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.
Highlights
Antimicrobial genes play an important role as a primary defense mechanism in all multicellular organisms
There are many antimicrobial peptides in nature. These peptides play a key role in the biological innate immune system and help the organism to resist the invasion of pathogenic bacteria; in spite of this, few antimicrobial peptides are used in clinical treatment[32]
Polypeptides are produced by ribosomes, such as ribosomally synthesized and post-translationally modified peptides (RiPPs), or by non-ribosomal peptide synthetases (NRPS)
Summary
Antimicrobial genes play an important role as a primary defense mechanism in all multicellular organisms. The E. coli expression system has become one of the most effective and widely used strategies for recombinant protein production due to its simple genetic operation, low cost, and fast growth[22], while the B. subtilis expression system has received extensive attention for its biosafety, clear genetic background, and strong ability to secrete p roteins[23,24] Both systems can be used to screen for antimicrobial peptides or antimicrobial genes, but our previous research indicates that the expression system of B. subtilis is more efficient than that of E. coli[19]. The resistance genes in Ae. tauschii can be retained in the new wheat varieties during the hybridization process to make use of these genes[28,29]
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