## 2 Results
### DksA Positively Regulated the Polymyrin B Resistance of Y. enterocolitica  
Polymyxin B is a cationic antimicrobial peptide antibiotic that targets the negatively charged phosphate group on the cell membrane of Gram-negative bacteria. By inserting into the fatty chain of the cell membrane, it destroys the structure of the cell membrane and increases the permeability of the cell membrane, leading to the leakage of small molecules  
Figure 3: Effect of DksA on _Y. enterocolitica_ cell membrane permeability. Outer (**A**) and inner (**B**) membrane permeability was evaluated by NPN and ONPG, respectively. The experiment was performed at least three times with five biological parallels. The data are presented as the mean \(\pm\) SD of at least three independent experiments. Asterisks indicate a significant difference. ***, \(p\) < 0.001.  
such as purines and nucleotides [35; 36]. In our previous study, we found that the minimum inhibitory concentration (MIC) of polymyxin B was 1 \(\upmu\)g/mL for the WT, YEND, and YEND-D strains [37]. However, due to the strong bactericidal effects against _Y. enterocolitiica_ of polymyxin B, it may not be apparent whether DksA plays a role in the bacteria's response to the antibiotic. Therefore, we conducted a more comprehensive analysis by measuring the growth conditions of the WT, YEND, and YEND-D strains at 0 MIC (0 \(\upmu\)g/mL), 1/8 MIC (0.125 \(\upmu\)g/mL), 1/4 MIC (0.25 \(\upmu\)g/mL), and 1/2 MIC (0.5 \(\upmu\)g/mL) to gain a more comprehensive understanding of the strains' response to polymyxin B (Figure 4 and Supplementary Table S2). At 30 h (the stationary phase), the growth gap between the YEND and WT was 15.88% in the absence of polymyxin B. However, the addition of 0.125 \(\upmu\)g/mL of polymyxin B resulted in an increase of the growth gap to 16.15%, which further rose to 17.35% when the concentration was increased to 0.25 \(\upmu\)g/mL. Notably, the highest growth difference of 20.35% was observed under 0.5 \(\upmu\)g/mL of polymyxin B. These results suggest that the DksA protein positively regulates the polymyxin B resistance of _Y. enterocolitiica_. DksA Deletion Rendered Y. enterocolitiica Prone to Intracellular Nuclei Acid and Protein Leakage When Exposed to Polymyxin B  
Previous findings have demonstrated that YEND has greater membrane integrity than WT, but exhibits lower resistance to polymyxin B, which suggests that the antibiotic causes more damage to the YEND cell membrane. To confirm this, the leakage of nucleic acid and protein under 1 MIC (1 \(\upmu\)g/mL), 2 MIC (2 \(\upmu\)g/mL), and 4 MIC (4 \(\upmu\)g/mL) of polymyxin B were measured. As shown in Figures 5 and 6, the amount of extracellular protein and nucleic acid detected gradually increased as the processing time continued to  
Figure 4: Growth conditions of _Y. enterocolitiica_ exposed to 0 \(\upmu\)g/mL (**A**), 0.125 \(\upmu\)g/mL (**B**), 0.25 \(\upmu\)g/mL (**C**), and 0.5 \(\upmu\)g/mL (**D**) polymyxin B supplemented with 0.02% L-arabinose. Data are mean OD\({}_{600}\) for five independent cultures and standard errors of the means. prolong, with the YEND exhibiting higher protein leakage compared to the WT at the same concentration of polymyxin B, suggesting that cell membrane integrity of the YEND strain is lower after polymyxin B treatment. Additionally, the amount of extracellular protein and nucleic acid, as well as the gap of the leakage amount between the YEND and WT were gradually increased as the concentration of polymyxin B increased, indicating that the YEND strain was more sensitive to polymyxin B. The above results verify that polymyxin B could damage the cell membrane structure of _Y. enterocolitia_, leading to the leakage of intracellular protein and nucleic acid, and that DksA helps _Y.