第五章 大腸桿菌 gspS 基因表現之調控
5.3.2 影響 gspS 表現的因素
已知以生物晶片分析時，BaeR 過量表現可誘發 gspS 的表現增強為原來的 13 倍(Nishino et al., 2005)，且 BaeR 可能對 envelope 氧還狀態的破壞有反應，進
而引發下游基因表現。
據此，將HY1002 培養於 37℃，待生長至對數生長期後，分別加入 H2O2 0 mM、1.0 mM、3.0 mM，在 30 分鐘、一小時及兩小時，分別取樣測 β-galactosidase 活性及菌液濃度OD600 。未加入H2O2前活性為70 units，加入 H2O2 1.0 mM 兩小 時後活性上升至268 units，表示 gspS-lacZ 可受 H2O2誘導。加入H2O2 3.0 mM 之 後，OD600由30 分鐘的 0.292 下降至兩小時的 0.198，表示 3 mM H2O2已超過細 菌的耐受範圍，造成細菌死亡。
為了更瞭解H2O2濃度與gspS-lacZ 表現之間的關係，將 HY1002 培養於 37℃，待生長至對數生長期後，分別加入 0、0.1、0.3、0.5、1.0 mM H2O2，在1 hr、2 hr 及 3 hr 後取樣分析 β-galactosidase 活性，結果如圖二十四。在 0 至 1.0 mM H2O2的濃度範圍內，三小時後活性都會上升，且H2O2濃度越高培養時間越長，
對gspS-lacZ 的誘導效果愈好。
(b)BaeR 對 gspS 的影響
生物晶片分析時，BaeR 過量表現可誘發 gspS 的表現增強，其為 in vitro 之 實驗數據，為瞭解在E. coli 中，BaeR 是否可誘導 gspS 表現，將 baeR 以 PCR 大 量複製，再與pBAD24 接合，得新載體 pBAD24-baeR，將 pBAD24-baeR 分別送 入HY1001，HY1002 及 HY1003，將 HY1001/pBAD24-baeR，
HY1002/pBAD24-baeR 及 HY1003/pBAD24-baeR 分別培養於 LB 及 LB/ 1%
arabinose，待生長至對數期後，取樣分析 β-galactosidase 活性 (表九)，
HY1001/pBAD24-baeR，HY1002/pBAD24-baeR 及 HY1003/pBAD24-baeR，以 arabinose 誘導使 BaeR 大量表現後，gspS-lacZ 的 β-galactosidase 活性均顯著升高，
由以上結果顯示，在E. coli 菌體內，大量的 BaeR 可以誘導 gspS 的表現，且活 性皆增加十倍以上。
另外，將可受 IPTG 誘導的載體 pJP24-baeR（from E.coli Strain National BioResource Project）送入 HY1002，得 HY1002/pJP24-baeR，將此菌培養於 37℃
至對數生長期後，加入1mM IPTG 誘導 pJP24-baeR，兩小時後取樣分析
β-galactosidase 活性，未以 IPTG 誘導者活性為 44 units，以 IPTG 誘導後活性升 至569 units，此結果亦符合前述，大量的 BaeR 可以誘導 gspS 的表現，且活性皆 增加十倍以上。
(c) BaeR 與 H2O2對gspS 的影響
為瞭解BaeR 是否參與 H2O2對gspS 的誘導，將 HY1002 及
HY1002/pJP24-baeR 兩菌培養至對數生長期後，分別加入 1 mM IPTG、1 mM H2O2
或1mM IPTG/ 1 mM H2O2，兩小時後取樣分析β-galactosidase 活性 (表十)，以 1 mM IPTG 培養前後，HY1002 的活性無顯著變化，HY1002/ pJP24-baeR 的活性 則由54 units 升至 522 units，此結果與前述(大量的 BaeR 可以誘導 gspS 的表現) 相符。以1 mM H2O2培養，HY1002 的活性由 94 units 上升至 348 units，亦符合 前述H2O2可誘導gspS-lacZ 的結果，但在 HY1002/pJP24-baeR，以 1 mM H2O2
培養後的活性由54 units 微升至 66 units，表示 HY1002 帶有 pJP24-baeR 時，以 H2O2培養兩小時無法誘導gspS-lacZ。
與加入 1 mM IPTG 比較，加入 1 mM IPTG/ 1 mM H2O2培養時，HY1002 的 活性由74 units 升為 222 units，OD600由1.110 降至 0.366，HY1002/pJP24-baeR 的活性由522 units 升為 586 units，OD600由0.524 降至 0.393，以上結果可知，已 有IPTG 使 pJP-baeR 大量表現而誘導 gspS-lacZ 時，再加上 H2O2，gspS-lacZ 活 性增加的幅度小，OD600降低的幅度也較小，顯示大量的BaeR 可能會減低 H2O2
對gspS-lacZ 的影響，且對細菌具有保護的效果。
為更進一步瞭解 BaeR、H2O2與gspS-lacZ 表現及細菌生長之間的關係，將 HY1002 與 HY1002/pJP24-baeR 兩菌培養於 37℃，待生長至對數生長期後，分別 加入1 mM IPTG 或 1 mM IPTG/ 1.5 mM H2O2，在培養期間每隔一小時取樣分析 菌液濃度，結果如圖二十五(A)(B)。
在觀察細菌生長後發現，HY1002 只加入 IPTG 後一小時，OD600由0.571 上 升至1.020，並且在實驗過程中持續上升正常生長，在加入 IPTG 與 H2O2後一小 時，OD600由0.556 增為 0.611，細菌生長極緩並且漸漸死亡。HY1002/pJP24-baeR
只加入IPTG 時，生長狀況亦正常，在加入 IPTG 與 H2O2的組別中，一小時後 OD600由0.446 增至 0.554，此組別生長雖較緩，但仍持續生長，細菌不會死亡，
由圖二十五(A)(B)可看出兩者顯著的差異。以上結果可確切證實，當 BaeR 被大 量表現時，具有保護作用。
為更加確認以上結果，將 HY1002 及 HY1002/pJP24-baeR 兩菌培養於 37℃，
待生長至對數生長期後，分別加入1.5 mM H2O2，在培養期間每隔一小時取樣偵 測菌液濃度OD600，結果如圖二十六(A)(B)。HY1002 受 H2O2影響，兩小時後 OD600由0.957 下降至 0.578，細菌大量死亡，其結果與前述實驗相符，在 HY1002/
pJP24-baeR，因有 BaeR 之存在，使細菌濃度大致持平無顯著死亡，在 H2O2加 入後兩小時，gspS-lacZ 表現開始明顯上升，使細菌較不受 H2O2影響而死亡，其 結果亦與前述實驗相符。
由以上實驗可知，gspS 可受過氧化物 H2O2誘導而大量表現，細菌內若有 BaeR，亦可使 gspS 表現大幅增加，且 BaeR 誘導 gspS 表現具有保護細菌不受 H2O2影響導致死亡的效果。
5.4 討論
細菌藉由將GSH 維持在還原態，以還原其他過氧化物來維持體內氧化還原 的平衡，多胺類如spermidine 等，可刺激調控蛋白生成，進而影響許多與生長或 抗氧化相關基因的表現，而GspdSH 的作用可能是調節 GSH 或 spermidine 含量。
gspS 可轉譯的蛋白為 glutathionylspermidine synthetase 及 amidase，其基因表現 應與細胞內GSH，spermidine 及 GspdSH 的平衡相關。 BaeR 可能受細胞膜的改 變或envelope 氧還狀態的破壞影響，進而引發下游基因表現，本研究中，大量 的BaeR 可誘導 gspS 表現增加十倍至十三倍。
Nishino 等人在 2005 年利用生物晶片分析 BaeR 可調控的基因時，經序列比 對的方式，分析出具有高度保守性的序列（highly conserved sequence）TTTTT
CTCCA TDATT GGC，稱之為 BaeR binding motif，並將 BaeR 可調控的基因中，
其上游具有BaeR binding motif 的基因稱之為 BaeR regulon (Nishino et al.,
2005)，這些 BaeR regulon 基因的上游區域中，與此 BaeR binding motif 相似度最 低為50%，但 Nishino 等人並未將 gspS 列入 BaeR regulon。另外，Hirakawa 等人 經實驗所得之BaeR 與啟動子結合區與 Nishino 等人以比對方式所得的 BaeR binding motif 序列稍有不同(Hirakawa et al., 2005)。
若以gspS start codon 的上游 300 bp 與 BaeR binding motif，以軟體 Bioedit 比對，在gspS start codon 上游 198 至 210 bp 處預測出 BaeR binding site 存在，與 Nishino 等人發表之 BaeR binding motif 相似度達 50%。以 gspS start codon 的上游 300 bp 與 Hirakawa 等人經實驗所得之 BaeR 與啟動子結合區比對，亦可得兩處 可能與BaeR 結合的區域，因此，gspS 上游區域中，可能有 BaeR binding site 存 在。
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