High cell densities favor lysogeny: induction of an H20 prophage is repressed by quorum sensing and enhances biofilm formation in Vibrio anguillarum

Abstract

Temperate ϕH20-like phages are repeatedly identified at geographically distinct areas as free phage particles or as prophages of the fish pathogen Vibrio anguillarum. We studied mutants of a lysogenic isolate of V. anguillarum locked in the quorum-sensing regulatory modes of low (ΔvanT) and high (ΔvanO) cell densities by in-frame deletion of key regulators of the quorum-sensing pathway. Remarkably, we find that induction of the H20-like prophage is controlled by the quorum-sensing state of the host, with an eightfold increase in phage particles per cell in high-cell-density cultures of the quorum-sensing-deficient ΔvanT mutant. Comparative studies with prophage-free strains show that biofilm formation is promoted at low cell density and that the H20-like prophage stimulates this behavior. In contrast, the high-cell-density state is associated with reduced prophage induction, increased proteolytic activity, and repression of biofilm. The proteolytic activity may dually function to disperse the biofilm and as a quorum-sensing-mediated antiphage strategy. We demonstrate an intertwined regulation of phage-host interactions and biofilm formation, which is orchestrated by host quorum-sensing signaling, suggesting that increased lysogeny at high cell density is not solely a strategy for phages to piggy-back the successful bacterial hosts but is also a host strategy evolved to take control of the lysis-lysogeny switch to promote host fitness.

Fig. 1. Free ФH20 particles accumulate in cultures of lysogens in a QS-dependent manner.

(a) The concentration of plaque forming units (PFU ml⁻¹) in cell-free spent supernatants from cultures of V. anguillarum isolate 90-11-287 wildtype (WT), and QS mutant strains (ΔvanT and ΔvanO) is shown relative to the concentration of bacterial colony forming units (CFU ml⁻¹) in the cultures at the time of phage harvest. Solid bars represent the average of all the low-cell-density data points from Fig. 1C (LCD: OD₆₀₀ < 0.05) and hatched bars represent the average of all the high-cell-density data points from Fig. 1C (OD₆₀₀ > 1.0). An asterisk indicates a significant difference between LCD and HCD conditions (Welch’s t test, α = 0.05) and error bars represent the standard error of the mean (N = 6–8 for LCD and N = 10–11 for HCD) (b) Optical densities (OD₆₀₀) of cultures of wildtype (WT) and QS mutant strains (ΔvanT and ΔvanO) were measured at 1 h intervals during growth from low to high cell density. (c) The corresponding concentrations of phage particles in the cultures (PFU ml⁻¹) were measured by plaque assay. For (b) and (c), symbols represent the averages of triplicate independent experiments. Time = 0 h indicates the time of the first measurement.

Fig. 2. An extracellular molecule(s) controls accumulation of ФH20-like phage particles.

(a) Optical densities (OD₆₀₀) of cultures of V. anguillarum wildtype, ΔvanT, and ΔvanO strains were measured at 2 h intervals over an 8 h period of incubation in the presence of cell-free spent supernatant obtained from a culture of wildtype V. anguillarum BA35 grown to mid-log phase. The depicted variation in cell density measured at the initial timepoint is largely due to the technical limitations of the spectrophotometer, as the values are at or below the detection limit of the instrument. (b) The corresponding abundances of phage particles per milliliter (PFU ml⁻¹) were quantified by plaque assay. Lines represent the averages of duplicate measurements represented as symbols.

Fig. 3. Similar adsorption rates of ϕH20-like phages to ΔH20, ΔvanT ΔH20, and ΔvanO ΔH20 mutant cells.

Exponentially growing cultures of the indicated strains at OD₆₀₀ = 0.6–0.9 were mixed with a lysate of ϕH20-like phage p41. At the indicated time points, unadsorbed phage enumerated by plaque assay. Shown is the average of three independent experiments. Error bars indicate the standard error of the mean (N = 3). Adsorption rate constants (4.0 × 10⁻¹¹ ± 0.8 × 10⁻¹¹ PFU × ml per minute per bacterium for the ΔH20 mutant that is wildtype with regards to the QS system, 4.2 × 10⁻¹¹ ± 1.7 × 10⁻¹¹ for the ΔvanT ΔH20 mutant, and 2.9 × 10⁻¹¹ ± 0.5 × 10⁻¹¹ for the ΔvanO ΔH20 mutant) are not significantly different (linear regression analysis with Tukey adjustment, P = 0.16, α = 0.05).

Fig. 4. Extracellular proteolytic activity reduces the number of infective phage particles.

Concentration of KVP40 (Myoviridae) and ϕH20 (Siphoviridae) was reduced post 48 h of incubation in cell-free spent supernatant (N_t) compared with incubation in growth medium (control, N_c). An asterisk indicates a significant reduction of infective phages compared with cell-free spent supernatants from the ΔH20 cultures (ANOVA and Sidak’s multiple comparison test, α = 0.05). Error bars represent standard error of the mean (N = 3).

Fig. 5. Biofilm formation and aggregation of V. anguillarum 90-11-287 is enhanced at low-cell density and affected by the H20-like prophage.

(a) Quantification of biofilm formation after 48 h of incubation. (b) Aggregates with a biovolume larger than 28 µm³ based on CLSM z-stack images in an area of 159.73 × 159.73 µm. An asterisk indicates a significant difference between the prophage-harboring and prophage-free mutants (ANOVA and Tukey’s multiple comparison test, α = 0.05) and error bars represent standard error of the mean (N = 3 for crystal violet assays and N = 4 for CLSM quantification).