## 2 Results  
DksA Positively Regulated the Expression Level of Genes Related to the Fatty Acid Degradation Pathway in Y. enterocolitica  
Bacteria degrade intracellular fatty acids through the \(\beta\)-oxidation pathway, which not only produces a substantial amount of energy and materials for cell metabolism, but also plays a vital role in maintaining the stability of phospholipids in the cell membrane structure. Our previous transcriptome analysis of the YEND strain revealed that the expression levels of fatty acid degradation pathway-related genes, specifically _fadA_ and _fadE_ (encoding acetyl-CoA acyltransferase and acyl-CoA dehydrogenase, respectively), were significantly reduced by over eight times compared to the WT strain, suggesting a potential link between DksA and fatty acid metabolism in _Y. enterocolitica_[32]. In this study, we further verified the expression levels of related genes using RT-qPCR (see Supplementary Table S1 for primers). As shown in Figure 1, the fatty acid synthesis pathway genes in YEND strains, such as _fabB_ and _fabF_, were up-regulated by approximately two-fold compared with that of the WT strain, while the expression levels of genes involved in the fatty acid degradation pathway, including _fadA_, _fadD_, _fadE_, _fadI_, and _fadJ_, were down-regulated by more than five times. These results suggest that the DksA protein represses the synthesis of fatty acids in _Y. enterocolitica_ while promoting the metabolism of fatty acids. Figure 1: Transcriptional changes of fatty acid metabolic genes of YEND. Total RNAs were extracted from mid-log phase _Y. enterocolitica_. The RNA-seq data is from our previous study, and the difference of RNA-seq data indicated the changes in fatty acid metabolic genes between YEND and WT strains (normalized to 166 RNA). Data are means from three independent RT-qPCRs. DksA Played a Crucial Role in Facilitating the Expeditious Proliferation of Y. enterocolitic at the Optimal Temperature  
_Y. enterocolitica_ is a foodborne pathogen known for its ability to survive and propagate at low temperatures, indicating its high tolerance to cold environments [33]. However, the DksA protein has been found to induce the degradation of fatty acids, which is likely to alter the fluidity of cellular membranes and the sensitivity to varying temperatures, ultimately affecting the bacterial growth dynamics. To verify this, the growth conditions of the WT strain, YEND strain, and complementing strain YEND-D at 4 \({}^{\circ}\)C 16 \({}^{\circ}\)C, 26 \({}^{\circ}\)C, and 37 \({}^{\circ}\)C were measured, and the results are shown in Figure 2. At 4 \({}^{\circ}\)C, all strains had an OD\({}_{600}\) value of approximately 0.3 after 24 h of growth, with no significant difference between the YEND and WT. However, as the temperature increased to 16 \({}^{\circ}\)C, 26 \({}^{\circ}\)C, and 37 \({}^{\circ}\)C, the growth rate of the YEND strain was consistently lower than that of the WT strain, while the YEND-D exhibited similar growth patterns to that of the WT strain at all tested temperatures. Therefore, this result confirmed the finding that DksA deletion would lead to deficient growth. Additionally, as the temperature increased, the growth gap between the WT and YEND strains gradually widened. As shown in Supplementary Figure S1, at 21 h (approximately the stable growth period of the strain), the biomass of YEND was similar to that of WT at 4 \({}^{\circ}\)C, with only a slight growth gap of \(-\)3.31%. However, this growth gap significantly increased to 11.08% at 16 \({}^{\circ}\)C and further expanded to 15.08% and 23.95% at 26 \({}^{\circ}\)C and 37 \({}^{\circ}\)C, respectively. These findings suggest that DksA plays a pivotal role in facilitating the rapid growth process of _Y. enterocolitica_ at optimal temperatures. Figure 2: Growth conditions of _Y. enterocolitica_ at 4 \({}^{\circ}\)C (**A**), 16 \({}^{\circ}\)C (**B**), 26 \({}^{\circ}\)C (**C**), 37 \({}^{\circ}\)C (**D**) supplemented with 0.02% L-Arabinose.