大腸桿菌轉錄因子CRP參與多重內部二次輸入節點前饋迴路控制系統之研究

Abstract

環單磷酸腺苷受體蛋白(cyclic adenosine monophosphate receptor protein；CRP) 是大腸桿菌中重要的轉錄因子，已知調控超過400個基因。前饋迴路控制系統（Feedforward loop; FFL）包含輸入（input node; X）、內部二次輸入（internal-secondary input node; Y）及輸出節點（output node; Z）共三角色。當X與Z之間受多個Y所調控，即為多重內部二次輸入節點前饋迴路控制系統（multi-Y FFL，MY）。已知大腸桿菌中有超過三分之二的轉錄因子（Transcription factors）會因特定環境或條件下表現或活化，並可能透過FFL執行不同基因之調控，但針對MY與基因調控關係的研究目前仍十分欠缺。目前資料庫中含有許多尚未被驗證之調控關係，若能進行驗證，有高度機率會形成MY。針對MY的文獻，僅停留在基礎理論，並無研究其生理功能及證實其存在與否。本研究利用EcoCyc資料庫，將其中之基因調控關係串聯成87組MY，並進一步驗證新發現含有三轉錄因子之MY：CRP-TFs-gltB。本研究首度證實MY確實存在於大腸桿菌中，可作為不同環境下基因表現緩衝系統。經過統計發現，在不同MY組態中，以I1出現頻率最高。統計結果也發現，大腸桿菌中之MY組態具備多樣性，最多可透過四個轉錄因子（包含CRP）形成MY進行基因控制，顯示大腸桿菌調控網絡的複雜度與多元性。

Cyclic adenosine monophosphate receptor protein (CRP) is a regulatory protein in bacteria. It is an important transcription factor, and it regulated more than 400 genes in E. coli. In 2004, Kashtan et. al. defined feed forward loop control systems (FFL). It contains three roles: input node (X), internal-secondary input node (Y), and output node (Z). There are two or more Y regulated to X and Z, which called "multi-Y FFL (MY).” It has been known over two-thirds of transcription factors can activate or repress genes by FFL in the special environments or conditions. Between there were few MY studies and related regulation researches reported in E. coli. There is no research for physiological function and confirmation of MY up to date. In this study, we analyzed 230 single FFL from the Ecocyc and Regulon DB database, and we further linked 87 groups of MY systems in E. coli. The functions are associated with biofilm formation and carbohydrate transport in C1 group. The functions are associated with glutamate metabolic process, cellular monovalent inorganic cation homeostasis and pH regulation in C2 group. Also, the functions are associated with L-glutamate biosynthetic process and deoxyribonucleotide catabolic process in C3 group. In C4 group, the functions are associated with organic substance transport. In I1 group, the functions are associated with carbohydrate catabolic process, localization and aerobic electron transport chain. In I2 group, the functions are associated with cellular homeostasis and chemical homeostasis in I2 group. In I4 group, the functions are associated with carbohydrate transport and metabolic process in I4 group. After statistics, we find that ‘C1+I1’ and ‘I1+I1+I4’ are the highest frequency to appear in different types of MY, and the gene’s functions are including iron ion homeostasis, cellular metabolic process and transport. In addition, we find the new MY regulated by three transcription factors (CRP-GadE and Fur - gltB) and MY has physiological function. Therefore, we confirm the presence of this MY in this study, and it can be a buffer system in different environments. In addition, the types of MY are diversity in E. coli by statistics. Genes regulated by the four TFs of MY (including CRP) in E. coli, and the networks of gene regulation are complicate and diversified in E. coli.