Characterizing the Staphylococcus aureus fatty acid degradation operon

Staphylococcus aureus can supplement its endogenous fatty acid synthesis pathway (FASII) with exogenous fatty acids it acquires from the environment through the fatty acid kinase (Fak) complex. While S. aureus has been thought to not degrade fatty acids, it does possess a potential fadXDEBA locus that contains all the genes necessary for ?-oxidation. Using mRNA analysis, we determined that the fadXDEBA operon can be found on one polycistronic mRNA. Moreover, we identified the fadX promoter and a putative binding site within this region that is consistent with negative regulation by the metabolism-responsive regulator, Carbon Catabolite Protein A (CcpA). Indeed, in the absence of glucose or CcpA, we saw the fadXDEBA operon was derepressed. S. aureus is annotated to lack the crotonase domain of FadB; however, new analysis indicates it is present. To test the functionality of the S. aureus FadB, we performed complementation assays with E. coli fad mutants using minimal media supplemented with single fatty acids. We were able to restore growth of E. coli fad mutants when providing safadBA genes on a plasmid and demonstrate that the SaFadB crotonase domain is required for complementation. Together, these data demonstrate the SaFadBA proteins are functional within a well characterized fatty acid degradation system and the fadXDEBA operon is under strong catabolite repression. Overall design: Six samples in total, three biological replicates of both wild type Staphylococcus aureus and an isogenic fakA mutant.

金黄色葡萄球菌（Staphylococcus aureus）可通过脂肪酸激酶（fatty acid kinase, Fak）复合物从环境中获取外源性脂肪酸，以补充其自身的内源性脂肪酸合成途径（FASII）。既往认为金黄色葡萄球菌无法降解脂肪酸，但该菌实则携带包含β-氧化全部必需基因的潜在fadXDEBA基因座。通过mRNA分析，我们证实fadXDEBA操纵子可整合至一条多顺反子mRNA上。此外，我们在该区域内鉴定出fadX启动子及一处推定结合位点，其与代谢响应调节因子碳分解代谢物蛋白A（Carbon Catabolite Protein A, CcpA）介导的负调控特征相符。实验证实，在缺失葡萄糖或CcpA时，fadXDEBA操纵子会发生去阻遏。尽管金黄色葡萄球菌的注释信息显示其缺乏FadB的巴豆酸酶结构域（crotonase domain），但新的分析表明该结构域实际存在。为验证金黄色葡萄球菌FadB的功能，我们利用添加单一脂肪酸的基础培养基，对大肠杆菌（E. coli）fad突变体开展互补实验。当通过质粒携带saFadBA基因时，我们成功恢复了大肠杆菌fad突变体的生长，并证实SaFadB的巴豆酸酶结构域是完成互补所必需的。综上，本研究数据表明SaFadBA蛋白在特征明确的脂肪酸降解系统中具备功能，且fadXDEBA操纵子受到强烈的分解代谢物阻遏调控。总体实验设计：共设置6个样本，野生型金黄色葡萄球菌与同基因fakA突变体各设置3个生物学重复。