Institute for Medical Biology, Polish Academy of Sciences, Lodz, Poland.
Centre National de la Recherche Scientifique FRE 3689, Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé, Université de Montpellier, Montpellier, France.
Sci Rep. 2017 Feb 16;7:42692. doi: 10.1038/srep42692.
Malonyl-coenzyme A (CoA) is a crucial extender unit for the synthesis of mycolic and other fatty acids in mycobacteria, generated in a reaction catalyzed by acetyl-CoA carboxylase. We previously reported on the essentiality of accD6 encoding the functional acetyl-CoA carboxylase subunit in Mycobacterium tuberculosis. Strikingly, the homologous gene in the fast-growing, non-pathogenic Mycobacterium smegmatis - (accD6) appeared to be dispensable, and its deletion did not influence the cell lipid content. Herein, we demonstrate that, despite the difference in essentiality, accD6 and accD6 encode proteins of convergent catalytic activity in vivo. To identify an alternative, AccD6-independent, malonyl-CoA synthesis pathway in M. smegmatis, a complex genetic approach combined with lipid analysis was applied to screen all five remaining carboxyltransferase genes (accD1-accD5) with respect to their involvement in mycolic acid biosynthesis and ability to utilize acetyl-CoA as the substrate for carboxylation. This approach revealed that AccD1, AccD2 and AccD3 are not essential for mycolic acid biosynthesis. Furthermore, we confirmed in vivo the function of AccD4 as an essential, long-chain acyl-CoA carboxyltransferase, unable to carboxylate short-chain substrate. Finally, our comparative studies unambiguously demonstrated between-species difference in in vivo ability of AccD5 carboxyltransferase to utilize acetyl-CoA that influences AccD6 essentiality in pathogenic and non-pathogenic mycobacteria.
丙二酰辅酶 A（CoA）是分枝杆菌中合成类脂酸和其他脂肪酸的重要延伸单位，由乙酰辅酶 A 羧化酶催化生成。我们之前报道过编码功能乙酰辅酶 A 羧化酶亚基的 accD6 在结核分枝杆菌中的必需性。引人注目的是，在生长迅速、非致病性的耻垢分枝杆菌中，同源基因（accD6）似乎是可有可无的，其缺失并不影响细胞脂质含量。在此，我们证明了尽管在必需性上存在差异，但 accD6 和 accD6 在体内编码具有趋同催化活性的蛋白质。为了鉴定耻垢分枝杆菌中替代的、AccD6 独立的丙二酰辅酶 A 合成途径，我们采用了一种复杂的遗传方法结合脂质分析，筛选了其余五个羧基转移酶基因（accD1-accD5），以确定它们是否参与了分枝菌酸生物合成以及是否能够利用乙酰辅酶 A 作为羧化的底物。这种方法表明 AccD1、AccD2 和 AccD3 对于分枝菌酸生物合成不是必需的。此外，我们在体内证实了 AccD4 作为必需的长链酰基辅酶 A 羧基转移酶的功能，它不能羧化短链底物。最后，我们的比较研究明确证明了在物种间 accD5 羧基转移酶利用乙酰辅酶 A 的体内能力存在差异，这影响了致病性和非致病性分枝杆菌中 AccD6 的必需性。
