DBTL Cycle 1
This cycle contains most of the experimental design, including the selection of targets, enzymes, chassis, and vector, and the successful construction of the recombinant vectors. This cycle ended with the unsuccessful electroporation transformation of Bacillus.
1. Design
1.1 The aquatic pathogen: Aeromonas hydrophila
China has a vast aquaculture industry and is the largest aquaculture producer globally (FAO, 2023). In our hometown of Zhejiang, China, A. hydrophila is the most prevalent aquatic pathogen, causing motile aeromonad septicemia (MAS) in aquatic animals (Nielsen et al., 2001). The opportunistic bacterium typically invades through wounds or damaged tissues (Stratev & Odeyemi, 2016). Additionally, it can lead to human gastroenteritis if contaminated food is consumed (Fleckenstein et al., 2021).
The virulence of A. hydrophila is primarily regulated by a mechanism known as quorum sensing. The bacterium produces acyl-homoserine lactone (AHL), specifically N-butanoyl-L-homoserine lactone (C4-HSL). When the concentration of C4-HSL reaches a certain threshold, it activates genes responsible for producing virulence factors (Coquant et al., 2020; Garde et al., 2010). These factors include toxins like aerolysin and hemolysin, extracellular proteases such as serine proteases, and biofilm formation (Khajanchi et al., 2009; Swift et al., 1997). These virulence factors facilitate infection by damaging host cells and forming protective biofilms. By disrupting quorum sensing, a process known as quorum quenching, it is possible to inhibit the production of these virulence factors, thereby reducing the ability of A. hydrophila to infect aquatic animals (Chen et al., 2013; Chu et al., 2014).
Figure 1. Quorum sensing and quenching in A. hydrophila. A. C4-HSL freely diffuses through the membrane and binds to receptors. The receptors dimerize and function as a transcription factor to trigger target gene expression (Coquant et al., 2020); B. C4-HSL is degraded by strains producing AHL lactonases, leading to quorum quenching.
1.2 The enzymes: AHL lactonases
As discussed, our goal is to inhibit quorum sensing in A. hydrophila, which is induced by C4-HSL. To achieve this, we decided to use enzymes to degrade this molecule. These quorum-quenching (QQ) enzymes include AHL lactonases, AHL acylases, and oxidoreductases (Chen et al., 2013). AHL lactonases, which are the most abundant in nature, can be sourced from various bacteria (Dong et al., 2002; Uroz et al., 2008; Wang et al., 2010; Zhang et al., 2002).
Among the potential gene donors, we selected species that are either commonly used or non-pathogenic to humans, animals, and plants to minimize safety concerns. Bacillus subtilis is non-pathogenic and well-studied as a model bacterium in laboratories (Errington & Aart, 2020; Su et al., 2020). Agrobacterium tumefaciens C58 is a disarmed lab strain of a plant pathogen (Gelvin, 2003; Goodner et al., 2001). Microbacterium testaceum is an endophytic bacterium, while Arthrobacter and Solibacillus silvestris are soil bacteria (Dsouza et al., 2015; Krishnamurthi et al., 2009; Morohoshi et al., 2012; Morohoshi et al., 2011; Wang et al., 2010). None of these species have known reports to cause disease in healthy animals. As a result, the AHL lactonase genes we chose are the following: AiiA from B. subtilis BS-1 (noted as BsAiiA) (Pan et al., 2008), YtnP from B. subtilis WB600 (BsYtnP) (Schneider et al., 2012), AttM from A. tumefaciens C58 (AtAttM) (Carlier et al., 2003), AiiM from M. testaceum StLB037 (MtAiiM) (Wang et al., 2010), AhlD from Arthrobacter sp. IBN110 (AsAhlD) (Park et al., 2003), and AhlS from S. silvestris StLB214 (SsAhlS) (Morohoshi et al., 2012). The goal of this research is to find the best enzyme (or enzyme combination) among these AHL lactonases to inhibit quorum sensing in A. hydrophila.
|
Gene Name |
Gene Donor |
Part Accession Number |
|
AiiA |
Bacillus subtilis BS-1 |
BBa_K5208000 |
|
YtnP |
Bacillus subtilis WB600 |
BBa_K5208001 |
|
AttM |
Agrobacterium tumefaciens C58 |
BBa_K5208002 |
|
AiiM |
Microbacterium testaceum StLB037 |
BBa_K5208003 |
|
AhlD |
Arthrobacter sp. IBN110 |
BBa_K5208004 |
|
AhlS |
Solibacillus silvestris StLB214 |
BBa_K5208005 |
Table 1. Genes of AHL lactonases used in this project.
1.3 The chassis: Bacillus subtilis WB600
Generally speaking, B. subtilis is an excellent chassis for several reasons: it is non-pathogenic to both animals and plants, has been widely used in research and industry, is reliable in large-scale production, offers ease of genetic manipulation, and can achieve high levels of secretory protein expression, all of which make it an efficient platform for producing recombinant proteins (Su et al., 2020; Urdaci et al., 2004).
What is more advantageous for our project is that plenty of studies have shown B. subtilis to be a probiotic in aquaculture, improving feed digestibility, boosting the immune system, preventing microbial diseases, and reducing water pollution (Hlordzi et al., 2020; Monier et al., 2023; Neissi et al., 2024; Olmos et al., 2020; Olmos & Paniagua-Michel, 2014). Reports have shown that some wild strains of B. subtilis have the ability to inhibit the growth of A. hydrophila (Nayak et al., 2023). Moreover, B. subtilis produces endogenous quorum-quenching enzymes and has been proven capable of expressing exogenous AHL lactonases as well (Pan et al., 2008; Schneider et al., 2012).
We chose to use B. subtilis WB600, a modified strain with six major protease genes knocked out. With minimal intrinsic protease activity, WB600 enhances the yield and stability of recombinant proteins (Wu et al., 1991). It shows potential for efficiently producing quorum-quenching enzymes to inhibit A. hydrophila.
1.4 The plasmid: pHT43
After selecting the chassis for our experiment, the next step is to determine an appropriate plasmid. We chose the pHT43 plasmid vector (Figure 2).
Figure 2. Plasmid map of pHT43.
This plasmid is a B. subtilis-E. coli shuttle expression vector that contains a strong inducible Pgrac promoter for B. subtilis (Phan et al., 2006), followed by a ribosome binding site (RBS) and a SPamyQ secretion signal peptide. Linking the AHL lactonase genes to the secretion peptide ensures efficient extracellular expression (Mu et al., 2018).
In pHT43, the selection marker for E. coli is ampicillin, while for B. subtilis, it is chloramphenicol. Therefore, it is crucial to confirm that B. subtilis WB600 is susceptible to chloramphenicol before using pHT43. We plated an overnight culture of B. subtilis WB600 on LB agar containing different concentrations of chloramphenicol, ranging from 0 to 68 µg/mL (Figure 3). The results showed that WB600 is sensitive to chloramphenicol, with a minimum inhibitory concentration of 51 µg/mL, which is much higher than the commonly reported 5-10 µg/mL in other studies (Hanif et al., 2017; Karimi et al., 2018). As a result, pHT43 is suitable for our strain. This experiment also helped determine the optimal chloramphenicol concentration for future B. subtilis transformation.
Figure 3. A-F. B. subtilis WB600 overnight growth on LB plates. White areas show bacterial growth. A. Control; B. LB containing 11.3 µg/mL chloramphenicol; C. LB containing 22.6 µg/mL chloramphenicol; D. LB containing 34 µg/mL chloramphenicol; E. LB containing 51 µg/mL chloramphenicol; F. LB containing 68 µg/mL chloramphenicol.
The following are the designed recombinant vectors, each carrying an AHL lactonase.
Figure 4. Maps of recombinant vectors. A. Map of pHT43-BsAiiA; B. Plasmid map of pHT43-BsYtnP; C. Map of pHT43-MtAiiM; D. Plasmid map of pHT43-AtAttM; E. Map of pHT43-AsAhlD; F. Plasmid map of pHT43-SsAhlS.
2. Build
2.1 Recombinant vector construction
Since we were unable to obtain the gene donor, Arthrobacter sp. IBN110, we requested GENEWIZ (Suzhou, Jiangsu, China) to chemically synthesize the AsAhlD gene and insert it into pHT43.
Then, BsAiiA (796 bp), BsYtnP (813 bp), MtAiiM (799 bp), SsAhlS (877 bp), and AtAttM (912 bp) were acquired by PCR (Phusion High-Fidelity PCR Master Mix, Thermo Fisher, Waltham, MA, USA) using 1 uL liquid bacteria culture (B. subtilis BS-1, B. subtilis WB600, M. testaceum StLB037, S. silvestris StLB214, A. tumefaciens C58) as the template respectively (Figure 5). The first step of PCR (initial denaturation) was elongated to 10 min to ensure cell lysis and the release of the genomic DNA template.
Figure 5. Gel electrophoresis of PCR products. Lane 1: BsAiiA (796 bp); Lane 2: BsYtnP (813 bp),; Lane 3: MtAiiM (799 bp); Lane 4: SsAhlS (877 bp); Lane 5: AtAttM(912 bp).
The pHT43 plasmid was linearized by PCR. Gibson assembly (ClonExpress Ultra One Step Cloning Kit, Vazyme, Nanjing, Jiangsu, China) was used to assemble the linearized plasmid with each AHL lactonase gene. The recombinant vectors pHT43-BsAiiA, pHT43-BsYtnP, pHT43-MtAiiM, pHT43-AtAttM, and pHT43-SsAhlS were transformed into E. coli DH5α.
Colony PCR was performed to ensure successful vector construction, followed by sequencing verification (Figure 6).
Figure 6. Gel electrophoresis of colony PCR products for verification of successful vector construction and transformation. Lanes 1-3: pHT43-BsAiiA (1177 bp); Lanes 4-6: pHT43-BsYtnP (1194 bp); Lanes 7-9: pHT43-MtAiiM (1180 bp); Lanes 10-12: pHT43-SsAhlS (1258 bp); Lanes 13-15: pHT43-AtAttM (1293 bp).
2.2 Transformation of B. subtilis
B. subtilis is a Gram-positive bacterium with a thick cell wall, making it more challenging for exogenous plasmids to enter compared to Gram-negative bacteria like E. coli (Chen & Dubnau, 2004). The two primary transformation methods are electroporation and chemical methods. Chemical transformation involves complex media (Bennallack et al., 2014; Spizizen, 1958), so we initially chose electroporation, which employs a straightforward electric shock method.
We utilized a MicroPulser Electroporator (Bio-Rad, Hercules, CA, USA) and followed the established electroporation protocols (McDonald et al., 1995; Xue et al., 1999).
3. Test
As we followed the electroporation protocols, colony growth on selective plates was observed, but no positive transformants were detected in colony PCR tests. We repeated the transformation several times with tuned voltage, plasmid amount, and buffer concentrations. Yet, results remained consistently unsuccessful.
4. Learn
Interestingly, B. subtilis appeared to develop antibiotic resistance (we tried kanamycin and chloramphenicol) even in negative controls without plasmid introduction. Literature suggests that cellular stress can induce antibiotic tolerance in bacteria (Nguyen et al., 2011; Poole, 2012; Yang et al., 2006). We hypothesize that the electric shock-induced stress responses in B. subtilis, allowing growth on selective plates and leading to false positive results.
Given the unresolved issues with electroporation and time constraints, we decided to switch to chemical transformation.
DBTL Cycle 2
This cycle included the successful transformation of B. subtilis using the Paris method. It ended with none of the B. subtilis strains inhibiting the growth of A. hydrophila. As a result, we were forced to explore alternative ways to assess the effects of the engineered B. subtilis on the virulence factors of A. hydrophila.
1. Design
B. subtilis transformation was performed using the Paris method, which utilizes Spizizen’s minimal medium (Bennallack et al., 2014; Spizizen, 1958).
2. Build
Minimal media GMI and GMII were prepared. B. subtilis in LB was subcultured into GMI and then transferred to GMII to achieve a nutrient-deficient state, which enhances the permeability of the cell membrane and cell wall.
Colony PCR was conducted to confirm successful B. subtilis transformation. Positive transformants were verified (Figure 7).
The Paris method proved to be a highly efficient approach for B. subtilis transformation and was simpler than anticipated. Unlike electroporation, which led to misleading results, this method showed no evidence of negative transformants acquiring antibiotic resistance, giving us a false positive rate of almost 0%.
Figure 7. Gel electrophoresis of colony PCR products for verification of successful B. subtilis transformation. Lane 1: pHT43-BsAiiA (1177 bp); Lane 2: pHT43-BsYtnP (1194 bp); Lane 3: pHT43-MtAiiM (1180 bp); Line 4: pHT43-AsAhlD (1246 bp); Lane 5: pHT43-SsAhlS (1258 bp); Lane 6: pHT43-AtAttM (1293 bp).
3. Test
3.1 The zone of inhibition test
We tested whether our engineered B. subtilis could directly inhibit the growth of A. hydrophila using the agar well diffusion method. Wells were punched into LB agar plates containing 10% overnight culture of A. hydrophila, and IPTG-induced B. subtilis cultures were loaded into the wells. Inhibition would be indicated by clear zones around the wells. The experiment was performed in triplicate.
Results indicated that none of the B. subtilis strains inhibited the growth of A. hydrophila (Figure 8).
Figure 8. 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.
4. Learn
We were surprised at first and went for literature research. It turned out that this outcome, although disappointing, was reasonable since quorum quenching does not kill bacteria but influences the expression of certain virulence factors (Khajanchi et al., 2009; Swift et al., 1997). This is also consistent with some previous studies on A. hydrophila (Chen et al., 2020; Chu et al., 2014).
Since direct growth inhibition of A. hydrophila was not feasible, we shifted our focus to testing the degradation of AHL and the inhibition of virulence factors of A. hydrophila using our engineered B. subtilis.
DBTL Cycle 3
This cycle involved organizing and developing a standard procedure for AHL-induced quorum sensing research. The successful results showed that our engineered B. subtilis degraded C4-HSL and inhibited the quorum sensing of A. hydrophila.
1. Design
1.1 Synthetic AHL degradation test
As discussed, since direct inhibition of A. hydrophila growth was not feasible, we shifted our focus to testing AHL degradation by engineered B. subtilis.
To accomplish this, we conducted extensive literature research to identify a simple method for AHL detection.We found Chromobacterium subtsugae CV026, formerly known as C. violaceum CV026 (Harrison & Soby, 2020), a widely used biosensor for AHL (specifically C4- to C8-HSL) in quorum sensing studies (Chu et al., 2014; Park et al., 2003; Santos et al., 2021; Tang et al., 2015). This strain uses its intrinsic quorum sensing ability to detect AHL in the environment and activates genes for producing a purple pigment, violacein (McClean et al., 1997). To conclude, the purple color shows the presence of AHL.
Our experiment design utilized the agar well diffusion method. Wells should be punched into LB agar plates containing synthetic C4-HSL and C. subtsugae CV026. IPTG-induced B. subtilis cultures should be loaded into the wells. The AHL lactonases secreted by the bacteria will diffuse into the agar, degrading the C4-HSL. Consequently, areas around the wells lacked the characteristic purple color.
1.2 Natural AHL degradation test
The goal is to confirm that our engineered B. subtilis works on our real bacterial target. C. subtsugae CV026 should be again used as the biosensor for C4-HSL. Plates should be prepared with molten LB agar mixed with CV026. Wells should be loaded with a mixture of A. hydrophila and B. subtilis. CV026 in the agar can detect the C4-HSL synthesized by A. hydrophila in the wells and respond by producing purple pigment, forming a purple ring around the wells. If B. subtilis secretes AHL lactonases, the C4-HSL synthesized by A. hydrophila will be degraded, resulting in a smaller purple area.
1.3 Biofilm reduction test
Biofilms are crucial in bacterial infections as they form protective barriers that allow bacteria to resist external threats such as antibiotics and immune responses. Biofilms also increase the infectious potential of bacteria by forming in water systems, as well as on the skin and wounds of animals (Sharma et al., 2023; Vestby et al., 2020). In A. hydrophila, biofilm formation is regulated by quorum sensing, so we tested it in the presence of quorum-quenching enzymes (Lynch et al., 2002; Sun et al., 2021).
The crystal violet assay can be used to assess biofilm formation. Crystal violet is a basic dye that binds non-specifically to negatively charged molecules on the cell surface and within the biofilm extracellular matrix (Kamimura et al., 2022; Wilson et al., 2017). To test the effect of engineered B. subtilis on A. hydrophila biofilm formation, A. hydrophila should be incubated in partial B. subtilis culture supernatants under static conditions for 48 hours. The biofilm should be then stained with crystal violet, and the dyed materials should be homogenized to measure absorbance at 570 nm. To control for bacterial growth, OD600 immediately after the 48-hour incubation should also be measured. The relative biofilm formation should be normalized by calculating OD570/OD600 (Cam & Bicek, 2023; Parker et al., 2017).
1.4 Extracellular protease reduction test
Extracellular proteases are another virulence factor regulated by quorum sensing in A. hydrophila. The proteases, including metalloprotease, serine proteases, and elastases, contribute to virulence by degrading host structural proteins and inducing inflammation, helping the bacterium invade host tissues (Rasmussen-Ivey et al., 2016; Wang et al., 2019). The activity of the extracellular proteases of A. hydrophila cultured in partial B. subtilis supernatants should be measured using a protease activity assay kit.
2. Build
The engineered B. subtilis strains expressing exogenous AHL lactonases have been successfully constructed in the previous cycle. The setups for AHL and quorum-related tests were prepared as described in the Design.
3. Test
3.1 Synthetic AHL degradation test
First, we tested whether the AHL lactonases we introduced into B. subtilis WB600 degrade pure AHL following the method described in Design. This experiment was performed in triplicate.
Figure 9. 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.
Results showed that all B. subtilis strains exhibited some ability to degrade C4-HSL, including the original WB600 strain and a commercially available aquaculture probiotic strain. For the engineered strains, the strain carrying the empty pHT43 vector and the one expressing AtAttM both showed no increase in AHL degradation compared to the unmodified WB600 strain. This is consistent with previous reports that AttM primarily targets longer AHLs, such as 3-Oxo-C8-HSL (Carlier et al., 2003; Haudecoeur et al., 2009).
Among the strains that showed increased AHL degradation, MtAiiM, AsAhlD, and SsAhlS exhibited strong activity, while BsYtnP was even stronger, and BsAiiA was the strongest by far (Figure 9A-D). The robust expression of BsYtnP and BsAiiA is likely because these genes are derived from B. subtilis and introduced back into the same species, making them cisgenes. Cisgenes offer several advantages, including reduced risk of genomic incompatibility, increased likelihood of correct protein folding and post-translational modifications, and natural alignment with the host’s codon usage preferences. These factors contribute to higher expression levels and enhanced enzyme activity of BsYtnP and BsAiiA (Ryffel, 2014; Vasudevan et al., 2023).
After identifying the best single strains, we aimed to find the optimal enzyme combination. This involves potential enzyme-enzyme interactions, which can be categorized into three main types: promotion, inhibition, and no interaction (Hu et al., 2013; Prochaska & Piekutowski, 1994; Van Dyk & Pletschke, 2012). In our experiment, we mixed the three most potent strains in pairs. Unfortunately, the results were somewhat disappointing: all mixed group data points fell between the values of the two individual strains. This indicates that no enzyme-enzyme interactions occurred among the AHL lactonases we tested (Figure 9E).
Figure 10. Degradation of C4-HSL by AHL lactonase. The dashed line on the left shows where the enzyme breaks the bond (Chen et al., 2013; Dong et al., 2000).
Upon reviewing the literature, we found that AHL lactonases deactivate AHL by cleaving its homoserine lactone ring (Figure 10) (Chen et al., 2013; Dong et al., 2000).
Since all the lactonases share the same substrate and target the same bond, it is understandable that no unusual interactions occurred, and the enzymes operated independently.
In conclusion, in this experiment, B. subtilis WB600 expressing BsAiiA demonstrated the strongest enzyme activity for C4-HSL degradation.
3.2 Natural AHL degradation test
Then, we conducted another AHL degradation test employing natural C4-HSL synthesized by A. hydrophila following the method described in Design. This experiment was repeated three times, and all operations involving A. hydrophila (Risk Group 2) were carried out in Class II biosafety cabinets in a BSL-2 laboratory.
Figure 11. 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.
Results were consistent with Test 1, showing limited AHL degradation by WB600, pHT43, AtAttM, and commercial strains. In contrast, the five strong AHL lactonases, BsAiiA, BsYtnP, MtAiiM, AsAhlD, and SsAhlS, exhibited complete degradation in this experiment (Figure 11). It is important to note that "complete" may not be entirely accurate since the detection limit for C4-HSL by C. subtsugae CV026 is 1 µM, but this is the extent observable from the experiment.
To conclude, this experiment confirmed that our engineered B. subtilis can degrade C4-HSL derived from A. hydrophila, supporting its potential for real-life applications.
3.3 Biofilm reduction test
The impact of engineered B. subtilis on biofilm formation by A. hydrophila was measured using the method outlined in the Design section. Since the biofilms on the tube walls are fragile and may be inadvertently removed during washing, the results exhibit a large variance. To ensure data reliability, the experiment was repeated five times.
Figure 12. 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.
The results showed that the quorum-quenching enzymes BsAiiA, BsYtnP, MtAiiM, AsAhlD, and SsAhlS significantly reduced A. hydrophila biofilm formation by approximately 80% (all p < 0.001), with BsAiiA demonstrating the most significant reduction at 85% (Figure 12C). In mixed enzyme tests, all combinations exhibited similar results to the individual enzymes, confirming that no enzyme-enzyme interactions affected the outcomes (Figure 12D).
In conclusion, our engineered B. subtilis significantly reduces biofilm formation in A. hydrophila and presents a promising approach for preventing and treating A. hydrophila infections in aquaculture.
3.4 Extracellular protease reduction test
The activity of the extracellular proteases of A. hydrophila cultured in partial B. subtilis supernatants was measured using the Neutral Protease (NP) Activity Assay Kit (Sangon Biotech, Shanghai, China). This experiment was conducted in three independent trials.
Figure 13. 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 the quorum-quenching enzymes BsAiiA, BsYtnP, MtAiiM, AsAhlD, and SsAhlS significantly decreased the activity of A. hydrophila extracellular proteases (all p < 0.05), with BsAiiA causing the most substantial reduction (Figure 12A). In mixed enzyme assays, the results were consistent with other tests, indicating that no enzyme-enzyme interactions happened (Figure 13B).
In this test, although our intention was to measure the changes in protease levels of A. hydrophila, since A. hydrophila was cultured in the supernatants of the B. subtilis cultures, we could not distinguish whether the proteases originated from B. subtilis or A. hydrophila. This test was only possible by using a protease-deficient B. subtilis strain, for example, WB600. The strain’s low baseline protease activity ensured that most of the proteases measured originated from A. hydrophila. On the contrary, the commercial B. subtilis group exhibited extremely high levels of protease activity compared to other groups (and therefore not included in the diagram), suggesting that most of the proteases were from B. subtilis itself rather than A. hydrophila.
4. Learn
We proved that even the original B. subtilis WB600 strain exhibited some ability to degrade C4-HSL. We learned that by introducing BsAiiA, BsYtnP, MtAiiM, AsAhlD, and SsAhlS, the C4-HSL degrading ability of WB600 increased dramatically, whereas AtAttM does not degrade C4-HSL efficiently. Additionally, we showed that BsAiiA, BsYtnP, and MtAiiM functioned independently without enzyme-enzyme interactions, and mixing two enzymes did not yield superior results compared to the individual enzymes. We also confirmed that quorum quenching does not inhibit the growth of A. hydrophila, but it does significantly reduce biofilm formation and extracellular protease activity. In conclusion, we found that B. subtilis WB600 expressing AiiA is the most effective quorum quenching strain against A. hydrophila, making it a promising probiotic for aquaculture applications.
Due to iGEM’s policy discouraging animal experiments on vertebrates, we chose not to collect direct mortality data on aquatic animals. Instead, we found studies employing similar strategies to control A. hydrophila have conducted animal experiments, so we performed a survival analysis using their mortality data (Chen et al., 2020; Chu et al., 2014). Specifically, we referenced a study that used a quorum-quenching Bacillus strain (Bacillus sp. QSI-1) to observe death rate changes in A. hydrophila-infected zebrafish (Figure 14).
Figure 14. Survival rates of A. hydrophila-infected zebrafish with or without Bacillus sp. QSI-1 (Chu et al., 2014).
The results showed a significant difference in zebrafish survival rates between the two groups (p = 0.02), suggesting that the quorum-quenching strain effectively protects against A. hydrophila infection. This result further validates that developing our engineered B. subtilis is a feasible approach for protecting aquatic animals from A. hydrophila.
5. Future plans
Our first plan focuses on biosafety measures for engineered B. subtilis. Although both the bacterium and the introduced enzymes are generally safe, preventing environmental leakage is still critical. We aim to introduce a suicide switch that activates under dry and low-temperature conditions, ensuring that the engineered bacteria can survive only in aquaculture ponds, which are maintained at warm temperatures even in winter. The bacteria will die when exposed to non-aquatic environments due to dry conditions or natural water bodies during winter due to low temperatures.
To achieve this, we plan to use the TreA promoter and the Des promoter from B. subtilis. The TreA promoter is activated by osmotic stress and dehydration (Sniezko et al., 1998; Xiao et al., 2024), and the Des promoter responds to low temperatures (Cybulski et al., 2004; Thuy Le & Schumann, 2007). We will place the MazF-MazE toxin-antitoxin suicide system under the control of these promoters. This well-established toxin-antitoxin system has been validated by several iGEM teams, including 2010_ Newcastle, 2023_Freiburg, 2023_ GEMS-Taiwan, etc. This design should ensure that the bacteria die under temperature and osmotic stress, effectively limiting their survival outside of controlled aquaculture environments.
Second, we plan to measure the influence of our engineered B. subtilis on other virulence factors of A. hydrophila, including aerolysin and hemolysin, which are two critical toxins involved in the bacterium’s pathogenicity (Khajanchi et al., 2009; Swift et al., 1997). This year, we were unable to acquire the red blood cells necessary for testing these two toxins.
Finally, we plan to test the engineered B. subtilis against other aquatic bacterial pathogens, including Edwardsiella tarda and Vibrio alginolyticus (Irshath et al., 2023), as well as any other pathogens that utilize quorum sensing for virulence factor expression. By doing so, we can explore broader applications for our bacteria in real-world settings.
References
Bennallack, P. R., Burt, S. R., Heder, M. J., Robison, R. A., & Griffitts, J. S. (2014). Characterization of a novel plasmid-borne thiopeptide gene cluster in Staphylococcus epidermidis strain 115. J Bacteriol, 196(24), 4344-4350. https://doi.org/10.1128/JB.02243-14
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
Carlier, A., Uroz, S., Smadja, B., Fray, R., Latour, X., Dessaux, Y., & Faure, D. (2003). The Ti plasmid of Agrobacterium tumefaciens harbors an attM-paralogous gene, aiiB, also encoding N-Acyl homoserine lactonase activity. Appl Environ Microbiol, 69(8), 4989-4993. https://doi.org/10.1128/aem.69.8.4989-4993.2003
Chen, B., Peng, M., Tong, W., Zhang, Q., & Song, Z. (2020). The Quorum Quenching Bacterium Bacillus licheniformis T-1 Protects Zebrafish against Aeromonas hydrophila Infection. Probiotics Antimicrob Proteins, 12(1), 160-171. https://doi.org/10.1007/s12602-018-9495-7
Chen, F., Gao, Y., Chen, X., Yu, Z., & Li, X. (2013). Quorum quenching enzymes and their application in degrading signal molecules to block quorum sensing-dependent infection. Int J Mol Sci, 14(9), 17477-17500. https://doi.org/10.3390/ijms140917477
Chen, I., & Dubnau, D. (2004). DNA uptake during bacterial transformation. Nat Rev Microbiol, 2(3), 241-249. https://doi.org/10.1038/nrmicro844
Chu, W., Zhou, S., Zhu, W., & Zhuang, X. (2014). Quorum quenching bacteria Bacillus sp. QSI-1 protect zebrafish (Danio rerio) from Aeromonas hydrophila infection. Sci Rep, 4, 5446. https://doi.org/10.1038/srep05446
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
Cybulski, L. E., del Solar, G., Craig, P. O., Espinosa, M., & de Mendoza, D. (2004). Bacillus subtilis DesR functions as a phosphorylation-activated switch to control membrane lipid fluidity. J Biol Chem, 279(38), 39340-39347. https://doi.org/10.1074/jbc.M405150200
Dong, Y. H., Gusti, A. R., Zhang, Q., Xu, J. L., & Zhang, L. H. (2002). Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species. Appl Environ Microbiol, 68(4), 1754-1759. https://doi.org/10.1128/AEM.68.4.1754-1759.2002
Dong, Y. H., Xu, J. L., Li, X. Z., & Zhang, L. H. (2000). AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proc Natl Acad Sci U S A, 97(7), 3526-3531. https://doi.org/10.1073/pnas.97.7.3526
Dsouza, M., Taylor, M. W., Turner, S. J., & Aislabie, J. (2015). Genomic and phenotypic insights into the ecology of Arthrobacter from Antarctic soils. BMC Genomics, 16(1), 36. https://doi.org/10.1186/s12864-015-1220-2
Errington, J., & Aart, L. T. V. (2020). Microbe Profile: Bacillus subtilis: model organism for cellular development, and industrial workhorse. Microbiology (Reading), 166(5), 425-427. https://doi.org/10.1099/mic.0.000922
FAO. (2023). World Food and Agriculture – Statistical Yearbook 2023. Rome Retrieved from https://openknowledge.fao.org/handle/20.500.14283/cc8166en
Fleckenstein, J. M., Matthew Kuhlmann, F., & Sheikh, A. (2021). Acute Bacterial Gastroenteritis. Gastroenterol Clin North Am, 50(2), 283-304. https://doi.org/10.1016/j.gtc.2021.02.002
Garde, C., Bjarnsholt, T., Givskov, M., Jakobsen, T. H., Hentzer, M., Claussen, A., Sneppen, K., Ferkinghoff-Borg, J., & Sams, T. (2010). Quorum sensing regulation in Aeromonas hydrophila. J Mol Biol, 396(4), 849-857. https://doi.org/10.1016/j.jmb.2010.01.002
Gelvin, S. B. (2003). Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool. Microbiol Mol Biol Rev, 67(1), 16-37, table of contents. https://doi.org/10.1128/MMBR.67.1.16-37.2003
Goodner, B., Hinkle, G., Gattung, S., Miller, N., Blanchard, M., Qurollo, B., Goldman, B. S., Cao, Y., Askenazi, M., Halling, C., Mullin, L., Houmiel, K., Gordon, J., Vaudin, M., Iartchouk, O., Epp, A., Liu, F., Wollam, C., Allinger, M., . . . Slater, S. (2001). Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58. Science, 294(5550), 2323-2328. https://doi.org/10.1126/science.1066803
Hanif, M. U., Gul, R., Hanif, M. I., & Hashmi, A. A. (2017). Heterologous Secretory Expression and Characterization of Dimerized Bone Morphogenetic Protein 2 in Bacillus subtilis. Biomed Res Int, 2017, 9350537. https://doi.org/10.1155/2017/9350537
Harrison, A. M., & Soby, S. D. (2020). Reclassification of Chromobacterium violaceum ATCC 31532 and its quorum biosensor mutant CV026 to Chromobacterium subtsugae. AMB Express, 10(1), 202. https://doi.org/10.1186/s13568-020-01140-1
Haudecoeur, E., Tannieres, M., Cirou, A., Raffoux, A., Dessaux, Y., & Faure, D. (2009). Different regulation and roles of lactonases AiiB and AttM in Agrobacterium tumefaciens C58. Mol Plant Microbe Interact, 22(5), 529-537. https://doi.org/10.1094/MPMI-22-5-0529
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
Hu, J., Arantes, V., Pribowo, A., & Saddler, J. N. (2013). The synergistic action of accessory enzymes enhances the hydrolytic potential of a "cellulase mixture" but is highly substrate specific. Biotechnol Biofuels, 6(1), 112. https://doi.org/10.1186/1754-6834-6-112
Irshath, A. A., Rajan, A. P., Vimal, S., Prabhakaran, V. S., & Ganesan, R. (2023). Bacterial Pathogenesis in Various Fish Diseases: Recent Advances and Specific Challenges in Vaccine Development. Vaccines (Basel), 11(2). https://doi.org/10.3390/vaccines11020470
Kamimura, R., Kanematsu, H., Ogawa, A., Kogo, T., Miura, H., Kawai, R., Hirai, N., Kato, T., Yoshitake, M., & Barry, D. M. (2022). Quantitative Analyses of Biofilm by Using Crystal Violet Staining and Optical Reflection. Materials (Basel), 15(19). https://doi.org/10.3390/ma15196727
Karimi, M., Behzadian, F., Rouhaninejad, H., & Yari, S. (2018). A Feasibility Study to Evaluate Bacillus subtilis as a Host for Producing Recombinant Human Parathyroid Hormone. Avicenna J Med Biotechnol, 10(3), 147-151.
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
Krishnamurthi, S., Chakrabarti, T., & Stackebrandt, E. (2009). Re-examination of the taxonomic position of Bacillus silvestris Rheims et al. 1999 and proposal to transfer it to Solibacillus gen. nov. as Solibacillus silvestris comb. nov. Int J Syst Evol Microbiol, 59(Pt 5), 1054-1058. https://doi.org/10.1099/ijs.0.65742-0
Lynch, M. J., Swift, S., Kirke, D. F., Keevil, C. W., Dodd, C. E., & Williams, P. (2002). The regulation of biofilm development by quorum sensing in Aeromonas hydrophila. Environ Microbiol, 4(1), 18-28. https://doi.org/10.1046/j.1462-2920.2002.00264.x
McClean, K. H., Winson, M. K., Fish, L., Taylor, A., Chhabra, S. R., Camara, M., Daykin, M., Lamb, J. H., Swift, S., Bycroft, B. W., Stewart, G., & Williams, P. (1997). Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology (Reading), 143 ( Pt 12), 3703-3711. https://doi.org/10.1099/00221287-143-12-3703
McDonald, I. R., Riley, P. W., Sharp, R. J., & McCarthy, A. J. (1995). Factors affecting the electroporation of Bacillus subtilis. J Appl Bacteriol, 79(2), 213-218. https://doi.org/10.1111/j.1365-2672.1995.tb00937.x
Monier, M. N., Kabary, H., Elfeky, A., Saadony, S., El-Hamed, N. N. B. A., Eissa, M. E. H., & Eissa, E.-S. H. (2023). The effects of Bacillus species probiotics (Bacillus subtilis and B. licheniformis) on the water quality, immune responses, and resistance of whiteleg shrimp (Litopenaeus vannamei) against Fusarium solani infection. Aquaculture International, 31(6), 3437-3455. https://doi.org/10.1007/s10499-023-01136-1
Morohoshi, T., Tominaga, Y., Someya, N., & Ikeda, T. (2012). Complete genome sequence and characterization of the N-acylhomoserine lactone-degrading gene of the potato leaf-associated Solibacillus silvestris. J Biosci Bioeng, 113(1), 20-25. https://doi.org/10.1016/j.jbiosc.2011.09.006
Morohoshi, T., Wang, W. Z., Someya, N., & Ikeda, T. (2011). Genome sequence of Microbacterium testaceum StLB037, an N-acylhomoserine lactone-degrading bacterium isolated from potato leaves. J Bacteriol, 193(8), 2072-2073. https://doi.org/10.1128/JB.00180-11
Mu, D., Lu, J., Qiao, M., Kuipers, O. P., Zhu, J., Li, X., Yang, P., Zhao, Y., Luo, S., Wu, X., Jiang, S., & Zheng, Z. (2018). Heterologous signal peptides-directing secretion of Streptomyces mobaraensis transglutaminase by Bacillus subtilis. Appl Microbiol Biotechnol, 102(13), 5533-5543. https://doi.org/10.1007/s00253-018-9000-y
Nayak, A., Harshitha, M., Dubey, S., Munang'andu, H. M., Chakraborty, A., Karunasagar, I., & Maiti, B. (2023). Evaluation of Probiotic Efficacy of Bacillus subtilis RODK28110C3 Against Pathogenic Aeromonas hydrophila and Edwardsiella tarda Using In Vitro Studies and In Vivo Gnotobiotic Zebrafish Gut Model System. Probiotics Antimicrob Proteins. https://doi.org/10.1007/s12602-023-10127-w
Neissi, A., Majidi Zahed, H., & Roshan, R. (2024). Probiotic performance of B. subtilis MS. 45 improves aquaculture of rainbow trout Oncorhynchus mykiss during acute hypoxia stress. Sci Rep, 14(1), 3720. https://doi.org/10.1038/s41598-024-54380-7
Nguyen, D., Joshi-Datar, A., Lepine, F., Bauerle, E., Olakanmi, O., Beer, K., McKay, G., Siehnel, R., Schafhauser, J., Wang, Y., Britigan, B. E., & Singh, P. K. (2011). Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria. Science, 334(6058), 982-986. https://doi.org/10.1126/science.1211037
Nielsen, M. E., Hoi, L., Schmidt, A. S., Qian, D., Shimada, T., Shen, J. Y., & Larsen, J. L. (2001). Is Aeromonas hydrophila the dominant motile Aeromonas species that causes disease outbreaks in aquaculture production in the Zhejiang Province of China? Dis Aquat Organ, 46(1), 23-29. https://doi.org/10.3354/dao046023
Olmos, J., Acosta, M., Mendoza, G., & Pitones, V. (2020). Bacillus subtilis, an ideal probiotic bacterium to shrimp and fish aquaculture that increase feed digestibility, prevent microbial diseases, and avoid water pollution. Arch Microbiol, 202(3), 427-435. https://doi.org/10.1007/s00203-019-01757-2
Olmos, J., & Paniagua-Michel, J. (2014). Bacillus subtilis A Potential Probiotic Bacterium to Formulate FunctionalFeeds for Aquaculture. Journal of Microbial & Biochemical Technology, 6, 1-5.
Pan, J., Huang, T., Yao, F., Huang, Z., Powell, C. A., Qiu, S., & Guan, X. (2008). Expression and characterization of aiiA gene from Bacillus subtilis BS-1. Microbiol Res, 163(6), 711-716. https://doi.org/10.1016/j.micres.2007.12.002
Park, S. Y., Lee, S. J., Oh, T. K., Oh, J. W., Koo, B. T., Yum, D. Y., & Lee, J. K. (2003). AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria. Microbiology (Reading), 149(Pt 6), 1541-1550. https://doi.org/10.1099/mic.0.26269-0
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
Phan, T. T., Nguyen, H. D., & Schumann, W. (2006). Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis. Protein Expr Purif, 46(2), 189-195. https://doi.org/10.1016/j.pep.2005.07.005
Poole, K. (2012). Stress responses as determinants of antimicrobial resistance in Gram-negative bacteria. Trends Microbiol, 20(5), 227-234. https://doi.org/10.1016/j.tim.2012.02.004
Prochaska, L. J., & Piekutowski, W. V. (1994). On the synergistic effects of enzymes in food with enzymes in the human body. A literature survey and analytical report. Med Hypotheses, 42(6), 355-362. https://doi.org/10.1016/0306-9877(94)90152-x
Rasmussen-Ivey, C. R., Figueras, M. J., McGarey, D., & Liles, M. R. (2016). Virulence Factors of Aeromonas hydrophila: In the Wake of Reclassification. Front Microbiol, 7, 1337. https://doi.org/10.3389/fmicb.2016.01337
Ryffel, G. U. (2014). Transgene flow: facts, speculations and possible countermeasures. GM Crops Food, 5(4), 249-258. https://doi.org/10.4161/21645698.2014.945883
Santos, R. A., Monteiro, M., Rangel, F., Jerusik, R., Saavedra, M. J., Carvalho, A. P., Oliva-Teles, A., & Serra, C. R. (2021). Bacillus spp. Inhibit Edwardsiella tarda Quorum-Sensing and Fish Infection. Mar Drugs, 19(11). https://doi.org/10.3390/md19110602
Schneider, J., Yepes, A., Garcia-Betancur, J. C., Westedt, I., Mielich, B., & López, D. (2012). Streptomycin-induced expression in Bacillus subtilis of YtnP, a lactonase-homologous protein that inhibits development and streptomycin production in Streptomyces griseus. Appl Environ Microbiol, 78(2), 599-603. https://doi.org/10.1128/aem.06992-11
Sharma, S., Mohler, J., Mahajan, S. D., Schwartz, S. A., Bruggemann, L., & Aalinkeel, R. (2023). Microbial Biofilm: A Review on Formation, Infection, Antibiotic Resistance, Control Measures, and Innovative Treatment. Microorganisms, 11(6). https://doi.org/10.3390/microorganisms11061614
Sniezko, I., Dobson-Stone, C., & Klein, A. (1998). The treA gene of Bacillus subtilis is a suitable reporter gene for the archaeon Methanococcus voltae. FEMS Microbiology Letters, 164(2), 237-242. https://doi.org/10.1111/j.1574-6968.1998.tb13092.x
Spizizen, J. (1958). Transformation of Biochemically Deficient Strains of Bacillus Subtilis by Deoxyribonucleate. Proc Natl Acad Sci U S A, 44(10), 1072-1078. https://doi.org/10.1073/pnas.44.10.1072
Stratev, D., & Odeyemi, O. A. (2016). Antimicrobial resistance of Aeromonas hydrophila isolated from different food sources: A mini-review. J Infect Public Health, 9(5), 535-544. https://doi.org/10.1016/j.jiph.2015.10.006
Su, Y., Liu, C., Fang, H., & Zhang, D. (2020). Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine. Microb Cell Fact, 19(1), 173. https://doi.org/10.1186/s12934-020-01436-8
Sun, B., Luo, H., Jiang, H., Wang, Z., & Jia, A. (2021). Inhibition of Quorum Sensing and Biofilm Formation of Esculetin on Aeromonas Hydrophila. Front Microbiol, 12, 737626. https://doi.org/10.3389/fmicb.2021.737626
Swift, S., Karlyshev, A. V., Fish, L., Durant, E. L., Winson, M. K., Chhabra, S. R., Williams, P., Macintyre, S., & Stewart, G. S. (1997). Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules. J Bacteriol, 179(17), 5271-5281. https://doi.org/10.1128/jb.179.17.5271-5281.1997
Tang, K., Su, Y., Brackman, G., Cui, F., Zhang, Y., Shi, X., Coenye, T., & Zhang, X. H. (2015). MomL, a novel marine-derived N-acyl homoserine lactonase from Muricauda olearia. Appl Environ Microbiol, 81(2), 774-782. https://doi.org/10.1128/AEM.02805-14
Thuy Le, A. T., & Schumann, W. (2007). A novel cold-inducible expression system for Bacillus subtilis. Protein Expr Purif, 53(2), 264-269. https://doi.org/10.1016/j.pep.2006.12.023
Urdaci, M. C., Bressollier, P., & Pinchuk, I. (2004). Bacillus clausii probiotic strains: antimicrobial and immunomodulatory activities. J Clin Gastroenterol, 38(6 Suppl), S86-90. https://doi.org/10.1097/01.mcg.0000128925.06662.69
Uroz, S., Oger, P. M., Chapelle, E., Adeline, M. T., Faure, D., & Dessaux, Y. (2008). A Rhodococcus qsdA-encoded enzyme defines a novel class of large-spectrum quorum-quenching lactonases. Appl Environ Microbiol, 74(5), 1357-1366. https://doi.org/10.1128/AEM.02014-07
Van Dyk, J. S., & Pletschke, B. I. (2012). A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes--factors affecting enzymes, conversion and synergy. Biotechnol Adv, 30(6), 1458-1480. https://doi.org/10.1016/j.biotechadv.2012.03.002
Vasudevan, S. N., Pooja, S. K., Raju, T. J., & Damini, C. S. (2023). Cisgenics and intragenics: boon or bane for crop improvement. Front Plant Sci, 14, 1275145. https://doi.org/10.3389/fpls.2023.1275145
Vestby, L. K., Grønseth, T., Simm, R., & Nesse, L. L. (2020). Bacterial Biofilm and its Role in the Pathogenesis of Disease. Antibiotics (Basel), 9(2). https://doi.org/10.3390/antibiotics9020059
Wang, W. Z., Morohoshi, T., Ikenoya, M., Someya, N., & Ikeda, T. (2010). AiiM, a novel class of N-acylhomoserine lactonase from the leaf-associated bacterium Microbacterium testaceum. Appl Environ Microbiol, 76(8), 2524-2530. https://doi.org/10.1128/AEM.02738-09
Wang, Y., Wang, X., Ali, F., Li, Z., Fu, Y., Yang, X., Lin, W., & Lin, X. (2019). Comparative Extracellular Proteomics of Aeromonas hydrophila Reveals Iron-Regulated Secreted Proteins as Potential Vaccine Candidates. Front Immunol, 10, 256. https://doi.org/10.3389/fimmu.2019.00256
Wilson, C., Lukowicz, R., Merchant, S., Valquier-Flynn, H., Caballero, J., Sandoval, J., Okuom, M., Huber, C., Brooks, T. D., Wilson, E., Clement, B., Wentworth, C. D., & Holmes, A. E. (2017). Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review. Res Rev J Eng Technol, 6(4).
Wu, X. C., Lee, W., Tran, L., & Wong, S. L. (1991). Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J Bacteriol, 173(16), 4952-4958. https://doi.org/10.1128/jb.173.16.4952-4958.1991
Xiao, F., Zhang, Y., Zhang, L., Li, S., Chen, W., Shi, G., & Li, Y. (2024). Advancing Bacillus licheniformis as a Superior Expression Platform through Promoter Engineering. Microorganisms, 12(8). https://doi.org/10.3390/microorganisms12081693
Xue, G.-P., Johnson, J. S., & Dalrymple, B. P. (1999). High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis. Journal of Microbiological Methods, 34(3), 183-191. https://doi.org/https://doi.org/10.1016/S0167-7012(98)00087-6
Yang, S., Lopez, C. R., & Zechiedrich, E. L. (2006). Quorum sensing and multidrug transporters in Escherichia coli. Proc Natl Acad Sci U S A, 103(7), 2386-2391. https://doi.org/10.1073/pnas.0502890102
Zhang, H. B., Wang, L. H., & Zhang, L. H. (2002). Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens. Proc Natl Acad Sci U S A, 99(7), 4638-4643. https://doi.org/10.1073/pnas.022056699