Two Interacting ATPases Protect Mycobacterium tuberculosis from Glycerol and Nitric Oxide Toxicity

The Mycobacterium tuberculosis H37Rv genome was sequenced and an-notated over 20 years ago, yet roughly half of the protein-coding genes still lack a predicted function. We characterized two genes of unknown function, rv3679 and rv3680, for which inconsistent findings regarding their importance for virulence in mice have been reported. We confirmed that the rv3679 and rv3680 operon (rv3679-80) deletion mutant (delta rv3679-80) was virulent in mice and discovered that the delta rv3679-80 mutant suffered from a glycerol-dependent recovery defect on agar plates following mouse infection. Glycerol also exacerbated killing of the delta rv3679-80 mutant by nitric oxide. Rv3679 and Rv3680 have previously been shown to form a complex with ATPase activity, and we demonstrate that the ability of M. tuberculosis to cope with elevated levels of glycerol and nitric oxide requires intact ATP-binding motifs in both Rv3679 and Rv3680. Inactivation of glycerol kinase or Rv2370c, a pro-tein of unknown function, suppressed glycerol-mediated toxicity in the delta rv3679-80 mutant. Glycerol catabolism led to increased intracellular methylglyoxal pools, and the delta rv3679-80 mutant was hypersusceptible to extracellular methylglyoxal, suggest-ing that glycerol toxicity in the delta rv3679-80 mutant is caused by methylglyoxal. Rv3679 and Rv3680 interacted with Rv1509, and Rv3679 had numerous additional interactors including proteins of the type II fatty acid synthase (FASII) pathway and mycolic acid-modifying enzymes linking Rv3679 to fatty acid or lipid synthesis. This work provides experimentally determined roles for Rv3679 and Rv3680 and stimu-lates future research on these and other proteins of unknown function. p IMPORTANCE A better understanding of the pathogenesis of tuberculosis requires a better understanding of gene function in M. tuberculosis. This work provides the first functional insight into the Rv3679/Rv3680 ATPase complex. We demonstrate that M. tuberculosis requires this complex and specifically its ATPase activity to resist glycerol and nitric oxide toxicity. We provide evidence that the glycerol-derived metabolite methylglyoxal causes toxicity in the absence of Rv3679/Rv3680. We further show that glycerol-dependent toxicity is reversed when glycerol kinase (GlpK) is inacti-vated. Our work uncovered other genes of unknown function that interact with Rv3679 and/or Rv3680 genetically or physically, underscoring the importance of un-derstanding uncharacterized genes.