未來需製作 yeeN 及 yebC 同時突變之 double mutants 突變株，進一步確認 大腸桿菌中 DUF 28 基因在生長、氧化及抗生素逆境上有何影響。
針對不同菌種中 DUF28 蛋白質之功能可利用 heterologous complementation 進行跨菌種或跨物種間表現基因，進一步推測有 DUF28 蛋白質的菌種或物種，
其 DUF28 蛋白質是否與大腸桿菌中的 DUF28 蛋白質有相似 ( 參與調節氧化逆 境 ) 的功能。
大腸桿菌 yeeN 基因參與調節氧化逆境，檢測 YeeN 蛋白質於氧化逆境之表 現量，驗證細胞處於逆境中會促使 YeeN 蛋白質聚集。對於 yeeN 可能參與之調 控路徑，可利用即時定量 RT-qPCR 檢測氧化逆境及調節氫氧根離子相關基因表現，
推測其調節氧化逆境之相關機制，並針對氧化磷酸化相關基因進一步做測試。可 藉由 relA 之互補株測定其中 yeeN 之表現量驗證 relA 是否直接調節 yeeN。對於 ppGpp 是否與參與調節 yeeN 基因表現可利用測定細胞內在正常環境下之 ppGpp 的含量，並藉由添加 ppGpp 檢測 yeeN 基因表現量有何變化，進一步找出 ppGpp 與 yeeN 基因調節上有何關聯。
對於其他可能位於上游調節之基因突變株參與泳動力及氧化逆境之調節，可 進行更多功能性的研究做進一步探討。
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50
續表一
δ subdivion 11/17 1 ~ 2
ε subdivion 7/20 0 ~1
γ subdivion Enterobacteriales 14/49 0 ~2 Aeromonadales 1/2 1 Alteromonadales 7/23 1 ~ 2 Cardiobacteriales 1/1 1 Chromatiales 3/3 1 Legionellales 2/4 1 Methylococcales 1/1 2 Oceanospirillales 4/4 1 ~ 2 Pasteurellales 4/11 1 Pseudomonadales 3/19 1 ~2 Thiotrichales 2/9 1 Vibrionales 2/8 1 ~ 2 Xanthomonadales 2/10 1 Spirochaetes Spirochaetales 3/12 1 Thermotogae Thermotogales 3/6 1
利用 PEC (Prolifling of E. coli chromosome)生物資訊網站(http://tinyurl.com/2cp8sh3) 進行分析。a. 本表中所分析同一目的菌種 / 菌系個數。b. 在該目的菌種中 DUF28 同源蛋白的個數。
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表二、搜尋微陣列資料庫中大腸桿菌 yebC 及 yeeN 於不同逆境處理之基因表現量
Treatment / Experiment
Expression level a
(fold increase or decrease)
Referencesc
yebC yeeN
kanamycin or w/ spectinomycin 2.46 3.55 (1) ampicillin or w/ spectinomycin 2.45 2.23 ~ 3.83 (1)
norfloxacin -2.17 ~ 2.46 2.43 ~ 16.17 (1)，(2)，(3) norfloxacin or w/ spectinomycin --- b 19.66 ~ 35.99 (1)
ofloxacin --- 7.89 (4)
novobiocin --- 15.77 ~ 17.28 (2)
rifampin --- -2.00 (3)
0.1MPa vs 30MPa --- -3.70 (5) 37℃ vs 28℃or 37℃ vs 33℃ 2.04 -5.78~ -2.22 (6) hydrogen peroxide (H2O2) --- -2.73 ~ -2.35 (7) No UV vs UV 2.14 ~ 2.67 --- (8)
a. yebC 或 yeeN 基因表現量在處理逆境後之改變倍率。
b. --- 表示在該文獻中 yebC 或 yeeN 基因表現量並未有兩倍以上或以下之變化。採 用微陣列資料庫(NCBI 及 Ecogene database http://ecogene.org/EcoarrayStat.php)搜 尋並分析。
c. references 以編號代表內容如下，
(1) Kohanski M.A., et al.,2007 (2) Peter B.J., et al., 2004 (3) Shaw K.J., et al., 2003 (4) Kaldalu N., et al., 2004 (5) Ishii A., et al., 2005 (6) Gadgil M., et al., 2005 (7) Zheng M., et al., 2001 (8) Courcelle J., et al., 2001。
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表三、生物資訊搜尋微陣列資料庫中大腸桿菌 yebC 及 yeeN 之基因表現量
a. yebC 或 yeeN 基因在該突變株中表現量改變之倍率。
b. 在該突變株中 yebC 或 yeeN 基因表現量有兩倍以上之變化。
Experiment Gene description Fold of expression change a
Ref.d
yebC yeeN Purine metabolism
ΔrelA ppGpp synthetase I/GTP pyrophosphokinase
-7.9~26 (1)，(2) ΔrelAspoT bifunctional (p)ppGpp synthetase II/
pyrophosphohydrolase
2.14 -3.1 (1)，(3) Homologous recombination
ΔrecA (also stress related)
DNA strand exchange and recombination protein
Two component systems
ΔglnG fused DNA-binding response regulator in two-component regulatory system with GlnL
2.1 (7)
ΔbaeR DNA-binding response regulator in two-component regulatory system with BaeS
15 (8)
Unknown
ΔnorR DNA-binding transcriptional activator
-2.2 (9) Δhns global DNA-binding transcriptional
dual regulator H-NS
6.3 (10)
ΔgyrB DNA gyrase, subunit B 2.7~ 4.7 (11) Δfur DNA-binding transcriptional dual
regulator of siderophore biosynthesis and transport
2. ~ 2.3 (12)
ΔparC DNA topoisomerase IV, subunit A ↓c ↓ (13)
53
c. 在該突變株中 yebC 或 yeeN 基因表現量有兩倍以下之變化。
利用微陣列資料庫(NCBI 及 Ecogene database http://ecogene.org/EcoarrayStat.php ) 搜尋並分析。
d. references 以編號代表內容如下，
(1) Traxler M.F., et al., 2008 (2) Sabina J., et al.,2003 (3) Aberg A., et al.,2009 (4) Jeong K. S., et al.,2006 (5) Domka J., et al., 2006 (6) Hagiwara D., et al., 2004 (7) Zimmer D.P., et al., 2000 (8) Nishino K., et al., 2005 (9) Mukhopadhyay P., et al., 2004 (10) Hommais F., et al., 2001 (11) Peter B.J., et al., 2004 (12) Abed N., et al., 2007 (13) Sangurdekar D. P., et al., 2006。
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圖一、不同菌種 DUF28 蛋白質之親緣關係及胺基酸序列相似性
a. 菌種名稱之縮寫，若該菌種中有兩個 DUF28 同源蛋白分別以 1、2 表示。菌種 全名分別為 sboy：Shigella boydii、ypes：Yersinia pestis、sone：Shwanella oneidensis、
paer：Pseudomonas aeruginosa、psyr：Pseudomonas syringae、reut：Ralstonia eutropha、
rso：Ralstonia solanacearum、ngon：Neisseria gonorrhoeae、xcam：Xanthomonas
ctetcampestris pv. Campestris、ctet：Clostridium tetani、lbre：Lactobacillus brevis、
spyo：Streptococcus pyogenes、bcer：Bacillus cereus、saur：Staphylococcus aureus、
55
mflo：Mesoplasma florum、fjoh：Flavobacterium johnsoniae、ccon：Campylobacter
concisus、ctra：Chlamydia trachomatis、atum：Agrobacterium tumefaciens、ecan：
Ehrlichia canis、syne：Synechococcus sp.、aqeo：Aquifex aeolicus、tlet：Thermotoga lettingae、maqu：Mycobacterium avium、dole：Desulfococcus oleovorans。
b. YebC 的胺基酸序列與其他菌種之 DUF28 胺基酸序列其相同度 (%)。
c. YeeN 的胺基酸序列與其他菌種之 DUF28 胺基酸序列其相同度 (%)。
以 http://www.ebi.ac.uk/Tools/msa/clustalw2/網站分析親緣關係圖，利用 Vector NTI 軟體比較胺基酸序列相同及相似度。
56
圖二、大腸桿菌 DUF28 蛋白質胺基酸序列比對
利用 Bioeditor 軟體分析 YeeN 及 YebC 胺基酸序列相同及相似度。黑色表示相同胺 基酸，灰色表示具有相似特性之胺基酸。
57
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圖四、大腸桿菌ΔyebC 及 ΔyeeN 突變株對 paraquat 生長抑制濃度之測試
在液態貧瘠培養基(M9 medium)中加入不同濃度的 paraquat 藥劑，測試突變株與野 生株之生長抑制情形，本實驗共重複三次以上共取 25 個數值計算其與未處理組相 除之相對生長量、平均及標準誤(SE)，*為顯著差異(P<0.05)，**為高度顯著差異 (P<0.01) 。
0.5 1 2 4
0.0 0.2 0.4 0.6 0.8
1.0 WT
yebC
yeeNparaquat (M)
Relative growth
**
**
**
**
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60
WT
ΔyeeN WT(pUFR
047)
ΔyeeN(pUF R04
7)
ΔyeeN(pUF Rye
eNF)
ΔyeeN(pUF Rye
bCF)
bCF)