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Plasticity of Regulation of Mannitol Phosphotransferase System Operon by CRP-cAMP Complex in Vibrio cholerae 被引量:4
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作者 ZHOU Yan Yan ZHANG Hong Zhi +3 位作者 LIANG Wei Li ZHANG Li Juan ZHU Jun KAN Biao 《Biomedical and Environmental Sciences》 SCIE CAS CSCD 2013年第10期831-840,共10页
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 (... 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 Tot 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. choleroe 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 intl. 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. choleroe, indicating an elaborate and subtle CRP activation mechanism. 展开更多
关键词 Cyclic AMP receptor protein crp-camp complex crp-binding sites Vibrio cholerae
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碳代谢总体调控蛋白CRP对肺炎克氏杆菌nif启动子的抑制作用 被引量:4
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作者 李稚婷 孙义成 +1 位作者 毛贤军 王忆平 《科学通报》 EI CAS CSCD 北大核心 2002年第15期1133-1139,共7页
在肠道细菌中,碳代谢总体调控蛋白CRP(也称为cAMP受体蛋白)通过PTS系统介导葡萄糖效应,调控σ70依赖型碳源分解代谢操纵子的转录活性。当自生(联合)固氮的肺炎克氏杆菌(Klebsiellapneumoniae)以不同的碳水化合物作为惟一碳源生长... 在肠道细菌中,碳代谢总体调控蛋白CRP(也称为cAMP受体蛋白)通过PTS系统介导葡萄糖效应,调控σ70依赖型碳源分解代谢操纵子的转录活性。当自生(联合)固氮的肺炎克氏杆菌(Klebsiellapneumoniae)以不同的碳水化合物作为惟一碳源生长时,其固氮活性明显不同,且这种不同碳源对固氮活性的影响发生在基因表达调控水平上,在近缘的大肠杆菌遗传背景下的进一步研究表明,在cAMP存在时,CRP对肺炎克氏杆菌所有的σ54依赖型nif启动子(nifB,nifE,nifF,nifH,nifJ,nifLA和nifU)都有抑制作用,序列分析结果表明,CRP的抑制作用与其在这些启动子上游的DNA顺式作用元件无直接相关性,对肺炎克氏杆菌crp基因的分离、测序结果显示,其编码的CRP蛋白与已知的大肠杆菌CRP蛋白在序列与功能上高度保守,因此认为不同碳源代谢对固氮基因的调控作用可能是由CRP-cAMP复合体介导的。这一研究工作将碳代谢调控系统和固氮调控系统通过CRP-cAMP耦联在一起,具有重要的生理学意义。 展开更多
关键词 碳代谢总体调控蛋白crp 肺炎克氏杆菌 抑制作用 crp-camp nif启动子 基因调控 固氮作用 基因转录活性
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cAMP receptor protein (CRP) downregulates Klebsiella pneumoniae nif promoters in Escherichia coli 被引量:1
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作者 Zhiting Li Yicheng Sun +1 位作者 Xianjun Mao Yiping Wang 《Chinese Science Bulletin》 SCIE EI CAS 2002年第19期1622-1628,共7页
In enteric bacteria, in response to the PTS system, the cAMP receptor protein (CRP) mediates the glucose effect, via regulating σ70-dependent catabolic genes at transcriptional level. In this study, it is observed th... In enteric bacteria, in response to the PTS system, the cAMP receptor protein (CRP) mediates the glucose effect, via regulating σ70-dependent catabolic genes at transcriptional level. In this study, it is observed that the nitrogen fixation capacity of Klebsiella pneumoniae varies strongly when cells are grown on different carbohydrates, and this carbon effect occurs at the level of nif gene expression. Here we show that CRP can repress σ54-dependent nif promoters (nifB, nifE, nifF, nifH, nifJ, nifLA and nifU), in a cAMP dependent fashion, in closed related E. coll background. Sequence analysis of these nif promoters indicates that there is no direct correlation between the fold of CRP-cAMP-mediated inhibition and the upstream cis elements at the promoters. In addition, the crp gene of K. pneumoniae has been isolated and sequenced, which is structural and functional highly homologous to that of E. coli. This suggests that CRP-cAMP-mediated inhibition on the nif promoters could be the reason for 展开更多
关键词 crp-camp nif PROMOTER regulation of gene expression NITROGEN fixation.
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Upstream CRP-binding site is not essential for CRP-cAMP-mediated inhibition on the nifU promoter 被引量:1
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作者 LI Zhiting ZHANG Weijia WANG Yiping 《Chinese Science Bulletin》 SCIE EI CAS 2002年第21期1821-1825,共5页
When the NifA-mediated activation of Kleb-siella pneumontae nifU promoter is recreated in Escherichia coli, it has been observed that CRP-cAMP has an inhibitory effect on the nifU promoter. Sequence analysis indicates... When the NifA-mediated activation of Kleb-siella pneumontae nifU promoter is recreated in Escherichia coli, it has been observed that CRP-cAMP has an inhibitory effect on the nifU promoter. Sequence analysis indicates that there is a strong CRP-binding site located upstream of the nifU promoter, overlapping completely with a previously identified Nif A-binding site. In vitro gel retardation analysis indicates that this putative CRP-binding site has similar affinity for CRP, when compared with that at the lac promoter, suggesting that CRP could effectively compete with NifA for such a binding site under physiological conditions. When this putative CRP-binding site on nifU was mutated, in vitro gel retardation analysis indicates that CRP can no longer bind to the mutant promoter. However, when constitutively expressed NifA is used as the activator, CRP-cAMP-mediated inhibitory effect on this mutant nifU promoter has no significant difference when compared with that obtained from its wild-type promoter. 展开更多
关键词 crp-camp the nifU PROMOTER gene regulation.
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碳代谢总体调控蛋白CRP对nifU启动子的抑制作用不依赖于该启动子上游CRP与NifA竞争的靶位点
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作者 李稚婷 张维佳 王忆平 《科学通报》 EI CAS CSCD 北大核心 2002年第16期1242-1246,共5页
在大肠杆菌中,碳代谢总体调控蛋白CRP抑制肺炎克氏杆菌nifU启动子的表达.序列分析表明,nifU启动子上游有一个CRP靶位点,且该位点与已知的NifA靶位点重合.体外凝胶阻滞实验结果表明,CRP对该位点的亲和性与对乳糖操纵子的CRP靶位点相似,... 在大肠杆菌中,碳代谢总体调控蛋白CRP抑制肺炎克氏杆菌nifU启动子的表达.序列分析表明,nifU启动子上游有一个CRP靶位点,且该位点与已知的NifA靶位点重合.体外凝胶阻滞实验结果表明,CRP对该位点的亲和性与对乳糖操纵子的CRP靶位点相似,说明在生理条件下,CRP可与NifA激活蛋白竞争该靶位点.该位点突变的体外凝胶阻滞实验结果表明,CRP不再与突变的nifU启动子结合.体内活性检测结果显示,在组成性表达NifA蛋白的激活下,CRP对上游CRP靶位点突变的nifU启动子依然有抑制作用,其程度与野生型启动子无明显差异.因此,CRP对nifU启动子的抑制作用不依赖于该启动子上游特异的CRP靶位点,暗示CRP与Eσ54RNA聚合酶间的直接相互作用在该抑制效应中起着主要作用. 展开更多
关键词 碳代谢总体调控蛋白crp 抑制作用 靶位点 crp-camp nifU启动子 基因调控 肺炎克氏杆菌 抑制机理
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