Date Published: November 13, 2012
Author(s): Rei Watanabe, Noriyuki Doukyu.
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.
Whole-cell biocatalysts are beneficial in the biotransformation involved in their internal cofactor regeneration and in bioconversions requiring multi-step metabolic pathways. Whole-cell biocatalysts are available in two-phase systems consisting of organic solvent and an aqueous medium, that are potentially advantageous for the bioconversion of hydrophobic and/or toxic organic compounds (Schmid et al.
Sardessai and Bhosle 2004
; Heipieper et al.
). The use of a second organic phase improves productivity levels and product recovery, unlike the case with conventional media whose substrate solubility is poor. One of the main limitations in the application of whole-cell biocatalysts in two-phase systems is the instability of biocatalysts due to the toxicity of organic solvents toward the cells. When microorganisms are incubated in the presence of a large amount of an organic solvent, the extent of growth inhibition is inversely correlated with the log POW of the solvent (
Inoue and Horikoshi 1989
). Hydrophobic organic solvents with a log POW of 2 to 5 bind to the cells and disrupt the cell membrane (Sikkema et al.
; Aono et al.
In the present study, we isolated cyclohexane-tolerant mutants from cyclohexane-sensitive E. coli K-12 strain JA300 and investigated whether or not these mutants carried mutations in regulatory genes marR, soxR, and acrR. Most of the mutants carried mutations in marR. 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 (marR109 mutation). These MarR mutations lie within the region spanning amino acids 61 to 121 in MarR, which are required for DNA binding activity (Alekshun et al.
). The L78 and R94 residues are highly conserved amino acids in many homologs, although the amino acid at the position of G116 is not conserved (Alekshun et al.
). The marR109 mutation led to the truncation of 35 C-terminal amino acids in MarR. A previous study showed that the C-terminal region contributes to dimer formation (Notka et al.
). In any case, the MarR mutations found in the present study are considered to lead to the loss of repressive function. Three mutants isolated in this study carried different mutations in acrR. Two mutations in acrR caused amino acid substitutions in AcrR at the amino acid positions of M1 and A41. The mutation in the translation initiation codon (M1I) will cause the complete inhibition of AcrR translation. The A41 residue positioned within the helical region (α3) forms part of a typical helix-turn-helix motif involved in DNA binding (Li et al.
). Thus, the A41D mutation will abolish AcrR’s repressive activity. Many IS elements have been shown to activate or inactivate the expression of neighboring genes. In strain CH7, the IS5 inserted within acrR seemed to activate the expression of acrAB through the disruption of transcriptional repression by AcrR. The alteration of organic solvent tolerances of several E. coli mutants by IS integration has been reported. E. coli OST4251 became sensitive to n-hexane because IS2 and IS5 became integrated upstream from the imp/ostA gene, which is involved in organic solvent sensitivity (Abe et al.
; Ohtsu et al.
). The hypersensitivity of an E. coli acrB disruptant to organic solvents was suppressed by the integrational activation of the acrEF operon with an IS1 or IS2 element (Kobayashi et al.
The authors declare that they have no competing interests.