Studies in membrane topology and function of inner membrane component, MexA,of antibiotic extrusion pump of Pseudomonas aeruginosa

Pseudomonas aeruginosa, a main causative agent of opportunistic infections, shows intrinsic and multiantibiotic resistance.该细菌产生抗生素耐药性的主要原因似乎是 mex 操纵子 (mexA-mexl3-oprM) 中编码的能量依赖性抗生素挤出泵 (MexAB-OprM 泵) 的存在。为了深入了解Mex泵的抗生素挤出模式，有必要研究泵各亚基的结构和功能。因此，我分析了膜融合蛋白家族成员MexA的膜拓扑和定位，MexA被认为将内膜泵MexB与外膜通道OprM连接起来。我采用基因融合技术，使用无信号序列β-内酰胺酶作为报告基因来分析MexA的膜拓扑，得到了78个框内MexA-β-内酰胺酶融合体。所有携带每个融合基因的转化体均表现出氨苄青霉素抗性，表明信号序列之外的几乎所有 MexA 区域都位于周质中。 MexA was also found to be modified by lipid(s) at its cysteine residue in the consensus sequence for lipoproteins.

Pseudomonas aeruginosa, a main causative agent of opportunistic infections, shows intrinsic and multiantibiotic resistance. The major reason for the antibiotic resistance in this bacterium appeared to be the presence of an energy dependent antibiotic extrusion pump (MexAB-OprM pump) encoded in the mex operon (mexA-mexl3-oprM). To get insight into the mode of antibiotic extrusion by the Mex pump, it is essential to study on the structure and function of each subunit of the pump. Therefore, I analyzed the membrane topology and localization of MexA, a member of membrane fusion protein family, which is thought to link inner membrane pump, MexB, to an outer membrane channel, OprM.I employed the gene fusion technique to analyze the membrane topology of MexA using signal sequenceless beta-lactamase as a reporter and got 78 in-frame MexA-beta--lactamase fusions. All transformants harbored each fusion gene showed ampicillin resistance, indicating that almost all regions of MexA beyond the signal sequence reside in the periplasm. MexA was also found to be modified by lipid(s) at its cysteine residue in the consensus sequence for lipoproteins.