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.

Cell Membrane-Interrupting Antimicrobial Peptides from Isatis indigotica Fortune Isolated by a Bacillus subtilis Expression System

“…For the isolation of novel AMPs, we established a new, sensitive, and high-throughput strategy based on the damaging or killing effects of peptides against B. subtilis host cells [25]. A drawback of this strategy is that strong AMPs will kill B. subtilis cells too rapidly to detect the clones, and therefore, only AMPs with weak killing effects are preferred.…”

“…Initial screening and confirmation were performed as described in a previous study [25]. Briefly, an overnight culture was plated onto fresh LB plates supplemented with kanamycin (10 mg/L) and incubated at 37 • C to observe the phenotype.…”

“…Two factors contribute to a bottleneck in plant resistance breeding-the scarcity of plant resistance genes and the fact that resistance genes are easily overcome by pathogens. To overcome the scarcity of plant resistance genes, we established an antimicrobial gene isolation method using a B. subtilis expression system [25]. To avoid the propensity for resistance genes to be overcome by pathogens, we designed a model in which pathogen-independent, nonself-recognition triggered, and heterosis-based fresh resistance can be generated in F1 hybrids [26].…”

Antimicrobial Potential of Genes from Garlic (Allium sativumL.)

“…By using this strategy, a number of antimicrobial genes were screened from garlic to reveal its further potentials. For example, in case of B. subtilis expression system, a total of 48 antimicrobial genes were screened, including AsR 416, while AsRE 67 was identified by using E. coli expression system [51].…”

“…Fusarium spp. and then PI (propidium iodide) staining was performed [51]. PI is fluorescent agent that has the ability to bind with DNA through broken cell membrane.…”