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Arbitrium communication controls phage lysogeny through non-lethal modulation of a host toxin–antitoxin defence system

Nature Microbiology volume 9, pages 150–160 (2024)Cite this article

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Abstract

Temperate Bacillus phages often utilize arbitrium communication to control lysis/lysogeny decisions, but the mechanisms by which this control is exerted remains largely unknown. Here we find that the arbitrium system of Bacillus subtilis phage ϕ3T modulates the host-encoded MazEF toxin–antitoxin system to this aim. Upon infection, the MazF ribonuclease is activated by three phage genes. At low arbitrium signal concentrations, MazF is inactivated by two phage-encoded MazE homologues: the arbitrium-controlled AimX and the later-expressed YosL proteins. At high signal, MazF remains active, promoting lysogeny without harming the bacterial host. MazF cleavage sites are enriched on transcripts of phage lytic genes but absent from the phage repressor in ϕ3T and other Spβ-like phages. Combined with low activation levels of MazF during infections, this pattern explains the phage-specific effect. Our results show how a bacterial toxin–antitoxin system has been co-opted by a phage to control lysis/lysogeny decisions without compromising host viability.

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Fig. 1: ϕ3T codes for two MazE-like antitoxins, AimX and YosL.
Fig. 2: MazF strongly promotes ϕ3T lysogenization.
Fig. 3: ϕ3T is hyper-sensitive to MazF activity.
Fig. 4: RNA-seq analysis of ϕ3T infection on different genetic backgrounds reveals an extended cleavage motif and phage-specific cleavage patterns.
Fig. 5: MazF cleavage site patterns are evolutionary conserved.

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Data availability

All data for Figs. 1–3 and Extended Data Figs. 1–3 can be found in Supplementary Information. Data on the phages used for the analysis shown in Fig. 5 are provided in Extended Data File 1, and the phylogenetic trees shown in Fig. 5 and Extended Data Figs. 9 and 10 are available in a Newick format in Supplementary Information. RNA-seq data used for making Fig. 4 and Extended Data Figs. 4–8 were deposited in the SRA repository, BioProject ID PRJNA1025859.

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Code would be provided upon request.

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Acknowledgements

We thank P. Culviner and M. Laub for advice on the RNA-seq experiment and its analysis. We thank S. Pollak, J. Jones, A. Stern, D. Burstein and members of the Eldar lab for fruitful discussions and comments on the paper and N. Ben-Tal for suggestions about structural modelling. The Eldar lab is funded by a European Research Council grant 724805 and by Israel Science Foundation grant 2228/21. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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  1. These authors contributed equally: Polina Guler, Shira Omer Bendori.

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  1. Shmunis School of Biomedicine and Cancer Research, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel

    Polina Guler, Shira Omer Bendori, Tom Borenstein, Nitzan Aframian & Avigdor Eldar

  2. Department of Biochemistry and Molecular Biology, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel

    Amit Kessel

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Contributions

P.G., S.O.B., N.A., T.B. and A.E. were involved in the conceptualization of the study. P.G. and S.O.B. performed the experiments. P.G., S.O.B., N.A., A.K., T.B. and A.E. analysed the data and formulated the theoretical predictions. P.G., S.O.B., N.A. and A.E. wrote the original draft. P.G., S.O.B., N.A., A.K., T.B. and A.E. made the figures. P.G., S.O.B., N.A., T.B. and A.E. edited the paper.

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Correspondence to Avigdor Eldar.

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Extended data

Extended Data Fig. 1 Infection and growth properties of wild-type and AimX variants.

(a) Phage yield for wild-type ϕ3T and ϕ3T;ΔaimX. Plaque forming units (PFU) was measured two hours after infection at MOI of 0.1. Error bars mark SEM over three biological repeats (b–c) Optical density as a function of time of cultures infected with phage ϕ3T at MOI of 0.1 with (dashed line, brighter color) or without (solid line, darker colors) the addition of the arbitrium peptide (SAIRGA). Bacterial strains are (b) Wild-type (blue) and Phs-aimX (green), (c) Phs-aimXstop (blue) and Phs-aimXrecoded (green). (d) Optical density as a function of time of cultures of the indicated strains with no infection. (e) Optical density as a function of time of cultures infected with unmarked phage ϕ3T (blue) or spectinomycin marked phage ϕ3T at MOI of 0.1 with (dashed line, brighter color) or without (solid line, darker colors) the addition of the arbitrium peptide (SAIRGA). (f) Optical density as a function of time of wild-type culture grown in LB (gray) or in LB with the addition of 2% w/v xylose (cyan). (g) DNA sequence of aimXORF and of aimXrecoded together with the amino acid sequence. (h) As in Fig. 1b, but with the addition to the comparison of the AimX homologue taken from a lysogenic phage within the genome of Bacillus velezensis strain NAU-B3 (AimX protein name: BAPNAU_1633, NCBI accession number: CDH95414.1). Lines and shaded regions in b-f mark mean and SEM of three biological repeats. Note that in some cases the SEM is too small for the shaded region to be visible.

Extended Data Fig. 2 Mutations and manipulation of expression of aimX and yosL.

(a–d) expression of antitoxin mutants and variants with or without MazF. Growth curves of cells induced with the antitoxin (100μM IPTG, light blue), toxin (2% xylose, purple), none (grey) or both (light orange) for (a) AimXY18A, (b) AimX homologue of a phage residing in B. velezensis NAU-B3 (see Extended Data Fig. 1h), (c) Full length YosL and (d) YosLY82A. Note that the MazF only (purple) and MazF and mutant antitoxin (light orange) lines overlap in panels (a,d). (e–g) Growth curves of (e) YosL, (f) YosLY82A and (g) YosLN-term as a function of time, induced with different levels of IPTG, as indicated in the legend. All growth experiments were done on multiple biological repeats. Lines mark mean and shaded areas mark the biological standard error of three biological repeats on different days,where in each day the curve is an average over multiple technical repeats. Note that the growth curve in panel (b) was done on a different plate reader than other curves due to technical limitations. (h, i) growth curves of different strains infected by ϕ3T at MOI of 0.1, either in the absence (solid line) or presence (dashed line) of the ϕ3T arbitrium peptide (SAIRGA). The following strains were used; (h) wild-type (blue, same as in Fig. 2a), ΔmazF (green). (i) ΔmazF;PmazEF-mazEF (blue), ΔmazF;PmazEF-mazEFT48A (green). (j) Lysogen numbers as in Fig. 2c, but infected cells were plated 20 minutes after infection. All measures were done in triplicates or more. Lines and shaded in h,i mark mean amd SEM of three biological repeats. Mean and error bars in j mark logarithmic mean and SEM done over three biological repeats.

Extended Data Fig. 3 MazEF is activated by yopMNO, inhibited by aimX,yosL and non-abortively eliminates phages lacking the antitoxins.

(a–c) Infection curves of strain ϕ3T(vir) (blue) compared with mutant strains (green) (a) ϕ3T(vir);ΔaimX, (b) ϕ3T(vir);ΔyosL, (c) ϕ3T(vir);ΔaimX;ΔyosL. The ϕ3T(vir) graph is the same for all panels. Each of the phage strains infected either wild-type (solid) or ΔmazEF (dashed) bacterial hosts. Line mark the mean and shaded region marks SEM over three biological repeats. (d) An extension of Fig. 3b in the main manuscript. Shown in addition to the data in the main manuscript are the plaque titer results for single gene deletion of yopM and yopO on the background of ϕ3T(vir);ΔaimX;ΔyosL and the results of infection of yopMN, yopMNO deletion on a bacterial strain backgropund with constitutive active MazF (ΔmazEF;Pxyl-mazF) induced with 0.01% w/v xylose (MazF++ condition, orange). The latter results suggest that yopMNO are only involved in releasing MazF and not in perturbing its activity. (e, f) Plaque (e) and colony (f) counts 2 hours after infection of a ϕ3T(vir);ΔaimX;ΔyosL and ϕ3T(vir) strains at a MOI of 4 on wild-type (green) and ΔmazEF (blue) bacteria. For PFU counts, shown also the results of infection of a complemented mazEF mutant. Colony forming units were also measured for a mock infection (marked Mock). Error bars in d-f mark SEM over 3-4 biological repeats.

Extended Data Fig. 4 Chromosomal gene expression in different conditions.

(a) expression of MazF under different conditions. Shown is the logarithmic mean average expression. (b) Gene expression in the ΔmazEF strain infected with the double mutated phage, ϕ3T;ΔaimX;ΔyosL (abbreviated Δ2, x-axis) against six other samples whose conditions are shown on the y-axis. ϕ3T(vir);ΔaimX;ΔyosL is abbreviated as Δ3. The latter phage was used eventually only during calculations of MazF cleavage motif and shows very similar expression patterns as with the Δ2 phage. Each dot shows the average coverage of each gene, averaged over two repeats for each sample. mazF and mazE are marked in some of the cases, as well as the pyr and pst operon genes. The R2 coefficient between the two samples is indicated in each panel. See Extended Data File 1 for more details.

Extended Data Fig. 5 Analysis of MazF binding site through identification of ‘cleavage ratio valleys’.

(a) three examples for cleavage ratio valleys. Shown are the expression levels (top) and cleavage ratio (bottoms) in three loci over a window of 600bp centered on a local smoothened minimum of cleavage ratio. Expression is shown as base 2 logarithm. Expression is in the reverse direction and genes in the reverse direction within the window are indicated with a thick red line and their names. (Top) Separate expression levels of MazF++ (red) and ΔmazEF (blue) infected with the same phage. (Bottom) shown are the smoothened (blue) and actual (red) cleavage ratios, which is the difference between the base 2 logarithm of expression of MazF++ sample and a ΔmazEF sample infected with the same phage. (b) Bar graph showing the total number of ‘cleavage ratio valleys’, with at least one ACA site in them and with exactly one ACA site. (c) Position weight matrix for different positions with respect to the constant ACA in positions −1 to +1 extracted from the valleys identified in the cleavage ratio profile. This is an extension of Fig. 4b which shows only positions −2 to 3. Indicated are mean and SEM based on bootstrapping of the data for 1000 times (see methods). Dots with colors corresponding to nucleotide letters show the score of each nucleotide. (d) Shown are the position weight matrices extracted from the valley analysis with modifications to three major parameters of the analysis – valley depth, width and minimum level of ΔmazEF expression accounted, with variation of up to 30% to the value of each parameter (methods). Dots with colors corresponding to nucleotide letters show the score of each nucleotide.

Extended Data Fig. 6 Distribution of average cleavage ratio for different sequences of the form XACAXX.

(a) Each panel shows the mean cleavage ratio over the chromosome (left) and ϕ3T (right) transcripts, where different samples (detailed on the left) are compared to the corresponding ΔmazEF mutant data. As in Fig. 4c, each dot marks the average expression ratio of a given sequence as a function of its score based on the position weight matrix given in Fig. 4b. The right most dot corresponds to UACAUA. The MazF activity parameter Δ is shown in the top left panel and defined as the difference between the average mean value of all sequences of the form XACAXX (except from UACAUA) and the mean cleavage ratio value of UACAUA. (b) The value of Δ for different samples on the chromosome (dark gray) and on ϕ3T (light gray). Inset: focus on the difference between Δ measured on the chromosome for infection of the wild-type by ϕ3T;ΔaimX;ΔyosL and mock infection. Note that Δ is not zero also for mock infection, suggesting some residual MazF activity level in the non-infected wild-type. (c) the fraction of reads covering ϕ3T out of all the reads of a given sample. Shown are the mean of two repeats of each sample.

Extended Data Fig. 7 Anti-correlation between cleavage mean ratio and number of MazF cut-sites over ϕ3T transcriptome.

The relative expression between MazF++ and ΔmazEF samples and the mean MazF cleavage site (UACAUA) density averaged over a moving window of varying size, were calculated for every position within the (a) forward expression right region and (b) reverse expressing left region of ϕ3T. Shown are the kernel distribution (black lines) and mean (blue circle in the forward and red circle in the reverse) for bases that has a given value of site density. The number of such bases (in thousands), over which density is averaged, is marked below each distribution. Note that the average density is discrete by definition. The panels show the distributions for averaging the site density over the indicated window sizes of 1,2,4,5 kbp for both the forward and reverse regions. R, the correlation coefficient between the expression ratio and the density of each bp in the relevant region is indicated in each panel. (c) Control calculations done for 1kbp window for the left region and 5kbp window for the right region, but for density of UACAUG sites, instead of the MazF sites.

Extended Data Fig. 8 Phage Expression during infection of the wild-type.

(a) Shown are the expression in base 2 logarithm of phage ϕ3T 20 minutes post infection of the wild-type (blue), ΔmazEF (green) and MazF++ condition (red). Right (forward) major transcript is shown in solid line while left (reverse) major transcript is shown in dashed line. (b) as in Extended Data Fig. 7, but here the expression ratio is defined as the difference in log2 expression between wild-type and ΔmazEF strains. Shown are results for an averaging window of 1kbp for the left region and 4kbp for the right region. Gray dots marks the values of individual bases.

Extended Data Fig. 9 AimR phylogeny and MazF coverage with overlaps.

(a) A phylogenetic tree of AimR proteins of Spβ-like phages. Shown is the tree of 123 unique protein sequences (from an altogether 321 AimR sequences). Branch colors indicate the associated AimP signal (see legend). External color mark whether the phage codes for AimX (orange), or not (gray). The AimX coding phages are monophyletic in this phylogeny, most likely because the genes coevolve. Phages SPβ and ϕ3T are indicated. (b) Same as Fig. 5b, with the difference that density calculation now also includes sites of permuted MazF motif sequences which overlap with an identified MazF site. Note that this strongly increases the density of several permuted sequences (especially in yosM and yotM) with high overlap with the consensus MazF sequence. The sequence of these sites is shown and the overlap with the MazF consensus underlined. The correct MazF motif remains the one with the highest density of sites.

Extended Data Fig. 10 MazF consensus site location in yonO (RNA polymerase gene) in different Spβ-like phages.

Top: phylogenetic tree of yonO nucleotide diversity. Sequences were aligned using the protein sequence. Shown are 167 different sequences out of 321 sequences (repeated nucleotide sequences were excluded). AimX encoding strains are marked in orange. Note the large phylogenetic distribution of AimX+ strains, suggesting recurrent recombination between this gene and the aimR locus (compare with Extended Data Fig. 9a). SPβ and ϕ3T location on the tree are indicated. Bottom: for each strain, blue dots indicate the positions of MazF sites along the aligned nucleotide sequence of the strain. No single site is conserved throughout the whole phylogeny.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3.

Reporting Summary

Supplementary Table 1

An Excel file with four sheets: (1) data on chromosomal gene expression and MazF sites; (2) data on phi3T gene expression, MazF sites, conservation and other properties; (3) mean MazF density for all SPβ genes over all phages that contain a homologue; (4) data on SPβ phages used for the analysis of MazF cleavage site conservation.

Supplementary Table 2

An Excel file with three sheets: (1) primer list; (2) plasmid list, including construction details; (3) strain list.

Supplementary Data 1

An Excel file with 45 sheets showing 15 plate reader records. Used to produce plate reader figures Figs. 1d–f and 2a and Extended Data Figs. 1b–f, 2a,c–I and 3a–c. Each record contains three sheets: (1) ResultsSheet—raw results sheet from the plate reader; (2) Conditions—list of conditions used in this experiment; (3) plate_setup—the setup of conditions on the 96-well plate of the plate reader. Note: contains also conditions that were not used in the final version of the paper.

Supplementary Data 2

An Excel file with 15 sheets showing 5 plate reader records. Used to produce plate reader figure Fig. 1a. Each record contains three sheets, as in Supplementary Data 1.

Supplementary Data 3

An Excel file with nine sheets showing three plate reader records. Used to produce plate reader figure Extended Data Fig. 2b. Each record contains three sheets, as in Supplementary Data 1.

Supplementary Data 4

Excel file containing the data needed to make Figs. 2b,c and 3a,b,e,f and Extended Data Figs. 1a, 2j and 3d–f.

Supplementary Data 5

Three Newick format phylogenetic trees appearing in Fig. 5a and Extended Data Figs. 9 and 10. The names of the leaves contain the number of the phage appearing in Extended Data Table 1, sheet 4.

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Guler, P., Bendori, S.O., Borenstein, T. et al. Arbitrium communication controls phage lysogeny through non-lethal modulation of a host toxin–antitoxin defence system. Nat Microbiol 9, 150–160 (2024). https://doi.org/10.1038/s41564-023-01551-3

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