Supplemental Figure S2. Circular Dichroism Spectra Form E. Coli Acra Mutant Proteins For

Supplemental Figures

Supplemental Figure S1. Expression of AcrA in E. colistrain BW25113ΔacrAused for Table 1. The hexa-histidine-tagged AcrA was visualized using anti-His-antibody (His) in western blotting, and S1 protein was shown as a loading control.

Supplemental Figure S2. Circular dichroism spectra form E. coli AcrA mutant proteins for Table 1.

All E. coliAcrA proteins (residues 26 – 397) were purified using Ni-NTA affinity chromatography and subsequently dialyzed against 20 mM Tris buffer containing 150 mM NaCl. Wild type and mutant AcrA proteins (R128D, L132D, and S139D) show similar spectra, confirming that mutant AcrA proteins are correctly folded.

SupplementalFigure S3. Expression of AcrA in E. colistrain BW25113ΔacrAused for Table 2. The hexa-histidine-tagged AcrA was visualized using anti-His-antibody (His) in western blotting, and S1 protein was shown as a loading control.

SupplementalFigure S4. Expression of AcrA and TolC in E. colistrain BW25113 ∆acrA ∆tolC210::Tn10used for Table 3. The hexa-histidine-tagged AcrA and TolC were visualized using anti-His-antibody (His) in western blotting, and S1 protein was shown as a loading control.

Supplemental Figure S5. Expression of AcrA cysteine variants in E. colistrain BW25113 ∆acrAused for Table S2. The hexa-histidine-tagged AcrA was visualized using anti-His-antibody (His) in western blotting, and S1 protein was shown as a loading control.

Supplemental Table S1. Constructed plasmids in this study

Plasmid / Cloning sites / Primer / Sequence
pAcrA-MacA hybrid 1 / NdeI/ XbaI / acrA-F(Nde I) / 5` ACATATGAACAAAAACAGAGGGT
Hybrid-6 / 5`TCGTTTGTGCCAGACGTTGCTGACGGGAATACGTCACCCGCGCCAGATTGGCTGCAGCCTGGGCTTT
Hybrid-5 / 5`GCAACGTCTGGCACAAACGAAGGCTGTTTCACAGCAGGATCTCGACCAGGCTCTGGCTGATGCGCAA
acrA-R(Xba I) / 5` GGGTCTAGATTAATGATGATGATGATGATGAGACTTGGACTGTTCAGG
pAcrA-MacAhybrid 2 / NdeI/ XbaI / acrA-F(Nde I) / 5` ACATATGAACAAAAACAGAGGGT
5`CGTTTGTGCCAGACGTTGCTGACGGGAATACGTCACCCGCGCCAGGGCTGCAGCCTGGGCTTTCGC
H2-(-N120)-R
Hybrid-5 / 5`GCAACGTCTGGCACAAACGAAGGCTGTTTCACAGCAGGATCTCGACCAGGCTCTGGCTGATGCGCAA
5` GGGTCTAGATTAATGATGATGATGATGATGAGACTTGGACTGTTCAGG
acrA-R(XbaI)
pAcrA-MacA hybrid 3 / NdeI/ XbaI / acrA-F(NdeI) / 5` ACATATGAACAAAAACAGAGGGT
Hybrid-6 / 5`TCGTTTGTGCCAGACGTTGCTGACGGGAATACGTCACCCGCGCCAGATTGGCTGCAGCCTGGGCTTT
H3-(-Q145)-F / 5`GCAACGTCTGGCACAAACGAAGGCTGTTTCACAGCAGGATCTCGACGCTCTGGCTGATGCGCAACAG
acrA-R(XbaI) / 5` GGGTCTAGATTAATGATGATGATGATGATGAGACTTGGACTGTTCAGG
pAcrA-MacA hybrid 4 / Nde I/ XbaI / acrA-F(NdeI) / 5` ACATATGAACAAAAACAGAGGGT
H2-(-N120)-R / 5`CGTTTGTGCCAGACGTTGCTGACGGGAATACGTCACCCGCGCCAGGGCTGCAGCCTGGGCTTTCGC
H3-(-Q145)-F / 5`GCAACGTCTGGCACAAACGAAGGCTGTTTCACAGCAGGATCTCGACGCTCTGGCTGATGCGCAACAG
acrA-R(XbaI) / 5` GGGTCTAGATTAATGATGATGATGATGATGAGACTTGGACTGTTCAGG
pAcrA-MacA hybrid 5 / NdeI/ XbaI / acrA-F(NdeI) / 5` ACATATGAACAAAAACAGAGGGT
acrA-macA-H-R / 5` CTGATAGAGAGAGACACCTGC
hybrid-9 / 5` GCAGGTGTCTCTCTCTATCAGATTGATCCTGAACAGGCTGAAAAC
hybrid-4 / 5` GCTAATCGGAGAGGTGACTTTAGTGTAATCGAGATTGGTTTT
hybrid-3 / 5` AAAGTCACCTCTCCGATTAGC
acrA-R(XbaI) / 5` GGGTCTAGATTAATGATGATGATGATGATGAGACTTGGACTGTTCAGG
pAcrA1 / NdeI/ XbaI / acrA-F(NdeI) / 5` ACATATGAACAAAAACAGAGGGT
acrA-R(XbaI) / 5` GGGTCTAGATTAATGATGATGATGATGATGAGACTTGGACTGTTCAGG
pAcrA1-R128D / NdeI/ XbaI / acrA-F(NdeI) / 5` ACATATGAACAAAAACAGAGGGT
acrA-R-R128D / 5` CAGTTTCTGATAATCATTCACCGTC
acrA-F-R128D / 5` GACGGTGAAGTATTATCAGAAACTG
acrA-R(XbaI) / 5`GGGTCTAGATTAATGATGATGATGATGATGAGACTTGGACTGTTCAGG
pAcrA1-L132D / NdeI/ XbaI / acrA-F(NdeI) / 5` ACATATGAACAAAAACAGAGGGT
acrA-R-L132D / 5` ACCGAGATCTTTCTGATAACG
acrA-F-L132D / 5` CGTTATCAGAAAGATCTCGGT
acrA-R(XbaI) / 5`GGGTCTAGATTAATGATGATGATGATGATGAGACTTGGACTGTTCAGG
pAcrA1-S139D / NdeI/ XbaI / acrA-F(NdeI) / 5` ACATATGAACAAAAACAGAGGGT
acrA-R-S139D / 5` CTCTTGCTTATCGATGTACTG
acrA-F-S139D / 5` CAGTACATCGATAAGCAAGAG
acrA-R(XbaI) / 5` GGGTCTAGATTAATGATGATGATGATGATGAGACTTGGACTGTTCAGG
pTolC1 / NdeI/ XbaI / tolC-F(NdeI) / 5` ACATATGAAGAAATTGCTCCCCAT
tolC-R(XbaI) / 5` GGGTCTAGATCAATGATGATGATGATGATGGTTACGGAAAGGGTT
pTolC1-M / NdeI/ XbaI / tolC-F(NdeI) / 5` ACATATGAAGAAATTGCTCCCCAT
tolC-M1 / 5`TCGTACTGTGCGCGGGCGTTCTGCACGTCGGTGATCGCTACCAGGCCCACGTTAAAACGTTGGGTGGT
tolC-466F / 5` AAC GCC CGC GCA CAG TAC GA
tolC-KpnI-R / 5` ATA CTG GGT ACC AGC GGC AC
tolC-KpnI-F / 5` GTG CCG CTG GTA CCC AGT AT
tolC-M2 / 5`TACAACGGGTGGCGCATCCAACACATCAACAATGGTACGCGTACCGACCGAGTAGCCCGCTTCCAT
tolC-1120F / 5`GAT GCG ACC ACC ACG TTG TA
tolC-R(XbaI) / 5` GGGTCTAGATCAATGATGATGATGATGATGGTTACGGAAAGGGTT
pTolC1-V146D / NdeI/ XbaI / tolC-F(NdeI) / 5` ACATATGAAGAAATTGCTCCCCAT
tolC-R-V146D / 5` TACCAGGCCGTCGTTAAAACG
tolC-F-V146D / 5` CGTTTTAACGACGGCCTGGTA
tolC-R(XbaI) / 5` GGGTCTAGATCAATGATGATGATGATGATGGTTACGGAAAGGGTT
pTolC1-I151D / NdeI/ XbaI / tolC-F(NdeI) / 5` ACATATGAAGAAATTGCTCCCCAT
tolC-R-I151D / 5` CACGTCGGTGTCCGCTACCAG
tolC-F-I151D / 5` CTGGTAGCGGACACCGACGTG
tolC-R(XbaI) / 5` GGGTCTAGATCAATGATGATGATGATGATGGTTACGGAAAGGGTT
pTolC1-K345E / NdeI/ XbaI / tolC-F(NdeI) / 5` ACATATGAAGAAATTGCTCCCCAT
tolC-R-K345E / 5` TACGGCTTGTTCGTAGGCGTT
tolC-F-K345E / 5` AACGCCTACGAACAAGCCGTA
tolC-R(XbaI) / 5` GGGTCTAGATCAATGATGATGATGATGATGGTTACGGAAAGGGTT
pTolC1-V364D / NdeI/ XbaI / tolC-F(NdeI) / 5` ACATATGAAGAAATTGCTCCCCAT
tolC-R-V364D / 5` ACGCGTACCGTCCGAGTAGCC
tolC-F-V364D / 5` GGCTACTCGGACGGTACGCGT
tolC-R(XbaI) / 5` GGGTCTAGATCAATGATGATGATGATGATGGTTACGGAAAGGGTT
pTolC1-I369D / NdeI/ XbaI / tolC-F(NdeI) / 5` ACATATGAAGAAATTGCTCCCCAT
tolC-R-I369D / 5` CACATCAACGTCGGTACGCGT
tolC-F-I369D / 5` ACGCGTACCGACGTTGATGTG
tolC-R(XbaI) / 5` GGGTCTAGATCAATGATGATGATGATGATGGTTACGGAAAGGGTT
pAcrAB1 / NotI/DraIII/
XbaI / acrAB+1(NotI) / 5` CGCGGCCGCACGATAATATAAACG CAG CA
acrAB-R(DraIII) / 5` ACCACTTCGTGAATAGAGATTTTCACGA
acrAB-F(DraIII) / 5` TCGTGAAAATCTCTATTCACGAAGTGGT
acrAB-T(Xba1) / 5` TCTAGAATCAATGATGATCGACAGTAT
acrAB(XbaI)-R / 5` TCTAGAATCAATGATGATCGACAGTAT
pAcrAB(His)1 / NotI/DraI/
XbaI / acrAB+1(NotI) / 5` CGCGGCCGCACGATAATATAAACG CAG CA
acrAB-R(DraIII) / 5` ACCACTTCGTGAATAGAGATTTTCACGA
acrAB-F(DraIII) / 5` TCGTGAAAATCTCTATTCACGAAGTGGT
acrB(his-C) / 5` GTCTAGAATCAATGATGATGATGATGATGATCGACAGTATGGC / acrAB+1(Not1) / 5` CGCGGCCGCACGATAATATAAACG CAG CA
pPro-AcrA-R128D / NcoI/XhoI / acrAp-F(NcoI)
acrAp-R-R128D / 5’GGGCCATGGACGACGACAAACAGGCCC
5’CAGTTTCTGATAATCATTCACCGTC / acrAB-R(DraⅢ) / 5` ACCACTTCGTGAATAGAGATTTTCACGA
acrAp-F-R128D
acrAp-R(XhoI) / 5’GACGGTGAATGATTATCAGAAACTG
5’GGGCTCGAGTTAAGACTTGGACTGTTCAGGC
pPro-AcrA-L132D / NcoI/XhoI / acrAp-F(NcoI)
acrAp-R-L132D / 5’GGGCCATGGACGACGACAAACAGGCCC
5’ACCGAGATCTTTCTGATAACG / acrAB-F(DraⅢ) / 5` TCGTGAAAATCTCTATTCACGAAGTGGT
acrAp-F-L132D
acrAp-R(XhoI) / 5’CGTTATCAGAAAGATCTCGGT
5’GGGCTCGAGTTAAGACTTGGACTGTTCAGGC
pPro-AcrA-S138D / NcoI/XhoI / acrAp-F(NcoI)
acrAp-R-S138D / 5’GGGCCATGGACGACGACAAACAGGCCC
5’CTCTTGCTTATCGATGTACTG / acrB(his-C) / 5` GTCTAGAATCAATGATGATGATGATGATGATCGACAGTATGGC
acrAp-F-S138D
acrAp-R(XhoI) / 5’CAGTACATCGATAAGCAAGAG
5’GGGCTCGAGTTAAGACTTGGACTGTTCAGGC
pAcrA2-A79C / NotI/XbaI / acrA-F(NotI) / 5’ TTGCGGCCGCATGAACAAAAACAGAGGGT
acrA-R-A79C / 5’ACTGTCGTATGTACACTGATAGGTCGC
acrA-F-A79C / 5’GCGACCTATCAGTGTACATACGACAGT
acrA-R(XbaI) / 5` GGGTCTAGATTAATGATGATGATGATGATGAGACTTGGACTGTTCAGG
pAcrA2-D87C / NotI/XbaI / acrA-F(NotI) / 5’ TTGCGGCCGCATGAACAAAAACAGAGGGT
acrA-R-D87C / 5’ GGCTTTCGCCAGACAACCTTTCGCACT
acrA-F-D87C / 5’ AGTGCGAAAGGTTGTCTGGCGAAAGCC
acrA-R(XbaI) / 5` GGGTCTAGATTAATGATGATGATGATGATGAGACTTGGACTGTTCAGG
pAcrA2-L100C / NotI/XbaI / acrA-F(NotI) / 5’ TTGCGGCCGCATGAACAAAAACAGAGGGT
acrA-R-L100C / 5’ ACGATTCACCGTACATTGCGCGATATT
acrA-F-L100C / 5’ AATATCGCGCAATGTACGGTGAATCGT
acrA-R(XbaI) / 5` GGGTCTAGATTAATGATGATGATGATGATGAGACTTGGACTGTTCAGG
pAcrA2-R104C / NotI/XbaI / acrA-F(NotI) / 5’ TTGCGGCCGCATGAACAAAAACAGAGGGT
acrA-R-R104C / 5’CAGTTTCTGATAACAATTCACCGTCAA
acrA-F-R104C / 5’ TTGACGGTGAATTGTTATCAGAAACTG
acrA-R(XbaI) / 5` GGGTCTAGATTAATGATGATGATGATGATGAGACTTGGACTGTTCAGG
pAcrA2-E118C / NotI/XbaI / acrA-F(NotI) / 5’ TTGCGGCCGCATGAACAAAAACAGAGGGT
acrA-R-E118C / 5’ AGCCTGATCGTAACATTGCTTACTGAT
acrA-F-E118C / 5’ ATCAGTAAGCAATGTTACGATCAGGCT
acrA-R(XbaI) / 5` GGGTCTAGATTAATGATGATGATGATGATGAGACTTGGACTGTTCAGG
pAcrA2-Q112C / NotI/XbaI / acrA-F(NotI) / 5’ TTGCGGCCGCATGAACAAAAACAGAGGGT
acrA-R-Q112C / 5’CTTACTGATGTAACAAGTACCGAGCAG
acrA-F-Q112C / 5’ CTGCTCGGTACTTGTTACATCAGTAAG
acrA-R(XbaI) / 5` GGGTCTAGATTAATGATGATGATGATGATGAGACTTGGACTGTTCAGG

Supplemental TABLES2. Antibiotic sensitivities of bacterial strains expressing various AcrA cysteine variantsa

AcrA / null / Wild-type / A79C / D87C / L100C / R104C / E118C / Q112C
MIC (µg/ml)b
Novobiocin / 6.25 / 100 / 50 / 50 / 50 / 6.25 / 12.5 / 25

aThe degree of resistance of E. coli strain BW25113ΔacrA that expressed AcrA cysteine variantstovancomycin was measured as MIC.The level of AcrA protein expressed is shown in Supplemental Fig. S5.

b Novobiocin concentrations used to measure MICs were 0, 0.78, 1.56, 3.13, 6.25, 12.50, 25.00, 50.00, and 100.00 μg/ml.

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