[Incorporation of phosphatidylcholine into Escherichia coli membrane affects secretion of penicillin beta-lactamase].
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-beta-D-thiogalactoside (IPTG) for 4-8 hours at 37 degrees C.


METHODS
Ampicillin inhibitionof E. coli Top10 pcs+ was tested at first, and then beta-lactamase activity in periplasm was examined. Finally Western blot was used to detect the amount of beta-lactamase in both bacterial periplasm and cytoplasm.


RESULTS
Antibiotic tests showed that high concentrations of ampicillin inhibited the growth of E. coli Top100 pcs+ with an IC50 of 70-800 microg/mL. Active assays revealed that the beta-lactamase activity in periplasm was only 1/5 of that for the control strain E. coli Top10/p(tac)85. Western blotting confirmed that the low activity of beta-lactamase in E. coli Top10 pcs+ resulted from a lower amount of beta-lactamase in its periplasm.


CONCLUSIONS
Our results demonstrated that the phospatidylcholine incorporated into bacterial membrane retarded secretion of Escherichia coli penicillin beta-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.
Cai
Xueli
X
Faculty of Life Sciences, Hubei University, Wuhan 430062, China. shirley-635@163.com
Li
Yang
Y
Xuan
Wenjing
W
Wang
Xinguo
X
D000667:Ampicillin / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000166:cytology* / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D010406:Penicillins / Q000378:metabolism*; D019897:Periplasm / Q000187:drug effects / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism*; D001618:beta-Lactamases / Q000378:metabolism*
D010406:Penicillins; D010713:Phosphatidylcholines; D000667:Ampicillin; D001618:beta-Lactamases