标题: | 大肠杆菌内调控转录因子CRP新发现之前馈式调控回路研究 Identification of new CRP feed-forward loops in Escherichia coli |
作者: | 林政宪 曾庆平 分子医学与生物工程研究所 |
关键字: | 环腺苷磷酸受体蛋白;基因调控网路模组;前馈式回路;阿拉伯糖代谢系统;cyclic-AMP receptor protein (CRP);regulatory motif;feed-forward loop (FFL);arabinose metabolism system. |
公开日期: | 2016 |
摘要: | 中文摘要 环磷酸腺苷受体蛋白(cAMP receptor protein; CRP)是大肠杆菌内一种重要的全面性转录因子,CRP已知可调控超过400个基因,CRP也能藉由其他转录因子形成前馈式调控回路(feed forward loops,FFLs)。根据CRP与其他转录因子及目标基因之间的调控关系FFLs可以被分成八种形式。FFLs对大肠杆菌适应环境中的变化有很大贡献,例如先前的FFLs相关研究中发现coherent type 1 FFLs具有延迟回应环境讯息的功能,而incoherent type 1 FFLs则具有加速基因表现至最大量的功能。 先前研究新发现了37个受CRP调控转录因子,在本次研究中将此新发现受CRP调控转录因子的资料,利用资料库与文献探查,用生物资讯CRP结合序列预测工具和EMSA与real-time PCR实验更新后,共发现有39个新受CRP调控转录因子。这些新的39个受CRP调控转录因子总共可调控663个基因,其中有176个为已知受CRP调控的基因,利用microarray进行筛选后发现在176个基因中有144个基因受CRP显着的调控。资料库中未记录受CRP调控的基因中利用生物资讯工具进行筛选会有226个基因(包含在120个操纵子中)可能受CRP调控,透过EMSA与real-time PCR验证后,这些基因中有109个(包含在69个操纵子中)会受CRP显着调控且能与CRP直接结合并形成371个新发现受CRP调控的转录因子的CRP FFLs。将CRP FFLs根据不同形式进行基因功能分析(GO term analysis),了解不同形式的CRP FFLs在大肠杆菌中的相关生物功能,并与先前研究中已知受CRP调控的转录因子形成的CRP FFLs的结果作比较,发现在新扩增的CRP FFLs中有之前的资料中没有的新的生物功能,例如coherent type 1 CRP FFLs不只与碳水化合物的运输和代谢与细菌型鞭毛依赖性的细胞运动肉硷(carnitine) 的代谢有关等功能有关,也与肉乳糖(galactarate)与醛糖二酸(aldaric acid)的代谢有关。而incoherent type 1 CRP FFLs不只与单生物体(single-organism)的运输、碳水化合物的代谢、有机羟基化合物(organic hydroxy compound)的代谢、次级代谢产物(secondary metabolite)的生物合成与蛋白质、肽、酰胺的运输有关,也与细胞呼吸、无氧呼吸与回应生物外源性刺激等功能有关。同时也发现有些生物功能会与多种CRP FFLs有关,可能是因为这些生物功能与大肠杆菌的生存有重要关系,因此需要多种调控模式可在不同的生存条件下也能正常运作。例如coherent type 1 CRP FFLs、coherent type 4 CRP FFLs与incoherent type 1 CRP FFLs都会与细胞呼吸有关。而在multi-node CRP FFLs分析中发现在可以形成CRP FFLs的基因中,约有98.14%的基因可以形成multi-output FFLs,此结果与先前的研究是一致。约有41.53%的基因可以形成multi-Y FFLs,且这些基因也都能形成multi-output FFLs。根据结果可以发现在multi-Y FFLs中随着Y的数目的增加,FFLs的数目会越少,而在本次研究中发现的multi-Y FFLs中Y的数目最多为8个。根据研究的结果在multi-Y FFLs中虽然只有2-Y CRP FFLs与3-Y CRP FFLs可以找到有特定的生物功能,它们大多数的生物功能都与能量产生有关,例如细胞呼吸、无氧呼吸、磷酸依赖性糖磷酸转移酵素系统和三羧酸循环等。此外有最多Y的CRP FFLs会与大肠杆菌的酸耐受系统有关,表示此生物功能可能是大肠杆菌中最重要的功能,因此需要最复杂的调控模式进行调控,可在各种环境中都能正常运作。 为了解CRP在大肠杆菌生理功能中的调控,以大肠杆菌的阿拉伯醣源代谢系统进行研究,利用counter selection的实验将大肠杆菌中的araC上的CRP结合位突变,并将此建立好的突变菌株与野生菌株(MG1655)作cAMP添加实验,分析在不同浓度的cAMP下目标基因araB与转录因子araC的表现变化,从实验中可以得知CRP在大肠杆菌的阿拉伯醣源代谢系统中为主要的调控因子,CRP可以调控转录因子AraC与araBAD operon结合的形式,以控制araBAD的活化或抑制。并藉由调控此碳源代谢系统,在多种碳源存在下优先选择特定碳源来利用。 Abstract Cyclic–AMP receptor protein (CRP) is one of the most important global transcription factors (TFs) in Escherichia coli. It can regulate other transcription factors to form the regulatory motif called feed forward loops (FFLs). FFLs are classified into eight types of the different regulatory relationships of CRP with nother transcription factors, and target genes. In the previous study, we used the bioinformatics method to select the candidates and find 37 novel CRP-regulated transcription factors. In this study, we updated the information of CRP-regulated transcription factors and found 39 novel CRP-regulated transcription factors. We then used the same method to find 226 novel CRP-regulated genes in 120 operons regulated by these 39 CRP-regulated transcription factors. We used electrophoretic mobility shift assay (EMSA) and real-time PCR (Q-PCR) to confirm the regulatory relationships of each gene. The total 109 novel CRP-regulated genes were identified. The distributions of total CRP FFLs in this study were including 120 coherent type 1 FFLs, 101 coherent type 2 FFLs, 71 coherent type 3 FFLs, 108 coherent type 4 FFLs, 213 incoherent type 1 FFLs, 65 incoherent type 2 FFLs, 53 incoherent type 3 FFLs, and 89 incoherent type 4 FFLs. These results indicated that the distributions of CRP FFLs were not even. The coherent type 1 and incoherent type 1 were the most type of CRP FFLs. The distributions of the coherent type 1, coherent type 2, and coherent type 4 FFLs were more than before. We compared the findings in this study with the results of database and known CRP-regulated TFs mediated FFLs in the previous study. We found some novel biology function of CRP FFLs, for example the coherent type 1 CRP FFLs is not only associated with bacterial-type flagellum-dependent cell motility, carbohydrate transport and metabolic process, but also associated with, galactarate catabolic process and aldaric acid metabolic process. The incoherent type 1 CRP FFLs is not only associated with single-organism transport, organic hydroxy compound metabolic process, secondary metabolite biosynthetic process,carbohydrate catabolic process, and protein, peptide, amide transport but also associated with aerobic respiration, anaerobic respiration, and response to xenobiotic stimulus. However, we did not find the novel biology function of incoherent type 2 CRP FFLs in this study. We also found some biology function that can be associated with more than one type of CRP FFLs. For example, the function of cellular respiration is associated with coherent type 1 CRP FFLs, coherent type 4 CRP FFLs, and incoherent type 1 CRP FFLs. These function may be important for the surviving of E. coli and they need more than one regulatory motif to control in different condition. In the results of multi-node CRP FFLs analysis, we found that about 98.14% genes forming CRP FFLs formed multi-output FFLs in this study. This result is consistent with the previous study and that 41.53% genes forming CRP FFLs formed multi-Y FFLs and these genes can also form multi-output FFLs. The most number of Y in multi-Y FFLs was 8 and the numbers of genes were fewer as the numbers of Y were more. In the gene ontology enrichment analysis, we only found the significant biology function of 2-Y CRP FFLs and 3-Y CRP FFLs. The majority of these biology function was associated with energy production such as cellular respiration, anaerobic respiration, phosphoenolpyruvate-dependent sugar phosphotransferase system, energy derivation by oxidation of organic compounds, oxidation-reduction process, and tricarboxylic acid cycle. The genes of multi-Y FFLs with the most Y were associated with acid resistance in E. coli. These results suggest that the function of acid resistance was the most important function in E. coli which need most complex regulatory motif to regulate in various conditions. Finally, we analysis the CRP regulation in E. coli arabinose metabolic system with the expression profile of target gene araB and transcription factor(TF) araC in cAMP dose response experiment between wildtype strain (MG1655) and CRP binding site of TF mutation strain(araC*) we constructed. We found that CRP was the key regulator in arabinose metabolism system. CRP can regulate the DNA-binding between araC regulator and araBAD operon and caused the activation or inhibition of araBAD to selectively utilize the substrates from a mixture of different carbon sources. |
URI: | http://etd.lib.nctu.edu.tw/cdrfb3/record/nctu/#GT070357106 http://hdl.handle.net/11536/139633 |
显示于类别: | Thesis |