Identification and Characterization of Genes Related to Ampicillin Antibiotic Resistance in <i>Zymomonas mobilis</i>

Abstract

Antibiotics can inhibit or kill microorganisms, while microorganisms have evolved antibiotic resistance strategies to survive antibiotics. Zymomonas mobilis is an ideal industrial microbial chassis and can tolerate multiple antibiotics. However, the mechanisms of antibiotic resistance and genes associated with antibiotic resistance have not been fully analyzed and characterized. In this study, we investigated genes associated with antibiotic resistance using bioinformatic approaches and examined genes associated with ampicillin resistance using CRISPR/Cas12a−based genome−editing technology. Six ampicillin−resistant genes (ZMO0103, ZMO0893, ZMO1094, ZMO1650, ZMO1866, and ZMO1967) were identified, and five mutant strains ZM4∆0103, ZM4∆0893, ZM4∆1094, ZM4∆1650, and ZM4∆1866 were constructed. Additionally, a four−gene mutant ZM4∆ARs was constructed by knocking out ZMO0103, ZMO0893, ZMO1094, and ZMO1650 continuously. Cell growth, morphology, and transformation efficiency of mutant strains were examined. Our results show that the cell growth of ZM4∆0103 and ZM4∆ARs was significantly inhibited with 150 μg/mL ampicillin, and cells changed to a long filament shape from a short rod shape. Moreover, the transformation efficiencies of ZM4∆0103 and ZM4∆ARs were decreased. Our results indicate that ZMO0103 is the key to ampicillin resistance in Z. mobilis, and other ampicillin−resistant genes may have a synergetic effect with it. In summary, this study identified and characterized genes related to ampicillin resistance in Z. mobilis and laid a foundation for further study of other antibiotic resistance mechanisms.

Keywords

• <i>Zymomonas mobilis</i>
• antibiotic resistance
• ampicillin
• genome editing
• CRISPR−Cas12a
• resistance selection markers