Kilstrup M, Martinussen J
Department of Microbiology, Technical University of Denmark, DK2800 Lyngby, Denmark.
J Bacteriol. 1998 Aug;180(15):3907-16. doi: 10.1128/JB.180.15.3907-3916.1998.
A purR::pGh9:ISS1 mutant of Lactococcus lactis was obtained following transposon mutagenesis of strain MG1363 and selection for purine auxotrophs. After determination of the nucleotide sequence and deduction of the purR reading frame, the PurR product was found to be highly similar to the purR-encoded repressor from Bacillus subtilis. The wild-type purR gene complemented the purine auxotrophy of a purR::ISS1 mutant, and it was shown that the purR::ISS1 mutation lowered the level of transcription from the purine-regulated L. lactis purD promoter. In a parallel study on the regulation of purC and purD expression in L. lactis (M. Kilstrup, S. G. Jessing, S. B. Wichmand-Jorgensen, M. Madsen, and D. Nilsson, J. Bacteriol. 180:3900-3906, 1998), we identified regions (PurBox sequences: AWWWCCGAACWWT) upstream of the promoters with a central G residue at exactly position -76 relative to the transcriptional start site. The PurBox sequences were found to be required for high-level promoter activity and purine regulation. We identified a PurBox sequence overlapping the -35 region of the L. lactis purR promoter and found, by studies of a purR-lacLM fusion plasmid, that purR is autoregulated. Because of the high degree of similarity of the PurR proteins from B. subtilis and L. lactis, we looked for PurBox sequences in the promoter regions of the PurR-regulated genes in B. subtilis and identified a perfectly matching PurBox sequence in the purA promoter region and slightly degenerate PurBox-like sequences in the promoter regions for the pur operon and the purR gene. Interestingly, the PurBox in the pur operon of B. subtilis is located almost identically, with respect to the promoter, to the PurBox sequences located in front of purC and purD in L. lactis. We present a hypothesis to explain how an ancestral PurR protein in B. subtilis could have evolved from an activator of the pur operon into a repressor which regulates transcription initiation from the same pur promoter by using the same PurR binding site and a similar response toward its effectors.
对乳酸乳球菌MG1363菌株进行转座子诱变并筛选嘌呤营养缺陷型后，获得了一株purR::pGh9:ISS1突变体。在确定核苷酸序列并推导purR阅读框后，发现PurR产物与枯草芽孢杆菌中由purR编码的阻遏蛋白高度相似。野生型purR基因可弥补purR::ISS1突变体的嘌呤营养缺陷，并且表明purR::ISS1突变降低了受嘌呤调控的乳酸乳球菌purD启动子的转录水平。在一项关于乳酸乳球菌中purC和purD表达调控的平行研究中（M. Kilstrup、S. G. Jessing、S. B. Wichmand-Jorgensen、M. Madsen和D. Nilsson，《细菌学杂志》180:3900 - 3906，1998年），我们在启动子上游鉴定出区域（PurBox序列：AWWWCCGAACWWT），相对于转录起始位点，其中心G残基恰好位于-76位置。发现PurBox序列是高水平启动子活性和嘌呤调控所必需的。我们鉴定出一个与乳酸乳球菌purR启动子的-35区域重叠的PurBox序列，并通过对purR-lacLM融合质粒的研究发现，purR是自我调控的。由于枯草芽孢杆菌和乳酸乳球菌的PurR蛋白高度相似，我们在枯草芽孢杆菌中PurR调控基因的启动子区域寻找PurBox序列，并在purA启动子区域鉴定出一个完全匹配的PurBox序列，在pur操纵子和purR基因的启动子区域鉴定出略有退化的PurBox样序列。有趣的是，枯草芽孢杆菌pur操纵子中的PurBox相对于启动子的位置，与乳酸乳球菌中位于purC和purD之前的PurBox序列几乎相同。我们提出一个假说，以解释枯草芽孢杆菌中一个祖先PurR蛋白如何从pur操纵子的激活剂进化为一个阻遏蛋白，该阻遏蛋白通过使用相同的PurR结合位点并对其效应物有类似反应来调控来自同一个pur启动子的转录起始。
