Macfarlane E L, Kwasnicka A, Ochs M M, Hancock R E
Department of Microbiology, 300, 6174 University Boulevard, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3.
Mol Microbiol. 1999 Oct;34(2):305-16. doi: 10.1046/j.1365-2958.1999.01600.x.
Rapid adaptation to environmental challenge is essential for the survival of many bacterial species, and is often effectively mediated by two-component regulatory systems. Part of the adaptive response of Pseudomonas aeruginosa to Mg2+ starvation is overexpression of the outer-membrane protein OprH and increased resistance to the polycationic antibiotic polymyxin B. Two overlapping open reading frames that encoded proteins with high similarities to the PhoP-PhoQ two-component regulatory system of Salmonella typhimurium were identified downstream of the oprH gene. A P. aeruginosa PhoP-null mutant, H851, was constructed by means of a phoP:xylE-GmR transcriptional fusion, and shown to be deficient in OprH expression. In contrast, an analogous PhoQ-null mutant, H854 (phoQ:xylE-GmR), exhibited constitutive overexpression of OprH. Normal Mg2+-regulated OprH expression could be restored in both mutants by complementation with a plasmid carrying the phoP and phoQ genes. Measurement of the catechol-2,3-dioxygenase activity, expressed from the xylE transcriptional fusion in strains H851 and H854, indicated that PhoP-PhoQ is involved in the regulation of phoP-phoQ as well as oprH. Reverse transcription polymerase chain reaction experiments and Northern blot analysis revealed linkage of oprH, phoP and phoQ into an operon that was demonstrated to be under the joint control of PhoP-PhoQ and Mg2+ ion concentration. In addition, studies of the polymyxin B resistance of the two mutant strains, H851 and H854, indicated that PhoP-PhoQ is involved in regulating P. aeruginosa polymyxin resistance in response to external Mg2+ concentrations.
快速适应环境挑战对许多细菌物种的生存至关重要，并且通常由双组分调节系统有效介导。铜绿假单胞菌对镁离子饥饿的部分适应性反应是外膜蛋白OprH的过表达以及对聚阳离子抗生素多粘菌素B的抗性增加。在oprH基因下游鉴定出两个重叠的开放阅读框，它们编码的蛋白质与鼠伤寒沙门氏菌的PhoP-PhoQ双组分调节系统具有高度相似性。通过phoP:xylE-GmR转录融合构建了铜绿假单胞菌PhoP缺失突变体H851，并显示其在OprH表达方面存在缺陷。相反，类似的PhoQ缺失突变体H854（phoQ:xylE-GmR）表现出OprH的组成型过表达。通过用携带phoP和phoQ基因的质粒互补，可以在两个突变体中恢复正常的镁离子调节的OprH表达。对菌株H851和H854中xylE转录融合表达的儿茶酚-2,3-双加氧酶活性的测量表明，PhoP-PhoQ参与了phoP-phoQ以及oprH的调节。逆转录聚合酶链反应实验和Northern印迹分析揭示了oprH、phoP和phoQ连接成一个操纵子，该操纵子被证明受PhoP-PhoQ和镁离子浓度的共同控制。此外，对两个突变菌株H851和H854的多粘菌素B抗性研究表明，PhoP-PhoQ参与调节铜绿假单胞菌对多粘菌素的抗性以响应外部镁离子浓度。
