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

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.