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The AcrAB-TolC efflux pump is involved in maintaining intrinsic organic solvent tolerance in  Escherichia coli.  Mutations in regulatory genes such as  marR ,  soxR , and  acrR  are known to increase the expression level of the AcrAB-TolC pump. To identify these mutations in organic solvent tolerant  E .  coli , eight cyclohexane-tolerant  E .  coli  JA300 mutants were isolated and examined by DNA sequencing for mutations in  marR ,  soxR , and  acrR . Every mutant carried a mutation in either  marR  or  acrR . Among all mutants, strain CH7 carrying a nonsense mutation in  marR  (named  marR109 ) and an insertion of IS 5  in  acrR , exhibited the highest organic solvent-tolerance levels. To clarify the involvement of these mutations in improving organic solvent tolerance, they were introduced into the  E. coli  JA300 chromosome by site-directed mutagenesis using λ red-mediated homologous recombination. Consequently, JA300 mutants carrying  acrR ::IS 5 ,  marR109 , or both were constructed and named JA300  acrR IS, JA300  marR , or JA300  acrR IS  marR , respectively. The organic solvent tolerance levels of these mutants were increased in the following order: JA300 < JA300  acrR IS < JA300  marR  < JA300  acrR IS  marR . JA300  acrR IS  marR  formed colonies on an agar plate overlaid with cyclohexane and  p -xylene (6:4 vol/vol mixture). The organic solvent-tolerance level and AcrAB-TolC efflux pump-expression level in JA300  acrR IS  marR  were similar to those in CH7. Thus, it was shown that the synergistic effects of mutations in only two regulatory genes,  acrR  and  marR , can significantly increase organic solvent tolerance in  E. coli .

Contributions of mutations in acrR and marR genes to organic solvent tolerance in Escherichia coli