Quorum Sensing N-acyl Homoserine Lactones-SdiA Suppresses Escherichia coli- Pseudomonas aeruginosa Conjugation through Inhibiting traI Expression

Abstract

Conjugation is a key mechanism for horizontal gene transfer and plays an important role in bacterial evolution, especially with respect to antibiotic resistance. However, little is known about the role of donor and recipient cells in regulation of conjugation. Here, using an Escherichia coli (SM10λπ)-Pseudomonas aeruginosa (PAO1) conjugation model, we demonstrated that deficiency of lasI/rhlI, genes associated with generation of the quorum sensing signals N-acyl homoserine lactones (AHLs) in PAO1, or deletion of the AHLs receptor SdiA in the donor SM10λπ both facilitated conjugation. When using another AHLs-non-producing E. coli strain EC600 as recipient cells, deficiency of sdiA in donor SM10λπ hardly affect the conjugation. More importantly, in the presence of exogenous AHLs, the conjugation efficiency between SM10λπ and EC600 was dramatically decreased, while deficiency of sdiA in SM10λπ attenuated AHLs-inhibited conjugation. These data suggest the conjugation suppression function of AHLs-SdiA chemical signaling. Further bioinformatics analysis, β-galactosidase reporter system and electrophoretic mobility shift assays characterized the binding site of SdiA on the promoter region of traI gene. Furthermore, deletion of lasI/rhlI or sdiA promoted traI mRNA expression in SM10λπ and PAO1 co-culture system, which was abrogated by AHLs. Collectively, our results provide new insight into an important contribution of quorum sensing system AHLs-SdiA to the networks that regulate conjugation.

Keywords: N-acyl homoserine lactones; P. aeruginosa; SdiA; antibiotic resistance; conjugation.

Figure 1

The quorum sensing system of P. aeruginosa inhibits conjugation between P. aeruginosa and E. coli. (A) Deficiency of the AHLs synthase genes rhlI or lasI in P. aeruginosa (PAO1) resulted in the absence of C4-HSL or both 3-oxo-C12-HSL and C4-HSL, respectively. The 3-oxo-C12-HSL and C4-HSL in the cell-free supernatants were extracted with ethyl acetate and redissolved in methanol, followed by HPLC-MS/MS analysis. (B) Deficiency of lasI or rhlI in P. aeruginosa abolished production of the downstream toxin of rhlI system pyocyanin. PAO1, PAO1ΔlasI and PAO1ΔrhlI were cultured in the presence or absence of 3-oxo-C12-HSL or C4-HSL as indicated for 30 h. (C) Deficiency of lasI or rhlI in P. aeruginosa significantly promoted SM10λπ-PAO1 conjugation; this effect could be abrogated by supplementation with exogenous 3-oxo-C12-HSL or C4-HSL. SM10λπ and PAO1 (10⁷ CFU/mL each) were mated at 37°C for 6 h in the presence or absence of 40 μM of C4-HSL or 3-oxo-C12-HSL. Ctrl, control; C12, 3-oxo-C12-HSL; C4, C4-HSL. Values are mean ± SEM of at least three independent experiments; ^***P < 0.001.

Figure 2

AHL inhibits SM10λπ-PAO1 conjugation via a mechanism dependent on SdiA of E. coli. (A) Deficiency of sdiA in E. coli significantly promoted SM10λπ-PAO1 conjugation. E. coli SM10λπ and P. aeruginosa PAO1 (10⁷ CFU/mL each) were mated at 37°C for 6 h. (B) Deficiency of sdiA in SM10λπ did not significantly affect SM10λπ-EC600 conjugation, but could rescue 3-oxo-C12-HSL and C4-HSL (C4/12)-inhibited conjugation. SM10λπ was cultured in the presence of DMSO or 40 μM C4-HSL and 3-oxo-C12-HSL for 6 h, followed by conjugation with EC600. Values are mean ± SEM of at least three independent experiments; ns, not significant, ^***P < 0.001.

Figure 3

The AHL-SdiA interaction represses the expression of TraI in E. coli. (A) EMSA verified the interaction of SdiA protein with the putative SdiA-box of the traI promoter. Biotin-labeled SdiA-box (50 fmol) was added to each reaction with 0, 2, 4, 8, or 16 pmol E. coli His-SdiA fusion protein (lane 1 to lane 5, respectively). The specificity analysis (lane 6 to lane 11) was performed in the presence (−) or absence (+) of His-SdiA fusion protein (8 pmol) and biotin-labeled SdiA-box (50 fmol, lanes 7), 200-fold unlabeled SdiA-box (lanes 8), anti-His antibody (lanes 9), or 200-fold SdiA-box full or spacer mutant competitors (lane 10, 11). (B) Activity of β-galactosidase reporters containing the predicted SdiA-box in genomic regions upstream of traI under various conditions. E.coli BW25113 or E.coli BW25113ΔsdiA were cultured with SM10λπ in the presence of DMSO (Ctrl) or 40 μM C4-HSL and 3-oxo-C12-HSL for 6 h, followed by β-galactosidase activity analysis. (C) Deficiency of sdiA promoted traI expression in E. coli. (D) Deficiency of lasI/rhlI promoted traI expression in E. coli. For (C,D), 10⁷ CFU/mL (each) of the indicated E. coli donor and recipient P. aeruginosa cells were mated at 37°C for 6 h, followed by real-time PCR analysis. The rpoD gene of E. coli was used as an internal control. (E) Exogenous 3-oxo-C12-HSL or C4-HSL inhibited traI expression in E. coli. PAO1ΔlasI or PAO1ΔrhlI were cultured with SM10λπ in the presence of DMSO (Ctrl) or 40 μM C4-HSL and 3-oxo-C12-HSL for 6 h, followed by real-time PCR analysis. The rpoD gene of E. coli was used as an internal control. Ctrl, control; C12, 3-oxo-C12-HSL; C4, C4-HSL. Values are mean ± SEM of at least three independent experiments; ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.