Statistical analysis - General Experimental Procedures

CHAPTER 6 Experimental Section

6.2 General Experimental Procedures

6.2.25 Statistical analysis

Two-tailed Student’s t test was used to determine the significance of the differences between the survivals in polymyxin B assay/phagocytosis, the levels of β-galactosidase activity and CPS amounts. P values less than 0.01 were considered statistically significant.

6.3 Table

Table 6.1. Bacterial strains used in this study

Strain Description Reference or source

K. pneumponiae

CG43 Clinical isolate (36)

CG43-101 CG43-101 (37)

CG43S3 CG43 Sm^r (130)

ΔlacZ CG43S3ΔlacZ Sm^r (142)

ΔrcsB (B2202) CG43S3ΔrcsB Sm^r (130)

ΔrmpA2 (R2035) CG43S3ΔrmpA2 Sm^r (130)

ΔgalU (U9451) CG43S3ΔgalU Sm^r Laboratory stock

Δfur CG43S3Δfur Sm^r This study

ΔphoP CG43S3ΔphoP Sm^r This study

ΔpmrA CG43S3ΔpmrA Sm^r This study

ΔpmrD CG43S3ΔpmrD Sm^r This study

ΔpmrF CG43S3ΔpmrF Sm^r This study

ΔrmpA CG43S3ΔrmpA Sm^r This study

ΔrstA CG43S3ΔrstA Sm^r This study

ΔrstB CG43S3ΔrstB Sm^r This study

Δugd CG43S3Δugd Sm^r This study

Δwza CG43S3Δwza Sm^r This study

ΔlacZΔfur CG43S3ΔlacZΔfur Sm^r This study

117

Strain Description Reference or source

ΔlacZΔphoP CG43S3ΔlacZΔphoP Sm^r This study

ΔlacZΔpmrA CG43S3ΔlacZΔpmrA Sm^r This study

ΔlacZΔpmrD CG43S3ΔlacZΔpmrD Sm^r This study

ΔlacZΔrmpA CG43S3ΔlacZΔrmpA Sm^r This study

ΔlacZΔrmpA2 CG43S3ΔlacZΔrmpA2 Sm^r This study

ΔpmrAΔphoP CG43S3ΔpmrAΔphoP Sm^r This study

ΔrcsBΔphoP CG43S3ΔrcsBΔphoP Sm^r This study

ΔrcsBΔpmrA CG43S3ΔrcsBΔpmrA Sm^r This study

ΔrcsBΔpmrD CG43S3ΔrcsBΔpmrD Sm^r This study

ΔrcsBΔpmrF CG43S3ΔrcsBΔpmrF Sm^r This study

ΔrmpAΔrmpA2 CG43S3ΔrmpAΔrmpA2 Sm^r This study

ΔrstAΔgalU CG43S3ΔrstAΔgalU Sm^r This study

ΔrstAΔrstB CG43S3ΔrstAΔrstB Sm^r This study

E. coli

BL21(DE3) F^- ompT hsdSB(rB -mB

-) gal dcm trxB15::kan (DE3) Novagen

BL21(DE3) [pLysS] F^- ompT hsdSB(rB -mB

-) gal dcm trxB15::kan (DE3) [pLysS] Novagen

JM109 endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB⁺ Δ(lac-proAB) e14- [F' traD36 proAB⁺ lacI^q lacZΔM15]

hsdR17(rK -mK

+) New England Biolabs

S17-1λpir hsdR recA pro RP4-2 (Tc::Mu; Km::Tn7)(λpir) (218)

XL1-Blue MRF’ Kan Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F’ proAB

lacI^qZ ΔM15 Tn5 (Kan^r)] Stratagene

Table 6.1. (continued)

118

Table 6.2. Plasmids used in this study

Plasmid Description Reference or source

yT&A Ap^r, T/A-type PCR cloning vector, Yeastern

pBT Cm^r, bait plasmid, p15A origin of replication, lac-UV5 promoter, λ-cI open reading frame Stratagene pTRG Tc^r, target plasmid, ColE1 origin of replication, lac-UV5 promoter, RNAPα open reading frame Stratagene

pET30a-c Km^r, His-tagged protein expression vector Novagen

pBT-LGF2 Cm^r, control plasmid containing a fragment encoding the yeast transcriptional activator Gal4

fused with λ-cI Stratagene

pTRG-GAL11^P Tc^r, control plasmid containing a fragment encoding a mutant form of Gal11 protein, called

Gal11^P, fused with RNAPα Stratagene

pGEX-5X-1 Ap^r, GST-tagged protein expression vector GH Hleathcare

pACYC184 Cm^r, Tc^r, plasmid with p15A origin of replication (35)

pKAS46 Ap^r, Km^r, suicide vector, rpsL (218)

pRK415 Tc^r, shuttle vector, mob⁺ (121)

pYC084 Tc^r, 2.1-kb HindIII/BamHI fragment containing the entire rmpA2 locus cloned into pRK415 (130)

placZ15 Cm^r, promoter selection vector, lacZ⁺ (142)

pOrf12 Cm^r, 500-bp fragment containing the region upstream of Klebsiella K2 cps orf1-orf2 cloned into

placZ15 (142)

pOrf315 Cm^r, 900-bp fragment containing the region upstream of Klebsiella K2 cps orf3-orf15 cloned into

placZ15 (142)

pOrf1617 Cm^r, 300-bp fragment containing the region upstream of Klebsiella K2 cps orf16-orf17 cloned

into placZ15 (142)

119

Plasmid Description Reference or source pHY048 Cm^r, 450-bp fragment containing the upstream region of the K. pneumoniae rstA gene cloned

into placZ15 This study

pHY050 Cm^r, 200-bp fragment containing the upstream region of the K. pneumoniae rstA gene cloned

into placZ15 This study

pHY053 Cm^r, 150-bp fragment containing the upstream region of the K. pneumoniae rstA gene cloned

into placZ15 This study

pHY083 Km^r, Ap^r, the Porf1-2::lacZ reporter cassette cloned into pKAS46 This study pHY115 Ap^r, 550-bp fragment containing a pmrD allele cloned intoyT&A This study pHY118 Ap^r, 300-bp fragment containing the upstream region of the K. pneumoniae pmrD gene cloned in

the same direction to lacZα in yT&A, This study

pHY290 Ap^r, 299-bp fragment encoding residues 1-100 of RmpA cloned in-frame with lacZα into yT&A This study pHY291 Ap^r, 299-bp fragment encoding residues 1-100 of RmpA cloned in the opposite direction to lacZα

into yT&A This study

pHY305 Ap^r, 347-bp fragment encoding residues 1-116 of RmpA2 cloned in-frame with lacZα into yT&A This study pHY306 Ap^r, 347-bp fragment encoding residues 1-116 of RmpA2 cloned in the opposite direction to

lacZα into yT&A This study

pACYC184-RcsB Tc^r, 1.1-kb EcoRI fragment containing full-length RcsB-His and the upstream T7 promoter

region from pET30b-RcsB cloned into pACYC184 This study

pBT-PmrA Cm^r, 669-bp fragment encoding full-length PmrA cloned into pBT This study pBT-RcsB Cm^r, 648-bp fragment encoding full-length RcsB cloned into pBT This study pET30b-PhoP Km^r, 711-bp fragment encoding full-length PhoP cloned into pET30b This study pET30b-PhoPN Km^r, 447-bp fragment encoding residues 1-149 of PhoP cloned into pET30b This study pET30b-PmrBC Km^r, 828-bp fragment encoding residues 90-365 of PmrB cloned into pET30b This study Table 6.2. (continued)

120

Plasmid Description Reference or source pET-PmrA Km^r, 669-bp fragment encoding full-length PmrA cloned into pET29b Courtesy of Dr.

Chinpan Chen pET-PmrD Km^r, 243-bp fragment encoding full-length PmrD cloned into pET29b Courtesy of Dr.

Chinpan Chen pET30b-RcsB Km^r, 648-bp fragment encoding full-length RcsB cloned into pET30b This study pET30c-Fur Km^r, 450-bp fragment encoding full-length Fur cloned into pET30c This study pGEX-RcsA Ap^r, 621-bp fragment encoding full-length RcsA cloned into pGEX-5X-1 This study pGEX-RmpA Ap^r, 585-bp fragment encoding full-length RmpA cloned into pGEX-5X-1 This study pGEX-RmpAN Ap^r, 252-bp fragment encoding residues 1-84 of RmpA cloned into pGEX-5X-1 This study pGEX-RmpA2 Ap^r, 636-bp fragment encoding full-length RmpA2 cloned into pGEX-5X-1 This study placZ15-PiroB Cm^r, 450-bp fragment containing the upstream region of the K. pneumoniae iroBCD genes

cloned into placZ15 This study

placZ15-PiucA Cm^r, 700-bp fragment containing the upstream region of the K. pneumoniae iucABCD genes

cloned into placZ15 This study

placZ15-PpmrD Cm^r, 350-bp fragment containing the upstream region of the K. pneumoniae pmrD gene cloned

into placZ15 This study

placZ15-PpmrH Cm^r, 500-bp fragment containing the upstream region of the K. pneumoniae pbgP genes cloned

into placZ15 This study

placZ15-PrmpA Cm^r, 500-bp fragment containing the upstream region of the K. pneumoniae rmpA gene cloned

into placZ15 This study

placZ15-PrmpA2 Cm^r, 500-bp fragment containing the upstream region of the K. pneumoniae rmpA2 gene cloned

into placZ15 This study

pRK415-Fur Tc^r , 0.8-kb fragment containing a fur allele cloned into pRK415 This study Table 6.2. (continued)

121

Plasmid Description Reference or source pRK415-PhoP Tc^r , 900-bp fragment containing a phoP allele cloned into pRK415 This study pRK415-PmrA Tc^r , 1.1-kb fragment containing a pmrA allele cloned into pRK415 This study pRK415-PmrD Tc^r , 550-bp fragment containing a pmrD allele cloned into pRK415 This study pRK415-PmrDK6A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDV8A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDQ9A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDD10A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDS23A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDG24A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDL26A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDM28A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDE31A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDT46A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDY53A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDT69A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDY71A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDW76A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDT77A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDN78A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrDD80A Tc^r , 550-bp fragment containing a pmrD allele encoding PmrDK6A cloned into pRK415 This study pRK415-PmrF Tc^r , 1.3-kb fragment containing a pmrF allele cloned into pRK415 This study Table 6.2. (continued)

122

Plasmid Description Reference or source pRK415-RcsB Tc^r , 1.2-kb fragment containing an rcsB allele cloned into pRK415 This study pRK415-RmpA Tc^r , 1.1-kb fragment containing an rmpA allele cloned into pRK415 This study pRK415-RmpAN Tc^r , 1.1-kb fragment containing the rmpA locus encoding residues 1-84 of RmpA cloned into

pRK415 This study

pRK415-RmpA2 Tc^r , 1.2-kb fragment containing an rmpA2 allele cloned into pRK415 This study pTRG-PmrD Tc^r, 243-bp fragment encoding full-length PmrD cloned into pTRG This study pTRG-RcsA Tc^r, 621-bp fragment encoding full-length RcsA cloned into pTRG This study pTRG-RmpAN Tc^r, 252-bp fragment encoding residues 1-84 of RmpA cloned into pTRG This study pTRG-RmpA2 Tc^r, 636-bp fragment encoding full-length RmpA2 cloned into pTRG This study Table 6.2. (continued)

123

Table 6.3. Oligonucleotide primers used in this study

Primer Sequence^a Enzyme

cleaved Complementary position

CH009 5'-AGCGTGACGAGACCTGCCCA-3' None -802 relative to the K2 cps orf1 start codon CH010 5'-GGCTGCGGGCGTAAGAGAAC-3' None +217 relative to the lacZ start codon CY007 5'-TCTAGAGGCAGGTTGGCTCTTCAGTC-3' XbaI +489 relative to the fur start codon CY008 5'-GGATCCATGAAGACAGCCAGCCGGA-3' BamHI -389 relative to the fur start codon CY0010 5'-GGATCCGATTCCGCATGACTGACAAC-3' BamHI -8 relative to the fur start codon CY0011 5'-AAGCTTGGCAGGTTGGCTCTTCAGTC-3' HindIII +489 relative to the fur start codon GSPrmpA01 5’-TTAGGATAAAACCGCCCCCCCCCCGAAAC-3’ None +254 relative to the rmpA start codon GSPrmpA02 5’-CATTTTGTACCCTCCCCATTTCCCTGA-3’ None +180 relative to the rmpA start codon KP1760-1 5’-GGAATTCCATATGAAAATCTTAGTCATTGAA-3’ NdeI +1 relative to the pmrA start codon KP1760-2 5’-CCGCTCGAGCTATTCCGTGTCGATGTTGTT-3’ XhoI +672 relative to the pmrA start codon KP3573-1 5’-GGAATTCCATATGGAGTGGTGGGTAAAAAAA-3’ NdeI +1 relative to the pmrD start codon KP3573-2 5’-CCGCTCGAGTTTGTCGGCGTTTGTCCAACG-3’ XhoI +243 relative to the pmrD start codon pET30f2 5’-TTGAATTCTAGGTTGAGGCCGTTGAGCA-3’ EcoRI +673 to +692 region on pET30a pET30r2 5’-CAAGAATTCAAACCCCTCAAGACCCGT-3’ EcoRI +31 to +47 region on pET30a phoP01 5’-CGCTCGCCGTTCGGATCCTG-3’ BamHI -171 relative to the phoP start codon phoP02 5’-GCAACGGTACCTTCATCAGCGC-3’ KpnI +729 relative to the phoP start codon phoP05 5’-GTAATGACAGCGGGAAGATATG-3’ None +753 relative to the phoP start codon phoP06 5’-CAGCCGTTTATATTTTGCGT-3’ None -25 relative to the phoP start codon pirop01 5'-GGATCCGATTTCAGTACGGCATGGAC-3' BamHI -379 relative to the iroB start codon pirop02 5'-AGATCTACGGGAAACGCCTGTGCCA-3' BglII +77 relative to the iroB start codon

124

Primer Sequence^a Enzyme

cleaved Complementary position

piucp01 5'-GGATCCAGAGGGTGATTTGCCAGCAT-3' BamHI -611 relative to the iucA start codon piucp02 5'-AGATCTGGAAGCACTGAGCAGCCACA-3' BglII +109 relative to the iucA start codon pmrA06 5’-GAGCCATGGTCTATTCCGTG-3’ NcoI +682 relative to the pmrA start codon pmrA10 5’-ACTCGAGCCATGGTCTATTCCGTG-3’ XhoI +1 relative to the pmrA start codon pmrA11 5’-AATGCGGCCGCAATGAAAATCTTAGTC-3’ NotI +672 relative to the pmrA start codon pmrAp03 5’-CAATTGGATCCAGGGCTGTAC-3’ BamHI -424 relative to the pmrA start codon pmrBe03 5’-TGGATCCTCGCAAGATCACCCGCC-3’ BamHI +283 relative to the pmrB start codon pmrBe04 5’-CAAGCTTATGGGTGCTGACGTTCTGAC-3’ HindIII +1095 relative to the pmrB start codon pmrDe02 5’-CGAGCTCGTGTTATTTGTCGGCGTTTGTC-3’ SacI +250 relative to the pmrD start codon pmrDe15 5’-AAAGCGGCCGCGATGGAGTGGTGGGTAAAAAAAGTA-3’ NotI +1 relative to the pmrD start codon pmrDe16 5’-TTTCTCGAGTGTGTTATTTGCCGGCGTTT-3’ XhoI +243 relative to the pmrD start codon pmrDp01 5’-TGGATCCTTCATGACGCTCTCTC-3’ BamHI -278 relative to the pmrD start codon pmrDp02 5’-CGCACAGATCTGAAGCACGAC-3’ BglII +75 relative to the pmrD start codon pmrDp05 5’-TTACCCACCACTCCATATGTTTTTTA-3’ NdeI +10 relative to the pmrD start codon pmrDK6Af 5’-ACAGATGGAGTGGTGGGTAGCAAAAGTACAGGACAACGCT-3’ None -4 to +36 relative to the pmrD start codon pmrDK6Ar 5’-AGCGTTGTCCTGTACTTTTGCTACCCACCACTCCATCTGT-3’ None -4 to +36 relative to the pmrD start codon pmrDQ9Af 5’-GGAGTGGTGGGTAAAAAAAGTAGCGGACAACGCTTCGGC-3’ None +2 to +41 relative to the pmrD start codon pmrDQ9Ar 5’-GCCGAAGCGTTGTCCGCTACTTTTTTTACCCACCACTCC-3’ None +2 to +41 relative to the pmrD start codon pmrDD10Af 5'-GGGTAAAAAAAGTACAGGCCAACGCTTCGGCCAGCC-3' None +11 to +47 relative to the pmrD start codon pmrDD10Ar 5'- GGCTGGCCGAAGCGTTGGCCTGTACTTTTTTTACCC-3' None +11 to +47 relative to the pmrD start codon pmrDS23Af1 5’-CGTCGTGCTTCAGGCCGGTGCGCT-3’ None +54 to +78 relative to the pmrD start codon Table 6.3. (continued)

125

Primer Sequence^a Enzyme

cleaved Complementary position

pmrDS23Ar1 5’-AGCGCACCGGCCTGAAGCACGACG-3’ None +54 to +78 relative to the pmrD start codon pmrDG24Af 5’-GTGCTTCAGAGCGCTGCGCTGGAGATG-3’ None +58 to + 85 relative to the pmrD start codon pmrDG24Ar 5’-CATCTCCAGCGCAGCGCTCTGAAGCAC-3’ None +58 to + 85 relative to the pmrD start codon pmrDL26Af1 5’-CAGAGCGGTGCGGCGGAGATGATCG-3’ None +64 to +89 relative to the pmrD start codon pmrDL26Ar1 5’-CGATCATCTCCGCCGCACCGCTCTG-3’ None +64 to +89 relative to the pmrD start codon pmrDM28Af1 5’-CGGTGCGCTGGAGGCGATCGCAGAGATTGAA-3’ None +69 to +100 relative to the pmrD start codon pmrDM28Ar1 5’-TTCAATCTCTGCGATCGCCTCCAGCGCACCG-3’ None +69 to +100 relative to the pmrD start codon pmrDE31Af 5’-CTGGAGATGATCGCAGCGATTGAAGCCTGTCGC-3’ None +76 to +109 relative to the pmrD start codon pmrDE31Ar 5’-GCGACAGGCTTCAATCGCTGCGATCATCTCCAG-3’ None +76 to +109 relative to the pmrD start codon pmrDT46Af 5’-GAAGGGGATAAACTCGCTCCGCTGGCCG-3’ None +121 to +149 relative to the pmrD start codon pmrDT46Ar 5’-CGGCCAGCGGAGCGAGTTTATCCCCTTC-3’ None +121 to +149 relative to the pmrD start codon pmrDY53Af 5’-CGCTGGCCGATGCGCGCGCTTGTCTGAATAATAATCC-3’ None +140 to +177 relative to the pmrD start codon pmrDY53Ar 5’-GGATTATTATTCAGACAAGCGCGCGCATCGGCCAGCG-3’ None +140 to +177 relative to the pmrD start codon pmrDT69Af1 5’-GAAGATCCGCAACGCCGCTCATTATAG-3’ None +189 to +216 relative to the pmrD start codon pmrDT69Ar1 5’-CTATAATGAGCGGCGTTGCGGATCTTC-3’ None +189 to +216 relative to the pmrD start codon pmrDY71Af 5’-GAAGATCCGCAACGCCACTCATGCTAGCAGCGAGCG-3’ None +189 to +225 relative to the pmrD start codon pmrDY71Ar 5’-CGCTCGCTGCTAGCATGAGTGGCGTTGCGGATCTTC-3’ None +189 to +225 relative to the pmrD start codon pmrDT77Af 5’-GCAGCGAGCGTTGGGCAAACGCCGACAAA-3’ None +215 to +244 relative to the pmrD start codon pmrDT77Ar 5’-TTTGTCGGCGTTTGCCCAACGCTCGCTGC-3’ None +215 to +244 relative to the pmrD start codon pmrDN78Af1 5’-GCAGCGAGCGTTGGACAGCCGCCGGCAAATAA-3’ None +215 to +247 relative to the pmrD start codon pmrDN78Ar1 5’-TTATTTGCCGGCGGCTGTCCAACGCTCGCTGC-3’ None +215 to +247 relative to the pmrD start codon Table 6.3. (continued)

126

Primer Sequence^a Enzyme

cleaved Complementary position

pmrDD80Af 5’-GCGTTGGACAAACGCCGCCAAATAACACAGATCTG-3’ None +222 to +257 relative to the pmrD start codon pmrDD80Ar 5’-CAGATCTGTGTTATTTGGCGGCGTTTGTCCAACGC-3’ None +222 to +257 relative to the pmrD start codon pmrHp01 5’-TCTGGATCCTGGTCATTAATTGCCCGGC-3’ BamHI -425 relative to the pmrH start codon

pmrHp02 5’-CTTAGATCTCGCTCATCATCATCCTGTTC-3’ BglII +34 relative to the pmrH start codon ppmrF01 5’-GATGGAAAAGCTGAAGGCGATGG-3’ None -161 relative to the pmrF start codon ppmrF02 5’-CAGCGATATCATACCCGGCGTC-3’ EcoRV +1116 relative to the pmrF start codon rcsAe04 5'-GTTTGTTTCACTCGAGGCGCATATTTACC-3' XhoI +631 relative to the rcsA start codon rcsAe07 5’-CTAGCGGCCGCGATGTCAACGATGATTATGGATT -3’ NotI +1 relative to the rcsA start codon rcsAe08 5’-CTAGGATCCCCATGTCAACGATGATTATGGATT-3’ BamHI +1 relative to the rcsA start codon rcsBc01 5’-CCCGGATCCAACTGCGGGTCAACTTT-3’ BamHI -398 relative to the rcsB start codon rcsBc02 5’-CCCGGATCCTTGTCTGTCCAAGCCGGTCA-3’ BamHI +781 relative to the rcsB start codon rcsBe01 5’-GGCCGCCTTATACCATATGAACACTA-3’ NdeI +1 relative to the rcsB start codon rcsBe02 5’-CCCTCGAGCTCTTTGTCCGTCGCGCTC-3’ XhoI +648 relative to the rcsB start codon rcsBe04 5’-CTCGCGGCCGCGATGAACACTATGAACGTAATTAT-3’ NotI -1 relative to the rcsB start codon rmpA06 5’-TTACCTAAATACTTGGCATGAGC-3’ None +592 relative to the rmpA start codon rmpA07 5’-CAAGGATCCAAAGCATAGTGTT-3’ BamHI -17 relative to the rmpA start codon rmpAc01 5’-CCCGGATCCAGAAACAGACAGTATTACTAAGCGAA-3’ BamHI -384 relative to the rmpA start codon rmpAe04 5’-CCCTTTTTTACCTCGAGAATACTTGGCATGA-3’ XhoI +585 relative to the rmpA start codon rmpAe05 5’-CTTGCGGCCGCGGTGTTGACTGATGATTATTTTTTTTA-3’ NotI +1 relative to the rmpA start codon rmpAe07 5’-CTTGGATCCCCGTGTTGACTGATGATTATTTTTTTTA-3’ BamHI -2 relative to the rmpA start codon rmpAg05 5’-TTGTGTTGACTGATGATTATTTT-3’ None +1 relative to the rmpA start codon Table 6.3. (continued)

127

Primer Sequence^a Enzyme

cleaved Complementary position

rmpAg06 5’-TTTACATTTCCTTGCAT-3’ None +299 relative to the rmpA start codon rmpAp04 5'-CCCAGATCTCAGTCAACACGGTGCTTTAC-3' BglII +10 relative to the rmpA start codon rmpAp05 5’-CCCGGATCCAACTCGCCCCTCCCCACAC-3’ BamHI -308 relative to the rmpA start codon rmpAp11 5’-CCAGGATCCTACCGTGATTGATTGAATTTTTA-3’ BamHI -184 relative to the rmpA start codon rmpAp12 5’-GTCGGATCCATCGCCAAATAACTC-3’ BamHI -479 relative to the rmpA start codon rmpAp13 5’-TCAATTAATTGCAAACACGC-3’ None -226 relative to the rmpA start codon rmpAt01 5’-ACAGAGGTAGTCCAGTTAACA-3’ None +61 relative to the rmpA start codon rmpA2g01 5’-TTATGGAAAAATATATTTACTT-3’ None +1 relative to the rmpA2 start codon rmpA2g02 5’-TTTAAATTTCCTTGCATGTT-3’ None +347 relative to the rmpA2 start codon rmpA2p06 5’-CCCGGATCCCACTTAGTCCTGTGTC-3’ BamHI -391 relative to the rmpA2 start codon rmpA2p07 5’-GATGGATCCCTAGGTATTTGATGTGCAC-3' BamHI +639 relative to the rmpA2 start codon rmpA2p08 5’-GATCTCGAGGGTATTTGATGTGCAC-3’ XhoI +639 relative to the rmpA2 start codon rmpA2p14 5’-CCCGCGGCCGCGATGGAAAAATATATTTACTT-3’ NotI -1 relative to the rmpA2 start codon rmpA2p17 5’-CCCGGATCCCCATGGAAAAATATATTTACTT-3’ BamHI +1 relative to the rmpA2 start codon rstAp01 5’-GCAGGATCCCGGTGAAATAC-3’ BamHI -378 relative to the rstA start codon rstAp02 5’-CCAGATCTTGCTTGCCGAG-3’ BglII +79 relative to the rstA start codon rstAp03 5’-CCCGGATCCCTTAACAGTGA-3’ BamHI -127 relative to the rstA start codon rstAp04 5’-GAGTAATGGCGGGTAAAATAAGTG-3’ None -61 relative to the rstA start codon RTrmpA01 5’-TGATGGATCAAAGTTACTGT-3’ None -70 relative to the rmpA start codon RTrmpA02 5’-TCCCTGAATAAAAAATCCTGCTGTC-3’ None +160 relative to the rmpA start codon Yu05 5’-CCTTCACATCCCCTCCCCTT-3’ None +614 relative to the rmpA start codon Table 6.3. (continued)

128

Primer Sequence^a Enzyme

cleaved Complementary position

Yu06 5’-GTCGGATCCATCGCCAAATAA-3’ None -479 relative to the rmpA start codon 23SF 5’-AGCGACTAAGCGTACACGGTGG-3’ None +4 relative to the rrnB start codon 23SR 5’-GATGTTTCAGTTCCCCCGGTTC-3’ None +200 relative to the rrnB start codon

a The nucleotide sequence recognized by each restriction enzyme listed are underlined; the boldface sequences indicate the altered nucleotides for the generation of point mutation in the PmrD coding sequence.

Table 6.3. (continued)

129

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在文檔中克雷白氏肺炎桿菌CG43雙分子系統與多黏菌素B抗性及莢膜多醣體生合成之調控 (頁 131-165)