Quorum Sensing and Metabolic State of the Host Control Lysogeny-Lysis Switch of Bacteriophage T1

Abstract

Bacterial viruses, or bacteriophages, are highly abundant in the biosphere and have a major impact on microbial populations. Many examples of phage interactions with their hosts, including establishment of dormant lysogenic and active lytic states, have been characterized at the level of the individual cell. However, much less is known about the dependence of these interactions on host metabolism and signal exchange within bacterial communities. In this report, we describe a lysogenic state of the enterobacterial phage T1, previously known as a classical lytic phage, and characterize the underlying regulatory circuitry. We show that the transition from lysogeny to lysis depends on bacterial population density, perceived via interspecies autoinducer 2. Lysis is further controlled by the metabolic state of the cell, mediated by the cyclic-3',5'-AMP (cAMP) receptor protein (CRP) of the host. We hypothesize that such combinations of cell density and metabolic sensing may be common in phage-host interactions.IMPORTANCE The dynamics of microbial communities are heavily shaped by bacterium-bacteriophage interactions. But despite the apparent importance of bacteriophages, our understanding of the mechanisms controlling phage dynamics in bacterial populations, and particularly of the differences between the decisions that are made in the dormant lysogenic and active lytic states, remains limited. In this report, we show that enterobacterial phage T1, previously described as a lytic phage, is able to undergo lysogeny. We further demonstrate that the lysogeny-to-lysis decision occurs in response to changes in the density of the bacterial population, mediated by interspecies quorum-sensing signal AI-2, and in the metabolic state of the cell, mediated by cAMP receptor protein. We hypothesize that this strategy enables the phage to maximize its chances of self-amplification and spreading in bacterial population upon induction of the lytic cycle and that it might be common in phage-host interactions.

Keywords: Escherichia coli; bacteriophage lysis; cyclic AMP; quorum sensing.

FIG 1

E. coli ATCC 14155 carries an AI-2- and sugar-inducible prophage. (A) Optical densities of E. coli ATCC 14155 cultures (each dot represents individual culture) grown alone or in a 1:1 mixture with E. coli W3110 or W3110 ΔluxS bacteria or with 10 μl E. coli W3110 wild-type bacteria or ΔluxS cell-free supernatants. (B) Transmission electron microscopy (TEM) of phage particles found in E. coli ATCC 15144 lysates. Scale bar, 200 nm. Note that the different levels of brightness in the four quadrants of the image represent an artifact of the TEM imaging system. (C) Prophage induction in E. coli ATCC 14155 by AI-2 and sugar influx. Single dots represent individual cultures. glu, glucose; 2-DG, 2-deoxy-d-glucose. Means of results of a minimum of 12 independent replicates are shown; error bars represent standard deviations. P values were calculated using the Mann-Whitney test (****, P < 0.0001; ***, P < 0.0005; *, P < 0.05; ns, not significant).

FIG 2

T1 phage-encoded transcription regulator Pir (Orf23) controls AI-2- and sugar-dependent prophage induction. (A) Pir is a functional transcription regulator. Activities of hol, dam, pir, and recE promoters controlling gfp expression (measured by flow cytometry and expressed in arbitrary units [AU]) in the absence (pir⁻) or presence (pir⁺) of plasmid-harbored pir were measured in E. coli W3110 by flow cytometry. (B) pir and hol were upregulated during AI-2- and glucose-mediated prophage induction. Activities of pir and hol promoters were measured at the onset of visible cell lysis by flow cytometry. (C) Activities of pir and hol promoters in a phage-free background strain, BL21(DE3), were measured by flow cytometry 2 h after addition of 30 μM DPD/AI-2 or 0.2% glucose. (D) Effect of CRISPRi-mediated inhibition of pir expression on lysis of E. coli ATCC 15144. Single dots represent optical densities of individual E. coli cultures (CRISPRi⁻ aTc⁻) carrying a CRISPRi system without (CRISPRi⁺ aTc⁻) or with (CRISPRi⁺ aTc⁺) induction of dCas9 protein expression, measured 2 h after addition of 10 μl E. coli W3110 cell-free supernatant. aTc, anhydrotetracycline. (E) hol promoter activity measured by flow cytometry in the setup described in the panel D legend. Single dots represent hol promoter activities in individual cultures. Means of results from a minimum of three independent replicates are shown; error bars represent standard deviations. P values were calculated using the Mann-Whitney test (****, P < 0.0001; ***, P < 0.0005; **, P < 0.005; ns, not significant).

FIG 3

Glucose uptake activates pir expression via cAMP-CRP. (A) Deletion of E. coli adenylate cyclase (cyaA) results in increased pir expression combined with insensitivity to glucose. The effect of cyaA deletion can be partially complemented by addition of 250 mM cAMP to the growth medium. Activity of pir promoter controlling gfp expression was measured by flow cytometry. Results are expressed in arbitrary units (AU). Single dots represent pir promoter activities in individual cultures. Means of results from a minimum of four independent replicates are shown; error bars represent standard deviations. P values were calculated using the Mann-Whitney test (****, P < 0.0001; **, P < 0.005; *, P < 0.05; ns, not significant). (B) Regulation of pir promoter controlling gfp by cAMP-CRP in an in vitro transcription-translation system (see Materials and Methods for details). GFP fluorescence was measured in a plate reader, and values were normalized to the fluorescence intensity at the time point 0 s. Synthetic constitutive promoter J23101 was used as a negative control. Means of results of three independent replicates are shown; error bars represent standard deviations.

FIG 4

Model of T1 prophage induction in response to AI-2-mediated signaling and sugar influx sensing. Phage-encoded Pir is a transcription regulator controlling expression of genes required for T1 prophage induction. In the absence of external stimuli, low pir expression is ensured by the self-inhibitory activity of Pir along with inhibition by Orf65 and by cAMP-CRP. Activation by glucose, likely mediated by PTS, relieves CRP inhibition of pir expression. This in turn results in Pir-dependent activation of lytic cycle and accumulation of holin (hol), followed by cell lysis and phage release. Expression of pir is also upregulated in response to AI-2 by an as-yet-unknown mechanism, similarly initiating prophage induction. Phage-carried genes are marked in green; host-encoded CRP is marked in pink. Regulatory effects are shown by solid lines (transcriptional regulation) or dashed lines (posttranslational regulation). Positive and negative regulatory effects are indicated by lines with arrowheads and by lines with blunt ends, respectively.