AcrB, AcrD, and MdtABC multidrug efflux systems are involved in enterobactin export in Escherichia coli

Abstract

Escherichia coli produces the iron-chelating compound enterobactin to enable growth under iron-limiting conditions. After biosynthesis, enterobactin is released from the cell. However, the enterobactin export system is not fully understood. Previous studies have suggested that the outer membrane channel TolC is involved in enterobactin export. There are several multidrug efflux transporters belonging to resistance-nodulation-cell division (RND) family that require interaction with TolC to function. Therefore, several RND transporters may be responsible for enterobactin export. In this study, we investigated whether RND transporters are involved in enterobactin export using deletion mutants of multidrug transporters in E. coli. Single deletions of acrB, acrD, mdtABC, acrEF, or mdtEF did not affect the ability of E. coli to excrete enterobactin, whereas deletion of tolC did affect enterobactin export. We found that multiple deletion of acrB, acrD, and mdtABC resulted in a significant decrease in enterobactin export and that plasmids carrying the acrAB, acrD, or mdtABC genes restored the decrease in enterobactin export exhibited by the ΔacrB acrD mdtABC mutant. These results indicate that AcrB, AcrD, and MdtABC are required for the secretion of enterobactin.

Figure 1. RP HPLC analysis of enterobactin release from the wild-type strain and the ΔtolC mutant.

(A) Chromatograms of enterobactin prepared from the supernatants of the wild-type strain and the ΔtolC mutant. Peaks were identified using HPLC-grade standards. (B) The amount of enterobactin exported by ΔtolC mutant relative to that exported by the wild-type was calculated using the peak areas. The peak area corresponding to enterobactin released from the wild-type strain was defined as 100%.

Figure 2. RP HPLC analysis of enterobactin released from deletion mutants of the RND-type efflux system genes.

The amounts of enterobactin exported by deletion mutants calculated using each peak area are shown. The peak area corresponding to enterobactin released from the wild-type strain was defined as 100%. The data corresponds to mean values from three independent replicates. The bars indicate standard deviations. Asterisks indicate statistically significant differences (p<0.01) according to two-tailed Student’s t-tests.

Figure 3. Requirement of AcrB, AcrD, and MdtABC drug efflux systems for enterobactin export.

Enterobactin was prepared from the supernatants of cultures of each multiple RND transporter mutant and analyzed by RP HPLC. The amount of enterobactin exported by each strain, calculated using each peak area, is shown. The amount of enterobactin released from the wild-type strain was defined as 100%. The data correspond to mean values from three independent replicates. The bars indicate standard deviations. Asterisks indicate statistically significant differences (p<0.01) according to the two-tailed Student’s t-tests.

Figure 4. Complementation of enterobactin release from the ΔacrB acrD mdtABC mutant using plasmids carrying acrB, acrD, or mdtABC genes.

The amounts of enterobactin exported by deletion mutants, calculated using peak areas, are shown. The amount of enterobactin released from the wild-type strain harboring an empty vector was defined as 100%. The data corresponds to mean values from three independent replicates. The bars indicate standard deviations. Asterisks indicate statistically significant differences (p<0.01) determined using the two-tailed Student’s t-tests.

Figure 5. Proposed model of enterobactin export in E. coli.

Enterobactin is synthesized in the cytoplasm and exported to the periplasm by EntS. The RND transporters AcrB, AcrD, and MdtABC capture enterobactin in the periplasm and then export it to the growth medium throughout the outer membrane channel TolC.