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

mailto:[email protected]

mailto:[email protected]


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

2 Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc.


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

Molecular Cell



(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.


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.

Molecular Cell




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.

Molecular Cell



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



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).


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



Received: June 14, 2013

Revised: July 29, 2013

Accepted: August 22, 2013

Published: October 3, 2013

REFERENCES

Balaban, N.Q., Merrin, J., Chait, R., Kowalik, L., and Leibler, S. (2004).

Bacterial persistence as a phenotypic switch. Science 305, 1622–1625.

Bigger, J.W. (1944). Treatment of staphylococcal infections with penicillin by

intermittent sterilisation. Lancet 294, 497–500.

Black, D.S., Irwin, B., andMoyed, H.S. (1994). Autoregulation of hip, an operon

that affects lethality due to inhibition of peptidoglycan or DNA synthesis.

J. Bacteriol. 176, 4081–4091.

Blower, T.R., Salmond, G.P., and Luisi, B.F. (2011). Balancing at survival’s

edge: the structure and adaptive benefits of prokaryotic toxin-antitoxin part-

ners. Curr. Opin. Struct. Biol. 21, 109–118.

Bokinsky, G., Baidoo, E.E., Akella, S., Burd, H., Weaver, D., Alonso-Gutierrez,

J., Garcıa-Martın, H., Lee, T.S., and Keasling, J.D. (2013). HipA-triggered

growth arrest and b-lactam tolerance in Escherichia coli are mediated by

RelA-dependent ppGpp synthesis. J. Bacteriol. 195, 3173–3182.

Castro-Roa, D., and Zenkin, N. (2012). In vitro experimental system for analysis

of transcription-translation coupling. Nucleic Acids Res. 40, e45.

Castro-Roa, D., Garcia-Pino, A., van Nuland, N.A.J., Loris, R., and Zenkin, N.

(2013). The Fic protein Doc uses an inverted substrate to phosphorylate

and inactivate EF-Tu. Nat. Chem. Biol., in press. http://dx.doi.org/10.1038/

nchembio.1364.

Christensen-Dalsgaard, M., Overgaard, M., Winther, K.S., and Gerdes, K.

(2008). RNA decay by messenger RNA interferases. Methods Enzymol. 447,

521–535.

Clark, D.J., and Maalø, O. (1967). DNA replication and the division cycle in

Escherichia coli. J. Mol. Biol. 23, 99–112.

Correia, F.F., D’Onofrio, A., Rejtar, T., Li, L., Karger, B.L., Makarova, K.,

Koonin, E.V., and Lewis, K. (2006). Kinase activity of overexpressed HipA is

required for growth arrest and multidrug tolerance in Escherichia coli.

J. Bacteriol. 188, 8360–8367.

English, B.P., Hauryliuk, V., Sanamrad, A., Tankov, S., Dekker, N.H., and Elf, J.

(2011). Single-molecule investigations of the stringent response machinery in

living bacterial cells. Proc. Natl. Acad. Sci. USA 108, E365–E373.

Eriani, G., Delarue, M., Poch, O., Gangloff, J., andMoras, D. (1990). Partition of

tRNA synthetases into two classes based on mutually exclusive sets of

sequence motifs. Nature 347, 203–206.

Evdokimov, A., Voznesensky, I., Fennell, K., Anderson, M., Smith, J.F., and

Fisher, D.A. (2009). New kinase regulation mechanism found in HipBA: a bac-

terial persistence switch. Acta Crystallogr. D Biol. Crystallogr. 65, 875–879.

Francklyn, C.S., First, E.A., Perona, J.J., and Hou, Y.M. (2008). Methods for

kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases.

Methods 44, 100–118.

Garcia-Pino, A., Balasubramanian, S., Wyns, L., Gazit, E., De Greve, H.,

Magnuson, R.D., Charlier, D., van Nuland, N.A., and Loris, R. (2010).

Allostery and intrinsic disorder mediate transcription regulation by conditional

cooperativity. Cell 142, 101–111.

Gerdes, K., and Maisonneuve, E. (2012). Bacterial persistence and toxin-anti-

toxin loci. Annu. Rev. Microbiol. 66, 103–123.

Gerdes, K., Christensen, S.K., and Løbner-Olesen, A. (2005). Prokaryotic

toxin-antitoxin stress response loci. Nat. Rev. Microbiol. 3, 371–382.

Han, J.S., Lee, J.J., Anandan, T., Zeng, M., Sripathi, S., Jahng, W.J., Lee, S.H.,

Suh, J.W., and Kang, C.M. (2010). Characterization of a chromosomal toxin-

antitoxin, Rv1102c-Rv1103c system in Mycobacterium tuberculosis.

Biochem. Biophys. Res. Commun. 400, 293–298.

Keren, I., Shah, D., Spoering, A., Kaldalu, N., and Lewis, K. (2004). Specialized

persister cells and the mechanism of multidrug tolerance in Escherichia coli.

J. Bacteriol. 186, 8172–8180.

Kern, D., and Lapointe, J. (1979). Glutamyl transfer ribonucleic acid synthetase

of Escherichia coli. Study of the interactions with its substrates. Biochemistry

18, 5809–5818.

Kern, D., and Lapointe, J. (1981). The catalytic mechanism of glutamyl-tRNA

synthetase of Escherichia coli. A steady-state kinetic investigation. Eur. J.

Biochem. 115, 29–38.

Kitagawa, M., Ara, T., Arifuzzaman, M., Ioka-Nakamichi, T., Inamoto, E.,

Toyonaga, H., and Mori, H. (2005). Complete set of ORF clones of

Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive):

unique resources for biological research. DNA Res. 12, 291–299.

Korch, S.B., and Hill, T.M. (2006). Ectopic overexpression of wild-type and

mutant hipA genes in Escherichia coli: effects on macromolecular synthesis

and persister formation. J. Bacteriol. 188, 3826–3836.

Korch, S.B., Henderson, T.A., and Hill, T.M. (2003). Characterization of the

hipA7 allele of Escherichia coli and evidence that high persistence is governed

by (p)ppGpp synthesis. Mol. Microbiol. 50, 1199–1213.

Kussell, E., Kishony, R., Balaban, N.Q., and Leibler, S. (2005). Bacterial persis-

tence: amodel of survival in changing environments.Genetics 169, 1807–1814.

Levin, B.R., and Rozen, D.E. (2006). Non-inherited antibiotic resistance. Nat.

Rev. Microbiol. 4, 556–562.

Lewis, K. (2010). Persister cells. Annu. Rev. Microbiol. 64, 357–372.

Magnusson, L.U., Farewell, A., and Nystrom, T. (2005). ppGpp: a global regu-

lator in Escherichia coli. Trends Microbiol. 13, 236–242.

Maisonneuve, E., Shakespeare, L.J., Jørgensen, M.G., and Gerdes, K. (2011).

Bacterial persistence by RNA endonucleases. Proc. Natl. Acad. Sci. USA 108,

13206–13211.

Maisonneuve, E., Castro-Camargo, M., and Gerdes, K. (2013). (p)ppGpp

Controls Bacterial Persistence by Stochastic Induction of Toxin-Antitoxin

Activity. Cell 154, 1140–1150.

Moyed, H.S., and Bertrand, K.P. (1983). hipA, a newly recognized gene of

Escherichia coli K-12 that affects frequency of persistence after inhibition of

murein synthesis. J. Bacteriol. 155, 768–775.

Ofir-Birin, Y., Fang, P., Bennett, S.P., Zhang, H.M., Wang, J., Rachmin, I.,

Shapiro, R., Song, J., Dagan, A., Pozo, J., et al. (2013). Structural switch of

lysyl-tRNA synthetase between translation and transcription. Mol. Cell 49,

30–42.

Pedersen, K., Christensen, S.K., and Gerdes, K. (2002). Rapid induction and

reversal of a bacteriostatic condition by controlled expression of toxins and

antitoxins. Mol. Microbiol. 45, 501–510.

Perla-Kajan, J., Lin, X., Cooperman, B.S., Goldman, E., Jakubowski, H.,

Knudsen, C.R., and Mandecki, W. (2010). Properties of Escherichia coli EF-

Tu mutants designed for fluorescence resonance energy transfer from tRNA

molecules. Protein Eng. Des. Sel. 23, 129–136.

Schumacher, M.A., Piro, K.M., Xu, W., Hansen, S., Lewis, K., and Brennan,

R.G. (2009). Molecular mechanisms of HipA-mediated multidrug tolerance

and its neutralization by HipB. Science 323, 396–401.

Sekine, S., Nureki, O., Dubois, D.Y., Bernier, S., Chenevert, R., Lapointe, J.,

Vassylyev, D.G., and Yokoyama, S. (2003). ATP binding by glutamyl-tRNA

synthetase is switched to the productive mode by tRNA binding. EMBO J.

22, 676–688.

Shah, D., Zhang, Z., Khodursky, A., Kaldalu, N., Kurg, K., and Lewis, K. (2006).

Persisters: a distinct physiological state of E. coli. BMC Microbiol. 6, 53.

Singh, R., Barry, C.E., 3rd, and Boshoff, H.I. (2010). The three RelE homologs

of Mycobacterium tuberculosis have individual, drug-specific effects on

bacterial antibiotic tolerance. J. Bacteriol. 192, 1279–1291.

Srivatsan, A., and Wang, J.D. (2008). Control of bacterial transcription, trans-

lation and replication by (p)ppGpp. Curr. Opin. Microbiol. 11, 100–105.

Vazquez-Laslop, N., Lee, H., and Neyfakh, A.A. (2006). Increased persistence

in Escherichia coli caused by controlled expression of toxins or other unrelated

proteins. J. Bacteriol. 188, 3494–3497.

Zaher, H.S., and Green, R. (2009). Quality control by the ribosome following

peptide bond formation. Nature 457, 161–166.


http://dx.doi.org/10.1038/nchembio.1364

http://dx.doi.org/10.1038/nchembio.1364

molecular mechanism of bacterial persistence by hipa

Documents