tRNA-abhängige Biosynthesis of Phospholipids in Pseudomonas aeruginosa

细菌膜的组成会随着环境条件的变化而发生永久性的变化，从而有效地完成它们的生物学任务。为此，例如通过在磷脂酰甘油(PG)上添加赖氨酸来共价修饰脂类，从而改变膜的净负电荷。这种脂质修饰介导了革兰氏阳性细菌对阳离子抗菌肽(CAMP)的耐药性。最近，我们在革兰氏阴性杆菌中发现了丙氨酸与丙三醇的tRNA依赖加成反应，形成了一种新的磷脂丙氨酰-磷脂酰甘油(A-PG)，介导了对两种正电荷抗菌剂β-内酰胺类抗生素、高浓度H+和乳酸的耐药性。在这里，我们希望利用纯化的重组铜绿假单胞菌酶和先进的生化和生物物理技术来阐明相应的丙氨酰-磷脂酰甘油合成酶(A-PGS)依赖tRNA的酶机制和底物识别模式。晶体结构将提供对相关结构-功能关系的理解。具有普遍重要性的新的生物化学原理是可望的。此外，还将阐明A-PG组与L-PG组的生理和生物物理作用。最后对碱性条件对油脂组成的影响进行了类比分析。

Bacterial membranes undergo permanent changes in their composition in response to varying environmental conditions to efficiently fulfil their biological tasks. For this purpose lipids are covalently modified for instance by adding lysine onto phosphatidylglycerol (PG), thereby altering the net negative charge of the membrane. This lipid modification mediates resistance to cationic antimicrobial peptides (CAMPs) in Gram positive bacteria. Recently we discovered the tRNA-dependent addition of alanine to PG with the formation of a new phospholipid alanyl-phosphatidylglycerol (A-PG) in the Gram negative pathogen Pseudomonas aeruginosa mediating resistance against two positively charged antibacterial agents, a β-lactam antibiotic, high concentrations of H+ and lactate. Here we want to elucidate the tRNA-dependent enzyme mechanism and the mode of substrate recognition for the corresponding alanyl-phosphatidylglycerol synthase (A-PGS) using purified recombinant P. aeruginosa enzyme and advanced biochemical and biophysical techniques. A crystal structure will provide the understanding of the involved structure-function relationships. Novel biochemical principles of general importance are expected. Furthermore the physiological and biophysical role of A-PG formation versus L-PG formation will be elucidated. Finally the influence of alkaline conditions on the lipid composition will be analyzed analogously.