Standley Melissa S, Million-Weaver Samuel, Alexander David L, Hu Shuai, Camps Manel
Department of Microbiology and Environmental Toxicology, University of California, Santa Cruz, Santa Cruz, CA, 95064, USA.
College of Engineering, University of Wisconsin-Madison, Madison, 53706, USA.
Curr Genet. 2019 Feb;65(1):179-192. doi: 10.1007/s00294-018-0858-0. Epub 2018 Jun 16.
ColE1-like plasmid vectors are widely used for expression of recombinant genes in E. coli. For these vectors, segregation of individual plasmids into daughter cells during cell division appears to be random, making them susceptible to loss over time when no mechanisms ensuring their maintenance are present. Here we use the plasmid pGFPuv in a recA relA strain as a sensitized model to study factors affecting plasmid stability in the context of recombinant gene expression. We find that in this model, plasmid stability can be restored by two types of genetic modifications to the plasmid origin of replication (ori) sequence: point mutations and a novel 269 nt duplication at the 5' end of the plasmid ori, which we named DAS (duplicated anti-sense) ori. Combinations of these modifications produce a range of copy numbers and of levels of recombinant expression. In direct contradiction with the classic random distribution model, we find no correlation between increased plasmid copy number and increased plasmid stability. Increased stability cannot be explained by reduced levels of recombinant gene expression either. Our observations would be more compatible with a hybrid clustered and free-distribution model, which has been recently proposed based on detection of individual plasmids in vivo using super-resolution fluorescence microscopy. This work suggests a role for the plasmid ori in the control of segregation of ColE1 plasmids that is distinct from replication initiation, opening the door for the genetic regulation of plasmid stability as a strategy aimed at enhancing large-scale recombinant gene expression or bioremediation.
ColE1 样质粒载体广泛用于在大肠杆菌中表达重组基因。对于这些载体，在细胞分裂过程中单个质粒向子细胞的分离似乎是随机的，这使得在没有确保其维持的机制时，它们容易随时间丢失。在这里，我们使用 recA relA 菌株中的质粒 pGFPuv 作为敏感模型，来研究在重组基因表达背景下影响质粒稳定性的因素。我们发现在这个模型中，质粒稳定性可以通过对质粒复制起点（ori）序列进行两种类型的基因修饰来恢复：点突变和在质粒 ori 的 5' 端的一个新的 269 nt 重复序列，我们将其命名为 DAS（重复反义）ori。这些修饰的组合产生了一系列的拷贝数和重组表达水平。与经典的随机分布模型直接矛盾的是，我们发现质粒拷贝数增加与质粒稳定性增加之间没有相关性。增加的稳定性也不能通过重组基因表达水平的降低来解释。我们的观察结果与最近基于使用超分辨率荧光显微镜在体内检测单个质粒而提出的混合聚集和自由分布模型更相符。这项工作表明质粒 ori 在控制 ColE1 质粒的分离中具有不同于复制起始的作用，为将质粒稳定性的基因调控作为一种旨在增强大规模重组基因表达或生物修复的策略打开了大门。
