Poole K, Gotoh N, Tsujimoto H, Zhao Q, Wada A, Yamasaki T, Neshat S, Yamagishi J, Li X Z, Nishino T
Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada.
Mol Microbiol. 1996 Aug;21(4):713-24. doi: 10.1046/j.1365-2958.1996.281397.x.
OprJ, overproduced in nfxB multidrug-resistant strains of Pseudomonas aeruginosa, and OprK, overproduced in the multidrug-resistant strain K385, were demonstrated to be immunologically cross-reactive using an OprJ-specific monoclonal antibody. Treatment of the purified proteins with trypsin or chymotrypsin yielded virtually indistinguishable digestion patterns, and the N-terminal sequence of two trypsin fragments was identical for both proteins, indicating that OprJ and OprK share identity. The N-terminal amino acid sequences were used to facilitate cloning of the oprJ gene on a 5kbp Kpnl fragment and a 10 kbp BamHl fragment. Nucleotide sequencing of portions of these fragments revealed that oprJ was the terminal gene in a putative three-gene operon, mexC-mexD-oprJ. The predicted mexC-mexD-oprJ gene products exhibit homology to the MexA-MexB-OprM components of the multidrug-resistance efflux pump of P. aeruginosa (43-46% identity). Consistent with an implied role for mexC-mexD-oprJ in drug efflux, the mexC-mexD-oprJ-hyperexpressing strain K385 showed reduced accumulation of a variety of antibiotics as compared with its parent strain, and this drug 'exclusion' was abrogated by energy inhibitors. The mexC and oprJ products are putative lipoproteins of a molecular mass of 40,707 and 51,742 Da, respectively, while mexD was predicted to encode a protein of 111 936 Da. Sequencing upstream of mexC revealed the presence of the nfxB gene transcribed divergently from the efflux genes. Overproduction of OprJ and the attendant multiple-antibiotic resistance of strain K385 was shown to result from a point mutation in nfxB, resulting in a H87-->R change in the predicted NfxB polypeptide. OprJ overproduction and multidrug resistance in K385 was reversed by the cloned nfxB gene, suggesting that nfxB encodes a repressor of mexC-mexD-oprJ expression. Consistent with this, the cloned nfxB gene repressed synthesis of a mexC-lacZ fusion in Escherichia coli. nfxB also repressed expression of a nfxB-lacZ fusion, indicating that NfxB negatively regulates its own expression. These data indicate that the multidrug resistance of nfxB strains is due to overexpression of an efflux operon, mexC-mexD-oprJ, encoding components of a second efflux pump in P. aeruginosa.
在铜绿假单胞菌的nfxB多药耐药菌株中过量产生的OprJ以及在多药耐药菌株K385中过量产生的OprK，使用OprJ特异性单克隆抗体证明它们具有免疫交叉反应性。用胰蛋白酶或糜蛋白酶处理纯化后的蛋白质产生了几乎无法区分的消化模式，并且两种蛋白质的两个胰蛋白酶片段的N端序列相同，表明OprJ和OprK具有一致性。N端氨基酸序列被用于促进oprJ基因在一个5kbp的KpnI片段和一个10kbp的BamHI片段上的克隆。对这些片段部分的核苷酸测序显示oprJ是一个假定的三基因操纵子mexC-mexD-oprJ中的末端基因。预测的mexC-mexD-oprJ基因产物与铜绿假单胞菌多药耐药外排泵的MexA-MexB-OprM组分具有同源性（一致性为43%-46%）。与mexC-mexD-oprJ在药物外排中的潜在作用一致，mexC-mexD-oprJ高表达菌株K385与其亲本菌株相比，多种抗生素的积累减少，并且这种药物“排除”被能量抑制剂消除。mexC和oprJ产物分别是分子量为40707Da和51742Da的假定脂蛋白，而mexD预计编码一个111936Da的蛋白质。对mexC上游的测序显示存在与外排基因反向转录的nfxB基因。已表明OprJ的过量产生以及菌株K385随之而来的多重抗生素耐药性是由nfxB中的一个点突变导致的，该突变导致预测的NfxB多肽中H87→R的变化。K385中OprJ的过量产生和多药耐药性被克隆的nfxB基因逆转，这表明nfxB编码mexC-mexD-oprJ表达的阻遏物。与此一致的是，克隆的nfxB基因抑制了大肠杆菌中mexC-lacZ融合体的合成。nfxB也抑制了nfxB-lacZ融合体的表达，表明NfxB负向调节其自身的表达。这些数据表明nfxB菌株的多药耐药性是由于一个外排操纵子mexC-mexD-oprJ的过表达，该操纵子编码铜绿假单胞菌中第二个外排泵的组分。
