耻垢分枝杆菌MSMEG_4259基因基因组维护功能研究

邓茗芝 许原原 吕亮东

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微生物与感染 ›› 2022, Vol. 17 ›› Issue (5) : 273-281.

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耻垢分枝杆菌MSMEG_4259基因基因组维护功能研究

邓茗芝¹,许原原¹,吕亮东²

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Functional study on the genome maintenance of Mycolicibacterium smegmatis MSMEG_4259

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摘要

MSMEG_4259基因及其同源基因广泛存在于分枝杆菌属中。蛋白序列分析显示MSMEG_4259包含DEDDh核酸外切酶结构域以及一个类似UvrC的核酸内切酶结构域，提示其可能参与DNA复制或修复。为了探究MSMEG_4259的生理功能，我们利用同源重组的方法在耻垢分枝杆菌（Mycolicibacterium smegmatis）构建了MSMEG_4259基因敲除菌株。通过测定了野生株和ΔMSMEG_4259菌株的生长曲线，发现缺失MSMEG_4259不影响耻垢分枝杆菌的正常生长。采用波动试验测定了菌株在对数生长期以及在H2O2处理后的利福霉素耐药突变频率，结果显示ΔMSMEG_4259菌株的自发突变频率都较野生株升高1.9倍（P<0.05），该表型能够通过表达MSMEG_4259回补，在H2O2处理条件下，野生株和ΔMSMEG_4259菌株的诱发突变频率较未处理组分别提高3.8倍和2.4倍（P<0.001），说明MSMEG_4259参与耻垢分枝杆菌基因组的维护。通过测定利福霉素耐药突变菌株的rpoB基因进行测序以及突变谱分析，发现ΔMSMEG_4259菌株的AT>CG突变比例上升。荧光定量PCR结果显示野生株的MSMEG_4259在H2O2处理后上调约7倍，提示该基因参与DNA氧化损伤应答。通过测定野生株和ΔMSMEG_4259在氧氟沙星、叔丁基过氧化氢和紫外线处理下的存活率，发现MSMEG_4259不参与耻垢分枝杆菌对上述DNA损伤剂的耐受。本研究揭示MSMEG_4259参与耻垢分枝杆菌基因组维护的生理功能及相关特性，为进一步研究其同源基因在结核分枝杆菌致病和耐药中的作用机制奠定了基础。

Abstract

MSMEG_4259 and its homologs are widely present in Mycobacterium, and protein sequence analysis revealed that MSMEG_4259 contains a DEDDh 3'-5' exonuclease domain as well as a GIY-YIG domain of nucleotide excision repair endonucleases UvrC, suggesting its possible involvement in DNA replication or repair. To investigate the physiological function of MSMEG_4259, MSMEG_4259 knockout strain were generated in Mycolicibacterium smegmatis using a homologous recombination method. We measured the growth curves of the wild type and the knockout strains and found no difference, suggesting that MSMEG_4259 is not an essential gene for growth of Mycolicibacterium smegmatis in eutrophic culture conditions. We measured the mutation frequencies of rifamycin-resistant mutation in these strains during the logarithmic growth phase as well as after H2O2 treatment by using a fluctuation test. The results showed that, during the logarithmic growth period, the mutation frequencies in the MSMEG_4259 knockout strains were increased 1.9-fold (P<0.05) compared to that of the wild type. The mutator phenotype of knockout strain was able to be complemented by expression of the wild-type MSMEG_4259. After H2O2 treatment, the mutation frequencies of the wild type and ΔMSMEG_4259 were elevated 3.7- and 2-fold (P<0.001) compared to untreated. These results showed that MSMEG_4259 is involved in the maintenance of genome stability. The results of real-time fluorescence quantitative PCR (qRT-PCR) showed that the transcription of MSMEG_4259 was upregulated 7-fold in wild type upon H2O2 treatment, suggesting that MSMEG_4259 may be involved in the response to DNA oxidative damage. We measured the survival of wild strain and ΔMSMEG_4259 under treatment with ofloxacin, tert-butyl hydrogen peroxide and UV and found that MSMEG_4259 was not involved in the tolerance of Mycolicibacterium smegmatis to these DNA damaging agents. This study reveals the physiological functions of MSMEG_4259 in Mycolicibacterium smegmatis and lays the foundation for further studies on the role of its homolog in the pathogenesis and drug resistance of Mycobacterium tuberculosis.

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邓茗芝许原原吕亮东. 耻垢分枝杆菌MSMEG_4259基因基因组维护功能研究[J]. 微生物与感染. 2022, 17(5): 273-281

Functional study on the genome maintenance of Mycolicibacterium smegmatis MSMEG_4259[J]. Journal of Microbes and Infections. 2022, 17(5): 273-281

中图分类号： R378.91

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