Part:BBa_K5208012

Pgrac-SPamyQ-AttM-Term

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

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 390
  
• 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 (AttM), 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. AttM BBa_K5208002 is an enzyme that catalyzes the hydrolysis of AHL. By expressing AttM in the probiotic Bacillus subtilis and introducing the engineered bacteria into fish ponds, quorum sensing in A. hydrophila may 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 AttM 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 expressing AttM did not inhibit the growth of A. hydrophila (Figure 1).

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.

Results showed that B. subtilis WB600 expressing AtAttM did not significantly increase the AHL degradation ability (p > 0.1) (Figure 2).

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 potent 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.

Results exhibited no C4-HSL degradation by AttM (p > 0.3) (Figure 3).

Biofilm reduction test

The crystal violet assay was used to assess biofilm formation. To test the effect of B. subtilis expressing AttM 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. Biofilm formation was quantified using OD570/OD600.

Results showed that B. subtilis WB600 expressing AttM failed to reduce A. hydrophila biofilm formation (p > 0.2) (Figure 4B).

Extracellular protease reduction test

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

Figure 5. The activity of the extracellular proteases of A. hydrophila cultured in partial B. subtilis supernatants.

The results demonstrated that AttM did not affect the activity of A. hydrophila extracellular proteases (p > 0.8) (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

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