Flores Francisco J, Rincón Javier, Martín Juan F
Area de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain.
Microb Cell Fact. 2003 May 19;2(1):5. doi: 10.1186/1475-2859-2-5.
The bioavailability of iron is quite low since it is usually present as insoluble complexes. To solve the bioavailability problem microorganisms have developed highly efficient iron-scavenging systems based on the synthesis of siderophores that have high iron affinity. The systems of iron assimilation in microorganisms are strictly regulated to control the intracellular iron levels since at high concentrations iron is toxic for cells. Streptomyces pilosus synthesizes the siderofore desferrioxamine B. The first step in desferrioxamine biosynthesis is decarboxylation of L-lysine to form cadaverine, a desferrioxamine B precursor. This reaction is catalyzed by the lysine decarboxylase, an enzyme encoded by the desA gene that is repressed by iron. RESULTS: The binding of the DmdR (acronym for divalent metal dependent repressor) to the desA promoter in presence of Fe2+ or other divalent ions has been characterized. A 51 bp DNA fragment of the desA promoter containing the 9 bp inverted repeat was sufficient for binding of the DmdR repressor, as observed by the electrophoretic mobility shift assay. The desA mobility shift was prevented by neutralizing DmdR with anti-DmdR antibodies or by chelating the divalent metal in the binding reaction with 2,2'-dipyridyl. Binding to the desA promoter was observed with purified DmdR repressors of Streptomyces coelicolor or Rhodococcus fascians suggesting that there is a common mechanism of iron-regulation in actinomycetes. The complete desA promoter region was coupled using transcriptional fusions to the amy reporter gene (encoding alpha-amylase) in low copy or multicopy Streptomyces vectors. The iron-regulated desA promoter was induced by addition of the iron chelating agent 2,2'-dipyridyl resulting in a strong expression of the reporter gene. CONCLUSIONS: The iron-regulated desA promoter can be used for inducible expression of genes in Streptomyces species, as shown by de-repression of the promoter when coupled to a reporter gene.
铁的生物利用度相当低，因为它通常以不溶性复合物的形式存在。为了解决生物利用度问题，微生物基于具有高铁亲和力的铁载体的合成，开发了高效的铁清除系统。由于高浓度的铁对细胞有毒，微生物中的铁同化系统受到严格调控以控制细胞内铁水平。多毛链霉菌合成铁载体去铁胺B。去铁胺生物合成的第一步是L-赖氨酸脱羧形成尸胺，尸胺是去铁胺B的前体。该反应由赖氨酸脱羧酶催化，该酶由受铁抑制的desA基因编码。结果：已对二价金属依赖性阻遏物（DmdR）在存在Fe2+或其他二价离子的情况下与desA启动子的结合进行了表征。如通过电泳迁移率变动分析所观察到的，包含9 bp反向重复序列的desA启动子的51 bp DNA片段足以用于DmdR阻遏物的结合。通过用抗DmdR抗体中和DmdR或通过在结合反应中用2,2'-联吡啶螯合二价金属来阻止desA迁移率变动。用天蓝色链霉菌或 fascians红球菌的纯化DmdR阻遏物观察到与desA启动子的结合，这表明放线菌中存在铁调节的共同机制。使用转录融合将完整的desA启动子区域与低拷贝或多拷贝链霉菌载体中的amy报告基因（编码α-淀粉酶）偶联。通过添加铁螯合剂2,2'-联吡啶诱导铁调节的desA启动子，导致报告基因的强烈表达。结论：如与报告基因偶联时启动子的去阻遏所表明的，铁调节的desA启动子可用于链霉菌物种中基因的诱导表达。
