Modular toolkit to facilitate molecular manipulations in mycobacteria

Gene expression in mycobacteria relies on both integrative and episomal <i>Escherichia coli</i>-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 <i>E. coli</i>-mycobacterium shuttle vectors. Each vector features N- or C-terminal tags: SBP-HA-S, 3×-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 <i>Mycobacterium smegmatis</i> by expressing RbpA, an RNA polymerase-binding protein. Fluorescent tags confirmed Wag31's polar localization, whereas co-expression with FtsZ illustrated their distinct localizations. The system's flexibility was further shown by co-expressing four <i>Mycobacterium tuberculosis</i> 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.IMPORTANCEMolecular 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.