Stringent Response Factor DksA Contributes to Fatty Acid Degradation Function to Influence Cell Membrane Stability and Polymyxin B Resistance of Yersinia enterocolitica

Abstract

DksA is a proteobacterial regulator that binds directly to the secondary channel of RNA polymerase with (p)ppGpp and is responsible for various bacterial physiological activities. While (p)ppGpp is known to be involved in the regulation and response of fatty acid metabolism pathways in many foodborne pathogens, the role of DksA in this process has yet to be clarified. This study aimed to characterize the function of DksA on fatty acid metabolism and cell membrane structure in Yersinia enterocolitica. Therefore, comparison analysis of gene expression, growth conditions, and membrane permeabilization among the wide-type (WT), DksA-deficient mutant (YEND), and the complemented strain was carried out. It confirmed that deletion of DksA led to a more than four-fold decrease in the expression of fatty acid degradation genes, including fadADEIJ. Additionally, YEND exhibited a smaller growth gap compared to the WT strain at low temperatures, indicating that DksA is not required for the growth of Y. enterocolitica in cold environments. Given that polymyxin B is a cationic antimicrobial peptide that targets the cell membrane, the roles of DksA under polymyxin B exposure were also characterized. It was found that DksA positively regulates the integrity of the inner and outer membranes of Y. enterocolitica under polymyxin B, preventing the leakage of intracellular nucleic acids and proteins and ultimately reducing the sensitivity of Y. enterocolitica to polymyxin B. Taken together, this study provides insights into the functions of DksA and paves the way for novel fungicide development.

Keywords: DksA; Yersinia enterocolitica; fatty acid metabolism; polymyxin B.

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 16sRNA). Data are means from three independent RT-qPCRs.

Figure 2

Growth conditions of Y. enterocolitica at 4 °C (A), 16 °C (B), 26 °C (C), 37 °C (D) supplemented with 0.02% L-arabinose. Data are mean OD₆₀₀ values for three independent experiments and standard errors of the means.

Figure 3

Effect of DksA on Y. enterocolitica cell membrane permeability. Outer (A) and inner (B) membrane permeability was evaluated by NPN and ONPG, respectively. The experiment was performed at least three times with five biological parallels. The data are presented as the mean ± SD of at least three independent experiments. Asterisks indicate a significant difference. ***, p < 0.001.

Figure 4

Growth conditions of Y. enterocolitica exposed to 0 μg/mL (A), 0.125 μg/mL (B), 0.25 μg/mL (C), and 0.5 μg/mL (D) polymyxin B supplemented with 0.02% L-arabinose. Data are mean OD₆₀₀ for five independent cultures and standard errors of the means.

Figure 5

Protein leakage of Y. enterocolitica strains exposed to 1 μg/mL (A), 2 μg/mL (B), 4 μg/mL (C) polymyxin B supplemented with 0.02% L-arabinose. The experiment was performed at least three times with three biological parallels. The data are presented as the mean ± SD of at least three independent experiments.

Figure 6

Nucleic acid leakage of Y. enterocolitica strains exposed to 1 μg/mL (A), 2 μg/mL (B), 4 μg/mL (C) polymyxin B supplemented with 0.02% L-arabinose. The experiment was performed at least three times with three biological parallels. The data are presented as the mean ± SD of at least three independent experiments.

Figure 7

Outer and inner membrane permeability of Y. enterocolitica exposed to polymyxin B. (A) Outer membrane permeability exposed to1 μg/mL (1 MIC) and 0.5 μg/mL (0.5 MIC) polymyxin B was evaluated by NPN. (B) Inner membrane permeability exposed to1 μg/mL (1 MIC), 2 μg/mL (2 MIC), and 4 μg/mL (4 MIC) polymyxin B was evaluated by ONPG. The data are presented as the mean ± SD of at least three independent experiments.