在圖十六(B)中可以發現 mrkH 啟動子活性並不高，推測可能有其他轉錄因 子抑制其活性，因此企圖利用跳躍子突變技術(圖十七 A)篩選出此抑制因子，首 在 LacZ 基因缺損株放入帶有 mrkH 啟動子的質體，再利用接合作用轉入 Tn5 跳 躍子，藉量測 LacZ 活性來篩選增加 mrkH 啟動子活性的突變株。經篩選約 10000 顆突變株，挑出 LacZ 活性較高的菌落 10 顆，重新培養滴在馬康基氏瓊脂上與 帶有 mrkH 啟動子質體的菌比較，卻沒有發現明顯變紅的菌落(圖十七 B)，而利 用 X-gal 篩選也無明顯深藍的菌落(圖十七 C)。
20
21
22
過頭抑制 Fur 的表現，SoxRS 也可能在低鐵環境扮演抑制 Fur 的角色；在低含氧 量下，RcsB 可能與 RmpA2 結合活化 MrkA 表現，c-di-GMP 是否直接參與活化 mrkH 啟動子，MrkH 及 MrkI 的角色仍待釐清。
23
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一、本研究所使用的菌株
細菌菌株 基因型或相關特性 來源或參考文獻
Escherichia coli
JM109 recA1 glnV44 endA1 thi-1 relA1 gyrA96 mcrB+
Laboratory stock Δ(lac-proAB) e14-[F' traD36 proAB+ lac1q
lacZ
ΔM15]hsdR17(rKmK+)
S17-1 λpir hsdR-M+ recA pro thi Tpr
Smr(RP4-2-Tc::Mu; Km::Tn7)( λpir)
Klebsiella pneumonia
CG43S3 Clinical isolate of K2 serotype, Smr Laboratory stock Δfur CG43S3 Δfur, Smr Laboratory stock ΔrcsB CG43S3 ΔrcsB, Smr Laboratory stock ΔryhB CG43S3 ΔryhB, Smr Laboratory stock ΔfurΔryhB CG43S3 ΔfurΔryhB, Smr Laboratory stock ΔfurΔrcsB CG43S3 ΔfurΔrcsB, Smr Laboratory stock ΔmrkA CG43S3 ΔmrkA , Smr Laboratory stock ΔmrkH CG43S3 ΔmrkH, Smr Laboratory stock ΔrmpA CG43S3ΔrmpA Smr Laboratory stock ΔrmpA2 CG43S3ΔrmpA2 Smr Laboratory stock ΔfurΔrmpA CG43S3ΔfurΔrmpA Smr Laboratory stock ΔfurΔrmpA2 CG43S3ΔfurΔrmpA2 Smr Laboratory stock ΔfimA CG43S3ΔfimA Smr Laboratory stock Δhfq CG43S3Δhfq Smr Laboratory stock ΔcpxRA CG43S3ΔcpxRA Smr Laboratory stock ΔlacZ CG43S3 ΔlacZ, Smr Laboratory stock ΔlacZΔfur CG43S3 ΔlacZΔfur, Smr Laboratory stock ΔlacZΔmrkH CG43S3 ΔlacZΔmrkH, Smr Laboratory stock ΔlacZΔfurΔryhB CG43S3ΔlacZΔfurΔryhB Smr Laboratory stock ΔsoxRS CG43S3 ΔsoxRS, Smr Laboratory stock ΔmrkI CG43S3 ΔmrkI, Smr Laboratory stock ΔlacZΔmrkI CG43S3 ΔlacZΔmrkI, Smr Laboratory stock ΔlacZΔrcsB CG43S3 ΔlacZΔrcsB, Smr Laboratory stock
33
表 二、本研究所使用和建構的質體
質體 基因型或相關特性
pRK415 broad-host-range IncP cloning vector, mob+, Tcr pLacZ15 promoter selection vector, lacZ+, Cmr
pETQ33 Protein overexpression vector, IPTG inducible, Kmr pmrkA-P1
(PL-mrkA)
Cmr, 551-bp fragment of the upstream region of mrkA cloned into placZ15
pYdeH (pAW47) Kmr, 894-bp fragment encoding YdeH, from E. coli W3110, cloned into pETQ
pYdeH*
(pAW71) Kmr, 894-bp fragment encoding YdeHAADEF cloned into pETQ pfur Cmr,placZ15 carrying the fur promoter region
pmrkH Cmr,407-bp fragment of the upstream region of mrkH cloned into placZ15
pRK415-ydeh Tcr,894-bp fragment encoding YdeH, from E. coli W3110 pUT mini-Tn5 pUT containing luxCDABE cassette cloned into of mini-Tn5
transposon upstream of Kmr gene
34
(A)
(B)
(C)
(D)
(E)
GAPDH
35
圖 一、在不同培養液之條件下 small RNA RyhB 對第三型纖毛 MrkA 蛋白的生 成影響
所有菌株皆以 37℃、16 小時搖晃培養，之後回收菌液以加熱破菌，並且定量 50 g/100 l。(A) LB 培養液；(B) LB 加入 Deferoxamine； (C) LB 加入 2, . 2-dipyridyl (Dip)； (D) M9 培養液； (E) DMEM 培養液。
36
圖 二、small RNA RyhB 對 mrkA 啟動子活性的影響
菌株先在 LB 培養液培養 37℃、16 小時，之後 40 倍稀釋到 M9 培養液中至 OD600 0.8 再進行啟動子活性實驗
37
圖 三、ryhB 基因缺損對 fur 基因缺損株生物膜生成的影響
圖 三、ryhB 基因缺損對 fur 基因缺損株生物膜生成的影響