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

Rei Watanabe,Noriyuki Doukyu

doi:10.1186/2191-0855-2-58

Abstract

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 IS5 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::IS5, marR109, or both were constructed and named JA300 acrRIS, JA300 marR, or JA300 acrRIS marR, respectively. The organic solvent tolerance levels of these mutants were increased in the following order: JA300 < JA300 acrRIS < JA300 marR < JA300 acrRIS marR. JA300 acrRIS 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 acrRIS 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.

Highlights

Whole-cell biocatalysts are beneficial in the biotransformation involved in their internal cofactor regeneration and in bioconversions requiring multi-step metabolic pathways
Isolation of cyclohexane-tolerant mutants of E. coli JA300 E. coli K-12 strain JA300 is tolerant to an organic solvent with a logPow value of more than 4 and is highly sensitive to cyclohexane (Aono 1998)
Three of the seven mutations found in marR caused amino acid substitutions in MarR at the amino acid positions of L78, R94, and G116, and four of the seven mutations led to a translation termination codon at the position of E109

Summary

Introduction

Whole-cell biocatalysts are beneficial in the biotransformation involved in their internal cofactor regeneration and in bioconversions requiring multi-step metabolic pathways. In E. coli, the AcrAB-TolC efflux pump belonging to the RND family has been shown to provide intrinsic tolerance to organic solvents (Tsukagoshi and Aono 2000). Mutations conferring a multidrug resistance phenotype have been found in the genes marR, soxR, and acrR among clinical and veterinary E. coli isolates (Wang et al 2001; Webber and Piddock 2001; Komp Lindgren et al 2003; Fernandes et al 2003; Karczmarczyk et al 2011 ) Some of those studies suggested that organic solvent tolerance is correlated with these mutations in these isolates (Wang et al 2001; Komp Lindgren et al 2003). To clarify the effects of mutations on the tolerance phenotype, it is necessary to reconstruct selected mutations in one type of strains in various combinations

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Conclusion