利用含有负选择标记 rpsL 的热敏质粒,在细胞内分枝杆菌 ATCC13950 株中构建基因敲除突变体。

IF 1.9 4区 医学 Q4 IMMUNOLOGY
Yoshitaka Tateishi, Akihito Nishiyama, Yuriko Ozeki, Sohkichi Matsumoto
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引用次数: 0

摘要

背景:过去 20 年来,非结核分枝杆菌疾病已在全球范围内出现。然而,目前有关构建非结核分枝杆菌基因敲除突变体的成熟技术的报道很少。因此,非结核分枝杆菌的基因重组技术还需要进一步研究:我们构建了载体 pPR23LHR,它含有核糖体蛋白 S12 基因(rpsL+)作为显性阴性选择标记,土霉素(Hyg)和 lacZ 盒作为阳性选择标记。我们构建了蛋白酶体基因的基因敲除突变体,通过功能基因组分析,我们发现这些基因是细胞内分枝杆菌缺氧小泡形成所必需的。基因敲除突变体显示缺氧小泡形成受损,这与之前使用蛋白酶体抑制剂环氧米星得出的数据一致:我们的研究结果表明,rpsL+ 是非结核分枝杆菌基因重组的高效显性阴性选择标记。我们采用对温度敏感的 rpsL+ 方法构建基因敲除突变体将有助于进行功能测试,以验证非结核分枝杆菌的毒力因子和非结核分枝杆菌疾病的发病机制。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Construction of knockout mutants in Mycobacterium intracellulare ATCC13950 strain using a thermosensitive plasmid containing negative selection marker rpsL+

Construction of knockout mutants in Mycobacterium intracellulare ATCC13950 strain using a thermosensitive plasmid containing negative selection marker rpsL+

Background

Nontuberculous mycobacterial disease has emerged worldwide over the past 20 years. However, there are currently few reports on the established technique for constructing knockout mutants of nontuberculous mycobacteria. Therefore, gene recombination techniques for nontuberculous mycobacteria require further research.

Results

We constructed vector pPR23LHR that harbors the ribosomal protein S12 gene (rpsL+) as a dominant negative selection marker and the hygromycin (Hyg) and lacZ cassettes as positive selection markers. We constructed knockout mutants of proteasomal genes, which we found to be required for hypoxic pellicle formation in Mycobacterium intracellulare by functional genomic analysis. The knockout mutants showed impaired hypoxic pellicle formation, consistent with previous data using epoxomicin, a proteasomal inhibitor.

Conclusions

Our findings demonstrate that rpsL+ is an efficient dominant negative selection marker for gene recombination in nontuberculous mycobacteria. Our temperature-sensitive rpsL+ method for the construction of knockout mutants will facilitate functional assays to validate the virulence factors of nontuberculous mycobacteria and the pathogenesis of nontuberculous mycobacterial disease.

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来源期刊
Microbiology and Immunology
Microbiology and Immunology 医学-免疫学
CiteScore
5.20
自引率
3.80%
发文量
78
审稿时长
1 months
期刊介绍: Microbiology and Immunology is published in association with Japanese Society for Bacteriology, Japanese Society for Virology, and Japanese Society for Host Defense Research. It is peer-reviewed publication that provides insight into the study of microbes and the host immune, biological and physiological responses. Fields covered by Microbiology and Immunology include:Bacteriology|Virology|Immunology|pathogenic infections in human, animals and plants|pathogenicity and virulence factors such as microbial toxins and cell-surface components|factors involved in host defense, inflammation, development of vaccines|antimicrobial agents and drug resistance of microbes|genomics and proteomics.
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