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.

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

doi:10.3389/fmicb.2018.02143

Abstract

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.

Highlights

Nitrogen metabolism is important for all bacteria, from harmless soil organisms to pathogens, including various mycobacteria
Unlike the other glnA genes, glnA2 is sometimes thought to be responsible for synthesis of Dglutamine and possibly iso-glutamine, but still, a deletion mutant could grow in un-supplemented media and replicate in mice with no apparent defect as compared to wt (Tullius et al, 2003; Harth et al, 2005)
We discovered that a point mutation in the glnA2 gene of M. smegmatis (Msmeg_4294, homolog of Rv2222c) rendered the bacterium temperaturesensitive, and decided to use this finding to explore the role of glnA2 in M. smegmatis

Summary

Introduction

Nitrogen metabolism is important for all bacteria, from harmless soil organisms to pathogens, including various mycobacteria. The M.smegI133F mutant was temperature sensitive, as it grew normally at 32◦C, but not at 42◦C, suggesting the function of GlnA2 was essential to normal growth, contrary to the situation in Mtb (Figure 1B).

Results

Conclusion