Two antimicrobial genes from Aegilops tauschii Cosson identified by the Bacillus subtilis expression system

Abstract

Antimicrobial genes play an important role as a primary defense mechanism in all multicellular organisms. We chose Bacillus subtilis as a target pathogen indicator and transferred the Aegilops tauschii Cosson cDNA library into B. subtilis cells. Expression of the candidate antimicrobial gene can inhibit B. subtilis cell growth. Using this strategy, we screened six genes that have an internal effect on the indicator bacteria. Then, the secreted proteins were extracted and tested; two genes, AtR100 and AtR472, were found to have strong external antimicrobial activities with broad-spectrum resistance against Xanthomonas oryzae pv. oryzicola, Clavibacter fangii, and Botrytis cinerea. Additionally, thermal stability tests indicated that the antimicrobial activities of both proteins were thermostable. Furthermore, these two proteins exhibited no significant hemolytic activities. To test the feasibility of application at the industrial level, liquid fermentation and spray drying of these two proteins were conducted. Powder dilutions were shown to have significant inhibitory effects on B. cinerea. Fluorescence microscopy and flow cytometry results showed that the purified protein impaired and targeted the cell membranes. This study revealed that these two antimicrobial peptides could potentially be used for replacing antibiotics, which would provide the chance to reduce the emergence of drug resistance. Antibiotics have been widely used worldwide in recent decades to control many pathogens. Due to the excessive use of antibiotics, many bacteria have already developed antibiotic resistance mechanisms 1,2. Meanwhile, pathogen resistance has become a serious global threat, especially in intensive care units, where resistance often occurs 3,4. The increasing number of multi-drug-resistant pathogens has led to a growing demand for new antibiotics; however, even if new antibiotics are identified, resistance cannot be avoided because of antibiotic overuse, which is the primary cause of antibiotic resistance 5,6. With the increasing use of antibiotics, there is an urgent need to produce something with a negative impact against resistance mechanisms; thus, new therapeutic agents to replace antibiotics must be found. Antimicrobial peptides (AMPs) are potential substitutes for antibiotics because of their broad-spectrum resistance and rare resistant variants 7. These peptides have broad activity against a variety of bacteria, fungi, viruses, parasites, and cancer cells; furthermore, almost all organisms have a variety of broad-spectrum antimicrobial peptides 8. An antimicrobial peptide is an amphiphilic, cationic, and small protein in organisms as well as an important component of the innate immune system 9. The natural immunity of many organisms relies on the invasive power of antimicrobial peptides against different microbes 10. Since some antimicrobial peptides are biologically safe and are not susceptible to drug resistance, more and more of these have been discovered and excavated 11-14 , and the discovery and application of a...

Antimicrobial Peptides from Plants: A cDNA-Library Based Isolation, Purification, Characterization Approach and Elucidating Their Modes of Action

“…Meanwhile, different enzymes (trypsin, lipase, pepsin and amylase at 30 • C, protease E, and peptidase K at 57 • C for 1 h) are used to test the sensitivity of peptides. The agar diffusion test is performed to characterize the stability of peptides on different Gram-positive and Gram-negative bacteria [36,39,70]. Importantly, we have verified that pure antimicrobial peptides are stable and resistant to enzymes at higher temperatures.…”

“…It has long been used successfully to express many therapeutic molecules, such as some industrial enzymes, e.g., lipases, proteases, and amylases, because of its comparatively simple structure of the cell, rapid growth, short fermentation duration, and strong ability to disperse proteins immediately into the outer membrane [97,98]. Recently, the Key Lab of Crop Disease Monitoring and Safety Control in Hubei Province, Huazhong Agricultural University, Wuhan, Hubei, China, proposed a new method to isolate resistance genes by using the B. subtilis expression system, which showed a significant potential against the superbug bacterial and fungal plant pathogens [36,39,70]. The details of this approach are discussed below.…”

“…Using the EasyPure ® Plasmid MiniPrep Kit (TransGen, No. 1 North Yongtaizhuang Road, Haidian District, Beijing-100192, China), E. coli cell plasmids are isolated and then transformed into B. subtilis SCK6 cells [36,38]. The pBE-S vector containing a subtilisin promotor (aprE-P) and a peptide secretion signal (aprE-SP) are used in this proposed method.…”

“…As a product, it is insoluble, tightly packed, forms denatured inclusion bodies, is biologically inactive, and needs further folding change, solubilization, and downstream purification [34,35]. On the other hand, the B. subtilis system releases peptides into its extracellular space and has broad-spectrum antimicrobial gene expression capability [36][37][38][39][40]. The products of this expression system are biologically active and require simple downstream processing [41,42].…”

Isolation of Antimicrobial Genes from Oryza rufipogon Griff by Using a Bacillus subtilis Expression System with Potential Antimicrobial Activities

“…Some recent studies have shown that antimicrobial peptides (AMPs) such as AtR472 derived from Aegilops tauschii Cosson, the cationic antimicrobial gene HCAP-18 expressed in Chinese cabbage, and peptide SM-985 from Teosinte (Zea mays ssp. mexicana) have significant inhibitory effects on many plant pathogenic bacteria [8][9][10]. Besides, antimicrobial peptides have also been used in many fields because of their excellent bacteriostatic effects [11][12][13][14].…”