Bacterial gene transfer studied at the single molecule level

大分子通过纳米尺寸的膜孔的运输是细胞生物学中普遍存在的主题。例子包括前体蛋白线性输入线粒体和细菌基因转移过程中的DNA运输。在前一个项目阶段，我们获得了强有力的证据表明，细菌转化过程中的DNA吸收是由一个易位棘轮机制驱动的;在周质中的ComE分子伴侣的结合偏向DNA扩散通过膜孔在DNA吸收的方向。在生物物理学教科书中已经讨论了移位棘轮机制，但据我们所知，很少有实验数据表明这种基本的分子马达产生力。在下一个项目阶段，我们建议通过将单分子工具与分子生物学相结合来表征易位棘轮的生物物理特性。特别是，我们将评估分子伴侣解离常数的变化如何影响速度，力的产生，和电动机的热力学效率。我们将通过用其他DNA结合蛋白取代天然伴侣ComE来产生具有人工易位棘轮的细菌菌株，进一步仔细检查易位棘轮模型。这种方法将使我们能够系统地调整解离常数和DNA上结合位点的密度。最后，我们将研究转运棘轮通过外膜转运DNA到细胞质中的马达的耦合。

Transport of macromolecules through nanometer-sized membrane pores is a ubiquitous theme in cell biology. Examples include the linear import of precursor proteins into mitochondria and DNA transport during bacterial gene transfer. During the previous project phase, we obtained strong evidence that the uptake of DNA during bacterial transformation is driven by a translocation ratchet mechanism; binding of ComE chaperones in the periplasm biases DNA diffusion through a membrane pore in the direction of DNA uptake. The translocation ratchet mechanism has been discussed in biophysical textbooks, but to our knowledge, there is little experimental data on force generation by this basic molecular motor. In the next project phase, we propose characterizing the biophysical properties of the translocation ratchet by combining single molecule tools with molecular biology. In particular, we will assess how variation of the chaperone dissociation constants affects velocity, force generation, and thermodynamic efficiency of the motor. We will further scrutinize the translocation ratchet model by generating bacterial strains with an artificial translocation ratchet by replacing the native chaperones ComE by other DNA-binding proteins. This approach will allow us to systematically tune the dissociation constants and the density of binding sites on the DNA. Finally, we will investigate the coupling of the translocation ratchet transporting DNA through the outer membrane to the motor that transports DNA into the cytoplasm.