Transcriptomic Analysis Reveals Key Roles of (p)ppGpp and DksA in Regulating Metabolism and Chemotaxis in Yersinia enterocolitica

Abstract

The stringent response is a rapid response system that is ubiquitous in bacteria, allowing them to sense changes in the external environment and undergo extensive physiological transformations. However, the regulators (p)ppGpp and DksA have extensive and complex regulatory patterns. Our previous studies demonstrated that (p)ppGpp and DksA in Yersinia enterocolitica positively co-regulated motility, antibiotic resistance, and environmental tolerance but had opposite roles in biofilm formation. To reveal the cellular functions regulated by (p)ppGpp and DksA comprehensively, the gene expression profiles of wild-type, ΔrelA, ΔrelAΔspoT, and ΔdksAΔrelAΔspoT strains were compared using RNA-Seq. Results showed that (p)ppGpp and DksA repressed the expression of ribosomal synthesis genes and enhanced the expression of genes involved in intracellular energy and material metabolism, amino acid transport and synthesis, flagella formation, and the phosphate transfer system. Additionally, (p)ppGpp and DksA inhibited amino acid utilization (such as arginine and cystine) and chemotaxis in Y. enterocolitica. Overall, the results of this study unraveled the link between (p)ppGpp and DksA in the metabolic networks, amino acid utilization, and chemotaxis in Y. enterocolitica and enhanced the understanding of stringent responses in Enterobacteriaceae.

Keywords: (p)ppGpp; DksA; Yersinia enterocolitica; chemotaxis; stringent response.

Figure 1

Expression analysis between samples. (A) Principal component analysis of twelve samples. (B) Correlation analysis of twelve samples. (C) Venn diagram of overlapping DEGs between ΔdksA, ΔrelAΔspoT, and ΔdksAΔrelAΔspoT.

Figure 2

Functional enrichment analysis of DEGs based on the KEGG database. (A) ΔdksA; (B) ΔrelAΔspoT; and (C) ΔdksAΔrelAΔspoT.

Figure 3

Validation of candidate DEGs by RT-qPCR. (A) Gene transcription in ΔdksA. (B) Gene transcription in ΔrelAΔspoT. (C) Gene transcription in ΔdksAΔrelAΔspoT. The data are the means and SEM from three independent RT-qPCRs.

Figure 4

The model for the cellular processes of ΔdksA. Red, green, and black indicate significant upregulation (FC > 2 and p-adjust < 0.05), significant downregulation (FC < 0.5 and p-adjust < 0.05), and no significant regulation (0.5 ≤ FC ≤ 2 or p-adjust ≥ 0.05), respectively.

Figure 5

The model for the cellular processes of ΔrelAΔspoT. Red, green, and black indicate significant upregulation (FC > 2 and p-adjust < 0.05), significant downregulation (FC < 0.5 and p-adjust < 0.05), and no significant regulation (0.5 ≤ FC ≤ 2 or p-adjust ≥ 0.05), respectively.

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 OD₆₀₀ for five independent cultures and the standard errors of the means.

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 ± SD of at least three biological repeats, and the error bars indicate standard deviations. An asterisk indicates a significant difference (*** p < 0.0001).