Communication between viruses guides lysis–lysogeny decisions

Journal name:
Nature
Volume:
541,
Pages:
488–493
Date published:
DOI:
doi:10.1038/nature21049
Received
Accepted
Published online

Abstract

Temperate viruses can become dormant in their host cells, a process called lysogeny. In every infection, such viruses decide between the lytic and the lysogenic cycles, that is, whether to replicate and lyse their host or to lysogenize and keep the host viable. Here we show that viruses (phages) of the SPbeta group use a small-molecule communication system to coordinate lysis–lysogeny decisions. During infection of its Bacillus host cell, the phage produces a six amino-acids-long communication peptide that is released into the medium. In subsequent infections, progeny phages measure the concentration of this peptide and lysogenize if the concentration is sufficiently high. We found that different phages encode different versions of the communication peptide, demonstrating a phage-specific peptide communication code for lysogeny decisions. We term this communication system the ‘arbitrium’ system, and further show that it is encoded by three phage genes: aimP, which produces the peptide; aimR, the intracellular peptide receptor; and aimX, a negative regulator of lysogeny. The arbitrium system enables a descendant phage to ‘communicate’ with its predecessors, that is, to estimate the amount of recent previous infections and hence decide whether to employ the lytic or lysogenic cycle.

At a glance

Figures

  1. Effect of conditioned media on the infection dynamics of phage phi3T.
    Figure 1: Effect of conditioned media on the infection dynamics of phage phi3T.

    a, Preparation protocol of control and conditioned media. b, Growth curves of B. subtilis 168 infected with phi3T at MOI = 0.1, in control and conditioned media. c, Growth curves of B. subtilis strain 3610 (wild type (WT)) and its oppD mutant derivative DS4979 (oppD::kan) infected by phi3T at MOI = 0.1. In b and c, data represent an average of 3 biological replicates, each with 3 technical replicates; error bars represent s.e. d, Semi-quantitative PCR assay for phage lysogeny during an infection time course of B. subtilis 168 with phi3T. No DNA, control without DNA; WT, DNA from uninfected culture; lysogen, genomic DNA of a phi3T lysogen.

  2. The arbitrium peptide and its receptor.
    Figure 2: The arbitrium peptide and its receptor.

    a, The arbitrium locus in the phage genome. b, Growth curves of B. subtilis 168 infected by phi3T at MOI = 0.1, in LB media supplemented with synthesized SAIRGA peptide. Numbers represent peptide concentrations. Average of 3 biological replicates, each with 3 technical replicates. c, Sequencing-based quantitative determination of the fraction of lysogenized bacteria. Top, Schematics of the analysis. Per cent of lysogeny was calculated as the fraction of reads spanning the phage–bacteria integration junction out of total reads covering this junction. Integration junction is red; integrated phage is green. Bottom, the percentage of lysogenized bacteria during infection of B. subtilis 168 with phi3T at MOI = 2. Average of three biological replicates, error bars denote s.e. Synthesized SAIRGA peptide was added at 1 μM. d, Microscale thermophoresis analysis of the binding between purified AimR and synthesized SAIRGA or GMPRGA peptides. Average and s.e. of three replicates. Fnorm, normalized fluorescence.

  3. Accumulation of arbitrium peptide during an infection time course.
    Figure 3: Accumulation of arbitrium peptide during an infection time course.

    B. subtilis 168 culture was grown to optical density (OD) = 0.5 and then infected at t = 0 by phi3T at MOI = 0.001. PFU, plaque-forming units; CFU, colony-forming units, sampled from the uninfected control. For peptide, PFU and OD shown is average of triplicate (except for t = 30, where it is duplicate), with error bars representing s.e. CFU was measured in duplicates, and error bars represent the two measured points around the average. a, Growth of infected and uninfected bacteria, and phage, during the infection time course. b, Accumulation of peptide during the infection course as compared to bacteria and phage.

  4. A peptide communication code guiding lysogeny in Bacillus phages.
    Figure 4: A peptide communication code guiding lysogeny in Bacillus phages.

    a, Selected instances of AimR homologues in sequenced genomes. Locus tags are indicated for AimR homologues, along with the percentage of amino acid sequence identity to the phi3T AimR. Mature arbitrium peptide is indicated below the AimP homologue. b, Distribution of arbitrium peptides among 112 homologues of AimP. c, Amino acid profile of arbitrium peptide types. Bits denote information content. d, Growth curves of B. subtilis BEST7003 infected by SPbeta at MOI = 0.1, in LB media supplemented with synthesized GMPRGA peptide. The BEST7003 strain was used for SPbeta infection as the B. subtilis 168 strain is naturally immune to SPbeta. e, Growth curves of B. subtilis BEST7003 infected by SPbeta at MOI = 0.1, in LB media supplemented with synthesized SAIRGA peptide. f, Growth curves of B. subtilis 168 infected by phi3T at MOI = 0.1, in LB media supplemented with synthesized GMPRGA peptide. Data in df represent average of 3 biological replicates, each with 3 technical replicates; error bars represent s.e.

  5. DNA binding and transcription regulation in the arbitrium system.
    Figure 5: DNA binding and transcription regulation in the arbitrium system.

    a, ChIP–seq of His-tagged AimR 15 min after infection with or without 1 μM of SAIRGA peptide. Shown is the ratio, along the phage genome, between sequenced pulled-down DNA during infection without the peptide and DNA pulled-down when the peptide was present in the medium. b, Same as a; shown is a zoomed-in region in the phage genome. c, Gel-filtration results of purified AimR with or without the presence of either SAIRGA or GMPRGA peptide. Inset presents a calibration curve for the gel filtration using proteins of known sizes. d, Expression of the AimX gene during infection. Data presented for individual biological replicates. eg, RNA-seq coverage of the arbitrium locus at 5 min (e), 10 min (f) and 20 min (g) after infection. h, Growth curves of wild-type and dCas9-silenced bacterial strains during phi3T-infection. Strains were infected at t = 0 at MOI = 0.1. Shown is average of 3 biological replicates, each with 3 technical replicates; error bars represent s.e.

  6. Mechanistic model for communication-based lysis-lysogeny decisions.
    Figure 6: Mechanistic model for communication-based lysis-lysogeny decisions.

    a, Dynamics of arbitrium accumulation during infection of a bacterial culture by phage. b, At the first encounter of a phage with a bacterial population, the early genes aimR and aimP are expressed immediately upon infection. AimR, as a dimer, activates AimX expression. AimX is an inhibitor of lysogeny, possibly as a regulatory non-coding RNA, directing the phage to a lytic cycle. At the same time AimP is expressed, secreted and processed extracellularly to produce the mature peptide. c, At later stages of the infection dynamics, the arbitrium peptide accumulates in the medium and is internalized into the bacteria by the OPP transporter. Now, when the phage infects the bacterium, the expressed AimR receptor binds the arbitrium molecules and cannot activate the expression of AimX, leading to lysogeny preference.

  7. Specificity of conditioned media.
    Extended Data Fig. 1: Specificity of conditioned media.

    Conditioned media were prepared using initial infection of B. subtilis 168 by four phages: phi3T, phi105, phi29 and rho14. Presented are growth curves of B. subtilis 168 in the different media, infected with each phage at MOI = 0.1: phi3T (a), phi105 (b), phi29 (c) and rho14 (d). Data represent average of 3 replicates, and error bars represent s.e.

  8. Proteinase K treatment reduces the effect of conditioned medium.
    Extended Data Fig. 2: Proteinase K treatment reduces the effect of conditioned medium.

    Growth curves of B. subtilis 168 infected by phi3T at MOI = 0.1, in control and conditioned media, with and without pre-treatment with proteinase K. Data represent average of 3 technical replicates, and error bars represent s.e.

  9. Mass spectrometry verifies the presence of the SAIRGA peptide in conditioned medium.
    Extended Data Fig. 3: Mass spectrometry verifies the presence of the SAIRGA peptide in conditioned medium.

    Presented are extracted ion chromatograms of targeted mass spectrometry analysis experiment. B2, Y3, Y4 and Y5 refer to fragmentation products of the peptides made by the instrument in the tandem mass spectrometry process, with the expected m/z ion masses indicated. The y axis represents the ion intensity of each fragment ion (arbitrary units). a, Reference synthesized SAIRGA peptide at 100nM concentration in LB. b, Control medium from B. subtilis 168. c, phi3T-infected B. subtilis 168. d, B. subtilis 168 expressing dCas9 with a spacer targeting aimP. e, phi3T-infected B. subtilis 168 expressing dCas9 with a spacer targeting aimP. Arrowhead depicts the expected retention time of the SAIRGA peptide (a, c).

  10. 5 aa versions of the 6 aa arbitrium peptide do not guide lysogeny.
    Extended Data Fig. 4: 5 aa versions of the 6 aa arbitrium peptide do not guide lysogeny.

    Growth curves of B. subtilis 168 infected by phi3T at MOI = 0.1, in LB media supplemented with synthesized SAIRGA, AIRGA or SAIRG peptide. Shown is average of 3 biological replicates, each with 3 technical replicates. Error bars represent s.e.

  11. Exposure of AimR to SAIRGA peptide reduces propensity for dimerization.
    Extended Data Fig. 5: Exposure of AimR to SAIRGA peptide reduces propensity for dimerization.

    Purified AimR was eluted from a gel-filtration column, dialysed, then mixed with SAIRGA peptide (final concentration, 100 μM) dissolved in water or with equal amount of volume without peptide and incubated at room temperature for 5 min. The protein samples were then mixed for 30 min with different concentrations of the crosslinker BS3 bis(sulfosuccinimidyl)suberate. Presented are electrophoresis results analysed using the TapeStation instrument (Agilent Technologies), showing that in the absence of peptide, purified AimR tends to preferentially be crosslinked into dimers, whereas in the presence of the SAIRGA peptide, dimerization is substantially reduced.

  12. Phage gene expression 20 min after infection.
    Extended Data Fig. 6: Phage gene expression 20 min after infection.

    Each dot represents a single phage gene. Axes represent average RNA-seq read count per gene from 3 replicates, after normalization to control for RNA-seq library size. x axis, expression when phage infection was in the presence of 1 μM of SAIRGA peptide; y axis, no peptide.

  13. Expression of the arbitrium locus in phage SPbeta during infection.
    Extended Data Fig. 7: Expression of the arbitrium locus in phage SPbeta during infection.

    RNA-seq coverage of the arbitrium locus at 20 min after infection, with (black) or without (green) 1 μM of synthesized GMPRGA peptide in the medium. RNA-seq coverage was normalized to the number of reads mapped to the phage genome in each RNA-seq library. Shown are two replicates of the experiment. The x axis represents the position (in bp) on the phage genome.

  14. Infection experiments with phi3T ΔaimP and derived conditioned media.
    Extended Data Fig. 8: Infection experiments with phi3T ΔaimP and derived conditioned media.

    ac, Growth curves of B. subtilis 168 infected with phi3T or phi3T ΔaimP in conditioned medium derived from phi3T (a); conditioned medium derived from phi3T ΔaimP (b); and control medium (c). Each medium was generated in biological replicate, and the two replicates are presented separately. Each curve represents the average of three technical replicates, and error bars represent s.e.

Tables

  1. Signal for cleavage by extracellular proteases in the AimP and Phr pre-pro-peptide
    Extended Data Table 1: Signal for cleavage by extracellular proteases in the AimP and Phr pre-pro-peptide

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Author information

  1. These authors contributed equally to this work.

    • Zohar Erez &
    • Ida Steinberger-Levy

Affiliations

  1. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel

    • Zohar Erez,
    • Ida Steinberger-Levy,
    • Maya Shamir,
    • Shany Doron,
    • Avigail Stokar-Avihail,
    • Sarah Melamed,
    • Azita Leavitt,
    • Gil Amitai &
    • Rotem Sorek
  2. Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, Israel

    • Ida Steinberger-Levy
  3. Israel Structural Proteomics Center (ISPC), Faculty of Biochemistry, Weizmann Institute of Science, Rehovot 7610001, Israel

    • Yoav Peleg &
    • Shira Albeck
  4. de Botton Institute for Protein Profiling, The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot 7610001, Israel

    • Alon Savidor

Contributions

Z.E. directly performed or was involved in all experiments unless otherwise stated. I.S.L. performed conditioned media and proteinase K assays. S.D. annotated phi3T genome. A.S. analysed the mass spectrometry results. Y.P. and S.A. expressed and purified AimR–6×His. A.S.A., A.L. and S.M. constructed strains. G.A. performed microscale thermophoresis, crosslinking and ChIP–seq experiments. M.S. performed RNA-seq experiments. R.S. supervised the project.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Reviewer Information Nature thanks P. Fineran and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Specificity of conditioned media. (241 KB)

    Conditioned media were prepared using initial infection of B. subtilis 168 by four phages: phi3T, phi105, phi29 and rho14. Presented are growth curves of B. subtilis 168 in the different media, infected with each phage at MOI = 0.1: phi3T (a), phi105 (b), phi29 (c) and rho14 (d). Data represent average of 3 replicates, and error bars represent s.e.

  2. Extended Data Figure 2: Proteinase K treatment reduces the effect of conditioned medium. (188 KB)

    Growth curves of B. subtilis 168 infected by phi3T at MOI = 0.1, in control and conditioned media, with and without pre-treatment with proteinase K. Data represent average of 3 technical replicates, and error bars represent s.e.

  3. Extended Data Figure 3: Mass spectrometry verifies the presence of the SAIRGA peptide in conditioned medium. (116 KB)

    Presented are extracted ion chromatograms of targeted mass spectrometry analysis experiment. B2, Y3, Y4 and Y5 refer to fragmentation products of the peptides made by the instrument in the tandem mass spectrometry process, with the expected m/z ion masses indicated. The y axis represents the ion intensity of each fragment ion (arbitrary units). a, Reference synthesized SAIRGA peptide at 100nM concentration in LB. b, Control medium from B. subtilis 168. c, phi3T-infected B. subtilis 168. d, B. subtilis 168 expressing dCas9 with a spacer targeting aimP. e, phi3T-infected B. subtilis 168 expressing dCas9 with a spacer targeting aimP. Arrowhead depicts the expected retention time of the SAIRGA peptide (a, c).

  4. Extended Data Figure 4: 5 aa versions of the 6 aa arbitrium peptide do not guide lysogeny. (92 KB)

    Growth curves of B. subtilis 168 infected by phi3T at MOI = 0.1, in LB media supplemented with synthesized SAIRGA, AIRGA or SAIRG peptide. Shown is average of 3 biological replicates, each with 3 technical replicates. Error bars represent s.e.

  5. Extended Data Figure 5: Exposure of AimR to SAIRGA peptide reduces propensity for dimerization. (141 KB)

    Purified AimR was eluted from a gel-filtration column, dialysed, then mixed with SAIRGA peptide (final concentration, 100 μM) dissolved in water or with equal amount of volume without peptide and incubated at room temperature for 5 min. The protein samples were then mixed for 30 min with different concentrations of the crosslinker BS3 bis(sulfosuccinimidyl)suberate. Presented are electrophoresis results analysed using the TapeStation instrument (Agilent Technologies), showing that in the absence of peptide, purified AimR tends to preferentially be crosslinked into dimers, whereas in the presence of the SAIRGA peptide, dimerization is substantially reduced.

  6. Extended Data Figure 6: Phage gene expression 20 min after infection. (53 KB)

    Each dot represents a single phage gene. Axes represent average RNA-seq read count per gene from 3 replicates, after normalization to control for RNA-seq library size. x axis, expression when phage infection was in the presence of 1 μM of SAIRGA peptide; y axis, no peptide.

  7. Extended Data Figure 7: Expression of the arbitrium locus in phage SPbeta during infection. (193 KB)

    RNA-seq coverage of the arbitrium locus at 20 min after infection, with (black) or without (green) 1 μM of synthesized GMPRGA peptide in the medium. RNA-seq coverage was normalized to the number of reads mapped to the phage genome in each RNA-seq library. Shown are two replicates of the experiment. The x axis represents the position (in bp) on the phage genome.

  8. Extended Data Figure 8: Infection experiments with phi3T ΔaimP and derived conditioned media. (211 KB)

    ac, Growth curves of B. subtilis 168 infected with phi3T or phi3T ΔaimP in conditioned medium derived from phi3T (a); conditioned medium derived from phi3T ΔaimP (b); and control medium (c). Each medium was generated in biological replicate, and the two replicates are presented separately. Each curve represents the average of three technical replicates, and error bars represent s.e.

Extended Data Tables

  1. Extended Data Table 1: Signal for cleavage by extracellular proteases in the AimP and Phr pre-pro-peptide (101 KB)

Supplementary information

Excel files

  1. Supplementary Table 1 (187 KB)

    This file shows the Homologs of AimR-AimP in sequenced genomes. Positions of AimR homologs are provided, as well as the corresponding full pro-pre-peptide sequence and the mature peptide sequences.

  2. Supplementary Table 2 (38 KB)

    This file contains the annotation of the phi3T genome.

Additional data