Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA.
Microbiol Spectr. 2024 Jun 4;12(6):e0397323. doi: 10.1128/spectrum.03973-23. Epub 2024 May 3.
Plasmids play important roles in microbial ecosystems, serving as carriers of antibiotic resistance and virulence. In the laboratory, they are essential tools for genetic manipulation and recombinant protein expression. We uncovered an intriguing survival phenotype in a fraction of the bacterial population while using plasmid-mediated arabinose-inducible gene expression to monitor the production of toxic ParE proteins. This phenotype was not correlated with changes to the plasmid sequence and could not be rescued by increasing arabinose uptake. Instead, survival correlates with a marked reduction in plasmid copy number (PCN). Reduced PCN is reproducible, not a function of the pre-existing population, and can be sequentially enriched by continual passage with induction. The reduction in PCN appears to allow mitigation of toxicity from the expression of ParE proteins while balancing the need to maintain a threshold PCN to withstand selection conditions. This indicates an adaptive cellular response to stressful conditions, likely by altering the regulation of plasmid replication. Furthermore, this survival mechanism appears to not be limited to a specific bacterial strain of or ParE toxin family member, suggesting a generalized response. Finally, bacterial whole genome sequencing indicated an N845S residue substitution in DNA polymerase I, which correlates with the observed reduction in PCN and has been previously reported to impact plasmid replication. Further understanding this molecular mechanism has broader implications for this adaptive response of the dynamics of plasmid-mediated gene expression, microbial adaptation, and genetic engineering methodologies.
This research has increased our understanding of how bacteria respond to the pressure from plasmid-borne toxic genes, such as those found in toxin-antitoxin systems. Surprisingly, we found that bacteria survived toxic ParE protein expression by reducing the number of these plasmids in the cells. This discovery reveals another way in which bacteria can balance toxin expression with antibiotic selection to attenuate the effects of deleterious genes. This insight is not only valuable for understanding bacterial survival strategies but may also influence the development of better tools in biotechnology, where plasmids are often used to study the functional roles of genes.
质粒在微生物生态系统中发挥着重要作用，是抗生素抗性和毒力的载体。在实验室中，它们是遗传操作和重组蛋白表达的重要工具。当我们使用质粒介导的阿拉伯糖诱导基因表达来监测有毒 ParE 蛋白的产生时，我们在细菌群体的一部分中发现了一种有趣的生存表型。这种表型与质粒序列的变化无关，并且不能通过增加阿拉伯糖摄取来挽救。相反，生存与质粒拷贝数（PCN）的显著降低相关。PCN 的降低是可重复的，不是现有群体的功能，并且可以通过连续诱导传代来依次富集。PCN 的降低似乎可以减轻 ParE 蛋白表达的毒性，同时平衡维持足以承受选择条件的阈值 PCN 的需要。这表明这是一种适应细胞对压力条件的反应，可能通过改变质粒复制的调节来实现。此外，这种生存机制似乎不仅限于特定的 或 ParE 毒素家族成员的细菌菌株，表明这是一种普遍的反应。最后，细菌全基因组测序表明 DNA 聚合酶 I 中的 N845S 残基取代与观察到的 PCN 降低相关，并且先前已经报道过该取代会影响质粒复制。进一步了解这种分子机制对质粒介导基因表达动力学、微生物适应和遗传工程方法的这种适应性反应具有更广泛的意义。
这项研究增加了我们对细菌如何应对来自质粒携带的毒性基因（如毒素-抗毒素系统中发现的基因）的压力的理解。令人惊讶的是，我们发现细菌通过减少细胞中这些质粒的数量来存活有毒 ParE 蛋白的表达。这一发现揭示了细菌平衡毒素表达与抗生素选择以减轻有害基因影响的另一种方式。这一见解不仅对理解细菌的生存策略有价值，而且可能会影响生物技术中更好的工具的发展，其中质粒经常用于研究基因的功能作用。
