## 3 Discussions
However, it is important to note that DksA, as a global regulator, influences bacterial life activities in various ways. Therefore, the growth defect may arise from multiple factors. Our previous study has also revealed that DksA positively regulates amino acid transport and utilization pathways [37]. Fatty acids constitute a primary component of phospholipids in the cell membrane structure, and the metabolism of fatty acids is closely related to the fluidity of cell membranes [43]. In this study, it was found that the YEND exhibits better growth in a low-temperature environment, and the growth gap between the WT strain and YEND strain gradually increased with temperature rise, indicating that the DksA is imperative for the rapid growth at optimal temperature. In _Shewanella baltica_, fatty acid degradation proteins were shown to positively regulate membrane fluidity, while fatty acid synthesis proteins negatively regulate it [44]. Given the reduced expression level of fatty acid degradation genes and increased expression level of fatty acid synthesis genes in the YEND strain, it is speculated that its cell membrane fluidity is inferior to that of the WT strain. And the negative impact of low temperature on the fluidity of the YEND cell membrane becomes limited. Additionally, the knockout of DksA would also lead to the upregulation of ribosomal proteins _rpA_, _rpIK_, _rpmA_, _rpmG_, and _rpmH_, as well as the transcription factors IF-1 and EF-Ts, which have been reported to promote cold adaptation of bacteria. All these reasons ultimately contribute to the insensitivity of the YEND strain to low temperature environments. The periplasmic space of Gram-negative bacteria separates two layers of lipid membranes. The outer membrane (OM) consists of an inner leaflet mainly containing glycerophospholipids and an outer leaflet mainly containing lipid A, and its structural integrity directly affects the infectivity of bacteria. The inner membrane (IM) is composed of a phospholipid bilayer membrane and is used to isolate cellular metabolism and biosynthesis processes. Most proteins involved in electron transfer, protein export, and signal transduction are present in the IM, ensuring the basic life activities of bacteria [45; 46]. It was shown that integrity of the inner and outer membranes of the YEND strain was higher than that of the WT strain, resulting in lower protein leakage. The specific impact of changes in fatty acid metabolism pathways on cell membrane structures, and whether there are other regulatory factors for DksA to affect cell membrane structure, are deed necessary to understand the roles of DksA in membrane integrity. Polymycin B is a cationic antimicrobial peptide antibiotic that has the ability to target the cell membrane and lyse both the OM and IM to release macromolecules, such as nucleic acid and cellular proteins, resulting in bacterial death. Previous studies have revealed that the WT strain and DksA-deficient strain are highly susceptible to polymyxin B, which can inhibit the growth of _Y. entriocolitica_ at a concentration of 1 \(\mu\)g/mL. This study has demonstrated that the growth gap at the stationary phase decreases as the polymyxin B concentration increases, indicating that the DksA protein positively regulates the ability of _Y. entriocolitica_ to resist polymyxin B. In addition, measurements of the leakage of cellular nucleic acid and protein in response to polymyxin B treatment indicate that the YEND is more severely lysed, whereas DksA can maintain the stability of the cell membrane under polymyxin B treatment. The integrity of the inner membrane and outer membrane also confirmed this. High concentrations of polymyxin B can cause damage to the inner and outer membranes of _Y. entercolitica_, with the degree of damage being higher in the YEND strain compared to the WT strain. Since the YEND has higher cell membrane integrity in the absence of polymyxin B, it is suggested that DksA plays a positive role in response to the bactericidal effect of polymyxin B.