Phosphatidic acid phosphatase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. PAP activity has a key role in the regulation of PA flux towards TAG or glycerophospholipid synthesis. In this work we have characterized two Mycobacterium smegmatis genes encoding for functional PAP proteins. Disruption of both genes provoked a sharp reduction in de novo TAG biosynthesis in early growth phase cultures under stress conditions. In vivo labeling experiments demonstrated that TAG biosynthesis was restored in the ∆PAP mutant when bacteria reached exponential growth phase, with a concomitant reduction of phospholipid synthesis. In addition, comparative lipidomic analysis showed that the ∆PAP strain had increased levels of odd chain fatty acids esterified into TAGs, suggesting that the absence of PAP activity triggered other rearrangements of lipid metabolism, like phospholipid recycling, in order to maintain the wild type levels of TAG. Finally, the lipid changes observed in the ∆PAP mutant led to defective biofilm formation. Understanding the interaction between TAG synthesis and the lipid composition of mycobacterial cell envelope is a key step to better understand how lipid homeostasis is regulated during Mycobacterium tuberculosis infection.

磷脂酸磷酸酶 (PAP) 催化磷脂酸 (PA) 去磷酸化，产生二酰甘油 (DAG)，这是三酰甘油 (TAG) 生物合成的脂质前体。 PAP 活性在调节 PA 流向 TAG 或甘油磷脂合成方面发挥着关键作用。在这项工作中，我们鉴定了两个编码功能性 PAP 蛋白的耻垢分枝杆菌基因。两个基因的破坏都会导致早期生长阶段培养物在应激条件下的从头 TAG 生物合成急剧减少。体内标记实验表明，当细菌达到指数生长期时，ΔPAP 突变体中 TAG 生物合成得到恢复，同时磷脂合成减少。此外，比较脂质组学分析表明，ΔPAP 菌株酯化为 TAG 的奇数链脂肪酸水平增加，这表明 PAP 活性的缺失引发了脂质代谢的其他重排，例如磷脂循环，以维持野生型水平标签。最后，在 ΔPAP 突变体中观察到的脂质变化导致生物膜形成缺陷。了解TAG合成与分枝杆菌细胞包膜脂质组成之间的相互作用是更好地了解结核分枝杆菌感染期间如何调节脂质稳态的关键步骤。