大腸桿菌內調控轉錄因子CRP新發現之前饋式調控迴路研究

Abstract

中文摘要 環磷酸腺苷受體蛋白(cAMP receptor protein; CRP)是大腸桿菌內一種重要的全面性轉錄因子，CRP已知可調控超過400個基因，CRP也能藉由其他轉錄因子形成前饋式調控迴路(feed forward loops，FFLs)。根據CRP與其他轉錄因子及目標基因之間的調控關係FFLs可以被分成八種形式。FFLs對大腸桿菌適應環境中的變化有很大貢獻，例如先前的FFLs相關研究中發現coherent type 1 FFLs具有延遲回應環境訊息的功能，而incoherent type 1 FFLs則具有加速基因表現至最大量的功能。 先前研究新發現了37個受CRP調控轉錄因子，在本次研究中將此新發現受CRP調控轉錄因子的資料，利用資料庫與文獻探查，用生物資訊CRP結合序列預測工具和EMSA與real-time PCR實驗更新後，共發現有39個新受CRP調控轉錄因子。這些新的39個受CRP調控轉錄因子總共可調控663個基因，其中有176個為已知受CRP調控的基因，利用microarray進行篩選後發現在176個基因中有144個基因受CRP顯著的調控。資料庫中未記錄受CRP調控的基因中利用生物資訊工具進行篩選會有226個基因(包含在120個操縱子中)可能受CRP調控，透過EMSA與real-time PCR驗證後，這些基因中有109個(包含在69個操縱子中)會受CRP顯著調控且能與CRP直接結合並形成371個新發現受CRP調控的轉錄因子的CRP FFLs。將CRP FFLs根據不同形式進行基因功能分析(GO term analysis)，了解不同形式的CRP FFLs在大腸桿菌中的相關生物功能，並與先前研究中已知受CRP調控的轉錄因子形成的CRP FFLs的結果作比較，發現在新擴增的CRP FFLs中有之前的資料中沒有的新的生物功能，例如coherent type 1 CRP FFLs不只與碳水化合物的運輸和代謝與細菌型鞭毛依賴性的細胞運動肉鹼(carnitine) 的代謝有關等功能有關，也與肉乳糖(galactarate)與醛糖二酸(aldaric acid)的代謝有關。而incoherent type 1 CRP FFLs不只與單生物體(single-organism)的運輸、碳水化合物的代謝、有機羥基化合物(organic hydroxy compound)的代謝、次級代謝產物(secondary metabolite)的生物合成與蛋白質、肽、酰胺的運輸有關，也與細胞呼吸、無氧呼吸與回應生物外源性刺激等功能有關。同時也發現有些生物功能會與多種CRP FFLs有關，可能是因為這些生物功能與大腸桿菌的生存有重要關係，因此需要多種調控模式可在不同的生存條件下也能正常運作。例如coherent type 1 CRP FFLs、coherent type 4 CRP FFLs與incoherent type 1 CRP FFLs都會與細胞呼吸有關。而在multi-node CRP FFLs分析中發現在可以形成CRP FFLs的基因中，約有98.14%的基因可以形成multi-output FFLs，此結果與先前的研究是一致。約有41.53%的基因可以形成multi-Y FFLs，且這些基因也都能形成multi-output FFLs。根據結果可以發現在multi-Y FFLs中隨著Y的數目的增加，FFLs的數目會越少，而在本次研究中發現的multi-Y FFLs中Y的數目最多為8個。根據研究的結果在multi-Y FFLs中雖然只有2-Y CRP FFLs與3-Y CRP FFLs可以找到有特定的生物功能，它們大多數的生物功能都與能量產生有關，例如細胞呼吸、無氧呼吸、磷酸依賴性糖磷酸轉移酵素系統和三羧酸循環等。此外有最多Y的CRP FFLs會與大腸桿菌的酸耐受系統有關，表示此生物功能可能是大腸桿菌中最重要的功能，因此需要最複雜的調控模式進行調控，可在各種環境中都能正常運作。 為了解CRP在大腸桿菌生理功能中的調控，以大腸桿菌的阿拉伯醣源代謝系統進行研究，利用counter selection的實驗將大腸桿菌中的araC上的CRP結合位突變，並將此建立好的突變菌株與野生菌株(MG1655)作cAMP添加實驗，分析在不同濃度的cAMP下目標基因araB與轉錄因子araC的表現變化，從實驗中可以得知CRP在大腸桿菌的阿拉伯醣源代謝系統中為主要的調控因子，CRP可以調控轉錄因子AraC與araBAD operon結合的形式，以控制araBAD的活化或抑制。並藉由調控此碳源代謝系統，在多種碳源存在下優先選擇特定碳源來利用。

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.