The involvement of multiple ABC transporters in daunorubicin efflux in Streptomyces coeruleorubidus

Abstract

Streptomyces genus produces a large number of antibiotics, which are always synthesized by specific biosynthetic gene clusters (BGCs). To resist autotoxicity, transporters encoded by genes located within BGC occasionally pump antibiotic along with transporter encoded by gene located outside BGC. Daunorubicin is an anthracycline antibiotic biosynthesized by Streptomyces species, playing a crucial role in the treatment of leukaemia. In existing studies, only one two-component ATP-binding cassette (ABC) transporter, encoded by drrA1-drrB1 (abbreviated as drrAB1) and located within the daunorubicin BGC, has been proven to extrude daunorubicin. In this work, two other two-component ABC transporters, encoded by drrAB2 and drrAB3 and located outside the cluster, were found to play the complementary role in daunorubicin efflux in S. coeruleorubidus. Disruption of three drrABs resulted in a 94% decrease in daunorubicin production. Furthermore, drrAB2 is regulated by the TetR family regulator DrrR1, responding to the intracellular accumulation of daunorubicin and suggesting its role in stress response and self-resistance. Although the homologues of DrrAB1 are only found in three anthracycline BGCs, the homologues of DrrAB2 and DrrAB3 are spread in many Streptomyces strains which do not contain any known anthracycline BGC. This indicates that DrrAB2 and DrrAB3 may recognize and extrude a broader range of substrates besides daunorubicin, thus playing a more extensive role in cellular detoxification.

FIGURE 1

Disruption of drrAB1 impairs the production of DNR in S. coeruleorubidus. DNR titre (A), HPLC analysis (B), and biomass (C) of the S. coeruleorubidus WT strain and the ΔdrrAB1 mutant. The relevant strains were cultured in NDYE medium for 120 h. Significance markers indicate the comparison between WT and ΔdrrAB1, analysed using the Mann–Whitney test. *denotes p < 0.05, and ns denotes no significance.

FIGURE 2

Comparative transcriptomic analysis between the WT strain and the ΔDNR mutant of S. coeruleorubidus. (A) Colour comparison of the substrate mycelium cultured for 5 days. (B) Volcano map of differentially expressed genes. (C) The FPKM values of drrABs in the WT strain and the ΔDNR mutant. (D) RT‐qPCR data of the drrABs in the WT and the ΔDNR mutant. The relevant strains were cultured in fermentation medium for 48 h to exponential growth phase. Then the mycelia in fermentation broth were collected for RNA‐seq and RT‐qPCR. Significance markers indicate the comparison between WT and ΔDNR, analysed using the Mann–Whitney test. **denotes p < 0.01, and ns denotes no significance.

FIGURE 3

DrrAB1, DrrAB2 and DrrAB3 participate in DNR efflux. (A) Deletion of drrABs reduces the DNR production in S. coeruleorubidus. (B) HPLC analysis of the ΔdrrAB mutants. (C) log2Foldchange of the expression levels of drrABs and dnrI of the mutants compared with that of the WT strain. (D) DNR resistance assay. The cultures were diluted to OD600 values of 2 per ml, and then 5 μL of bacterial suspensions were spotted onto plates and incubated for 5 days. Significant differences between mutant strains and the wild‐type were determined by One‐way ANOVA, where **denotes p < 0.01 and **** denotes p < 0.0001.

FIGURE 4

Complementation of drrAB1/2/3. (A) The change in DNR titre after complementation with drrAB1, drrAB2, and drrAB3. Statistical significance was analysed using the Mann–Whitney test. **indicates p < 0.01. (B) DNR resistance assay, 1 μM oxytetracycline was added to each plate. The bacterial suspensions were diluted to OD₆₀₀ values of 2, 0.2, 0.02, and 0.002 per ml, 5 μL of bacterial suspensions of different concentrations were spotted onto plates and incubated for 5 days. To facilitate understanding, it is important to note that daunorubicin is naturally red in colour. OTC, oxytetracycline. The flat triangle arrows above the images in Figure 4B indicate that lower cell concentrations were spotted from left to right.

FIGURE 5

DrrR1 responds to the intracellular accumulation of DNR/DOX and negatively regulates drrAB2 expression. (A) Change of the expression levels of drrAB2 (blue line chart) and DNR titre (red bar chart) in the WT strain during fermentation. (B) Change of the expression level of drrAB2 after adding DNR. The mycelia for RT‐qPCR were collected from the NDYE broth at 0 h, 1 h, 3 h, and 7 h after adding DNR. (C) Deletion of drrR1 results in the overexpression of drrAB2. (D) The symmetrical palindrome sequence (DrrR1‐BS) located in the intergenic region between drrR1 and drrAB2. (E) DrrR1 binds to the intergenic region between drrR1 and drrAB2. Biotin‐DNA represents the biotin‐labelled 110 bp intergenic region between drrR1 and drrAB2, DNA represents the unlabelled 110 bp intergenic region between drrR1 and drrAB2, mutant‐DNA represents the unlabelled intergenic region between drrR1 and drrAB2 with removal of DrrR1‐BS. (F) Effect of DOX and DNR on the binding of DrrR1 to DrrR1‐BS.

FIGURE 6

Four Streptomyces strains contain the homologues of all three DrrBs and the conserved binding motif of DrrR1 homologues. (A) Comparison of the DrrAB1 homologue containing BGCs from different organisms that contain all three DrrBs. (B) The conserved binding motif of DrrR1 homologues.