## 2 Results
### DlsA and ppGpp Positively Affect Bacterial Chemotaxis  
The biosynthesis of flagella is a vital process that requires a significant amount of resources and energy, enabling bacteria to achieve motility, competitiveness, and virulence [32; 33]. Results obtained from RNA-Seq analysis indicated that in the absence of DksA, the expression of most genes involved in flagellar synthesis was elevated, except for _fliC_, _fliC_2, and _fliC_3 (Figure 4). In our previous study, we confirmed that the motility and flagella formation abilities in \(\Delta\)_dksA_ were repressed. Combined with this phenotypic  
Figure 6: Growth characteristics of WT and mutant strains in liquid medium. (**A**) LBNS; (**B**) LBNS supplemented with 1.16 g/L arginine; (**C**) LBNS supplemented with 0.1 g/L cystine; (**D**) LBNS supplemented with 1.28 g/L histidine; (**E**) LBNS supplemented with 0.38 g/L arginine, 1.31 g/L glutamic acid, and 0.42 g/L histidine; (**F**) LBNS supplemented with 0.29 g/L arginine, 0.1 g/L cystine, 0.98 g/L glutamic acid, and 0.32 g/L histidine. The data are the means OD\({}_{600}\) for five independent cultures and the standard errors of the means.  
result, DksA is determined to be the key factor in filament synthesis. Although flagellar assembly in \(\Delta relA\Delta spoT\) was not a significant functional group in the KEGG enrichment analysis (\(p\)-value = 0.2584), 15 genes involved in the flagellar assembly were perturbed by (p)ppGpp knockout. Among these genes, only _flhD_ was significantly upregulated (FC > 2), whereas the remaining genes (_fliN_, _flgl_, _flgkH_, _flgC_, _flgC_, _fliC_, _fliC_, _fliC_, _fliC_, and _fliC_) were significantly downregulated (FC < 2). The decreased expression of these flagellar genes may be responsible for the decreased motility and flagellar formation ability of \(\Delta relA\Delta spoT\)[27]. In addition, the expression levels of these genes in \(\Delta k\Delta relA\Delta spoT\) were more similar to those in \(\Delta k\Delta k\Delta\) than in \(\Delta relA\Delta spoT\), indicating that DksA plays a more critical role in the regulation of flagellar biosynthesis during stringent response.  
Flagellar synthesis is critical for the chemotactic ability of bacteria to move to a more favorable environment and confer a selective advantage. The Rcs phosphorelay system was found to be overexpressed in the functional group of \(\Delta k\Delta A\), which inhibits the chemotactic ability of _Y. enterocolitica_[34]. These studies imply that a stringent response has a significant regulatory effect on the chemotactic ability of _Y. enterocolitica_. To validate this, the chemotaxis ability of the mutant strains was evaluated using swimming and competitive capillary assays. As shown in Figure 7, the swimming diameter of the mutant strain was smaller than that of the WT for the three carbon sources of glucose, glycerol, and malic acid. Moreover, the mutant strains accounted for 26.7% or less of the total number of colonies after incubation (Figure 7C). The experimental results demonstrate that DksA and (p)ppGpp play positive roles in _Y. enterocolitica_ motility, flagella formation ability, and chemotaxis.  
Figure 7: Comparison of the chemotactic responses of WT and mutant strains in three carbon resources. (**A**) Quantification of swim diameters. (**B**) Images of a swim plate. (**C**) Competitive chemotactic responses of WT and mutant strains. The data are presented as the mean \(\pm\) SD of at least three biological repeats, and the error bars indicate standard deviations. An asterisk indicates a significant difference (*** \(p\) < 0.0001).