Molecular Mechanism of Bacterial Persistence by HipA
Transcript of Molecular Mechanism of Bacterial Persistence by HipA
Please cite this article in press as: Germain et al., Molecular Mechanism of Bacterial Persistence by HipA, Molecular Cell (2013), http://dx.doi.org/10.1016/j.molcel.2013.08.045
Molecular Cell
Short Article
Molecular Mechanismof Bacterial Persistence by HipAElsa Germain,1,2 Daniel Castro-Roa,1,2 Nikolay Zenkin,1,* and Kenn Gerdes1,*1Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Baddiley-Clark Building, Richardson
Road, NE2 4AX Newcastle upon Tyne, UK2These authors contributed equally to this work*Correspondence: [email protected] (N.Z.), [email protected] (K.G.)
http://dx.doi.org/10.1016/j.molcel.2013.08.045
SUMMARY
HipA of Escherichia coli is a eukaryote-like serine-threonine kinase that inhibits cell growth and inducespersistence (multidrug tolerance). Previously, it wasproposed that HipA inhibits cell growth by the phos-phorylation of the essential translation factor EF-Tu.Here, we provide evidence that EF-Tu is not a targetof HipA. Instead, a genetic screen reveals that theoverexpression of glutamyl-tRNA synthetase (GltX)suppresses the toxicity of HipA. We show that HipAphosphorylates conserved Ser239 near the activecenter of GltX and inhibits aminoacylation, a uniqueexample of an aminoacyl-tRNA synthetase beinginhibited by a toxin encoded by a toxin-antitoxinlocus. HipA only phosphorylates tRNAGlu-boundGltX, which is consistent with the earlier finding thatthe regulatory motif containing Ser239 changesconfiguration upon tRNA binding. These results indi-cate that HipA mediates persistence by the genera-tion of ‘‘hungry’’ codons at the ribosomal A site thattrigger the synthesis of (p)ppGpp, a hypothesis thatwe verify experimentally.
INTRODUCTION
Bacteria can enter a physiological state, called persistence or
multidrug tolerance, in which lethal antibiotics do not kill them
(Bigger, 1944). Persistence is a phenotype expressed by almost
all bacteria, including major pathogens, and is believed to
contribute to the intractability of chronic and relapsing infections
(Levin and Rozen, 2006; Lewis, 2010). Experiments employing
E. coli as the model organism indicated that persister cells
form stochastically by switching into and out of slow growth
(Balaban et al., 2004), a phenotype that was argued to be advan-
tageous in changing environments (Kussell et al., 2005). Impor-
tantly, descendants of persister cells are as sensitive as their
ancestors toward the bactericidal antibiotic used, demonstrating
that bacterial persistence is a noninherited epigenetic character-
istic (Lewis, 2010).
The isolation of high-persistence (hip) mutants in E. coli indi-
cated that persistence can have a genetic basis (Moyed andBer-
trand, 1983). E. coli cells carrying hipA7, the most thoroughly
analyzed hip mutant, exhibited a dramatic 100- to 1,000-fold in-
crease in persistence (Korch et al., 2003). A low level of induction
of hipA induced a bacteriostatic condition that could be counter-
acted by HipB encoded by the gene just upstream to hipA (Korch
and Hill, 2006). Moreover, HipA and HipB formed a tight complex
that autoregulated the transcription of the hipBA operon (Black
et al., 1994). Due to these observations, it was suggested that
hipBA constitutes a type II toxin-antitoxin (TA) locus (Korch
et al., 2003), a suggestion that was later confirmed experimen-
tally (Korch and Hill, 2006; Schumacher et al., 2009).
Most TA loci encode two components, a toxin whose ectopic
production inhibits cell growth and an antitoxin (RNA or protein)
that counteracts toxin expression or activity (Blower et al., 2011;
Gerdes and Maisonneuve, 2012). In type II TA loci, which are
almost ubiquitous in bacteria and archaea, the antitoxins are
proteins that interact with and neutralize the activity of the toxins
(Gerdes et al., 2005). Most type II TA loci encode inhibitors of
translation whose ectopic expression induces a static, drug-
tolerant condition from which the cells can be resuscitated by
the induction of the antitoxin-encoding gene, consistent with
a role in persistence (Correia et al., 2006; Han et al., 2010;
Maisonneuve et al., 2011; Pedersen et al., 2002; Singh et al.,
2010; Vazquez-Laslop et al., 2006). E. coli has 11 canonical
type II TA loci, ten of which encode toxins that inhibit transla-
tion by mRNA cleavage (Gerdes and Maisonneuve, 2012).
Many type II TA operon mRNAs were significantly increased in
persister cells (Keren et al., 2004; Shah et al., 2006), and the
progressive deletion of these TA loci led to a gradual reduction
in persistence, convincingly arguing that there is a causal
connection between type II TA loci and persistence (Maison-
neuve et al., 2011).
Because hipA of E. coli was the first ‘‘persister’’ gene to be
discovered, it has been of considerable interest in understanding
how HipA inhibits cell growth and confers persistence. The
ectopic induction of hipA induced growth arrest and strongly in-
hibited replication, transcription, and translation (Korch and Hill,
2006). Interestingly, HipA exhibits a eukaryotic serine-threonine
kinase-like fold and has kinase activity (Correia et al., 2006;
Schumacher et al., 2009). On the basis of in vitro experiments,
it was suggested that HipA inhibits translation by the phosphor-
ylation of EF-Tu (Schumacher et al., 2009). However, such phos-
phorylation did not explain the strong inhibition of replication and
transcription seen after the induction of hipA (Korch and Hill,
2006; Schumacher et al., 2009). Moreover, hipA induction
Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc. 1
Figure 1. HipA Does Not Inhibit Translation by the Phosphorylation
of EF-Tu
(A) Translation in vitro with an S30 extract without HipA (lanes 1–3) or with HipA
(lanes 4–6). 0.1 mMHipA was incubated with the S30 extract for 10 min before
the reaction was started by the addition of a DNA-template-encoding lucif-
erase.
(B) A scheme of in vitro translation system reconstituted from purified
components and stages 0.6 mM when HipA was added to the system: during
the initiation step (blue), before formation of the ternary complex (EF-
Tu,GTP,Phe-tRNAPhe) (purple), and to the preformed ternary complex (red).
Bottom, the synthesis ofMet-Phe dipeptide in the absence or presence ofHipA
added during the different stages explained above (lanes 3–5). As a control, the
same experiment was performed by adding 0.2 mMEF-Tu kinase Doc (Castro-
Roa et al., 2013); shown are no Doc added (lane 6) and Doc added before
ternary complex formation (lane 7). Dipeptides were analyzed by thin-layer
electrophoresis and autoradiography (Castro-Roa and Zenkin, 2012).
(C) Analysis of purified GST-EF-Tu (0.13 mM) after incubation for 45 min with
(0.1 mM) HipA and 0.1 mM g[32P]ATP by SDS-PAGE (left) and autoradiography
(right). Only the autophosphorylation of HipA was observed (lanes 7–9) in
comparison to the positive control with GST-EF-Tu phosphorylated by Doc
kinase (lane 11). Experiments were reproduced at least three times.
See also Figure S1.
Molecular Cell
HipA Inhibits Glutamyl-tRNA Synthetase
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stimulated RelA-dependent synthesis of (p)ppGpp (Bokinsky
et al., 2013), thus raising the possibility that HipA has additional
cellular targets.
Here, we re-examine the molecular mechanism underlying
HipA-induced persistence and show that the target of HipA is
glutamyl-tRNA synthetase (GltX), which is inactivated by phos-
phorylation by HipA. Our results explain previous enigmatic
physiological effects seen after the induction of hipA.
RESULTS
HipA Does Not Inhibit Translation by thePhosphorylation of EF-TuHipA inactivates itself by autophosphorylation (Correia et al.,
2006), which does not occur in the presence of HipB (Evdokimov
et al., 2009). Therefore, we purified HipA in complex with HipB,
as described previously (Christensen-Dalsgaard et al., 2008).
After purification, MALDI-TOF mass spectrometry analysis
revealed that more than 65% of the HipA molecules were non-
phosphorylated (Figure S1A).
We tested whether HipA could affect translation in vitro. As
seen in Figure 1A, HipA efficiently inhibited the synthesis of lucif-
erase in a cell-free translation system on the basis of a crude S30
cell extract. To identify the natural target of HipA, we used an
in vitro translation system assembled from purified components
(Castro-Roa and Zenkin, 2012). The mRNA used in this system
encoded the dipeptide Met-Phe (MF) in its initial sequence.
Translation was initiated by the addition of purified ribosomes,
initiation factors IF-1, IF-2, IF-3, and [35S]-fMet-tRNAfMet. Then,
initiated ribosomes were allowed to elongate by one codon via
the addition of preformed ternary complex EF-Tu:GTP:Phe-
tRNAPhe and the elongation factor EF-G in the presence of
GTP. The short peptide products of the reaction were analyzed
by thin layer electrophoresis (Castro-Roa and Zenkin, 2012;
Zaher and Green, 2009). HipA was added to the reactions at
three different stages: (1) during initiation complex formation
(blue in Figure 1B), (2) before ternary complex formation (purple
in Figure 1B), and (3) to the preformed ternary complex (TC)
Figure 2. GltX Counteracts HipA Toxicity, and HipA-Induced Persis-
tence Depends on (p)ppGpp
(A) Strains MG1655DhipAB harboring pBAD30 (bla, vector plasmid carrying an
arabinose-inducible promoter) or pEG3 (pBAD30::hipA) were transformedwith
pCA24N (cat, vector plasmid carrying an IPTG-inducible promoter) or
pCA24N::gltX. The resulting four strains were plated on nutrient agar plates
containing ampicillin (50 mg/ml), chloramphenicol (50 mg/ml), and arabinose
(0.2%) without (left) or with (right) 50 mM IPTG, which induced gltX. As seen, the
presence of the gltX-encoding plasmid suppressed HipA toxicity with and
without IPTG (due to a slight leakiness of the IPTG-inducible promoter on the
high-copy plasmid); as expected, the suppression of HipA toxicity was
stronger with gltX induction (+IPTG).
(B) Growth curves of MG1655DhipBA containing the plasmids are indicated.
Overnight cultures were diluted 1,000-fold in fresh rich medium with ampicillin
(50 mg/ml), chloramphenicol (50 mg/ml), and 100 mMof IPTG (in order to induce
gltX) and incubated at 37�C. The arrow indicates that hipA was induced at
OD600 = 0.2 via the addition of 0.2% arabinose.
(C) Levels of hipA induced persistence in WT, DrelA, and D(relA spoT) strains.
Exponentially growing cultures of MG1655 and its relA and relA spoT
deletion derivatives containing pBAD30 control plasmid (�) or pEG4
(pBAD30::hipA) (+) were exposed to 2 mg/ml of ciprofloxacin (OD600 z0.5).
The transcription of hipA was induced for 30 min before the addition of
ciprofloxacin (t = 0). This panel shows the percentage of survival after 5 hr
of antibiotic treatment (log scale). The bars show the averages of at least
three independents experiments, and error bars indicate SD. The difference
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(red in Figure 1B). As a positive control, we used Doc, a kinase
that inhibits translation by the phosphorylation of EF-Tu
(Castro-Roa et al., 2013) (Figure 1B). Surprisingly, although
Doc efficiently inhibited the formation of the MF dipeptide (Fig-
ure 1B, lane 7), HipA had no effect on the reaction in any of the
setups (Figure 1B, lanes 3–5).
The above result contradicts the previously proposedmodel in
which HipA inactivates translation by the phosphorylation of EF-
Tu (Schumacher et al., 2009). Furthermore, to test this earlier
model, we analyzed the phosphorylation of EF-Tu by HipA using
g[32P]ATP. Given that HipA and EF-Tu migrate similarly in SDS-
PAGE, we used a functional GST-tagged version of EF-Tu to
improve separation (Perla-Kajan et al., 2010). As seen from Fig-
ure 1C, no phosphorylation of GST-EF-Tu by HipAwas observed
(Figure 1C, lane 9). In contrast, GST-EF-Tu was efficiently phos-
phorylatedby theDockinase (Castro-Roaet al., 2013) (Figure 1C,
lane 11). Next, we decided to follow the phosphorylation state of
EF-Tu in vivo. We simultaneously overproduced HipA and EF-Tu
(in a strain lacking hipBA in order to avoid the neutralization of
HipA by the endogenous antitoxin HipB). Then, EF-Tu purified
before or after HipA overproduction was analyzed bymass spec-
trometry. As seen from Figure S1B (available online), we did not
detect phosphorylation of EF-Tu. Altogether, our results show
that HipA does not phosphorylate EF-Tu and that EF-Tu is not
the target of HipA during the inactivation of translation.
Overproduction of Glutamyl-tRNA SynthetaseCounteracts HipATo investigate the molecular mechanism of HipA-mediated inhi-
bition of cell growth, we selected for genes that, in multiple
copies, would suppress HipA toxicity. We pooled a collection
of plasmids obtained from the ASKA library containing most of
the 4,120 E. coli genes, each cloned into the high-copy-number
vector pCA24N downstream of the isopropyl b-D-1-thiogalacto-
pyranoside (IPTG)-inducible PT5-lac promoter (Kitagawa et al.,
2005). As described in the Experimental Procedures and Supple-
mental Information, we found only one plasmid, pCA24N::gltX,
that suppressed HipA-mediated growth inhibition (Figures 2A
and 2B). The gltX gene encodes glutamyl-tRNA synthetase
(Kern and Lapointe, 1979), a type IB tRNA charging enzyme
(Eriani et al., 1990). Our result raised the possibility that HipA
inhibits translation by targeting GltX. Genes encoding EF-Tu
(tufA or tufB) or other tRNA synthetases were not found in this
genetic screen, consistent with the observation that none of
them, when tested individually, counteracted HipA toxicity (Fig-
ures S2A and S2B).
HipA Phosphorylates GltX In Vivo at Ser239
To test the possibility that HipA phosphorylates GltX, we purified
GltX from a HipA-overproducing strain (see above) before and
after hipA induction and analyzed the samples with MALDI-
TOF mass spectrometry. As seen in Figure S3A, the induction
of hipA increased the molecular weight (MW) of purified GltX
by 80 Da, which corresponds to the substitution of hydrogen
between DrelA and D(relA spoT) strains was not significant (Student’s t test;
p = 0.2; n = 3).
See also Figure S2.
Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc. 3
Figure 3. Phosphorylation of GltX In Vitro by HipA at Ser239 Requires
tRNAGlu
(A) The phosphorylation of GltX in vitro. 6 mM GltX, 0.1 mM g[32P]ATP, and
66 mMATP were incubated with or without 0.2 mMHipA, 1.6 mMGlu, or 1.5 mM
tRNAGlu for 45min. In reactions where HipAwas present, HipAwas added first.
(B) Structures of the conserved KLSKR motif containing Ser239 phosphory-
lated by HipA. Shown are the ATP-bound form (green; PDB 1N75) and the
ATP-Glu-tRNAGlu bound form (blue; PDB 1N77) of GltX. Rearrangement upon
tRNAGlu binding is shown with a line. The numbering corresponds to amino
acids of E. coli GltX.
(C) In vitro aminoacylation activity of GltXWT, GltX-P, and GltXS239D. Amino-
acylation was tested in vitro in a reaction mixture containing 2 mMATP, 0.6 mM
GltX (WT, HipA treated or GltXS239D), 0.2 mg/ml tRNA, 100 mM glutamate cold,
and [3H]-Glu (240 counts min�1 pmol�1) and, for experiments where GltX was
phosphorylated, 0.6 mMHipA was used. The reaction was performed for 3 min
at 37�C and quenched by precipitation in 5% TCA (Francklyn et al., 2008;
Kern and Lapointe, 1981). Error bars indicate the SD of three independent
experiments.
(D) GltXS239D was not phosphorylated by HipA in vitro. 6 mMGltX, 0.1 mM g[32P]
ATP, and 66 mM ATP were incubated with or without 0.2 mMHipA, 1.6 mMGlu,
or 1.5 mM tRNAGlu for 45 min. In reactions where HipA was present, HipA was
added first.
See also Figure S3.
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Please cite this article in press as: Germain et al., Molecular Mechanism of Bacterial Persistence by HipA, Molecular Cell (2013), http://dx.doi.org/10.1016/j.molcel.2013.08.045
with a H2PO3- group. These results suggested that GltX was
phosphorylated by HipA in vivo. Liquid chromatography tandem
mass spectrometry (LC-MS/MS) analysis revealed that GltX was
indeed phosphorylated at Ser239 (Figure S3B).
Next, we analyzed the phosphorylation of GltX by HipA in vitro
by using g[32P]-ATP (Figure 3A). Components were mixed prior
to the addition of ATP in order to avoid the autophosphorylation
of HipA (it was still autophosphorylated after the reaction with
GltX was over; Figure 3A, compare lane 3 to lane 7). Non-ra-
dio-labeled ATP was added in order to suppress the hydrolysis
of g[32P]-ATP by GltX. Surprisingly, negligible phosphorylation
of GltX was observed (Figure 3A, lane 4). Ser239 is a part of the
highly conserved flexible loop that changes conformation upon
tRNAGlu binding (but not upon the binding of either Glu or ATP)
(Sekine et al., 2003). We hypothesized that the phosphorylation
of GltX by HipA depended on this conformational change.
To test this hypothesis, we compared the phosphorylation
of GltX:ATP, GltX:ATP:Glu, GltX:ATP:tRNAGlu, and GltX:ATP:
tRNAGlu:Glu complexes. In accord with our proposal, the phos-
phorylation of GltX took place only when tRNAGlu was present
in the reaction (Figure 3A, lanes 5–7).Consistently, as seen
from the crystal structures, Ser239, which is shielded by several
positively charged residues in the free GltX, becomes more
exposed upon tRNAGlu binding (Figure 3B). Note that only cata-
lytic amounts of HipA were required for GltX phosphorylation,
consistent with the high toxicity of the protein.
HipA-Dependent Phosphorylation of GltX Inhibits ItsAminoacylation ActivityWe tested whether HipA-mediated phosphorylation inhibited
GltX catalysis of tRNA aminoacylation in vitro. Aminoacylation
reactions containing saturating concentrations of ATP and
[3H]-glutamate to follow the charging of tRNAGlu were assembled
with native GltX and HipA-treated GltX (phosphorylated form),
respectively. The resulting [3H]-Glu-tRNAGlu was precipitated
on filter discs presoaked in 5% trichloroacetic acid. The discs
were thoroughly washed in order to remove free [3H]-Glu,
Figure 4. Molecular Model Explaining HipA-Induced (p)ppGpp Syn-
thesis and Persistence
(A) HipA is absent (or inactivated by HipB), glutamyl tRNA synthetase is active,
and translation proceeds normally.
(B) HipA is active, GltX is inhibited by phosphorylation, and, therefore, un-
charged tRNAGlu accumulates. Uncharged tRNAGlu loads at empty ribosomal
A sites (‘‘hungry’’ codons) that trigger the activation and release of RelA. The (p)
ppGpp level increases and the stringent response is mounted. The model
explains the highly pleiotropic cellular effects observed after hipA induction.
HipA, pink; GltX, yellow; phosphate group, red; ribosome, gray. Charged and
uncharged tRNAs are shown as sticks with or without filled circles, respec-
tively. mRNA is shown as a wavy line. E, P, and A symbolize the ribosomal
tRNA binding sites.
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desalted, and dried, and the bound [3H]-Glu (as a measure of
aminoacylation) was assessed by scintillation counting. As
seen in Figure 3C, native GltX was able to aminoacylate tRNAGlu.
However, when treated with HipA, the aminoacylation efficiency
of GltX was dramatically reduced, suggesting that HipA inhibited
GltX aminoacyl-tRNA synthetase activity (Figure 3C). These re-
sults were also obtained qualitatively by thin-layer chromatog-
raphy of the glutamate released after hydrolysis of the ester
bond linking it to the tRNAGlu (Figure S3C). This result is consis-
tent with the position of Ser239 in the loop that forms part of the
active center of GltX (Figure 3B; see the Discussion). To investi-
gate the effect of phosphorylation at Ser239 further, we con-
structed a GltX mutant, which had Ser239, substituted with
aspartate. Aspartate in this position would mimic phosphory-
lated Ser. The mutant GltXS239D was not phosphorylated by
HipA in vitro (Figure 3D), suggesting that Ser239 was the sole
site of phosphorylation by HipA. Furthermore, this mutant was
inactive in the aminoacylation of tRNAGlu (Figure 3C), supporting
the idea that the negative charge of aspartate at the critical
Ser239 is incompatible with efficient catalytic activity of GltX.
HipA-Induced Persistence Depends on (p)ppGppHipA overproduction inhibits cell growth and simultaneously
induces a high level of persistence (Korch and Hill, 2006). The
inhibition of Glu-tRNAGlu caused by HipA would result in an
increased concentration of uncharged tRNAGlu loading at the A
site of the ribosome and, as a result, in synthesis of the alarmone
(p)ppGpp by RelA (Bokinsky et al., 2013). Such a scenario would
not be expected if EF-Tu was a target of HipA. We tested
whether HipA-mediated persistence depended on RelA and/or
SpoT, the two (p)ppGpp synthetases of E. coli. As expected,
the overproduction of HipA increased the persistence of the
wild-type (WT) strain by 120-fold (Figure 2C). The deletion of
relA or relA and spoT resulted in a dramatic reduction of HipA-
mediated persistence (increases of only 4- and 2-fold, respec-
tively). We suggest that HipA-induced persistence is mediated
by (p)ppGpp. Therefore, the result supports our model in which
HipA inactivates GltX, and thus generates, ‘‘hungry’’ codons
that trigger (p)ppGpp production.
DISCUSSION
HipA of E. coli is a kinase with a serine-threonine kinase fold
(Correia et al., 2006) that has been proposed to inhibit translation
by the phosphorylation of the essential translation factor EF-Tu
(Schumacher et al., 2009). However, our results do not support
this model. Instead, we present strong evidence that HipA in-
hibits GltX by the phosphorylation of Ser239. Ser239 belongs to
the conserved KLSKR motif, which is the characteristic
sequence motif of ATP-binding sites of type I aminoacyl-tRNA
synthetases (Sekine et al., 2003). This motif forms a loop that
participates in the binding of the catalytic ATP, which, in the
absence of tRNAGlu, binds in a catalytically inactive configura-
tion. The binding of tRNAGlu changes the conformation of the
KLSKR motif, which allows the a-phosphate of ATP to align for
a nucleophilic attack by Glu. The conformational change of the
KLSKR motif makes Ser239 more exposed, which is consistent
with our observation that HipA can transfer phosphate to
Ser239 only when GltX is in complex with tRNAGlu. The critical
role of the KLSKR motif in the regulation of the formation of ami-
noacyl-adenylate explains how the phosphorylation of Ser239 (or
a negative charge of Asp in this position) may lead to the catalytic
inactivation of GltX.
The pleiotropic physiological effects of HipA expression was
not clearly explained by previous models. Below, we discuss a
molecular model explaining cellular consequences of the inhibi-
tion of GltX by HipA. In the absence of HipA, GltX is active, trans-
lation proceeds normally, and RelA is sequestered via its binding
to the ribosome (Figure 4A). When HipA is present and active,
GltX is inhibited by phosphorylation with the consequence that
uncharged tRNAGlu accumulates. This, in turn, increases the
frequency of hungry A site codons (Figure 4B). Consequently,
Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc. 5
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uncharged tRNAGlu enters the ribosomal A site and triggers the
activation and release of RelA (English et al., 2011). RelA activa-
tion leads to an increased (p)ppGpp level that conjures the
inhibition of translation, transcription, replication, and cell-wall
synthesis, thereby leading to slow growth, multidrug tolerance,
and persistence (Magnusson et al., 2005; Maisonneuve et al.,
2013; Srivatsan and Wang, 2008). The fact that hipA induction
leads to a dramatic increase in [(p)ppGpp] was shown recently
by Bokinsky et al. (2013), and the mechanism behind this obser-
vation is now explained by our model.
Recently, eukaryotic Lys-tRNA synthetase was shown to
switch its function upon its phosphorylation in response to envi-
ronmental stimulus (Ofir-Birin et al., 2013). The nonphosphory-
lated form acts as a common tRNA synthetase to aminoacylate
tRNALys, whereas the phosphorylated one is transported to the
nucleus, where it acts as a transcription factor. This raises the
intriguing possibility that the phosphorylation of GltX may also
switch the function of the enzyme from aminoacylation. Whether
there is a switch between functions or whether phosphorylation
just inactivates GltX, the ability of cells to revive from HipA-
induced dormancy suggests the existence of a phosphatase
that would dephosphorylate GltX. Additional investigations are
required in order to test these hypotheses.
EXPERIMENTAL PROCEDURES
Bacterial strains and plasmids are listed in Table S1. DNA oligonucleotides are
listed in Table S2. For more details, see the Supplemental Experimental
Procedures.
Media and Antibiotics
Luria-Bertani broth was prepared as described (Clark andMaalø, 1967). When
required, the medium was supplemented with 50 mg/ml ampicillin or 50 mg/ml
chloramphenicol. Expression of protein from plasmids carrying pBAD pro-
moter was induced by 0.2% arabinose and repressed by 0.2% glucose.
Multicopy Suppression of HipA Toxicity by the Overproduction of
Glutamyl-tRNA Synthetase
A pooling mixture of the ASKA plasmid library (Kitagawa et al., 2005)
was transformed into a hipBA deletion strain harboring plasmid pEG3
(pBAD30::hipA) containing an arabinose-inducible promoter. In order to
induce the expression of ASKA plasmid-encoded genes, plasmids that
produced colonies in the presence of IPTG were analyzed.
HipA and Doc Purification
HipA was purified in complex with HipB as described in Christensen-Dals-
gaard et al. (2008) with a few modifications. The entire protocol is described
in the Supplemental Experimental Procedures. Doc kinase was purified as
described previously (Garcia-Pino et al., 2010).
EF-Tu and GltX Purification
Overexpressed EF-Tu or GltXwere purified before or after the induction of hipA
with Ni-NTA affinity chromatography as described in detail in the Supple-
mental Experimental Procedures.
Phosphorylation of EF-Tu In Vitro
HipA (0.1 mM) phosphorylation was performed in the presence or absence of
0.13 mM EF-Tu and 0.1 mM (unless otherwise specified) of g[32P]ATP (3,000
Ci/mmol; Hartmann Analytic) in a 30 ml final volume of ternary complex buffer
(50mMTris-HCl [pH 7.4], 40mMNH4Cl, 10mMMgCl2, and 1mMdithiothreitol
(DTT)) for 45 min. The reaction was stopped by the addition of 1 vol Laemmli
loading buffer, resolved by SDS-PAGE, and revealed by phosphorimaging
(GE Healthcare).
6 Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc.
Phosphorylation of GltX In Vitro
GltX (6 mM) was mixed in aminoacylation buffer (1 mM DTT, 10 mM KCl, 16 mM
ZnSO4, and 20 mM MgCl2) with 0.2 mM HipA, 66 mM ATP (nonradioactive),
0.1 mM g[32P]ATP, 1.5 mM tRNAGlu, and 1.6 mM glutamic acid. The reaction
was incubated at 37�C for 45min and was stopped by the addition of 1 volume
of Laemmli buffer, resolved by SDS-PAGE, and revealed by phosphorimaging
(GE Healthcare).
Translation In Vitro with Cell-Free Extract
In vitro translation was performed with an S30 kit (Promega) following the
manufacturer’s guidelines, the exception being that 16 mg/ml total tRNA
and 1 mM ATP with or without 0.1 mM HipA were added and incubated at
37�C for 10 min before the plasmid pBESTluc was added for the times indi-
cated in Figure 1A.
Preparation of Ternary Complexes for Translation In Vitro System
Translation in vitro assembled from purified components was performed, and
products were analyzed exactly as described in Castro-Roa and Zenkin (2012)
(see the Supplemental Information for full details). HipA (0.6 mM) or Doc
(0.2 mM) were added to the ternary complex or initiation complex formation
reactions as described in Figure 1B.
Aminoacylation Assay
For quantitatively testing aminoacylation of tRNAGlu by WT GltX, phosphory-
lated GltX by HipA or GltX phosphomutant, an aminoacylation reaction
(100 ml) containing aminoacylation buffer 13, 2 mM ATP, 0.6 mM GltX (WT,
HipA-treated or GltXS239D), 0.2 mg/ml tRNA, 100 mM glutamate cold, and
[3H]-Glu (240 counts min�1 pmol�1) and, for experiments where GltX was
phosphorylated, 0.6 mM HipA was added, and the reaction was initiated by
the addition of amino acid (Kern and Lapointe, 1981). The aminoacylation
reaction was initiated upon the addition of the enzyme (for WT and GltXS239D)
and incubated for 3 min at 37�C. The reaction was terminated by spotting 10 ml
on Whatmann 3 MM Chr filter paper presoaked in 5% TCA. The filter was
immediately immersed in 15 ml of ice-cold 5% TCA for 15 min. After three
washings of 15 ml of 5% TCA with incubation for 15 min in order to remove
free glutamic acid, the filters were desalted in 95%ethanol for 20min and dried
overnight, and the remaining radioactivity was measured with a scintillation
counter (Francklyn et al., 2008).
Measurement of Persistence
Persistence was measured as described previously (Maisonneuve et al., 2011)
and in more detail in the Supplemental Experimental Procedures.
Mass Spectrometry Analysis
MALDI TOF analysis was performed as described in the Supplemental Exper-
imental Procedures. LC MS/MS analysis was performed on purified GltX after
HipA overproduction in DhipBA strain (additional information is provided in the
Supplemental Experimental Procedures).
SUPPLEMENTAL INFORMATION
Supplemental Information contains Supplemental Experimental Procedures,
three figures, and two tables and can be found with this article online at
http://dx.doi.org/10.1016/j.molcel.2013.08.045.
ACKNOWLEDGMENTS
We thank members of the Gerdes and Zenkin groups for stimulating discus-
sions. We also thank Joe Gray for help with mass spectrometry analysis,
Abel Garcia-Pino and Remy Loris for purified Doc, Loranne Agius for scintilla-
tion counter and Charlotte R.Knudsen for pGEX-FX-tufA plasmid coding for
GST-EF-Tu. This work was funded by a European Research Council Advanced
Investigator Grant [294517, ‘‘PERSIST’’] to K.G., a European Research Council
Starting Grant (202994, ‘‘MTP’’), and a UK Biotechnology and Biological Sci-
ences Research Council Grant to N.Z.
Molecular Cell
HipA Inhibits Glutamyl-tRNA Synthetase
Please cite this article in press as: Germain et al., Molecular Mechanism of Bacterial Persistence by HipA, Molecular Cell (2013), http://dx.doi.org/10.1016/j.molcel.2013.08.045
Received: June 14, 2013
Revised: July 29, 2013
Accepted: August 22, 2013
Published: October 3, 2013
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