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Table 4 Bacterial strains and plasmids used in this study

From: Characterization of AcrD, a Resistance-Nodulation-Cell Division-type multidrug efflux pump from the fire blight pathogen Erwinia amylovora

Plasmid or strain Relevant characteristics or genotype a Reference or source
Plasmid   
pJET1.2 Apr, rep (pMB1) from pMBI responsible for replication Thermo scientific
pCAM-MCS Apr, pCAM140-derivative without mini-Tn5, contains the MCS of pBluescript II SK (+) [16]
pFCm1 Apr, Cmr, source of Cmr cassette flanked by FRT sequences [43]
pCAM-Km Kmr, variant of the gene replacement vector pCAM-MCS, Apr replaced by Kmr This study
pCAM-Km.acrD-Cm Kmr, Cmr, contains a 1.1-kb fragment of acrD from E. amylovora Ea1189, insertion of 1153-bp Cm-FRT cassette from pFCm1 in BamHI site This study
pBlueScript II SK(+) Apr, ColE1 origin Stratagene
pBlueScript II KS(+) Apr, ColE1 origin Stratagene
pBBR1MCS Cmr, ColE1 origin [44]
pBlueKS.acrD Apr, contains a 3.1-kb fragment carrying acrD of E. amylovora Ea1189 under control of lac promoter This study
pBlueKS.acrD-ext Apr, contains a 3.5-kb fragment carrying acrD of E. amylovora Ea1189 including promoter region under control of lac promoter This study
pBlueSK.acrD Apr, contains a 3.1-kb fragment carrying acrD of E. amylovora Ea1189 in opposite orientation with respect to lac promoter This study
pBlueSK.acrD-ext Apr, contains a 3.5-kb fragment carrying acrD of E. amylovora Ea1189 including promoter region in opposite orientation with respect to lac promoter This study
pBBR.egfp.TIR Cmr, contains the TIR-egfp-T0 cassette in pBBR1MCS in opposite orientation with respect to lac promoter [16]
pBBR.acrD-Pro.egfp Cmr, contains a 206-bp fragment carrying the promoter region of acrD, transcriptional fusion of acrD with egfp This study
pBBR.acrA-Pro.egfp Cmr, contains a 133-bp fragment carrying the promoter region of acrA, transcriptional fusion of acrA with egfp This study
pBlueSK.baeR Apr, contains a 0.7-kb fragment carrying baeR of E. amylovora Ea1189 under control of lac promoter This study
pET-28a(+) Kmr, f1 origin Novagen
pET28a.baeR Kmr, contains a 0.7-kb fragment carrying baeR of E. amylovora Ea1189, C-terminal translational fusion with His-tag This study
pCP20 Cmr, Apr, contains yeast Flp recombinase gene, rep (pSC101) responsible for temperature-sensitive replication [45]
pBAD24 Apr, pMB1 origin, araC [46]
pBAD24.baeR Apr, contains a 0.7-kb fragment carrying baeR of E. amylovora Ea1189 under control of PBAD promoter This study
Strain   
Escherichia coli   
XL1-Blue endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 F'[ ::Tn10 proAB+ lacIq Δ(lacZ)M15] hsdR17(rK - mK +) Stratagene
TG1 K-12 supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5, (rK -mK -) [47]
KAM3 acrAB mutant of TG1 [48]
BL21(DE3) F ompT gal dcm lon hsdSB(rB - mB -) (λDE3) Novagen
S17-1 TpR SmR recA, thi, pro, hsdR-M+RP4: 2-Tc:Mu: Km [49]
S17-1 λ-pir λpir phage lysogen of S17-1 [49]
DH5α λ-pir sup E44, ΔlacU169 (ΦlacZΔM15), recA1, endA1, hsdR17, thi-1, gyrA96, relA1, λpir phage lysogen D. Lies, Caltech
Erwinia amylovora   
Ea1189 Wild type GSPBb
Ea1189-3 Kmr, acrB mutant carrying Kmr cassette in the acrB gene [16]
Ea1189.acrD acrD mutant This study
  1. aApr, ampicillin resistance; Cmr, chloramphenicol resistance; Kmr, kanamycin resistance.
  2. bGSPB, Göttinger Sammlung Phytopathogener Bakterien, Göttingen, Germany.