Part:BBa_K5208011

Pgrac-SPamyQ-YtnP-Term

This is an expression cassette consisting of a strong inducible promoter Pgrac, a secretion signal peptide SPamyQ, the AHL lactonase YtnP, and a terminator.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 771
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Aeromonas hydrophila is an aquatic pathogen that poses a significant threat to the aquaculture industry. Its virulence is primarily regulated by quorum sensing, a mechanism triggered by acyl-homoserine lactones (AHLs), particularly N-butanoyl-L-homoserine lactone (C4-HSL) (Coquant et al., 2020; Hlordzi et al., 2020).

In this expression cassette, Pgrac BBa_K1628202 is a strong promoter, and SPamyQ BBa_K1074014 is a signal peptide that ensures the efficient secretion of the AHL lactonase. YtnP BBa_K5208001 is an enzyme that catalyzes the hydrolysis of AHL. By overexpressing YtnP in the probiotic Bacillus subtilis and introducing the engineered bacteria into fish ponds, quorum sensing in A. hydrophila can be disrupted, reducing its virulence and safeguarding aquatic animals. Finally, BBa_K4934022 is the terminator.

Characterization

2024 Hangzhou-BioX Team characterized this part for its quorum-quenching ability against A. hydrophila

The zone of inhibition test

Our first test aimed to determine if B. subtilis WB600 overexpressing YtnP directly inhibits the growth of A. hydrophila. Our experiment utilized the agar well diffusion method. We prepared LB agar plates containing 10% overnight culture of A. hydrophila and loaded B. subtilis cultures into the wells.

Figure 1. The inhibition test of A. hydrophila was conducted with wells loaded with B. subtilis. LB served as the negative control.

Results indicated that B. subtilis WB600 overexpressing YtnP did not inhibit the growth of A. hydrophila (Figure 1), since quorum quenching does not kill bacteria but only influences the expression of certain virulence factors (Khajanchi et al., 2009).

Synthetic AHL degradation test

Wells were punched into LB agar plates containing synthetic C4-HSL and Chromobacterium subtsugae CV026 (the biosensor for AHL, turns purple when detecting AHL). IPTG-induced B. subtilis cultures were loaded into the wells. The AHL lactonases secreted by the bacteria diffused into the agar, degrading the C4-HSL. Consequently, areas around the wells lacked the characteristic purple color.

Figure 2. A. 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; B. AHL degradation levels of each strain were measured in the width of the colorless ring; C. AHL degradation levels of mixed sample groups. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Results showed that B. subtilis WB600 overexpressing YtnP significantly increased the AHL degradation ability (p < 0.001) (Figure 2B).

For the mixed tests combining YtnP with other AHL lactonases (AiiA and AiiM), all data points for the mixed groups fell between the values of the individual strains (Figure 2C). This suggests that no enzyme-enzyme interactions occurred among the AHL lactonases tested.

Natural AHL degradation test

Our experiment utilized the agar well diffusion method. Plates were prepared with molten LB agar mixed with CV026. Wells were loaded with a mixture of A. hydrophila and B. subtilis. CV026 in the agar detected the C4-HSL synthesized by A. hydrophila in the wells and responded by producing purple pigment, forming a purple ring around the wells. If B. subtilis secretes AHL lactonases, the C4-HSL synthesized by A. hydrophila would be degraded, resulting in a smaller purple area.

Figure 3. A. Natural AHL degradation test on LB plates involved loading wells with a mixture of A. hydrophila and B. subtilis. Purple rings indicated the presence of C4-HSL; B. 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.

Results exhibited complete AHL degradation by YtnP (Figure 3).

Biofilm reduction test

The crystal violet assay was used to assess biofilm formation. To test the effect of B. subtilis overexpressing YtnP on A. hydrophila biofilm formation, A. hydrophila was incubated in partial B. subtilis culture supernatants under static conditions for 48 hours. The biofilm was then stained with crystal violet, and the dyed materials were homogenized to measure absorbance at 570 nm. To control for bacterial growth, we also measured OD600 immediately after the 48-hour incubation. The relative biofilm formation was normalized by calculating OD570/OD600 (Cam & Bicek, 2023; Parker et al., 2017).

Figure 4. A. 48-hour A. hydrophila biofilm stained with crystal violet; B. Homogenized dye, prepared for OD570 measurement; C. Biofilm formation was quantified using OD570/OD600; D. Biofilm formation in mixed sample groups. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Results showed that B. subtilis WB600 overexpressing YtnP significantly reduced A. hydrophila biofilm formation by 79.2% (p < 0.001) (Figure 4B).

In mixed enzyme tests, all combinations containing YtnP exhibited similar results to the individual enzymes, confirming that no enzyme-enzyme interactions affected the outcomes (Figure 4C).

Extracellular protease reduction test

We measured the activity of the extracellular proteases of A. hydrophila cultured in B. subtilis YtnP supernatants using the Neutral Protease (NP) Activity Assay Kit (Sangon Biotech, Shanghai, China).

Figure 5. 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 demonstrated that YtnP significantly decreased the activity of A. hydrophila extracellular proteases (p < 0.05) (Figure 5A). In mixed enzyme assays, the results were consistent with other tests, indicating that no enzyme-enzyme interactions (Figure 5B).

References

Cam, S., & Bicek, S. (2023). The effects of temperature, salt, and phosphate on biofilm and exopolysaccharide production by Azotobacter spp. Arch Microbiol, 205(3), 87. https://doi.org/10.1007/s00203-023-03428-9

Coquant, G., Grill, J. P., & Seksik, P. (2020). Impact of N-Acyl-Homoserine Lactones, Quorum Sensing Molecules, on Gut Immunity. Front Immunol, 11, 1827. https://doi.org/10.3389/fimmu.2020.01827

Hlordzi, V., Kuebutornye, F. K. A., Afriyie, G., Abarike, E. D., Lu, Y., Chi, S., & Anokyewaa, M. A. (2020). The use of Bacillus species in maintenance of water quality in aquaculture: A review. Aquaculture Reports, 18, 100503. https://doi.org/https://doi.org/10.1016/j.aqrep.2020.100503

Khajanchi, B. K., Sha, J., Kozlova, E. V., Erova, T. E., Suarez, G., Sierra, J. C., Popov, V. L., Horneman, A. J., & Chopra, A. K. (2009). N-acylhomoserine lactones involved in quorum sensing control the type VI secretion system, biofilm formation, protease production, and in vivo virulence in a clinical isolate of Aeromonas hydrophila. Microbiology (Reading), 155(Pt 11), 3518-3531. https://doi.org/10.1099/mic.0.031575-0

Parker, A., Cureoglu, S., De Lay, N., Majdalani, N., & Gottesman, S. (2017). Alternative pathways for Escherichia coli biofilm formation revealed by sRNA overproduction. Mol Microbiol, 105(2), 309-325. https://doi.org/10.1111/mmi.13702