Department of Molecular Biosciences, Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas, United States of America.
PLoS Genet. 2024 Sep 16;20(9):e1011306. doi: 10.1371/journal.pgen.1011306. eCollection 2024 Sep.
Organelles and endosymbionts have naturally evolved dramatically reduced genome sizes compared to their free-living ancestors. Synthetic biologists have purposefully engineered streamlined microbial genomes to create more efficient cellular chassis and define the minimal components of cellular life. During natural or engineered genome streamlining, deletion of many non-essential genes in combination often reduces bacterial fitness for idiosyncratic or unknown reasons. We investigated how and to what extent laboratory evolution could overcome these defects in six variants of the transposon-free Acinetobacter baylyi strain ADP1-ISx that each had a deletion of a different 22- to 42-kilobase region and two strains with larger deletions of 70 and 293 kilobases. We evolved replicate populations of ADP1-ISx and each deletion strain for ~300 generations in a chemically defined minimal medium or a complex medium and sequenced the genomes of endpoint clonal isolates. Fitness increased in all cases that were examined except for two ancestors that each failed to improve in one of the two environments. Mutations affecting nine protein-coding genes and two small RNAs were significantly associated with one of the two environments or with certain deletion ancestors. The global post-transcriptional regulators rnd (ribonuclease D), csrA (RNA-binding carbon storage regulator), and hfq (RNA-binding protein and chaperone) were frequently mutated across all strains, though the incidence and effects of these mutations on gene function and bacterial fitness varied with the ancestral deletion and evolution environment. Mutations in this regulatory network likely compensate for how an earlier deletion of a transposon in the ADP1-ISx ancestor of all the deletion strains restored csrA function. More generally, our results demonstrate that fitness lost during genome streamlining can usually be regained rapidly through laboratory evolution and that recovery tends to occur through a combination of deletion-specific compensation and global regulatory adjustments.
细胞器和内共生体与自由生活的祖先相比，其基因组大小自然显著缩小。合成生物学家有意设计了精简的微生物基因组，以创建更高效的细胞底盘，并定义细胞生命的最小组成部分。在自然或工程基因组精简过程中，许多非必需基因的缺失常常会降低细菌的适应能力，但原因却各不相同或未知。我们研究了实验室进化如何以及在何种程度上可以克服六种转座子缺失的鲍曼不动杆菌 ADP1-ISx 变体中的这些缺陷，这六种变体中的每一种都缺失了不同的 22-42kb 区域，还有两种菌株缺失了 70kb 和 293kb 区域。我们在化学定义的最小培养基或复杂培养基中对 ADP1-ISx 和每个缺失菌株的复制种群进行了约 300 代的进化，并对终点克隆分离株的基因组进行了测序。除了两个祖先在两种环境中的一个中都未能得到改善的情况外，所有被检测的情况都观察到了适应性的增加。影响九个蛋白编码基因和两个小 RNA 的突变与两种环境之一或特定的缺失祖先显著相关。全局转录后调控因子 rnd（核糖核酸酶 D）、csrA（RNA 结合碳储存调节因子）和 hfq（RNA 结合蛋白和伴侣）在所有菌株中经常发生突变，尽管这些突变对基因功能和细菌适应性的影响因祖先缺失和进化环境而异。该调控网络中的突变可能补偿了所有缺失菌株的 ADP1-ISx 祖先中早期转座子缺失如何恢复 csrA 功能。更一般地说，我们的结果表明，在基因组精简过程中失去的适应性通常可以通过实验室进化迅速恢复，并且恢复往往是通过缺失特异性补偿和全局调节调整的组合来实现的。
