CONTROL OF ESCHERICHIA-COLI SUPEROXIDE-DISMUTASE (SODA AND SODB) GENES BY THE FERRIC UPTAKE REGULATION (FUR) LOCUS
CONTROL OF ESCHERICHIA-COLI SUPEROXIDE-DISMUTASE (SODA AND SODB) GENES BY THE FERRIC UPTAKE REGULATION (FUR) LOCUS
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DOI：
10.1128/jb.172.4.1930-1938.1990
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发表时间：
1990-04-01
影响因子：
3.2
通讯作者：
FEE, JA
中科院分区：
文献类型：
--
作者：
NIEDERHOFFER, EC;NARANJO, CM;FEE, JA
铁吸收调节（毛皮）基因产物参与调控表达的锰和铁的含超氧化物歧化酶基因的大肠杆菌。对编码自染色体phi（sodA“-”lacZ）融合体的β-半乳糖苷酶活性的检查表明，金属化的Fur蛋白充当sodA（锰超氧化物歧化酶[MnSOD]）的转录阻遏物。凝胶阻滞试验证明了纯Mn 2（+）-Fur蛋白与含有sodA启动子的DNA片段的高亲和力结合。这些数据和铁盒序列在其启动子中的存在强烈表明sodA是铁摄取调节子的一部分。与Fur-细胞相比，在低拷贝或高拷贝质粒上携带的sodB '-'lacZ融合基因在Fur+细胞中产生约2 - 3倍的β-半乳糖苷酶活性;活性水平仅微弱地依赖于生长期，并且在延长的静止期期间没有变化。在对数生长期和过夜培养的sodA和毛皮sodA背景的FeSOD活性的测量显示，几乎没有FeSOD活性在毛皮-菌株中表达，而野生型水平在毛皮+细胞中表达。携带多拷贝质粒pHS 1 -4（sodB+）的Fur+和Fur-细胞在Fur背景中表达的FeSOD活性约为7倍，非变性电泳凝胶染色表明Fur-细胞中FeSOD蛋白的合成大大减少。凝胶阻滞实验表明，Mn 2（+）-Fur对sodB启动子片段的亲和力显著高于随机DNA序列，但显著低于sodA启动子片段。这些观察结果表明，SOdB的明显的正调控并不完全是由于全（金属化）毛皮本身与SOdB启动子的直接相互作用。然而，sodB基因似乎也是铁摄取调节子的一部分，但不是以经典的铁依赖性抑制方式。
The ferric uptake regulation (fur) gene product participates in regulating expression of the manganese- and iron-containing superoxide dismutase genes of Escherichia coli. Examination of beta-galactosidase activity coded from a chromosomal phi(sodA'-'lacZ) fusion suggests that metallated Fur protein acts as a transcriptional repressor of sodA (manganese superoxide dismutase [MnSOD]). Gel retardation assays demonstrate high-affinity binding of pure, Mn2(+)-Fur protein to DNA fragments containing the sodA promoter. These data and the presence of an iron box sequence in its promoter strongly suggest that sodA is part of the iron uptake regulon. An sodB'-'lacZ fusion gene borne on either a low- or high-copy plasmid yielded approximately two- to threefold more beta-galactosidase activity in Fur+ compared with Fur- cells; the levels of activity depended only weakly on the growth phase and did not change during an extended stationary phase. Measurement of FeSOD activity in logarithmic growth phase and in overnight cultures of sodA and fur sodA backgrounds revealed that almost no FeSOD activity was expressed in Fur- strains, whereas wild-type levels were expressed in Fur+ cells. Fur+ and Fur- cells bearing the multicopy plasmid pHS1-4 (sodB+) expressed approximately sevenfold less FeSOD activity in the fur background, and staining of nondenaturing electrophoretic gels indicates that synthesis of FeSOD protein was greatly reduced in Fur- cells. Gel retardation assays show that Mn2(+)-Fur had a significantly higher affinity for the promoter fragment of sodB compared with that of random DNA sequences but significantly lower than for the promoter fragment of sodA. These observations suggest that the apparent positive regulation of sodB does not result exclusively from a direct interaction of holo (metallated) Fur itself with the sodB promoter. Nevertheless, the sodB gene also appears to be part of the iron uptake regulon but not in the classical manner of Fe-dependent repression.