Niederhoffer E C, Naranjo C M, Bradley K L, Fee J A
Isotope and Structural Chemistry Group (INC-4), Los Alamos, National Laboratory, New Mexico 87545.
J Bacteriol. 1990 Apr;172(4):1930-8. doi: 10.1128/jb.172.4.1930-1938.1990.
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.
铁摄取调节（fur）基因产物参与调控大肠杆菌含锰和铁的超氧化物歧化酶基因的表达。对染色体上phi（sodA'-'lacZ）融合编码的β-半乳糖苷酶活性的检测表明，金属化的Fur蛋白作为sodA（锰超氧化物歧化酶[MnSOD]）的转录阻遏物。凝胶阻滞分析表明，纯的Mn2(+)-Fur蛋白与含有sodA启动子的DNA片段有高亲和力结合。这些数据以及其启动子中存在铁盒序列强烈表明sodA是铁摄取调节子的一部分。携带低拷贝或高拷贝质粒的sodB'-'lacZ融合基因在Fur+细胞中产生的β-半乳糖苷酶活性比Fur-细胞高约两到三倍；活性水平仅微弱依赖于生长阶段，并且在延长的稳定期内没有变化。对数生长期以及sodA和fur sodA背景的过夜培养物中FeSOD活性的测量表明，Fur-菌株中几乎不表达FeSOD活性，而Fur+细胞中表达野生型水平。携带多拷贝质粒pHS1-4（sodB+）的Fur+和Fur-细胞在fur背景中表达的FeSOD活性约低七倍，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。凝胶阻滞分析表明，与随机DNA序列相比，Mn2(+)-Fur对sodB启动子片段的亲和力明显更高，但明显低于对sodA启动子片段的亲和力。这些观察结果表明，sodB的明显正调控并非完全源于全（金属化）Fur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阻遏方式。
