Journal: PLoS Pathogens
Article Title: Cyclic di-GMP-dependent Signaling Pathways in the Pathogenic Firmicute Listeria monocytogenes
Figure Lengend Snippet: Role of the c-di-GMP-induced EPS in biofilm formation, cell aggregation, and tolerance of L. monocytogenes to disinfectants and desiccation. A : Biofilm formation of L. monocytogenes in 96-well polystyrene plates (measured using a Crystal violet dye-binding assay). Cultures were grown for 6 days at 30°C in LB (top panel) or LB supplemented with 3% glycerol (bottom panel). Shown are average results from two biological replicates, where each strain was grown in six wells in a replicate (i.e., six technical replicates). Black circle, wild type; red square, Δ pdeB/C/D ; green triangle, Δ pdeB/C/D Δ pssC ; blue cross, Δ pdeB/C/D Δ pssE . B : EPS-dependent L. monocytogenes cell aggregation (clumping) in HTM medium. Overnight cultures grown in BHI were inoculated into HTM liquid medium at A 600 of 0.01 and incubated at 30°C with gentle shaking (rotary shaker, 125 rpm) for 48 h. 1, ΔpdeB/C/D ; 2, wild type; 3, ΔpdeB/C/D ΔpssC ; 4, ΔpdeB/C/D ΔpssE ; C : Protective role of the c-di-GMP-inducible EPS in disinfection. Aliquots of the HTM-grown cultures were mixed with disinfectant solutions for 10 min at room temperature. Disinfection was stopped by adding a D/E neutralizing broth (Difco); the cultures were vortexed vigorously (5 min) with glass beads to break clumps and plated on BHI agar. Colonies were enumerated after a 48-h growth at 37°C. SH, sodium hydrochloride (1600 ppm); HP, hydrogen peroxide (200 mM); BC, benzalkonium chloride (100 ppm). White background, EGD-e; black, ΔpdeB/C/D ; grey, ΔpdeB/C/D ΔpssC . SH, sodium hypochlorite; HP, hydrogen peroxide; BC, benzalkonium chloride. The absence of the bar for the EGD-e strain treated with SH indicates the lack of survivors. D : Protective role of the c-di-GMP-inducible EPS in desiccation. Aliquots of overnight cultures grown in HTM at 37°C were spun down, the supernatants were removed, and cell pellets were stored in desiccators at room temperature for the indicated periods. The pellets were rehydrated, vortexed with glass beads for better suspension and plated on BHI agar. The numbers of surviving colonies after incubation at 37°C for 24 h are plotted. In panels C and D, bars denote mean values for data from three biological replicates. *, significantly different (p
Article Snippet: L. monocytogenes was grown in Brain Heart Infusion (BHI) medium (Difco), HTM (minimal medium containing 3% glucose) or LB, supplemented with appropriate antibiotics at 25, 30, 37 or 42°C, as indicated.
Techniques: Binding Assay, Incubation