Seow Vui Yin, Tsygelnytska Olga, Biais Nicolas
Brooklyn College of the City University of New York, Brooklyn, NY, United States.
The Graduate Center of the City University of New York, New York, NY, United States.
Front Microbiol. 2023 Jun 20;14:1178128. doi: 10.3389/fmicb.2023.1178128. eCollection 2023.
Natural transformation, or the uptake of naked DNA from the external milieu by bacteria, holds a unique place in the history of biology. This is both the beginning of the realization of the correct chemical nature of genes and the first technical step to the molecular biology revolution that sees us today able to modify genomes almost at will. Yet the mechanistic understanding of bacterial transformation still presents many blind spots and many bacterial systems lag behind power horse model systems like in terms of ease of genetic modification. Using as a model system and using transformation with multiple DNA molecules, we tackle in this paper both some aspects of the mechanistic nature of bacterial transformation and the presentation of new molecular biology techniques for this organism. We show that similarly to what has been demonstrated in other naturally competent bacteria, can incorporate, at the same time, different DNA molecules modifying DNA at different loci within its genome. In particular, co-transformation of a DNA molecule bearing an antibiotic selection cassette and another non-selected DNA piece can lead to the integration of both molecules in the genome while selecting only through the selective cassette at percentages above 70%. We also show that successive selections with two selection markers at the same genetic locus can drastically reduce the number of genetic markers needed to do multisite genetic modifications in . Despite public health interest heightened with the recent rise in antibiotic resistance, the causative agent of gonorrhea still does not possess a plethora of molecular techniques. This paper will extend the techniques available to the Neisseria community while providing some insights into the mechanisms behind bacterial transformation in . We are providing a suite of new techniques to quickly obtain modifications of genes and genomes in the Neisserial naturally competent bacteria.
自然转化，即细菌从外部环境中摄取裸露的DNA，在生物学史上占有独特的地位。这既是认识基因正确化学本质的开端，也是分子生物学革命的第一步技术突破，使我们如今几乎能够随意修改基因组。然而，对细菌转化的机制理解仍存在许多盲点，而且许多细菌系统在基因修饰的便捷性方面落后于像[具体模型系统名称]这样的主流模型系统。本文以[具体模型系统名称]为模型系统，并利用多个DNA分子进行转化，探讨了细菌转化机制本质的一些方面，以及针对该生物体的新分子生物学技术。我们发现，与其他天然感受态细菌中所证明的情况类似，[具体细菌名称]能够同时整合不同的DNA分子，在其基因组内的不同位点对DNA进行修饰。特别是，携带抗生素选择盒的DNA分子与另一个未选择的DNA片段的共转化，可导致两个分子都整合到基因组中，同时仅通过选择盒进行选择，整合率高于70%。我们还表明，在同一基因位点使用两个选择标记进行连续选择，可大幅减少在[具体细菌名称]中进行多位点基因修饰所需的遗传标记数量。尽管近期抗生素耐药性上升引发了公众对公共卫生的关注，但淋病病原体仍然缺乏大量的分子技术。本文将扩展奈瑟菌属群体可用的技术，同时深入了解[具体细菌名称]中细菌转化背后的机制。我们提供了一套新技术，可快速在奈瑟菌属天然感受态细菌中实现基因和基因组的修饰。
