Regulation of acrAB expression by cellular metabolites in Escherichia coli

Abstract

Objectives: Multidrug efflux pumps mediate resistance to antibiotics and other toxic compounds. We studied the role of AcrAB-TolC, the main efflux pump in Escherichia coli, in regulating gene expression.

Methods: Deletion mutants, an acrABp-lacZ fusion and reverse transcription-real-time quantitative PCR experiments were used to study the role of AcrAB-TolC and metabolism in regulating gene expression of the acrAB operon and its transcriptional regulators.

Results: Deletion of the acrB gene increased the expression of the acrAB operon. A similar induction of acrAB was found when acrA or tolC was deleted, and when the pump function was inhibited using phenylalanine-arginine-β-naphthylamide. The induction of acrAB in the ΔacrB strain was totally (AcrR or SoxS) or partially (SoxR or MarA) prevented when the genes for these acrAB regulators were also deleted. The expression of soxS and marA, but not of acrR, was increased in the ΔacrB strain, which also showed altered expression of many other genes related to different cellular processes, including motility. Deletion of the metabolic genes entA and entE (enterobactin biosysnthesis), glpX (gluconeogenesis), cysH (cysteine biosynthesis) and purA (purine biosynthesis) also prevented activation of the acrAB promoter in the ΔacrB strain. Addition of the enterobactin biosynthesis intermediate metabolite 2,3-dihydroxybenzoate induced the expression of acrAB.

Conclusions: These results together suggest a model in which the AcrAB-TolC pump effluxes cellular metabolites that are toxic and/or have a signalling role. If the pump is inactivated or inhibited, these metabolites would accumulate, inactivating AcrR and/or up-regulating soxS and marA expression, ultimately triggering the up-regulation of acrAB expression to restore homeostasis.

Keywords: AcrAB-TolC; acrR; gene regulation; marA; multidrug efflux; soxS.

Figure 1.

Effect of AcrAB-TolC inactivation or inhibition on acrAB expression. (a) acrABp-lacZ expression in the wild-type and ΔacrB strains measured by β-galactosidase assay using cells grown in different culture media. Statistically significant differences between both strains in each medium are shown as **P < 0.01 or ***P < 0.0001. The exact acrB⁻/acrB⁺ ratios—i.e. the induction of acrAB expression in the ΔacrB mutant compared with the wild-type—are shown above each pair of bars. (b) Effect of deletion of different components of the AcrAB-TolC pump on acrABp-lacZ expression measured using cells grown in LB. Significant differences between each mutant and the wild-type are shown as **P < 0.01 or ***P < 0.0001. (c) Cells were grown in LB medium in the presence of increasing concentrations of the efflux pump inhibitor PAβN to measure its effect on acrABp-lacZ expression. Statistically significant differences between the wild-type and the ΔacrB mutant both treated with the same concentration of PAβN were found at concentrations <100 µM (***P < 0.0001) but not ≥100 µM (P > 0.09). (a–c) All results are presented as average ± SEM (n = 3–4) and are shown normalized to acrABp-lacZ expression in the wild-type strain grown in LB. WT, wild-type.

Figure 2.

Effect of deletion of acrAB regulators on ΔacrB-mediated induction of acrABp-lacZ expression. Known regulators of acrAB were each deleted in both the wild-type (acrB⁺, light grey) and ΔacrB (acrB⁻, dark grey) parental strains to assess their role in ΔacrB-mediated induction of acrABp-lacZ expression; i.e. their effect on the acrB⁻/acrB⁺ ratio, which is shown above each corresponding pair of bars. The experiments were performed using cells grown in LB medium. The results are presented as average ± SEM (n = 4) and are shown normalized to acrABp-lacZ expression in the wild-type (acrB⁺ parental) strain. Statistically significant differences between the acrB⁻/acrB⁺ acrABp-lacZ ratio in each mutant compared with the ratio (2.3) of the parental strains are shown as *P < 0.02 or ***P < 0.0001.

Figure 3.

Effect of deletion of acrB on the expression of the acrAB regulators acrR, soxS and marA. The expression in exponential phase cultures grown in LB of the regulators involved in the feedback regulation of acrAB expression (soxS, marA and acrR), and of the control gene gapA, was measured by RT–qPCR in the wild-type and ΔacrB strains to assess whether it was affected by a lack of AcrAB-TolC. The results are presented as average ± SEM (n = 4) and are shown normalized to the expression of each gene in the wild-type strain. Statistically significant differences between the wild-type and ΔacrB strains are shown as **P < 0.01 or ***P < 0.0001. WT, wild-type.

Figure 4.

Effect of deletion of 29 metabolic genes on ΔacrB-mediated induction of acrAB expression. Twenty-nine metabolic genes were each deleted in both the wild-type (acrB⁺, light grey) and ΔacrB (acrB⁻, dark grey) parental strains to assess their role in ΔacrB-mediated induction of acrABp-lacZ expression; i.e. their effect on the acrB⁻/acrB⁺ ratio, which is shown above each corresponding pair of bars. The experiments were performed using cells grown in LB medium. The results are presented as average ± SEM (n = 4) and are shown normalized to acrABp-lacZ expression in the wild-type (acrB⁺ parental) strain. Data are shown only for the five metabolic genes (entA, entE, cysH, purA and glpX) whose inactivation had a significant effect (P < 0.02) on ΔacrB-mediated induction of acrAB expression (on the acrB⁻/acrB⁺ ratio). The full list of genes tested was: fes, entA, entB, entC, entE and entF (enterobactin metabolism); trpA, trpC, trpD and trpE (tryptophan biosynthesis); aroA (chorismate metabolism); idcA, acnA and acnB (tricarboxylic acid cycle); cysH (cysteine biosynthesis); metE (methionine biosynthesis); purA, purC and purH (purine biosynthesis); glpA, glpB, glpC, glpD, glpF, glpK, glpQ, glpR and glpT (glycerol and glycerol 3-phosphate metabolism, transport and regulation); and glpX (gluconeogenesis). WT, wild-type.

Figure 5.

Effect of externally added cellular metabolites on acrAB expression. (a) Wild-type cells were grown in LB supplemented with the metabolites DHB (4 mM; a similar concentration to that used in Chubiz and Rao) or F1,6P (5 mM) to measure their effect on acrABp-lacZ expression. (b) The effect of DHB was also measured in strains deleted for marA or marR. The results are presented as average ± SEM (n = 4) and are shown normalized to acrABp-lacZ expression in each strain grown without metabolite. Statistically significant differences for each strain between cells grown with and without metabolite are shown as *P < 0.02 or ***P < 0.0001. WT, wild-type.

Figure 6.

Proposed model for the regulation of acrAB expression by cellular metabolites. As detailed more extensively in the Discussion section, our results suggest that DHB and other unknown cellular metabolites normally excreted by the AcrAB-TolC pump accumulate in the ΔacrB strain, inactivating AcrR and inducing the expression of soxS and marA, ultimately up-regulating the expression of acrAB to restore homeostasis. Functional interactions are represented as arrows for activation/induction or as ‘┴’ for repression. Continuous lines indicate known interactions and broken lines indicate hypothetical interactions. DHB and two other putative metabolites are depicted as small shapes.