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Mycobacterium tuberculosis  is a major health threat, necessitating novel drug targets. Protein synthesis in bacteria uses initiator tRNA i  charged with formylated methionine residue. Deletion of the formylase gene, tRNA fMet -formyl transferase ( fmt ), causes severe growth-retardation in  E .  coli  and in  S .  pneumoniae , but not in  P .  aeruginosa  or  S .  aureus .  fmt  was predicted to be essential in  M .  tuberculosis  by transposon library analysis, but this was never formally tested in any mycobacteria. We performed a targeted deletion of  fmt  in  M .  smegmatis  as well as Mtb-complex ( M .  bovis ). In both cases, we created a mero-diploid strain, deleted the native gene by two-step allelic exchange or specialized-phage transduction, and then removed the complementing gene to create full deletion mutants. In  M .  smegmatis  a full deletion strain could be easily created. In contrast, in  M .  bovis -BCG, a full deletion strain could only be created after incubation of 6 weeks, with a generation time ~2 times longer than for wt bacteria. Our results confirm the importance of this gene in pathogenic mycobacteria, but as the deletion mutant is viable, validity of  fmt  as a drug target remains unclear. Our results also refute the previous reports that  fmt  is essential in  M .  tuberculosis -complex.

The gene fmt, encoding tRNAfMet-formyl transferase, is essential for normal growth of M. bovis, but not for viability