Stress-driven temporal production of phage tail-like particles (tailocins) in Dickeya dadantii strain 3937

Abstract

Tailocins are bacteriocins resembling bacteriophage tails. Previously, we reported the production of tailocins in the plant pathogen Dickeya dadantii 3937, the synthesis of which was upregulated upon treatment with mitomycin C. In this study, we investigated how mitomycin treatment over time influences the expression of tailocin-related genes, the accumulation of tailocin particles, and the survival of producer cells. The expression of tailocin P2D1 structural genes peaks two hours after the addition of mitomycin C as measured with an RT-qPCR assay. Simultaneous measurements of tailocin titer revealed that the concentration of the particles in the culture supernatant peaked 6 h after induction and remained stable for at least 18 h. Progressive accumulation of P2D1 that occurred from 2 to 6 h after mitomycin C treatment was associated with a substantial decrease in viable cells of the tailocin-producing strain (ca. 100,000-fold). Decreased cell viability upon tailocin production indicates that they are released from the cells upon cell lysis. Likewise, we found new potent inducers, viz., hydrogen peroxide and antibiotics affecting DNA replication and repair (viz. norfloxacin and ciprofloxacin), that can increase tailocin yield in D. dadantii 3937.

Keywords: Erwinia chrysanthemi; Bacteriophage; Phage tail-like particles; Real-time qPCR.

Fig. 1

Production of tailocins by D. dadantii 3937 depending on the applied concentration of mitomycin C, the growth medium, and the type of inducer. Tailocin titer was expressed as relative tailocin activity in arbitrary units (AU), with 1 AU defined as the reciprocal of the highest dilution that caused a visible plaque on a lawn of susceptible strain M. paradisiaca IFB 0117 (NCPPB 2511). In all panels, data points show mean values, and error bars represent standard deviations. Statistically significant differences between groups for analyses in panels A and C were determined using Tukey’s pairwise comparisonfollowed by Copenhaver Holland post hoc analysiswhile for analyses in panels B and D, these were determined by Kruskal-Wallis testfollowed by Dunn’s post hoc test. In all panels, groups labeled with the same letter are not significantly different (α = 0.05). Panel (A) depicts the titer of P2D1 tailocins depending on the applied concentration of mitomycin C. Panel (B) shows tailocin titer when testing the inducive potential of selected antibiotics: norfloxacin 0.016 µg mL^− 1; ciprofloxacin 0.016 µg mL^− 1; ampicillin 0.004 µg mL^− 1; chloramphenicol 4 µg mL^− 1. Treatment with mitomycin C (1 µg mL^− 1) was used as a reference. Control – culture with no potential inducer added. Panel (C) shows tailocin yield following induction with different concentrations of hydrogen peroxide: 0.5, 1, 5, and 10 mM, with mitomycin treatment as reference (1 µg mL^− 1). Control – culture with no potential inducer added. Panel (D) shows tailocin yield in different growth media when induced with mitomycin C (1 µg mL^− 1): TSB – Trypticase Soya Broth; M9 – M9 minimal medium with 0.4% glucose; PDB – Potato Dextrose Broth.

Fig. 2

Tailocin yield and viable bacterial cell count as a function of time following the addition of inducer. (A) Survival of D. dadantii strain 3937 over time, measured in colony-forming units (CFU). Data points represent the viable bacterial cell count in cultures treated with mitomycin C at a concentration of 1 µg mL^− 1. The counts are shown at various intervals, illustrating the decline in bacterial viability as the cells produce and release tailocins.(B) The titer of P2D1 tailocins isolated from the culture of D. dadantii strain 3937 at different time points (0–24 h) post induction with mitomycin C (1 µg mL^− 1). The x-axis indicates the incubation time of bacterial cells with the inducer, after which tailocins were harvested for activity against M. paradisiaca IFB 0117 (NCPPB 2511). Cultures unexposed to mitomycin C were used as controls. Points represent the mean activity (mean AU) of measurements from 3 independent experiments. Error bars indicate standard deviation ranges. S represents the start of the culture, and Mitomycin C indicates the time of spiking the culture with mitomycin C. In panel (B), significant differences between Mitomycin C-treated samples collected at different time points were determined using the Mann-Whitney U test. Groups labeled with the same letter are not significantly different (α = 0.05). Small letters are used for Mitomycin C-treated samples and capital letters for control. Asterisks (*) indicate statistically significant differences (p < 0.05; Mann-Whitney U test between experimental and control samples collected at the same time points.

Fig. 3

Expression of P2D1-encoding structural genes in D. dadantii 3937 at different time points following induction with mitomycin C. The graphs depict the relative expression levels of three target genes: fiber (DDA3937_RS12070), tube (DDA3937_RS12115), and sheath (DDA3937_RS12110), in samples collected after specified incubation times following the addition of mitomycin C (1 µg mL^{[- [}). Control samples consist of bacteria that were not treated with the antibiotic. The fold change (log2) in gene expression for the target genes was calculated relative to the control samples collected at time 0. Statistically significant differences among the experimental groups were determined using one-way ANOVA, followed by Tukey’s Honest Significant Difference post hoc analysis. Groups marked with the same letter do not differ significantly (α = 0.05).

Fig. 4

Temporal response of D. dadantii 3937 to mitomycin C. The figure illustrates the dynamics of strain 3937 in response to stress induced by mitomycin C. The graph presents the number of viable bacterial cells producing P2D1 tailocins, the relative activity of the produced tailocins against M. paradisiaca IFB 0117 (NCPPB 2511), and the relative expression levels of genes encoding structural proteins of the tailocins. The cultures were treated with mitomycin C at a concentration of 1 µg mL^{[- [}, with the inducer added to the bacterial culture at time 0.