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.
Highlights
The emerging resistance of pathogenic microbes against antibiotics or their hosts has created a basic need for the development of novel and unique classes of antimicrobial compounds and antimicrobial genes[1,2]
In an effort to identify a simple and fast system to screen antimicrobial genes, we developed a strategy that is different from traditional approaches and can efficiently isolate and purify antimicrobial proteins from almost all kinds of organisms. cDNA libraries were constructed using two different expression vectors, pBE-S and pET22-(b), which were transformed into B. subtilis and E. coli expression systems, respectively
In view of the bottleneck problem of finding new classes of antimicrobial genes, we constructed A. sativum cDNA libraries by using two different expression systems, B. subtilis and E. coli. By comparing their screening efficiency and antimicrobial activities, we concluded that the B. subtilis expression system is more suitable for screening antimicrobial peptides/proteins
Summary
The emerging resistance of pathogenic microbes against antibiotics or their hosts has created a basic need for the development of novel and unique classes of antimicrobial compounds and antimicrobial genes[1,2]. Antimicrobial peptides encoded by these pathogen infection upregulated host genes are relatively small (
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