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表一：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 E. coli:
JM-109 RecA1 supE44 endA1 hsdR17 gyrA96 rolA1 thi Δ(lac-proAB)
Laboratory stock S17-1λpir Tpr Smr recA, thi, pro, hsdR－M＋
[PR4-2-Tc::Mu:Kmr Tn7](pir)
(18)
K. pneumoniae:
CG43 K2 serotype (52)
CG43S3 rspl mutant, Smr (52) CG43S3ΔrcsB rcsB mutant in CG43S3, Smr (36)
CG43S3ΔrcsA rcsA mutant in CG43S3, Smr Laboratory stock
Z01 lacZ mutant in CG43S3, Smr (39)
Z01ΔrcsB rcsB mutant in Z01, Smr Laboratory stock
Z01ΔkvhA kvhA mutant in Z01, Smr Laboratory stock
CG43S3Δcfa cfa mutant in CG43S3, Smr This study CG43S3ΔyfdX yfdX mutant in CG43S3, Smr This study
表二：本研究所使用的質體
質體 相關特性 來源或
參考文獻
yT＆A PCR cloning vector，Apr Yeastern Biotech
Co.
pKAS46 Suicide vector，rpsL，Kmr Apr (62)
pLacZ15 A derivative of pYC016 (35), containing a promoterless lacZ from K. pneumoniae
CG43S3, Cmr (39)
pRK415 broad-host-range IncP cloning vector, mob＋, Tcr (33)
pKvhAcTA 698 bp PCR product carrying kvhA cloned into yT&A, Apr (65) pKvhAcPP BamHI/EcoRI digested fragment of pKvhAcTA cloned into pCPP45, Tcr (65)
pA15 467 bp PCR product carrying putative kvhA promoter cloned into pLacZ15, Cmr (38) pF15 379 bp PCR product carrying putative kvhA promoter cloned into pLacZ15, Cmr (38) pE15 180 bp PCR product carrying putative kvhA promoter cloned into pLacZ15, Cmr (38) pCPP45 broad-host-range cloning vector with the partition region from RK2 plasmid, Tcr Dr. David Bauer at
Cornell University
pHY123 rcsB complement plasmid, Tcr Laboratory stock
質體 相關特性 來源或 參考文獻
pHY064 424 bp PCR product carrying putative rcsB promoter cloned into pLacZ15, Cmr Laboratory stock PhdeD-hdeB1 417 bp PCR product carrying putative hdeD-hdeB1 promoter cloned into pLacZ15, Cmr Laboratory stock
PyfdX 417 bp PCR product carrying putative yfdX promoter cloned into pLacZ15, Cmr Laboratory stock Pcfa 322 bp PCR product carrying putative cfa promoter cloned into pLacZ15, Cmr This Study
PhdeB2 409 bp PCR product carrying putative hdeB2 promoter cloned into pLacZ15, Cmr This Study
PydeP 330 bp PCR product carrying putative ydeP promoter cloned into pLacZ15, Cmr This Study PyhiO 396 bp PCR product carrying putative yhiO promoter cloned into pLacZ15, Cmr This Study pCH012 two about 1kb DNA fragments flanking the yfdX cloned into yT＆A, Apr This Study
pCH015 EcoRI/XbaI digested fragment of pCH012 cloned into pKAS46, Kmr Apr This Study pCH013 two about 1kb DNA fragments flanking the cfa cloned into yT＆A, Apr This Study
pCH014 EcoRI/XbaI digested fragment of pCH013 cloned into pKAS46, Kmr Apr This Study pCH017 283 bp PCR product carrying putative yfdX promoter cloned into yT&A, Apr This Study
PyfdX-2 BamHI/BglII digested fragment of pCH017 cloned into pLacZ15, Cmr This Study
pCH018 216 bp PCR product carrying putative yfdX promoter cloned into yT&A, Apr This Study
PyfdX-3 BamHI/BglII digested fragment of pCH018 cloned into pLacZ15, Cmr This Study
PkvhA-1 409 bp PCR product carrying putative kvhA promoter cloned into pLacZ15, Cmr This Study
PkvhA-2 276 bp PCR product carrying putative kvhA promoter cloned into pLacZ15, Cmr This Study
表三：本研究所使用的引子
引子 序列
CH001 5'-CCCAGATCTCACCCCACTCTTCCCACGCG-3' CH002 5'-CCCAGATCTCTCATAGCTTCTCCATCACGCCC-3' CH003 5'-GACGGATCCGATTATCGCATTCATGGGGGC-3' CH005 5'-CCCGGATCCCTGACCTGAGCCTGGCCC-3'
CH006 5'-CCTAGATCTGGTCATCTCTTTACTCCTGCTGC-3' CH007 5'-CCCGGATCCAGCCATAGTGTTACTCCTTCCA-3' CH008 5'-CCTAGATCTCTGATCATACCTCCTCCCCG-3' CH015 5'-CCACCGCGGCGCTACTCATT-3'
CH024 5'-AGAAGGCCACCGGGGTCATG-3' CH027 5'-AGCAGACCGGCTCCGGACT-3' CH028 5'-AACGTCCTGATCTACGCCGC-3'
CH031 5'-GTATCGACAAAACCCATCGCC-3'
CH033 5'-GACGGATCCGGGCTAAACGCATTTAAGGTG-3' CH034 5'-GACGGATCCGCCTGACGCTGAATAAAAAGC-3' CH035 5'-CCTAGATCTATCACCAAACGCAGCCAGC-3'
表四：可能由RcsB所調控的抗酸基因
灰色部分表示選出的6 個基因
基因 功能描述 AR 系統 在克雷白氏肺炎桿菌中
是否有同源基因
RcsB 結合序列數目
adiA arginine decarboxylase III 是 1
appB cytochrome oxidase bd-II, subunit II ─a 是 0
cfa cyclopropane fatty acid synthetase I 是 5
gadA glutamate decarboxylase II 否 Xb
gadB glutamate decarboxylase II 否 Xb
gadC putative glutamate:GABA antiporter II 否 Xb
hdeA acid-resistance protein I 否 Xb
hdeB2 acid-resistance protein I 是 4
hdeD acid-resistance membrane protein ─a 是 4
ydeP putative anaerobic dehydrogenases ─a 是 1
ydgK putative oxidoreductase ─a 是 0
yfdX hypothetical protein ─a 是 4
yhiO universal stress protein UspB ─a 是 3
yjdE (adiC) putative arginine:agmatine antiproter III 是 1
a：不屬於任何一套 AR 系統
表五：CG43S3 與CG43S3ΔrcsB在中性環境下表現量有差異的蛋白質
Match ID pI Mr (kDa) Expressional fold change 167 5.60 107 ↓1.50 215 5.56 92 ↓1.24 332 5.20 72 ↓1.28 412 6.26 65 ↑1.10 491 6.36 57 ↑1.30 553 6.29 51 ↑1.10 821 6.04 34 ↑1.21
表六：CG43S3 與CG43S3ΔrcsB在酸性環境下表現量有差異的蛋白質
Match ID pI Mr (kDa) Expressional fold change 772 5.98 25 ↓2.18 817 5.23 21 ─a 832 5.05 19 ─a 879 5.92 14 ↓1.13 946 5.98 10 ↓1.22 972 4.70 9 ↓1.90 973 4.59 9 ↓1.52 1064 5.08 18 ↑1.14 a：只在 CG43S3 中出現的蛋白質
(A) (B)
圖一：rcsB基因缺損降低克雷白氏肺炎桿菌的抗酸能力
細菌在 LB 培養液隔夜培養後，20 倍稀釋至新鮮的 LB 培養液 中，再培養3 個小時至 OD600約為0.6~0.8，之後將菌體移至 pH 4.4 的 LB 培養液中，適應 1 小時後，再移至 pH 3.0 的 M9 培養 液中，培養1 小時 (A)或 45 分鐘 (B)後，取適當菌液稀釋塗盤 後，計算菌數；存活率是根據在 45 分鐘或 1 小時後，每毫升存 活的菌數和初始菌數的比值。pHY123 為 rcsB 互補質體。
survival rate (%)
10 20 30 40 50
Δrcs B[pR
K415] Δrcs
B[pH Y123]
survival rate (%)
10 20 30 40 50 60
CG43S3 ΔrcsB