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Dormant phages communicate via arbitrium to control exit from lysogeny

Nature Microbiology volume 7, pages 145–153 (2022)Cite this article

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Abstract

Temperate bacterial viruses (phages) can transition between lysis—replicating and killing the host—and lysogeny, that is, existing as dormant prophages while keeping the host viable. Recent research showed that on invading a naïve cell, some phages communicate using a peptide signal, termed arbitrium, to control the decision of entering lysogeny. Whether communication can also serve to regulate exit from lysogeny (known as phage induction) is unclear. Here we show that arbitrium-coding prophages continue to communicate from the lysogenic state by secreting and sensing the arbitrium signal. Signalling represses DNA damage-dependent phage induction, enabling prophages to reduce the induction rate when surrounded by other lysogens. We show that in certain phages, DNA damage and communication converge to regulate the expression of the arbitrium-responsive gene aimX, while in others integration of DNA damage and communication occurs downstream of aimX expression. Additionally, signalling by prophages tilts the decision of nearby infecting phages towards lysogeny. Altogether, we find that phages use small-molecule communication throughout their entire life cycle to sense the abundance of lysogens in the population, thus avoiding lysis when they are likely to encounter established lysogens rather than permissive uninfected hosts.

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Fig. 1: Arbitrium signalling is active during lysogeny and represses DNA damage-dependent prophage induction.
Fig. 2: In the SPβ phage, DNA damage and communication are integrated downstream of aimX expression.
Fig. 3: In phage ϕ106, DNA damage and communication jointly control aimX expression.
Fig. 4: Prophage signalling biases infections towards lysogeny.

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Data availability

All data generated in this study are provided as source data with this paper. The data analysed in Extended Data Fig. 3d are available from Nicolas et al.16.

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Acknowledgements

We thank R. Sorek, S. Pollak, J. Jones, J. van-Gestel and I. Lev for fruitful discussions and comments on the manuscript. The Eldar lab is funded by a European Research Council grant no. 724805. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. Valeria Lipsman

    Present address: Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel

  2. These authors contributed equally: Nitzan Aframian, Shira Omer Bendori.

Authors and Affiliations

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

    Nitzan Aframian, Shira Omer Bendori, Stav Kabel, Polina Guler, Erica Manor, Kholod Msaeed, Valeria Lipsman, Ilana Grinberg, Alaa Mahagna & Avigdor Eldar

  2. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

    Avigail Stokar-Avihail

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Contributions

N.A., S.O.B., P.G. and A.E. were involved in the conceptualization of the study. N.A., S.O.B., S.K., P.G., A.S.-A. and A.M. performed the experiments. N.A., S.O.B., P.G., S.H. and A.E. analysed the data and formulated the theoretical predictions. N.A. and A.E. wrote the original draft. N.A., S.H., P.G. and A.E. created the figures. N.A. and A.E. edited the manuscript. S.K., E.M., K.M., V.L. and I.G. provided essential resources.

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Correspondence to Avigdor Eldar.

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Peer review information Nature Microbiology thanks Joseph Bondy-Denomy and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Sensitivity of the aimX-YFP reporter to addition of AimP in SPβ lysogens and the fate of lysogen infection.

(a) Doubling time in minimal medium was measured by fitting the growth curve to an exponent. Shown is the mean of doubling times for four different experiments. Error bar marks the standard error. (b) Lysogens coding for the aimX-YFP reporter were diluted 106-fold and grown overnight with the indicated levels of externally supplemented AimP peptide. Expression level and optical density were measured at different times. For low optical density, optical density was back-extrapolated from optical densities at later times and from the time passed by using a doubling time of 50 minutes (see A, as in Fig. 1b). Dashed lines show a tri-linear fit on the log-log scale of the data to two horizontal lines and an increasing line. The y-value of the horizontal lines of all fits is equal to the autofluorescence level (bottom line) and to the mean expression of cells with no signal (marked by diamond markers). The slope and intersect of the monotonously increasing lines are subject to fitting. (c) SPβ adsorption to lysogens and non-lysogens is equal. Shown are levels of SPβ phages, measured as PFU/ml, in the medium right after phage addition to medium with no cells and 15 minutes after addition of the same phage doses to medium containing either Non-lysogens or ΔaimR SPβ lysogens. Shown are geometric means of n = 4 biological repeats and error bars represent standard error of the log. The level of PFU is significantly lower in medium containing cells by a factor of ~30 (P-value of two-tailed paired t-test on the log of the PFU is shown in figure), but is not significantly different between the two cell types (P = 0.75, paired t-test on the log of the PFU).

Source data

Extended Data Fig. 2 Impact of signaling on cellular growth curves as measured by plate reader.

(a) Further examples for plate reader experiments showing the same strains and conditions as shown in Fig. 1c of the main text. The second panel is the same as the panel shown in Fig. 1c. A dashed line marks the time-point at which OD levels were used for the statistical analysis shown in (C). (B) Plate reader experiments of additional strains and conditions. Shown are growth curves for the wild-type and ΔaimP lysogens with and without MMC, and for the ΔaimP lysogen with MMC and AimP. See legend for the color of the curve of each strain. (c) Shown are the mean and standard error of 5 biological repeats for each strain and condition, as well as individual measurements of optical density at T = 700 minutes. P-values obtained by two-tailed paired t-test Are shown in the figure. Note that all differences between strains without MMC are non-significant. (d) Shown are graphs of optical density as a function of time for four cultures of ϕ3T lysogens grown in LB broth, as described in the legend. Solid lines mark the mean of three replicates done on the same multi-well plate. The region around each line indicates the standard error of the mean. ϕ3T AimP is the ϕ3T arbitrium peptide (SAIRGA), and SPβ AimP is the peptide GMPRGA.

Source data

Extended Data Fig. 3 Gene expression upon addition of MMC to strain PY79 and related strains.

(a) qRT-PCR results for SOS genes recA and yneA. Shown are relative expression of the two genes under three different times after the addition of MMC (0,20 and 45 minutes) when 10 μM of AimP are either added or not. Expression is normalized to 45 minutes after MMC addition with no addition of AimP. Shown are geometric means of n = 3 biological repeats. Error bars correspond to standard errors of the log. (b) YFP and BFP expression upon addition of MMC. Shown are fluorescence levels (on a log scale) for different reporters and strains as a function of time from addition of MMC. An aimX-YFP reporter in wild-type (deep red) and in ΔaimP (blue) lysogens, a PdinC-YFP reporter in a non-lysogen strain containing PBSX (purple), and a Pveg-BFP constitutive reporter plotted on a single curve for all the strains (cyan). Individual points are biological repeats, solid lines are linear best fit (with y data on a log10 scale) and shaded area is the boundary of error. (c) RT-PCR of aimX-YFP under different conditions. A ΔaimP SPβ lysogen carrying the aimX-YFP reporter was assayed for its expression using RT-PCR (methods), either with or without the addition of 0.5 μg/ml MMC and 10μM SPβ AimP. Measurements were taken either 13 or 24 minutes after the addition of MMC/AimP/Mock. Addition of MMC does not significantly alter aimX-YFP expression levels. Shown are geometric means of n = 3 biological repeats. Error bars correspond to standard errors of the log. (d) Analysis of public RNAseq data of strain 168, after addition of mitomycin C (MMC). Shown are the RNAseq expression data from16, for selected phage genes (and one non-phage gene, dinC) for 5 conditions – t = 0 minutes (−0), t = 45 minutes no MMC (−45), t = 90 minutes no MMC (−90), t = 45 minutes after MMC addition (+45), t = 90 minutes after MMC addition (+90). Genes are divided into flour main categories based on their functional dependence. Gene name in the expression database and its current name or function are annotated. n = 3 biological repeats, P-values obtained by two-sided paired t-test are shown in figure. Error bars indicate standard error of the log. Specifically, note that aimX, aimR and aimP are not significantly different from the control 45 minutes after MMC addition and are marginally higher at 90 minutes. Note that strain 168 carries the mobile element ICEBs1 and contains several additional differences from the PY79 strain background we use in this work.

Source data

Extended Data Fig. 4 Characteristics of phage ϕ106 and its host.

(a) Comparison of phages ϕ105 and ϕ106. We use the following sequences for comparison. ϕ105 is analyzed using its directed sequence file at the NCBI accession number NC_048631. ϕ106 is defined here as the lysogenic segment of Bacillus subtilis subsp. inaquosorum strain KCTC 13429 (NCBI accession number NZ_CP029465) from locus tag DKG76_14125 to locus tag DKG76_14370. ϕ106 proteins were blasted against ϕ105 proteins. Marked are proteins with high (red, Expectation<10−20), medium (black, 10−10 < Expectation<10−20) and few low homology genes within the lytic module (gray, 10−5 < Expectation<10−10). There is little or no homology out of the region annotated here as the lytic module. Protein annotation within the lytic module is based on phage ϕ105 annotation. The lysogenic module of phage ϕ105 including the recombinase ImmRA105 and Rap gene are shown28. Homologs of these genes are absent from ϕ106. Also shown is the putative lysogenic module of ϕ106 analyzed in this work, which is absent from phage ϕ105. (b) Coverage statistics of ϕ106 genomic region in paired-end deep sequencing data of DNAase-protected DNA from lysate of Bacillus subtilis subsp. inaquosorum strain KCTC 13429. ~34,000 pairs of reads matched into the shown region (positions 2,820,000 to2,900,000 within this strain’s genome). A shift in coverage occurs immediately at the beginning and end of the phage. Shown are the coverage (gray) and smoothened coverage over a moving window of 1kbp (black). 72 pairs of reads matched two different sides of the phage, suggesting that the phage genome was excised and circularized to form the attP site. Their locations are shown as green and red dots. (c) qRT-PCR results for SOS genes recA and yneA. Shown are relative expression of the two genes of B. subtilis subsp. Inaquosorum KCTC 13429 under three different times after the addition of MMC (0,45 and 70 minutes) when 10 μM of AimP are either added or not. Shown are geometric means of n = 3 biological repeats. Error bars correspond to standard errors of the log. Expression is normalized to 70 minutes after MMC addition with no addition of AimP.

Source data

Extended Data Fig. 5 aimRPXϕ106-YFP construct and its expression as a function of time in three relevant genetic background.

(a) schematic structure of the aimRPXϕ106-YFP reporter. The YFP genes starts where the putative cI gene ends in the native genome of the phage. (b-d) YFP expression from the aimRPXϕ106-YFP construct as a function of time after addition of MMC and/or AimP (or mock additions), divided by fluorescence of a Pveg-BFP constitutive reporter within the same cells. Circles represent individual measurements (median YFP level from a flow cytometry measurement). Lines of the same color show linear best fit and shaded area show the boundary of error. Each strain was measured with three or four independent time series taken on different days. The legend in (C) is true for all panels. The three panels correspond to the same strains described in Fig. 3c-e of the main work and the illustrations describing them are identical; (B) aimRPXϕ106-YFP reporter based on the wild-type sequence of phage ϕ106. (C) as in (A) but with overexpression of a non-cleavable defective lexA mutant. (D) a mutant aimRPXϕ106-YFP reporter with substitution in two base-pairs within the LexA binding site. Figure 3c-e shows the data presented here for 90 minutes.

Source data

Extended Data Fig. 6 Pveg-BFP and PdinC-YFP expression upon addition of MMC with and without overexpression of a non-cleavable LexA mutant.

(a, b) Expression as a function of time of a constitutive Pveg-BFP reporter for the four conditions shown also in Supplementary Fig. 5. The two strains are the ones presented in panels A, B of Supplementary Fig. 5 correspondingly. Note the change in BFP expression upon addition in MMC between panels A, B, indicating the impact of LexA(ind-) allele expression on growth (c) Expression of a PdinC-YFP reporter as a function of time after addition of MMC in a genetic background including the IPTG-inducible Phs-lexA(ind-) allele. Shown are results with 0 IPTG (no induction, orange) and 100μM (full induction, purple). Circles represent individual measurements, lines represent best linear fits and shaded area the boundary of error.

Source data

Extended Data Fig. 7 Consistency and diversity of the ϕ106 family putative lysogeny module.

Left: Shown is the phylogenetic tree of 8 different AimR clade 1 proteins (out-grouped by AimR from phage SPβ). Each of the eight corresponding aimR genes has an adjacent aimP gene coding for the putative mature arbitrium AimP signal shown to the right of the corresponding tree leaf. Right: the genomic organization of the arbitrium locus is shown for each AimR gene. Colored arrows represent genes, according to the annotation at the top. Black arrows correspond to additional genes. Green line marks the position of the putative LexA binding site. The ϕ106 system used in this work is specifically marked (strain inaquosorum). The NCBI accession number of the AimR proteins (and the genome GCF number) as shown in the phylogeny, from top to bottom, are: WP_003237457 (003148415), WP_101605414 (002850535), ARW33040 (001747445), WP_049627412 (004119735), WP_047936208 (001023595), WP_003220312 (005218185), API45091 (002982175), WP_017417251 (003860445), WP_181217268 (013620725).

Extended Data Fig. 8 Raw flow cytometry data and gating procedure.

YFP reporters were used to measure aimX expression in SPβ (Fig. 1a,b, Extended Data Figs. 2b and 4b) and ϕ106 phages (Figs. 2b, 3c-e, Extended Data Fig. 5b-d). Populations were marked with either BFP or RFP constitutive reporters. Constitutive reporter expression allowed us to distinguish between population in co-cultures (Fig. 1a, b) and to normalize for constitutive reporter expression (Figs. 2b, 3, Extended Data Fig. 5b-d). The following gating scheme was used: a. cells were initially gated on their forward and side-scattering to exclude non-canonical elements. b. cells were then gated on their BFP or RFP. c. finally, YFP expression histograms and medians were calculated for an individual population. For Figs. 2b, 3c-e and Extended Data Fig. 5b-d, median of YFP expression was divided by the median of constitutive BFP expression.

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Aframian, N., Omer Bendori, S., Kabel, S. et al. Dormant phages communicate via arbitrium to control exit from lysogeny. Nat Microbiol 7, 145–153 (2022). https://doi.org/10.1038/s41564-021-01008-5

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