AUTHOR=Rakovitsky Nadya , Bar Oz Michal , Goldberg Karin , Gibbons Simon , Zimhony Oren , Barkan Daniel 

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

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.02143

DOI=10.3389/fmicb.2018.02143

ISSN=1664-302X

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