Tover Andres, Zernant Jana, Chugani Sudha A, Chakrabarty Ananda M, Kivisaar Maia
Department of Genetics, Institute of Molecular and Cell Biology, Estonian Biocentre and Tartu University, 51010 Tartu, Estonia1.
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois, USA2.
Microbiology (Reading). 2000 Jan;146 ( Pt 1):173-183. doi: 10.1099/00221287-146-1-173.
The promoter of the plasmid-borne pheBA genes encoding enzymes for phenol degradation resembles the catBCA promoter and is activated by CatR, the regulator of the chromosomally encoded catechol-degradative catBCA genes in Pseudomonas putida. In this study, site-directed mutagenesis of the pheBA promoter region was performed. The interrupted inverted repeat sequence of the CatR recognition binding site (RBS) of the pheBA promoter is highly homologous to that of the catBCA promoter. However, the RBS was shown not to be the sole important feature for high-affinity binding of CatR to this site. Mutagenesis of the activation binding site (ABS) of CatR, which overlaps the -35 hexamer sequence TTGGAT of the promoter, revealed that the two G nucleotides in this sequence are important for promoter activity but not for CatR binding. All other substitutions made in the ABS negatively affected both the promoter activity and CatR binding. The spacer sequence of the pheBA and catBCA promoters between the -10 and -35 hexamers is 19 bp, which is longer than optimal. However, reducing the spacer region of the pheBA promoter was not sufficient for CatR-independent promoter activation. An internal binding site (IBS) for CatR is located downstream of the transcriptional start site of the catBCA genes and it negatively regulates the operon. A similar IBS was identified in the case of the pheBA operon and tested for its functionality. The results indicate a conservation of CatR-mediated regulation mechanisms between the pheBA promoter and the catBCA promoter. This universal mechanism of CatR-mediated transcriptional activation could be of great importance in enabling catechol-degrading bacteria to expand their substrate range via horizontal transfer of the phenol degradative genes.
编码苯酚降解酶的质粒携带的pheBA基因的启动子类似于catBCA启动子，并被CatR激活，CatR是恶臭假单胞菌中染色体编码的儿茶酚降解catBCA基因的调节因子。在本研究中，对pheBA启动子区域进行了定点诱变。pheBA启动子的CatR识别结合位点（RBS）的中断反向重复序列与catBCA启动子的高度同源。然而，RBS并非CatR与该位点高亲和力结合的唯一重要特征。与启动子的-35六聚体序列TTGGAT重叠的CatR激活结合位点（ABS）的诱变表明，该序列中的两个G核苷酸对启动子活性很重要，但对CatR结合不重要。在ABS中进行的所有其他替换均对启动子活性和CatR结合产生负面影响。pheBA和catBCA启动子在-10和-35六聚体之间的间隔序列为19 bp，长于最佳长度。然而，缩短pheBA启动子的间隔区域不足以实现不依赖CatR的启动子激活。CatR的一个内部结合位点（IBS）位于catBCA基因转录起始位点的下游，并对操纵子起负调节作用。在pheBA操纵子的情况下也鉴定出了类似的IBS并测试了其功能。结果表明，pheBA启动子和catBCA启动子之间存在CatR介导的调节机制的保守性。这种CatR介导的转录激活的普遍机制对于使儿茶酚降解细菌能够通过苯酚降解基因的水平转移来扩大其底物范围可能非常重要。
