tracking measurements in cells selectively depleted of DNA and/or mRNA, we determined the contribution to crowding of mRNA, DNA, and remaining cellular components, i.e., mostly proteins and ribosomes. To estimate this contribution to crowding, we quantified the difference of the particle’s diffusion coefficient in conditions with and without those macromolecules. We found that the contributions of the three classes of components were of comparable magnitude, being largest in the case of proteins and ribosomes. We further found that the contributions of mRNA and DNA to crowding were significantly larger than expected based on their volumetric fractions alone. Finally, we found that the crowding contributions change only slightly with the growth conditions. These results reveal how various cellular components partake in crowding of the cytoplasm and the consequences this has for the mobility of large macromolecular complexes.

中文翻译：

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不同大分子对 40 nm 颗粒在大肠杆菌中扩散的贡献

由于蛋白质、核酸和其他大分子的浓度很高，细菌细胞质通常被描述为一个拥挤的环境。然而，目前尚不清楚这些大分子中的每一个单独影响大分子复合物的迁移性的程度，以及这如何取决于生长条件。在这项研究中，我们试图量化不同生长条件下细胞质中外源 40 nm 荧光颗粒所经历的拥挤情况。通过在选择性耗尽 DNA 和/或 mRNA 的细胞中进行单粒子跟踪测量，我们确定了 mRNA、DNA 和剩余细胞成分（即主要是蛋白质和核糖体）拥挤的贡献。为了估计对拥挤的影响，我们量化了在有和没有这些大分子的条件下颗粒扩散系数的差异。我们发现三类成分的贡献大小相当，其中蛋白质和核糖体的贡献最大。我们进一步发现，mRNA 和 DNA 对拥挤的贡献明显大于仅根据其体积分数所预期的。最后，我们发现拥挤贡献仅随着生长条件而略有变化。这些结果揭示了各种细胞成分如何参与细胞质的拥挤以及这对大分子复合物的流动性的影响。