## 3 Discussions  
DksA is a transcriptional regulator that directly binds to RNAP secondary channels and plays an indispensable role in stringent response. It is generally believed that DksA functions to amplify (p)ppGpp signals during transcriptional regulation. (p)ppGpp inhibits the rRNA promoter by approximately three- to four-fold through the \(\beta^{\prime}\)-\(\omega\) interface site, whereas DksA binding to the secondary channel of RNAP can result in a twenty-fold inhibition [18]. In addition, DksA has been found to act in transcriptional regulation independently of (p)ppGpp. In _Haemophilus ducreyi_ and _Y. enterocolitis_, DksA has been shown to govern approximately 17% and 22% of the entire open reading frames, respectively, involving transcription, macromolecular synthesis, energy metabolism, amino acid transport and utilization [38]. In our previous study, it was shown that the expression levels of several fatty acid metabolism genes, including _fadA_, _fadE_, and _fadI_, significantly decreased in DksA-deficient strains, suggesting a potential regulatory role of DksA in fatty acid metabolism and cell membrane integrity [32]. These results raise intriguing questions regarding the effect of DksA proteins on fatty acid metabolism in _Y. enterocolitis_ and may have practical implications for the development of disinfectants targeting DksA in the food industry. Here, it was confirmed that fatty acid metabolic genes were regulated by DksA using RT-qPCR. There was an approximately twofold decrease in the expression of fatty acid syntheses genes, such as _fabB_, _fabF_, and _fabR_, while several fatty acid degradation genes, including _fadA_, _fadD_, _fadE_, _fadI_, and _fadI_, showed a more than fourfold decrease, with _fadA_ exhibiting a significant ninefold decrease. Previous studies have extensively explored the effect of (p)ppGpp on fatty acid metabolism. Specifically, during the amino acid starvation, (p)ppGpp readsupts metabolic flux and regulates the expression of central metabolic genes, leading to the redistribution of acetic acid or \(\beta\)-oxidation originally used for fatty acid synthesis to the synthesis of glutamate precursors pyruvate and \(\alpha\)-ketoglutate [39]. In addition, fatty acid starvation induces the production of uncharged ACP, thereby inducing SpoT to synthesize a large amount of (p)ppGpp and turn on the stringent response [40; 41]. Nevertheless, in _Y. enterocolitia_, only eight genes of fatty acid metabolism were significantly regulated (FC > 2), and KEGG analysis did not reveal significant enrichment. It is thus  
Figure 7: Outer and inner membrane permeability of _Y. enterocolitia_ exposed to polymyxin B. **(A)** Outer membrane permeability exposed to1 \(\upmu\)g/mL (1 MIC) and 0.5 \(\upmu\)g/mL (0.5 MIC) polymyxin B was evaluated by NPN. **(B)** Inner membrane permeability exposed to1 \(\upmu\)g/mL (1 MIC), 2 \(\upmu\)g/mL (2 MIC), and 4 \(\upmu\)g/mL (4 MIC) polymyxin B was evaluated by ONPG. The data are presented as the mean \(\pm\) SD of at least three independent experiments. presumed that DksA functions independently from (p)ppGpp in regulating fatty acid metabolism pathways. FadR is a transcriptional regulator that controls the expression of fatty acid synthesis genes, such as _fabABHDG_. However, there was no significant change in the expression level of FadR in the YEND, and only a slight change in the relative expression level of FabABDG, suggesting that its regulation of fatty acid metabolism is not achieved through FadR, which requires additional research for detailed clarification. The degradation pathway of fatty acids is not only a significant source of energy but also generates an essential intermediate product, acetyl-CoA, which functions as a critical intermediary in the metabolic process [42]. The YEND strain was found to have a growth defect, possibly due to insufficient energy and material caused by the downregulation of the fatty acid degradation genes.