Phage integration alters the respiratory strategy of its host

  1. Jeffrey N Carey
  2. Erin L Mettert
  3. Daniel R Fishman-Engel
  4. Manuela Roggiani
  5. Patricia J Kiley
  6. Mark Goulian  Is a corresponding author
  1. Perelman School of Medicine, University of Pennsylvania, United States
  2. University of Pennsylvania, United States
  3. University of Wisconsin, United States
5 figures, 1 table and 6 additional files

Figures

Bacteriophage HK022 integrates between the signaling genes torS and torT, disrupting regulation of torCAD and a metabolic bet-hedging strategy.

(A) HK022 integrates as a prophage at an integration site (attBHK022) between torS and torT, separating torS from the IscR binding site that represses its transcription. TorS regulates torCAD by phosphorylating and dephosphorylating the transcription factor TorR, which in its phosphorylated state activates transcription from the torCAD promoter. To phosphorylate TorR, TorS must interact with TMAO-bound TorT; in the absence of this interaction, TorS dephosphorylates TorR. When oxygen is present, transcription of torS and torT is repressed to an extremely low level by IscR, and stochasticity in the ratio of TorS to TorT leads to noisy torCAD transcription (Carey et al., 2018). (B) The HK022 prophage shuts off aerobic transcription of torCAD but leaves anaerobic expression intact. Distributions of single-cell fluorescence are shown for strains carrying a fluorescent reporter of torCAD transcription. Data are shown for an HK022 lysogen (DFE12) and a non-lysogen (MMR8) grown in the presence or absence of oxygen. Each circle represents a fluorescence measurement made in an individual cell. To facilitate qualitative comparisons between distributions, density curves (shown in gray) were generated from single-cell measurements (see Materials and methods). Data are pooled from three independent experiments, with the vertical red lines indicating the population mean fluorescence for each experiment. a.u., arbitrary units. (C,D) Most cells carrying the HK022 prophage fail to grow following rapid oxygen depletion. Each circle represents an individual cell monitored for growth following an aerobic-to-anaerobic transition. The same data are presented on a linear scale (C) for easier comparison with (B) and on a log scale (D) for clearer resolution of individual points. The HK022 lysogen (JNC173) constitutively expresses CFP to distinguish it from the non-lysogen (JNC174), which constitutively expresses mCherry. Both strains carry the YFP reporter of torCAD transcription and lack fhuA, the gene encoding the HK022 receptor. Growth is quantified as the ratio of microcolony area approximately 5 hr after oxygen depletion to the area of the parent cell at the time of depletion. Data are shown for a single representative experiment.

https://doi.org/10.7554/eLife.49081.003
Figure 1—source data 1

Fluorescence measurements for Figure 1B.

https://doi.org/10.7554/eLife.49081.004
Figure 1—source data 2

Fluorescence and growth measurements for Figure 1C, D.

https://doi.org/10.7554/eLife.49081.005
The HK022 prophage increases torS transcription and has no effect on torT transcription.

Aerobic and anaerobic transcription of torS (A) and torT (B) was measured by β-galactosidase assays in strains carrying torS-lacZ or torT-lacZ operon fusions, with or without the HK022 prophage (strains JNC166, JNC169, JNC163, and JNC168). Each circle represents a measurement obtained from an independent experiment, and the horizontal lines indicate average values. (C) Overexpression of torT restores aerobic torCAD expression in an HK022 lysogen. The distributions of single-cell fluorescence are shown for strains carrying a fluorescent reporter of torCAD transcription. The strains are an HK022 lysogen (DFE12) and a non-lysogen (MMR8) containing a plasmid for torT overexpression (pMR26) or an empty vector control (pDSW206), grown in the presence or absence of oxygen. Expression of torT from the plasmid is driven by a weakened trc promoter without added inducer. Each circle represents a fluorescence measurement made in an individual cell. To facilitate qualitative comparisons between distributions, density curves (shown in gray) were generated from single-cell measurements (see Materials and methods). Data are pooled from three independent experiments, with the vertical red lines indicating the population mean fluorescence for each experiment. a.u., arbitrary units.

https://doi.org/10.7554/eLife.49081.006
Figure 2—source data 1

β-Galactosidase measurements for Figure 2A.

https://doi.org/10.7554/eLife.49081.007
Figure 2—source data 2

β-Galactosidase measurements for Figure 2B.

https://doi.org/10.7554/eLife.49081.008
Figure 2—source data 3

Fluorescence measurements for Figure 2C.

https://doi.org/10.7554/eLife.49081.009
Figure 3 with 1 supplement
Transcription of torS in an HK022 lysogen originates from within the prophage.

(A) Sequence of the torS-adjacent HK022 integration site (attLHK022) in an HK022 lysogen. B, O, and P’ indicate the bacterial, overlap, and phage segments of the integration site, respectively (Campbell, 1992; Yagil et al., 1989). The location of the Ω element terminator insertion in strain JNC175 is indicated. In this strain, transcription reading toward torS from within the HK022 prophage is blocked by the Ω element. The transcription start site, TSSHK022, was mapped by in vitro transcription and primer extension, shown in (C) and (D). The previously inferred torS GTG start codon is outlined, and the experimentally confirmed ATG start codon is indicated as the start of the torS coding sequence. (B) Aerobic and anaerobic transcription of torS was measured by β-galactosidase assays in strains carrying a torS-lacZ operon fusion. Strains contained the wild-type HK022 prophage (JNC169), the prophage with an Ω element (JNC175), or had no prophage at the integration site (JNC166). Each circle represents a measurement obtained from an independent experiment, and the horizontal lines indicate average values. (C) In vitro transcription from plasmids containing sequence upstream of torS from the HK022 lysogen (‘HK022+’, pPK13256) or the non-lysogen (‘no phage’, pPK12669) shows that different transcripts are produced when the prophage is present or absent. Transcription using non-lysogen sequence produces two distinct transcripts, suggesting two transcription start sites for torS. RNA-1 is a control transcript for in vitro transcription and gel loading that is generated from a σ70-regulated promoter in pPK13256 or pPK12669. (D) Primer extension was performed to map the transcription start site of the in vitro ‘HK022+’ transcript shown in (C). The position of the start site is indicated in (A). (E) Primer extension was performed to map the transcription start sites of the in vitro ‘no phage’ transcripts shown in (C). The positions of the start sites are indicated in (F). (F) Sequence upstream of torS in a non-lysogen, with the torS transcription start sites depicted. Transcripts originating from TSS2 can begin at the underlined G or A position, as indicated by the adjacent bands in (E).

https://doi.org/10.7554/eLife.49081.010
Figure 3—source data 1

β-Galactosidase measurements for Figure 3B.

https://doi.org/10.7554/eLife.49081.012
Figure 3—figure supplement 1
Identification of the torS start codon.

(A) Two start codons have been proposed for the torS gene: an upstream GTG start (UniProt Consortium, 2019) and a downstream ATG start (Jourlin et al., 1996). The GTG start codon appears to be too close to the 5’ end of the TSS2 transcript to permit ribosome binding and translation. (B) To determine which of the proposed start codons is used in vivo, we constructed strains containing chromosomally encoded lacZ translational fusions to each codon (PK13196 and PK13199). β-Galactosidase activity was only detected for the lacZ fusion to the ATG start codon, suggesting that this codon is the primary, or perhaps exclusive, start codon for torS. Each circle represents a measurement obtained from an independent experiment, and the horizontal lines indicate average values.

https://doi.org/10.7554/eLife.49081.011
Figure 3—figure supplement 1—source data 1

β-Galactosidase measurements.

https://doi.org/10.7554/eLife.49081.013
A wild E. coli strain carrying a prophage at attBHK022 shows an oxygen-dependent torCAD expression pattern similar to that of the HK022-infected laboratory strain.

A fluorescent reporter of torCAD transcription was introduced into the Crohn’s disease-associated E. coli strain NRG 857C and used to measure expression during aerobic and anaerobic growth. NRG 857C naturally carries a prophage at the HK022 integration site and displays a qualitatively similar pattern of torCAD expression as HK022-infected MG1655 (see Figure 1B). Distributions of single-cell fluorescence are shown for the NRG 857C PtorCAD-yfp strain (DFE34), with each circle representing a fluorescence measurement made in an individual cell. To facilitate qualitative comparisons between distributions, density curves (shown in gray) were generated from single-cell measurements (see Materials and methods). Data are pooled from two independent experiments, with the vertical red lines indicating the population mean fluorescence for each experiment. a.u., arbitrary units.

https://doi.org/10.7554/eLife.49081.014
Model of how bacteriophage HK022 reprograms the regulation of torCAD expression during lysogeny.

In cells lacking the HK022 prophage, IscR repression of torS and torT during aerobic growth leads to very low TorS and TorT abundance. High variability in the ratio of TorS to TorT results in noisy torCAD transcription (top left). In the absence of oxygen, IscR repression of torS and torT is relieved, decreasing variability in the TorS-to-TorT ratio and noise in torCAD transcription (bottom left) (Carey et al., 2018). In HK022 lysogens, a prophage-encoded promoter drives high torS expression. IscR still represses torT during aerobic growth, and the resulting excess of TorS relative to TorT shuts down torCAD transcription (top right) (see Figure 1A). In the absence of oxygen, IscR repression of torT is relieved, and torCAD transcription is restored (bottom right).

https://doi.org/10.7554/eLife.49081.016

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Escherichia coli)torSNAEcoCyc:G6514; UniProt:P39453
Strain, strain background (Escherichia virus HK022)HK022PMID: 4569213RefSeq:NC_002166Dr. Max E. Gottesman (Columbia University)
Strain, strain background (E. coli)DFE12this paperMG1655 attBλ::(cat PtorCAD-yfp) ompA-cfp (HK022)n
Strain, strain background (E. coli)DFE34this paperNRG 857C ΔlacIZY::PtorCAD-yfp-FRT-kan-FRT
Strain, strain background (E. coli)JNC151this paperMG1655 (HK022)n
Strain, strain background (E. coli)JNC163PMID: 29502970MG1655 ΔlacZYA::FRT-cat-FRT torT-lacZ-FRT-kan-FRT ΔtorR
Strain, strain background (E. coli)JNC166PMID: 29502970MG1655 ΔlacZYA::FRT torS-lacZ-FRT-kan-FRT
Strain, strain background
(E. coli)
JNC168this paperMG1655 ΔlacZYA::FRT-cat-FRT (HK022)n torT-lacZ-FRT-kan-FRT ΔtorR
Strain, strain background (E. coli)JNC169this paperMG1655 ΔlacZYA::FRT torS-lacZ-FRT-kan-FRT (HK022)n
Strain, strain background (E. coli)JNC173this paperMG1655 ΔfhuA::FRT-kan-FRT attBλ::(cat PtorCAD-yfp) ompA-cfp (HK022)n
Strain, strain background (E. coli)JNC174this paperMG1655 ΔfhuA::FRT-kan-FRT attBλ::(cat PtorCAD-yfp) ΔxylAFG::PtetA-mcherry-FRT
Strain, strain background (E. coli)JNC175this paperMG1655 ΔlacZYA::FRT torS-lacZ-FRT-kan-FRT (HK022)n attLHK022::Ω
Strain, strain background (E. coli)MG1655Coli Genetic Stock CenterCGSC:7740; RefSeq:NC_000913
Strain, strain background (E. coli)MMR8PMID: 25825431MG1655 attBλ::(cat PtorCAD-yfp) ompA-cfp
Strain, strain background (E. coli)NRG 857CPMID: 21108814RefSeq:NC_017634Dr. Alfredo G. Torres (UTMB)
Strain, strain background (E. coli)PK13196this paperMG1655 lacZ::kan-PtorS-(GTG)lacZ ΔiscR::FRT
Strain, strain background (E. coli)PK13199this paperMG1655 lacZ::kan-PtorS-(ATG)lacZ ΔiscR::FRT
Recombinant DNA reagentpDSW206PMID: 9882665ori(pBR322) lacIq amp Ptrc attenuated promoter.
Dr. Jon Beckwith (Harvard University)
Recombinant DNA reagentpMR26PMID: 25825431pDSW206 torT
Recombinant DNA reagentpPK7179PMID: 15659690ori(pBR322) ter(spf) amp RNA-1
Recombinant DNA reagentpPK12669this paperpPK7179 with −152 to +28 bp relative
to the torS ATG start codon from MG1655 in XhoI/BamHI sites
Recombinant DNA reagentpPK13256this paperpPK7179 with −231 to +28 bp relative to the torS ATG start codon from JNC151 in XhoI/BamHI sites
Sequence-based reagentnative torSthis paper32P-labeled DNA oligonucleotide: 5’-TTAACAGCGCCATCAG-3’
Sequence-based reagentHK022/torSthis paper32P-labeled DNA oligonucleotide: 5’-GGGTCAGGGTTAAATTCACGG-3’
Peptide, recombinant proteinE. coli σ70 RNA polymerase holoenzymeNew England BiolabsNEB:M0551S
Commercial assay or kitHiSpeed Plasmid Maxi KitQiagenQiagen:12662
Commercial assay or kitMMLV Reverse Transcriptase 1st-Strand cDNA Synthesis KitLucigenLucigen:MM070150
Commercial assay or kitSequenase Version 2.0 DNA Sequencing KitUSBUSB:70770
Software, algorithmBLASTPMID: 23609542RRID:SCR_004870
Software, algorithmClermonTypingPMID: 29916797v. 1.4.0
Software, algorithmggridgesComprehensive R Archive NetworkRRID:SCR_003005v. 0.5.0
Software, algorithmMauvePMID: 20593022RRID:SCR_012852v. 2015-02-25
Software, algorithmMUSCLEPMID: 15034147v. 3.8.1551
Software, algorithmRR Foundation for Statistical ComputingRRID:SCR_001905v. 3.4.4
Software, algorithmSnapGeneGSL BiotechRRID:SCR_015052v. 5.0b3

Additional files

Source data 1

FASTA sequence alignment file.

Source data for Supplementary file 2.

https://doi.org/10.7554/eLife.49081.017
Supplementary file 1

Fully sequenced E. coli isolates carrying prophages at the HK022 integration site.

https://doi.org/10.7554/eLife.49081.018
Supplementary file 2

Sequence alignment of the genomic region around the prophage-encoded torS transcription start site from all fully sequenced E. coli strains carrying a prophage at the HK022 integration site.

https://doi.org/10.7554/eLife.49081.019
Supplementary file 3

Strains used in this study.

https://doi.org/10.7554/eLife.49081.020
Supplementary file 4

Plasmids used in this study.

https://doi.org/10.7554/eLife.49081.021
Transparent reporting form
https://doi.org/10.7554/eLife.49081.022

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  1. Jeffrey N Carey
  2. Erin L Mettert
  3. Daniel R Fishman-Engel
  4. Manuela Roggiani
  5. Patricia J Kiley
  6. Mark Goulian
(2019)
Phage integration alters the respiratory strategy of its host
eLife 8:e49081.
https://doi.org/10.7554/eLife.49081