{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T16:58:27Z","timestamp":1790787507602,"version":"4.1.0"},"reference-count":27,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T00:00:00Z","timestamp":1777334400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T00:00:00Z","timestamp":1777334400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/100018809","name":"Universidade Federal De Pelotas","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100018809","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Mol Biol Rep"],"published-print":{"date-parts":[[2026,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      <jats:italic>Mycobacterium bovis<\/jats:italic>\n                      BCG is widely used as a vaccine vector due to its safety, genetic stability, and immunomodulatory properties. However, current expression systems for BCG transformation rely on antibiotic resistance genes, raising biosafety concerns. Therefore, alternative strategies\u2014such as the use of auxotrophic strains\u2014are highly desirable. Our group previously employed BioBrick\u2122 technology to construct the pUP500 plasmid series containing various mycobacterial promoters. We engineered these plasmids for the auxotrophic complementation of BCG \u0394LeuD, a leucine-auxotrophic strain, using an antibiotic-free and BioBrick\u2122-compatible approach.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods and Results<\/jats:title>\n                    <jats:p>\n                      The auxotrophic complementation cassette pAN-\n                      <jats:italic>leuD<\/jats:italic>\n                      was cloned into pUP500 plasmids. Subsequently, the\n                      <jats:italic>egfp<\/jats:italic>\n                      gene was inserted downstream of the promoters, the kanamycin resistance gene was removed, and fluorescence was assessed by flow cytometry, qPCR and confocal microscopy in BCG grown in 7H9 medium and inside macrophages. Auxotrophic complementation was confirmed by growth of transformants in non-selective medium and analysis of\n                      <jats:italic>leuD<\/jats:italic>\n                      expression, and plasmid stability was evaluated along six BCG generations. Strains harboring plasmids containing the pAN and HspX promoters exhibited stronger fluorescence levels both\n                      <jats:italic>in vitro<\/jats:italic>\n                      and within macrophages. In contrast, the 18\u00a0kDa promoter demonstrated marked activation specifically in the intracellular environment, driving robust gene expression inside macrophages. These expression patterns were significantly higher compared to strains carrying the Ag85B or Hsp60 promoters, as well as to the negative control. Except for the Hsp60 construct, all plasmids exhibited robust\n                      <jats:italic>in vitro<\/jats:italic>\n                      stability across six consecutive passages, supporting their potential as reliable expression systems for heterologous antigens in therapeutic and vaccine applications.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>We developed five BioBrick\u2122-compatible plasmids with distinct promoters for auxotrophic complementation and antigen expression in BCG \u0394LeuD, eliminating the need for antibiotic-based selection. This standardized plasmid series reduces vector-related variability in comparative studies and provides a diverse promoter repertoire that may enhance the stability and efficacy of recombinant BCG \u0394LeuD strains, representing a valuable advancement for prophylactic and therapeutic vaccine development.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1007\/s11033-026-11830-x","type":"journal-article","created":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T08:59:58Z","timestamp":1777366798000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["A versatile plasmid platform for auxotrophic complementation in attenuated Mycobacterium bovis BCG"],"prefix":"10.1007","volume":"53","author":[{"given":"Andriele Bonemann","family":"Madruga","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jady Duarte","family":"Nogueira","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mara Andrade Colares","family":"Maia","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natasha Rodrigues","family":"de Oliveira","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Eduarda","family":"Ehlert","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valentina Gessinger","family":"Ferreira","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bruna Silveira","family":"Pacheco","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabiana Kommling","family":"Seixas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tiago Veiras","family":"Collares","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Izani Bonel","family":"Acosta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ant\u00f4nio Sergio","family":"Varela","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Odir Ant\u00f4nio","family":"Dellagostin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tha\u00eds Larr\u00e9 Oliveira","family":"Bohn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,4,28]]},"reference":[{"key":"11830_CR1","doi-asserted-by":"publisher","first-page":"830","DOI":"10.1134\/S0026893322060036","volume":"56","author":"DK Armianinova","year":"2022","unstructured":"Armianinova DK, Karpov DS, Kotliarova MS, Goncharenko AV (2022) Genetic Engineering in Mycobacteria. Mol Biol 56:830\u2013841. https:\/\/doi.org\/10.1134\/S0026893322060036","journal-title":"Mol Biol"},{"key":"11830_CR2","doi-asserted-by":"publisher","first-page":"1853","DOI":"10.1007\/s00253-012-3920-8","volume":"93","author":"T Wang","year":"2012","unstructured":"Wang T, Ma X, Zhu H, Li A, Du G, Chen J (2012) Available methods for assembling expression cassettes for synthetic biology. Appl Microbiol Biotechnol 93:1853\u20131863. https:\/\/doi.org\/10.1007\/s00253-012-3920-8","journal-title":"Appl Microbiol Biotechnol"},{"key":"11830_CR3","doi-asserted-by":"publisher","first-page":"5","DOI":"10.1186\/1754-1611-2-5","volume":"2","author":"RP Shetty","year":"2008","unstructured":"Shetty RP, Endy D, Knight TF (2008) Engineering BioBrick vectors from BioBrick parts. J Biol Eng 2:5. https:\/\/doi.org\/10.1186\/1754-1611-2-5","journal-title":"J Biol Eng"},{"key":"11830_CR4","doi-asserted-by":"publisher","first-page":"1275","DOI":"10.1007\/s00253-011-3633-4","volume":"92","author":"JE Vick","year":"2011","unstructured":"Vick JE, Johnson ET, Choudhary S, Bloch SE, Lopez-Gallego F, Srivastava P, Tikh IB, Wawrzyn GT, Schmidt-Dannert C (2011) Optimized compatible set of BioBrick\u2122 vectors for metabolic pathway engineering. Appl Microbiol Biotechnol 92:1275\u20131286. https:\/\/doi.org\/10.1007\/s00253-011-3633-4","journal-title":"Appl Microbiol Biotechnol"},{"key":"11830_CR5","doi-asserted-by":"publisher","first-page":"e9841","DOI":"10.7717\/peerj.9841","volume":"8","author":"I Matsumura","year":"2020","unstructured":"Matsumura I (2020) Methylase-assisted subcloning for high throughput BioBrick assembly. PeerJ 8:e9841. https:\/\/doi.org\/10.7717\/peerj.9841","journal-title":"PeerJ"},{"key":"11830_CR6","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1111\/1567-1364.12171","volume":"15","author":"R Chao","year":"2015","unstructured":"Chao R, Yuan Y, Zhao H (2015) Recent advances in DNA assembly technologies. FEMS Yeast Res 15:1\u20139. https:\/\/doi.org\/10.1111\/1567-1364.12171","journal-title":"FEMS Yeast Res"},{"key":"11830_CR7","doi-asserted-by":"publisher","first-page":"574591","DOI":"10.1155\/2011\/574591","volume":"2011","author":"K Matsuo","year":"2011","unstructured":"Matsuo K, Yasutomi Y (2011) Mycobacterium bovis bacille calmette-gu\u00e9rin as a vaccine vector for global infectious disease control. Tuberc Res Treat 2011:574591. https:\/\/doi.org\/10.1155\/2011\/574591","journal-title":"Tuberc Res Treat"},{"key":"11830_CR8","doi-asserted-by":"publisher","first-page":"6495","DOI":"10.1016\/j.vaccine.2009.08.044","volume":"27","author":"RG Bastos","year":"2009","unstructured":"Bastos RG, Borsuk S, Seixas FK, Dellagostin OA (2009) Recombinant Mycobacterium bovis BCG. Vaccine 27:6495\u20136503. https:\/\/doi.org\/10.1016\/j.vaccine.2009.08.044","journal-title":"Vaccine"},{"key":"11830_CR9","doi-asserted-by":"publisher","unstructured":"Dellagostin OA, Borsuk S, Oliveira TL, Seixas FK, Dellagostin OA, Borsuk S, Oliveira TL, Seixas FK (2022) Auxotrophic Mycobacterium bovis BCG: Updates and perspectives. Vaccines 10. https:\/\/doi.org\/10.3390\/vaccines10050802","DOI":"10.3390\/vaccines10050802"},{"key":"11830_CR10","doi-asserted-by":"publisher","first-page":"474","DOI":"10.1016\/j.tube.2007.07.006","volume":"87","author":"S Borsuk","year":"2007","unstructured":"Borsuk S, Mendum TA, Fagundes MQ, Michelon M, Cunha CW, McFadden J, Dellagostin OA (2007) Auxotrophic complementation as a selectable marker for stable expression of foreign antigens in Mycobacterium bovis BCG. Tuberculosis 87:474\u2013480. https:\/\/doi.org\/10.1016\/j.tube.2007.07.006","journal-title":"Tuberculosis"},{"key":"11830_CR11","doi-asserted-by":"publisher","first-page":"101851","DOI":"10.1016\/j.tube.2019.07.002","volume":"119","author":"TL Oliveira","year":"2019","unstructured":"Oliveira TL, Stedman A, Rizzi C, Dorneles J, da Cunha CEP, Junior ASV, Dellagostin OA, McFadden J (2019) A standardized BioBrick toolbox for the assembly of sequences in mycobacteria. Tuberculosis 119:101851. https:\/\/doi.org\/10.1016\/j.tube.2019.07.002","journal-title":"Tuberculosis"},{"key":"11830_CR12","doi-asserted-by":"publisher","first-page":"e51396","DOI":"10.1371\/journal.pone.0051396","volume":"7","author":"C Rizzi","year":"2012","unstructured":"Rizzi C, Bianco MV, Blanco FC, Soria M, Gravisaco MJ, Montenegro V, Vagnoni L, Buddle B, Garbaccio S, Delgado F, Leal KS, Cataldi AA, Dellagostin OA, Bigi F (2012) Vaccination with a BCG Strain Overexpressing Ag85B Protects Cattle against Mycobacterium bovis Challenge. PLoS ONE 7:e51396. https:\/\/doi.org\/10.1371\/journal.pone.0051396","journal-title":"PLoS ONE"},{"key":"11830_CR13","doi-asserted-by":"publisher","first-page":"1543","DOI":"10.1007\/s00253-012-4416-2","volume":"97","author":"KR Begnini","year":"2013","unstructured":"Begnini KR, Rizzi C, Campos VF, Borsuk S, Schultze E, Yurgel VC, Nedel F, Dellagostin OA, Collares T, Seixas FK (2013) Auxotrophic recombinant Mycobacterium bovis BCG overexpressing Ag85B enhances cytotoxicity on superficial bladder cancer cells in vitro. Appl Microbiol Biotechnol 97:1543\u20131552. https:\/\/doi.org\/10.1007\/s00253-012-4416-2","journal-title":"Appl Microbiol Biotechnol"},{"key":"11830_CR14","doi-asserted-by":"publisher","first-page":"13105","DOI":"10.1073\/pnas.0801143105","volume":"105","author":"ST Park","year":"2008","unstructured":"Park ST, Kang C-M, Husson RN (2008) Regulation of the SigH stress response regulon by an essential protein kinase in Mycobacterium tuberculosis. Proc Natl Acad Sci 105:13105\u201313110. https:\/\/doi.org\/10.1073\/pnas.0801143105","journal-title":"Proc Natl Acad Sci"},{"key":"11830_CR15","doi-asserted-by":"publisher","first-page":"867195","DOI":"10.3389\/fimmu.2022.867195","volume":"13","author":"L Moraes","year":"2022","unstructured":"Moraes L, Trentini MM, Fousteris D, Eto SF, Chudzinski-Tavassi AM, Leite LC, de Kanno C AI (2022) CRISPR\/Cas9 approach to generate an auxotrophic BCG strain for unmarked expression of LTAK63 adjuvant: a tuberculosis vaccine candidate. Front Immunol 13:867195. https:\/\/doi.org\/10.3389\/fimmu.2022.867195","journal-title":"Front Immunol"},{"key":"11830_CR16","doi-asserted-by":"publisher","first-page":"1999","DOI":"10.1099\/00221287-148-7-1999","volume":"148","author":"MA Medeiros","year":"2002","unstructured":"Medeiros MA, Dellagostin OA, Arm\u00f4a GRG, Degrave WM, de Mendon\u00e7a-Lima L, Lopes MQ, Costa JF, Mcfadden J, McIntosh D (2002) Comparative evaluation of Mycobacterium vaccae as a surrogate cloning host for use in the study of mycobacterial genetics. Microbiology 148:1999\u20132009. https:\/\/doi.org\/10.1099\/00221287-148-7-1999","journal-title":"Microbiology"},{"key":"11830_CR17","doi-asserted-by":"publisher","first-page":"407","DOI":"10.1007\/s11274-014-1794-x","volume":"31","author":"K Dokladda","year":"2015","unstructured":"Dokladda K, Billamas P, Palittapongarnpim P (2015) Different behaviours of promoters in Mycobacterium tuberculosis H37Rv and H37Ra. World J Microbiol Biotechnol 31:407\u2013413. https:\/\/doi.org\/10.1007\/s11274-014-1794-x","journal-title":"World J Microbiol Biotechnol"},{"key":"11830_CR18","doi-asserted-by":"publisher","first-page":"818","DOI":"10.1002\/iub.1862","volume":"70","author":"RB Abramovitch","year":"2018","unstructured":"Abramovitch RB (2018) Mycobacterium tuberculosis reporter strains as tools for drug discovery and development. IUBMB Life 70:818\u2013825. https:\/\/doi.org\/10.1002\/iub.1862","journal-title":"IUBMB Life"},{"key":"11830_CR19","doi-asserted-by":"publisher","first-page":"e190347","DOI":"10.1590\/0074-02760190347","volume":"115","author":"LV Nascimento","year":"2020","unstructured":"Nascimento LV, Santos CC, Leite LC, Nascimento IP (2020) Characterisation of alternative expression vectors for recombinant Bacillus Calmette-Gu\u00e9rin as live bacterial delivery systems. Mem Inst Oswaldo Cruz 115:e190347. https:\/\/doi.org\/10.1590\/0074-02760190347","journal-title":"Mem Inst Oswaldo Cruz"},{"key":"11830_CR20","doi-asserted-by":"publisher","first-page":"1785","DOI":"10.1099\/13500872-141-8-1785","volume":"141","author":"OA Dellagostin","year":"1995","unstructured":"Dellagostin OA, Esposito G, Eales L-J, Dale JW, McFadden J (1995) Activity of mycobacterial promoters during intracellular and extracellular growth. Microbiology 141:1785\u20131792. https:\/\/doi.org\/10.1099\/13500872-141-8-1785","journal-title":"Microbiology"},{"key":"11830_CR21","doi-asserted-by":"publisher","first-page":"2240","DOI":"10.1128\/AEM.03677-15","volume":"82","author":"AI Kanno","year":"2016","unstructured":"Kanno AI, Goulart C, Rofatto HK, Oliveira SC, Leite LC, McFadden J (2016) New recombinant Mycobacterium bovis BCG expression vectors: improving genetic control over mycobacterial promoters. Appl Environ Microbiol 82:2240\u20132246. https:\/\/doi.org\/10.1128\/AEM.03677-15","journal-title":"Appl Environ Microbiol"},{"key":"11830_CR22","doi-asserted-by":"publisher","first-page":"283","DOI":"10.1054\/tube.2002.0374","volume":"82","author":"M Al-Zarouni","year":"2002","unstructured":"Al-Zarouni M, Dale JW (2002) Expression of foreign genes in Mycobacterium bovis BCG strains using different promoters reveals instability of the hsp60 promoter for expression of foreign genes in Mycobacterium bovis BCG strains. Tuberculosis 82:283\u2013291. https:\/\/doi.org\/10.1054\/tube.2002.0374","journal-title":"Tuberculosis"},{"key":"11830_CR23","doi-asserted-by":"publisher","first-page":"357370","DOI":"10.1155\/2010\/357370","volume":"2010","author":"J Joseph","year":"2010","unstructured":"Joseph J, Fern\u00e1ndez-Lloris R, Pezzat E, Saubi N, Cardona P-J, Mothe B, Gatell JM (2010) Molecular Characterization of heterologous HIV-1gp120 gene expression disruption in mycobacterium bovis BCG host strain: a critical issue for engineering mycobacterial based-vaccine vectors. BioMed Res Int 2010:357370. https:\/\/doi.org\/10.1155\/2010\/357370","journal-title":"BioMed Res Int"},{"key":"11830_CR24","doi-asserted-by":"publisher","first-page":"282","DOI":"10.2174\/157016210791208686","volume":"8","author":"R Chapman","year":"2010","unstructured":"Chapman R, Chege G, Shephard E, Stutz H, Williamson A-L (2010) Recombinant Mycobacterium bovis BCG as an HIV vaccine vector. Curr HIV Res 8:282\u2013298. https:\/\/doi.org\/10.2174\/157016210791208686","journal-title":"Curr HIV Res"},{"key":"11830_CR25","doi-asserted-by":"publisher","first-page":"1798","DOI":"10.1080\/21645515.2017.1316911","volume":"13","author":"A Mahant","year":"2017","unstructured":"Mahant A, Saubi N, Eto Y, Guitart N, Gatell JM, Hanke T, Joseph J (2017) Preclinical development of BCG.HIVA2auxo.int, harboring an integrative expression vector, for a HIV-TB Pediatric vaccine. Enhancement of stability and specific HIV-1 T-cell immunity. Hum Vaccines Immunother 13:1798\u20131810. https:\/\/doi.org\/10.1080\/21645515.2017.1316911","journal-title":"Hum Vaccines Immunother"},{"key":"11830_CR26","doi-asserted-by":"publisher","first-page":"123","DOI":"10.1590\/0074-02760160360","volume":"112","author":"C Rizzi","year":"2017","unstructured":"Rizzi C, Peiter AC, Oliveira TL, Seixas ACP, Leal KS, Hartwig DD, Seixas FK, Borsuk S, Dellagostin OA (2017) Stable expression of Mycobacterium bovis antigen 85B in auxotrophic M. bovis bacillus Calmette-Gu\u00e9rin. Mem Inst Oswaldo Cruz 112:123\u2013130. https:\/\/doi.org\/10.1590\/0074-02760160360","journal-title":"Mem Inst Oswaldo Cruz"},{"key":"11830_CR27","doi-asserted-by":"publisher","first-page":"6865","DOI":"10.1007\/s00253-017-8439-6","volume":"101","author":"TL Oliveira","year":"2017","unstructured":"Oliveira TL, Rizzi C, Dellagostin OA (2017) Recombinant BCG vaccines: molecular features and their influence in the expression of foreign genes. Appl Microbiol Biotechnol 101:6865\u20136877. https:\/\/doi.org\/10.1007\/s00253-017-8439-6","journal-title":"Appl Microbiol Biotechnol"}],"container-title":["Molecular Biology Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s11033-026-11830-x.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1007\/s11033-026-11830-x","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s11033-026-11830-x.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T22:51:44Z","timestamp":1781650304000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/10.1007\/s11033-026-11830-x"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,28]]},"references-count":27,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2026,12]]}},"alternative-id":["11830"],"URL":"https:\/\/doi.org\/10.1007\/s11033-026-11830-x","relation":{},"ISSN":["0301-4851","1573-4978"],"issn-type":[{"value":"0301-4851","type":"print"},{"value":"1573-4978","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,28]]},"assertion":[{"value":"5 August 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"13 April 2026","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"28 April 2026","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"The authors declare no conflicts of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval"}},{"value":"Not applicable.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consents"}}],"article-number":"679"}}