大腸桿菌Thioredoxin C32, P34與G92點突變對噬菌體感染之分析

Abstract

E. coli thioredoxin在E. coli中除了參與本身生理的氧化還原反應以外, 同時也可幫助噬菌體T7､f1､M13的生長。在噬菌體T7中, thioredoxin為T7 DNA polymerase的次單元(subunit), 可提高DNA複製時的延展性(processivity); 在線形噬菌體f1､M13中, thioredoxin參與噬菌體組裝子代(assembly)的過程。本實驗中, 我們分別在thioredoxin C32､P34､G92位置建構了18隻E. coli thioredoxin突變株, 分析它們對於噬菌體T3/T7､f1､M13生長的影響, 來探討thioredoxin 突變的胺基酸位置, 在與T7 gene 5蛋白或與f1 gene I蛋白交互作用時的情形。本實驗設計在pET-32c-stop質體上進行thioredoixn基因的定點突變, 再將質體送入不會產生thioredoxin蛋白的E. coli菌株SK3967及A179(1-3)中, 分別進行噬菌體T3/T7及f1､M13的感染實驗, 由所得的實驗結果：感染平盤效率(E.O.P)做進一步的分析。我們推測thioredoxin蛋白無論與T7 gene 5蛋白或與f1 gene I蛋白交互作用時, 其胺基酸Gly92位置應是重要的接觸點, 尤其在噬菌體f1感染實驗中, 發現任何胺基酸皆無法取代Glycine; 與f1只有些微差異的噬菌體M13只勉強可以在G92A突變株中生長, 而在T3/T7感染實驗中, G92A突變株可以使T3/T7在37℃中正常生長, 以及G92S突變株可稍微支持T3/T7生長以外, 其他G92突變株同樣皆無法支持噬菌體T3/T7生長。至於thioredoxin活性中心的C32､P34位置雖然包容性較大, 仍可置換少數幾個胺基酸, 但胺基酸的side chain不宜過大, 且32位置應以極性胺基酸, 34位置應維持疏水性胺基酸較佳。另外我們也發現有些thioredoxin突變株因為培養方式的不同, 使得菌株生長時間變長, 導致噬菌體感染的E.O.P上升, 推測應是thioredoxin的表現量因生長速度變慢而增加, 所以thioredoxin突變株抑制噬菌體生長的效果減弱。

E.coli thioredoxin is a general disulfide reductant in E. coli and is also required for phage growth. For phage T7, thioredoxin is an essential subunit of phage T7 DNA polymerase. For phage f1 and M13, thioredoxin participates in the assembly of filamentous phage. In this study, we used site-directed mutagenesis to construct thioredoxin mutants at residues G92, P34, C32. Eighteen thioredoxin mutants were produced and were inserted into pET-32c-stop. The plasmids contain trxA mutant gene were transformed into E. coli SK3967 and A179(1-3) for phage infection. The residue Glycine 92 of thioredoxin is more important for interaction of thioredoxin with T7 gene 5 protein or f1 gene I protein then residues Proline 34 or Cysteine 32. In phage infection, any thioredoxin G92 mutant can’t support f1 growth, whereas only G92A mutant and its double mutants can support T3/T7 growth like wild-type thioredoxin at 37℃. Residues 32, 34 of thioredoxin could be replaced to the smaller residues. Residue 32 is suit for hydrophilic and residue 34 should be hydrophobic. We found that culture E. coli in TA broth or incubator made the generation time longer than culture in LA broth or shaker and the efficiency of plating of phage rose because the expression of thioredoxin increased.