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

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.

MarA, SoxS and Rob of Escherichia coli – Global Regulators of Multidrug Resistance, Virulence and Stress Response

“…Oh and co-workers [107] showed that marR deletion in E. coli , together with deletion of fadR , which changes the composition of the inner membrane to make it less permeable to organic solvents, caused an increase in growth and cell viability with organic solvents, and also decreased organic solvent accumulation within the bacteria. In a similar vein, Watanabe and Doukyu [108] identified mutations in AcrR and MarR that maximize the tolerance of the bacteria to organic solvents. However, in a different biofuel study [109], overexpression of the TRs (MarA, SoxS and Rob) in strains that overproduce free fatty acids (FFAs) that are potential precursors for high density biofuels did not lead to an increased tolerance for FFAs.…”

Efflux systems in bacteria and their metabolic engineering applications

“…After all, E. coli possesses its own hydrocarbon tolerance mechanisms dependent on the soxS, marA and robA stress-responsive genes. For example, constitutive activation of marA along with deletion of acrR , the repressor for acrAB multidrug efflux system, is known to confer resistance to lethal concentrations of cyclohexane ( log P ow = 3.44) (Oh et al, 2012; Watanabe and Doukyu, 2012). …”

“…coli , however, little is known about how these molecules are exiting the cells (Alonso-Gutierrez et al, 2013). Overexpression of marA , a stress response activator in E. coli , confers resistance to geraniol, a monoterpene alcohol (Shah et al, 2013) and activation of marA leads to upregulation of the AcrAB-TolC efflux system suggesting that AcrAB-TolC mediates the extrusion of terpenes in E. coli (Watanabe and Doukyu, 2012). Given this, and using an E. coli strain that produces the sesquiterpene amorphadiene or the diterpene kaurene, Wang et.…”

Emerging tools, enabling technologies, and future opportunities for the bioproduction of aromatic chemicals

“…In E. coli , acrAB is known to be regulated by multiple transcription factors including marA, marR, acrR, and soxR . Recently it was shown that the simultaneous introduction of a nonsense mutation in marR and insertion into acrR could be used to enhance tolerance to multiple solvents including cyclohexane and p‐xylene, likely by upregulating the acrAB expression . Mingardon et al ., meanwhile, recently demonstrated that the presence of acrAB is important for optimal production of styrene in E. coli , however, directed evolution did not improve its function on styrene, or resulting styrene tolerance/production .…”