## 3 Discussion
However, the decreased expression level of amino acid transporters in the mutant strains also made it difficult to absorb external amino acids (Figures 4 and 5). To verify that the limitation in amino acids is a possible factor for the growth defects, the growth condition of _Y. enterocolitica_ in LBNS medium supplemented with single or mixed amino acids was measured. The results showed that the addition of arginine reduced the growth gap between the WT and the \(\Delta\)_xelAAspoT_ strain in the stationary phase from 33.50% to 9.11% (Table S4), indicating that (p)ppGpp is required for arginine transport and utilization in _Y. enterocolitica_. In addition,system utilization may respond to the defective growth in \(\Delta\)_dksA_, which could minimize the growth gap with the WT. Chemotaxis, the motility response to chemical stimuli in the external environment, is critical for bacteria to find food and stay away from harmful environments [41]. The flagella are the main motility organ of bacteria, and their synthesis is the basis of bacterial chemotaxis [42; 43]. However, flagellar synthesis is an extremely energy-intensive process that requires approximately 2% of the energy in _E. coli_[44]. Knockout of (p)ppGpp and DksA leads to a decrease in energy and substance metabolism. Therefore, (p)ppGpp and DksA are likely to regulate bacterial flagellar synthesis and bacterial chemotaxis. It has been reported that flagellar biosynthesis was inhibited in both \(\Delta\)_dksA_ and \(\Delta\)_relAASpoT_. Interestingly, in \(\Delta\)_dksA_, only the expression of _fliC_ decreased, whereas the expression of other synthetic genes increased or did not change significantly, indicating that DksA inhibits flagellar biosynthesis by repressing filament synthesis (Figure 4). Although most of the genes related to flagellar synthesis were downregulated in \(\Delta\)_relAASpoT_, these genes in \(\Delta\)_dksArelAASpoT_ exhibited a similar trend in \(\Delta\)_dksA_. This may be due to the critical role of DksA in regulating the synthesis of flagella in _Y. enterocolitica_ than (p)ppGpp and the fact that an independent regulatory mechanism exists between them. Because of the impaired flagellar synthesis, it is not surprising that the mutant strains have decreased chemotaxis. Notably, increased expression of the regulator of capsule synthesis (RCS) phosphorelay system was observed, which has been reported to reduce the chemotaxis of _Y. enterocolitica_ in glucose, glycerol, and malate. Swimming and competition experiments in this study obtained similar results (Figure 6), indicating that an indirect mechanism may be involved in the regulation of bacterial flagellar synthesis genes by DksA. In summary, this study revealed the regulation of stringent response factors (p)ppGpp and DksA on gene expression and cell function in _Y. enterocolitica_, focusing on the role of stringent response in bacterial ribosome synthesis and cell metabolism. Results showed that DksA and (p)ppGpp could positively promote the utilization and transport of amino acids, as well as bacterial flagellar synthesis and chemotactic ability. Since these phenotypes are critical for bacterial viability, colonization, and virulence, these findings demonstrated that DksA and (p)ppGpp could be potential targets for developing new disinfectants or agents.