Identification and Characterization of Genes Related to Ampicillin Antibiotic Resistance in Zymomonas mobilis

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: CRISPR−Cas12a; Zymomonas mobilis; ampicillin; antibiotic resistance; genome editing; resistance selection markers.

Figure 1

Confirmation of ampicillin−resistant (AR) knockout strains in Z. mobilis ZM4 by PCR. The mutants of ZM4∆0103 (A), ZM4∆0893 (B), ZM4∆1650 (C), ZM4∆1094 (D), ZM4∆1866 (E), and ZM4∆ARs (F) were confirmed by colony PCR using their corresponding primers. The sizes of PCR products (bp) of WT and knockout strains were 4021, 2247 (ZM4∆0103); 3314, 2232 (ZM4∆0893); 3122, 2361 (ZM4∆1650); 3746, 2221 (ZM4∆1094); 3768, 2080 (ZM4∆1866).

Figure 2

The specific growth rate of ampicillin−resistant (AR) gene knockout strains cultured under 0 and 150 μg/mL of ampicillin. Three replicates were performed for the experiment. When the mutant could not grow under the condition, the sample is marked “ND” (not detected). * represents a significant difference with p−value < 0.05. ** represents a significant difference with p−value < 0.01. *** represents a significant difference with p−value < 0.001. ns represents no significant difference.

Figure 3

Cell morphology of strains ZM4, ZM4∆0103, and ZM4∆ARs cultured in RM and RMA100 was observed by light microscopy. The numbers with error value in each image represent the average cell size (μm) analyzed with ImageJ software. Numbers in the lower right corner of each represent the scale. RM and RMA100 represent the different RMG5 media with 0 and 100 μg/mL of ampicillin.

Figure 4

Electroporation efficiency of ZM4, ZM4∆0103, and ZM4∆ARs using plasmids of pEZ15A (~3 kb) and pE39−MVA (~10 kb). Three replicates were performed for the experiment. The error bar represents standard deviation (SD). When transformation of a plasmid was below the limit of detection (0.00001), the sample is marked “ND” (not detected). *** represents a very significant difference (p−value < 0.001).