{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,2]],"date-time":"2026-01-02T05:12:55Z","timestamp":1767330775654,"version":"3.48.0"},"reference-count":36,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T00:00:00Z","timestamp":1767139200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Key Research and Development Program of China","award":["2020YFA0908300"],"award-info":[{"award-number":["2020YFA0908300"]}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["31970049"],"award-info":[{"award-number":["31970049"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibiotics"],"abstract":"<jats:p>Background: The global incidence of multidrug-resistant and extensively drug-resistant tuberculosis continues to rise. The ribosome serves as a target for multiple antimicrobials, making functional research on it hold great significance. Methods: Using homologous recombination combined with a multiple serine integrase-mediated site-specific recombination system, we replaced the two endogenous rRNA operons in Mycobacterium smegmatis MC2 155 with a single copy of the single rRNA operon from Mycobacterium tuberculosis H37Rv, constructing the M. smegmatis BRkoA strain. We assessed growth kinetics at 37 \u00b0C, cold sensitivity at lower temperatures, transcriptional levels by RT-qPCR, 70S ribosome integrity through cryo-EM, and antimicrobial susceptibility by microdilution assays. Results: The BRkoA strain was successfully constructed. It exhibited markedly slower growth compared to the wild-type strain. Cold-sensitivity assays indicated potential ribosome assembly defects, while transcriptional analysis suggested altered rRNA processing and modification. Cryo-EM analysis further demonstrated the absence of specific ribosomal proteins in the BRkoA 70S ribosome. Moreover, BRkoA displayed reduced susceptibility tendency to several ribosome-targeting antibiotics, including kanamycin, amikacin, paromomycin, gentamicin, and linezolid. Conclusions: Replacement of the two endogenous rrn operons in M. smegmatis with a single copy of the single M. tuberculosis rrn operon using a serine integrase-mediated recombination system caused growth impairment and decreased sensitivity tendency to several ribosome-targeting antimicrobials. These findings suggest that ribosome structural variation contributes to intrinsic drug resistance mechanisms.<\/jats:p>","DOI":"10.3390\/antibiotics15010030","type":"journal-article","created":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T18:10:51Z","timestamp":1767204651000},"page":"30","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Heterologous Substitution of Mycobacterium tuberculosis rRNA in Mycobacterium smegmatis and Its Impact on Antimicrobial Susceptibility"],"prefix":"10.3390","volume":"15","author":[{"given":"Qianwen","family":"Yue","sequence":"first","affiliation":[{"name":"Collaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan University, Shanghai 200438, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chan","family":"Shan","sequence":"additional","affiliation":[{"name":"Collaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan University, Shanghai 200438, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Arslan","family":"Habib","sequence":"additional","affiliation":[{"name":"Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200438, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guoping","family":"Zhao","sequence":"additional","affiliation":[{"name":"Collaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan University, Shanghai 200438, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaoming","family":"Ding","sequence":"additional","affiliation":[{"name":"Collaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan University, Shanghai 200438, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"555","DOI":"10.1016\/j.tim.2018.02.012","article-title":"Mycobacterium tuberculosis","volume":"26","author":"Koch","year":"2018","journal-title":"Trends Microbiol."},{"key":"ref_2","unstructured":"WHO (2025). Global Tuberculosis Report 2025, World Health Organization."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1146\/annurev.biochem.66.1.679","article-title":"Ribosomes and translation","volume":"66","author":"Green","year":"1997","journal-title":"Annu. Rev. Biochem."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"242","DOI":"10.1038\/nrm2352","article-title":"A structural understanding of the dynamic ribosome machine","volume":"9","author":"Steitz","year":"2008","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"979","DOI":"10.1016\/j.cell.2014.10.052","article-title":"The ribosome emerges from a black box","volume":"159","author":"Ramakrishnan","year":"2014","journal-title":"Cell"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e20420","DOI":"10.7554\/eLife.20420","article-title":"Ribosomal mutations promote the evolution of antibiotic resistance in a multidrug environment","volume":"6","author":"Gomez","year":"2017","journal-title":"eLife"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1016\/j.tube.2016.03.005","article-title":"Strategies of genome editing in mycobacteria: Achievements and challenges","volume":"98","author":"Choudhary","year":"2016","journal-title":"Tuberculosis"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1255","DOI":"10.1016\/j.jiph.2020.06.023","article-title":"Features of the biochemistry of Mycobacterium smegmatis, as a possible model for Mycobacterium tuberculosis","volume":"13","author":"Sundarsingh","year":"2020","journal-title":"J. Infect. Public Health"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"472","DOI":"10.1016\/S0966-842X(01)02168-0","article-title":"Mycobacterium smegmatis: An absurd model for tuberculosis?","volume":"9","author":"Reyrat","year":"2001","journal-title":"Trends Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1016\/j.mib.2009.11.006","article-title":"To catch a killer. What can mycobacterial models teach us about Mycobacterium tuberculosis pathogenesis?","volume":"13","author":"Shiloh","year":"2010","journal-title":"Curr. Opin. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"333","DOI":"10.2323\/jgam.53.333","article-title":"Evaluation of Mycobacterium smegmatis as a possible surrogate screen for selecting molecules active against multi-drug resistant Mycobacterium tuberculosis","volume":"53","author":"Chaturvedi","year":"2007","journal-title":"J. General. Appl. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1111\/j.1574-6968.1989.tb03558.x","article-title":"The cell envelope of Mycobacterium smegmatis: Cytochemistry and architectural implications","volume":"52","author":"Mamadou","year":"1989","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"780","DOI":"10.1038\/s41429-020-0320-7","article-title":"Application of Mycobacterium smegmatis as a surrogate to evaluate drug leads against Mycobacterium tuberculosis","volume":"73","author":"Lelovic","year":"2020","journal-title":"J. Antibiot."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1016\/j.celrep.2017.06.029","article-title":"The Complete Structure of the Mycobacterium smegmatis 70S Ribosome","volume":"20","author":"Hentschel","year":"2017","journal-title":"Cell Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"10884","DOI":"10.1093\/nar\/gkx785","article-title":"Structural insights into species-specific features of the ribosome from the human pathogen Mycobacterium tuberculosis","volume":"45","author":"Yang","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1016\/j.pbiomolbio.2019.12.001","article-title":"Unique structural features of the Mycobacterium ribosome","volume":"152","author":"Kushwaha","year":"2020","journal-title":"Prog. Biophys. Mol. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"667","DOI":"10.1099\/13500872-142-3-667","article-title":"The rRNA operons of Mycobacterium smegmatis and Mycobacterium tuberculosis: Comparison of promoter elements and of neighbouring upstream genes","volume":"142","author":"Gonzalez","year":"1996","journal-title":"Microbiology"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"697","DOI":"10.1093\/abbs\/gmz054","article-title":"Efficient circular gene knockout system for Burkholderiales strain DSM 7029 and Mycobacterium smegmatis mc2 155","volume":"51","author":"Lei","year":"2019","journal-title":"Acta Biochim. Biophys. Sin."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1820","DOI":"10.1038\/nprot.2015.115","article-title":"Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange","volume":"10","author":"Hmelo","year":"2015","journal-title":"Nat. Protoc."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"660","DOI":"10.1101\/gad.7.4.660","article-title":"A cold-sensitive mutation in 16S rRNA provides evidence for helical switching in ribosome assembly","volume":"7","author":"Dammel","year":"1993","journal-title":"Genes. Dev."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"256","DOI":"10.1016\/j.tibs.2009.01.011","article-title":"Deconstructing ribosome construction","volume":"34","author":"Connolly","year":"2009","journal-title":"Trends Biochem. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1016\/S0968-0004(03)00169-5","article-title":"RNA, the first macromolecular catalyst: The ribosome is a ribozyme","volume":"28","author":"Steitz","year":"2003","journal-title":"Trends Biochem. Sci."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Kotta-Loizou, I. (2021). Wellcome Trust\u2013Funded Monographs and Book Chapters. The Lifecycle of Ribosomal RNA in Bacteria. RNA Damage and Repair, Springer Nature.","DOI":"10.1007\/978-3-030-76571-2"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1146\/annurev-biochem-062917-011942","article-title":"Ribosome-Targeting Antibiotics: Modes of Action, Mechanisms of Resistance, and Implications for Drug Design","volume":"87","author":"Lin","year":"2018","journal-title":"Annu. Rev. Biochem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1038\/nrmicro3155","article-title":"Ribosome-targeting antibiotics and mechanisms of bacterial resistance","volume":"12","author":"Wilson","year":"2014","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2264","DOI":"10.1128\/IAI.00417-16","article-title":"A Mutation in the 16S rRNA Decoding Region Attenuates the Virulence of Mycobacterium tuberculosis","volume":"84","author":"Watanabe","year":"2016","journal-title":"Infect. Immun."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3192","DOI":"10.1128\/AAC.49.8.3192-3197.2005","article-title":"Molecular analysis of cross-resistance to capreomycin, kanamycin, amikacin, and viomycin in Mycobacterium tuberculosis","volume":"49","author":"Maus","year":"2005","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"340","DOI":"10.1038\/35030019","article-title":"Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics","volume":"407","author":"Carter","year":"2000","journal-title":"Nature"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"S242","DOI":"10.1093\/infdis\/135.Supplement_2.S242","article-title":"Discovery, chemistry, and activity of amikacin","volume":"134","author":"Kawaguchi","year":"1976","journal-title":"J. Infect. Dis."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Pertschy, B., and Zierler, I. (2024). Ribosomal Proteins in Ribosome Assembly. Biomolecules, 15.","DOI":"10.3390\/biom15010013"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1038\/nmeth.4193","article-title":"MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy","volume":"14","author":"Zheng","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1038\/nmeth.4169","article-title":"cryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination","volume":"14","author":"Punjani","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e42166","DOI":"10.7554\/eLife.42166","article-title":"New tools for automated high-resolution cryo-EM structure determination in RELION-3","volume":"7","author":"Zivanov","year":"2018","journal-title":"eLife"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1605","DOI":"10.1002\/jcc.20084","article-title":"UCSF Chimera\u2014A visualization system for exploratory research and analysis","volume":"25","author":"Pettersen","year":"2004","journal-title":"J. Comput. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"861","DOI":"10.1107\/S2059798319011471","article-title":"Macromolecular structure determination using X-rays, neutrons and electrons: Recent developments in Phenix","volume":"75","author":"Liebschner","year":"2019","journal-title":"Acta Crystallogr. D Struct. Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1038\/nprot.2007.521","article-title":"Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances","volume":"3","author":"Wiegand","year":"2008","journal-title":"Nat. Protoc."}],"container-title":["Antibiotics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6382\/15\/1\/30\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,2]],"date-time":"2026-01-02T05:10:29Z","timestamp":1767330629000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6382\/15\/1\/30"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,31]]},"references-count":36,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["antibiotics15010030"],"URL":"https:\/\/doi.org\/10.3390\/antibiotics15010030","relation":{},"ISSN":["2079-6382"],"issn-type":[{"type":"electronic","value":"2079-6382"}],"subject":[],"published":{"date-parts":[[2025,12,31]]}}}