Braaten B A, Platko J V, van der Woude M W, Simons B H, de Graaf F K, Calvo J M, Low D A
Department of Pathology, University of Utah Medical Center, Salt Lake City 84132.
Proc Natl Acad Sci U S A. 1992 May 15;89(10):4250-4. doi: 10.1073/pnas.89.10.4250.
The methylation blocking factor gene (mbf) in Escherichia coli is required for specific methylation inhibition of two DNA GATC sites upstream of the papBA pilin promoter and transcriptional activation of pap. Complementation and mutational analysis using pap-lac and ilvIH-lac operon fusions indicates that the mbf gene is identical to a recently described global regulatory gene lrp (leucine-responsive regulatory protein) that acts as a positive regulator of some genes and a negative regulator of others in E. coli. DNA sequence analysis of an mbf::mTn10 insertion showed that the mbfDNA sequence was identical to lrp. Thus Lrp inhibits DNA methylation at specific GATC sites. We also show that Lrp positively regulates transcription of the fan operon, which encodes K99 pili of diarrheagenic E. coli. Purified Lrp was found to bind to DNA fragments encompassing the pap and fan promoters, which is consistent with previous results indicating that Lrp controls gene expression by binding to regulatory DNA sites. Exogenous leucine significantly reduced fan transcription and K99 pili expression, similar to results obtained with the ilvIH operon. However, pap gene expression was unresponsive to leucine, which distinguishes pap from other lrp-regulated genes whose expression is modulated by leucine.
大肠杆菌中的甲基化阻断因子基因（mbf）对于抑制papBA菌毛素启动子上游两个DNA GATC位点的特异性甲基化以及激活pap的转录是必需的。使用pap - lac和ilvIH - lac操纵子融合进行的互补和突变分析表明，mbf基因与最近描述的全局调节基因lrp（亮氨酸响应调节蛋白）相同，lrp在大肠杆菌中对一些基因起正调节作用，对另一些基因起负调节作用。对mbf::mTn10插入进行的DNA序列分析表明，mbfDNA序列与lrp相同。因此，Lrp抑制特定GATC位点的DNA甲基化。我们还表明，Lrp正向调节fan操纵子的转录，该操纵子编码致腹泻大肠杆菌的K99菌毛。发现纯化的Lrp与包含pap和fan启动子的DNA片段结合，这与先前的结果一致，即Lrp通过与调节性DNA位点结合来控制基因表达。外源性亮氨酸显著降低了fan转录和K99菌毛表达，这与ilvIH操纵子的结果相似。然而，pap基因表达对亮氨酸无反应，这使pap与其他受lrp调节且其表达受亮氨酸调节的基因区分开来。
