Light activation of staphylococcus aureus toxin YoeBSa 1 reveals guanosine-specific endoribonuclease activity

Article

pubs.acs.org/biochemistry

Light Activation of Staphylococcus aureus Toxin YoeB_Sa1Reveals

Guanosine-Speciﬁc Endoribonuclease Activity

Amy S. Larson and Paul J. Hergenrother*

Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States

S

* Supporting Information

ABSTRACT: The Staphylococcus aureus chromosome harbors

two homologues of the YefM-YoeB toxin−antitoxin (TA)

system. The toxins YoeB_Sa1and YoeB_Sa2possess ribosome-

dependent ribonuclease (RNase) activity in Escherichia coli.

This activity is similar to that of the E. coli toxin YoeB_Ec, an

enzyme that, in addition to ribosome-dependent RNase

activity, possesses ribosome-independent RNase activity in

vitro. To investigate whether YoeB_Sa1is also a ribosome-independent RNase, we expressed YoeB_Sa1using a novel strategy and

characterized its in vitro RNase activity, sequence speciﬁcity, and kinetics. Y88 of YoeB_Sa1was critical for in vitro activity and cell

culture toxicity. This residue was mutated to o-nitrobenzyl tyrosine (ONBY) via unnatural amino acid mutagenesis. YoeB_Sa1

Y88ONBY could be expressed in the absence of the antitoxin YefM_Sa1in E. coli. Photocaged YoeB_Sa1-Y88ONBY displayed UV

light-dependent RNase activity toward free mRNA in vitro. The in vitro ribosome-independent RNase activity of YoeB_Sa1

-

Y88ONBY, YoeB_Sa1-Y88F, and YoeB_Sa1-Y88TAG was signiﬁcantly reduced or abolished. In contrast to YoeB_Ec, which cleaves

RNA at both adenosine and guanosine with a preference for adenosine, YoeB_Sa1cleaved mRNA speciﬁcally at guanosine. Using

this information, a ﬂuorometric assay was developed and used to determine the kinetic parameters for ribosome-independent

RNA cleavage by YoeB_Sa1

.

oxin−antitoxin (TA) systems are genetic modules that are

chromosomally encoded TA systems is somewhat controversial,

with at least 13 proposed roles, including junk DNA, selﬁsh

genes, stabilization of mobile genetic elements, anti-addiction

elements, gene regulation, growth control/stress response,

persistence, growth arrest, programmed cell death, phage

defense, bioﬁlm formation, virulence, and phenotypic bist-

ability.^8,9However, it is generally accepted that cellular stress

modulates transcription at the TA locus and stimulates

degradation of the antitoxin, releasing the toxin to act on its

cellular target and arrest growth until conditions become more

favorable.⁵Upon the cessation of stress, the antitoxin is

replenished, inactivating the toxin and allowing the cell to

resume normal growth.¹⁰The growth arrest and eventual cell

death resulting from toxin overexpression have led to the

proposal that artiﬁcial toxin activation could provide an

eﬀective antibacterial strategy.¹¹⁻¹⁴

The Gram-positive pathogen Staphylococcus aureus is the

leading cause of bloodstream, lower respiratory tract, and skin

and soft tissue infections worldwide.¹⁵A recent comparative

genomic analysis identiﬁed between one and seven Type II TA

loci in the sequenced genomes of 14 S. aureus strains, including

YefM-YoeB_Sa1and YefM-YoeB_Sa2(previously identiﬁed as

Axe1-Txe1 and Axe2-Txe2, respectively)^16,17that are homo-

logues of the YefM-YoeB_EcTA system from Escherichia coli.¹⁸

The toxin YoeB_Ecpossesses ribosome-independent RNase

T

found in almost all free-living prokaryotes.¹TA loci

encode a protein toxin and a protein or RNA antitoxin and are

grouped into ﬁve types on the basis of the mechanism by which

the antitoxin counteracts the activity of the toxin.²⁻⁴In Type II

TA systems, the antitoxin is a protein that binds to the toxin

and prevents it from interacting with its cellular target. The

activity of Type II toxins is regulated by the diﬀerential

susceptibility of the toxin and the antitoxin to proteolysis. The

antitoxin is more labile than the toxin and must be continuously

expressed to maintain cellular levels capable of inhibiting the

toxin. Toxin inhibition is relieved by the activity of cellular

proteases, which degrade the labile antitoxin and shift the

antitoxin:toxin ratio to favor the free toxin, which can then act

on its cellular target.⁵The targets of TA system toxins include

DNA replication, protein translation, and cell wall biosynthesis.

Many toxins are ribonucleases (RNases) that inhibit translation

by cleaving RNA in a ribosome-dependent or -independent

fashion.²

TA systems were originally discovered on plasmids, where

they function as a postsegregational killing mechanism to

maintain a plasmid in a bacterial population. If a daughter cell

inherits a plasmid containing a TA locus during replication,

both the antitoxin and the toxin will be expressed to form a

stable, innocuous complex, and the cell will survive. However, if

the plasmid is not inherited, rapid proteolytic degradation of

the antitoxin will release the toxin to act on its cellular target

and kill the cell, thereby eliminating plasmid-free cells from the

population.⁶TA systems were subsequently discovered on the

chromosomes of many bacteria and archaea.^1,7The function of

Received: June 22, 2013

Revised: November 26, 2013

Published: November 26, 2013

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dx.doi.org/10.1021/bi4008098 | Biochemistry 2014, 53, 188−201

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activity in vitro as well as ribosome-dependent RNase activity in

vitro and in cells.^19,20YoeB_Ecbinds in the A site of the 50S

ribosomal subunit, causing cleavage of mRNA transcripts three

bases downstream of the start codon.²⁰It is unknown whether

YoeB_Ec-mediated mRNA cleavage on the ribosome results from

direct catalysis by YoeB_Ec, from enhancement of the latent

ribonucleolytic activity of the ribosome upon binding of

YoeB_Ec, or from some combination of the two.²⁰

A signiﬁcant challenge when studying toxic proteins is the

diﬃculty associated with their expression and puriﬁcation in E.

coli. Herein, we present a novel solution to this problem by

incorporating a noncanonical amino acid, a photocaged

tyrosine derivative, to replace a tyrosine that is critical for the

activity and toxicity of YoeB_Sa1. Using protein produced

through this method, the substrate speciﬁcity of YoeB_Sa1was

unveiled, allowing the creation of a ﬂuorogenic substrate that

can be used to monitor YoeB_Sa1activity.

the modiﬁcation that lysis was performed by vortexing

resuspended cells at maximal speed for 5 × 1 min pulses at

room temperature with recovery for 1−5 min on ice between

pulses. Isolated RNA was treated with DNase I and puriﬁed

with reagents from the Total RNA Kit I (Omega Bio-Tek)

according to the RNA Cleanup protocol from the RNeasy Mini

Handbook (Qiagen). Puriﬁed total RNA (10 ng) was used in

RT-PCR and in PCR with Platinum Taq DNA Polymerase

(Invitrogen) to detect DNA contamination. RT-PCR was

performed with primers YefM-YoeB_Sa1-F and YefM-YoeB_Sa1-R

or YefM-YoeB_Sa2-F and YefM-YoeB_Sa2-R using the Superscript

III One-Step RT-PCR System with Platinum Taq (Invitrogen)

as previously reported²²with the following modiﬁcations: the

annealing temperature was increased to 55 °C, and the number

of cycles was reduced to 35. PCR products were separated by

electrophoresis on 1% agarose gels and stained with ethidium

bromide.

Construction of Plasmids. The yoeB_Sa1open reading

frame (ORF) was ampliﬁed via PCR from the total DNA of

MRSA S4²¹with primers YoeB_Sa1-NdeI-F and YoeB_Sa1-Y88F-

HindIII-R or YoeB_Sa1-Y88TAG-HindIII-R and cloned into the

corresponding sites of pET-28a (Novagen) to create pET-28a-

yoeB_Sa1-Y88F and pET-28a-yoeB_Sa1-Y88TAG, respectively. The

yef M-yoeB_Sa1ORF was ampliﬁed via PCR from the total DNA

EXPERIMENTAL PROCEDURES

■

Bacterial Strains. MRSA clinical isolates were from a

previously published collection.²¹E. coli DH5α and NiCo21-

(DE3) were used for cloning and protein expression,

respectively.

Primers. All primers used for polymerase chain reaction

(PCR), reverse transcription PCR (RT-PCR), cloning, and site-

directed mutagenesis were synthesized by Integrated DNA

Technologies (IDT) and are listed in Table S1 of the

Supporting Information.

PCR Analysis of Clinical Isolates. Total DNA was

previously puriﬁed from a diverse collection of 78 clinical

isolates of MRSA.²¹Primers YefM-YoeB_Sa1-F and YefM-

YoeB_Sa1-R or YefM-YoeB_Sa2-F and YefM-YoeB_Sa2-R were used

to amplify the yef M-yoeB_Sa1or yefM-yoeB_Sa2loci, respectively,

from the total DNA. Primers YefM_Sa1-NdeI-F, YefM_Sa1-XhoI-R,

YoeB_Sa1-NdeI-F, YoeB_Sa1-HindIII-R, YefM_Sa2-NdeI-F, YefM_Sa2

HindIII-R, YoeB_Sa2-NdeI-F, and YoeB_Sa2-HindIII-R were used

to amplify the yefM_Sa1, yoeB_Sa1, yefM-yoeB_Sa1, yefM_Sa2, yoeB_Sa2

and yef M-yoeB_Sa2operons from the total DNA of strains

NRS27 and NRS76. PCR ampliﬁcation was performed on a

DNA thermocycler using previously described reaction

conditions.^21,22PCR products were separated by electro-

phoresis on 1% agarose gels and stained with ethidium

bromide.

Sequencing Analysis. Approximately 10% of the PCR

products generated via PCR ampliﬁcation of the yef M-yoeB_Sa1

and yefM-yoeB_Sa2loci were sequenced by the University of

Illinois W. M. Keck Center for Comparative and Functional

Genomics. Sequence data were analyzed using BioEdit version

7.0.5.3. The sequences were aligned using CLUSTAL W²³and

used as query sequences to search the BLAST database to verify

the identity of the PCR products and homology to known

genes.²⁴

RT-PCR Analysis of Clinical Isolates. MRSA isolates were

streaked from glycerol stocks on Brain Heart Infusion (BHI)

agar. Single colonies from freshly streaked plates were

inoculated into 10 mL of BHI medium and incubated

aerobically at 37 °C with shaking at 250 rpm overnight (14−

16 h). Overnight cultures were diluted 1:100 in 10 mL of BHI

medium and incubated aerobically at 37 °C with shaking at 250

rpm until the A₆₀₀reached 0.6−1.0. Logarithmically growing

cultures were harvested by centrifugation at 3220g for 10 min at

4 °C. Total RNA was puriﬁed using the FastRNA Pro Blue Kit

(Qbiogene) according to the manufacturer’s instructions with

of MRSA S4²¹with primers YefM_Sa1-NdeI-F and YoeB_Sa1

-

HindIII-R and cloned into the corresponding sites of pET-28a.

Because a single nonsilent point mutation was found in the

sequence for YefM_Sa1, Quikchange site-directed mutagenesis

was performed with primers YefM_Sa1-QC-F and YefM_Sa1-QC-R

to create pET-28a-yefM-yoeB_Sa1. Site-directed mutagenesis was

carried out with the Quikchange site-directed mutagenesis kit

(Stratagene) according to the manufacturer’s instructions with

the modiﬁcation that E. coli DH5α was used as the host strain.

The yefM_Sa1ORF was ampliﬁed via PCR from the total DNA

of MRSA NRS3 (Network on Antimicrobial Resistance in S.

aureus) with primers YefM_Sa1-NdeI-F and YefM_Sa1-XhoI-R and

cloned into the corresponding sites of pET-28a to create pET-

28a-yefM_Sa1. All clones were conﬁrmed by sequencing. The

following antibiotic concentrations were used: 50 μg/mL

kanamycin (pET-28a), 35 μg/mL chloramphenicol (pEVOL-

ONBY), and 100 μg/mL ampicillin (Pentaprobes).

ONBY Synthesis. o-Nitrobenzyl tyrosine (ONBY) was

synthesized according to a previously published method²⁵with

some modiﬁcations. Two grams (11 mmol) of ^L-tyrosine was

stirred with 1.9 g (7.6 mmol) of CuSO₄·5H₂O in 20 mL of 1 M

NaOH at 60 °C for 20 min. The reaction was cooled to room

temperature, quenched with hydrochloric acid, ﬁltered, and

washed with water. The resulting solid was stirred with 1.5 g

(11 mmol) of K₂CO₃and 1.8 g (8.3 mmol) of o-nitrobenzyl

bromide in 60 mL of 75% aqueous dimethylformamide (DMF)

at room temperature in the dark for 3 days. The resulting solid

was ﬁltered; washed with 75% aqueous DMF, water, 75%

aqueous acetone, and ice-cold acetone; stirred in 100 mL of 1

M HCl for 2 h; ﬁltered; stirred in 100 mL of 1 M HCl for 1 h;

ﬁltered; and washed with water and acetone. The overall yield

was 1.8 g (5.7 mmol, 69%).

-

,

Puriﬁcation of YoeB_Sa1-Y88F and YoeB_Sa1-Y88TAG.

pET-28a-yoeB_Sa1-Y88F or pET-28a-yoeB_Sa1-Y88TAG was intro-

duced into E. coli NiCo21(DE3). A single colony from a freshly

streaked plate was inoculated into LB medium supplemented

with kanamycin and grown at 37 °C with shaking at 250 rpm

overnight (14−16 h). The overnight culture was diluted 1:100

and grown at 37 °C with shaking at 250 rpm until the A₆₀₀

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reached 0.4−0.6. Protein expression was induced with 1 mM

IPTG at 37 °C with shaking at 250 rpm for 4 h. The culture

was harvested by centrifugation at 6000g for 5 min at 4 °C. Cell

pellets were frozen at −20 °C, thawed on ice for 30 min, and

resuspended in 10 mL of cold lysis buﬀer [20 mM Tris, 500

mM NaCl, and 60 mM imidazole (pH 7.9)]. Cells were lysed

by sonication on ice at 40% amplitude for 5 min with a 1 s

pulse. The lysate was cleared by centrifugation at 35000g for 30

min at 4 °C. The supernatant was batch-loaded onto 1 mL of

1:1 Ni-NTA agarose (Qiagen) at 4 °C for 30 min with

inversion. The resin was washed with 20 mL of cold lysis buﬀer

and eluted with 10 mL of cold elution buﬀer A [50 mM Tris,

500 mM NaCl, 500 mM imidazole, 1 mM TCEP, and 10%

glycerol (pH 7.9)]. The eluted fraction was concentrated to

∼0.5 mL using an Ultra-15 Centrifugal Filter Unit with an

Ultracel-3 membrane (Amicon), 0.2 μm ﬁltered, and further

puriﬁed on a HiLoad 16/60 Superdex 75 PG column (GE

Healthcare) using fast performance liquid chromatography

(FPLC) buﬀer A [50 mM Tris, 200 mM NaCl, 1 mM TCEP,

and 10% glycerol (pH 7.9)]. Fractions containing pure protein

were pooled and concentrated to ∼0.5−1 mL. Purity was

assessed by sodium dodecyl sulfate−polyacrylamide gel electro-

phoresis (SDS−PAGE) using 4−20% TGX Mini-PROTEAN

gels (Bio-Rad). The concentration was determined by

densitometry and by the bicinchoninic acid (BCA) assay

(Pierce) performed according to the manufacturer’s instruc-

tions, with the modiﬁcation that lysozyme standards were used

instead of bovine serum albumin (BSA) standards.

ESI-MS. Five micrograms of YefM_Sa1, YoeB_Sa1-Y88F,

YoeB_Sa1-Y88TAG, or YoeB_Sa1-Y88ONBY was precipitated

according to a previously published method.²⁶YoeB_Sa1

-

Y88ONBY was exposed to 312 nm light on a UV trans-

illuminator for 0−600 s prior to precipitation. Precipitated, air-

dried protein was analyzed by electrospray ionization mass

spectrometry (ESI-MS) at the University of Illinois Mass

Spectrometry Laboratory.

Agarose Gel RNase Activity Assay. pET-28a-yef M-

yoeB_Sa1was digested with XhoI (NEB) at 37 °C for 3 h. The

fully linearized plasmid was extracted once with a 24.5:24.5:1

phenol/chloroform/isoamyl alcohol mixture and twice with

chloroform, precipitated with 3 M NaOAc (pH 5.3) and 100%

EtOH, and resuspended in nuclease-free water. One microgram

of linearized plasmid was used as the template for standard

RNA synthesis with the T7 High Yield RNA Synthesis Kit

(NEB) according to the manufacturer’s instructions. RNA was

puriﬁed with reagents from Total RNA Kit I (Omega Bio-Tek)

according to the RNA Cleanup protocol from the RNeasy Mini

Handbook (Qiagen). RNase assays were performed according

to a modiﬁed published method.¹⁹For the light dependence of

decaging, 10 pmol of YoeB_Sa1-Y88ONBY in 50 mM Tris and

5% glycerol (pH 8.0) was exposed to UV light for 0−5 min,

followed by the addition of 320 ng of yef M-yoeB_Sa1RNA with

Human Placental RNase Inhibitor (NEB) at a ﬁnal

concentration of 1 unit/μL. Reactions were incubated at 37

°C for 2 h and quenched by addition of 1 μg of proteinase K

(Invitrogen) and incubation at 37 °C for 15 min. Eleven

microliters of RNA loading dye I (95% formamide, 18 mM

EDTA, 0.025% SDS, and 0.025% bromophenol blue) was then

added, and samples were incubated at 95 °C for 5 min

immediately prior to electrophoresis on 1.2% agarose, 0.5×

TBE [45 mM Tris-borate and 1 mM EDTA (pH 8.3)], 0.1 μg/

mL ethidium bromide gels. For the inhibition of YoeB_Sa1by

YefM_Sa1, YoeB_Sa1(10, 20, or 30 pmol) in 50 mM Tris and 5%

glycerol (pH 8.0) was exposed to UV light for 3 min. YefM_Sa1

(0, 10, 20, or 30 pmol) was then added. Following incubation

at 37 °C for 30 min to allow formation of the YefM-

YoeB_Sa1complex, 320 ng of yefM-yoeB_Sa1RNA was added with

Human Placental RNase Inhibitor at a ﬁnal concentration of 1

unit/μL. Reactions were incubated at 37 °C for 1 h, quenched,

and analyzed as described above. For the time course of

Puriﬁcation of YoeB_Sa1-Y88ONBY. pET-28a-yoeB_Sa1

-

Y88TAG and pEVOL-ONBY were introduced into E. coli

NiCo21(DE3). A single colony from a freshly streaked plate

was inoculated into LB medium supplemented with kanamycin

and chloramphenicol and grown at 37 °C with shaking at 250

rpm overnight (14−16 h). The overnight culture was diluted

1:100, 100 mM ONBY dissolved in 1 M NaOH was added to a

ﬁnal concentration of 1 mM, and the culture was grown at 37

°C with shaking at 250 rpm in the dark. When the A₆₀₀of the

culture reached 0.5−0.6, arabinose was added to a ﬁnal

concentration of 0.2% to induce expression of ONBY-aaRS.

When the A₆₀₀of the culture reached 1.0−1.2, IPTG was added

to a ﬁnal concentration of 1 mM to induce expression of

YoeB_Sa1-Y88ONBY. Expression was allowed to proceed at 37

°C for 15 h. The culture was harvested, and YoeB_Sa1-Y88ONBY

was puriﬁed in the dark as described above for YoeB_Sa1-Y88F

and YoeB_Sa1-Y88TAG.

Puriﬁcation of YefM_Sa1. pET-28a-yefM_Sa1was introduced

into E. coli NiCo21(DE3), and protein expression and

puriﬁcation were performed as described for YoeB_Sa1-Y88F

and YoeB_Sa1-Y88TAG, with the modiﬁcation that YefM_Sa1was

eluted with 10 mL of cold elution buﬀer B [20 mM Tris, 500

mM NaCl, and 500 mM imidazole (pH 7.9)]. Gel ﬁltration was

performed twice using FPLC buﬀer B [20 mM Tris and 500

mM NaCl (pH 7.9)] to remove RNase contamination.

Fractions corresponding to the second major peak from the

ﬁrst gel ﬁltration step were pooled, concentrated, and subjected

to a second gel ﬁltration step. Fractions corresponding to the

second major peak from the second gel ﬁltration step were

pooled, concentrated, and used in experiments. Purity was

assessed by SDS−PAGE using 4−20% TGX Mini-PROTEAN

gels (Bio-Rad). Protein concentration was determined by the

BCA assay (Pierce) performed according to the manufacturer’s

instructions using BSA standards.

YoeB_Sa1and YefM_Sa1RNase activity, 10 pmol of YoeB_Sa1

-

Y88ONBY, YoeB_Sa1-Y88F, YoeB_Sa1-Y88TAG, or YefM_Sa1was

incubated with 320 ng of yefM-yoeB_Sa1RNA in the presence of

1 unit/μL Human Placental RNase Inhibitor in 50 mM Tris

and 5% glycerol (pH 8.0). One set of reactions containing

YoeB_Sa1-Y88ONBY was exposed to UV light for 3 min prior to

the addition of RNA. Reactions were incubated at 37 °C for 1−

20 h, quenched, and analyzed as described above.

Polyacrylamide Gel RNase Activity Assay. Quikchange

site-directed mutagenesis was performed with primers

PP1QC1-F and PP1QC1-R to insert a G into Pentaprobe 1

and with primers PP1QC2-F and PP1QC2-R, PP2QC-F and

PP2QC-R, PP4QC-F and PP4QC-R, PP6QC-F and PP6QC-R,

PP7QC-F and PP7QC-R, PP8QC-F and PP8QC-R, PP9QC-F

and PP9QC-R, PP10QC-F and PP10QC-R, and PP12QC-F

and PP12QC-R (Table S1 of the Supporting Information) to

introduce XbaI sites into Pentaprobes 1, 2, 4, 6−10, and 12,

respectively. Site-directed mutagenesis was carried out with the

Quikchange site-directed mutagenesis kit (Stratagene) accord-

ing to the manufacturer’s instructions, with the modiﬁcation

that E. coli DH5α was used as the host strain. Pentaprobe

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plasmids were digested with XbaI (NEB) at 37 °C for 3 h. Fully

linearized plasmids were puriﬁed by phenol/chloroform

extraction as described above. RNA was synthesized and

puriﬁed as described above. RNase assays were performed

according to a modiﬁed published method.²⁷One microgram

of YoeB_Sa1-Y88ONBY was incubated with 1 μg of Pentaprobe

RNA in 50 mM Tris (pH 8.0) for 0.5−2 h at 37 °C. Reactions

were quenched by addition of 1 μg of proteinase K and

incubation at 37 °C for 15 min. An equal volume of RNA

loading dye II (95% formamide, 5 mM EDTA, and 0.025%

bromophenol blue) was then added, and the reactions were

incubated at 95 °C for 5 min to denature the RNA. The

products were visualized by electrophoresis on 8% poly-

acrylamide TBE−urea gels [89 mM Tris-borate, 2 mM EDTA,

and 8 M urea (pH 8.3)] poststained with ethidium bromide.

Matrix-Assisted Laser Desorption Ionization (MALDI)

RNase Activity Assay. PP7-1, PP7-2, and PP7-3 were

Calibration Plot. Fluorescence values for 1:1 molar

mixtures of the cleavage products at 30 min after addition of

YoeB_Sa1were used to construct a calibration plot of picomoles

of cleaved substrate versus relative ﬂuorescence units (RFU). A

separate calibration plot was constructed each time the

ﬂuorometric assay was performed.

Kinetic Analysis. In some of the progress curves, an

increase in ﬂuorescence was not observed until ∼15 min after

the addition of YoeB_Sa1. However, the increase in measured

ﬂuorescence was linear over the majority of the remainder of

the assay. Consequently, initial rates were calculated from the

linear portion of the progress curves between 20 and 30 min.

Fluorescence values were corrected by subtracting the

ﬂuorescence measured at 20 min from all subsequent time

points. Corrected ﬂuorescence values were converted to

picomoles of cleaved substrate using the slope from the

calibration plot, and linear regression was performed using

Microsoft Excel to obtain initial velocities. Linear velocities

were plotted versus substrate concentration, and the resulting

data points were ﬁt with the Hill equation using OriginPro

version 8.0.

synthesized by Genscript. Ten micrograms of YoeB_Sa1

-

Y88ONBY was incubated with 2 μg of oligonucleotide for 1

or 2 h at 37 °C. Reactions were quenched by addition of 10 μg

of proteinase K (Invitrogen) and incubation at 37 °C for 30

min followed by precipitation with 5 M NH₄OAc (ﬁnal

concentration of 2 M) and 3 volumes of 100% EtOH at −80

°C. Pellets were washed with ice-cold 70% EtOH, resuspended

in 1 μL of H₂O, and analyzed by matrix-assisted laser

desorption ionization mass spectrometry (MALDI-MS) at the

University of Illinois Mass Spectrometry Laboratory.

High-Performance Liquid Chromatography (HPLC)

RNase Activity Assay. The ﬂuorogenic chimeric oligonucleo-

tide substrate 5′-6-FAM-AACrArArArArGrArArAAATT-

IABkFQ-3′ (6-FAM, 6-carboxyﬂuorescein ﬂuorophore;

IABkFQ, Iowa Black ﬂuorophore quencher) and cleavage

products 5′-6-FAM-AACrArArArArG-3′ and 5′-rArArAAATT-

IABkFQ-3′ were synthesized by IDT. YoeB_Sa1(20 μM) was

incubated with oligonucleotide (30 μM) in buﬀer at 25 °C for 5

h. HPLC was performed using an Alliance HPLC System

(e2695 Separations Module, Waters) with detection at 260 nm

(2489 UV/visible Detector, Waters). The full-length oligonu-

cleotide was separated from the cleavage products on a

YMCbasic S5 column (4.6 mm × 150 mm, 5 μm, Waters)

using a linear gradient from 100 mM triethylammonium acetate

(TEAA) (pH 7.0) to a mixture of 50 mM TEAA and 50%

acetonitrile (pH 7.0) over 25 min. Peak fractions were

collected, concentrated to 5−10 μL, and analyzed by

MALDI-MS at the University of Illinois Mass Spectrometry

Laboratory.

RESULTS

■

Prevalence, Conservation, and Transcription of yef M-

yoeB_Sa1and yef M-yoeB_Sa2in MRSA Clinical Isolates. The

artiﬁcial activation of TA systems is of signiﬁcant interest as an

intriguing antibacterial strategy.¹¹⁻¹⁴YoeB_Sa1and YoeB_Sa2

speciﬁcally present outstanding targets for this approach, as

expression of YoeB_Sa1and YoeB_Sa2in the absence of their

respective antitoxins, YefM_Sa1and YefM_Sa2, induces growth

arrest in E. coli.²⁸For artiﬁcial activation of YoeB_Sa1or YoeB_Sa2

to be an eﬀective antibacterial strategy, YefM-YoeB_Sa1and

YefM-YoeB_Sa2must be present and functional in clinical isolates

of S. aureus. Thus, the presence, conservation, and transcription

of yef M-yoeB_Sa1and yef M-yoeB_Sa2were investigated in a

collection of 78 clinical isolates of MRSA from three Illinois

hospitals and the Network on Antimicrobial Resistance in S.

aureus (NARSA). Multiple-locus variable number of tandem

repeats analysis previously conﬁrmed that these isolates were

not clonal.²¹

The total DNA of these isolates was probed for the presence

of yefM-yoeB_Sa1and yefM-yoeB_Sa2using PCR with intragenic

speciﬁc primers (Figure 1A). The yef M-yoeB_Sa1and yefM-

yoeB_Sa2genes were present in 99% (77 of 78) of the clinical

isolates (Table S2 of the Supporting Information). Approx-

imately 10% of the PCR products were subjected to DNA

sequencing, and the sequences were aligned with the reference

genome from the S. aureus COL strain using CLUSTAL W²³

(Figures S1 and S2 of the Supporting Information) and

compared with yef M-yoeB_Sa1and yef M-yoeB_Sa2loci from

published S. aureus genomes using BLAST.²⁴The yefM-yoeB_Sa1

PCR products were >97% identical with the yefM-yoeB_Sa1genes

in 29 of the 30 genomes containing that locus, while the yefM-

yoeB_Sa2PCR products were at least 96% identical with the

yef M-yoeB_Sa2genes in all 26 genomes containing that locus.

PCR products of ∼1.3 and ∼2.5 kb resulted from attempts to

detect yefM-yoeB_Sa1and yef M-yoeB_Sa2, respectively, in NRS27.

DNA sequence analysis revealed that the product ampliﬁed by

yef M-yoeB_Sa1-speciﬁc primers spanned a phage transcriptional

repressor and a DNA polymerase III α subunit, while the

product ampliﬁed by yefM-yoeB_Sa2-speciﬁc primers spanned an

ornithine cyclodeaminase, a siderophore biosynthesis protein,

and a multidrug resistance eﬄux pump. Sites of partial

Fluorometric Assay. Wells of a black 384-well plate were

ﬁlled with 15 μL of 0.5−20 μM intact ﬂuorogenic substrate or

cleavage products diluted in assay buﬀer [200 mM sodium

phosphate (pH 6.0) and 5% glycerol] and allowed to

equilibrate for 30 min at room temperature. YoeB_Sa1

-

Y88ONBY was diluted to 10 μM in assay buﬀer, exposed to

UV light at 312 nm for 3 min, and added to wells containing

intact substrate or cleavage products to a ﬁnal concentration of

5 μM. Fluorescence was measured once every minute for 100

min using a Criterion Analyst AD instrument (Molecular

Devices) with 485

15 nm excitation and 530

15 nm

emission ﬁlters and a 505 nm cutoﬀ dichroic mirror. The

ﬂuorophore was excited with a 1000 W continuous xenon arc

lamp with 10 reads per well. Three separately puriﬁed batches

of YoeB_Sa1-Y88ONBY were each assayed in technical triplicate

on two diﬀerent days. Results are the average of the average

rate for each batch of protein.

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biology techniques than yefM-yoeB_Sa2, a number of strategies

were explored in eﬀorts to obtain pure, functional YoeB_Sa1

.

The most common method employed to obtain a functional

TA system toxin for in vitro characterization involves co-

expression of the antitoxin with the toxin, puriﬁcation of the

resulting TA complex, isolation of the toxin by denaturation of

the complex, and subsequent refolding. Active YoeB_Echas been

obtained by dissociation from YefM_Ecin 6 M guanidine

hydrochloride (GuHCl) followed by refolding via gradual

dialysis into HEPES buﬀer.¹⁹YoeB_Ecand YoeB_Sa1share a high

degree of sequence homology, suggesting that a similar method

could allow puriﬁcation of functional YoeB_Sa1. The yefM-

yoeB_Sa1operon was therefore cloned into pET-28a with a

hexahistidine tag at the N-terminus of YefM_Sa1to facilitate

puriﬁcation, and the complex was expressed in E. coli and

puriﬁed using Ni-NTA resin. A variety of conditions were

screened to identify an optimal method for denaturing,

purifying, and refolding YoeB_Sa1. In each case, the YefM-

YoeB_Sa1complex was denatured by extended dialysis against

either 6 M GuHCl or 8 M urea, YefM_Sa1was extracted with Ni-

NTA resin, and the presence and purity of the remaining

YoeB_Sa1were assessed using SDS−PAGE. If 6 M GuHCl was

used as the denaturant, the recaptured YefM_Sa1was highly pure,

but YoeB_Sa1was not recovered in the unbound fraction,

probably because of the low initial ratio of YoeB_Sa1to YefM_Sa1

and the necessity of diluting and precipitating samples

containing GuHCl for SDS−PAGE. In contrast, when 8 M

urea was used as the denaturant, YoeB_Sa1was recovered, but a

signiﬁcant amount of residual YefM_Sa1was also present. Eﬀorts

to refold the impure toxin by dilution or gradual dialysis

resulted in substantial precipitation. These unpromising results

led to the abandonment of denaturing puriﬁcation and

refolding as a viable approach to obtaining large quantities of

Figure 1. yef M-yoeB_Sa1and yef M-yoeB_Sa2are prevalent and transcribed

in MRSA clinical isolates. (A) Locations of homology for primers used

in PCR and RT-PCR. Primer sequences were designed from the S.

aureus COL genome. (B) RT-PCR analysis of yef M-yoeB_Sa1(Sa1, top

two panels) and yef M-yoeB_Sa2(Sa2, bottom two panels) transcription

in MRSA clinical isolates. Lane M contains a DNA ladder [1517, 1200,

1000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp (NEB)]. Plus

and minus signs denote the inclusion and exclusion, respectively, of

reverse transcriptase. Clinical isolates are identiﬁed by strain number.

complementarity were identiﬁed between the yefM-yoeB_Sa1- and

yefM-yoeB_Sa2-speciﬁc primers and these genes, which could

allow ampliﬁcation in the absence of yef M-yoeB_Sa1and yef M-

yoeB_Sa2. To conﬁrm the absence of yef M-yoeB_Sa1and yef M-

yoeB_Sa2in NRS27, the yefM_Sa1, yoeB_Sa1, yef M-yoeB_Sa1, yefM_Sa2

,

pure, active YoeB_Sa1

.

yoeB_Sa2, and yef M-yoeB_Sa2operons were individually ampliﬁed

using primers designed to clone each of the full genes. PCR

products were detected in the positive control strain NRS76,

but not in NRS27. These results indicate that the yef M-yoeB_Sa1

and yefM-yoeB_Sa2genes are not present in NRS27.

To investigate the transcription of yefM-yoeB_Sa1and yef M-

yoeB_Sa2in MRSA, RT-PCR was performed on total RNA from

eight isolates using the same primers used in the PCR screen.

yefM-yoeB_Sa1and yefM-yoeB_Sa2were transcribed as bicistronic

messages in each of the strains (Figure 1B). No PCR products

were detected in the absence of reverse transcriptase, indicating

the absence of DNA contamination. Taken together, these

results suggest that yefM-yoeB_Sa1and yefM-yoeB_Sa2are wide-

spread, conserved, and transcribed in clinical MRSA isolates.

Thus, an activator of YoeB_Sa1or YoeB_Sa2with antibacterial

activity could have broad eﬃcacy against S. aureus.

Photocaging Allows Expression of YoeB_Sa1in E. coli.

YoeB_Ecexhibits ribosome-dependent RNase activity in vitro and

in E. coli.²⁰YoeB_Sa1and YoeB_Sa2were found to exhibit RNase

activity similar to that of YoeB_Ecupon overexpression in E. coli,

suggesting that they are ribosome-dependent RNases with the

same mechanism of action as YoeB_Ec.²⁸Intriguingly, YoeB_Ec

also possesses “residual” ribosome-independent RNase activ-

ity.¹⁹Evaluation of the possibility that YoeB_Sa1and YoeB_Sa2

might also possess such activity necessitates the expression and

An alternative to denaturing puriﬁcation of the toxin from

the TA complex is to use a commercially available protease to

selectively degrade the labile antitoxin from the TA complex,

leaving the toxin unscathed.²⁷The YefM-YoeB_Sa1complex was

expressed and puriﬁed as described above and subjected to

digestion with trypsin. Analysis of the digest products by SDS−

PAGE suggested that YoeB_Sa1remained intact while YefM_Sa1

was rapidly and selectively degraded. However, the YoeB_Sa1

protein obtained from trypsin digestion of YefM-YoeB_Sa1did

not bind to YefM_Sa1in pull-down experiments. Native PAGE of

the digestion time course suggested that a fragment of YefM_Sa1

remained bound to YoeB_Sa1, prohibiting complex formation

and indicating that this approach could not be used to obtain

pure YoeB_Sa1

.

The yoeB_Sa1gene was then cloned at the C-terminus of

glutathione S-transferase (GST) in hopes that a larger fusion

partner would reduce the toxicity of YoeB_Sa1by preventing it

from binding to the ribosome. However, all sequenced clones

contained frameshift mutations introducing premature stop

codons in the yoeB_Sa1gene. Two rounds of site-directed

mutagenesis were performed to correct these mutations.

Additional premature stop codons or inactivating mutations

were introduced in each round, suggesting that low levels of

full-length GST-YoeB_Sa1, produced by leaky expression from

the T7 promoter even in the absence of T7 RNA polymerase,

retained the ability to bind to the ribosome, inhibit translation,

induce growth arrest, and prevent colony formation. Only

mutant clones encoding premature stop codons were not toxic.

These results hinted that the C-terminal region of YoeB_Sa1is

puriﬁcation of full-length, functional YoeB_Sa1and YoeB_Sa2

.

However, YoeB_Sa1and YoeB_Sa2inhibit translation initiation and

induce growth arrest in E. coli,²⁸eﬀectively preventing

overexpression and characterization. As yef M-yoeB_Sa1proved

to be more amenable to manipulation using standard molecular

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Figure 2. ESI-MS analysis of YoeB_Sa1mutants. (A) YoeB_Sa1-Y88TAG (calculated molecular mass of 12307 Da and observed molecular mass of

12303 Da). (B) YoeB_Sa1-Y88F (calculated molecular mass of 12454 Da and observed molecular mass of 12450 Da). (A and B) Gel inset lanes:

Kaleidoscope prestained standards (Bio-Rad), expression culture lysate immediately prior to induction with IPTG, and expression culture lysate 4 h

postinduction (from left to right, respectively). The asterisk indicates the band corresponding to YoeB_Sa1-Y88TAG in panel A and YoeB_Sa1-Y88F in

panel B. (C) YoeB_Sa1-Y88ONBY prior to exposure to UV light (calculated molecular mass of 12606 Da and observed molecular mass of 12574 Da).

(D−H) YoeB_Sa1-Y88ONBY exposed to UV light for 1, 2, 3, 4, and 5 min, respectively (calculated molecular mass of 12470 Da and observed

molecular mass of 12468−12469 Da).

necessary for activity and toxicity. As the C-terminal residues

H83 and Y84 of YoeB_Ecare required for both in vitro RNase

activity and toxicity in E. coli,¹⁹the inability to clone the full-

length yoeB_Sa1gene at the C-terminus of GST suggested that

the analogous H87 and Y88 in YoeB_Sa1have similar roles.

Mutation of the C-terminal Y84 of YoeB_Ecto phenylalanine

or alanine was previously found to diminish both in vitro RNase

activity and toxicity in E. coli,¹⁹suggesting that similar

mutations to the homologous C-terminal Y88 of YoeB_Sa1

might suﬃciently reduce toxicity to allow expression in E.

coli. To investigate the contribution of Y88 to the toxicity of

YoeB_Sa1, the TAT codon for Y88 in YoeB_Sa1was changed to

TAG for the amber stop codon (Y88TAG) or to TTT for

phenylalanine (Y88F). The genes encoding these mutants were

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cloned into pET-28a and expressed in E. coli. The resulting

proteins were puriﬁed with yields of 5 mg of YoeB_Sa1-Y88TAG

and 2.5 mg of YoeB_Sa1-Y88F per liter of culture. ESI mass

spectrometry (ESI-MS) showed that the molecular masses of

these mutants matched the predicted values [YoeB_Sa1-Y88TAG,

expected molecular mass of 12307 Da and observed molecular

mass of 12303 Da (Figure 2A); YoeB_Sa1-Y88F, expected

molecular mass of 12454 Da and observed molecular mass of

12450 Da (Figure 2B)]. These results indicated that Y88

contributes to YoeB_Sa1toxicity in E. coli and suggested that a

nonpermanent modiﬁcation of the structure of Y88 might

suﬃciently alleviate the toxicity of YoeB_Sa1to allow expression

of the full-length protein in E. coli.

Decaged YoeB_Sa1Is a Ribosome-Independent RNase.

The ribosome-independent RNase activity of YoeB_Sa1was

assessed with respect to yefM-yoeB_Sa1RNA in vitro. Decaging of

YoeB_Sa1-Y88ONBY resulted in UV light-dependent degradation

of the RNA (Figure 3A). In agreement with ESI-MS data

Unnatural amino acid (UAA) mutagenesis allows the

incorporation of a noncanonical amino acid site-speciﬁcally

into a protein via the use of an aminoacyl-tRNA synthetase

(aaRS)/tRNA pair that is orthogonal to the host organism’s

translational machinery.²⁹⁻³¹The photocaged UAA o-nitro-

benzyl tyrosine (ONBY) has been incorporated into a number

of proteins in which a tyrosine is critical for enzymatic

activity.³²⁻³⁶Mutation of tyrosine to ONBY “cages” the

enzyme by inhibiting catalysis, substrate binding, or both.

Irradiation with UV light between 300 and 365 nm releases the

photocaging o-nitrobenzyl group,³⁷revealing the free tyrosine

and activating the enzyme for catalysis. As Y88 contributes to

the toxicity and, presumably, the activity of YoeB_Sa1, it was

hypothesized that mutation of Y88 to ONBY would allow

expression of an inactive, nontoxic YoeB_Sa1variant that could

be activated by UV light following expression and puriﬁcation.

The pEVOL plasmids for UAA mutagenesis encode an

optimized amber (TAG) stop codon suppressor tRNA and two

copies of an evolved UAA-speciﬁc Methanocaldococcus

jannaschii aaRS, one under the control of a constitutive glnS′

promoter and the other under the control of an arabinose-

inducible araBAD promoter, which allows the expression level

of the aaRS to be ﬁnely tuned.³⁸As Y88 is the last residue in

YoeB_Sa1, it would be challenging to purify ONBY-containing

YoeB_Sa1from prematurely truncated YoeB_Sa1. A variety of

expression conditions were therefore screened by ESI-MS for

maximal incorporation of ONBY into YoeB_Sa1. Maximal

incorporation was achieved when 1 mM ONBY was present

in the culture medium from the beginning of the expression,

when aaRS expression was induced in early logarithmic growth

phase, and when YoeB_Sa1expression was induced in late

logarithmic or early stationary phase and allowed to proceed

Figure 3. RNase activity of YoeB_Sa1mutants. (A) RNase activity of

YoeB_Sa1-Y88ONBY exposed to UV light for 0, 5, 10, 20, 30, 60, 120,

180, 240, or 300 s and incubated with yefM-yoeB_Sa1RNA for 2 h.

Buﬀer controls were incubated with yef M-yoeB_Sa1RNA for 0 or 2 h.

(B) Inhibition of YoeB_Sa1by YefM_Sa1. YoeB_Sa1-Y88ONBY was exposed

to UV light for 3 min. YefM_Sa1and YoeB_Sa1were mixed and incubated

at 37 °C for 30 min to allow formation of the YefM-YoeB_Sa1complex

prior to incubation with yef M-yoeB_Sa1RNA at 37 °C for 1 h. A minus

sign designates 0 pmol of protein, a plus sign designates 10 pmol of

protein, and right triangles designate increasing amounts of protein

overnight at 37 °C. Using this method, 1 mg of pure YoeB_Sa1

-

Y88ONBY was obtained per liter of culture. ESI-MS revealed

that the mass of the full-length caged protein was ∼30 Da lower

than predicted [YoeB_Sa1-Y88ONBY, expected molecular mass

of 12606 Da and observed molecular mass of 12574 Da (Figure

2C)]. This discrepancy is attributed to reduction of the nitro

group of ONBY to an amino group, a phenomenon that has

been observed when ESI-MS is performed in protic solvents,

resulting in a loss of 30 Da that corresponds to the observed

mass shift.³⁹Importantly, very little truncated YoeB_Sa1-Y88TAG

was present (expected molecular mass of 12307 Da and

observed molecular mass of 12305 Da). UV light-induced loss

from 10 to 30 pmol. (C) RNase activity of YoeB_Sa1, YoeB_Sa1

-

Y88ONBY, YoeB_Sa1-Y88F, YoeB_Sa1-Y88TAG, and YefM_Sa1. Ten

picomoles of protein was incubated with yefM-yoeB_Sa1RNA for 0, 1,

2, 4, 8, 12, 16, or 20 h. Lane M contains an ssRNA ladder [1000, 500,

300, 150, 80, and 50 nucleotides (NEB)]. Lane B contains buﬀer and

RNA at 20 h. In the top panel only, YoeB_Sa1-Y88ONBY was exposed

to UV light for 3 min.

showing that the extent of decaging did not increase with

additional UV exposure after 3 min (Figure 2F), the RNase

activity of YoeB_Sa1-Y88ONBY also did not increase with

additional UV exposure after 3 min (Figure 3A). YoeB_Sa1

activity was inhibited by the addition of an equimolar amount

of YefM_Sa1(Figure 3B), demonstrating that the observed

RNase activity was due speciﬁcally to YoeB_Sa1and not to RNase

of the o-nitrobenzyl moiety was followed by ESI-MS [YoeB_Sa1

-

Y88, expected molecular mass of 12470 (Figure 2D−H)]. The

extent of decaging increased as the extent of UV exposure was

increased up to 3 min, after which no further decaging was

observed (Figure 2F−H).

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Figure 4. YoeB_Sa1cleaves RNA at guanosine residues. (A) RNase activity of YoeB_Sa1toward Pentaprobes 1, 4, 7, and 10. Lane M contains an ssRNA

ladder [1000, 500, 300, 150, 80, and 50 nucleotides (NEB)]. In the buﬀer lanes, RNA was incubated in buﬀer alone for 0 or 2 h. In the −UV lanes,

YoeB_Sa1-Y88ONBY was incubated with RNA for 2 h. In the +UV lanes, YoeB_Sa1-Y88ONBY was exposed to UV light for 2 min and incubated with

RNA for 0.5, 1, 1.5, or 2 h. (B) Primary sequences of Pentaprobe 7 and Pentaprobe 7 oligonucleotides PP7-1, PP7-2, and PP7-3. YoeB_Sa1cleavage

sites identiﬁed by MALDI-MS are denoted with arrows. (C) YoeB_Sa1consensus cleavage sequence calculated by WebLogo.⁷⁴

contamination. This result also suggests that YefM_Sa1inhibits

YoeB_Sa1at a 1:1 ratio, which is identical to the ratio of YefM_Ec

to YoeB_Ecrequired to inhibit YoeB_Ecin a similar in vitro assay.¹⁹

Comparison of the activity of decaged YoeB_Sa1with that of

YoeB_Sa1mutants and YefM_Sa1reveals that YoeB_Sa1-Y88ONBY

retains weak in vitro RNase activity, YoeB_Sa1-Y88F degrades

RNA very slowly, and no detectable RNase activity is observed

for YoeB_Sa1-Y88TAG or YefM_Sa1(Figure 3C).

YoeB_Ec, which cleaves RNA after purine residues with a

preference for adenosine.¹⁹

To identify the speciﬁc sequence recognized and cleaved by

YoeB_Sa1within Pentaprobe 7, cleavage of three overlapping

oligonucleotides spanning Pentaprobe 7 (Figure 4B) was

assessed by MALDI-MS. YoeB_Sa1cleaved each oligonucleotide

selectively after guanosine, leaving a 3′-cyclic phosphate on the

5′-RNA product. This suggests that YoeB_Sa1is a guanosine-

speciﬁc RNase in vitro and furthermore that the mechanism of

YoeB_Sa1RNA cleavage involves activation of the 2′-OH for

nucleophilic attack of the 3′-phosphodiester bond. Fragments

corresponding to both the 5′- and 3′-products were observed

for 5 of the 9 guanosines in PP7-1, 6 of 13 in PP7-2, and 1 of 4

in PP7-3. Most of the noncleaved guanosines were near the

termini of the oligonucleotides, which reduces the likelihood of

observing cleavage at these sites by MALDI because of the 2

kDa lower mass limit of the detector and a reduction in MALDI

sensitivity with an increase in oligonucleotide length. YoeB_Sa1

may also require a minimum number of residues 5′ or 3′ from

the cleavage site to bind and cleave RNA and thus may skip

guanosines near the termini of an oligonucleotide.

YoeB_Sa1Cleaves RNA after Guanosine Residues. The

Pentaprobes are a set of 12 plasmids that together encode every

possible combination of ﬁve nucleotides in sequences of ∼100

nucleotides per plasmid.⁴⁰The Pentaprobes have been used to

determine the sequence speciﬁcity of RNases with recognition

sequences of ﬁve or fewer nucleotides. Cleavage of each

Pentaprobe by the RNase of interest is assessed by PAGE.

Once cleaved Pentaprobes are identiﬁed, deconvolution occurs

via MALDI-MS analysis of cleavage of small oligonucleotides.²⁷

The RNase activity of YoeB_Sa1toward each of the 12

Pentaprobe RNA transcripts was assessed by denaturing

PAGE. YoeB_Sa1was least active toward Pentaprobe 1 and

most active toward Pentaprobes 4, 7, and 10 (Figure 4A; other

Pentaprobes not shown). Cleavage of Pentaprobe 7 was the

most dramatic, with rapid and signiﬁcant degradation of the

full-length parent band and the appearance of a number of

smaller discrete product bands, indicating that YoeB_Sa1cleaves

this Pentaprobe at multiple sites. The sequence of Pentaprobe 7

is rich in purine residues (Figure 4B), suggesting that the

sequence speciﬁcity of YoeB_Sa1might be similar to that of

In the consensus sequence cleaved by YoeB_Sa1, adenosine

residues precede and follow the guanosine at the cleavage site

(Figure 4C). This may be an artifact of oligonucleotide design

and selection, as most of the guanosines in Pentaprobe 7 are

preceded by one or more adenosines. However, it is also

possible that YoeB_Sa1prefers to cleave after guanosines in the

midst of purine-rich sequences. The fact that YoeB_Sa1cleaved

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Figure 5. Kinetic analysis of YoeB_Sa1using the ﬂuorometric assay. (A) Fluorogenic substrate design. “r” designates RNA nucleotides. (B) HPLC

traces of products of YoeB_Sa1activity: black for 30 μM intact substrate, green for 30 μM 5′-cleavage product, cyan for 30 μM 3′-cleavage product, and

blue for 30 μM substrate cleaved by 20 μM YoeB_Sa1after 5 h at 25 °C. Peak fractions from the blue trace were analyzed by MALDI-MS and are

labeled with the observed molecular masses of the 5′- and 3′-cleavage products (calculated molecular masses of 3115.0 Da and 2602.8 Da,

●

respectively). (C) Representative set of progress curves from one experiment. (D) Initial slopes of ﬂuorometric assay curves ( ) with a Hill ﬁt ().

Error bars represent the standard deviation (n = 3 separately puriﬁed batches of YoeB_Sa1, each assayed in technical triplicate on two diﬀerent days;

plotted data are the average of the average for each batch).

PP7-3 at only one of the three internal guanosines suggests that

the ability of YoeB_Sa1to cleave free mRNA may be inhibited by

secondary structure, as PP7-3 had more predicted secondary

structure than PP7-1 and PP7-2, which may have limited the

ability of YoeB_Sa1to access other potential cleavage sites in

PP7-3.

standards (Figure 5B), suggesting that cleavage occurred

primarily at guanosine, as predicted. MALDI-MS analysis of

fractions collected from the elution peaks conﬁrmed that the

majority of cleavage events occurred at guanosine (Figure S3 of

the Supporting Information). These results corroborate the

guanosine speciﬁcity of YoeB_Sa1ribosome-independent RNase

activity.¹⁹

To determine the kinetic parameters of YoeB_Sa1as a baseline

for future high-throughput screening eﬀorts, the RNase activity

of YoeB_Sa1toward the ﬂuorogenic substrate was assessed in

384-well plate format. YoeB_Sa1(5 μM) was incubated with a

range of concentrations of the ﬂuorogenic substrate (0.5−20

μM) to produce a set of progress curves (representative data

from one replicate of one experiment shown in Figure 5C). For

each experiment, a calibration curve was constructed by mixing

the two chimeric products resulting from YoeB_Sa1cleavage at a

1:1 molar ratio in the presence of YoeB_Sa1(5 μM). The

relationship between the measured ﬂuorescence and the

concentration of the cleavage products was linear up to 1.5

μM. This calibration curve was used to convert relative

ﬂuorescence units (RFU) at 530 nm to picomoles of cleaved

substrate. Ideally, initial rates would be measured immediately

following addition of YoeB_Sa1to the substrate. However, in

some experiments, a signiﬁcant increase in ﬂuorescence was not

Kinetic Characterization of YoeB_Sa1RNase Activity.

With the objective of developing a YoeB_Sa1substrate that could

be used to assess the activation of this RNase in a high-

throughput setting, a ﬂuorogenic oligonucleotide substrate was

designed based on the data presented in Figure 4. An analogous

design was previously used for the creation of a substrate for

the ribosome-independent RNase MazF.^41,42One of the

sequences from PP7 that was cleaved by YoeB_Sa1was converted

into a 15-mer chimeric oligonucleotide with eight internal RNA

residues surrounded by DNA nucleotides for stability (Figure

5A). Cleavage of this substrate was predicted to occur after the

central guanosine residue. A 6-carboxyﬂuorescein ﬂuorophore

(excitation at 495 nm and emission at 520 nm) and an Iowa

Black FQ quencher (absorbance maximum at 530 nm) were

appended to the 5′- and 3′-ends, respectively, of the

oligonucleotide. In the intact substrate, proximity allows the

quencher to absorb the ﬂuorescence emitted by the

ﬂuorophore. Cleavage of the substrate increases the distance

between the ﬂuorophore and the quencher, producing an

increase in ﬂuorescence that can be monitored spectrophoto-

metrically.

observed until ∼15 min after the addition of YoeB_Sa1

.

Consequently, the rates plotted in Figure 5D were determined

from the linear portion of the progress curves between 20 and

30 min.

The ability of YoeB_Sa1to cleave this substrate was assessed by

HPLC. The retention times of the products from YoeB_Sa1

activity overlapped with those of the independently synthesized

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Traditional Michaelis−Menten enzyme kinetics are charac-

terized by a hyperbolic rate, V, versus substrate concentration,

[S], curve that can be ﬁt by the equation

Information). As a result, the activity measured in the

ﬂuorometric assay is the sum of the rates at which YoeB_Sa1

cleaves after all of the RNA residues in the substrate. The

preference for guanosine over adenosine suggests that cleavage

after guanosine is faster than cleavage after adenosine. Thus, at

nonsaturating substrate concentrations, the observed rate will

be inﬂuenced primarily by cleavage after guanosine. As the

substrate concentration increases, the proportion of cleavage

events that are catalyzed after adenosine rather than guanosine

will also increase. Eventually, at supersaturating substrate

concentrations, the proportion of cleavage events at adenosine

will increase to the degree that it will cause a measurable

decrease in the overall rate, as observed in Figure 5D.

Consequently, V_maxfor guanosine-speciﬁc substrate cleavage

cannot be determined from the kinetic data. However,

assuming that cleavage after adenosine makes a limited

contribution to the measured rate at low substrate concen-

trations, ﬁtting the kinetic data at substrate concentrations

below those at which a measurable decrease in rate occurs will

allow extrapolation to an estimate for V_max. As it is possible that

cleavage after adenosine makes a non-negligible contribution to

the overall rate even at low substrate concentrations, this

approach will provide minimal estimates only for V_maxand K_m.

The Hill equation (eq 3) was originally derived to describe

the cooperative binding of ligands to a protein containing

multiple binding sites when one or more ligands are already

bound:

V

_max[S]

V =

K_m+ [S]

(1)

where V_maxis the maximal reaction velocity and K_mis the

Michaelis constant, equal to the substrate concentration at

which half-maximal activity is observed. This equation describes

a hyperbolic rate that increases linearly when [S] is low and

asymptotically approaches V_maxat high values of [S].^43,44The

rate data in Figure 5D reveal that the kinetics of YoeB_Sa1RNase

activity toward the ﬂuorogenic substrate are complex, with a

sigmoidal increase in rate up to 10 μM substrate, followed by a

marked decrease in rate at 15 and 20 μM substrate. The shape

of the rate data suggests that the activity of YoeB_Sa1is

inﬂuenced by positive cooperativity at low substrate concen-

trations and by substrate inhibition at high substrate

concentrations. However, careful consideration of the assay

conditions and the nature of the ﬂuorogenic substrate points to

alternative explanations for the apparent cooperativity and

substrate inhibition.

In traditional Michaelis−Menten enzyme kinetics, the

substrate must be present in signiﬁcant excess relative to the

enzyme so that the substrate concentration is not substantially

reduced by formation of the enzyme−substrate complex.⁴⁵In

the ﬂuorometric assay, however, YoeB_Sa1(5 μM) is present at a

concentration similar to that of the ﬂuorogenic substrate (0.5−

20 μM) in each reaction. Rather than Michaelis−Menten

conditions, these enzyme and substrate concentrations

resemble those of mutual depletion systems, in which

formation of the enzyme−substrate complex signiﬁcantly

reduces the concentrations of both the enzyme and the

substrate.⁴⁶Consequently, the classical Michaelis−Menten

equation (eq 1) cannot be used to ﬁt the kinetic data of

YoeB_Sa1, but this does not necessarily mean that YoeB_Sa1does

not follow Michaelis−Menten kinetics. Interestingly, it has been

shown that when the concentration of enzyme, [E]_T, is equal to

or greater than the K_m, enzymes that follow Michaelis−Menten

kinetics appear to have cooperative activity. The magnitude of

the apparent cooperativity increases as [E]_Tincreases relative to

K_m, causing a concomitant increase in the substrate

concentration ([S]_T)_0.5at which half-maximal velocity is

observed.⁴⁷This increase is given by eq 2:

[L]^h

(K_A)^h+ [L]^h

θ =

(3)

where θ is the ratio of occupied binding sites to total binding

sites, [L] is the ligand concentration, K_Ais the association

constant, and h is the Hill coeﬃcient, which quantiﬁes the

degree of cooperativity.⁴⁸The maximum possible value of h for

a perfectly cooperative protein is equal to the number of ligand-

binding sites. An h >1 indicates positive cooperativity, where

the rate of ligand binding is enhanced by already bound ligands.

An h <1 indicates negative cooperativity, where the rate of

ligand binding is reduced by already bound ligands. An h equal

to 1 indicates no cooperativity, where the rate of ligand binding

is unaﬀected by already bound ligands. If h = 1, the Hill

equation (eq 3) reduces to a form similar to that of the

Michaelis−Menten equation (eq 1).⁴⁴

In addition to ligand-binding proteins, many enzymes exhibit

cooperative enhancements or reductions in rate that result from

conformational changes induced by enzyme oligomerization or

the binding of allosteric eﬀectors. Another smaller but growing

class of monomeric enzymes with single ligand-binding sites

also exhibits cooperative behavior caused by conformational

transformations between two or more enzyme forms with

diﬀering substrate aﬃnities. If the rate of conformational

interconversion is slower than the rate of the reaction, substrate

binding cannot reach equilibrium, resulting in nonhyperbolic,

non-Michaelis−Menten kinetics.⁴⁹

Therefore, an alternative to mutual depletion as an

explanation for the sigmoidal kinetics of YoeB_Sa1is that its

activity is cooperative, which would require oligomerization,

allosteric eﬀector binding, or a slow conformational change.

Comparison of the gel ﬁltration elution volume of YoeB_Sa1with

those of known standards indicates that YoeB_Sa1is monomeric

under the conditions employed in this assay. Furthermore, the

assay buﬀer contains no known allosteric eﬀectors, indicating

[E]_T

2

([S]_T)_0.5

=

+ K_m

(2)

Eq 2 can be used to calculate K_mfrom a plot of reaction

velocity versus substrate concentration for enzymes operating

under mutual depletion conditions.^46,47

However, the ability to calculate K_musing eq 2 depends on

knowledge of V_max, which is required to estimate ([S]_T)_0.5. The

reduction in rate observed at 15 and 20 μM substrate precludes

extrapolation to V_maxfrom the kinetic plot and suggests the

inﬂuence of substrate inhibition. However, this behavior is

likely an artifact of the substrate design, in which a central

guanosine RNA residue is ﬂanked on both sides by three or

four adenosine RNA residues (Figure 5A). These RNA residues

could not be removed without abolishing the activity of YoeB_Sa1

toward the substrate. Although YoeB_Sa1cleaves primarily after

guanosine, minor cleavage is also observed after the

neighboring adenosines (Figure S3 of the Supporting

197

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Biochemistry

Article

that oligomerization and eﬀector binding are unlikely to be

responsible for the sigmoidal shape of the kinetic data. On the

other hand, the crystal structures of YefM-YoeB_Ecand YoeB_Ec

reveal that YefM_Ecinduces a conformational change in the

active site of YoeB_Ec,¹⁹which suggests that this area may be

conformationally ﬂexible. Moreover, as the primary cellular

function of YoeB homologues appears to be ribosome-

dependent RNase activity,^{20,28,50−52}the conformation of

YoeB_Sa1that cleaves RNA in a ribosome-dependent fashion

may be distinct from the conformations that exist free in

solution and cleave free mRNA. Slow conformational change is

therefore a plausible explanation for the observed cooperativity

in the kinetics of YoeB_Sa1activity toward the ﬂuorogenic

substrate.

Regardless of whether the sigmoidal shape of the kinetic data

results from mutual depletion or monomeric cooperativity, a

modiﬁed version of the Hill equation can be used to provide a

minimal estimate for V_max. The simplest and most common

method applied in the analysis of the kinetics of cooperative

monomeric enzymes with single ligand-binding sites is a

combination of the Hill and Michaelis−Menten equations:

nuclease in an inactive, nontoxic form in E. coli,³⁶suggesting

that incorporation of a photocaged version of a critical catalytic

residue via UAA mutagenesis may provide a general strategy for

allowing expression and characterization of toxic enzymes,

provided that an appropriate aaRS/tRNA pair is available. For

example, Y87 in YoeB_Sa2is homologous to Y88 in YoeB_Sa1and

appears to be important for YoeB_Sa2toxicity in E. coli

(unpublished data). Consequently, incorporation of ONBY

into YoeB_Sa2using UAA mutagenesis may allow expression of

YoeB_Sa2in E. coli for in vitro characterization as well.

Furthermore, aaRS/tRNA pairs have also been developed for

photocaged cysteine, serine, and lysine.³¹Photocaged versions

of aspartate, glutamate, glycine, alanine, and histidine have been

synthesized,⁵³⁻⁵⁵but corresponding aaRS/tRNA pairs do not

yet exist. A list of residues identiﬁed by mutagenesis and

structural data to be responsible for the activity and/or toxicity

of a number of TA system toxins is provided in Table 1.

Table 1. Residues Responsible for the Activity and/or

Toxicity of Various TA System Toxins

residues implicated in activity

toxin

source(s)

and/or toxicity

V

_max[S]^h

(K_H)^h+ [S]^h

CcdB^57,58Aliivibrio fischeri, plasmid F Trp99, Gly100, Ile101

V =

Doc⁵⁹

Plasmid P1

Neisseria gonorrheae

Proteus spp.

E. coli

His66, Asp70

(4)

FitB⁶⁰

Asp5, Glu42, Asp104, Asp122

His92

where V_maxis the maximal reaction velocity, [S] is the substrate

concentration, K_His the substrate concentration at which half-

maximal activity is observed, and h is the Hill coeﬃcient.⁴⁹If

YoeB_Sa1is cooperative, h quantiﬁes the degree of cooperativity,

and K_His equivalent to K_m. On the other hand, if mutual

depletion occurs, h quantiﬁes the deviation from hyperbolic

Michaelis−Menten kinetics, and K_mcan be calculated using eq

2 with K_H= ([S]_T)_0.5. Eq 4 was therefore used to ﬁt the kinetic

data for substrate concentrations between 0.5 and 10 μM to

allow extrapolation to a minimal estimate for V_max. This gave a

HigB⁶¹

HipA⁶²

Kid⁶³

MazF⁶⁴

MqsR⁶⁵

PemK⁶⁶

PezT^67,68

Ser150, Asp209, Asp332

Asp75, Arg73, His17

Glu24, His28

E. coli

Lys56, Gln68, Tyr81, Lys96

Glu24, His59

Bacillus anthracis

Streptococcus pneumoniae

Lys45, Asp66, Thr118, Arg157,

Arg170

RelE⁵⁶

E. coli

Arg61, Arg81, Tyr87

VapC-3⁶⁹Mycobacterium tuberculosis

VapC-5⁷⁰M. tuberculosis

Asp9, Glu43, Asp99, Asp117

Asp26, Glu57, Asp115, Asp135

Asp7, Glu42, Asp98

V_maxof 1.05 0.03 pmol/min and a K_Hof 4.9 0.2 μM, with

VapC⁷¹

YafQ⁷²

Shigella flexneri

E. coli

an h of 2.10 0.09 (Figure 5D). Using K_H= ([S]_T)_0.5in eq 2

gives a K_mof 2.4 0.2 μM.

His50, His63, Asp67, Trp68,

Arg83, His87, Phe91

YoeB¹⁹

ζ⁷³

E. coli

Glu46, Arg65, His83, Tyr88

Lys46, Asp67

DISCUSSION

■

Streptococcus pyogenes

The YoeB_Sa1and YoeB_Sa2toxins from S. aureus were previously

found to induce growth arrest and to exhibit cellular RNase

activity identical to that of YoeB_Ecupon overexpression in E.

coli.²⁸As YoeB_Ecpossesses weak ribosome-independent RNase

activity with a preference for purine nucleotides in vitro,¹⁹the

possibility that YoeB_Sa1also has ribosome-independent RNase

activity was investigated.

Notably, each characterized toxin possesses one or more

residues corresponding to an accessible photocaged amino acid.

Introduction of aaRS/tRNA pairs speciﬁc for photocaged

aspartate and histidine could allow for the general application of

the photocaging strategy for expressing these proteins and

studying their activities and cellular roles in vitro and in cells.

YoeB_Ecbelongs to the RelE structural family. RelE is a

ribosome-dependent RNase toxin that is active only in the

presence of the ribosome. In the ribosome-bound structure of

RelE, Y87 of RelE stacks with the residue upstream of the

cleavage site, and its hydroxyl is the closest functional group to

the 2′-OH of the cleaved residue, suggesting a dual role in

substrate binding and catalysis. Y87 is hypothesized to act as a

general base through hydrogen bonding to a water molecule,

causing the deprotonation and activation of the 2′-OH for

attack of the 3′-phosphodiester bond.⁵⁶The role of Y88 in the

catalytic mechanism of YoeB_Sa1is currently unknown and may

be diﬀerent in the presence and absence of the ribosome.

Mutation of Y88 in YoeB_Sa1-Y88TAG, YoeB_Sa1-Y88F, and

YoeB_Sa1-Y88ONBY suﬃciently reduced cellular toxicity to

allow overexpression in E. coli (Figure 2). The relative yields

One of the most signiﬁcant obstacles to characterizing the

activities and determining the structures of toxic proteins is the

diﬃculty of overexpression in heterologous hosts such as E. coli.

Many TA system toxins cannot be obtained in suﬃcient

quantities for characterization because their overexpression

arrests growth and/or kills the cells. After the failure of multiple

strategies to provide pure, full-length, functional YoeB_Sa1, the

discovery that Y88 is required for YoeB_Sa1toxicity in E. coli led

to the utilization of UAA mutagenesis to replace this residue

with the photocaged amino acid ONBY, which reduced the

toxicity of YoeB_Sa1to a suﬃcient degree to allow overexpression

in E. coli.

To the best of our knowledge, this is the ﬁrst reported use of

in vivo UAA mutagenesis to facilitate the isolation and

characterization of a toxin from a TA system. Incorporation

of ONBY has also been used to overexpress a toxic zinc-ﬁnger

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Biochemistry

Article

of YoeB_Sa1-Y88TAG (5 mg/L) and YoeB_Sa1-Y88F (2.5 mg/L)

under identical expression conditions indicate that the Y88TAG

mutation renders YoeB_Sa1less toxic than the Y88F mutation,

which suggests that both the aromatic ring and hydroxyl

functional group of Y88 contribute to cellular RNase activity.

Similarly, YoeB_Sa1-Y88F and YoeB_Sa1-Y88ONBY retained the

capacity to cleave free mRNA in vitro, albeit at a signiﬁcantly

interactions with aromatic side chains. Ribosomal nucleotides

may supply these interactions to facilitate proper orientation

and cleavage of bound mRNA when YoeB binds in the

ribosome, as in the crystal structure of the RelE−ribosome

complex.⁵⁶The absence of the C-terminal extension and

ribosomal interactions may explain the low aﬃnity and slow

rate of the RNase activity of free YoeB_Sa1

.

Although YoeB_Ecwas originally found to have ribosome-

independent RNase activity in in vitro experiments,¹⁹the only

cellular RNase activity observed for YoeB homologues has been

ribosome-dependent.^{20,28,50−52}This suggests that the ribo-

some-independent RNase activity of YoeB_Sa1described here

may have little relevance in cells apart from the discovery of an

artiﬁcial activator capable of stabilizing a ribosome-independent

RNase conformation of YoeB_Sa1. Then activation of YoeB_Sa1

from the YefM−YoeB_Sa1complex in S. aureus could lead to

global nonspeciﬁc RNA degradation via the guanosine-speciﬁc

ribosome-independent RNase activity reported here, in

addition to translation inhibition via the ribosome-dependent

RNase activity described previously,²⁸causing growth arrest

and possibly eventual death. The ﬂuorometric assay developed

for YoeB_Sa1ribosome-independent RNase activity will facilitate

high-throughput screens of chemical and peptide libraries to

identify molecules capable of activating YoeB_Sa1via prevention

of YefM-YoeB_Sa1complex formation or disruption of the

complex.

reduced rate and to a much lesser extent than decaged YoeB_Sa1

,

while the RNase activity of YoeB_Sa1-Y88TAG was almost

completely abolished (Figure 3C). These results suggest that

both the aromatic ring and hydroxyl functional group of Y88

also contribute to the in vitro activity of YoeB_Sa1, although the

exact role played by each of these moieties in ribosome-

independent RNase activity remains unknown.

YoeB_Ecwas previously found to possess purine-speciﬁc

ribosome-independent RNase activity with a preference for

adenosine over guanosine.¹⁹In contrast, YoeB_Sa1preferentially

cleaves free mRNA after guanosine and to a much lesser extent

after adenosine (Figures 4B and 5B and Figure S3 of the

Supporting Information). This speciﬁcity was used to design a

chimeric ﬂuorogenic substrate to study the kinetics of YoeB_Sa1

activity. The resulting kinetic data show a sigmoidal increase in

rate at low substrate concentrations followed by a decrease at

high substrate concentrations. There are several plausible

explanations for these deviations from standard hyperbolic

Michaelis−Menten kinetics. First, the sigmoidal shape of the

kinetic data may be an artifact of the high enzyme

concentration used in these experiments,⁴⁷as it was necessary

for the concentrations of YoeB_Sa1and the ﬂuorogenic substrate

to be roughly equal (5 and 0.5−20 μM, respectively) to observe

YoeB_Sa1activity using the ﬂuorometric assay. Alternatively, a

slow transition between inactive and active enzyme con-

formations may be required for catalysis, resulting in a failure to

achieve a steady state of the bound substrate due to the faster

speed of enzymatic catalysis. The seeming substrate inhibition

is likely an artifact resulting from the unavoidable inclusion of

adenosine RNA residues bordering guanosine in the substrate.

Cleavage at these adenosine residues is hypothesized to cause a

measurable reduction in rate at high substrate concentrations,

necessitating the exclusion of these points from the kinetic

analysis. It should be noted that these explanations are not

mutually exclusive, and two or all three could conceivably be

involved in the complex kinetics of YoeB_Sa1activity toward the

ﬂuorogenic substrate. The Hill equation was used to ﬁt the

sigmoidal portion of the kinetic data to provide minimal

estimates for V_maxand K_m. Fitting the kinetic data with the Hill

equation (eq 4) for cooperative enzymatic activity gave a V_max

of 1.05 0.03 pmol/min, a K_Hof 4.9 0.2 μM, and a Hill

coeﬃcient h of 2.10 0.09 (Figure 5D). Using eq 2 to convert

K_Hto K_mgives a K_mof 2.4 0.2 μM.

ASSOCIATED CONTENT

■

S

* Supporting Information

Two tables listing the primers used in this study (Table S1) and

the results of the PCR screen for yef M-yoeB_Sa1and yefM-yoeB_Sa2

in MRSA clinical isolates (Table S2) as well as three ﬁgures

showing the sequence alignments of yefM-yoeB_Sa1(Figure S1)

and yefM-yoeB_Sa2(Figure S2) PCR products and the MALDI

spectra of the ﬂuorogenic substrate cleavage products (Figure

S3). This material is available free of charge via the Internet at

http://pubs.acs.org.

AUTHOR INFORMATION

■

Corresponding Author

*E-mail: hergenro@illinois.edu. Phone: (217) 333-0363.

Funding

This work was supported by National Institutes of Health

(NIH) Grant 2R01-GM068385 to P.J.H. A.S.L. was partially

supported by an NIH Chemistry-Biology Interface Training

Grant (National Institute of General Medical Sciences Grant

5T32-GM070421).

Notes

The authors declare no competing ﬁnancial interest.

The relatively low aﬃnity and slow rate observed for YoeB_Sa1

cleavage of free mRNA suggest that ribosome-independent

RNase activity is unlikely to make a signiﬁcant contribution to

the activity of YoeB_Sa1in cells. This is consistent with prior

evidence of only ribosome-dependent RNase activity upon

overexpression of YoeB_Sa1in E. coli.²⁸YoeB_Ecis structurally

homologous to RNase Sa from Streptomyces aureofaciens and

Barnase from Bacillus amyloliquefaciens, both of which are

guanosine-speciﬁc RNases,¹⁹and the catalytic Glu-Arg-His triad

found in these and other microbial guanosine-speciﬁc RNases is

conserved in YoeB_Ec, YoeB_Sa1, and YoeB_Sa2. However, YoeB

homologues lack a C-terminal extension that orients the

nucleotide downstream of the cleavage site via stacking

ACKNOWLEDGMENTS

■

We thank Prof. Peter Schultz at the Scripps Research Institute

(La Jolla, CA) for providing pEVOL-ONBY and Prof. Joel

Mackay at the University of Sydney (Sydney, Australia) for

providing the Pentaprobes. We also thank Prof. Alex Deiters

and Jessica Torres Kolbus at North Carolina State University

(Raleigh, NC) for advice regarding the synthesis of ONBY.

ABBREVIATIONS

■

TA, toxin−antitoxin; RNase, ribonuclease; ONBY, o-nitro-

benzyl tyrosine.

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REFERENCES

Article

(21) Williams, J. J., Halvorsen, E. M., Dwyer, E. M., DiFazio, R. M.,

and Hergenrother, P. J. (2011) Toxin-antitoxin (TA) systems are

prevalent and transcribed in clinical isolates of Pseudomonas aeruginosa

and methicillin-resistant Staphylococcus aureus. FEMS Microbiol. Lett.

322, 41−50.

(22) Moritz, E. M., and Hergenrother, P. J. (2007) Toxin-antitoxin

systems are ubiquitous and plasmid-encoded in vancomycin-resistant

enterococci. Proc. Natl. Acad. Sci. U.S.A. 104, 311−316.

(23) Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994)

CLUSTAL W: Improving the sensitivity of progressive multiple

sequence alignment through sequence weighting, position-specific gap

penalties and weight matrix choice. Nucleic Acids Res. 22, 4673−4680.

(24) Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman,

D. J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403−

410.

(25) Miller, J. C., Silverman, S. K., England, P. M., Dougherty, D. A.,

and Lester, H. A. (1998) Flash decaging of tyrosine sidechains in an

ion channel. Neuron 20, 619−624.

(26) Wessel, D., and Flugge, U. I. (1984) A method for the

quantitative recovery of protein in dilute solution in the presence of

detergents and lipids. Anal. Biochem. 138, 141−143.

(27) McKenzie, J. L., Duyvestyn, J. M., Smith, T., Bendak, K.,

Mackay, J., Cursons, R., Cook, G. M., and Arcus, V. L. (2012)

Determination of ribonuclease sequence-specificity using Pentaprobes

and mass spectrometry. RNA 18, 1267−1278.

(28) Yoshizumi, S., Zhang, Y., Yamaguchi, Y., Chen, L., Kreiswirth, B.

N., and Inouye, M. (2009) Staphylococcus aureus YoeB homologues

inhibit translation initiation. J. Bacteriol. 191, 5868−5872.

(29) Wang, L., Brock, A., Herberich, B., and Schultz, P. G. (2001)

Expanding the genetic code of Escherichia coli. Science 292, 498−500.

(30) Liu, C. C., and Schultz, P. G. (2010) Adding new chemistries to

the genetic code. Annu. Rev. Biochem. 79, 413−444.

(31) Davis, L., and Chin, J. W. (2012) Designer proteins:

Applications of genetic code expansion in cell biology. Nat. Rev.

Mol. Cell Biol. 13, 168−182.

(32) Deiters, A., Groff, D., Ryu, Y., Xie, J., and Schultz, P. G. (2006)

A genetically encoded photocaged tyrosine. Angew. Chem., Int. Ed. 45,

2728−2731.

(33) Chou, C., Young, D. D., and Deiters, A. (2009) A light-activated

DNA polymerase. Angew. Chem., Int. Ed. 48, 5950−5953.

(34) Edwards, W. F., Young, D. D., and Deiters, A. (2009) Light-

activated Cre recombinase as a tool for the spatial and temporal

control of gene function in mammalian cells. ACS Chem. Biol. 4, 441−

445.

(35) Chou, C., Young, D. D., and Deiters, A. (2010) Photocaged T7

RNA polymerase for the light activation of transcription and gene

function in pro- and eukaryotic cells. ChemBioChem 11, 972−977.

(36) Chou, C., and Deiters, A. (2011) Light-activated gene editing

with a photocaged zinc-finger nuclease. Angew. Chem., Int. Ed. 50,

6839−6842.

(37) Zhao, H., Sterner, E. S., Coughlin, E. B., and Theato, P. (2012)

o-Nitrobenzyl alcohol derivatives: Opportunities in polymer and

materials science. Macromolecules 45, 1723−1736.

(38) Young, T. S., Ahmad, I., Yin, J. A., and Schultz, P. G. (2010) An

enhanced system for unnatural amino acid mutagenesis in E. coli. J.

Mol. Biol. 395, 361−374.

■

(1) Pandey, D. P., and Gerdes, K. (2005) Toxin-antitoxin loci are

highly abundant in free-living but lost from host-associated

prokaryotes. Nucleic Acids Res. 33, 966−976.

(2) Yamaguchi, Y., Park, J. H., and Inouye, M. (2011) Toxin-antitoxin

systems in bacteria and archaea. Annu. Rev. Genet. 45, 61−79.

(3) Masuda, H., Tan, Q., Awano, N., Wu, K. P., and Inouye, M.

(2012) YeeU enhances the bundling of cytoskeletal polymers of MreB

and FtsZ, antagonizing the CbtA (YeeV) toxicity in Escherichia coli.

Mol. Microbiol. 84, 979−989.

(4) Wang, X., Lord, D. M., Cheng, H. Y., Osbourne, D. O., Hong, S.

H., Sanchez-Torres, V., Quiroga, C., Zheng, K., Herrmann, T., Peti,

W., Benedik, M. J., Page, R., and Wood, T. K. (2012) A new type V

toxin-antitoxin system where mRNA for toxin GhoT is cleaved by

antitoxin GhoS. Nat. Chem. Biol. 8, 855−861.

(5) Gerdes, K., Christensen, S. K., and Lobner-Olesen, A. (2005)

Prokaryotic toxin-antitoxin stress response loci. Nat. Rev. Microbiol. 3,

371−382.

(6) Gerdes, K., Rasmussen, P. B., and Molin, S. (1986) Unique type

of plasmid maintenance function: Postsegregational killing of plasmid-

free cells. Proc. Natl. Acad. Sci. U.S.A. 83, 3116−3120.

(7) Leplae, R., Geeraerts, D., Hallez, R., Guglielmini, J., Dreze, P., and

Van Melderen, L. (2011) Diversity of bacterial type II toxin-antitoxin

systems: A comprehensive search and functional analysis of novel

families. Nucleic Acids Res. 39, 5513−5525.

(8) Magnuson, R. D. (2007) Hypothetical functions of toxin-

antitoxin systems. J. Bacteriol. 189, 6089−6092.

(9) Schuster, C. F., and Bertram, R. (2013) Toxin-antitoxin systems

are ubiquitous and versatile modulators of prokaryotic cell fate. FEMS

Microbiol. Lett. 340, 73−85.

(10) 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.

(11) Engelberg-Kulka, H., Sat, B., Reches, M., Amitai, S., and Hazan,

R. (2004) Bacterial programmed cell death systems as targets for

antibiotics. Trends Microbiol. 12, 66−71.

(12) DeNap, J. C., and Hergenrother, P. J. (2005) Bacterial death

comes full circle: Targeting plasmid replication in drug-resistant

bacteria. Org. Biomol. Chem. 3, 959−966.

(13) Williams, J. J., and Hergenrother, P. J. (2008) Exposing plasmids

as the Achilles’ heel of drug-resistant bacteria. Curr. Opin. Chem. Biol.

12, 389−399.

(14) Williams, J. J., and Hergenrother, P. J. (2012) Artificial

activation of toxin-antitoxin systems as an antibacterial strategy. Trends

Microbiol. 20, 291−298.

(15) Diekema, D. J., Pfaller, M. A., Schmitz, F. J., Smayevsky, J., Bell,

J., Jones, R. N., and Beach, M. (2001) Survey of infections due to

Staphylococcus species: Frequency of occurrence and antimicrobial

susceptibility of isolates collected in the United States, Canada, Latin

America, Europe, and the Western Pacific region for the SENTRY

Antimicrobial Surveillance Program, 1997−1999. Clin. Infect. Dis. 32

(Suppl. 2), S114−S132.

(16) Donegan, N. P., and Cheung, A. L. (2009) Regulation of the

mazEF toxin-antitoxin module in Staphylococcus aureus and its impact

on sigB expression. J. Bacteriol. 191, 2795−2805.

(17) Donegan, N. P., Thompson, E. T., Fu, Z., and Cheung, A. L.

(2010) Proteolytic regulation of toxin-antitoxin systems by ClpPC in

Staphylococcus aureus. J. Bacteriol. 192, 1416−1422.

(18) Makarova, K. S., Wolf, Y. I., and Koonin, E. V. (2009)

Comprehensive comparative-genomic analysis of type 2 toxin-antitoxin

systems and related mobile stress response systems in prokaryotes.

Biol. Direct 4, 19.

(19) Kamada, K., and Hanaoka, F. (2005) Conformational change in

the catalytic site of the ribonuclease YoeB toxin by YefM antitoxin.

Mol. Cell 19, 497−509.

(20) Zhang, Y., and Inouye, M. (2009) The inhibitory mechanism of

protein synthesis by YoeB, an Escherichia coli toxin. J. Biol. Chem. 284,

6627−6638.

(39) Karancsi, T., and Slegel, P. (1999) Reliable molecular mass

determination of aromatic nitro compounds: Elimination of gas-phase

reduction occurring during atmospheric pressure chemical ionization.

J. Mass Spectrom. 34, 975−977.

(40) Bendak, K., Loughlin, F. E., Cheung, V., O’Connell, M. R.,

Crossley, M., and Mackay, J. P. (2012) A rapid method for assessing

the RNA-binding potential of a protein. Nucleic Acids Res. 40, e105.

(41) Wang, N. R., and Hergenrother, P. J. (2007) A continuous

fluorometric assay for the assessment of MazF ribonuclease activity.

Anal. Biochem. 371, 173−183.

(42) van Rensburg, J. J., and Hergenrother, P. J. (2013) Detection of

endogenous MazF enzymatic activity in Staphylococcus aureus. Anal.

Biochem. 443, 81−87.

200

dx.doi.org/10.1021/bi4008098 | Biochemistry 2014, 53, 188−201

Biochemistry

Article

(43) Michaelis, L., Menten, M. L., Johnson, K. A., and Goody, R. S.

(2011) The original Michaelis constant: Translation of the 1913

Michaelis-Menten paper. Biochemistry 50, 8264−8269.

Boelens, R. (2006) Model for RNA binding and the catalytic site of the

RNase Kid of the bacterial parD toxin-antitoxin system. J. Mol. Biol.

357, 115−126.

(64) Li, G. Y., Zhang, Y., Chan, M. C., Mal, T. K., Hoeflich, K. P.,

Inouye, M., and Ikura, M. (2006) Characterization of dual substrate

binding sites in the homodimeric structure of Escherichia coli mRNA

interferase MazF. J. Mol. Biol. 357, 139−150.

(65) Brown, B. L., Grigoriu, S., Kim, Y., Arruda, J. M., Davenport, A.,

Wood, T. K., Peti, W., and Page, R. (2009) Three dimensional

structure of the MqsR:MqsA complex: A novel TA pair comprised of a

toxin homologous to RelE and an antitoxin with unique properties.

PLoS Pathog. 5, e1000706.

(66) Agarwal, S., Mishra, N. K., Bhatnagar, S., and Bhatnagar, R.

(2010) PemK toxin of Bacillus anthracis is a ribonuclease: An insight

into its active site, structure, and function. J. Biol. Chem. 285, 7254−

7270.

(44) Nelson, D. L., Lehninger, A. L., and Cox, M. M. (2008)

Lehninger Principles of Biochemistry, 5th ed., W. H. Freeman, New York.

(45) Schnell, S., and Maini, P. K. (2003) A century of enzyme

kinetics: Reliability of the K_mand V_maxestimates. Comments on

Theoretical Biology 8, 169−187.

(46) Griffiths, J. R. (1979) Steady-state enzyme kinetics in mutual

depletion systems. Biochem. Soc. Trans. 7, 429−439.

(47) Laurent, M., and Kellershohn, N. (1984) Apparent co-

operativity for highly concentrated Michaelian and allosteric enzymes.

J. Mol. Biol. 174, 543−555.

(48) Hill, A. V. (1910) The possible effects of the aggregation of the

molecules of haemoglobin on its dissociation curves. J. Physiol. 40, iv−

vii.

(67) Khoo, S. K., Loll, B., Chan, W. T., Shoeman, R. L., Ngoo, L.,

Yeo, C. C., and Meinhart, A. (2007) Molecular and structural

characterization of the PezAT chromosomal toxin-antitoxin system of

the human pathogen Streptococcus pneumoniae. J. Biol. Chem. 282,

19606−19618.

(49) Porter, C. M., and Miller, B. G. (2012) Cooperativity in

monomeric enzymes with single ligand-binding sites. Bioorg. Chem. 43,

44−50.

(50) Halvorsen, E. M., Williams, J. J., Bhimani, A. J., Billings, E. A.,

and Hergenrother, P. J. (2011) Txe, an endoribonuclease of the

enterococcal Axe-Txe toxin-antitoxin system, cleaves mRNA and

inhibits protein synthesis. Microbiology 157, 387−397.

(51) Sevillano, L., Diaz, M., Yamaguchi, Y., Inouye, M., and

Santamaria, R. I. (2012) Identification of the first functional toxin-

antitoxin system in Streptomyces. PLoS One 7, e32977.

(52) Nolle, N., Schuster, C. F., and Bertram, R. (2013) Two

paralogous yef M-yoeB loci from Staphylococcus equorum encode

functional toxin-antitoxin systems. Microbiology 159 (Part 8), 1575−

1585.

(53) Wilcox, M., Viola, R. W., Johnson, K. W., Billington, A. P.,

Carpenter, B. K., Mccray, J. A., Guzikowski, A. P., and Hess, G. P.

(1990) Synthesis of photolabile precursors of amino-acid neuro-

transmitters. J. Org. Chem. 55, 1585−1589.

(54) Zhang, Z., Papageorgiou, G., Corrie, J. E. T., and Grewer, C.

(2007) Pre-steady-state currents in neutral amino acid transporters

induced by photolysis of a new caged alanine derivative. Biochemistry

46, 3872−3880.

(55) Nakayama, K., Heise, I., Gorner, H., and Gartner, W. (2011)

Peptide release upon photoconversion of 2-nitrobenzyl compounds

into nitroso derivatives. Photochem. Photobiol. 87, 1031−1035.

(56) Neubauer, C., Gao, Y. G., Andersen, K. R., Dunham, C. M.,

Kelley, A. C., Hentschel, J., Gerdes, K., Ramakrishnan, V., and

Brodersen, D. E. (2009) The structural basis for mRNA recognition

and cleavage by the ribosome-dependent endonuclease RelE. Cell 139,

1084−1095.

(57) Bahassi, E. M., Salmon, M. A., Van Melderen, L., Bernard, P.,

and Couturier, M. (1995) F plasmid CcdB killer protein: ccdB gene

mutants coding for non-cytotoxic proteins which retain their

regulatory functions. Mol. Microbiol. 15, 1031−1037.

(68) Sedwick, C. (2011) PezT: A bacterial suicide gene. PLoS Biol. 9,

e1001036.

(69) Min, A. B., Miallau, L., Sawaya, M. R., Habel, J., Cascio, D., and

Eisenberg, D. (2012) The crystal structure of the Rv0301-Rv0300

VapBC-3 toxin-antitoxin complex from M. tuberculosis reveals a Mg²⁺

ion in the active site and a putative RNA-binding site. Protein Sci. 21,

1754−1767.

(70) Miallau, L., Faller, M., Chiang, J., Arbing, M., Guo, F., Cascio,

D., and Eisenberg, D. (2009) Structure and proposed activity of a

member of the VapBC family of toxin-antitoxin systems. VapBC-5

from Mycobacterium tuberculosis. J. Biol. Chem. 284, 276−283.

(71) Dienemann, C., Boggild, A., Winther, K. S., Gerdes, K., and

Brodersen, D. E. (2011) Crystal structure of the VapBC toxin-

antitoxin complex from Shigella flexneri reveals a hetero-octameric

DNA-binding assembly. J. Mol. Biol. 414, 713−722.

(72) Armalyte, J., Jurenaite, M., Beinoraviciute, G., Teiserskas, J., and

Suziedeliene, E. (2012) Characterization of Escherichia coli dinJ-yaf Q

toxin-antitoxin system using insights from mutagenesis data. J.

Bacteriol. 194, 1523−1532.

(73) Meinhart, A., Alonso, J. C., Strater, N., and Saenger, W. (2003)

Crystal structure of the plasmid maintenance system ε/ζ: Functional

mechanism of toxin ζ and inactivation by ε₂ζ₂complex formation.

Proc. Natl. Acad. Sci. U.S.A. 100, 1661−1666.

(74) Crooks, G. E., Hon, G., Chandonia, J. M., and Brenner, S. E.

(2004) WebLogo: A sequence logo generator. Genome Res. 14, 1188−

1190.

(58) Loris, R., Dao-Thi, M. H., Bahassi, E. M., Van Melderen, L.,

Poortmans, F., Liddington, R., Couturier, M., and Wyns, L. (1999)

Crystal structure of CcdB, a topoisomerase poison from E. coli. J. Mol.

Biol. 285, 1667−1677.

(59) Magnuson, R., and Yarmolinsky, M. B. (1998) Corepression of

the P1 addiction operon by Phd and Doc. J. Bacteriol. 180, 6342−6351.

(60) Mattison, K., Wilbur, J. S., So, M., and Brennan, R. G. (2006)

Structure of FitAB from Neisseria gonorrhoeae bound to DNA reveals a

tetramer of toxin-antitoxin heterodimers containing pin domains and

ribbon-helix-helix motifs. J. Biol. Chem. 281, 37942−37951.

(61) Hurley, J. M., and Woychik, N. A. (2009) Bacterial toxin HigB

associates with ribosomes and mediates translation-dependent mRNA

cleavage at A-rich sites. J. Biol. Chem. 284, 18605−18613.

(62) 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.

(63) Kamphuis, M. B., Bonvin, A. M., Monti, M. C., Lemonnier, M.,

Munoz-Gomez, A., van den Heuvel, R. H., Diaz-Orejas, R., and

201

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Light activation of staphylococcus aureus toxin YoeBSa 1 reveals guanosine-specific endoribonuclease activity

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