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表一、本實驗所使用的菌株基因型
Strain Genotype or relevant property Reference or source
E.coli：
RecA1 supE44 endA1 hsdR17 gyrA96 rolA1 thiΔ(lac-proAB) Tpr Smr recA, thi, pro, hsdR－M＋ [RP4-2-Tc::Mu:Kmr Tn7]( pir )
K2 serotype rspL mutant,Smr
lacZ mutant in CG43S3
pmrA mutant in CG43S3-Z01 phoP mutant in CG43S3-Z01 rcsB mutant in CG43S3-Z01 ugd mutant in CG43S3 pmrH mutant in CG43S3 pmrF mutant in CG43S3 pmrA mutant in CG43S3 phoP mutant in CG43S3 pmrD mutant in CG43S3 pmrA/phoP mutant in CG43S3
Laboratory stock
CG43S3wza/pmrF CG43S3wza/pmrA CG43S3wza/phoP
wza and pmrF mutant in CG43S3 wza and pmrA mutant in CG43S3 wza and phoP mutant in CG43S3
This study This study This study
表二、本實驗所使用的質體構築表
Plasmid Relevant characteristic Reference or source
yT&A
PCR cloning vector, Apr
A derivative of pYC016, containing a promoterless lacZ from K.pneumoniae CG43S3 as the reporter, Cmr
Suicide vector, rspL, Apr, Kmr
Broad-host-range IncP cloning vector, Tcr
946-bp and 984-bp PCR product of the region upstream and downstream of pmrH cloned into yT&A
XbaI/SacI digested fragment of pYTHm034 cloned into pKAS46
1034-bp and 1037-bp PCR product of the region upstream and downstream of pmrF cloned into yT&A
EcoRI/XbaI digested fragment of pYTFm034 cloned into pKAS46
A 2.0-kb fragment containing a 500-bp deletion in wza locus cloned into pKAS46, A 2.0-kb fragment containing a 700-bp deletion in phoP locus cloned into pKAS46
Yeastern Biotech Co.
(34)
pHY025
A 2.0-kb fragment containing a 660-bp deletion in pmrA locus cloned into pKAS46 A 2.0-kb fragment containing a 500-bp deletion in pmrD locus cloned into pKAS46 A 2.0-kb fragment containing a 1100-bp deletion in ugd locus cloned into pKAS46 1277-bp PCR product carrying pmrF cloned into yT&A
HindIII/BamHI digested fragment of pFcYT cloned into pRK415 1105-bp PCR product carrying pmrA cloned into yT&A
HindIII/XbaI digested fragment of pAcYT cloned into pRK415 900-bp PCR product carrying phoP cloned into yT&A
BamHI/EcoRI digested fragment of pPcYT cloned into pRK415
BamHI/BglII digested fragment of 253-bp PCR product amplified by primer sets ugd03/ugd04 cloned into the BamHI site of pLacZ15
BamHI/BglII digested fragment of 477-bp PCR product amplified by primer sets pmrHp01/pmrHp02 cloned into the BamHI site of pLacZ15
1181-bp PCR product carrying rcsB cloned into pRK415
A 400-bp PCR product of cps porf16-17 promoter region cloned into yT&A A BglII fragment of porf162Ycloned into the BamHI site of pLacZ15
Laboratory stock
表三、本實驗所使用的引子序列
a，restriction enzyme site Primer Sequence
5’-CAA,TTG,GAT,CaCA,GGG,CTG,TAC-3’
5’-GAG,CCA,TGGa,TCT,ATT,CCG,TG-3’
5’-TGA,TTC,AGC,AGT,AGT,TCG,CCA,GCC-3’
5’-CGC,TCG,CCG,TTC,GGA,TCCa,TG-3’
5’-GCA,ACG,GTA,CCaT,TCA,TCA,GCG,C-3’
5'-GAT,GGA,AAA,GCT,GAA,GGC,GAT,GG-3' 5'-CAG,CGA,TATa,CAT,ACC,CGG,CGT,C-3' 5’-GCA,GGA,TCCa,ATA,ATG,GAA,GC-3’
5’-CGA,GAT,CTaA,GGG,CCA,CCA,C-3’
5’-TCT,GGA,TCCa,TGG,TCA,TTA,ATT,GCC,CGG,C-3’
5’-CTT,AGA,TCTa,CGC,TCA,TCA,TCA,TCC,TGT,TC-3’
5’-TTT,CTA,TGG,GCA,GAT,GGT,TG-3’
5’-GCT,GAC,TAT,CGG,GAA,GCA,TC-3’
圖一、不同菌種間 pmr 操縱子上游序列比較分析
比較克雷白氏菌、大腸桿菌、沙門氏菌及鼠疫桿菌間的 pmr 操縱子上游序 列，並標示-10 box 和-35 box，PhoP 結合位以空心方塊表示，PmrA 結合位以 實心方塊表示。
（A）
（B）
圖二、PCR 分析確認 pmrH 突變株
（A）pmrH 及上下游基因組成示意圖。引子 pmrH01/pmrH02 是用來確 pmrH 基因缺損。（B）利用引子 pmrH01/pmrH02 進行 PCR 分析確認 pmrH 基因缺 損。質體 pKAHm034 包含 pmrH 基因上下游序列以進行同源互換產生 pmrH 突變株。M：DNA 大小標準溶液，lane 1：K. pneumoniae CG43S3，lane 2：
pKAHm034，lane 3：K. pneumoniae CG43S3pmrH。
1.0 3.0 2.5 2.0
2 3
1.5
1 2 3
kb
M
（A）
（B）
圖三、PCR 分析確認 pmrF 突變株
（A）pmrF 及上下游基因組成示意圖。引子 pmrF01/pmrF02 是用來確認 pmrF 基因缺損。（B）利用引子 pmrF01/pmrF02 進行 PCR 分析確認 pmrF 基因缺 損。質體 pKAFm034 包含 pmrF 基因上下游序列以進行同源互換產生 pmrF 突變株。M：DNA 大小標準溶液，lane 1：K. pneumoniae CG43S3，lane 2：
pKAFm034，lane 3：K. pneumoniae CG43S3pmrF。
3.0 2.5 2.0
1 2 3
1.5 1.0 kb
M
圖四、K. pneumoniae CG43S3、CG43S3pmrH和CG43S3pmrF 生長曲線圖
將隔夜培養的菌液以1：100 比例更新培養於 LB、LB＋1 mM Fe3+及LB＋10 mM Mg2+離子，在 37℃下振 盪培養，每隔1 小時量測 OD600的吸光值。
（A）
（B）
圖五、pmrH、pmrF 和 ugd 基因缺損株（A）以及 pmrF 互補株（B）對多黏 菌素的抗性分析
隔夜培養的菌液以 1：200 的比例重新培養於含 1 mM Fe3+的 LB 培養液，在 37℃下振盪培養 3-4 小時。將菌液稀釋成 1-3 × 103 CFU/ml，加入多黏菌素使 最後濃度為 2 units/ml，在 37℃下振盪培養 1 小時後直接塗盤於 LB 培養基使 其長出單一菌落。存活率的計算是以多黏菌素作用後長出來的總菌數除以原 始下反應未和多黏菌數作用的總菌數的百分比。質體 pFcRK 為 pRK415 包含 pmrF 完整基因及其核醣體結合位序列。
（A）
（B）
圖六、pmrA、phoP 和 pmrD 基因缺損株以及互補株對多黏菌素的抗性分析 野生株和 pmrA、phoP 及 pmrD 基因缺損株（A）以及 pmrA、phoP 和 pmrD 互補株（B）在濃度 4 units/ml 的多黏菌素環境下的存活率。質體 pAcRK、
pPcRK 及 pHY212 分別為 pRK415 包含 pmrA、phoP 或 pmrD 完整基因及其核 醣體結合位序列。
圖七、pmrA/phoP基因缺損的pmrA或phoP互補株對多黏菌素的抗性分析 pmrA/phoP 突變株、pmrA/phoP 基因缺損的 pmrA 或 phoP 互補株在濃度 4 units/ml 的多黏菌素環境下的存活率。
（A）
（B） （C）
圖八、pmr 操縱子的基因組成示意圖和 PpmrH::lacZ 的活性測試
（A）利用 VectorNTI 軟體 （Invitrogen Vector NTI™ Advance）分析 pmr 操縱 子的基因組成，並標示 PpmrH::lacZ（pHY072）的建構方式。（B）隔夜培養 的菌液重新培養於LB、LB＋1 mM Fe3+及 LB＋10 mM Mg2+ 中 3-4 小時，並 測試 PpmrH::lacZ 的活性。（C）隔夜培養的菌液重新培養於 LB 培養液 3-4 小 時，並測量在野生株、pmrA 或 phoP 突變株中的 PpmrH 活性。
（A）
（B）
圖九、pmrF、pmrA 和 phoP 基因缺損株（A）及互補株（B）在 THP-1 細胞 株內的存活率測試
將 m.o.i＝30 的細菌加到已接種在 24 孔培養盤的細胞中，並培養在不含胎牛 血清及抗生素的 RPMI 1640 液培養 2 小時，加入 gentamicin 抗生素作用 1 小 時，之後分別在 0、4、8 小時加入 0.1％ 的 Triton X-100 作用 10 分鐘，將菌 液系列稀釋（1/100X）後直接塗盤於 LB 培養基使其長出單一菌落。回收率 的計算是以經細胞吞噬後回收的總菌數除以下反應的總菌數的百分比。
圖十、pmrF、pmrA 和 phoP 基因缺損株在 RAW264.7 細胞株內的存活率測 試
wza 野生株、pmrF、phoP 及 pmrA 突變株在受 RAW264.7 細胞株吞噬後 0、
4、8 小時的回收率。
圖十一、Pugd-1::lacZ 的活性測試
隔夜培養的菌液重新培養於 LB 培養液 3-4 小時，並測量在野生株、phoP、 pmrA 或 rcsB 突變株中的 Pugd-1活性。
（A）
（B）
（C）
圖十二、RT-PCR 分析確認 manC-manB-ugd 操縱子和 Pugd-2::lacZ 的活性測 試
（A ） ugd 及 上 下 游 基 因 組 成 示 意 圖 。 a 、 b 和 c 分 別 為 利 用 引 子 Rorf1601/Rorf1602、Rorf1701/Rorf1702 和 Rugd01/Rugd02 進行 PCR 所增殖
出 gnd-manC、manC-manB 和 manB-ugd 基因間的片段大小。Pugd-1::lacZ 和
Pugd-2::lacZ 分別表示兩個所建構的 ugd 啟動子部位。（B）RT-PCR 分析
manC、manB 和 ugd 基因間的區域，PCR 增殖的片段 a、b 和 c 分別為 1-3 行
（431 bp）、4-6 行（443 bp）和 7-9 行（510 bp）。第 1、4 和 7 行為 genomic DNA 的 PCR 產物，當做正對照組。第 2、5 和 8 行為 RNA 的 PCR 產物，當作負對照組。第 3、6 和 9 行為 cDNA 的 PCR 產物。（C）隔夜培 養的菌液重新培養於 LB 培養液 3-4 小時，並測量在野生株、phoP、pmrA 或 rcsB 突變株中的 Pugd-2活性。
圖十三、rcsB 基因缺損株對多黏菌素的抗性分析
野生株、rcsB 突變株及 rcsB 互補株在濃度 2、4 units/ml 的多黏菌素環境下的 存活率。質體 pHY123 為 pRK415 包含 rcsB 完整基因及其核醣體結合位序
野生株、rcsB 突變株及 rcsB 互補株在濃度 2、4 units/ml 的多黏菌素環境下的 存活率。質體 pHY123 為 pRK415 包含 rcsB 完整基因及其核醣體結合位序