Antagonistic interactions between phage and host factors control arbitrium lysis–lysogeny decision

Phages can use a small-molecule communication arbitrium system to coordinate lysis–lysogeny decisions, but the underlying mechanism remains unknown. Here we determined that the arbitrium system in Bacillus subtilis phage phi3T modulates the bacterial toxin–antitoxin system MazE–MazF to regulate the phage life cycle. We show that phi3T expresses AimX and YosL, which bind to and inactivate MazF. AimX also inhibits the function of phi3T_93, a protein that promotes lysogeny by binding to MazE and releasing MazF. Overall, these mutually exclusive interactions promote the lytic cycle of the phage. After several rounds of infection, the phage-encoded AimP peptide accumulates intracellularly and inactivates the phage antiterminator AimR, a process that eliminates aimX expression from the aimP promoter. Therefore, when AimP increases, MazF activity promotes reversion back to lysogeny, since AimX is absent. Altogether, our study reveals the evolutionary strategy used by arbitrium to control lysis–lysogeny by domesticating and fine-tuning a phage-defence mechanism.

Supplementary Table S3 Supplementary Table S5.Primers used in this study (continuation)

Supplementary Figure 3 . 4 . 5 . 6 .
Nucleotide and protein sequence comparison between aimX and aimX variant.An aimX variant was generated by modifying the nucleotide sequence without changing the encoded amino acid sequence.a, Nucleotide sequence alignment between aimX and the aimX variant.The upper line shows the wt aimX DNA sequence; the lower line shows the aimX variant DNA sequence.The conserved nucleotides are highlighted in blue.b, Protein sequence alignment between wt AimX and the AimX variant.The upper line shows the AimX protein sequence; the lower line shows the aimX variant protein sequence.The conserved amino acids are highlighted in blue.c, Complementation of the phi3T ∆aimR mutant in recipient.Strains lysogenic for phages phi3T ΔaimR were MC induced.The morphology of plaques following phi3T ∆aimR infection of strains 168 Δ6 expressing different versions of the aimX gene were photographed.Point mutations on AimX and phi3T_93 phage do not affect protein behaviour in solution.a, Size exclusion chromatography pattern for AimX WT and AimX M14D (His-tagged) in a Superdex 75 increase column.b, Size exclusion chromatography pattern for phi3T_93, and phi3T_93 L23D in a Shodex KW-402.5-4Fcolumn.c, Molecular weight in kDa for AimX, phi3T_93, phi3T_93 L23D and as calculated using SEC-MALS.Competition assays by native gel assay.Native gel mobility shift assays tested the binding preference of MazF between its partner antitoxin MazE and AimX.The pre-formed MazE-MazF complex was mixed with equimolar amounts of AimX and the formation of the MazE-MazF and AimX-MazF complexes at different times (from 0 to 20 minutes) were visualised by native gels showing the rapid disappearance of the MazE-MazF complex by the formation of the AimX-MazF complex.A representative of five gels is shown.Source Data Circular dichroism (CD) spectrum of recombinant MazE.The CD spectrum of recombinant MazE at pH 7.0 showed two negative peaks at 208 and 222 nm (upper panel) that after curve fitting with BeStSel software indicates a high content of alpha helix (lower panel) in agreement with the secondary structures of MazE determined by the DSSP form its crystal structure (PDM 4ME7 reporter vector containing β-gal gene, integrates at thrC.Modified MCS (EcoRI-NotI-HindIII-SpeI-NheI-SphI-BamHI) This study pDG1663+ Pspank B. subtilis reporter vector containing β-gal gene, integrates at thrC, containing IPTG-inducible Pspank promoter This study pJP2808 pDG1663+ Pspank -transcriptional terminator upstream aimX phi3T (TT) This study pJP2809 pDG1663+ Pspank -transcriptional terminator upstream aimX phi3T mutated version (G-C) (TT*) This study pJP2810 pDG1663+ aimP -aimX phi3T (No PRaimP) This study pJP2811 pDG1663+ PaimP -aimP -aimX phi3T (wt) This study pJP2812 pDG1663+ PaimP -aimP -aimX phi3T transcriptional terminator mutated version (G-C) (TT*) This study pJP2813 pDG1663+ PaimP -aimP -aimX phi3T mutant AimR binding site (ΔBS) This study pJP2814 pDG1663+ PaimP -aimP -aimX phi3T increased distance (Incr) This study pJP2815 pDG1663+ PaimP -aimP -aimX phi3T decreased distance (Decr) This study pJP2816 pDG1663+ Pspank -S784 -S785 SPβ (wt) This study pJP2817 pDG1663+ Pspank -S784 -S785 SPβ transcriptional terminator mutated version (G-T) (TT*) This study pJP2821 pDG1663+ PaimP -aimP -aimX phi3T AimR binding site SPβ This study pJP2822 aimX3T gene cloned in integration vector pDR110 This study pJP2823 aimX3T nucleotide variant gene cloned in integration vector pDR110 This study pJP2824 aimX3T L5* gene cloned in integration vector pDR110 This study pJP2825 aimX3T M14D gene cloned in integration vector pDR110 This study pJP3004 yosL3T gene cloned in integration vector pDR110 This study

Supplementary Table S4. Plasmids used in this study Plasmid Description Reference or source
4 pDG1663B.subtilis reporter vector containing β-gal gene, integrates at thrC

Table S5 .
Primers used in this study

Table S5 .
Primers used in this study(continuation)