CLONING AND SEQUENCE-ANALYSIS OF A GENE (PCHR) ENCODING AN ARAC FAMILY ACTIVATOR OF PYOCHELIN AND FERRIPYOCHELIN RECEPTOR SYNTHESIS IN PSEUDOMONAS-AERUGINOSA
CLONING AND SEQUENCE-ANALYSIS OF A GENE (PCHR) ENCODING AN ARAC FAMILY ACTIVATOR OF PYOCHELIN AND FERRIPYOCHELIN RECEPTOR SYNTHESIS IN PSEUDOMONAS-AERUGINOSA
复制标题
DOI：
10.1128/jb.175.18.5882-5889.1993
复制
发表时间：
1993-09-01
影响因子：
3.2
通讯作者：
POOLE, K
中科院分区：
文献类型：
--
作者：
HEINRICHS, DE;POOLE, K
铜绿假单胞菌K372在75-kDa铁绿脓菌螯铁蛋白受体蛋白和绿脓菌螯铁蛋白的产生中是缺陷的。克隆了恢复受体蛋白和绿脓菌螯铁蛋白生产的1.8 kb EcoRI-SalI片段。该片段的核苷酸测序揭示了一个888 bp的开放阅读框架，命名为pchR（绿脓菌螯铁蛋白），能够编码一个分子量为32，339-Da的296个氨基酸的蛋白质。通过使用基于噬菌体T7的表达系统，ca.从1.8-kb片段产生32 kDa，证实该开放阅读框确实表达。一个区域表现出同源性的共识毛皮结合位点的大肠杆菌被确定上游的pchR编码区重叠一个假定的启动子。此外，PchR的C-末端80个氨基酸残基与AraC的羧基末端显示出约50%的同源性（同一性，31%;保守性变化，19%），AraC是大肠杆菌中基因表达的已知转录激活因子。大肠杆菌、鼠伤寒沙门氏菌、弗氏柠檬酸杆菌和欧文氏菌。在PchR、AraC和AraC家族转录激活因子的许多其他成员的C-末端区域内，存在高度保守的17个残基结构域，事实上，其中两个残基被严格保持，另外两个残基仅表现出保守的变化，这表明在所有这些蛋白质中该区域具有共同的功能意义。这些数据与PchR作为铜绿假单胞菌中绿脓菌螯铁蛋白和铁绿脓菌螯铁蛋白受体合成的转录激活剂的作用一致。与此一致，通过体外诱变和基因置换获得的PchR突变体在铁绿脓菌螯铁蛋白受体和绿脓菌螯铁蛋白的产生中是缺陷的。
Pseudomonas aeruginosa K372 is deficient in the production of both the 75-kDa ferripyochelin receptor protein and pyochelin. A 1.8-kb EcoRI-SalI fragment which restored production of both the receptor protein and pyochelin was cloned. Nucleotide sequencing of the fragment revealed an open reading frame of 888 bp, designated pchR (pyochelin), capable of encoding a 296-amino-acid protein of a 32,339-Da molecular mass. By using a phage T7-based expression system, a protein of ca. 32 kDa was produced off the 1.8-kb fragment, confirming that this open reading frame was indeed expressed. A region exhibiting homology to the consensus Fur-binding site of Escherichia coli was identified upstream of the pchR coding region overlapping a putative promoter. In addition, the C-terminal 80 amino acid residues of PchR showed approximately 50% homology (identity, 31%; conserved changes, 19%) to the carboxy terminus of AraC, a known transcriptional activator of gene expression in E. coli, Salmonella typhimurium, Citrobacter freundii, and Erwinia chrysanthemi. Within the C-terminal region of PchR, AraC, and a number of other members of the AraC family of transcriptional activators, there exists a highly conserved 17-residue domain where, in fact, two residues are strictly maintained and two others exhibit only conserved changes, suggesting a common functional significance to this region in all of these proteins. These data are consistent with a role for PchR as a transcriptional activator of pyochelin and ferripyochelin receptor synthesis in P. aeruginosa. In agreement with this, a PchR mutant obtained by in vitro mutagenesis and gene replacement was deficient in production of the ferripyochelin receptor and pyochelin.