Department of Respiratory Diseases, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Department of Infectious Diseases, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.
BMC Microbiol. 2020 Feb 28;20(1):40. doi: 10.1186/s12866-020-01729-3.
Acinetobacter baylyi ADP1 is an ideal bacterial strain for high-throughput genetic analysis as the bacterium is naturally transformable. Thus, ADP1 can be used to investigate DNA mismatch repair, a mechanism for repairing mismatched bases. We used the mutS deletion mutant (XH439) and mutL deletion mutant (XH440), and constructed a mutS mutL double deletion mutant (XH441) to investigate the role of the mismatch repair system in A. baylyi.
We determined the survival rates after UV irradiation and measured the mutation frequencies, rates and spectra of wild-type ADP1 and mutSL mutant via rifampin resistance assay (Rif assay) and experimental evolution. In addition, transformation efficiencies of genomic DNA in ADP1 and its three mutants were determined. Lastly, the relative growth rates of the wild type strain, three constructed deletion mutants, as well as the rifampin resistant mutants obtained from Rif assays, were measured. All three mutants had higher survival rates after UV irradiation than wild type, especially the double deletion mutant. Three mutants showed higher mutation frequencies than ADP1 and favored transition mutations in Rif assay. All three mutants showed increased mutation rates in the experimental evolution. However, only XH439 and XH441 had higher mutation rates than the wild type strain in Rif assay. XH441 showed higher transformation efficiency than XH438 when donor DNA harbored transition mutations. All three mutants showed higher growth rates than wild-type, and these four strains displayed higher growth rates than almost all their rpoB mutants. The growth rate results showed different amino acid mutations in rpoB resulted in different extents of reduction in the fitness of rifampin resistant mutants. However, the fitness cost brought by the same mutation did not vary with strain background.
We demonstrated that inactivation of both mutS and mutL increased the mutation rates and frequencies in A. baylyi, which would contribute to the evolution and acquirement of rifampicin resistance. The mutS deletion is also implicated in increased mutation rates and frequencies, suggesting that MutL may be activated even in the absence of mutS. The correlation between fitness cost and rifampin resistance mutations in A. baylyi is firstly established.
鲍曼不动杆菌 ADP1 是一种理想的高通量遗传分析细菌株，因为它天然可转化。因此，ADP1 可用于研究 DNA 错配修复，这是一种修复错配碱基的机制。我们使用 mutS 缺失突变体 (XH439) 和 mutL 缺失突变体 (XH440)，并构建了 mutS mutL 双缺失突变体 (XH441)，以研究错配修复系统在鲍曼不动杆菌中的作用。
我们通过紫外线照射后存活率的测定和利福平抗性测定 (Rif 测定) 和实验进化来测量野生型 ADP1 和 mutSL 突变体的突变频率、速率和谱。此外，还测定了 ADP1 及其三个突变体的基因组 DNA 的转化效率。最后，测定了野生型菌株、三个构建的缺失突变体以及 Rif 测定中获得的利福平抗性突变体的相对生长率。与野生型相比，所有三个突变体在紫外线照射后具有更高的存活率，尤其是双缺失突变体。三个突变体在 Rif 测定中显示出比 ADP1 更高的突变频率，并且有利于转换突变。所有三个突变体在实验进化中显示出更高的突变率。然而，只有 XH439 和 XH441 在 Rif 测定中比野生型菌株具有更高的突变率。当供体 DNA 携带转换突变时，XH441 显示出比 XH438 更高的转化效率。所有三个突变体的生长速度都高于野生型，这四个菌株的生长速度都高于其几乎所有 rpoB 突变体。生长速率结果表明，rpoB 中的不同氨基酸突变导致利福平抗性突变体的适应性降低程度不同。然而，同一突变带来的适应性成本不会随菌株背景而变化。
我们证明了 mutS 和 mutL 的失活均增加了鲍曼不动杆菌中的突变率和频率，这有助于获得利福平抗性。mutS 的缺失也与更高的突变率和频率有关，这表明即使在没有 mutS 的情况下，MutL 也可能被激活。首次在鲍曼不动杆菌中建立了适应性成本与利福平抗性突变之间的相关性。
