The antibacterial mechanism of phenylacetic acid isolated from Bacillus megaterium L2 against Agrobacterium tumefaciens

Strains and culture medium

B. megaterium L2: provided by the Institute of Fungal Resources of Guizhou University, currently stored in the China Center for Type Culture Collection (CCTCC, NO. M2012381).

A. tumefaciens T-37: purchased from the Institute of Soil and Fertilizer, Chinese Academy of Agricultural Sciences, and preserved by the Institute of Fungal Resources Laboratory of the College of Life Sciences, Guizhou University.

Beef extract peptone medium (NA): peptone 10 g/L, NaCl 5 g/L, beef extract 3 g/L, agar powder 20g/L, distilled water 1000 mL, pH natural. All media were autoclaved at 121 °C for 15 min.

Experimental reagents and instruments

The experimental reagents and instruments used in this study are shown in Tables 1 and 2.

Isolation and purification of PAA

The steps of isolation and purification of PAA roughly include five steps. (i) The first is the large-scale fermentation of B. megaterium L2. (ii) The fermented bacterial cells were spray-dried to form a pulverized powder, and then extracted and concentrated by refluxing with ethanol at 80 °C to obtain the crude ethanol extract. (iii) Then, according to the antibacterial activity of the crude extract to the three indicator bacteria, fractional distillation was carried out with petroleum ether, ethyl acetate, n-butanol, and water to obtain the organic fraction with the highest activity, namely the ethyl acetate phase. (iv) The ethyl acetate extract was separated by silica gel column chromatography to obtain ten fractions of B1–B10. (v) Finally, the B4 fraction was separated by atmospheric pressure silica gel column chromatography (200-300 mesh) and recrystallization to obtain the monomeric compound PAA. For specific information, please refer to the literature (Xie et al., 2021).

Determination of IC₅₀ of PAA

Preparation of bacterial suspension: T-37 strain was activated and transferred into the NA medium, and the cultures were incubated at 30 °C with shaking at 150 r/min for the logarithmic growth phase (18 h). Then, the T-37 strain culture was centrifuged at 6,000 r/min for 5 min to prepare bacterial suspension with sterile water, and the final cell concentration was adjusted to 1 × 10⁸ CFU/mL approximately for further studies. PAA and chloramphenicol were dissolved in DMSO to make 10 mg/mL stock solutions. Then the suspension of the T-37 strain was inoculated into NA liquid medium at 10% inoculum volume, PAA was then added to NA liquid medium to final concentrations of 0.6, 0.8, 1.0, 1.2, and 1.4 mg/mL. The control was prepared as described above but in the absence of PAA. All samples were incubated at 30 °C, 150 r/min for 18 h, and the optical density (OD) value was measured at the maximum absorption wavelength of 400 nm. The sterile water was used as the negative control and chloramphenicol as the positive control, the inhibition rate was calculated according to the following formula, and the IC₅₀ values of PAA and chloramphenicol were calculated by GraphPad Prism 9 software. The experiment was repeated 3 times. ${\displaystyle Inhibitionrate\left(\text{\%}\right)=\frac{ODcontrol-ODtreatment}{ODcontrol}\times 100}$ where OD control is the OD value of the negative control; and OD treatment is the OD value of PAA and chloramphenicol treatment, respectively.

Effects of PAA on cell membrane permeability of T-37 pathogenic bacteria

This analysis was performed using the method described by Song et al. (2016), with some modifications. T-37 bacterial suspension (1 × 10⁸ CFU/mL) was inoculated into NA liquid medium at 10% inoculum volume, and the PAA was added to make the final concentration IC₅₀. The bacteria cultures were incubated at 30 °C, with shaking at 150 r/min. Samples were taken out after being treated for 0, 1, 2, 3, 4, and 5 h, then 5 mL of the bacterial culture was centrifuged at 4,000 r/min for 10 min, and the supernatant was taken to measure the relative conductivity. Sterile water and chloramphenicol were used as the negative control and positive control, respectively. All tests were performed in triplicate.

Effects of PAA on cell membrane integrity of T-37 pathogenic bacteria

Leakage of total soluble sugar concentration

The determination of the total sugar of the sample was conducted according to the method of Zhou et al. (2019). We draw first the standard curve using the concentration of glucose as the abscissa (mg/mL) and the absorbance as the ordinate. Then after, the bacteria suspension of T-37 was inoculated into NA liquid medium at 10% inoculum volume, and the PAA was added to make the final concentration IC₅₀. The bacteria cultures were incubated with shaking (150 r/min) at 30 °C. After 0, 2, 4, 6, 8, 10, and 12 h of incubation, aliquots of 1 mL were taken out and centrifuged, then took 50 µL of supernatant and added to 200 µL of anthrone reagent. The mixture was cooled on ice for 5 min and boiled for 10 min immediately. After it was naturally cooled to room temperature, the absorbance was measured with SpectroAmax TM 250 at 630 nm using sterile water as the negative control and chloramphenicol as the positive control. All tests were performed in triplicate.

SDS-PAGE analysis of total protein of T-37 bacteria

This analysis was performed using the method of Zhao et al. (2015), with some modifications. The bacterial suspension containing IC₅₀ concentration PAA was prepared according to the previous method. The bacteria cultures were incubated at 30  °C, with shaking at 150 r/min. After 0, 4, 8, and 12 h of incubation, 1 mL of bacterial culture was taken out, centrifuged at 4,000 r/min for 10 min, and then the supernatant was discarded, and the precipitate was washed twice with sterile water. Then added 40 µL of 2 × loading buffer into the bacterial suspension, heated at 100 °C for 10 min, and centrifuged to collect the supernatant. Sterile water and chloramphenicol were used as the negative control and positive control, respectively. The SDS–PAGE was performed with a 12% separating gel, 5% stacking gel, and 5 × Tris-glycine electrode buffer, the sample volume was 10 µL, the voltage was 85 V, the current was 100 mA, and the time was 1.5–2 h. Then the gel was stained with 0.5% Coomassie brilliant blue R-250 was stained overnight, and then decolorized with a decoloring agent (ethanol-acetic acid-water solution, 10:5:85, v/v) to obtain the separated protein bands.

Effects of PAA on phosphorus metabolism in T-37 bacteria

The determination of the phosphorus content of the sample was conducted according to the method of Zhai et al. (2006). Bacterial cells were collected by centrifugation at 4000 r/min for 10 min, washed twice with sterile water, and diluted the bacterial cells to OD₆₀₀ = 0.5. Added 0.5 mL bacterial culture and 0.5 mL glucose solution (1.0 mg/mL) to the test tube, mixed with 200 µL phosphoric acid standard solution (500 µg/mL), and then bacterial cells were mixed again with PAA to make the final concentration of IC₅₀. When cultured for 0, 2, 4, 6, 8, 10, and 12 h, 0.1 mL of bacterial culture was added to 1 mL of trichloroacetic acid-ferrous sulfate solution, which was reacted at room temperature for 10 min, and centrifuged at 4,000 r/min for 10 min. The ammonium molybdate solution (50 µL) was added to 0.2 mL of supernatant, shaken well, incubated at 30 °C with a water bath for 15 min, and cooled naturally to room temperature, then measured the absorbance with SpectroAmax TM 250 at 630 nm. Sterile water and chloramphenicol were used as the negative control and positive control, respectively. All tests were performed in triplicate.

Effects of PAA on the content of reactive oxygen species (ROS) in T-37 cells

The bacteria suspension of T-37 was inoculated into NA liquid medium at 10% inoculum volume and cultured at 30 °C for 18 h. The content of ROS was determined according to the kit instructions. Cells from the bacteria culture were collected by centrifuged for 5 min at 12,000 r/min, washed twice with 0.1 M phosphate buffer solution (PBS, pH 7.4), and dissolved with 10 µmol/L 2, 7-dichlorofluorescein diacetate (DCFH-DA). All samples were incubated at 37 °C for 30 min, shaken for 3–5 min, and then washed the cell pellet with PBS three times to remove extracellular residual DCFH-DA. Finally, added PAA to the prepared solution to make the final concentration of IC₅₀. Fluorescence intensity was detected by a fluorescence microscope every 10 min. The fluorescence results were quantitatively analyzed by Image J 17.0 software, and the fluorescence intensity was calculated according to the following formula. ${\displaystyle Fluorescenceintensity=\frac{Fluorescenceintensitywithinthesamplingarea}{Samplingarea}.}$

Effects of PAA on the activity of key enzymes of T-37 tricarboxylic acid

The bacteria suspension of the T-37 strain was inoculated into 100 mL of fresh culture medium at 10% inoculum volume in the presence of PAA at the IC₅₀ concentration, and the negative control was prepared as described above, but in the absence of PAA, chloramphenicol was used as the positive control, all samples were incubated at 30 °C for 18 h. After treatment, the activities of succinate dehydrogenase (SDH) and NAD-malate dehydrogenase (NAD-MDH) were determined by the SDH assay kit and NAD-MDH assay kit (Solarbio, Beijing, China) respectively according to the manufacturer’s guidance. The experiments were performed in triplicate.

Scanning electron microscopy (SEM) analysis

This analysis was performed using the method of Zhang et al. (2016), with some modifications. The bacterial suspension containing IC₅₀ concentration PAA was prepared according to the previous method. The control (without PAA) and samples were incubated at 30 °C, 150 rpm for 18 h, and centrifuged at 4,000 r/min for 10 min after cultivation, discarded the supernatant, and washed the precipitate twice with PBS for use. Scanning electron microscope (SEM) sample processing: the samples were fixed with 2.5% glutaraldehyde at 4 °C for 10 ∼12 h, washed twice with 0.1 M PBS, and then dehydrated in gradient ethanol solutions of 30%, 50%, 70%, 90%, and 100%. Then the 100% ethanol solution was replaced by tert-butanol and fixed for 10 min. Finally, all the samples were plated with a metal film in the vacuum evaporator and observed by SEM (Hitachi S-3400N, Hitachi, Japan).

Experimental data processing

Excel 2016 was used for data processing, and IBM SPSS Statistics 26 software was used for all statistical analyses. The data were analyzed by analysis of variance (ANOVA) and Duncan test, besides, the calculation of the IC₅₀ value and graphs were produced using Graph Pad Prism 9.0, and Image J 17.0 software was used for the conversion of fluorescence intensity and gray value. P < 0.05 was considered statistically significant.

IC₅₀ of PAA determination results

In this experiment, the inhibition rate of PAA on T-37 was determined at five concentrations of 0.6 mg/mL, 0.8 mg/mL, 1 mg/mL, 1.2 mg/mL and 1.4 mg/mL. Obtained results (Fig. 2) showed that at the inhibition rate of PAA reached 80.00 ± 0.02% at the mass concentration of 1.0 mg/mL and no significant difference were observed at concentration of 1.2 mg/mL and 1.4 mg/mL (P > 0.05). While the inhibition rate of chloramphenicol reached 87.76 ± 0.01% at the concentration of 0.2 mg/mL and was not significantly different from that at 0.4 mg/mL (P > 0.05). Concerning the IC₅₀ calculated by Graph Pad Prism 9 software was 0.8038 mg/mL and 0.0658 mg/mL respectively for the PAA and the chloramphenicol and those values were for subsequent experiments.

Examination of relative electric conductivity to indicate changes in the permeability of T-37 bacterial cell membranes

The results in Fig. 3 showed the effect of compound PAA on the membrane permeability of T-37 cells by relative electric conductivity assay. Our data suggested that there was less change in the relative electric conductivity during the whole experiment period in the negative control and positive control groups, and the relative electric conductivity of T-37 cells treated with PAA showed an upward trend at 0∼3 h, but it began to decrease for 4 h. Specifically, after treatment for 1 h, there was a significant difference in the extracellular relative electric conductivity between the PAA group and the negative group (P < 0.05), indicating that PAA began to destroy the cell membrane at this stage, and the intracellular electrolyte was released, leading to an increase in the relative electric conductivity. At 3 h, the relative electric conductivity of the PAA treatment group increased by 8.87% and 10.55% compared with the chloramphenicol treatment and the negative treatment group, respectively (P < 0.05). The relative electric conductivity of the bacteria cells treated with PAA decreased slowly at 4∼5 h, but it was still higher than in other treatment groups. These results indicated that PAA could affect the permeability of the T-37 cell membrane and release the cell contents, which led to the increase of electric conductivity in the extracellular environment.

Effects of PAA on cell membrane integrity of T-37 pathogenic bacteria

Leakage of T-37 bacterial nucleic acid and protein

The integrity of the cell membrane was determined by measuring the release of protein (OD₂₆₀) and nucleic acid (OD₂₈₀) of the supernatant of tested bacteria. The nucleic acid leakage of T-37 cells caused by PAA was shown in Fig. 4A. After treatment with PAA, the absorbance value increased linearly. At 2 h, the nucleic acid leakage of T-37 cells was not significantly different from the negative control, which indicated that the cell membrane was not seriously damaged and there was no obvious leakage of nucleic acid. Compared with the negative control, the leakage of nucleic acid treated with PAA increased by 1.32 times for 4 h (P < 0.05). In addition, after 10 h of incubation, the nucleic acid leakage of the PAA treatment group increased by 25% and 70.45% compared with the negative control and the positive control group, respectively, indicating that the cell membrane integrity of the T-37 cells treated with PAA was destroyed, resulting in the leakage of intracellular nucleic acids. Moreover, the protein leakage of T-37 cells caused by PAA was shown in Fig. 4B. The PAA treatment group was significantly higher than the negative treatment group at 2 h (P < 0.05), and the absorbance value showed an upward trend with the extension of the culture time, indicating that the cell membrane of T-37 cells had been destroyed at 2 h. At 10 h, the OD₂₈₀ of protein treated with PAA was 0.082 ± 0.005, which increased by 36.67% and 74.47% compared with the chloramphenicol treatment group and the negative treatment group (P < 0.05). The above results demonstrated that the integrity of the cell membrane was severely damaged at this time, resulting in a large amount of intracellular protein leakage.

Leakage of total soluble sugar in T-37 bacteria

The leakage of total soluble sugar in T-37 cells caused by PAA was shown in Fig. 5. The total sugar concentration in the culture medium of the blank treatment group showed a gradual downward trend with time, which can be described as the continuous consumption of sugar substances during cell growth. The leakage of total soluble sugar from T-37 cells treated with PAA and chloramphenicol increased as the treatment time up to 2 h with significant differences (P < 0.05), indicating that the T-37 cell membrane had been damaged at this time. And the concentration of total soluble sugar in the extracellular environment increased with the increase of treatment time and reached the maximum at 10 h (0.2430 ± 0.0047 mg/mL), which increased by 29.74% and 144.47% compared with the chloramphenicol group and the negative treatment group, respectively (P < 0.05). These findings indicated that the integrity of the cell membrane of pathogenic bacteria T-37 was severely damaged, and intracellular sugar substances leaked into the medium, increasing the concentration of total soluble sugars in the medium.

SDS-PAGE total protein profile of T-37 bacteria

The SDS-PAGE spectrum of the soluble protein of T-37 cells treated with PAA is shown in Fig. 6. The protein profiles of T-37 cell treated with PAA differed from those of the control. After treatment with PAA at IC₅₀ concentration for 4 h, the protein bands of the negative control group showed strong intensities, but the bacterial protein bands of the PAA treatment group got much fainter, and some even disappeared. Similar results were also found in bacteria cells treated with chloramphenicol. After treatment for 8 h, the protein bands in the PAA treatment group almost disappeared. The above results indicated that the reason for the disappearance of protein bands’ might be PAA interfered synthesis and expression of T-37 cell proteins, or resulted in the leakage of proteins from bacterial cells.

Phosphorus metabolism in T-37 bacteria

The overall metabolic function and growth of cells can be reflected by measuring phosphorus consumption in microbial metabolic activities. It can be seen from Fig. 7 that the phosphorus concentration in the three treatment groups showed a downward trend with the incubation time, indicating that the pathogenic bacteria T-37 consumed phosphorus for normal metabolic activities during the cultivation process. However, in the PAA and chloramphenicol treatment groups, the phosphorus consumption of T-37 cells decreased with the increase in incubation time, which indicated that phosphorus metabolism was severely affected by PAA.

Reactive oxygen species (ROS) content of T-37 bacteria

The effect of PAA on the ROS content of pathogenic bacteria T-37 was shown in Fig. 8. PAA can stimulate T-37 cells to produce more ROS, and the ROS content level in the cytoplasm of the PAA treatment group showed a trend of first increasing and then decreasing, reaching a peak value at 30 min of treatment, which was 1.112 ± 0.064, an increase of 45.42% compared with the negative treatment group (P < 0.05). After being incubated for 40 min, chloramphenicol reached the peak value, which was 0.861 ± 0.016, an increase of 16.75% compared with the negative treatment group (P < 0.05). In summary, PAA can increase the ROS content in T-37 cells, which may damage the cellular structure of T-37 cells.

MDH and SDH tricarboxylic acid key enzyme activities in T-37 bacteria

The antibacterial action of PAA may be partly due to its effect on key enzyme activities of T-37 cells. By measuring the two key enzyme activities of MDH and SDH in the tricarboxylic acid cycle (TCA cycle) of pathogenic bacteria T-37 treated with PAA, it can reflect the energy metabolism status. As we can see from Fig. 9A, the MDH enzyme activity of strain T-37 in the negative group was at a normal level. Compared with chloramphenicol and PAA treatment, the enzyme activity of MDH in T-37 cells was significantly different (P < 0.05). The changes in SDH enzyme activity in the negative control group and treatment groups (both PAA and chloramphenicol) exhibited the same trend (Fig. 9B). These results indicated that PAA can decrease effectively the enzymatic activities of MDH and SDH in the TCA cycle, resulting in the influence of the energy metabolism pathway of T-37 cells, thereby inhibiting its normal physiological metabolism.

Scanning electron microscope (SEM) observation of T-37 cells

SEM micrographs revealed changes in morphology and the outer surface of bacterial cells. Figure 10 showed the SEM photomicrographs of T-37 cells treated with and without PAA. As observed in Fig. 10A, untreated cells showed typical bacilliform morphology with a plump and smooth surface, no swelling and shrinkage, and were uniform in distribution, indicating that the cell membrane was intact and the cells were in normal condition. In contrast, T-37 cells treated with PAA at IC₅₀ concentration revealed a severely damaging effect on the cell morphology. It can be seen from Fig. 10B that adhesion occurs between the cells of strain T-37, the boundaries were blurred, the cells were deformed and tarnished, and the cells can be seen to be damaged and broken, some bacterial cells appeared dented, and the cells were obviously damaged, indicating that PAA could destroy the morphological structure of strain T-37 cells. These findings demonstrated that PAA treatment may result in damage to the T-37 strain cell wall and cytoplasmic membrane and further inhibit cell growth.