掺入大肠杆菌膜中的磷脂酰胆碱(PC)影响青霉素b-内酰胺酶的分泌

Incorporation of phosphatidylcholine into Escherichia coli membrane affects secretion of penicillin b-lactamase

摘要:

【目的】为了研究磷脂酰胆碱(PC)在原核生物细胞中的生物学作用, 探讨PC对细菌膜系统的功能的影响。【方法】使用ptac85质粒作载体, 将螺旋菌pcs基因导入E.coli Top10细胞构建了E.coli Top10 pcs+菌株, 并在特定的条件下培养细菌, 使细菌膜磷脂中合成30%左右的磷脂酰胆碱。然后再使用抗生素抗性分析、b-内酰胺酶的酶活测定以及Western blot杂交技术,分析质粒编码的b-内酰胺酶从细胞质到细胞间质的分泌情况。【结果】抗生素抗性分析发现, 高浓度的氨苄青霉素抑制E.coli Top10 pcs+细菌的生长的氨苄青霉素剂量低于对照组,其半致死剂量IC50在700~800mg/mL之间。酶活检测显示E.coli Top10 pcs+细菌周质内b-内酰胺酶的酶活性只有对照菌株的1/5, Western blot进一步分析发现周质内b-内酰胺酶的含量也为对照菌株的1/5。由此可见, 周质内低含量的b-内酰胺酶是导致E.coli Top10 pcs+ 细菌氨苄青霉素抗性降低的原因。【结论】掺入细菌膜磷脂双分子层的PC影响b-内酰胺酶通过Sec转运途径从细胞质分泌到细菌周质空间内, 提示细菌磷脂酰胆碱可能在调节蛋白转运和分泌方面起着重要的作用。

Abstract:

[Objective]To study the biological function of phosphatidylcholine in bacteria, the borrelial pcs gene was inserted into ptac85 plasmid. Then E. coli Top10 pcs+ was constructed via the transformation of the recombinant plasmid. Phosphatidylcholine (30%) in total phospholipids was achieved when the bacterial cells were incubated in Luria-Bertani (LB) medium supplemented with 1% choline and induced by 0.5 mmol/L isopropy-β-D-thiogalactoside (IPTG) for 4~8 hours at 37℃. [Methods] Ampicillin inhibitionof E. coli Top10 pcs+ was tested at first, and then b-lactamase activity in periplasm was examined. Finally Western blot was used to detect the amount of b-lactamase in both bacterial periplasm and cytoplasm. [Results] Antibiotic tests showed that high concentrations of ampicillin inhibited the growth of E. coli Top10 pcs+ with an IC50 of 70~800 mg/mL. Active assays revealed that the b-lactamase activity in periplasm was only 1/5 of that for the control strain E. coli Top10/ptac85. Western blotting confirmed that the low activity of b-lactamase in E. coli Top10 pcs+ resulted from a lower amount of b-lactamase in its periplasm. [Conclusion]Our results demonstrated that the phospatidylcholine incorporated into bacterial membrane retarded secretion of Escherichia coli penicillin b-lactamase from cytoplasm into periplasm, which suggested that phosphatidylcholine might play a role in the regulation of protein secretion in those bacteria able to synthesize phosphatidylcholine.