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

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.

Multi-omics Investigation into the Mechanism of Action of an Anti-tubercular Fatty Acid Analogue

“…GlnA is an essential Msm protein with a role as a global nitrogen metabolism regulator. 59 Nitrogen metabolism plays a central role in all bacteria, and our proteomics data identified the organo-nitrogen metabolic process as the third most altered biological process following treatment with DA-CB (Figure 6C). Proteins associated with ribosome, pyrimidine, purine, and amino acid biosynthesis were all altered due to DA-CB.…”

“…Some of the observed amino acid changes were also correlated with a downregulation in proteins related to the biosynthesis of amino acids, which included Tal (which also has a role in PPP), imidazole glycerol phosphate synthase subunit (HisF), ATP phosphoribosyltransferase (HisG), and glutamine synthase (GlnA) (Table ). GlnA is an essential Msm protein with a role as a global nitrogen metabolism regulator . Nitrogen metabolism plays a central role in all bacteria, and our proteomics data identified the organo-nitrogen metabolic process as the third most altered biological process following treatment with DA-CB (Figure C).…”

Research progress on GlnR-mediated regulation in Actinomycetes

“…smegmatis, Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium bovis are the most commonly studied Mycobacterium genus (Ergan et al, 2004;Amon et al, 2009;Schuenemann et al, 2013;Meehan et al, 2019; European Food Safety Authority and European Centre for Disease Prevention and Control, 2021). Different from S. coelicolor, glnA1 myc , glnA2 myc , glnA3 myc , and glnA4 myc are all encoding GS genes present in M. tuberculosis (Rakovitsky et al, 2018). In all mycobacteria, the GS genes glnA1 myc and glnA2 myc are present and have conserved genomic positions near glnE.…”