Similar and Divergent Roles of Stringent Regulator (p)ppGpp and DksA on Pleiotropic Phenotype of Yersinia enterocolitica

Abstract

Stringent response plays an important role in the response of Enterobacteriaceae pathogens to rapid environmental changes. It has been shown that synergistic and antagonistic actions exist between the signaling molecules (p)ppGpp and DksA in several foodborne pathogens; however, the biological function of these molecules and their interactions in Yersinia are still unclear. This study systematically investigated the role of stringent response in Yersinia enterocolitica, a typical foodborne Enterobacteriaceae pathogen, by deleting the (p)ppGpp and DksA biosynthesis genes. (p)ppGpp and DksA copositively regulated most phenotypes, such as motility, antibiotic resistance, and tolerance to oxidative stress, whereas they exhibited independent and/or divergent roles in the growth and biofilm synthesis of Y. enterocolitica. Gene expression analysis revealed that (p)ppGpp- and DksA-deficiency reduced the transcription of flagellar synthesis genes (fliC and flgD) and biofilm synthesis genes (bssS and hmsHFRS), which could potentially contribute to changes in motility and biofilm formation. These results indicate that stringent response regulators (p)ppGpp and DksA have a synergistic role and independent or even completely opposite biological functions in regulating genes and phenotypes of Y. enterocolitica. Our findings revealed the biofunctional relationships between (p)ppGpp and DksA and the underlying molecular mechanisms in the regulation of the pathogenic phenotype of Y. enterocolitica. IMPORTANCE The synergetic actions between the stringent response signaling molecules, (p)ppGpp and DksA, have been widely reported. However, recent transcriptomic and phenotypic studies have suggested that independent or even opposite actions exist between them. In this study, we demonstrated that the knockout of (p)ppGpp and DksA affects the polymorphic phenotype of Yersinia enterocolitica. Although most of the tested phenotypes, such as motility, antibiotic resistance, and tolerance to oxidative stress, were copositively regulated by (p)ppGpp and DksA, it also showed inconsistencies in biofilm formation ability as well as some independent phenotypes. This study deepens our understanding of the strategies of foodborne pathogens to survive in complex environments, so as to provide theoretical basis for the control and treatment of these microorganisms.

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

FIG 1

Growth characteristic of WT and mutant strains in LB (A), LBNS (B), and M63 minimum medium (C) supplemented with 0.02% l-arabinose. Data are mean OD₆₀₀ for three independent cultures and standard errors of the means.

FIG 2

The role of DksA and (p)ppGpp in stress resistance. Survival rate of WT, mutant strains and the complemented strain after challenge with pH 4.0 (A), pH 10.0 (B), or 0.5 M NaCl (C) for 60 min, or 1 mM H₂O₂ for 30 min (D) were determined. Data are mean ± SD of three biological repeats, each of which was perforemed with three technical replicates. An asterisk indicates a significant difference with ns, not significant; *, P < 0.01; **, P < 0.001; ***, P < 0.0001.

FIG 3

Motility assay of WT, mutant strains and the complemented strain in 0.35% LBNS agar plate. A 1.0-μL of each Y. enterocolitica culture was injected into the plate. The plates were incubated at 26°C for 48 h before photographing. (A) Quantification of swim diameters. (B) Images of swim plate. The results are mean of four independent plates, and the error bars indicate standard deviations. An asterisk indicates a significant difference with **, P < 0.001; ***, P < 0.0001.

FIG 4

Flagella biosynthesisof WT, mutant strains and the complemented strain. Strains grown to the midlog phase in LBNS medium were stained with phosphotungstic acid and the flagella were visualized and the average flagella numbers in a single cell were calculated. (A) Average number of flagella per cell in various 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. (B) Transmission electron microscopy pictures of the wild-type, mutant strains and the complemented strain. The scale bar represents 2 μm and the orangearrow in the picture refers to the flagella.

FIG 5

Biofilm formation of WT, mutant strains and the complemented strain in LBNS medium. Y. enterocolitica were cultured in LBNS media at a 24-well polystyrene microtiter plate. The biofilms were stained with crystal violet after 24, 48 and 72 h of incubation and measured at 595 nm. (A) Quantification of biofilm. (B) Images of purple color depth after 72 h of incubation. Data are mean of six biological repeats, and the error bars indicate standard deviations. An asterisk indicates a significant difference with *, P < 0.01; ***, P < 0.0001.

FIG 6

Congo red binding assay of WT, mutant strains and the complemented strain. The mass of Y. enterocolitica was mixed with Congo red for binding. After removing bacterial mass, the optical density of Congo red in supernatant was measured at 490 nm, each of which was normalized by relating to the value of 100 μg/mL Congo red at 490 nm. Data are mean of three biological repeats, and the error bars indicate standard deviations. An asterisk indicates a significant difference with ns, not significant; *, P < 0.01; **, P < 0.001; ***, P < 0.0001.

FIG 7

Transcriptional changes in the mutant strains of Y. enterocolitica. Y. enterocolitica were grown to the midlog phase in LBNS medium and the total RNA was extracted. The change in the abundance of the indicated transcripts (normalized to 16sRNA) in the YEND (A), YENR (B), YENRS (C), YENDRS (D) were determined by RT-qPCR. Data are means and SEM from three independent RT-qPCRs.

FIG 8

Schematic representations underscoring the possible mechanism(s) of stringent response regulator of Y. enterocolitica on pleiotropic phenotype. Red arrows and blue balls indicate positive or negative traits, respectively.