Part:BBa_K5208014
Pgrac-SPamyQ-AhlD-Term
This is an expression cassette consisting of a strong inducible promoter Pgrac, a secretion signal peptide SPamyQ, the AHL lactonase AhlD, and a terminator.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 827
Illegal BamHI site found at 941
Illegal XhoI site found at 526
Illegal XhoI site found at 640 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 523
Illegal BsaI.rc site found at 247
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. AhlD BBa_K5208004 is an enzyme that catalyzes the hydrolysis of AHL. By expressing AhlD 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 expressing AhlD 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.
Results indicated that B. subtilis WB600 expressing AhlD 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.
Results showed that B. subtilis WB600 expressing AhlD significantly increased the AHL degradation ability (p < 0.01) (Figure 2B).
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.
Results exhibited complete AHL degradation by AhlD (Figure 3).
Biofilm reduction test
The crystal violet assay was used to assess biofilm formation. To test the effect of B. subtilis expressing AhlD 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).
Results showed that B. subtilis WB600 expressing AhlD significantly reduced A. hydrophila biofilm formation by 79.9% (p < 0.001) (Figure 4B).
Extracellular protease reduction test
We measured the activity of the extracellular proteases of A. hydrophila cultured in B. subtilis AhlD supernatants using the Neutral Protease (NP) Activity Assay Kit (Sangon Biotech, Shanghai, China).
The results demonstrated that AhlD significantly decreased the activity of A. hydrophila extracellular proteases (p < 0.05) (Figure 5).
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
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