A Bacterial Isolate Capable of Quenching Both Diffusible Signal Factor- and N-Acylhomoserine Lactone-Family Quorum Sensing Signals Shows Much Enhanced Biocontrol Potencies

Abstract

N-Acylhomoserine lactone (AHL) and diffusible signal factor (DSF) molecules are two families of widely conserved quorum sensing (QS) signals. Quorum quenching (QQ) via enzymatic inactivation of QS signals is a promising strategy of biocontrol. In the search for biocontrol agent quenching both AHL and DSF signals, it has been recently identified that DSF-quenching biocontrol agent Pseudomonas sp. HS-18 contains at least three genes (aigA, aigB, and aigC) encoding AHL-acylases displaying strong AHL-acylase activities on various AHLs. Among them, AigA and AigC presented broad-spectrum enzyme activity against AHLs, while AigB preferred longer AHLs. Interestingly, transcriptional expression of aigC could be significantly induced by AHL signals. Heterologous expression of aigA-C in Burkholderia cenocepacia and Pseudomonas aeruginosa resulted in drastically decreased AHL accumulation, virulence factor production, biofilm formation, motility, and virulence on plants. Significantly, the two types of QQ mechanisms in HS-18 showed a strong and much desired synergistic effect for enhanced biocontrol potency against AHL- and DSF-dependent pathogens.

Keywords: AHL; DSF; Pseudomonas sp. HS-18; acylase; quorum quenching.

Figure 1

Phylogenetic analysis of AigA–C and previously characterized AHL-acylases. Phylogenetic analysis was conducted with the MEGA (version 5.05) program with neighbor-joining method (1000 bootstrap replicates) to show the phylogenetic relationship among AigA–C and the previously reported AHL-acylases (see text for references). The scale bar indicates the number of substitutions per residue. Bootstrap coefficients below 50% were not presented.

Figure 2

AHL QQ activity of strains HS-18, DH5α(aigA), DH5α(aigB), and DH5α(aigC) on various AHLs. (A) Relative AHL degradation rates of strain HS-18 on various AHL signals at 12 h and 36 h postincubation. (B) Relative AHL degradation rates of E. coli strains DH5α(aigA), DH5α(aigB), DH5α(aigC), and the vector control DH5α(pBBR1) on various AHLs at 36 h postincubation. Bacterial strains were incubated respectively with corresponding AHL signals in LB medium containing 50 mM MOPS. The same amount of AHLs was added to the same medium as the untreated controls. After incubation, the remaining AHLs in the culture supernatants were extracted with ethyl acetate, dried, and resuspended in the same volume of methanol, respectively. The remaining AHLs were detected by using biosensor A. tumefaciens NT1 or C. violaceum CV026 as described in Methods. The relative degradation rate was derived by calculating the ratio of distance of remaining AHLs and that of the corresponding untreated AHL control. Medium- and long-chain AHLs (C8-C14) were detected by biosensor NT1(traR, tra::lacZ749) and short-chain AHLs (C4-C6) were detected by biosensor CV026. Bars indicate the mean with SD of three independent repeats. Asterisks indicate the statistical significance (***, P < 0.001; ns, not significant).

Figure 3

Deletion of aigA–C did not affect the growth of strain HS-18 but impaired its QQ activity against AHL signal. (A) Growth curve of wild-type strain HS-18 and its aigA–C deletion mutant 3Δaig. (B) AHL degradation assay of strain HS-18 and mutant 3Δaig in LB medium containing 25 μM 3-O-C12-HSL. The bacterial cultures were incubated at 28 °C for 24 h prior to measurement of remaining AHL signals. LB broth containing same amount of 3-O-C12-HSL without bacterial inoculation was taken as a control. Statistical analyses were performed using the t-test and two-way analysis of variance (ANOVA). Statistics significance: ***, P < 0.001.

Figure 4

LC–MS analysis of degradation products of 3-O-C12-HSL by Aig enzymes. The MS profile of standard 3-O-C12-HSL without treatment showing the typical parental ion (m/z of M + H = 298.20) with HPLC retention time (RT) at 8.68 min (A). The MS profile of standard HSL showing the typical parental ion (m/z of M + H = 102.05) with HPLC RT at 1.5 min (B). The signal 3-O-C12-HSL was incubated with the protein sample from E. coli containing the vector as a control (C). The signal 3-O-C12-HSL was incubated with the protein samples from E. coli expressing aigA (D), aigB (E), and aigC (F), respectively. Chemical structures of 3-O-C12-HSL and its degradation products with their calculated M + H m/z shown below, respectively (G). Recombinant E. coli BL21(DE3) expressing aigA–C carried by vector pET32a was induced by 0.5 mM IPTG when OD₆₀₀ reached about 0.7, followed by incubation of 4 h at 37 °C with 200 rpm shaking. Crude enzyme solutions were obtained by sonication of the bacterial cells and 2 mM 3-O-C12-HSL was incubated with the crude enzyme solutions at 30 °C for 3 h. The mixture was then extracted with ethyl acetate, resuspended in methanol, and analyzed by LC–MS. The experiments were repeated at least twice with similar results.

Figure 5

RT-qPCR analysis of aigA–C transcript levels in strain HS-18. The transcript levels of aigC were measured using RNA extracted from strain HS-18 grown in the presence of AHL compared to that in the absence of AHL after incubation for 3 h (A), 6 h (B), and 9 h (C). (D) Relative transcript levels of aigA and aigC were compared to that of aigB in the presence of AHL. The final concentration of AHL signal 3-O-C12-HSL used in this experiment was 0.1 mM. The transcript levels of the aigA–C were normalized to the housekeeping gene rpoD. Statistical analyses were performed using two-way ANOVA and a t-test. Bars indicate the mean with SD of three independent repeats. Asterisks indicate the statistical difference (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant).

Figure 6

Expression of aigA–C in B. cenocepacia and P. aeruginosa reduced AHL accumulation. The aig genes were cloned under the control of lac promoter. (A) Expression of aigA–C in B. cenocepacia strain H111 reduced C8-HSL accumulation in culture supernatants. (B) Expression of aigA–C in P. aeruginosa strain PAO1 reduced accumulation of C4-HSL, and (C) 3-O-C12-HSL. Data are expressed as the mean with SD of three independent assays. Statistics significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 7

Effect of aigA–C on the AHL-mediated virulence traits in B. cenocepacia H111 and P. aeruginosa PAO1. Expression of aigA–C in strain H111 reduced its motility (A), biofilm formation (B), and extracellular protease production (C). Expression of aigA–C in strain PAO1 reduced swarming motility (D), and extracellular protease production (E). Statistical analyses were performed using t-test and two-way ANOVA. Data shown are the mean with SD. Statistics significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 8

Effect of AigA–C on the pathogenicity of B. cenocepacia H111 and P. aeruginosa PAO1. Virulence of B. cenocepacia H111 and its derivatives on onion (A). Virulence of PAO1 and its derivatives on cabbage (B) and lettuce (C). Comparison of the biocontrol effect of Pseudomonas sp. strain HS-18 (WT) and its derivatives on the infections caused by B. cenocepacia H111 (D) and by P. aeruginosa PAO1 (E). The maceration area on plant tissues caused by pathogens was analyzed using ImageJ software (version 1.52a). The relative disease incidence rate was presented as the percentage of maceration area by comparison with that of corresponding wild-type pathogen. Symbol: 3Δaig, aigA–C deletion mutant in strain HS-18; 4Δ: digA–D deletion mutant in strain HS-18; 4Δ3Δaig: aigA–C and digA–D deletion mutant of strain HS-18. Statistical analyses were performed using a t-test and two-way ANOVA. Bars indicate the mean with SD. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001.