Combined lactate- and phosphate-dependent cytoplasmic acidification is associated with Mycobacterium tuberculosis growth arrest at acidic pH

Mycobacterium tuberculosis (Mtb) cultured in minimal medium at acidic pH arrests growth when provided specific single carbon sources, including glycerol, propionate, and lactate, a phenomenon we refer to as acid growth arrest. To define the mechanisms of acid growth arrest on lactate, we selected transposon mutants that grew under these conditions. Five mutants carried insertions in phoT or pstC2 , which encode components of a phosphate ABC transporter. Depleting phosphate restored Mtb growth in minimal medium supplemented with lactate at acidic pH, demonstrating that growth arrest on lactate depended on phosphate. The combination of lactate and phosphate at acidic pH acidified the cytoplasm to below pH 6.7, whereas the phoT mutant maintained a cytoplasmic pH > 7.2. Lactate slightly decreased membrane potential in a dose-dependent manner in wild-type Mtb, whereas the phoT mutant maintained a higher membrane potential. Transcriptional profiling identified a lactate-associated PMF stress signature, including upregulation of electron transport chain genes. Together, these findings associate lactate- and phosphate-dependent cytoplasmic acidification with proton motive force (PMF) stress and acid growth arrest. The phoT mutant grown in lactate at acidic pH upregulated the senX3 / regX3 regulon. Using a regX3 mutant, we found that growth on lactate under low-phosphate conditions required regX3 . We propose a model in which (1) acidic pH, lactate, and phosphate together modulate cytoplasmic pH homeostasis and are associated with PMF stress and acid growth arrest; and (2) low phosphate induces SenX3-RegX3 activity, which may alter cell-envelope physiology through ESX-5- and PE/PPE-dependent pathways and promote growth on lactate at acidic pH.