Transcriptional Activation of the <i>mrkA</i> Promoter of the <i>Klebsiella pneumoniae</i> Type 3 Fimbrial Operon by the c-di-GMP-Dependent MrkH Protein

The Gram-negative bacterial pathogen Klebsiella pneumoniae forms biofilms to facilitate colonization of biotic and abiotic surfaces. The formation of biofilms by K. pneumoniae requires the expression of type 3 fimbriae: elongate proteinaceous filaments extruded by a chaperone-usher system in the bacterial outer membrane. The expression of the mrkABCDF cluster that encodes this fimbrial system is strongly positively regulated by MrkH, a transcriptional activator that responds to the second messenger, c-di-GMP. In this study, we analyzed the mechanism by which the MrkH protein activates transcriptional initiation from the mrkA promoter. A mutational analysis supported by electrophoretic mobility shift assays demonstrated that a 12-bp palindromic sequence (the MrkH box) centered at −78.5 is the binding site of MrkH. Deletion of half a turn, but not a full turn, of DNA located between the MrkH box and the mrkA promoter destroyed the ability of MrkH to activate mrkA transcription. In addition, a 10-bp AT-rich sequence (the UP element) centered at −63.5 contributed significantly to MrkH-dependent mrkA transcription. In vivo analysis of rpoA mutants showed that the R265 and E273 determinants in the C-terminal domain of RNA polymerase α subunit are needed for MrkH-mediated activation of mrkA transcription. Furthermore, results from mutagenesis of the mrkH gene suggest that the N-terminal region of the protein is involved in transcriptional activation. Taken together, our results suggest that MrkH activates mrkA expression by interacting directly with RNA polymerase, to overcome the inefficient transcriptional initiation caused by the presence of defective core promoter elements.革兰氏阴性细菌病原菌肺炎克雷伯菌（Klebsiella pneumoniae）可形成生物被膜（biofilms），以促进其在生物与非生物表面的定殖。该菌生物被膜的形成依赖于3型菌毛（type 3 fimbriae）的表达：这类由细菌外膜分子伴侣- usher系统（chaperone-usher system）分泌的细长蛋白质丝状结构。编码该菌毛系统的mrkABCDF基因簇的表达，受转录激活因子MrkH的强力正向调控；该转录激活因子可响应第二信使（second messenger）环二鸟苷酸（c-di-GMP）。本研究解析了MrkH蛋白从mrkA启动子处激活转录起始的分子机制。结合电泳迁移率变动分析实验（electrophoretic mobility shift assays）开展的突变分析显示，一段以-78.5位为中心的12 bp回文序列（MrkH结合盒，MrkH box）为MrkH的结合位点。在MrkH结合盒与mrkA启动子之间的DNA区段中，删除半圈DNA螺旋（而非整圈螺旋）会使MrkH丧失激活mrkA转录的能力。此外，一段以-63.5位为中心的10 bp AT富集序列（UP元件，UP element）对依赖MrkH的mrkA转录具有显著促进作用。对rpoA突变体（rpoA mutants）的体内分析表明，RNA聚合酶α亚基（RNA polymerase α subunit）C端结构域中的R265与E273残基位点，是MrkH介导的mrkA转录激活所必需的。此外，mrkH基因诱变实验的结果显示，该蛋白的N端区域（N-terminal region）参与了转录激活过程。综合以上研究结果，MrkH可通过直接与RNA聚合酶相互作用，克服因缺陷型核心启动子元件（core promoter elements）存在而导致的低效转录起始，从而激活mrkA的表达。