Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, Australia.
Electron Microscope Unit, Mark Wainwright Analytical Centre, The University of New South Wales, Sydney, NSW 2052, Australia.
ACS Synth Biol. 2020 Nov 20;9(11):3079-3090. doi: 10.1021/acssynbio.0c00323. Epub 2020 Oct 12.
Sequence overlap between two genes is common across all genomes, with viruses having high proportions of these gene overlaps. Genome modularization and refactoring is the process of disrupting natural gene overlaps to separate coding sequences to enable their individual manipulation. The biological function and fitness effects of gene overlaps are not fully understood, and their effects on gene cluster and genome-level refactoring are unknown. The bacteriophage φX174 genome has ∼26% of nucleotides involved in encoding more than one gene. In this study we use an engineered φX174 phage containing a genome with all gene overlaps removed to show that gene overlap is critical to maintaining optimal viral fecundity. Through detailed phenotypic measurements we reveal that genome modularization in φX174 causes virion replication, stability, and attachment deficiencies. Quantitation of the complete phage proteome across an infection cycle reveals 30% of proteins display abnormal expression patterns. Taken together, we have for the first time comprehensively demonstrated that gene modularization severely perturbs the coordinated functioning of a bacteriophage replication cycle. This work highlights the biological importance of gene overlap in natural genomes and that reducing gene overlap disruption should be an integral part of future genome engineering projects.
两个基因之间的序列重叠在所有基因组中都很常见，病毒中这些基因重叠的比例很高。基因组模块化和重构是破坏自然基因重叠以分离编码序列从而实现其单独操作的过程。基因重叠的生物学功能和适应度效应尚未完全了解，它们对基因簇和基因组水平重构的影响也未知。噬菌体 φX174 基因组约有 26%的核苷酸参与编码一个以上的基因。在这项研究中，我们使用一种经过工程改造的噬菌体 φX174，其基因组中去除了所有的基因重叠，以证明基因重叠对于维持最佳病毒繁殖力至关重要。通过详细的表型测量，我们揭示了 φX174 中的基因组模块化导致病毒粒子复制、稳定性和附着缺陷。在整个感染周期中对完整噬菌体蛋白质组的定量表明，30%的蛋白质显示出异常的表达模式。总之，我们首次全面证明了基因模块化严重扰乱了噬菌体复制周期的协调功能。这项工作突出了基因重叠在自然基因组中的生物学重要性，并且减少基因重叠的破坏应该成为未来基因组工程项目的一个组成部分。
