Antimicrobial genes from Allium sativum and Pinellia ternata revealed by a Bacillus subtilis expression system

Abstract

Antimicrobial genes are found in all classes of life. To efficiently isolate these genes, we used Bacillus subtilis and Escherichia coli as target indicator bacteria and transformed them with cDNA libraries. Among thousands of expressed proteins, candidate proteins played antimicrobial roles from the inside of the indicator bacteria (internal effect), contributing to the sensitivity (much more sensitivity than the external effect from antimicrobial proteins working from outside of the cells) and the high throughput ability of screening. We found that B. subtilis is more efficient and reliable than E. coli. Using the B. subtilis expression system, we identified 19 novel, broad-spectrum antimicrobial genes. Proteins expressed by these genes were extracted and tested, exhibiting strong external antibacterial, antifungal and nematicidal activities. Furthermore, these newly isolated proteins could control plant diseases. Application of these proteins secreted by engineered B. subtilis in soil could inhibit the growth of pathogenic bacteria. These proteins are thermally stable and suitable for clinical medicine, as they exhibited no haemolytic activity. Based on our findings, we speculated that plant, animal and human pathogenic bacteria, fungi or even cancer cells might be taken as the indicator target cells for screening specific resistance genes.

Figure 1

Killing effects of intracellular expression of antimicrobial genes on host cells. (a,b) The B. subtilis expression system showing autolysis from 12 to 60 h (a and b are the same culture dish). A drop of 2 µl of B. subtilis strains was placed on LB plates. Kanamycin was used as screening antibiotic. All colonies (a: I–VI) grew normally in the first 12 h, while after 48 h three different colonies (b: I–III) with intracellular expression of AsR36, AsR117 and AsR416 genes showed autolysis and three different colonies (IV–VI) showed no autolysis (control). (c,d) The E. coli strain harboring AsRE67 gene stained by trypan blue and bromophenol blue. The strain was spread on LB plates covered with membrane filters. Damaged host cells were stained blue (d: cell death was caused by IPTG-induced AsRE67 gene expression), and intact host cells were not able to be stained (c: control, without IPTG induction and cell death) after growth in the presence or absence of IPTG. (e,f) Scanning electron microscopy of B. subtilis, (e) control colonies no autolysis, and (f) autolyzed colonies (AsR416 protein intracellularly disrupted the host cells). (g,h) Scanning electron microscopy of E. coli, (g) unstained colonies as control, and stained colonies (h) with AsRE76 caused shrinking cells. Experiments were repeated three times under the same conditions, and similar results were observed.

Figure 2

Antimicrobial activities of screened proteins. (a–i) Antibacterial effects of proteins screened by the B. subtilis system, in which 20 mg proteins extracted by ammonium sulfate were dropped on filter papers, and inhibition zones appeared after 6–12 h. Indicator bacteria are B. subtilis 168 (a), B. anthraci (b), B. cereus (c), R. solanacearum (d), C. fangii (e), B. subtilis WB800 (f), C. michiganensis subsp. michiganense (g), B. subtilis 330-2 (h), and C. michiganensis subsp. insidiosus (i). AsR416 (I) and AsR36 (II) are tested proteins, As118 (III) and B. subtilis WB800 (IV) are controls. (j) Antibacterial effects of proteins screened by the E. coli expression system. AsRE67 (V) and AsRE39 (VI) proteins induced by IPTG, and AsRE67 (VII) without IPTG induction. (k) Antifungal effects of proteins screened by B. subtilis expression system. The P. capsici was inoculated in the center. AsR416 (I) and AsR36 (II) are tested proteins, and As118 (III) and B. subtilis WB800 (IV) are controls. Only the AsR416 protein had a significant effect against P. capsici. Experiments were repeated three times under the same conditions, and similar results were observed.

Figure 3

Protein thermal stability. AsR498 (a), AsR416 (b) and AsR117 (c) were confronted with B. subtilis 168 (background indicator bacteria) after heating at 4 °C (I), 30 °C (II), 50 °C (III), 70 °C (IV), and 100 °C (V) for 15 minutes. (d) Inhibition diameters of 3 proteins with temperature curves. Vertical bars shows SD. Data are the mean values from three individual experiments. (e) Western blot confirmed the expression of the purified AsR416 gene. The full-length blot was presented in Supplementary Fig. S3. Experiments were repeated three times under the same conditions, and similar results were observed.

Figure 4

The PI uptake assay. (a–f) Confocal laser scanning microscopy analysis of PI staining. The B. subtilis 168 cells (1 × 10⁸ CFU/ml) incubated with AsR416 protein (a–c) and PBS buffer without test protein (d–f). The B. subtilis 168 cells under white light (a,d) and under fluorescence (b,e). (c) Combination of a and b. (f) Combination of d and e. (g) Flow cytometry analysis of PI staining. The B. subtilis 168 cells incubated with PBS buffer (control) and AsR416 and observed under FACSVerse machine. The x-axis shows the relative fluorescence intensity. Experiments were repeated three times under the same conditions, and similar results were observed.

Figure 5

Percent inhibition of different proteins against P. capsici. Leaves after treatment with purified proteins of AsR498 (b), AsR416 (c) and AsR117 (d) at a concentration of 30 ng/μl. A disc of P. capsici was placed on tobacco leaves at 25 °C in darkness and humidity; lesion diameters were measured after 48 h. As118 (a) was used as control. Pictures were taken under UV light. (e) Percent inhibition against P. capsici. Vertical bars show SD. Data are the mean values from three individual experiments. Significance analysis was performed using the t-test. *p < 0.05 and **p < 0.01.