Kotaka Yuto, Hashimoto Masayuki, Lee Ken-Ichi, Kato Jun-Ichi
Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Tokyo, Japan.
Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan.
Front Microbiol. 2023 May 25;14:1189877. doi: 10.3389/fmicb.2023.1189877. eCollection 2023.
Characterizing genes that regulate cell growth and survival in model organisms is important for understanding higher organisms. Construction of strains harboring large deletions in the genome can provide insights into the genetic basis of cell growth compared with only studying wild-type strains. We have constructed a series of genome-reduced strains with deletions spanning approximately 38.9% of the chromosome. Strains were constructed by combining large deletions in chromosomal regions encoding nonessential gene groups. We also isolated strains Δ33b and Δ37c, whose growth was partially restored by adaptive laboratory evolution (ALE). Genome sequencing of nine strains, including those selected following ALE, identified the presence of several Single Nucleotide Variants (SNVs), insertions, deletions, and inversions. In addition to multiple SNVs, two insertions were identified in ALE strain Δ33b. The first was an insertion at the promoter region of , which increased cognate gene expression. The second was an insertion sequence (IS) present in , encoding the antitoxin in a toxin-antitoxin system, which decreased expression of . 5 strains of Δ37c independently isolated following ALE harboring multiple SNVs and genetic rearrangements. Interestingly, a SNV was identified in the promoter region of in all five strains, which increased expression and, we predict, rescued the attenuated Δ37b growth. Experiments using defined deletion mutants suggested that encodes a 3-phenylpropionate transporter protein and is involved in survival during stationary phase under oxidative stress. This study is the first to document accumulation of mutations during construction of genome-reduced strains. Furthermore, isolation and analysis of strains derived from ALE in which the growth defect mediated by large chromosomal deletions was rescued identified novel genes involved in cell survival.
在模式生物中鉴定调控细胞生长和存活的基因对于理解高等生物至关重要。与仅研究野生型菌株相比，构建基因组中存在大片段缺失的菌株能够为细胞生长的遗传基础提供深入见解。我们构建了一系列基因组精简菌株，其缺失片段跨越了约38.9%的染色体。这些菌株是通过将编码非必需基因组的染色体区域中的大片段缺失进行组合构建而成的。我们还分离出了菌株Δ33b和Δ37c，它们的生长通过适应性实验室进化（ALE）得到了部分恢复。对包括ALE后筛选出的菌株在内的9个菌株进行基因组测序，确定存在多个单核苷酸变异（SNV）、插入、缺失和倒位。除了多个SNV外，在ALE菌株Δ33b中还鉴定出两个插入。第一个是在 的启动子区域的插入，它增加了同源基因的表达。第二个是存在于 中的插入序列（IS），其在毒素-抗毒素系统中编码抗毒素，这降低了 的表达。ALE后独立分离出的5株Δ37c菌株含有多个SNV和基因重排。有趣的是，在所有五株菌株的 的启动子区域都鉴定出一个SNV，它增加了 的表达，并且我们预测，挽救了减弱的Δ37b生长。使用确定的缺失突变体进行的实验表明， 编码一种3-苯丙酸转运蛋白，并参与氧化应激下稳定期的存活。本研究首次记录了基因组精简菌株构建过程中突变的积累。此外，对ALE衍生菌株的分离和分析，其中由大片段染色体缺失介导的生长缺陷得到挽救，鉴定出了参与细胞存活的新基因。
