Miravet-Verde Samuel, Burgos Raul, Garcia-Ramallo Eva, Weber Marc, Serrano Luis
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona, 08003, Spain.
Department of Biology, Institute of Microbiology and Swiss Institute of Bioinformatics, ETH Zurich, Zurich, Switzerland.
Mol Syst Biol. 2025 Aug 13. doi: 10.1038/s44320-025-00133-1.
Essentiality studies have traditionally focused on coding regions, often overlooking other small genetic regulatory elements. To address this, we combined transposon libraries containing promoter or terminator sequences to obtain a high-resolution essentiality map of a genome-reduced bacterium, at near-single-nucleotide precision when considering non-essential genes. By integrating temporal transposon-sequencing data by k-means unsupervised clustering, we present a novel essentiality assessment approach, providing dynamic and quantitative information on the fitness contribution of different genomic regions. We compared the insertion tolerance and persistence of the two engineered libraries, assessing the local impact of transcription and termination on cell fitness. Essentiality assessment at the local base-level revealed essential protein domains and small genomic regions that are either essential or inaccessible to transposon insertion. We also identified structural regions within essential genes that tolerate transposon disruptions, resulting in functionally split proteins. Overall, this study presents a nuanced view of gene essentiality, shifting from static and binary models to a more accurate perspective. Additionally, it provides valuable insights for genome engineering and enhances our understanding of the biology of genome-reduced cells.
必要性研究传统上主要集中在编码区域，常常忽略其他小的基因调控元件。为了解决这个问题，我们将包含启动子或终止子序列的转座子文库结合起来，以获得一种基因组简化细菌的高分辨率必要性图谱，在考虑非必需基因时达到近单核苷酸精度。通过k均值无监督聚类整合时间转座子测序数据，我们提出了一种新颖的必要性评估方法，提供了关于不同基因组区域适应性贡献的动态和定量信息。我们比较了两个工程文库的插入耐受性和持久性，评估转录和终止对细胞适应性的局部影响。在局部碱基水平的必要性评估揭示了必需的蛋白质结构域和对转座子插入而言要么必需要么无法插入的小基因组区域。我们还在必需基因内鉴定出了耐受转座子破坏的结构区域，从而产生了功能上分裂的蛋白质。总体而言，这项研究呈现了基因必要性的细微差别观点，从静态和二元模型转向了更准确的视角。此外，它为基因组工程提供了有价值的见解，并增进了我们对基因组简化细胞生物学的理解。
