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.

中文翻译：

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基因组模块化揭示重叠基因拓扑结构是有效病毒繁殖所必需的

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以使其能够单独操作的过程。基因重叠的生物学功能和适应度效应尚不完全清楚，它们对基因簇和基因组水平重构的影响尚不清楚。噬菌体 φX174 基因组约有 26% 的核苷酸参与编码一种以上的基因。在这项研究中，我们使用了一个工程改造的 φX174 噬菌体，该噬菌体包含一个去除了所有基因重叠的基因组，以表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、和附件缺陷。整个感染周期中完整噬菌体蛋白质组的定量显示 30% 的蛋白质显示异常表达模式。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。这项工作强调了自然基因组中基因重叠的生物学重要性，并且减少基因重叠破坏应该是未来基因组工程项目的一个组成部分。