Cho You-Hee, Kim Eun-Ja, Chung Hye-Jung, Choi Jae-Hyun, Chater Keith F, Ahn Bo-Eun, Shin Jung-Ho, Roe Jung-Hye
Department of Life Science, Sogang University, Seoul 121-742, Korea.
J Bacteriol. 2003 Dec;185(23):6756-63. doi: 10.1128/JB.185.23.6756-6763.2003.
Paraquat (methyl viologen)-resistant mutants of Streptomyces coelicolor A3(2) that grew and sporulated normally in the presence of paraquat were isolated. Based on the positions of the mutant loci in the genetic map, we isolated the pqr (paraquat resistance) gene whose mutation (pqr501) caused a dominant paraquat-resistant phenotype. The pqr locus consists of two genes (pqrA and pqrB) that form a transcription unit. The pqrA gene encodes a protein with a TetR-like DNA-binding motif, and the pqrB gene encodes a putative efflux pump of the major facilitator superfamily. The pqr501 mutation was a base substitution changing arginine-18 to glutamine (R18Q) near the helix-turn-helix motif in PqrA. A pqrA null mutant exhibited similar paraquat resistance, and an increase in the amount of pqrA promoter-driven transcripts of about eightfold was observed for the pqrA501 mutant. These results suggest that PqrA is a negative regulator of its own operon. Deletion of the pqrAB operon caused cells to be very sensitive to paraquat, consistent with the prediction that PqrB may function as a paraquat-efflux pump. Purified PqrA protein specifically bound to the pqrA promoter region, whereas mutant R18Q protein did not, indicating that PqrA is a direct autoregulator of its own operon.
我们分离出了天蓝色链霉菌A3(2)的百草枯(甲基紫精)抗性突变体,这些突变体在百草枯存在的情况下能够正常生长并形成孢子。根据突变位点在遗传图谱中的位置,我们分离出了pqr(百草枯抗性)基因,其突变(pqr501)导致了显性的百草枯抗性表型。pqr位点由两个基因(pqrA和pqrB)组成,它们形成一个转录单元。pqrA基因编码一种具有类TetR DNA结合基序的蛋白质,pqrB基因编码一个假定的主要易化子超家族的外排泵。pqr501突变是一个碱基替换,将PqrA中靠近螺旋-转角-螺旋基序的精氨酸-18变为谷氨酰胺(R18Q)。一个pqrA缺失突变体表现出类似的百草枯抗性,并且在pqrA501突变体中观察到pqrA启动子驱动的转录本数量增加了约八倍。这些结果表明PqrA是其自身操纵子的负调节因子。pqrAB操纵子的缺失导致细胞对百草枯非常敏感,这与PqrB可能作为百草枯外排泵发挥作用的预测一致。纯化的PqrA蛋白特异性结合到pqrA启动子区域,而突变的R18Q蛋白则不能,这表明PqrA是其自身操纵子的直接自调节因子。