How T4 works

Rapid isolation and identification of bacteriophage T4-encoded
modifications of Escherichia coli RNA polymerase: a generic
method to study bacteriophage/host interactions
Lars F. Westblade1, Leonid Minakhin2, Konstantin Kuznedelov2, Alan J. Tackett1,3,
Emmanuel J. Chang1,4, Rachel A. Mooney5, Irina Vvedenskaya2, Qingjun Wang1, David
Fenyö1, Michael P. Rout1, Robert Landick5, Brian T. Chait1, Konstantin Severinov2, and Seth
A. Darst1,*
1The Rockefeller University, New York, NY 10021, USA;
2Waksman Institute for Microbiology and Department of Molecular Biology and Biochemistry, Rutgers, the
State University of New Jersey, Piscataway, NJ, 08854, USA;
3Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little
Rock, AR 72205, USA;
4Department of Natural Sciences, York College/City University of New York, Jamaica, NY, 11451;
5Departments of Biochemistry and Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA
Bacteriophages are bacterial viruses that infect bacterial cells and they have developed ingenious
mechanisms to modify the bacterial RNA polymerase. Using a rapid, specific, single-step
immunoisolation procedure to purify Escherichia coli RNA polymerase from bacteriophage T4
infected cells; we have identified bacteriophage T4-dependent modifications of the host RNA
polymerase. We suggest that this methodology is applicable for the identification of bacteriophagedependent
alterations of the host synthesis machinery.
Escherichia coli; bacteriophage; RNA polymerase; bacteriophage infection; immunoisolation;
In the bacterium Escherichia coli (Ec), transcription is dependent upon a single ∼400 kDa
multisubunit RNA polymerase (RNAP) that can exist in two forms: the catalytic core enzyme
(α2ββ′ω) and the promoter-specific holoenzyme (α2ββ′ωσ). Transcription can be divided into
three distinct stages: initiation, elongation, and termination. Regulation of RNAP can occur at
each stage of transcription by protein, RNA, and small molecule trans-acting factors that
associate with the enzyme to modulate its activity.
Bacteriophages (phages) are bacterial viruses dependent upon a host organism in order to
propagate; therefore, phages are endowed with mechanisms that subvert the host's cellular
*Corresponding author: Seth A. Darst; Telephone: (212) 327-7479; FAX: (212) 327-7477; E-mail: ude.rellefekcor|tsrad#ude.rellefekcor|tsrad
NIH Public Access
Author Manuscript
J Proteome Res. Author manuscript; available in PMC 2008 December 30.
Published in final edited form as:
J Proteome Res. 2008 March ; 7(3): 1244–1250. doi:10.1021/pr070451j.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
processes to serve the needs of the virus. Consequently, bacteriophages offer an exciting and
tractable system by which to study developmental processes. Recently, resurgence in the use
of bacteriophages for the treatment or prophylaxis of bacterial infectious diseases –
bacteriophage therapy – has increased the demands for understanding bacteriophage/host
interactions1. In particular, identification of phage proteins that bind to and inhibit essential
host enzymes should allow the identification of novel drug targets. Therefore, it is highly
desirable to have efficient methods for the identification of phage-encoded proteins that
associate with host proteins.
Many phages encode proteins that directly bind to and regulate the activity of the bacterial host
RNAP, and some phages encode enzymes that covalently modify the host transcriptional
apparatus. These phage-induced modifications ensure coordinated temporal transcription of
the phage genome and inhibition of host transcription2.
Bacteriophage T4 (T4) does not encode its own RNAP and relies on Ec RNAP for expression
of its genes. In order to appropriate the host RNAP, T4 employs several redundant mechanisms
that target Ec RNAP to the T4 genome2 and promote the transcription of T4 DNA. One such
mechanism involves the T4-encoded Alc protein that functions to increase the pool of host
RNAP available for transcribing T4 DNA by selectively terminating RNAP transcription on
Ec DNA without affecting transcription of T4 DNA3,4,5.
In this paper, we present a method for the rapid isolation of RNAP from phage-infected Ec
cells, followed by matrix-assisted laser desorption ionization mass spectrometric (MALDI MS)
analysis of proteins co-isolating with RNAP. This method allowed unequivocal identification
of most of the known T4-encoded proteins that bind directly to host RNAP, identification of
Alc whose binding to RNAP had not previously been reported, and also identified covalent
modifications of the host RNAP by T4-encoded enzymes. This general procedure should be
broadly applicable for the identification of phage-encoded proteins that interact with
components of the host macromolecular synthesis machinery, and will become increasingly
important in characterizing the many bacteriophage/host systems.
Experimental Section
Ec strains and growth conditions
A strain of Ec MG1655 encoding two Fc-binding repeats of the Protein A (2PrA) affinity
tag6 appended to the 3′ end of the rpoC gene (which encodes the RNAP β′ subunit) was
constructed as previously described7. Both the MG1655 Ec wild-type and MG1655 Ec rpoC::
2PrA strains exhibited almost identical growth curves in Luria-Bertani broth (LB; data not
shown) and doubling times (Ec wild-type, 25.3 +/− 1.6 minutes; Ec rpoC::2PrA, 28.5 +/− 2.1
All Ec strains were grown in LB at 37 °C with shaking. To prepare T4-infected biomass, wildtype
Ec and Ec rpoC::2PrA strains (4 L of each) at an A600 nm 0.5 – 0.6 were infected with T4
(alt-) at a multiplicity of infection of 10. Infection was halted 15 minutes post-infection by
rapidly cooling the samples in icy water baths. Cells were harvested by centrifugation, washed
once with ice-cold 10% (v/v) glycerol, and frozen as pellets in liquid nitrogen. Cells were
cryogenically disrupted with a Retsch MM301 mixer mill (Retsch) that was maintained at
liquid nitrogen temperature, and stored at minus 80 °C8.
Immunoisolation and mass spectrometric identification of protein complexes
Cryogenically lysed cells (1 g) were suspended in 5 ml of extraction buffer (20 mM Hepes (pH
7.4); 2 mM MgCl2; 150 mM NaCl) supplemented with 1 protease inhibitor cocktail tablet
(Roche Diagnostics). Suspended lysate was treated with 0.002 % (w/v) DNase I (Sigma-
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Aldrich) with agitation. The soluble fraction was isolated by centrifugation for 10 minutes; the
supernatant was decanted and subjected to another 10 minute centrifugation step. The
supernatant was incubated with 25 mg of Dynabeads (Invitrogen) cross-linked to rabbit IgG
(MP Biomedicals) with gentle agitation for 10 minutes. For β′-2PrA, and other affinity tagged
proteins, rapid immunoisolations are optimal (data not shown;9). Dynabeads were collected
with a magnet and washed with wash buffer (20 mM Hepes (pH 7.4); 150 mM NaCl). The β
′-2PrA-tagged protein and co-purifying proteins were eluted from the IgG-Dynabeads with 0.5
M NH4OH; 0.5 mM EDTA. The eluted proteins were frozen in liquid nitrogen and evaporated
to dryness in a SpeedVac (Thermo Savant). Dried protein samples were dissolved in SDSPAGE
loading buffer and heated at 95 °C. Samples were resolved by SDS-PAGEwith 4-12 %
(w/v) Bis-Tris polyacrylamide gels (Invitrogen), and bands due to proteins were visualized by
Coomassie blue staining. The entire gel lane was sliced into 1 mm slices and the proteins in
each gel slice were destained with a mixture of 50 % (v/v) acetonitrile and 50 mM ammonium
bicarbonate. After destaining, the proteins were reduced with 10 mM TCEP-HCl (Pierce) for
30 minutes and then alkylated with 50 mM iodoacetamide (Sigma-Aldrich) for 1 hour. Finally,
proteins in each gel slice were digested with Trypsin (Roche Diagnostics) at 37 °C for 4 hours.
Proteins were identified by MALDI-QqTOF single stage MS and MALDI-ion-trap MS2 as
previously described10,11. Single-stage and multi-stage mass spectrometric data were used
for protein identification with the programs Profound12 and Sonar MS/MS13, respectively.
Cloning and overexpression of Alc
Due to the toxicity of T4 Alc to Ec cells, a T7 RNAP-directed Alc overexpression plasmid was
constructed in several steps. First, using purified T4 genomic DNA as a template, the DNA
encoding Alc was amplified by the Polymerase Chain Reaction (PCR). The resultant PCR
product was blunt-end cloned into pT7Blue (Novagen) in an anti-sense orientation relative to
the T7 RNAP f10 and Ec RNAP lacUV5 promoters to reduce basal expression of Alc, creating
pT7-alcrev. Second, pT7-alcrev was cleaved with AfIIII; treated with Klenow DNA polymerase
to generate blunt ends, and digested with BamHI to obtain a fragment encoding Alc and the
ϕ10 and lacUV5 promoters. The resultant fragment was cloned into pET28a (Novagen) cleaved
with HindIII; treated with Klenow to generate blunt ends, and then cleaved with BamHI to
create pET28-alc(T7-lacUV5). Finally, a DNA fragment encoding only the lacUV5 promoter
was generated by digesting pUC19 with AflIII; followed by incubation with Klenow to generate
blunt-ends, and then cleaved with HindIII. The resultant lacUV5 fragment was cloned between
the NotI site, also treated with Klenow, and the HindIII sites of pET28-alc(T7-lacUV5), thus
creating pET28-alc(lacUV5). Thus, Alc overexpression is under the control of the plasmidencoded
T7 RNAP ϕ10 promoter, while the Ec RNAP lacUV5 promoter drives transcription
anti-sense to Alc reducing Alc toxicity.
The expression plasmid pET28-alc(lacUV5) was transformed into Ec cells harboring the paf
rpoB allele (3; which partially nullifies the toxicity of Alc) and the lambda (DE3) lysogen
encoding T7 RNAP, and transformants were selected in the presence of 50 μg/ml kanamycin.
Cultures were grown at 37 °C to an A600 nm ∼0.6 and induced with 1 mM IPTG for 14 hours
at 37 °C. Cells containing overexpressed proteins were harvested by centrifugation and stored
at minus 80°C.
Protein Purification
Recombinant Alc formed inclusion bodies and was solubilised by adding urea to a final
concentration of 8 M. A polyhistidine tag (derived from the vector) was appended to the aminoterminus
of Alc, thus Alc was purified by HiTrap Ni2+-charged affinity chromatography (GE
Healthcare) with 8 M urea present in all the column buffers. Purified (His)6-Alc was exchanged
into renaturation buffer (40 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 0.5 mM EDTA, 2 mM β-
mercaptoethanol, 5 % (v/v) glycerol) by dialysis and concentrated using centrifugal
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concentration units (Pall Life Sciences). Finally, the protein was exchanged into storage buffer
(20 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 1 mM EDTA, 0.5 mM DTT, 50 % (v/v) glycerol) by
dialysis and stored at minus 20 °C.
Wild-type Ec σ70 was purified as previously described14. Ec core enzyme and σ70-associated
holoenzyme were reconstituted in vitro14 and fractionated by anion-exchange chromatography
(Mono Q; GE Healthcare;15). Fractions containing core and σ70-associated enzyme were
pooled separately and stored as described previously15.
Analysis of Alc/RNAP interactions
Reactions (8 μl) containing 0.75 μg Alc and either 0.125 mg core RNAP, or 0.07 μg σ70-
associated holoenzyme in transcription buffer (30 mM Tris-HCl (pH 7.9), 40 mM KCl, 10 mM
MgCl2, 1 mM β-mercaptoethanol) were incubated at room temperature for 10 minutes.
Samples (4 μl of each reaction mixture) were resolved by non-denaturing PAGE on 4-15 %
(w/v) gradient polyacrylamide gel and the bands due to proteins stained with Commassie blue.
To determine the composition of the bands due to proteins separated by gel electrophoresis
under non-denaturing conditions, the bands were excised from the non-denaturing gel,
equilibrated in SDS-PAGE loading buffer, and their composition determined by
electrophoresis on a 10 % (w/v) SDS-PAGE denaturing gel.
Results and Discussion
Noncovalent modifications of Ec RNAP: Immunoisolation and mass spectrometric
identification of T4-encoded proteins that co-isolate with Ec RNAP
To identify T4-encoded Ec RNAP co-isolating proteins, we immunoisolated RNAP from T4-
infected Ec cells (Figure 1, lane 2); isolation was performed using the genomically 2PrA-tagged
β′ subunit of RNAP. The conditions of immunoisolation were sufficiently gentle and rapid to
maintain interactions with other cellular macromolecules8,9,16-19. As a control, parallel
isolation of proteins from T4-infected wild-type cells (untagged β′) was performed. Little or
no protein bands are present in the control, indicating that our conditions are stringent enough
to prevent non-specific association of most Ec and T4 proteins with the immunoaffinity matrix
(Figure 1, lane 1). The material from the tagged strain contained major bands corresponding
to the RNAP subunits (Figure 1, lane 2), indicating that the procedure allowed specific singlestep
purification of RNAP from whole-cell lysates.
Immunoisolated T4-encoded proteins were identified by mass spectrometry (Table 1). Traces
of contaminating proteins that were present in the untagged control sample that non-specifically
associated with the immunoaffinity matrix (lane 1) were computationally filtered out of the
dataset obtained from the tagged strain (lane 2). As is clearly seen in Figure 1, and as confirmed
by MS analysis, all RNAP subunits were present in the immunoisolated (lane 2) but were absent
from the untagged control (lane 1).
Analysis of the immunoisolated material from the tagged strain (lane 2) revealed the presence
of all T4-encoded proteins that are known to associate directly with RNAP: MotA, an activator
of T4 middle promoters20, gp55, a T4-encoded σ factor that controls the expression of T4 late
genes21,22; RpbA, a protein with unknown function; gp33, an activator of T4 late genes that
co-activates T4 late genes with σgp55; 22 and AsiA, a potent inhibitor of host-dependent
transcription23,24 host RNAP appropriator, and coactivator of T4 middle genes20.
We also identified the T4-encoded transcription termination factor Alc. Alc increases the pool
of host RNAP available for transcribing T4 DNA by selectively terminating RNAP
transcription on Ec DNA without affecting transcription of T4 DNA3,4,5. Based on genetic
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studies, Alc is thought to bind directly to the β subunit dispensable region 1 (bDR1) of Ec
RNAP, but no direct interaction between RNAP and Alc had been previously reported5,6.
Since the immunoisolation experiment indicated an Alc/RNAP interaction, we tested this result
using non-denaturing PAGE (Figure 2A). Under our conditions, purified core RNAP migrated
as a diffuse set of bands (lane 1), the σ70-holoenzyme migrated as a single band (lane 3), and
purified Alc migrated with the dye front (lane 5). From the gel it is clear that the bands labeled
c1 and c2 appear to have a slightly lower electrophoretic mobility than the corresponding bands
from the RNAP core sample, suggesting an interaction between core and Alc. With the mixture
containing Alc and σ70-holoenzyme (lane 4), two poorly resolved bands are observed, one
(labeled h2) has the same mobility as the σ70-holoenzyme alone, and another (h1) has a slightly
decreased mobility compared to the σ70-holoenzyme alone.
The labeled bands were excised from the non-denaturing gel and their protein content
established by denaturing SDS-PAGE (Figure 2B). It is apparent that the c1, c2, and h1 bands
contained, in addition to the expected RNAP subunits, a band due to Alc (lanes 4, 5, and 8,
respectively), thus establishing that Alc binds both core and σ70-holoenzyme. The material in
bands c3, c4, and h2 contained little or no Alc (lanes 6, 7, and 9, respectively). The h2 band
contained free σ70-holoenzyme; band c3 contained the RNAP assembly intermediate α2β, and
band c4 contained the free α subunit. Thus, it appears that Alc fails to bind to α2β despite the
fact that the genetically identified determinants for Alc binding are located in the β subunit3.
This result suggests that Alc binding to RNAP may require determinants in both the β and β′
Covalent modifications of Ec RNAP: T4-dependent ADP-ribosylation of the Ec RNAP α
ADP-ribosyltransferases catalyze the transfer of the ADP-ribosyl moiety from substrate ADPribosyl-
nicotinamide onto Arg or His residues of a target protein25. T4 encodes two ADPribosyltransferases,
Alt and ModA, that ADP-ribosylate Arg 265 of the RNAP α
subunit26-30. While ModA specifically modifies α-Arg 26530 of both α subunits, Alt modifies
α-Arg 265 on only one of the two α subunits, multiple sites within Ec RNAP, and other host
proteins31-33. The sites of Alt-dependent ADP-ribosylation within the β, β′, and σ70 subunits
remain to be elucidated. Residue α-Arg 265 is involved in contacting both DNA and transacting
factors34-36, thus, ADP-ribosylation of this key residue alters α/DNA and α/protein
interactions necessary for transcription activation.
To determine the facility of mass spectrometric analysis for elucidating posttranslational
modifications of RNAP, we sought to identify the ModA-dependent ADP-ribosylation of the
Ec α subunit. To do this, tryptic peptides of the α subunit immunoisolated from both uninfected
and T4-infected Ec cells were prepared and analyzed by MALDI MS. Using a subtractive
approach, the resulting mass spectra were scanned for peptides present in the T4-infected
sample, but absent from the uninfected sample. A peak due to a singly charged species at m/z
3838.0 was observed in the T4-infected sample but not the uninfected sample (Figure 3A), and
thus is likely due to T4-dependent modification of the α subunit. The theoretical m/z for a
singly protonated ADP-ribosylated tryptic peptide containing residues 244-271 is 3837.8. The
match of both the theoretical and observed masses suggests that the species at m/z 3838.0 is
due to α residues 244 to 271, encompassing an ADP-ribosyl group. This interpretation is further
corroborated by the missed cleavage at α-Arg 265, suggesting that ADP-ribsoylation of this
Arg residue may block tryptic cleavage.
Interrogation of this peptide ion using MALDI-ion-trap MS2 mass spectrometry yielded a
family of strong peaks corresponding to fragmentation of the ADP-ribosyl modification. Peaks
consistent with the neutral losses of adenosine monophosphate, adenosine diphosphate, and
ADP-ribose, combined with multiple water/ammonia losses were observed (Figure 3B).
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MS3 of a peptide ion at m/z 3237.0 (loss of ADP-ribose + water) yields a fragmentation pattern
consistent with residues 244-271 (Figure 3C). The expected preferential fragmentations
carboxy-terminal to acidic amino acid residues (Asp and Glu) and amino-terminal to Pro
residues are observed37. Our data reveal that mass spectrometry can be used to probe for phagedependent
covalent modifications of host proteins, and should allow the elucidation of those
sites within Ec RNAP that are ADP-ribosylated by T4 Alt.
We have immunoisolated Ec RNAP from T4-infected cells and analyzed the T4-encoded coisolating
proteins using MALDI MS. The T4 phage system has been studied extensively and
T4-encoded proteins that interact with and regulate the activity of host RNAP have been
identified over the years by painstaking genetic and biochemical approaches. Our results show
that optimized RNAP immunoisolations, coupled with mass spectrometric identification, allow
rapid identification of many T4 proteins known to interact with Ec RNAP, as well as the novel
identification of Alc as a binding partner, whose low affinity for RNAP prevented earlier
identification. In addition, this method is also suitable for the identification of phage-dependent
covalent modifications of the host transcription machinery, as demonstrated by our
comprehensive mass spectrometric analysis of an ADP-ribosylated peptide. The ADPribosylation
of proteins is an important process in both prokaryotes and eukaryotes. Indeed,
many bacterial exotoxins are ADP-ribosyltransferases38, and function to manipulate and
override host cellular processes. In eukaryotes, ADP-ribosylation is involved in regulating,
amongst other things, the activity of histones and membrane traffic39,40. Elucidation of the
MS2 and MS3 fragmentation characteristics of ADP-ribosylated peptides should facilitate the
definition of control mechanisms and signaling pathways that are dependent upon ADPribosylation.
Finally, we suggest that this methodology, coupled with phage genomic
sequencing, will become an important tool for probing novel host/phage systems.
We are grateful to Betty Kutter, Sohail Malik, Ian Molineux, and Richard Wolf for many stimulating discussions and
to Malcolm Twist for expert art work. This work was funded by grants from the National Institutes of Health: RR00862
(BTC), U54 RR022220 (MPR and BTC), R01 GM062427 (MPR), and GM61898 (SAD).
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Figure 1.
Immunoisolation of β′-2PrA and co-isolating proteins from T4-infected Ec cells. Complexes
were isolated via a PrA tag under conditions that co-isolated interacting proteins. Proteins were
resolved by denaturing SDS-PAGE, visualized by Coomassie blue staining, and analyzed by
MALDI MS. Lanes are loaded as follows: lane 1, Immunoisolated proteins from Ec wild-type
cells infected with T4; lane 2, immunoisolated proteins from EcrpoC::2PrA cells infected with
T4. RNAP subunits in the 2PrA-tagged sample (lane 2) are labeled.
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Figure 2.
Non-denaturing PAGE and denaturing SDS-PAGE were used to probe the associations of Alc
with the core enzyme and the σ70-holoenzyme. (A) Different combinations of Alc, core, and
σ70-holoenzyme were incubated together and resolved under non-denaturing conditions.
(B) Bands due to the proteins separated by non-denaturing PAGE, labeled in (A), were excised
and their composition revealed by denaturing SDS-PAGE.
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Figure 3.
Identification of T4-dependent ADP-ribosylation of RNAP α subunit Arg 265. Bands due to
the RNAP α subunit isolated from uninfected and T4-infected cells were excised, digested with
trypsin, and analysed by MALDI MS. The resulting mass spectra were aligned and scanned
for peptides present in the T4 infected sample, but absent from the uninfected sample. (A) Mass
spectrum of tryptic peptides due to the RNAP α subunit isolated from uninfected Ec rpoC::
2PrA cells (top panel) and Ec rpoC::2PrA cells infected with T4 (bottom panel). The
appearance of a peak at 3838.0 m/z, with a mass that corresponds to a singly protonated ADPribsoylated
peptide derived from α residues 244 to 271 in the spectrum of the infected phage
that is absent in the uninfected sample spectrum suggests that this peptide is ADP-ribosylated
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in vivo in a T4 dependent manner. (B) The mass spectrum of the MS2 analysis of the species
with an m/z of 3838.0 described in (A) illustrates neutral loss of AMP, ADP and ADP-ribose,
together with various water/ammonia neutral losses. This fragmentation is consistent with the
interpretation that the peptide ion at 3838.0 m/z is indeed an ADP-ribsoylated species. (C)
MS3 analysis of the ions in the MS2 spectrum with a nominal m/z of 3237.0 (corresponding to
the neutral loss of ADP-Ribose +HCN2 +water/ammonia) yielded backbone fragmentation
providing partial sequence of the peptide. The fragmentation pattern is consistent with the
interpretation that the original peptide ion at 3838.0 m/z is indeed derived from α residues 244
to 271. Observed are fragments corresponding to the preferential cleavage of the singlyprotonated
peptide ion carboxy-terminal to acidic residues and amino-terminal to Pro residues.
Together, the MS, MS2, and MS3 spectra in panels (A), (B) and (C) provide strong evidence
that α is ADP-ribosylated on an internal Arg residue within the tryptic peptide encoding
residues 244-271.
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Westblade et al. Page 13
Table 1
MotA 58 23.6 2.5×10−4 1.0×10−11
gp55 70 21.6 6.2×10−8 5.0×10−11
β′-PrA Alc 57 19.2 4.2×10−4 3.7×10−12
RpbA 81 14.8 8.8×10−8 1.8×10−5
gp33 35 12.9 2.5×10−1 6.2×10−1
AsiA 88 10.6 2.6×10−7 2.0×10−11
J Proteome Res. Author manuscript; available in PMC 2008 December 30.

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