Schmitt M P, Holmes R K
Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Infect Immun. 1991 Jun;59(6):1899-904. doi: 10.1128/iai.59.6.1899-1904.1991.
A regulatory gene (dtxR) responsible for iron-dependent repression of the toxin (tox) and siderophore genes in Corynebacterium diphtheriae was cloned and characterized. A DNA fragment carrying dtxR repressed expression of a tox-lacZ gene fusion in Escherichia coli DH5 alpha in a high-iron environment but not under low-iron conditions. A protein with mobility corresponding to approximately 28 to 29 kDa was identified as the product of the dtxR gene by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A shuttle vector designated pCM2.6 was constructed which carries the origin of replication from C. diphtheriae plasmid pNG2 and confers resistance to chloramphenicol in E. coli and C. diphtheriae. DNA fragments carrying dtxR were cloned into pCM2.6, and the hybrid shuttle plasmids were transformed by electroporation into wild-type C. diphtheriae C7(beta) and the regulatory mutant C7(beta)hm723, which produces toxin and siderophore constitutively under high-iron conditions. Expression of the cloned dtxR determinant did not affect the phenotype of C. diphtheriae C7(beta). In C. diphtheriae C7(beta)hm723, expression of cloned dtxR restored full repression of siderophore production and partial repression of diphtheria toxin production during growth in a high-iron environment.
克隆并鉴定了一种负责铁依赖性抑制白喉棒状杆菌毒素（tox）和铁载体基因的调控基因（dtxR）。携带dtxR的DNA片段在高铁环境下可抑制大肠杆菌DH5α中tox-lacZ基因融合体的表达，但在低铁条件下则无此作用。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定出一种迁移率约为28至29 kDa的蛋白质为dtxR基因的产物。构建了一种名为pCM2.6的穿梭载体，它携带白喉棒状杆菌质粒pNG2的复制起点，并赋予大肠杆菌和白喉棒状杆菌氯霉素抗性。将携带dtxR的DNA片段克隆到pCM2.6中，通过电穿孔将杂交穿梭质粒转化到野生型白喉棒状杆菌C7(β)和调控突变体C7(β)hm723中，后者在高铁条件下组成性地产生毒素和铁载体。克隆的dtxR决定簇的表达不影响白喉棒状杆菌C7(β)的表型。在白喉棒状杆菌C7(β)hm723中，克隆的dtxR的表达恢复了高铁环境下生长期间铁载体产生的完全抑制和白喉毒素产生的部分抑制。
