Ochsner U A, Vasil M L
Department of Microbiology, University of Colorado Health Science Center, Denver 80262, USA.
Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4409-14. doi: 10.1073/pnas.93.9.4409.
The expression of at least 24 distinct genes of Pseudomonas aeruginosa PAO1 is under direct control of the "ferric uptake regulator" (Fur). Novel targets of the Fur protein were isolated in a powerful SELEX (systematic evolution of ligands by exponential enrichment)-like cycle selection consisting of in vitro DNA-Fur interaction, binding to anti-Fur antibody, purification on protein G, and PCR amplification. DNA fragments obtained after at least three exponential enrichment cycles were cloned and subjected to DNA mobility-shift assays and DNase I footprint analyses to verify the specific interaction with the Fur protein in vitro. Iron-dependent expression of the corresponding genes in vivo was monitored by RNase protection analysis. In total, 20 different DNA fragments were identified which represent actual Pseudomonas iron-regulated genes (PIGs). While four PIGs are identical to already known genes (pfeR, pvdS, tonB, and fumC, respectively), 16 PIGs represent previously unknown genes. Homology studies of the putative proteins encoded by the PIGs allowed us to speculate about their possible function. Two PIG products were highly similar to siderophore receptors from various species, and three PIG products were significantly homologous to alternative sigma factors. Furthermore, homologs of the Escherichia coli ORF1-tolQ, nuoA, stringent starvation protein Ssp, and of a two-component regulatory system similar to the Pseudomonas syringae LemA sensor kinase were identified. The putative gene products of seven additional PIGs did not show significant homologies to any known proteins. The PIGs were mapped on the P.aeruginosa chromosome. Their possible role in iron metabolism and virulence of P. aeruginosa is discussed.
铜绿假单胞菌PAO1至少24个不同基因的表达受“铁摄取调节蛋白”（Fur）的直接控制。在一个类似强大的SELEX（指数富集配体的系统进化）循环选择中分离出Fur蛋白的新靶点，该循环选择包括体外DNA-Fur相互作用、与抗Fur抗体结合、蛋白G纯化和PCR扩增。经过至少三个指数富集循环后获得的DNA片段被克隆，并进行DNA迁移率变动分析和DNase I足迹分析，以验证体外与Fur蛋白的特异性相互作用。通过核糖核酸酶保护分析监测体内相应基因的铁依赖性表达。总共鉴定出20个不同的DNA片段，它们代表实际的铜绿假单胞菌铁调节基因（PIGs）。虽然四个PIGs分别与已知基因（pfeR、pvdS、tonB和fumC）相同，但16个PIGs代表以前未知的基因。对PIGs编码的推定蛋白进行同源性研究，使我们能够推测它们可能的功能。两种PIG产物与来自不同物种的铁载体受体高度相似，三种PIG产物与替代sigma因子显著同源。此外，还鉴定出大肠杆菌ORF1-tolQ、nuoA、严格饥饿蛋白Ssp的同源物，以及与丁香假单胞菌LemA传感器激酶类似的双组分调节系统的同源物。另外七个PIGs的推定基因产物与任何已知蛋白均无显著同源性。这些PIGs被定位在铜绿假单胞菌染色体上。讨论了它们在铜绿假单胞菌铁代谢和毒力中的可能作用。
