Regulation of prophage induction and lysogenization by phage communication systems

Abstract

Many viruses cause both lytic infections, where they release viral particles, and dormant infections, where they await future opportunities to reactivate.¹ The benefits of each transmission mode depend on the density of susceptible hosts in the environment.^2-4 Some viruses infecting bacteria use molecular signaling to respond plastically to changes in host availability.⁵ These viruses produce a signal during lytic infection and regulate, based on the signal concentration in the environment, the probability with which they switch to causing dormant infections.^5,6 We present an analytical framework to examine the adaptive significance of plasticity in viral life-history traits in fluctuating environments. Our model generalizes and extends previous theory⁷ and predicts that host density fluctuations should select for plasticity in entering lysogeny as well as virus reactivation once signal concentrations decline. Using Bacillus subtilis and its phage phi3T, we experimentally confirm the prediction that phages use signal to make informed decisions over prophage induction. We also demonstrate that lysogens produce signaling molecules and that signal is degraded by hosts in a density-dependent manner. Declining signal concentrations therefore potentially indicate the presence of uninfected hosts and trigger prophage induction. Finally, we find that conflict over the responses of lysogenization and reactivation to signal is resolved through the evolution of different response thresholds for each trait. Collectively, these findings deepen our understanding of the ways viruses use molecular communication to regulate their infection strategies, which can be leveraged to manipulate host and phage population dynamics in natural environments.

Keywords: arbitrium; induction; lysis-lysogeny; microbiology; phage; prophage.

Graphical abstract

Figure 1

Coevolutionary stable strategies for reactivation and lysogenization In a theoretical evolutionary model (see STAR Methods for details), fluctuating concentrations of signaling peptide select for plasticity in both lysogeny and reactivation. The joint evolution of these two traits is expected to yield very different reaction norms with arbitrium concentration. See also Figures S1–S3.

Figure 2

The arbitrium system modulates prophage reactivation (A) Plaque-forming units (PFUs) produced by prophage reactivation from phi3T (wild-type) and phi3T^aimR-N202A (signal non-responder) lysogens in the presence (1,000 nM) or absence of signaling molecules (12 h growth in LB). (B) phi3T receptor (ΔaimR), signal production (aimR), lysogeny regulator (ΔaimX), and arbitrium system (ΔaimRPX) deletion mutants (18 h growth in LB in the absence of synthetic signal). (C) phi3TΔaimP (signal non-producer) lysogens of hosts carrying the aimX gene under the control of a xylose promoter in LB containing 0% xylose (uninduced) or 0.2% xylose (induced). n = 6 in all treatments. Error bars represent standard error.

Figure 3

Infection and prophage signal responses (A) Lysogen formation from BEST7003 cultured in LB with increasing concentrations of signaling peptide and infected with phi3TΔaimP(spc) at MOI = 0.1 (40-min infection, n = 4). (B) PFUs produced by prophage reactivation from phi3TΔaimP(spc) (signal negative) lysogens cultured in LB with increasing concentrations of signaling peptide (8 h growth, n = 6). Error bars represent standard error.

Figure 4

Signal production, durability, and decay (A) Prophage induction in spent media of uninfected BEST7003, phi3TΔaimP lysogens, and phi3T lysogens (n = 4). (B) The durability of signaling peptides was quantified under different conditions: spent media of Bacillus subtilis BEST7003 extracted from early (3 h) low-density (∼0.3) cultures and late (8 h) high-density (∼1.8) cultures, supplemented with signaling peptide to 1,000 nM and incubated for 12 h. Signal decay was calculated by comparing initial and final signal concentrations. Signal concentrations were calculated using BEST7003:RPXgfp (a signal reporter containing the phi3T AimR-AimP-AimX locus genetically fused to a fluorescent reporter gene) and a calibration curve constructed using spent media supplemented with known concentrations of signaling peptide (n = 4; see STAR Methods for details). Error bars represent standard error. (C) Conceptual model of lysis and lysogeny and prophage induction as a function of signal production and decay. Lysogenic and lytic infections produce signal that is decayed at high cell densities. At high lysogen densities, constitutive signal production maintains the prophage state. An influx of susceptible cells, or invasion of a susceptible population, rapidly decays signal, triggering prophage induction. Subsequent lytic infections remove susceptible hosts from the population, increasing signal concentrations and triggering the switch to lysogeny. See also Figures S4A and S4B.