MetF-Dependent Methionine Biosynthesis Is Required for Mycobacterium tuberculosis Survival and In Vivo Persistence

Methionine biosynthesis is essential for Mycobacterium tuberculosis, but the in vivo relevance of individual enzymes remains unclear. We performed transposon sequencing in Mycobacterium bovis BCG under methionine-free conditions and identified metF as a major fitness determinant. To define enzyme-specific functions, ΔmetE and ΔmetF mutants were generated in M. tuberculosis and evaluated using in vitro growth assays and a murine infection model. The ΔmetE mutant showed vitamin B₁₂-dependent growth in minimal medium but maintained normal growth in mice, indicating that its in vitro requirement does not translate to an in vivo defect. In contrast, ΔmetF exhibited strict methionine auxotrophy in vitro and completely failed to survive or persist in murine lungs and spleens. Structural modeling showed that MetF is distinct from human homologs, supporting selective inhibition. These findings demonstrate that MetF, but not MetE, is indispensable for M. tuberculosis survival in vivo and identify MetF as a promising metabolic drug target.