CLONING AND CHARACTERIZATION OF THE FERRIC ENTEROBACTIN RECEPTOR GENE (PFEA) OF PSEUDOMONAS-AERUGINOSA
CLONING AND CHARACTERIZATION OF THE FERRIC ENTEROBACTIN RECEPTOR GENE (PFEA) OF PSEUDOMONAS-AERUGINOSA
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DOI：
10.1128/jb.175.2.317-324.1993
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发表时间：
1993-01-01
影响因子：
3.2
通讯作者：
POOLE, K
中科院分区：
文献类型：
--
作者：
DEAN, CR;POOLE, K
铜绿假单胞菌（Pseudomonas aeruginosa） K407是一种缺乏高亲和力80000分子量铁肠obactin受体蛋白（80K蛋白）的突变体，在含有乙二胺-二羟基苯基乙酸（EDDHA）和肠obactin的缺铁琥珀酸最小培养基上表现出生长不良（小菌落）。通过对该生长缺陷的互补，克隆了编码铁肠杆菌受体的基因。互补DNA随后定位到7.1千碱基对（kb）的SstI-HindIII片段，该片段能够恢复菌株K407中80K蛋白的合成，并指导大肠杆菌fepA菌株MT912和IR20外膜中相同分子量的高水平蛋白质的合成。此外，该片段补充了MT9l2中的fepA突变，恢复了含eddha培养基中的生长和肠杆菌依赖的Fe-55（3+）摄取。铜绿假单胞菌受体在大肠杆菌IR20中的表达受铁和肠杆菌素的调控。将互补DNA进一步定位到5.3 kb的SphI-SstI片段，然后进行缺失分析，获得菌株K407外膜中能够指导80K蛋白合成的最小片段。随后在菌株K407中分离出一个恢复受体生产的3.2 kb DNA片段。该片段还指导了大肠杆菌MT912中蛋白质的合成，但其水平远低于之前观察到的水平。对该片段进行核苷酸测序，发现一个2241 bp的开放阅读框（为铁肠杆菌假单胞菌命名为pfeA），能够编码一个分子量为80,967的746个氨基酸的蛋白。PfeA蛋白与大肠杆菌FepA蛋白同源性超过60%。与此一致的是，这两种蛋白表现出显著的免疫交叉反应性。
Pseudomonas aeruginosa K407, a mutant lacking a high-affinity 80,000-molecular-weight ferric enterobactin receptor protein (80K protein), exhibited poor growth (small colonies) on iron-deficient succinate minimal medium containing ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and enterobactin. The gene encoding the ferric enterobactin receptor was cloned by complementation of this growth defect. The complementing DNA was subsequently localized to a 7.1-kilobase-pair (kb) SstI-HindIII fragment which was able to restore synthesis of the 80K protein in strain K407 and also to direct the synthesis of high levels of a protein of the same molecular weight in the outer membranes of Escherichia coli fepA strains MT912 and IR20. Moreover, the fragment complemented the fepA mutation in MT9l2, restoring both growth in EDDHA-containing medium and enterobactin-dependent uptake of Fe-55(3+). Expression of the P. aeruginosa receptor in E. coli IR20 was shown to be regulated by both iron and enterobactin. The complementing DNA was further localized to a 5.3-kb SphI-SstI fragment which was then subjected to deletion analysis to obtain the smallest fragment capable of directing the synthesis of the 80K protein in the outer membrane of strain K407. A 3.2-kb DNA fragment that restored production of the receptor in strain K407 was subsequently isolated. The fragment also directed synthesis of the protein in E. coli MT912 but at levels much lower than those previously observed. Nucleotide sequencing of the fragment revealed an open reading frame (designated pfeA for Pseudomonas ferric enterobactin) of 2,241 bp capable of encoding a 746-amino-acid protein with a molecular weight of 80,967. The PfeA protein showed more than 60% homology to the E. coli FepA protein. Consistent with this, the two proteins showed significant immunological cross-reactivity.