Protein cysteine phosphorylation of SarA/MgrA family transcriptional regulators mediates bacterial virulence and antibiotic resistance.
ABSTRACT Protein posttranslational modifications (PTMs), particularly phosphorylation, dramatically expand the complexity of cellular regulatory networks. Although cysteine (Cys) in various proteins can be subject to multiple PTMs, its phosphorylation was previously considered a rare PTM with almost no regulatory role assigned. We report here that phosphorylation occurs to a reactive cysteine residue conserved in the staphylococcal accessary regulator A (SarA)/MarR family global transcriptional regulator A (MgrA) family of proteins, and is mediated by the eukaryotic-like kinase-phosphatase pair Stk1-Stp1 in Staphylococcus aureus. Cys-phosphorylation is crucial in regulating virulence determinant production and bacterial resistance to vancomycin. Cell wall-targeting antibiotics, such as vancomycin and ceftriaxone, inhibit the kinase activity of Stk1 and lead to decreased Cys-phosphorylation of SarA and MgrA. An in vivo mouse model of infection established that the absence of stp1, which results in elevated protein Cys-phosphorylation, significantly reduces staphylococcal virulence. Our data indicate that Cys-phosphorylation is a unique PTM that can play crucial roles in bacterial signaling and regulation.
- SourceAvailable from: utah.eduCell 07/1993; 73(5):857-71. · 31.96 Impact Factor
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ABSTRACT: The diversity of distinct covalent forms of proteins (the proteome) greatly exceeds the number of proteins predicted by DNA coding capacities owing to directed posttranslational modifications. Enzymes dedicated to such protein modifications include 500 human protein kinases, 150 protein phosphatases, and 500 proteases. The major types of protein covalent modifications, such as phosphorylation, acetylation, glycosylation, methylation, and ubiquitylation, can be classified according to the type of amino acid side chain modified, the category of the modifying enzyme, and the extent of reversibility. Chemical events such as protein splicing, green fluorescent protein maturation, and proteasome autoactivations also represent posttranslational modifications. An understanding of the scope and pattern of the many posttranslational modifications in eukaryotic cells provides insight into the function and dynamics of proteome compositions.Angewandte Chemie International Edition 01/2006; 44(45):7342-72. · 13.73 Impact Factor
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ABSTRACT: PCR reactions were carried out on the genomic DNA of M. xanthus, a soil bacterium capable of differentiation to form fruiting bodies, using oligonucleotides representing highly conserved regions of eukaryotic protein serine/threonine kinases. A gene (pkn1) thus cloned contains an ORF of 693 amino acid residues whose amino-terminal domain shows significant sequence similarity with the catalytic domain of eukaryotic protein serine/threonine kinases. The pkn1 gene was overexpressed in E. coli, and the gene product has been found to be autophosphorylated at both serine and threonine residues. The expression of pkn1 is developmentally regulated to start immediately before spore formation. When pkn1 is deleted, differentiation starts prematurely, resulting in poor spore production. These results indicate that the protein serine/threonine kinase plays an important role in the onset of proper differentiation.Cell 12/1991; 67(5):995-1006. · 31.96 Impact Factor
Protein cysteine phosphorylation of SarA/MgrA family
transcriptional regulators mediates bacterial virulence
and antibiotic resistance
Fei Suna, Yue Dingb, Quanjiang Jia, Zhongjie Liangb, Xin Denga, Catherine C. L. Wongc, Chengqi Yia, Liang Zhanga,
Sherrie Xiea, Sophie Alvarezd, Leslie M. Hicksd, Cheng Luob, Hualiang Jiangb, Lefu Lanb,1, and Chuan Hea,1
aDepartment of Chemistry and Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637;bShanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, China;cThe Scripps Research Institute, San Diego, CA 92121; anddDonald Danforth Plant Science Center, St. Louis,
Edited by Richard P. Novick, New York University School of Medicine, New York, NY, and approved July 27, 2012 (received for review April 8, 2012)
Protein posttranslational modifications (PTMs), particularly phos-
phorylation, dramatically expand the complexity of cellular regula-
tory networks. Although cysteine (Cys) in various proteins can be
subject to multiple PTMs, its phosphorylation was previously consid-
ered a rare PTM with almost no regulatory role assigned. We report
here that phosphorylation occurs to a reactive cysteine residue
conserved in the staphylococcal accessary regulator A (SarA)/MarR
family global transcriptional regulator A (MgrA) family of proteins,
and is mediated by the eukaryotic-like kinase-phosphatase pair Stk1-
Stp1 in Staphylococcus aureus. Cys-phosphorylation is crucial in reg-
ulating virulence determinant production and bacterial resistance to
vancomycin. Cell wall-targeting antibiotics, such as vancomycin and
ceftriaxone, inhibit the kinase activity of Stk1 and lead to decreased
Cys-phosphorylation of SarA and MgrA. An in vivo mouse model of
infection established that the absence of stp1, which results in ele-
vated protein Cys-phosphorylation, significantly reduces staphylo-
coccal virulence. Our data indicate that Cys-phosphorylation is
a unique PTM that can play crucial roles in bacterial signaling
Ser/Thr kinase PknB|transcriptional regulation
greatly expands the coding capacity of prokaryotic/eukary-
otic genomes, which leads to the production of much more di-
verse proteomes (1). Attaching different chemical groups, such
as phosphate, acetate, lipids, and carbohydrates to amino acid
residues in proteins allows these PTMs to fine-tune functions of
proteins in response to various signaling events.
Among various PTMs, the reversible protein phosphorylation is
the most widespread in signal transduction, which is a central
process to the regulation of nearly every aspect of cell life, in-
cluding growth, metabolism, motility, division, differentiation, or-
ganelle trafficking, and immunity, as well as learning and memory
behaviors in higher organisms (2). In a typical phosphorylation
event, a protein kinase transfers the γ-phosphate from ATP to
specific amino acids of a protein to initiate signal transduction; in
and Tyr residues (3, 4). In contrast, His/Asp-based phospho-relay
of two-component systems has been well recognized as a classic
mode of signal transduction in the bacterial world (5). However, it
wasn’t until recently that eukaryotic-like Ser/Thr/Tyr phosphory-
lation in bacteria started to receive significant attention (6–8).
Particularly, in pathogenic bacteria such as Mycobacterium tuber-
culosis and Staphylococcus aureus, eukaryotic-like Ser/Thr kinases
(and associated phosphatases) have often been shown to partici-
are known as Stks/Stps in bacteria; the kinase Stk1 has been called
as PknB or Stk, and the phosphatase Stp1 as Stp (9). Throughout
this study, we will use Stk1 for the kinase and Stp1 for the phos-
phatase because these names are more common in the literature.
Less commonlysineand argininephosphorylations have also been
studied (12). For example, in Bacillus subtilis, a specific arginine
ovalent posttranslational modification (PTM) of proteins
kinase McsB has been isolated and shown to phosphorylate argi-
nine residues of its binding partner CtsR (a stress-response tran-
scriptional regulator) in vitro (13).
Cysteine is one of the more reactive amino acid side chains in
processes such as enzymatic catalysis, metal binding, signal [reactive
oxgen/nitrogen species (ROS/RNS)] sensing, and protein folding
(disulfide formation). Thanks to its intrinsic reactivity, the Cys resi-
and oxidation (sulfenation, sulfination, S-nitrosylation, disulphide
formation, and so forth) (14, 16, 17). The latter case enables Cys to
in their cellular environment (18–21). Despite these well-appreci-
intermediates in the transport of carbohydrates by the bacterial
phosphoenolpyruvate-dependent phosphotransferase system (22,
phosphatase during catalysis (24). Because of its “rare occurrence”
regulatory role of Cys-phosphorylation as a type of PTM remains
elusive. Moreover, unlike conventional Ser/Thr/Tyr or His/Asp
phosphorylation, no specific kinase/phosphatase pair has ever been
recognized and designated for protein Cys-phosphorylation, not to
mention its contribution to cellular signaling.
Here we report the identification of Cys-phosphorylation on
a number of proteins, including several global transcriptional regu-
lators in S. aureus. We demonstrate that Cys-phosphorylation, me-
Stp1) pair in S. aureus (25–27), significantly impacts the function of
these proteins, thereby resulting in altered bacterial phenotypes.
Cys-Phosphorylation of SarA/MgrA Family Proteins (SarA, MgrA, and
SarZ) Mediated by Stk1-Stp1. S. aureus, a major human pathogen
that is the most common source of bacterial infections in the
communityandhospital, causesa widevarietyofdiseases,ranging
from minor skin infections to life-threatening blood infections
(28). The virulence of this organism is controlled by regulatory
the agr system and the staphylococcal accessary protein A (SarA)/
MarR family global transcriptional regulator A (MgrA) family
Author contributions: F.S.,L.L.,and C.H.designed research;F.S.,Y.D., Q.J., Z.L.,X.D., C.C.L.W.,
S.X., and L.L. performed research; F.S., Y.D., Q.J., Z.L., X.D., C.C.L.W., C.Y., L.Z., S.A., L.M.H.,
C.L., H.J., and L.L. contributed new reagents/analytic tools; F.S., Q.J., Z.L., L.L., and C.H. ana-
lyzed data; and F.S., L.L., and C.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or chuanhe@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| September 18, 2012
| vol. 109
| no. 38
global regulatory proteins, which can respond to changing host
microenvironments (29, 30). Recent studies have revealed that
thesole andconserved Cys residue in SarA,MgrA, andSarZ (Fig.
1) acts as a redox switch to modulate the regulatory functions of
these proteins (31–34). However, in some cases oxidation of Cys
cannot fully account for the observed phenotypes, which led us to
speculate that other PTMs might take place on these proteins to
modulate their regulatory functions (35) (Fig. 1). Recent studies
suggest that both MgrA and SarA could be subject to potential
Ser/Thr phosphorylation mediated by a eukaryotic-like kinase
Stk1 in S. aureus, and that the phosphorylation modulates the
DNA-binding activity of these proteins (35, 36). Stk1-Stp1 is the
only conserved pair of eukaryotic-like kinase-phosphatase in S.
SarZ using crude cell extract from S. aureus supplemented
As shown in Fig. 2A, SarA, MgrA, and SarZ were indeed
phosphorylated by cell extract isolated from the wild-type
S. aureus strain Newman. Intriguingly, we noted that the observed
phosphorylation occurred exclusively to the reduced forms of
SarA, MgrA, and SarZ, but not to their oxidized forms (Fig. S1 A
and B), which indicates that the phosphorylation of these pro-
teins is highly redox-dependent. Subsequently, we tested mutant
proteins with the conserved Cys residue mutated to Ser. No
protein phosphorylation was observed for all three mutant
proteins (Fig. 2A), indicating that the conserved reactive Cys is
very likely the site of the observed phosphorylation.
sole Ser/Thr kinase-phosphatase pair in S. aureus, we tested their
roles in the observed phosphorylation. An in-frame deletion
all. Cell extract from the Δstp1 strain complemented with plasmid
pYJ335::stp1 (Δstp1-C), expressing the wild-type Stp1, gave re-
duced protein phosphorylation compared with that of the Δstp1
mutant (Fig. 2B). Furthermore, the recombinant Stp1 expressed
from Escherichia coli was able to remove the phosphate group of
phosphorylated SarA (Fig. 2C), MgrA (Fig. S1C), and SarZ
(Fig. S1D), supporting its phosphatase role. Consistently, over-
expression of Stk1—the associated kinase of Stp1—in the wild-
type background increased the phosphorylation level of all three
proteins (Fig. 2D and Fig. S2A).
Surprisingly, when we performed the phosphorylation assays
in the presence of high concentrations of DTT (10 mM), a widely
used reducing agent in biochemical assays, we found that phos-
phorylation of both SarA and MgrA were almost abolished
(Fig. S2B). In contrast, the phosphorylation of the kinase Stk1
itself, which is known to be a Ser/Thr type phosphorylation, was
barely affected by the DTT treatment (Fig. S2B). Thus, the
the oxidation-sensing SarA/MgrA/SarZ. The N-terminal Cys residue, highly
conserved in SarA/MgrA/SarZ, is subject to oxidation by ROSs, thus leading to
dissociation from DNA. Phosphorylation of the same Cys residue mediated
by Stk1-Stp1 might also modulate their target gene regulation. (B) Sequence
alignment of SarZ, MgrA, and SarA. The Cys residue (indicated by an arrow)
and the surrounding conserved residues are highlighted in red. The align-
ment was performed with ClustalW2 (61).
S. aureus SarA/MgrA family protein. (A) Model of gene regulation by
The Cys-to-Ser substitution abolished the phosphorylation of SarA, MgrA,
and SarZ. WT, cell extract from the wild-type strain Newman; Δstp1, cell
extract from Δstp1 strain (an in-frame deletion mutant of stp1). Protein
bands stained with Coomassie blue after autoradiography were shown. (B)
The phosphatase Stp1 dephosphorylates phospho-SarA (lane 3). The phos-
pho-SarA was treated with Stp1 at 37 °C for 10 min before analysis. (C) The
phosphorylation activity of cell extract from Δstp1 was restored by com-
plementation with pYJ335::stp1 (lane 3). Δstp1-C, Δstp1/pYJ335::stp1. (D)
Overexpression of Stk1 enhanced phosphorylation of SarA (lane 3). stk1++,
cell extract from the wild-type strain carrying pYJ335::stk1, in which the
expression of stk1 was induced by anhydrotetracycline (1 μg/mL). (E) De-
activation of stk1 abolished phosphorylation of SarA (lane 3). stp1-I, cell
extract from the mutant with bursa aurealis transposon insertion in stp1 that
deactivates both stp1 and stk1.
Phosphorylation of SarA family proteins (SarA, MgrA, and SarZ). (A)
| www.pnas.org/cgi/doi/10.1073/pnas.1205952109Sun et al.
observed phosphorylation on the SarA/MgrA family proteins
represents a unique type of modification chemically distinct from
conventional Ser/Thr phosphorylation.
A bursa aurealis transposon insertion mutant of stp1 (stp1-I)
(37) that deactivates both stp1 and its downstream cotranscribed
stk1 (Fig. S3) was used for the in vitro phosphorylation assay. Cell
extract from this mutant strain significantly diminished phos-
phorylation of all three proteins (Fig. 2E), indicating that Stk1 is
essential for the observed protein phosphorylation. Collectively,
our phosphorylation results suggest that phosphorylation occurs
to the Cys residue conserved in the SarA/MgrA family proteins
and that the sole Stp1-Stk1 pair in S. aureus mediates this Cys-
Verification of Cys-Phosphorylation by LC-MS/MS. To further verify
Cys-phosphorylation of the SarA/MgrA family proteins, we per-
formed mass spectrometric characterization on the phosphory-
lated proteins. Despite the labile feature of phospho-Cys (38), we
successfully identified the phospho-Cys modification in both
phosphorylated SarA and phosphorylated MgrA (Fig. 3 and Fig.
S4). In the case of SarA, after trypsin digestion, one phospho-
peptide INDpCFELLSMVTYADKLK (observed m/z2182.0140)
was identified using the Mascot (v 2.3, MatrixScience) database
searchengine (Fig.3A) (39).Thedetected y6andy7fragmentions
showed that the phosphorylation was not on Thr-17 or Tyr-18,
whereas fragment ions b5and b6indicated that the phosphoryla-
tion was on Cys-9 but not on Ser-14. In the case of MgrA, one
phosphopeptide EQLpCFSLYNAQR (observed m/z 1550.6555)
(Fig. 3B) was also identified. The y7and y8fragment ions were
detected showing that neither Ser-14 nor Tyr-16 was phosphory-
was phosphorylated (+80 Da mass shift). A mis-cleaved peptide
from the same sequence GSHMNLKEQLpCFSLYNAQR (ob-
ion confirming phosphorylation on Cys-13 (Fig. S4).
Cys-Phosphorylation of the SarA/MgrA Family Proteins Is Blocked by
Oxidation and Alkylation. Protein modifications are known to con-
tribute to changes in cell physiology in response to particular sig-
nals. Pathogenicbacteria,suchasS.aureus, requirerapidresponse
toa hostile environment during pathogenesis. It is well established
that ROS serve beneficial roles for host defense, particularly when
macrophages and neutrophils produce a burst of oxidants, such as
H2O2, to kill invading microorganisms. The abrupt change of the
redox status, on the other hand, is also used by the pathogen as
a signal to adapt and evade the host defense. The reactive cysteine
in the SarA/MgrA family proteins is known to be redox active and
plays a significant role in responding to oxidative stress (40). As
hydrogen peroxide, the phosphorylation levels of both SarA and
MgrA were greatly reduced, supporting the finding that oxidation
blocks Cys-phosphorylation. Cys-oxidation in MgrA, SarZ, and
SarA is critical for gene expression regulation in S. aureus. The
same Cys residues can be phosphorylated, allowing the bacterium
to perhaps balance the oxidation-sensing pathways through the
same residues. Importantly, phosphorylation of the reactive Cys
residues in these global regulatory proteins enables the bacterium
to incorporate additional signaling pathways to affect/control
existing gene regulation networks.
We also tested nucleophilic alkylators that can alkylate the re-
active cysteine. Both iodoacetamide and maleimide significantly
diminished the phosphorylation of SarA and MgrA (Fig. S2 D and
F). In addition, we observed that the phosphorylated SarA is labile
(Fig. S2G), consistent with a previous report that phospho-Cys
displays unique liability toward iodine (I2) (24). All these data fur-
ther confirm cysteine phosphorylation in these proteins.
Cys-Phosphorylation Modulates the DNA-Binding Ability of the SarA/
MgrA Family Proteins. To investigate whether Cys-phosphorylation
modulates DNA-binding of SarA, we performed an EMSA with
apo-SarA and phospho-SarA. Phospho-SarA was purified using
phospho-protein purification kit (Qiagen) after the in vitro phos-
phorylation reaction. The hla promoter region contains a putative
LC-MS/MS spectrum of the phosphopeptide I6NpCFELLSMVTYADKLK23(ob-
served m/z 2182.0140 Da corresponding to apo-peptide theoretical mass of
of phospho-SarA. The b5and b6fragment ions corresponding to I6NDpCF and
I6NDpCFE, respectively (observed m/z 673.2051 and 802.2247 corresponding to
apo-fragment + 1 phosphate group 79.9437 Da) indicates the presence of
phospho-Cys rather than on Ser or Thr. The phospho-Cys-9-Phe-10 (pCF) frag-
ment is also highlighted by the mass difference of b5and b3fragment ions. (B)
1550.6555 Da corresponding to apo-peptide experimental mass of 1470.6967
Da + 1 phosphate group 79.9588 Da) obtained after trypsin digestion of
phospho-MgrA. The characteristic mass difference of the phospho-Cys-12 is
highlighted. Fragment ions y5and y7indicate that phosphorylation is not on
Ser-14 or Thr-16 and fragment y9shows a mass shift of 80 Da from the phos-
nomenclature. The phospho-fragments are colored red. Fragment ions arising
ions with the loss of ammonia (−17 Da) are marked with an asterisk (*).
LC-MS/MSidentificationofCys-phosphorylation ofSarAand MgrA.(A)
Sun et al.PNAS
| September 18, 2012
| vol. 109
| no. 38
SarA-binding sequence (Fig. S5E) (41) and therefore was chosen to
examine the effect of Cys-phosphorylation on the DNA-binding
sequence is specific as SarA failed to show any binding toward the
SarA [Kd(SarA) = ∼32 nM], phospho-SarA displayed a reduced
S5A). The treatment of phospho-SarA with the phosphatase Stp1
Cys-phosphorylation accounts for the attenuation of the DNA-
binding affinity of phospho-SarA. Attenuation of DNA binding by
Cys-phosphorylation was also observed on MgrA [Kd(MgrA) =
∼102 nM compared with Kd(MgrA∼P) = ∼500 nM] and SarZ [Kd
(SarZ) = ∼35 nM compared with. Kd(SarZ∼P) = ∼120 nM] (Fig.
S6). Because glutamate (Glu) has been widely used as a phospho-
of three mutant proteins, SarAC9E, MgrAC12E, and SarZC13E,
proteins exhibited reduced binding affinities to their cognate DNA
[Kd(SarAC9E) = ∼134 nM, Kd(MgrAC12E) = ∼480 nM, and Kd
(SarZC13E) = ∼110 nM] (Figs. S5C and S6 B and D).
SarA, MgrA, and SarZ are dimeric proteins, in which the sole
conserved Cys residue resides at the dimerization domain and is
involved in hydrogen-bonding interactions with residues from the
other monomer (31, 42, 43). To unveil how Cys-phosphorylation
and the Cys-to-Glu substitution could impact DNA binding, we
constructed initial structural models of wild-type Cys-phosphory-
the Protein Data Bank (PDB ID: 3HSE) as described in SI Ex-
perimental Procedures. Molecular dynamics simulations were fur-
ther performed on all three forms of SarZ to explore the detailed
molecular basis. Time evolutions of centroid distance between
chain A and B of dimeric SarZ from their initial conformation (t =
0) were recorded (Fig. S7B). As illustrated in Fig. S7A, the wild-
type SarZ displayed very little conformational change during mo-
lecular dynamics (MD) simulation and the distance between chain
A and B remained steady (Fig. S7B). However, a dramatic con-
formational change was observed in both SarZC13E and Cys-P-
SarZ during MD simulation (Fig. S7B). Particularly for Cys-P-
SarZ, the centroid distance between two monomers was sharply
shortened by up to 0.75 Å after 40-ns MD simulation (Fig. S7B)
and DNA binding domains of two chains almost clashed together
(Fig. S7A), indicating that Cys-phosphorylation introduces a sig-
nificant structural disturbance to SarZ dimerization. We attemp-
ted structural characterization of the Cys-to-Glu mutant proteins
underlying structural changes caused by Cys-phosphorylation. Al-
though we were unable to grow good quality crystals of SarAC9E
or MgrAC12E, we obtained the crystal structure of SarZC13E at
2.0 Å resolution (Fig. S8). This structure shows the presence of
three strong hydrogen-bonding interactions between Glu-13 and
two Tyr residues, Tyr-27 and Tyr-41, from the neighboring
monomer (Fig. S8A). In contrast, only two weak hydrogen bonds
exist in the wild-type SarZ (Fig. S8B). Consistent with the result
from MD simulations, the enhanced hydrogen-bonding inter-
in the DNA-binding domain of SarZ (Fig. S8C), with the DNA
recognition helix α4 of SarZC13E displaced by 6 Å compared with
that of the wild-type SarZ (Fig. S8D) (42). This observation sug-
gests that Cys-phosphorylation of the SarA/MgrA family proteins
induces changes at the dimeric interface, thereby modulating the
DNA-binding properties of these proteins.
Phosphorylation of SarA Modulates Its Regulatory Function. Toprobe
SarA (Δstp1-sarA/pYJ335::sarA) in the stp1-deficient strain. The
phosphorylated SarA could be directly detected using an anti-
phosphorylation antibody after purifying His6-SarA from the stp1-
deficient strain with Ni-NTA beads (Fig. 4A, lane 1). In contrast,
there was no detectable signal for His6-SarAC9S (with Cys-9 mu-
tated to Ser) obtained from the stp1-deficient strain expressing
Δstp1-sarA/pYJ335::sarAC9S (Fig. 4A, lane 2), indicating that the
SarA and Stp1 are positive regulators of α-hemolysin (hla) in
either the Δstp1 or sarA mutant strain displayed reduced hemolysis
compared with the wild-type strain, as indicated by zones of clear-
of sarA and stp1 (Δstp1-sarA/pYJ335, a double mutant with in-
frame, unmarked stp1 deletion and bursa aurealis transposon in-
sertion in sarA) led to further attenuated hemolysis (Fig. S9A). The
unable to restore hemolysis (Fig. 4B). However, theintroduction of
pYJ335-sarAC9S into Δstp1-sarA, with the expression of the Cys-
to-Ser SarAC9S mutant protein, significantly enhanced hemolytic
sarAC9E (a stable mimic of Cys-phosphorylated SarA) into Δstp1-
sarA failed to enhance hemolysis (Fig. 4B). Western blot analysis
showedthat the production of α-hemolysin isvery likelyaffected by
phosphorylation of SarA (Fig. 4C and Fig. S9B). Specifically, the
but wild-type SarA, as well as SarAC9E (a phosphomimetic mu-
tant), failed to restore the α-hemolysin level in the Δstp1-sarA
background presumably because of Cys-phosphorylation or Cys to
that Cys-phosphorylation of SarA contributes to the attenuation of
hemolysis observed for the Δstp1 strain (Fig. 4B and Fig. S9A),
which is consistent with the weakened binding of the Cys-phos-
phorylated SarA to the hla promoter region observed by EMSA.
SarA also impacts the susceptibility/resistance of S. aureus to
cell wall-targeting antibiotics. The sarA mutant strain in the
Newman background displayed enhanced resistance to vanco-
mycin (Fig. S9D), which could be complemented by expressing
SarA using the plasmid pYJ335::sarA induced by anhydrotetracy-
cline (aTc, 1 μg/mL) (Fig. S9D). In addition to the sarA mutant,
both Δstp1 (Fig. S9D) and Δstp1-sarA mutant strains exhibited
type Newman strain based on plate assays (Fig. 4D and Fig. S9D).
(Δstp1-sarA/pYJ335::sarA) or SarAC9S (Δstp1-sarA/pYJ335::sar-
AC9S), but not SarAC9E (Δstp1-sarA/pYJ335::sarAC9E), fully
restored bacterial susceptibility to vancomycin. Collectively, these
data support that phosphorylation of Cys-9 plays a critical role for
the regulatory function of SarA in bacterial antibiotic resistance.
To further prove that protein Cys-phosphorylation is affected by
cell wall-targeting antibiotics through Stk1, we examined the phos-
phorylation of SarA and MgrA mediated by Δstp1 cell extract in the
absence or presence of antibiotics. As shown in Fig. S10, Cys-phos-
phorylation of both proteins was significantly inhibited by cell wall
antibiotics vancomycin and ceftriaxone. A recent study in Strepto-
interfering with the function of S. mutans Stk1 (46). To determine
whether the reduced Cys-phosphorylation was caused by the in-
hibition of S. aureus Stk1 by antibiotics, we tested the autophos-
phorylation of Stk1 in vitro in the absence or presence of various
antibiotics including vancomycin, ceftriaxone, and erythromycin.
Intriguingly, as shown in Fig. S10C, the phosphorylation of Stk1 was
greatly inhibited by cell wall-acting antibiotics vancomycin and cef-
triaxone, but not by erythromycin, a protein synthesis inhibitor. This
observation agrees with the effects of antibiotics on Cys-phosphory-
lation of SarA (25). Taken together, these results suggest that Cys-
that affect Cys-phosphorylation in S. aureus.
Effect of Mutation of stp1 on the Virulence of S. aureus in a Mouse
Model of Abscess Formation. As global transcriptional regulators,
| www.pnas.org/cgi/doi/10.1073/pnas.1205952109 Sun et al.
Given that deletion of stp1 inevitably accumulates Cys-phosphory-
lation among these proteins (SarA, MgrA, and SarZ), thereby par-
effects resulting from mutation of any of these transcriptional reg-
ulators. A mouse infection model of abscess formation was used to
compare the infectivity of the Δstp1 deletion mutant with the wild-
type Newman. As expected, the Δstp1 strain was incompetent in
establishing infection in the mouse. As shown in Fig. 4 E and F,
dramatically reduced after 5 d. Compared with the Newman strain,
the Δstp1 strain showed a >2-log reduction (P = 5 × 10−6, t test) of
virulence in livers of the infected mice (Fig. 4E). The virulence at-
tenuation of Δstp1 in kidneys of the infected mice was even more
striking, exhibiting a >4-log reduction (P = 1 × 10−7, t test) of bac-
terial loads in comparison with wild-type Newman (Fig. 4F).
Ser/Thr/Tyr phosphorylations are ubiquitous in eukaryotes (2,
50). These types of protein phosphorylations dominate eukary-
otic signaling and regulation. Another common group of protein
phosphorylation is the bacterial two-component system that uses
His/Asp phosphorylations in signal relays in microbes (51, 52).
His/Asp phosphorylations are transient and unstable to almost
all enrichment and mass spectrometry detection procedures,
thus making direct observation and characterization of these
phosphorylations difficult and challenging (5). The presence of
eukaryotic-like Ser/Thr kinase-phosphatase pairs in bacteria has
fueled interests in elucidating their potential roles in bacterial
signaling and regulation (53–55). The Stk1-Stp1 pair is conserved
in Gram-positive bacteria and has been suggested to play global
regulatory roles through Ser/Thr phosphorylation/dephosphory-
lation (6, 9). We show here that this kinase/phosphatase pair
can phosphorylate Cys residues in different proteins in Gram-
positive bacteria. Notably, the observed Cys-phosphorylation of
global regulatory proteins exhibits significant regulatory func-
tions, indicating the existence of a unique type of PTM that im-
pacts biological signaling and regulation.
Despite the fact that cysteine is the most nucleophilic residue
among natural amino acids and is known to be subject to multiple
PTMs (56), Cys-phosphorylation was previously considered an
unusual PTM with only a few known examples as catalytic
intermediates in enzymatic reactions (24, 57, 58). We show here
that the conserved Cys residue in the global transcriptional factors
SarA, MgrA, and SarZ can be phosphorylated, which controls
virulence and other properties of S. aureus. Previous in vitro ki-
nase-mediated phosphorylation assays have indicated that the
(35, 36, 59). Our present study establishes Cys-phosphorylation as
mutated to Ser, the mutant proteins could hardly be phosphory-
major site of phosphorylation in the SarA/MgrA family proteins.
The labile nature of Cys-phosphorylation under common experi-
mental conditions might have contributed to previous lack of ob-
servation of this PTM on these regulatory proteins and other
biological systems (38). To further support the observation that
Cys-phosphorylation can be reversed by DTT, we used S-phos-
phocysteamine as a model substrate to test the stability of alkyl S-
phosphate toward DTT under conditions similar to those used for
the in vitro phosphorylation assay. As shown in Fig. S11, cyste-
amine, the dephosphorylated product, was observed after in-
cubating S-phosphocysteamine with high concentrations of DTT
(100–200 mM) and Mn2+(200 mM). High concentrations of DTT
above the physiological reduction range and excess of Mn2+are
required to reverse Cys-phosporylation, indicating stability of this
PTM under physiological conditions. It is also possible that this
when bacteria encounter stresses such as ROS/RNS that shift the
intracellular redox balance or through other signaling events that
lead to activation of Stk1 or deactivation of Stp1.
Cys-phosphorylation could be more prevalent than previously
thought and detected if appropriate experimental conditions are
global transcriptional regulators such as CymR (at the sole Cys-25
residue) (Fig. S12), a recently identified oxidation-sensing protein
responsible for cysteine metabolism regulation in S. aureus (60).
Given that all of the proteins we have shown to undergo Cys-phos-
phorylation are global regulators that control a broad spectrum of
genes and properties in S. aureus,this study may suggest a previously
kinases may have evolved to selectively phosphorylate cysteine over
serine (or vice versa), even despite the near isostructures of these
virulence and antibiotic resistance. (A) Western blot
SarA enriched with Ni-NTA beads from whole-cell
extract of Δstp1-sarA/pYJ335::sarA; SarAC9S, His6-
SarAC9S from Δstp1-sarA/pYJ335::sarAC9S; Control,
the recombinant SarA expressed by E. coli after
in vitro phosphorylation as a positive control. Phos-
pho-Ab, antiphospho-Thr antibody; His-tag, InVision
His-tag in-gel stain (Invitrogen). Δstp1-sarA, a double
mutant with in-frame, unmarked stp1 deletion and
bursa aurealis transposon insertion in sarA. (B) He-
molysis on the sheep blood agar. The strains tested
were spotted on 5% (vol/vol) sheep blood agar plate.
Zones of clearance indicate hemolysis. (C) Western
blot analysis of the production of α-hemolysin (Hla)
(see SI Experimental Procedures). (D) Vancomycin re-
sistance assays. Aliquots (10 μL) of the diluted over-
night cultures for each strain (5 × 106CFU/mL) were
(Upper) or with 1.6 μg/mL vancomycin (Lower). Both
plates were supplemented with 1 μg/mL anhydrote-
tracycline (aTc) to induce the expression of SarA. (E
aureus in a mouse model of abscess formation. The
wild-type strain Newman and the stp1 deletion mutant (Δstp1) were used to infect 10 mice each via retro-orbital injection. After 5 d, S. aureus colonization in
difference between mutant and wild-type strains was determined by Student t test (two-tailed). The limit of detection for organ infection is 100 CFU per organ.
Cys-phosphorylation modulates bacterial
Sun et al. PNAS
| September 18, 2012
| vol. 109
| no. 38
amino acids. The detailed molecular mechanism responsible for this
Cys-phosphorylation event remains to be further elucidated.
Detailed procedures are available in SI Experimental Procedures. See Table S1
for bacterial strains and plasmids used in this study and Table S2 for the list
of primers used. Table S3 shows data collection and refinement statistics for
the SarZC13E structure.
ACKNOWLEDGMENTS. We thank Drs. O. Schneewind and D. Missiakas for
providing transposon mutants and S. F. Reichard for editing the manuscript.
This work was financially supported by National Institutes of Health National
Program of the Chinese Academy of Sciences (L.L.); a Burroughs Wellcome Fund
Investigator in the Pathogenesis of Infectious Disease Award (to C.H.); and
the 863 Program 2012AA020302 (to H.J.). F.S. is a Scholar of the Chicago
Biomedical Consortium with support from The Searle Funds at The Chicago
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| www.pnas.org/cgi/doi/10.1073/pnas.1205952109 Sun et al.