The Unexpected Essentiality of <i>glnA2</i> in <i>Mycobacterium smegmatis</i> Is Salvaged by Overexpression of the Global Nitrogen Regulator <i>glnR</i>, but Not by L-, D- or Iso-Glutamine

Nitrogen metabolism plays a central role in the physiology of microorganisms, and Glutamine Synthetase (GS) genes are present in virtually all bacteria. In M. tuberculosis , four GS genes are present, but only glnA1 is essential, whereas glnA2 was shown to be non-essential for in-vitro as well as in-vivo growth and pathogenesis, and is postulated to be involved in D-glutamine and iso-glutamine synthesis. Whilst investigating the activity of an antimicrobial compound in M. smegmatis , we found a spontaneous temperature-sensitive mutant in glnA2 (I133F), and used it to investigate the role of glnA2 in M. smegmatis . We deleted the native glnA2 and replaced it with a mutated allele. This re-created the temperature sensitivity-as after 3-4 seemingly normal division cycles, glnA2 became essential for growth. This essentiality could not be salvaged by neither L, D- nor iso-glutamine, suggesting an additional role of glnA2 in M. smegmatis over its role in M. tuberculosis . We also found that overexpression of the global nitrogen regulator glnR enabled bypassing the essentiality of glnA2 , allowing the creation of a complete deletion mutant. The discrepancy between the importance of glnA2 in Mtb and M. smegmatis stresses the caution in which results in one are extrapolated to the other.