There were 62 strains of Bt used in this study which were isolated from Middle Tennessee. The Bt strains were grown in Luria Bertani (LB) broth and incubated overnight at 30˚C in a shaking incubator set at a speed of 200 rpm. In addition, LB agar was also used in the AHL-lactonase assay.

CV026, a mutant form of Chromobacterium violaceum, which produces N-acyl homoserine lactone to activate the gene responsible for a purple pigment, was used as biosensor. In contrast E. coli, was used in the study as a negative control. CV026 and E. coli were both grown in LB broth and incubated overnight at 30˚C in a shaking incubator set at a speed of 180 rpm.

Genetic features of AHL-lactonase of the Bt strains were examined. The DNA was extracted using a DNA purification kit from Sigma and the provided protocol was followed. Due to the cell wall thickness in Bt, proteinase-k and lysozyme were used in the first steps of DNA extraction. A Nanodrop spectrophotometer was used to measure, quantify, and assess the purity of the DNA nucleic acids in each Bt strain.

PCR was performed to determine the presence of AHL lactonase gene aiiA using the forward primer 5'-ATGGGATCCATGACAGTAAAGAAGCTTTAT-3' and reverse primer 5'-GTCGAATTCCTCAACAAGATACTCCTAATG-3' obtained from Thermo Fisher Scientific, USA. The PCR reaction was set up at a volume of 50 μl each reaction containing 45 μl supermix solution (Thermo Fisher Scientific, USA), 1 μl forward primer, 1 μl reverse primer, 1 μl of DNA, and 2 μl of ddH₂O. The PCR reaction was set to run 30 cycles as follows: (1) Denaturation at 94˚C for 30 s; (2) Annealing at 55˚C for 30 s; and (3) Extension at 72˚C for 60 s per cycle with the final extension set at 72˚C for 5 min.

After the PCR reaction was completed, gel electrophoresis of the products was performed. Prior to loading the reaction into the gel, the PCR reaction mixture was mixed with loading dye. Then agarose gel (1%) was prepared in 100 mL of TAE buffer. Ethidium bromide was added to the agarose and mixed well before it was poured into the tray. Once gel was solidified, PCR products were loaded into the wells where electrophoresis was set at 90 V for 40 min. Bands were expected to be seen around 800 bp under UV light and the ladder used in this experiment was 100 bp.

N-acyl homoserine lactone (AHL) was used a source of the quorum sensing molecule. Two types of AHL were tested in this study: N-hexanoyl homoserine lactone (C₆-HSL) and N-3-oxo-hexanoyl homoserine lactone (3-oxo-C₆-HSL) (Sigma-Aldrich, USA).

To determine the enzymatic activity of the AHL lactonase, CV026 was used to detect the presence of C₆-HSL which would produce the purple pigment in CV026. This was done using the LB agar plates containing wells in which 100 μL of a mixture of the AHL with or without Bt was placed.

To microcentrifuge tubes, overnight Bt culture, and a solution of C₆-HSL were added in a 1:1 volume to volume ratio and incubated at room temperature. The optical density of the Bt samples was measured at a wavelength of 600 nm (OD₆₀₀) and the absorbance was set at 1.0 for standardization and mixed with AHL as follows: 1) Bt culture (OD₆₀₀ of 1.0) with 10 μM C₆-HSL and 2) Bt culture (OD₆₀₀ of 1.0) with 5 μM C₆-HSL.

After a one-hour-incubation at room temperature, the mixture of Bt culture/AHL was centrifuged, and the supernatant was loaded in the LB agar wells after streaking with CV026. The plates were incubated at 30˚C overnight. E. coli was used instead of Bt as a negative control. AHL-lactonase activity was accessed by the lack of a purple pigment around the wells of the plates.

In this experiment the C₆-HSL was not directly incubated with the Bt culture. After overnight growth, the bacterial cells were centrifuged first, and then the supernatant which would contain AHL-lactonase was taken incubated with C₆-HSL at different temperatures; room temperature, 30˚C, 40˚C, 50˚C, and 60˚C. After an hour incubation of the supernatant and C₆-HSL, the mixture was loaded into the wells of plates streaked with CV026, the plates incubated at different temperature and the presence or absence of the purple pigment was observed.

Degraded AHL molecules by AHL-lactonase can be reformed back to the active molecule in an acidic environment. After Bt cultures were incubated with C₆-HSL and then centrifuged, HCL was added to the supernatant and incubated for 1 h at room temperature. The mixture was loaded into the LB agar wells of plates that were streaked with CV026. The plates were incubated at 30˚C for 24 h, and the presence or absence of the purple pigment was observed.

In addition to the activity of AHL lactonase on C₆-HSL in Bt, another test was performed on modified C₆-HSL which was 3-oxo-C₆-HSL. This test was run to assess whether AHL-lactonase in Bt strains had the ability to inactivate different AHLs. C₆-HSL was replaced by 3-oxo-C₆-HSL and the protocol was performed as described above.

Although clavulanate potassium has been used to inhibit β-lactamase from degrading beta lactam antibiotics, this study tested its action on AHL lactonase since both β-lactamase and AHL lactonase belong to the same superfamily. In addition to the mixture of Bt culture and AHL, clavulanate potassium was added to the mixture. This mixture was incubated for different periods of time at room temperature and then centrifuged. The supernatant was loaded into the agar wells streaked with CV026 and incubated at 30˚C for 24 h. The plates were then observed for the presence or absence of the purple pigment.

A total of 62 strains of Bacillus thuringiensis were screened for the aiiA gene using PCR. The strains were numbered 1 through 72; however, strains 31, 55, 59, 61, 64, 66, 68, 70, 71 and 72 did not grow. As a result, the study included 62 strains of Bt, among which 56 possessed the aiiA gene, and six strains did not possess the gene (Figure 1).

Although the PCR results showed that 56 strains possess the AHL lactonase gene, not all strains with the aiiA gene degraded C₆-HSL. As described below, the bioassay activity was categorized into three different groups based on the concentration of the bacterial culture as well as the concentration of C₆-HSL.

Group 1 assay was done to screen for the activity of lactonase in the Bt strains. The strains were grown overnight, when the optical density (OD₆₀₀) was determined to be between 2.0 and 2.2. At the beginning of the experiment different concentrations of C₆-HSL were tested and the best concentration was determined to be 10 μM and 5 μM. Once the screening was performed the assay showed that 45 strains out of the 56 strains completely degraded C₆-HSL at 10 μM (Figure 2).

In group 2, the concentration of the Bt strains was set an OD₆₀₀ of 1, and the concentration of C₆-HSL was 5 μM. The pigment was observed in this condition and the number of strains degrading C₆-HSL decreased to 20 strains inhibiting pigment formation.

The last group was performed by mixing 10 μM C₆-HSL with a low concentration of Bt cultures (OD₆₀₀ of 1.0). This category revealed that only 11 strains were able to degrade 10 μM C₆-HSL and inhibit the production of the pigment.

The degradation of C₆-HSL by AHL-lactonase can be repaired by lowering the solution’s pH with hydrochloric acid (HCl). The acidic environment restores the structure of C₆-HSL by lactone-ring closing that was cleaved by AHL-lactonase activity. This resulted in exposing the degraded C₆-HSL to HCl which restores the function of C₆-HSL and thus, regenerated pigment production in CV026. The result showed that as the volume of the HCl increases which consequently lowers the pH, the intensity of the pigment increases as well.

There are different reasons that involve the hydrolysis of the lactone ring and inactivating quorum sensing. Inhibiting the lactonase activity produced by the Bt strains was assessed by clavulanate potassium which is a beta-lactamase inhibitor and prevents antibiotic inactivation by bacterial lactamases. It was used as an inhibitor of AHL-lactonase activity since both AHL-lactonase and β-lactamase are structurally similar. Bt strains were incubated with both clavulanate potassium and C₆-HSL at the same time. The results indicated that clavulanate potassium was a suitable inhibitor of the lactonase activity because the lactonase could not hydrolyze C₆-HSL when incubated with clavulanate potassium.

There are different molecules of N-acyl homoserine lactone produced by microbes. These molecules are specific to a species and a role. Due to this variation in the AHL molecules, enzymatic activities of quorum sensing inhibitors are also specific to certain molecules. A slight variation could change the chemical characteristic of the molecules. For example, C₆-HSL is a non-polar molecule, while 3-oxo-C₆-HSL is a polar molecule. This experiment tested whether the lactonase could also degenerate 3-oxo-C₆-HSL as it degraded C₆-HSL. C₆-HSL was replaced with 3-oxo-C₆-HSL and tested for pigment production by CV026. It was found that CV026 turned purple, indicating that 3-oxo-C₆-HSL molecules were not degraded. These results show that the lactonase in the Bt strains have a specific activity on C₆-HSL but not on 3-oxo-C₆-HSL.

In this experiment the C₆-HSL was not incubated directly with the Bt culture. After the Bt strains were grown overnight, the bacterial cells were first centrifuged, and then the supernatant which contained the lactonase was incubated with C₆-HSL at different temperatures; room temperature, 30˚C, 40˚C, 50˚C, and 60˚C. Following one-hour incubation of the supernatant with C₆-HSL, the mixture was loaded into the wells of plates streaked with CV026. The outcome of this experiment found that there was no degradation of C₆-HSL when the mixture was incubated at room temperature or at 30˚C as the intensity of the pigment at these temperatures was identical to the control. However, as the temperature increased, basically at 40˚C and 50˚C, the pigment around the wells was faint due to slight degradation of C₆-HSL. The pigment disappeared completely when the mixture was incubated at 60˚C. This indicates that the supernatant contained active AHL lactonase; however, the enzyme showed activity only at higher temperatures as CV026 did not produce the pigment at a temperature of 60˚C.

In addition, different incubation periods (0, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min) of supernatant/C₆-HSL mixture were set to determine at what time the lactonase completely degenerates C₆-HSL. It was found that a slight degradation occurred during the beginning of the incubation periods (20 min and 30 min). However, complete degradation was established at 40 min and a longer period of incubation.

The AHL-lactonase, aiiA, gene is a common lactonase gene found in Gram-positive bacteria especially Bacillus species including B. cereus, B. thuringiensis, and B. mycoides. There are other AHL-lactonase genes found in different Gram-positive bacteria such as aiiM in Microbacterium testaceum [10] and qsdA in Rhodococcus erythropolis [23] as well as in Gram-negative bacteria such as aiiB in Agrobacterium tumefaciens [24] and ahlK in Klebsiella pneumoniae [25] . After performing PCR and gel electrophoresis, this study found that the majority, around 90%, of Bt isolates possess the aiiA gene. AHL-lactonase enzymes are not the same, that is, aiiA and AiiB enzymes belong to the metallo-β-lactamase family while AiiM and QsdA are related to a/β hydrolase family and phosphotriesterase family, respectively.

The AHL-lactonase from the different Bt isolates showed a variation in C₆-HSL degradation. Among the 56 strains with aiiA gene, 25 strains showed weak activity, 9 strains showed moderate activity, and 11 strains showed potent activity. The activity varies on the concentration of the bacterial cells based on the optical density as well as on the concentration of the C₆-HSL molecules (10 µM and 5 µM). A previous study also showed that some Bt strains showed intense degradation and others weak degradation [26] . Although the lactonase degraded C₆-HSL, it could not break down 3-oxo-C₆-HSL, indicating that the lactonase cannot hydrolyze an acyl group with a substitution in C-3, and it specifically hydrolyzes the C₆-HSL molecule.

AHL molecules are affected by the pH of the medium. Increasing the pH leads to the hydrolysis of the homoserine lactone similar to the AHL lactonase. On the other hand, lowering the pH to less than two leads to reformation of the hydrolyzed products to the original molecules [27] . In the findings of this work, the reformation of degraded AHL was demonstrated by purple pigment around CV026. When the hydrolysis product of lactonase was incubated with HCl and compared to the hydrolysis product of lactonase with no HCL the purple pigment was present. This reversible process by acidification is known as lactonolysis. Hydrolysis products of other enzymes such as AHL acylase or oxidoreductase cannot be restored by acidification. Thus, the hydrolysis product of lactonase is pH-dependent, and acidification can assure that the molecules were degraded by AHL lactonase [28] .

It is known that the hydrolyzed product by lactonase restores the degraded AHL into active AHL when the pH is <2. In this study, pH was presented in the ratio between HCl and the mixture (Bt supernatant/AHL). It was found that at low pH, the lactonolysis starts to establish and thus gives light pigment. When pH was, the pigment got darker, indicating more reformation. Lowering the pH by adding more HCl, for example, 1:2 or 1:1 would increase the chance of getting more active AHL, but it would increase a clear zone around the wells as CV026 bacterium cannot grow in a highly acidic environment.

Studies have shown that AHL-lactonase belongs to the metallo-β-lactamase family [29] . Due to the high similarity between the two enzymes, clavulanate potassium, which has been used to block the activity of β-lactamase, was used to inhibit AHL-lactonase activity. When clavulanate potassium was added to the mixture (Bt cell/AHL), it inhibited the AHL lactonase from degrading C₆-HSL and thus, CV026 turned to purple. Hence, clavulanate potassium was found to be a suitable inhibitor and can inhibit AHL lactonase. This ensures that degradation occurred by AHL lactonase and not by other enzymes. It was a good inhibitor to be used to block lactonase activity and to test whether other AHL enzymes exist in a particular organism.

This study showed that AHL lactonase was a temperature-dependent enzyme when the supernatant of the bacterial cells was used for the enzymatic activity. At a temperature of 20˚C and 30˚C, no enzymatic activity was observed as pigment appeared as dark as the control (AHL). When the mixture (Bt supernatant/AHL) was incubated at 40˚C, the pigment appeared faint, indicating slight hydrolysis of AHL. However, when the mixture was incubated at a high temperature (>60˚C), the pigment disappeared in each of Bt strains of this study. Therefore, this study showed that the activity of AHL lactonase depended upon temperature when the supernatant of the Bt culture was mixed with AHL molecules. In addition, the result showed that AHL-lactonase is a thermostable enzyme and can tolerate high temperature (>60˚C) without losing activity. Similarly, a previous study showed that AidB, another lactonase belonging to the metallo β-lactamase superfamily, was thermostable in the range 30˚C to 80˚C and the optimum temperature of AidB was 60˚C [30] .