Plasticity of regulation of mannitol phosphotransferase system operon by CRP-cAMP complex in Vibrio cholerae.
Plasticity of regulation of mannitol phosphotransferase system operon by CRP-cAMP complex in Vibrio cholerae.
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DOI：
10.3967/bes2013.006
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发表时间：
2013-10
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影响因子：
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通讯作者：
Yan Zhou;Hong zhi Zhang;W. Liang;Li Zhang;Jun Zhu;B. Kan
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文献类型：
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作者：
Yan Zhou;Hong zhi Zhang;W. Liang;Li Zhang;Jun Zhu;B. Kan
目的环腺苷酸受体蛋白（CRP）与环腺苷酸（cAMP）的复合物是原核生物中许多基因的重要转录调控因子。甘露醇通过磷酸转移酶系统（PTS）的转运受CRP-cAMP复合物调节。本研究的目的是研究CRP-cAMP复合物如何作用于霍乱弧菌El Tor生物型的甘露醇PTS操纵子mtl。方法通过同源重组产生CRP突变株，以评估CRP激活胆固醇弧菌El Tor的甘露醇PTS操纵子的需要。用电泳迁移率变动分析（EMSA）和报告质粒pBBRlux证实CRP-cAMP复合物对甘露醇PTS操纵子mtl的作用。结果在本研究中，我们证实了CRP是mtl操纵子激活所必需的。我们进一步通过实验鉴定了霍乱弧菌El Tor生物型的甘露醇PTS操纵子mtl上游启动子区域内的5个CRP结合位点，并发现这些位点显示出对CRP的不同亲和力，并对操纵子的激活提供不同的贡献。结论5个结合位点共同赋予CRP对甘露醇转运的强烈激活作用，提示CRP激活甘露醇的机制复杂而微妙。
OBJECTIVE The complex of the cyclic AMP receptor protein (CRP) and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems (PTS) is regulated by the CRP-cAMP complex. The aim of the study is to investigate how the CRP-cAMP complex acting on the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype. METHODS The crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. cholerae El Tor. Electrophoretic mobility shift assays (EMSA) and the reporter plasmid pBBRlux were used to confirm the role that the CRP-cAMP complex playing on the mannitol PTS operon mtl. RESULTS In this study, we confirmed that CRP is strictly needed for the activation of the mtl operon. We further experimentally identified five CRP binding sites within the promoter region upstream of the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype and found that these sites display different affinities for CRP and provide different contributions to the activation of the operon. CONCLUSION The five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. cholerae, indicating an elaborate and subtle CRP activation mechanism.