## 3 Discussion  
The stringent response is a sensory system widely present in bacteria that can regulate various cellular processes such as fatty acid metabolism and flagellar synthesis, thereby determining bacterial survival, virulence, and pathogenicity [35; 36]. This system works by altering the concentration of the intracellular secondary messenger (p)ppGpp with the participation of DksA. Although studies have confirmed that DksA is the main cofactor of (p)ppGpp during stringent response, (p)ppGpp- and DksA-deficient strains display differences at the genotypic and phenotypic levels [25; 37]. Our previous studies also showed that DksA and (p)ppGpp cooperatively regulate antibiotic resistance and environmental tolerance in _Y. enterocolitica_, whereas they exhibit dependent or independent roles in biofilm synthesis [27]. To comprehensively understand the regulatory function of the stringent response in _Y. enterocolitica_, WT and the mutant strains \(\Delta\)_dksA_, _AxelAAspoT_, and \(\Delta\)_dksAArelAAspoT_ were subjected to comparative transcriptome analysis and phenotypic characterization. Our study provides new insights into the regulation of ribosomal synthesis, energy metabolism, transporters, flagellar synthesis, two-component systems, and flagellar synthesis by (p)ppGpp and DksA in _Y. enterocolitica_. The interplay between (p)ppGpp and DksA has been widely explored in _E. coli_ and _X. citri_[9; 10]. DksA and (p)ppGpp bind together in the secondary channel of RNAP and positively or negatively affect the transcriptional activity of RNAP according to the properties that are intrinsic to the promoter [24]. In addition, (p)ppGpp has also been confirmed to directly bind to the promoters of the genes involved in amino acid biosynthesis, and DksA can also form a complex with DNAJ in response to external redox signals, indicating that (p)ppGpp and DksA also have regulatory effects independent of each other [38; 39]. In the present study, 18.57% of the total genes of _Y. enterocolitica_ were regulated by DksA, while 33.32% of the total genes of _AxelAAspoT_ were regulated, showing comprehensive and extensive regulation of the stringent response (Table 1). Among these genes, 496 were covered by DksA and (p)ppGpp, accounting for 61.92% of the total DEGs in \(\Delta\)_dksA_, indicating that the main function of DksA is to participate in (p)ppGpp. In addition, DksA and (p)ppGpp exhibited negative interplay with certain DEGs (such as the flagella synthesis gene _fliE_). Similar results were also reported by [PERSON] et al., who found that the deficiency of DksA and (p)ppGpp resulted in the different expression of the siderophore synthesis and utilization gene cluster in _X. citri_[10]. Identifying the interaction between DksA and (p)ppGpp and dissecting its influence on bacterial physiological activities requires further investigation. During the stringent response, cellular energy and material are rapidly reallocated from the synthesis of macromolecules for reproduction to produce factors that are crucial for survival. Reports have shown that (p)ppGpp or DksA is required for the growth of _E. coli_ and _S. enterica_[36; 40]. Our previous studies also showed that the maximum growth values of \(\Delta\)_dksA_ and \(\Delta\)_xelAAspoT_ in LBNS medium were weaker than those of the WT strain, and \(\Delta\)_xelAAspoT_ cannot even grow in M63 minimal medium (a minimum medium, an oligotrophic environment for bacteria) [27]. Due to the complexity of growth-related factors, it is difficult to explain the growth defect with a few specific cellular processes. Nevertheless, the decline in cellular carbon metabolism and amino acid utilization cannot be ignored. From the enrichment analysis of DEGs, the knockout of DksA or (p)ppGpp resulted in decreased expression of almost all genes in the tricarboxylic acid cycle (TCA cycle), thus reducing the level of intracellular material and energy metabolism, including the fatty acid metabolism and arginine synthesis pathways.