*2.3. Antibiotic Tolerance of Ampicillin*−*Resistant (AR) Gene Knockout Strains*

Previous studies reported that the concentration of ampicillin required for plate screening and liquid culture of the transformants in genetic engineering manipulation of *Z. mobilis* ZM4 was 300 µg/mL [19]. In this study, we set three ampicillin concentration gradients of 0, 150, and 300 µg/mL to cultivate the ampicillin−resistant (AR) gene knockout strains. As demonstrated in the Supplementary Materials Figure S4, the deletion of *ZMO1094* and *ZMO1650* individually did not decrease the ampicillin resistance. Specifically, the growth of ZM4∆1094 and ZM4∆1650 under different ampicillin concentrations was not inhibited compared with ZM4, whereas the deletion of *ZMO0893* and *ZMO1866* individually were effective. The growth of ZM4∆0893 and ZM4∆1866 was inhibited under 150 µg/mL ampicillin with a slower growth rate of 0.15 <sup>±</sup> 0.03 h−<sup>1</sup> and 0.05 <sup>±</sup> 0.01 h−<sup>1</sup> compared with the value of 0.24 <sup>±</sup> 0.02 h−<sup>1</sup> for wild−type ZM4 (Figure 2). The growth of the *ZMO0103* mutant ZM4∆0103 was the slowest among all strains with the lowest growth rate of 0.21 <sup>±</sup> 0.01 h−<sup>1</sup> under 0 µg/mL ampicillin, and ZM4∆0103 was inhibited by all concentrations of ampicillin, such as 150 µg/mL (Figure 2, Supplementary Materials Figure S4).

**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.

The same experiment was also carried out for ZM4∆ARs, a mutant strain with four ampicillin−resistant (AR) genes deleted. Under 0 µg/mL ampicillin, a long lag phase was observed with a lower growth rate of 0.15 <sup>±</sup> 0.01 h−<sup>1</sup> in ZM4∆ARs compared to ZM4∆0103 (Figure 2, Supplementary Materials Figure S4), and ZM4∆ARs also cannot grow at 150 µg/mL ampicillin. Combined with the growth of single−gene knockout strains under RMG5, we speculated that the deletion of *ZMO0103* is one of the major reasons for the poor growth of ZM4∆ARs, and other ampicillin−resistant (AR) genes may have a synergetic effect on ampicillin resistance. However, as previously reported, when the ampicillin concentration increased to 300 µg/mL, nearly all the strains could not grow. These results also demonstrate that the mutants ZM4∆0103 and ZM4∆ARs had a lower ampicillin tolerance concentration—150 µg/mL.

#### *2.4. Cell Morphology of Mutants Treated with Ampicillin*

Previous studies demonstrated that cell cultures in the presence of antibiotics resulted in abnormal cellular morphology in various degrees, such as elongated or distorted cell shapes [28,29]. To evaluate the morphological changes of ampicillin−resistant (AR) gene knockout strains during the ampicillin treatment, cell morphologies of three *Z. mobilis* strains (ZM4, ZM4∆0103, and ZM4∆Ars) were observed under light microscopy. The results show that ZM4∆0103 and ZM4∆ARs had longer rod shapes with various cell lengths and widths. The lengths of ZM4∆0103 and ZM4∆ARs were around 10.6 ± 7.2 µm and 11.3 ± 5.9 µm compared to that of wild−type ZM4 (3.2 ± 0.9 µm) in RMG5 without ampicillin (Figure 3). However, when strains were cultured in RMG5 with 100 µg/mL ampicillin (RMA100), the cellular morphology of ZM4∆0103 (16.1 ± 9.3 µm) and ZM4∆ARs (16.0 ± 7.8 µm) changed and was longer with a filament shape, while the length of ZM4 did not change much (4.1 ± 1.0 µm). This demonstrated that ampicillin was more stressful to ZM4∆0103 and ZM4∆ARs during cell growth, and ZM4∆0103 and ZM4∆ARs were more sensitive to ampicillin than ZM4. Similar morphological changes of *Z. mobilis* have been previously described when the cells were exposed to different stresses, such as high temperature [30], lignocellulosic hydrolysate inhibitory [31], high concentration of xylose [32], and salt conditions [33].

**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.
