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Table 3 Phenotypic and functional effects of selected marC, acrAB, mdh, and rph mutations

From: Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli

    0% i-BtOH 0.5% i-BtOH  
Locus Gene/mutation Clone Δμmax ΔODmax Δμmax ΔODmax Functional Effect
marC ΔmarC::kan (xylose media) X1,X2,X3 (IS1/IS5 insertion) -5.0 ± 0.5% -2.4 ± 0.1% 39 ± 2% 7.6 ± 0.5% -
  ΔmarC::kan (glucose media) G1,G3 (IS1 insertion) -3.2 ± 0.2% -7.8 ± 0.3% 20 ± 5% 40 ± 10% -
acrAB acrA 483735 +1:A X3.5 14 ± 1% 3.3 ± 0.1% 49 ± 9% 72.4 ± 0.8% Reduced EtBR efflux; 188 ± 7% increase in intracellular EtBr
  ΔacrA::kan N/A (control) 6.5 ± 0.6% 7.6 ± 0.5% 32 ± 5% 103 ± 1% Reduced EtBR efflux; 210 ± 10% increase in intracellular EtBr
  acrB 480665 G→A G3.2 11.2 ± 0.9% 2.1 ± 0.1% 22 ± 3% 31 ± 1% Reduced EtBR efflux; 21 ± 8% increase in intracellular EtBr
  ΔacrB::kan N/A (control) 5.7 ± 0.6% -3.6 ± 0.1% 8.2 ± 1.1% 64 ± 2% Reduced EtBR efflux; 340 ± 20% increase in intracellular EtBr
mdh mdh 3390936 +5:AACCT X3.5 -3.7 ± 0.3% -1 ± 0.1% 0.4 ± 0.1% 13 ± 2% Loss of function; no detectable mdh activity
  mdh 3390726 -1:C G3.2 2.3 ± 0.1% 4.4 ± 0.1% -8.1 ± 3% -1.2 ± 0.1% Loss of function; no detectable mdh activity
  Δmdh::kan N/A (control) -1.6 ± 0.2% 4 ± 0.1% 10.8 ± 1.6% 10 ± 1% Loss of function; no detectable mdh activity
rph rph 3823220 +4:GTCG G3.2 39 ± 2% 11.9 ± 0.1% 49 ± 16% -12.9 ± 0.1% -
  1. Selected point mutations in acrAB, mdh, and rph identified in isobutanol tolerant clones were reconstructed in the parent E. coli EcNR1 strain using ssDNA mediated mutagenesis. marC transposon insertions could not be produced with ssDNA mutagenesis; instead, we approximated the effect of transposon insertions by knocking out marC. Reconstructed mutants were phenotyped by measuring specific growth rate in minimal media with 0% and 0.5% (w/v) isobutanol; percent change in growth rate and maximum OD600 relative to the parent E. coli EcNR1 are reported. Functional effects of acrAB mutations were assessed with an in vivo ethidium bromide accumulation assay and mdh mutations were assessed by directly measuring malate dehydrogenase enzyme activity in cell lysates.