Quantitative essentiality in a reduced genome: a functional, regulatory and structural fitness map

Essentiality studies have traditionally focused on coding regions, often overlooking other small genetic regulatory elements. To address this, we obtained a high-resolution essentiality map at near-single-nucleotide precision of the genome-reduced bacterium Mycoplasma pneumoniae , combining transposon libraries containing promoter or terminator sequences. By integrating temporal transposon-sequencing data, we developed a novel method of essentiality assessment based on k-means unsupervised clustering, which provides 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.