Characterization and mutagenesis of fur gene from Burkholderia pseudomallei
Characterization and mutagenesis of fur gene from Burkholderia pseudomallei
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DOI：
10.1016/s0378-1119(00)00279-1
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发表时间：
2000-08-22
期刊：
影响因子：
3.5
通讯作者：
Mongkolsuk, S
中科院分区：
文献类型：
--
作者：
Loprasert, S;Sallabhan, R;Mongkolsuk, S
利用反向遗传技术从类鼻疽伯克霍尔德氏菌（Bp）中克隆了铁吸收调节基因（fur）的同源基因。2.2 kb的DNA片段的测序揭示了一个开放的阅读框架与细菌的皮毛蛋白具有广泛的同源性。克隆的基因编码一种16 kDa的蛋白质，该蛋白质与多克隆抗大肠杆菌毛皮血清发生交叉反应。通过引物延伸技术确定转录起始位点。毛皮的表达分析表明，没有增加毛皮的mRNA水平在各种压力和铁条件。一个积极的选择程序，涉及锰抗性突变体的分离，用于分离突变体，产生改变的毛皮蛋白。毛皮突变体的测序揭示了将保守氨基酸精氨酸-69转化为组氨酸的核苷酸变化（G至A）。毛皮错义突变体产生的铁载体的水平升高，可以补充携带Bp毛皮的多拷贝质粒。有趣的是，发现毛皮作为FeSOD和过氧化物酶的正调节剂发挥作用。该突变体的FeSOD和过氧化物酶活性降低，这可能是其致病性和在巨噬细胞中的存活的重要。(C)2000 Elsevier Science B. V.保留所有权利。
A homolog of the ferric uptake regulator gene (fur) was isolated from Burkholderia pseudomallei (Bp) by a reverse genetic technique. Sequencing of a 2.2 kb DNA fragment revealed an open reading frame with extensive homology to bacterial Fur proteins. The cloned gene encodes a 16 kDa protein that cross-reacts with a polyclonal anti-Escherichia coli Fur serum. The transcription start site was determined by the primer extension technique. Expression analysis of fur showed no increased fur mRNA levels in response to various stresses and iron conditions. A positive selection procedure involving the isolation of manganese-resistant mutants was used to isolate mutants that produce altered Fur proteins. Sequencing of a fur mutant revealed a nucleotide change (G to A) converting a conserved amino acid arginine-69 to histidine. The fur missense mutant produced an elevated level of siderophore that could be complemented by a multicopy plasmid carrying the Bp fur. Interestingly, Fur was found to play roles as a positive regulator of FeSOD and peroxidase. The mutant showed a decreased activity of FeSOD and peroxidase, which could be important in its pathogenicity and survival in macrophages. (C) 2000 Elsevier Science B.V. All rights reserved.