{"title":"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。","authors":"Yoshitaka Tateishi, Akihito Nishiyama, Yuriko Ozeki, Sohkichi Matsumoto","doi":"10.1111/1348-0421.13167","DOIUrl":null,"url":null,"abstract":"<div>\n \n \n <section>\n \n <h3> Background</h3>\n \n <p>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.</p>\n </section>\n \n <section>\n \n <h3> Results</h3>\n \n <p>We constructed vector pPR23LHR that harbors the ribosomal protein S12 gene (<i>rpsL</i><sup><i>+</i></sup>) as a dominant negative selection marker and the hygromycin (Hyg) and <i>lacZ</i> cassettes as positive selection markers. We constructed knockout mutants of proteasomal genes, which we found to be required for hypoxic pellicle formation in <i>Mycobacterium intracellulare</i> by functional genomic analysis. The knockout mutants showed impaired hypoxic pellicle formation, consistent with previous data using epoxomicin, a proteasomal inhibitor.</p>\n </section>\n \n <section>\n \n <h3> Conclusions</h3>\n \n <p>Our findings demonstrate that <i>rpsL</i><sup><i>+</i></sup> is an efficient dominant negative selection marker for gene recombination in nontuberculous mycobacteria. Our temperature-sensitive <i>rpsL</i><sup>+</sup> 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.</p>\n </section>\n </div>","PeriodicalId":18679,"journal":{"name":"Microbiology and Immunology","volume":null,"pages":null},"PeriodicalIF":1.9000,"publicationDate":"2024-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1348-0421.13167","citationCount":"0","resultStr":"{\"title\":\"Construction of knockout mutants in Mycobacterium intracellulare ATCC13950 strain using a thermosensitive plasmid containing negative selection marker rpsL+\",\"authors\":\"Yoshitaka Tateishi, Akihito Nishiyama, Yuriko Ozeki, Sohkichi Matsumoto\",\"doi\":\"10.1111/1348-0421.13167\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div>\\n \\n \\n <section>\\n \\n <h3> Background</h3>\\n \\n <p>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.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Results</h3>\\n \\n <p>We constructed vector pPR23LHR that harbors the ribosomal protein S12 gene (<i>rpsL</i><sup><i>+</i></sup>) as a dominant negative selection marker and the hygromycin (Hyg) and <i>lacZ</i> cassettes as positive selection markers. We constructed knockout mutants of proteasomal genes, which we found to be required for hypoxic pellicle formation in <i>Mycobacterium intracellulare</i> by functional genomic analysis. The knockout mutants showed impaired hypoxic pellicle formation, consistent with previous data using epoxomicin, a proteasomal inhibitor.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Conclusions</h3>\\n \\n <p>Our findings demonstrate that <i>rpsL</i><sup><i>+</i></sup> is an efficient dominant negative selection marker for gene recombination in nontuberculous mycobacteria. Our temperature-sensitive <i>rpsL</i><sup>+</sup> 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.</p>\\n </section>\\n </div>\",\"PeriodicalId\":18679,\"journal\":{\"name\":\"Microbiology and Immunology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.9000,\"publicationDate\":\"2024-07-23\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1348-0421.13167\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Microbiology and Immunology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1111/1348-0421.13167\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"IMMUNOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Microbiology and Immunology","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1111/1348-0421.13167","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"IMMUNOLOGY","Score":null,"Total":0}
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.
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.