Modular toolkit to facilitate molecular manipulations in mycobacteria

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• Gene expression in mycobacteria is essential for studying pathogens.
• Existing Escherichia coli-mycobacterium shuttle vectors lack flexibility for genetic manipulation.
• Limitations include restricted options for epitope tags, selectable markers, and fluorescent reporters.

Purpose of the Study:

• To create a modular toolkit of integrative and episomal shuttle vectors for enhanced gene expression in mycobacteria.
• To provide greater flexibility for incorporating various tags and reporters.
• To facilitate advanced genetic and cell biological studies in mycobacteria.

Main Methods:

• Developed 32 integrative and 8 episomal Escherichia coli-mycobacterium shuttle vectors.
• Incorporated N- or C-terminal tags (SBP-HA-S, 3×-FLAG, GFP, mCherry).
• Utilized four distinct phage systems with unique antibiotic resistance markers for integrative vectors.

Main Results:

• Successfully validated the system in Mycobacterium smegmatis, expressing RbpA.
• Demonstrated protein localization using fluorescent tags (Wag31, FtsZ).
• Enabled simultaneous co-expression of four Mycobacterium tuberculosis sigma factors and efficient two-step purification of protein complexes using SBP-HA-S tag.

Conclusions:

• The novel toolkit offers a versatile platform for gene complementation, protein localization, and multi-protein co-expression in mycobacteria.
• The SBP-HA-S tandem affinity tag facilitates efficient purification of native protein complexes.
• These tools will significantly aid mycobacterial research and pathogen studies.