{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T03:00:51Z","timestamp":1787626851796,"version":"build-2736575974"},"reference-count":51,"publisher":"American Society for Microbiology","issue":"4","license":[{"start":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T00:00:00Z","timestamp":1776902400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T00:00:00Z","timestamp":1776902400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100001843","name":"Science and Engineering Research Board","doi-asserted-by":"publisher","award":["CRG\/2018\/001294"],"award-info":[{"award-number":["CRG\/2018\/001294"]}],"id":[{"id":"10.13039\/501100001843","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["6044-SC23-26"],"award-info":[{"award-number":["6044-SC23-26"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Bacteriol"],"published-print":{"date-parts":[[2026,4,23]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Gene expression in mycobacteria relies on both integrative and episomal\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      -mycobacterium shuttle vectors. However, existing vectors have limited flexibility for adding epitope tags, selectable markers, and fluorescent reporters. To overcome this, we created a modular toolkit consisting of 32 integrative and eight episomal\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      -mycobacterium shuttle vectors. Each vector features N- or C-terminal tags: SBP-HA-S, 3\u00d7-FLAG, GFP, and mCherry. The integrative vectors contain four different phage systems (L5, Giles, MS6, and Tweety), each with a distinct antibiotic resistance marker, enabling stable integration at various chromosomal sites. We validated the system in\n                      <jats:italic toggle=\"yes\">Mycobacterium smegmatis<\/jats:italic>\n                      by expressing RbpA, an RNA polymerase-binding protein. Fluorescent tags confirmed Wag31\u2019s polar localization, whereas co-expression with FtsZ illustrated their distinct localizations. The system\u2019s flexibility was further shown by co-expressing four\n                      <jats:italic toggle=\"yes\">Mycobacterium tuberculosis<\/jats:italic>\n                      sigma factors (SigA, SigC, SigG, and SigH) simultaneously. Additionally, an SBP-HA-S tandem affinity tag facilitated efficient two-step purification of RbpA with RNA polymerase and related regulators, reducing background compared to FLAG pull-down. This toolkit provides a versatile, reliable platform for gene complementation, protein localization, multi-protein co-expression, and native complex purification, supporting advanced genetic and cell biological studies in mycobacteria.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>Molecular manipulation of pathogens is crucial for studying gene functions. Here, we have used existing knowledge and tools to create a new set of shuttle vectors with an expanded range of tags at both the N- and C-termini, along with antibiotics. This expansion allowed us to target four known integrative sites in mycobacteria and one episomal vector, resulting in the creation of 40 novel constructs. We have developed constructs that enable the simultaneous visualization of mCherry and GFP for co-localization studies. Importantly, for the first time in mycobacteria, we utilized the SBP-HA-S tandem affinity tag, which enables the retention of native protein complexes for pulldown studies. The tools generated in this study will be handy for mycobacterial research.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/jb.00474-25","type":"journal-article","created":{"date-parts":[[2026,3,16]],"date-time":"2026-03-16T13:00:27Z","timestamp":1773666027000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Modular toolkit to facilitate molecular manipulations in mycobacteria"],"prefix":"10.1128","volume":"208","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7110-9474","authenticated-orcid":false,"given":"Asis Kumar","family":"Khuntia","sequence":"first","affiliation":[{"name":"CSIR-Centre for Cellular and Molecular Biology","place":["Hyderabad, India"]},{"id":[{"id":"https:\/\/ror.org\/053rcsq61","id-type":"ROR","asserted-by":"publisher"}],"name":"Academy of Scientific and Innovative Research (AcSIR)","place":["Ghaziabad, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ankita","family":"Dabla","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02gezhp66","id-type":"ROR","asserted-by":"publisher"}],"name":"BRIC-National Institute of Immunology","place":["New Delhi, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yogita","family":"Kapoor","sequence":"additional","affiliation":[{"name":"CSIR-Centre for Cellular and Molecular Biology","place":["Hyderabad, India"]},{"id":[{"id":"https:\/\/ror.org\/053rcsq61","id-type":"ROR","asserted-by":"publisher"}],"name":"Academy of Scientific and Innovative Research (AcSIR)","place":["Ghaziabad, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amit","family":"Chakraborty","sequence":"additional","affiliation":[{"name":"CSIR-Centre for Cellular and Molecular Biology","place":["Hyderabad, India"]},{"id":[{"id":"https:\/\/ror.org\/053rcsq61","id-type":"ROR","asserted-by":"publisher"}],"name":"Academy of Scientific and Innovative Research (AcSIR)","place":["Ghaziabad, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Praveen Ebenezer","family":"Arputharaj","sequence":"additional","affiliation":[{"name":"CSIR-Centre for Cellular and Molecular Biology","place":["Hyderabad, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5682-4178","authenticated-orcid":true,"given":"Vinay Kumar","family":"Nandicoori","sequence":"additional","affiliation":[{"name":"CSIR-Centre for Cellular and Molecular Biology","place":["Hyderabad, India"]},{"id":[{"id":"https:\/\/ror.org\/053rcsq61","id-type":"ROR","asserted-by":"publisher"}],"name":"Academy of Scientific and Innovative Research (AcSIR)","place":["Ghaziabad, India"]},{"id":[{"id":"https:\/\/ror.org\/02gezhp66","id-type":"ROR","asserted-by":"publisher"}],"name":"BRIC-National Institute of Immunology","place":["New Delhi, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","unstructured":"World Health Organization. 2024. 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