Miwa Y, Nagura K, Eguchi S, Fukuda H, Deutscher J, Fujita Y
Department of Biotechnology, Faculty of Engineering, Fukuyama University, Hiroshima, Japan.
Mol Microbiol. 1997 Mar;23(6):1203-13. doi: 10.1046/j.1365-2958.1997.2921662.x.
Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis-acting catabolite-responsive element (cre) by a complex of CcpA, which is a member of the GalR-Lacl family of bacterial regulatory proteins, and the seryl-phosphorylated form of HPr (P-ser-HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the cre in the first gene (gntR) of the gnt operon (credown), this operon contains another cre located in the promoter region (creup). A translational gntR'-'lacZ fusion expressed under the control of various combinations of wild-type and mutant credown and creup was integrated into the chromosomal amyE locus, and then catabolite repression of beta-galactosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by creup was probably independent of catabolite repression exerted by credown; both creup and credown catabolite repression involved CcpA. Catabolite repression exerted by creup was independent of P-ser-HPr, and catabolite repression exerted by credown was partially independent of P-ser-HPr. DNase I footprinting experiments indicated that a complex of CcpA and P-ser-HPr did not recognize creup, in contrast to its specific recognition of credown. However, CcpA complexed with glucose-6-phosphate specifically recognized creup as well as credown, but the physiological significance of this complexing is unknown.
枯草芽孢杆菌分解代谢操纵子的分解代谢物阻遏被认为是通过一种负调控机制发生的，该机制涉及细菌调控蛋白GalR-Lacl家族成员CcpA与丝氨酰磷酸化形式的HPr（P-ser-HPr）形成的复合物对顺式作用分解代谢物反应元件（cre）的识别，最近关于gnt操纵子分解代谢物阻遏的研究证实了这一点。通过缺失和点突变对gnt启动子区域进行分析发现，除了gnt操纵子第一个基因（gntR）中的cre（credown）外，该操纵子在启动子区域还含有另一个cre（creup）。将在野生型和突变型credown和creup的各种组合控制下表达的翻译融合体gntR'-'lacZ整合到染色体amyE位点，然后检测所得整合体中β-半乳糖苷酶合成的分解代谢物阻遏情况。体内结果表明，creup施加的分解代谢物阻遏可能独立于credown施加的分解代谢物阻遏；creup和credown的分解代谢物阻遏都涉及CcpA。creup施加的分解代谢物阻遏独立于P-ser-HPr，而credown施加的分解代谢物阻遏部分独立于P-ser-HPr。DNase I足迹实验表明，与对credown的特异性识别相反，CcpA和P-ser-HPr的复合物不识别creup。然而，与6-磷酸葡萄糖复合的CcpA既特异性识别creup也识别credown，但这种复合的生理意义尚不清楚。
