Parts
Our project aimed to reduce the virulence of Aeromonas hydrophila, a harmful aquatic pathogen, to safeguard the aquaculture industry. This pathogen uses AHL molecules to activate quorum sensing, leading to the expression of virulence factors. We introduced six AHL lactonases from various bacteria donors into Bacillus subtilis. By degrading AHL, our engineered B. subtilis effectively inhibits the quorum sensing of A. hydrophila, significantly reducing both biofilm formation and extracellular protease activity, with the strain expressing AiiA showing the most pronounced effect.
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Name |
Type |
Description |
Length |
|
Basic |
AiiA |
753 bp |
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|
Basic |
YtnP |
771 bp |
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|
Basic |
AttM |
792 bp |
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|
Basic |
AiiM |
756 bp |
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|
Basic |
AhlD |
822 bp |
|
|
Basic |
AhlS |
834 bp |
|
|
Composite |
Pgrac-SPamyQ-AiiA-Term |
992 bp |
|
|
Composite |
Pgrac-SPamyQ-YtnP-Term |
1010 bp |
|
|
Composite |
Pgrac-SPamyQ-AttM-Term |
1031 bp |
|
|
Composite |
Pgrac-SPamyQ-AiiM-Term |
995 bp |
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|
Composite |
Pgrac-SPamyQ-AhlD-Term |
1061 bp |
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|
Composite |
Pgrac-SPamyQ-AhlS-Term |
1073 bp |
Part Collection
All 12 parts contributed to the degradation of AHL (C4-HSL) and the inhibition of quorum sensing in A. hydrophila. Therefore, they make up a part collection.
The quorum quenching ability of each component was evaluated through synthetic AHL degradation tests (Figure 1), natural AHL degradation tests (Figure 2), biofilm reduction tests (Figure 3), and extracellular protease reduction tests (Figure 4). To study the interactions between AHL lactonases and identify the most effective enzyme combination for quorum quenching, mixed pairs of the top three enzymes were tested additionally.
Figure 1. A-C. Synthetic AHL degradation test on LB plates. B. subtilis cultures were loaded in wells. Liquid LB was used as the negative control. Rings without the purple color indicated AHL degradation; D. AHL degradation levels of each strain were measured in the width of the colorless ring; E. AHL degradation levels of mixed sample groups. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
Figure 2. A-C. Natural AHL degradation test on LB plates involved loading wells with a mixture of A. hydrophila and B. subtilis. LB served as the negative control. A. hydrophila alone and pure C4-HSL served as positive controls. Purple rings indicated the presence of C4-HSL; D. The AHL degradation levels of each strain were measured in the percentage reduction in ring size compared to the wells with A. hydrophila alone. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
Figure 3. Crystal violet biofilm assay. A. hydrophila was cultured in B. subtilis culture supernatants for 48 hours to allow biofilm formation. A. 48-hour A. hydrophila biofilm stained with crystal violet; B. Homogenized dye, prepared for OD570 measurement; C. Biofilm formation in each group was quantified using OD570/OD600; D. Biofilm formation in mixed sample groups. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
Figure 4. A. The activity of the extracellular proteases of A. hydrophila cultured in partial B. subtilis supernatants; B. Extracellular protease activities in mixed sample groups. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
The results showed that introducing all genes except AttM (BsAiiA, BsYtnP, MtAiiM, AsAhlD, and SsAhlS) enhanced the C4-HSL degrading ability of B. subtilis WB600. Additionally, it was confirmed that BsAiiA, BsYtnP, and MtAiiM functioned independently, with no significant enzyme-enzyme interactions, since mixing these enzymes did not produce better results than using each enzyme individually. Among all groups, B. subtilis WB600 expressing AiiA is the most effective quorum quenching strain against A. hydrophila, with the most decrease in biofilm formation (85.0%) and extracellular protease activities (42.96%), making it a promising probiotic for aquaculture applications.
We also demonstrated that the expression of AHL lactonases in B. subtilis WB600 did not directly inhibit the growth of A. hydrophila, as quorum quenching does not kill bacteria but only affects the expression of specific virulence factors.
Figure 5. The inhibition test of A. hydrophila was conducted with wells loaded with B. subtilis. LB served as the negative control. Chloramphenicol (Cm+) served as the positive control. Clear rings around the wells indicated inhibition of A. hydrophila.
In the future, the iGEM community can expand our part collection by testing more quorum-quenching enzymes.