Molecular insights into the impact of oxidative stress on the quorum-sensing regulator protein LasR

Article abstract of DOI:10.1074/jbc.M116.719351

The LasR regulator protein functions at the top of the Pseudomonas aeruginosa quorum-sensing hierarchy and is implicated in promoting bacterial virulence. Of note is recent evidence that this transcription factor may also respond to oxidative stress. Here, all cysteines in LasR were inspected to deduce their redox sensitivity and to probe the connection between stress response and LasR activity using purified LasR and individual LasR domains. Cys⁷⁹ in the ligand binding domain of LasR appears to be important for ligand recognition and folding of this domain to potentiate DNA binding but does not seem to be sensitive to oxidative stress when bound to its native ligand. Two cysteines in the DNA binding domain of LasR do form a disulfide bond when treated with hydrogen peroxide, and formation of this Cys²⁰¹-Cys²⁰³ disulfide bond appears to disrupt the DNA binding activity of the transcription factor. Mutagenesis of either of these cysteines leads to expression of a protein that no longer binds DNA. A cell-based reporter assay linking LasR function with β-galactosidase activity gave results consistent with those obtained with purified LasR. This work provides a possible mechanism for oxidative stress response by LasR and indicates that multiple cysteines within the protein may prove to be useful targets for disabling its activity.

Full text of DOI:10.1074/jbc.M116.719351

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 22, pp. 11776–11786, May 27, 2016
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Molecular Insights into the Impact of Oxidative Stress on the
Quorum-Sensing Regulator Protein LasR*
Received for publication, February 2, 2016, and in revised form, March 29, 2016 Published, JBC Papers in Press, April 6, 2016, DOI 10.1074/jbc.M116.719351
Prapti Kafle1, Amanda N. Amoh1,2, Jocelyn M. Reaves2, Emma G. Suneby, Kathryn A. Tutunjian, Reed L. Tyson,
and Tanya L. Schneider3
From the Department of Chemistry, Connecticut College, New London, Connecticut 06320
The LasR regulator protein functions at the top of the Pseu-
domonas aeruginosa quorum-sensing hierarchy and is impli-
cated in promoting bacterial virulence. Of note is recent evi-
dence that this transcription factor may also respond to
oxidative stress. Here, all cysteines in LasR were inspected to
deduce their redox sensitivity and to probe the connection
between stress response and LasR activity using purified LasR
and individual LasR domains. Cys79 in the ligand binding
domain of LasR appears to be important for ligand recognition
and folding of this domain to potentiate DNA binding but does
not seem to be sensitive to oxidative stress when bound to its
native ligand. Two cysteines in the DNA binding domain of LasR
do form a disulfide bond when treated with hydrogen peroxide,
and formation of this Cys201-Cys203 disulfide bond appears to
disrupt the DNA binding activity of the transcription factor.
Mutagenesis of either of these cysteines leads to expression of a
protein that no longer binds DNA. A cell-based reporter assay
linking LasR function with ␤-galactosidase activity gave results
consistent with those obtained with purified LasR. This work
provides a possible mechanism for oxidative stress response by
LasR and indicates that multiple cysteines within the protein
may prove to be useful targets for disabling its activity.
LuxI-type synthases, and detected by LuxR-type regulator pro-
teins (8). The two major systems responsive to acylhomoserine
lactones (AHLs) in P. aeruginosa are LasI/LasR and RhlI/RhlR.
LasI and RhlI are LuxI-type synthases, whereas LasR and RhlR
are LuxR-type regulatory proteins. These AHL-dependent sys-
tems are also closely connected to signaling governed by 2-al-
kyl-4-quinolones in P. aeruginosa (9).
The regulator protein LasR, with its cognate synthase LasI,
functions at the top of the P. aeruginosa quorum-sensing hier-
archy (10). Like other LuxR-type proteins, this transcription
factor is composed of two domains (11). The amino-terminal
ligand binding domain (LBD) specifically recognizes its autoin-
ducer, N-(3-oxo-dodecanoyl)-L-homoserine lactone (3O-C12-
HSL), promoting protein folding and dimerization, whereas the
DNA binding domain (DBD) recognizes target sites to activate
downstream gene expression. There has been significant inter-
est in understanding LasR, as its inhibition represents a prom-
ising antivirulence strategy for the treatment of P. aeruginosa.
One prevalent mode of inhibition is the development of non-
native small molecules shown to disrupt LasR function through
the reduction of virulence factors produced (12–14). A number
of these analogs appear to inhibit LasR through competition
with the native 3O-C12-HSL because they are often structurally
similar.
Recent work has also revealed a somewhat unexpected role
for LasR in oxidative sensing (15). A proteome-wide screen of
P. aeruginosa to detect H2O2-sensitive cysteines identified
Cys79 of LasR. Subsequent experiments in P. aeruginosa indi-
cated that a LasR-C79A mutant strain showed decreased motil-
ity and proteolysis, highlighting Cys79 as important for LasR QS
function. The link between LasR and oxidative stress response
was supported by Northern hybridization analysis that revealed
that the RNA level of lasI, under LasR control, was reduced
after cells were treated with H2O2. Complementation studies
with a LasR deletion strain showed that LasR function was
restored when complemented with wild-type plasmid p-lasR
but diminished by about half when cells were treated with 5 mM
H2O2. Interestingly, complementation with mutant plasmid
p-lasR-C79S was only partially successful but did not change
significantly in the presence of oxidant. Complementation with
p-lasR-C79A resulted in negligible LasR activity even in the
absence of oxidant. Taken together, these findings suggest that
LasR responds to both its autoinducer and oxidative stress.
Numerous bacterial proteins have been shown to respond to
oxidative stress through redox-sensitive thiols, including the
OxyR, HypT, and MgrA transcription factors (16–18).
Although these proteins are important for regulating pathogen
The Gram-negative bacterium Pseudomonas aeruginosa is
an opportunistic human pathogen that establishes chronic
infections in immunocompromised patients, with persistent
pulmonary colonization in individuals with cystic fibrosis
(1–3). Like many bacteria, P. aeruginosa employs quorum-
sensing (QS)4 machinery to communicate local population
density through the exchange of self-produced signaling mole-
cules (4). QS contributes to the development of acute infections
by coordinating the production of multiple virulence factors,
including elastase and pyocyanin, and the formation of biofilms
(5–7). QS in P. aeruginosa is largely mediated by N-acyl L-ho-
moserine lactone autoinducer molecules, biosynthesized by
* This work was supported by a Cottrell College Science Award from the
Research Corporation for Science Advancement. The authors declare that
they have no conflicts of interest with the contents of this article.
1 Supported by Keck Foundation summer research fellowships.
2 Supported by NSF-S-STEM 1153371.
3 To whom correspondence should be addressed: Dept. of Chemistry, Con-
necticut College, 270 Mohegan Ave., New London, CT 06320. Tel.: 860-439-
2481; Fax: 860-439-2477; E-mail: tschneid@conncoll.edu.
4 The abbreviations used are: QS, quorum-sensing; AHL, acylhomoserine lac-
tone; LBD, ligand binding domain; 3O-C12-HSL, N-(3-oxo-dodecanoyl)-L-
homoserine lactone; DBD, DNA binding domain; DTNB, 5,5Ј-dithiobis(2-
nitrobenzoic acid); Tm, melting temperature; OP1, Operator 1.
11776 JOURNAL OF BIOLOGICAL CHEMISTRY
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Oxidative Stress and Quorum Sensing Regulator LasR
response to potentially damaging oxidants, they are not known
For expression of all proteins, BL21(DE3) cells were trans-
to be associated with QS. In addition to the recent finding that formed with the expression plasmid of interest and grown in
LasR may be governed by both oxidants and QS autoinducers, Luria broth medium supplemented with 30 ␮g/ml kanamycin.
several other bacterial regulators have been linked to a similar
dual function. Staphylococcus aureus AgrA has been shown to
form an intramolecular disulfide bond within its DBD when
oxidized, leading to a reduction in DNA binding affinity (19).
Additionally, Escherichia coli SdiA, a LuxR homolog like LasR,
forms an intermolecular disulfide bond within its DBD, cova-
lently linking two monomers and altering promoter binding
affinity when oxidized with the addition of 2 mM H2O2 in
EMSAs (20). These studies provide additional evidence for a
possible intersection between QS and oxidative sensing in bac-
terial regulator proteins.
For the expression of LasR and LBD, growth medium was addi-
tionally supplemented with 20 ␮M 3O-C12-HSL (Sigma-Al-
drich) or non-native AHLs. Cultures were grown to A600 ϭ 0.8
at 37 °C, protein expression was induced by the addition of iso-
propyl 1-thio-␤-D-galactopyranoside to 400 ␮M, and growth
continued for an additional 24 h at 23 °C. Cells were harvested
by centrifugation, with pellets stored at Ϫ80 °C.
Harvested cells resulting from 0.5 liter of culture were resus-
pended in 10 ml of lysis buffer (25 mM Tris (pH 7.5), 200 mM
NaCl, and 10% (v/v) glycerol). Lysis buffer was supplemented
with 1 mM tris(2-carboxyethyl)phosphine for the purification of
LasR and DBD. Resuspended cells were lysed by sonication and
clarified by centrifugation, and the resulting supernatant was
incubated with 0.25 ml of nickel-nitrilotriacetic acid-agarose
resin (Qiagen) for 2 h at 4 °C. The resin was decanted into a
column and washed with 5 ml of 5 mM imidazole in lysis buffer.
C-terminally His6-tagged LasR, LBD, or DBD protein was
eluted with a step gradient of 30, 60, 100, 250, and 500 mM
imidazole in lysis buffer. Fractions containing the desired pro-
tein as judged by SDS-PAGE were pooled and dialyzed against
dialysis buffer (50 mM sodium phosphate (pH 7.0), 200 mM
NaCl, and 10% (v/v) glycerol, with 1 mM DTT added for LasR
and DBD purification). LasR purified without DTT in dialysis
buffer was not an active DNA-binding protein.
In this work, we probed the mechanism for the reported LasR
sensitivity to oxidative stress to further characterize this impor-
tant transcriptional regulator at the biochemical level. Our
exploration of Cys79 revealed that this residue is important for
ligand recognition and protein folding, but it does not seem to
be sensitive to oxidative stress when LasR is autoinducer-bound
and folded. Instead, we have identified two cysteines in the DBD
of LasR, Cys201 and Cys203, which appear to modulate DNA
binding affinity through the formation of an intramolecular
disulfide bond in the presence of H2O2. Additionally, mutagen-
esis of either of these residues leads to poor protein overexpres-
sion and loss of DNA binding, underscoring their critical role in
functional LasR. Results from these in vitro assays with purified
protein were further supported by parallel studies using E. coli
transformed with a plasmid containing both the lasR gene and
a lasB-lacZ detection system (21). Experiments with this
reporter gene system also demonstrated the critical importance
of Cys201 and Cys203 for LasR function in a cell-based assay and
indicated the down-regulation of LasR activity in the presence
of H2O2. Taken together, our results add to the growing collec-
tive understanding of LasR, supporting a possible additional
role for this protein as an oxidative sensor and suggesting new
ways in which this pathogenic regulator may be handicapped.
Synthesis of Non-native AHLs—AHL derivatives were pre-
pared by coupling acyl carboxylic acids with L-homoserine lac-
tone in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide as described previously (23). Product identity and
purity were confirmed by 1H NMR (500 MHz Agilent DD2-500
NMR), with comparison to known characterization data for
each compound (24).
SDS-PAGE Analysis of LBD—To measure the impact of oxi-
dants on LBD dimerization, 25 ␮M LBD or LBD C79S was com-
bined with 2.5 mM H2O2, 2.5 mM cumene hydroperoxide, or
water as a control and incubated for 3 h at 25 °C. LBD and LBD
C79S samples were diluted in non-reducing SDS sample buffer
and separated using 12% polyacrylamide gels (Bio-Rad). Sam-
ples containing 100 mM DTT in sample buffer were incubated
for 10 min at 25 °C prior to electrophoresis. Detection, excision,
and elution of protein bands for mass spectrometric confirma-
tion of molecular weight followed established methods (25).
LBD expressed in the presence of non-native AHLs was also
diluted in non-reducing SDS sample buffer and resolved on a
12% polyacrylamide gel.
Experimental Procedures
Cloning, Expression, and Purification of LasR, LBD, and
DBD—The P. aeruginosa PA01 lasR gene was amplified by PCR
from a lasR-containing pDONR201 vector obtained from the
Harvard Medical School PlasmID Repository. Using incorpo-
rated NotI and NcoI restriction sites, the lasR insert was ligated
into the pET28b (Novagen) expression vector to yield plasR.
Similarly, the lasR fragment (nucleotides 1–519) correspond-
ing to the LasR LBD (amino acid residues 1–173) (22) was
amplified and cloned into the NdeI/NotI sites of the pET24b
vector to yield pLBD. The lasR fragment (nucleotides 523–720)
corresponding to amino acid residues 175–240 with added
N-terminal Met was amplified and cloned into the NotI/NcoI
DTNB Assay—LBD (25 ␮M) was combined with 0–200 ␮M
H2O2 in native (100 mM sodium phosphate and 1 mM EDTA
(pH 8.0)) or denaturing (6 M guanidine hydrochloride, 100 mM
sodium phosphate, and 1 mM EDTA (pH 8.0)) buffer and incu-
bated for 1 h at 25 °C. To detect free thiols, 180 ␮M 5,5Ј-dithio-
bis(2-nitrobenzoic acid) (DTNB) was added and incubated for
15 min at 25 °C, and the absorbance of each sample was mea-
sites of pET28b to yield pDBD. Generation of Cys79, Cys188
Cys201 and Cys203 mutants was achieved using the
QuikChange Lightning site-directed mutagenesis kit (Agilent
,
,
Technologies). All constructs were sequenced to confirm cor- sured at 412 nm. The concentration of 2-nitro-5-thiobenzoate,
rect gene insertion and desired mutations (DNA Analysis Facil- released upon reaction between free thiols and DTNB, was cal-
ity, Yale University).
culated using this absorbance and known extinction coeffi-
MAY 27, 2016•VOLUME 291•NUMBER 22
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Oxidative Stress and Quorum Sensing Regulator LasR
cients for 2-nitro-5-thiobenzoate under native and denaturing
conditions (26).
A
C79
LBD
C188
DCB2D01 C203
239
Circular Dichroism Spectroscopy—Far-UV CD spectra of
wild-type and mutant LBD (15 ␮M in 100 mM sodium phos-
phate (pH 7.0)) expressed in the presence of varied AHLs were
measured with a Jasco J-810 spectropolarimeter using a 0.1-cm
path length quartz cuvette. Data were acquired at 10 °C, and
spectra represent the average of four scans for each sample.
Temperature-dependent CD analysis at 222 nm from 10–90 °C
at a rate of 1 °C/min permitted determination of melting tem-
perature (Tm) using the Jasco Spectra Analysis software to fit
data.
1
173
B
Cys79
Cys79
Electrophoretic Mobility Shift Assays—Binding reactions
were performed in binding buffer (0.7 mM KH2PO4, 2.15 mM
Na2HPO4, 1.35 mM KCl, 20 mM NaCl, 0.5 mM EDTA, 0.05%
(v/v) Triton X-100, 5% (v/v) glycerol, and 10 ␮g/ml salmon
sperm DNA (pH 7.4)). A 43-mer DNA duplex containing the
lasB OP1 site (5Ј-ATCAAGGCTACCTGCCAGTTCTGGCA-
GGTTTGGCCGCGGGTTC-3Ј annealed to its complemen-
tary strand) with 5Ј modification of the top strand with fluores-
cein (6-FAM, Integrated DNA Technologies) was used for all
binding assays. For quantitative binding assays, serial dilutions
of LasR, LasR mutants, and DBD were incubated with 100 nM
lasB OP1 for 30 min at 25 °C before binding reactions were
applied to a nondenaturing 6% polyacrylamide DNA retarda-
tion gel (Invitrogen) with 0.5ϫ Tris borate-EDTA as the elec-
trophoresis running buffer. The amounts of complexed and
free DNA were visualized and quantified using a Gel Doc EZ
system (Bio-Rad). Equilibrium dissociation constants were
determined as described previously (27). Assays to test the
impact of H2O2 on DNA binding were performed under the
same conditions, with incubation with oxidant for 30 min at
25 °C either before or after incubation with target DNA as
noted. The low millimolar concentration of H2O2 used in these
EMSA assays is common practice for the in vitro evaluation of
the impact of oxidant on regulator protein-DNA binding affin-
ity (18–20, 28–30) and is required in part to overcome the 1
mM DTT necessary for the purification and storage of active
LasR. To evaluate recovery of binding with DTT, samples were
incubated for an additional 30 min at 25 °C in the presence of
FIGURE 1. Cysteine composition of LasR. A, domain structure of LasR with
the relative positions of all LasR cysteines shown. B, dimeric crystal structure
(22) of the LBD with 3O-C12-HSL bound by each monomer and both Cys79
residues shown. Within each monomer, the terminal carbon of 3O-C12-HSL is
proximal to Cys79 (4.3 Å), whereas the two cysteines in the dimeric complex
are 21.6 Å apart.
␤-Galactosidase Reporter Assays—XL10-Gold E. coli cells
(Agilent Technologies) were transformed with plasmid pKDT17 to
yield a reporter strain coupling the lasR gene with a lasB::lacZ
translational fusion (21). pKDT17 was a gift from Peter Green-
berg (Addgene plasmid 27503). Site-directed mutagenesis of
pKDT17 to convert LasR Cys79, Cys188, Cys201, and Cys203 to
serine was achieved using the QuikChange Lightning site-di-
rected mutagenesis kit (Agilent Technologies) and confirmed
by DNA sequencing (DNA Analysis Facility, Yale University).
Using this reporter strain, LasR activity was quantified as a
function of ␤-galactosidase activity as measured in Miller
assays (31) using the modified procedure reported previously
(32). Starting with a 1/1000-fold dilution of an overnight cul-
ture, the reporter strain was grown at 37 °C with shaking in LB
medium supplemented with ampicillin (200 ␮g/ml) to A600 ϭ
0.8–1.2. Cultures also included 20 ␮M 3O-C12-HSL and
10–100 ␮M H2O2 as noted. Subsequent dilution, permeabiliza-
tion, and ␤-galactosidase activity assays were performed as
described previously (32), with measured enzyme activity sim-
ilarly converted to Miller units to account for any variance in
culture density or reaction times.
excess reducing agent (25 mM DTT). DBD (64 ␮M) was reacted Results
with 45 mM iodoacetamide for 1 h at 25 °C to give alkylated
DBD for gel shift analysis.
Impact of Oxidative Stress on the LasR Ligand Binding
Domain—Previous work identified Cys79 of LasR, the sole cys-
teine within the LasR ligand binding domain (Fig. 1), as sensi-
tive to oxidative stress in a proteome-wide screen of P. aerugi-
nosa (15). This report provided evidence that LasR activity is
impacted by oxidative stress, particularly in cell-based assays,
but did not delineate the underlying protein chemistry of this
redox response. We were interested in probing this biochemi-
cal mechanism further and learning the specifics of how LasR
function may span both quorum-sensing and redox-sensing
pathways. To take a close look at Cys79, we expressed and puri-
fied the LasR LBD (amino acids 1–173) in the presence of its
native autoinducer, 3O-C12-HSL, yielding soluble protein.
Although there are numerous possible outcomes for the reac-
tion of a thiol such as Cys79 with an oxidizing agent, one con-
ceivable oxidative stress response mechanism is the formation
Mass Spectrometric Characterization of LasR—All mass
spectral analyses were performed by tandem HPLC-electros-
pray ionization mass spectrometry (LC-MS) using a Thermo
Finnigan Surveyor HPLC system and a Thermo Scientific LTQ
XL mass spectrometer. In preparation for LC-MS analysis, 2.5
␮M LasR C79S/C188S and LasR C79S each were incubated with
3 mM H2O2 for 2 h at 25 °C, followed by reaction with 45 mM
iodoacetamide for 1 h at 25 °C to cap any remaining free thiols.
Digestion of LasR C79S/C188S proceeded with the addition of
Glu-C (Promega) to 3.5 ng/␮l in 50 mM NH4HCO3 (pH 8.0)
with incubation at 37 °C for 18 h. Digested proteins were sub-
jected to LC-MS, with ions of interest selected and subjected to
collision-induced dissociation (MS/MS) to confirm peptide
identity.
11778 JOURNAL OF BIOLOGICAL CHEMISTRY
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Oxidative Stress and Quorum Sensing Regulator LasR
A
B
LasR
LBD C79S
+H2O2 +CHP +DTT
+H2O2 +CHP +DTT
250
150
100
75
50
37
25
20
15
C
250
150
100
D
Tm (°C)
WT 59.4
WT C12 C10 C8
O
O
O
O
O
O
N
H
O
O
C12 54.7
C10 47.6
C8 42.3
N
H
O
O
N
H
75
O
O
N
H
50
37
O
25
20
15
FIGURE2.ImpactofoxidativestressandunnaturalligandsonLasRLBD. A,SDS-PAGEof25␮M LBDundernon-reducingconditionsshowingbothmonomer
and dimer bands. Protein bands were excised from the third lane of a duplicate gel and subjected to LC-MS to confirm the predicted molecular weight of
monomer (20,453.0 Da observed and 20,451.1 Da calculated) and dimer (40,906.5 Da observed and 40,900.2 Da calculated) LBD. Dimer formation is enhanced
in the presence of 2.5 mM H2O2 and cumene hydroperoxide (CHP), whereas only monomer LBD is observed in the presence of 100 mM DTT or with the LBD C79S
mutant. B, DTNB quantification of free thiols under denaturing conditions illustrates the impact of 10–200 ␮M H2O2 on 25 ␮M LBD. Error bars denote standard
deviation for triplicate data. C, non-reducing SDS-PAGE of LBD expressed in the presence of 3O-C12-HSL (WT) and the non-native ligands C12-HSL, C10-HSL, and
C8-HSL. D, wavelength-dependent CD spectra comparing relative folding of 15 ␮M LBD expressed in the presence of varied AHLs. The thermal stability (Tm) of
each was also determined.
of a disulfide bond between two reactive thiols, here the linking of dimeric LBD as detected by LC-MS (40,899.8 Da observed
of two Cys79 residues. Interestingly, we saw evidence of some and 40,900.2 Da calculated). Similar experiments with H2O2
dimeric LBD when the protein was purified under non-reduc- were performed with native LBD, revealing neither the forma-
ing conditions and evaluated on a denaturing, non-reducing tion of dimeric LBD nor any other change in mass, suggesting
SDS-PAGE gel (Fig. 2A). This result suggests a disulfide bond that the thiol is largely protected from oxidation when the LBD
between monomers. Dimer formation was promoted in the is folded and ligand-bound. Additionally, no alkylation was
presence of oxidants such as H2O2 and cumene hydroperoxide observed by LC-MS when the native LBD was treated with
but reversed in the presence of the reducing agent DTT. Addi- excess iodoacetamide. The proximity of Cys79 to the terminal
tionally, mutagenesis of Cys79 to serine (LBD C79S) confirmed carbon of 3O-C12-HSL (22) (Fig. 1B) suggests that this thiol
that Cys79 was required for formation of the observed covalent might be important for ligand recognition, and others have
dimer. These results led us to probe this dimer further to deter- reported covalent modification of Cys79 when LBD is expressed
mine its importance for oxidative sensing by LasR. Because the in the presence of electrophilic probes derived from the native
two Cys79 in the known dimeric, autoinducer-bound LBD crys- AHL structure, placing reactive functional groups proximal to
tal structures (22, 33) are ϳ21 Å apart (Fig. 1B), a significant the thiol (34, 35). Cys79 appears to be most sensitive to oxidative
conformational change or unfolding of the protein would be stress when not bound to its native ligand, and perhaps the
necessary to permit the formation of a disulfide bond. Such a dimer observed by SDS-PAGE (Fig. 2A) reflects the reaction of
structural change, though, could impact the ability of LasR to two LBD monomers denatured by SDS gel sample buffer.
activate transcription.
Although we and others (22) have found that it is not possible
To further characterize the reactivity of the Cys79 thiol, we to purify soluble LBD expressed in the absence of ligand, we
completed a DTNB assay for free thiols under native and dena- moved toward an approximation of ligand-free LBD by incre-
turing conditions with 25 ␮M LBD. No labeling was observed mentally shortening the acyl chain length of ligands added to
for native LBD, but denaturation of the protein led to stoichio- LBD expression cultures. We successfully expressed and puri-
metric labeling of one thiol per monomer as anticipated. Addi- fied the LBD in the presence of a series of AHLs differing in acyl
tion of H2O2 from 10–200 ␮M readily protected the denatured chain length and presence of the 3-oxo group. Gel analysis (Fig.
LBD from labeling by DTNB (Fig. 2B), promoting the formation 2C) of these samples under non-reducing conditions revealed a
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Oxidative Stress and Quorum Sensing Regulator LasR
A
B
LasR
LasR C79S
+
3
+
+
+
6
+
+
+
+
H2O2
DTT
+
+
+
+
H2O2
DTT
IAM
1
2
4
5
7
8
9
10
+
5
1
2
3
4
(
)
LasR DNA
2
(DBD)2 DNA
DNA
DNA
H2O2 + C79S,
then DNA
DNA + LasR,
then H2O2
H2O2 + LasR,
then DNA
FIGURE 3. Oxidative stress impacts LasR-DNA binding. A, representative EMSA with 0.5 ␮M LasR and 5.0 ␮M LasR C79S. Target lasB OP1 is shown alone (lane
1). Both proteins shifted DNA in the absence of oxidant (lanes 2, 5, and 8), and the addition of 7.5 mM H2O2 significantly reduced DNA binding observed (lanes
3 and 9). The concentration of H2O2 used in these assays is similar to that reported in prior EMSA studies to evaluate the impact of oxidant on transcription
factor-DNA binding (18–20, 28–30). The incubation of LasR with DNA for 30 min prior to the addition of oxidant appears to protect LasR from oxidation,
preserving DNA binding (compare lanes 3 and 6). DNA binding for both proteins is restored with the addition of 25 mM DTT following incubation with H2O2
(comparelanes3and4forLasRandlanes9and10forLasRC79S). B, representativeEMSAwith14␮M LasRDBDindicatesbindingofthisdomaintolasBOP1(lane
2) that is disrupted in the presence of 7.5 mM H2O2 (lane 3) but restored with the addition of 25 mM DTT (lane 4). DBD reacted with iodoacetamide (IAM) prior
to the binding assay did not show DNA binding (lane 5).
trend toward dimer formation as AHL acyl chains shortened, reversible. These results suggest that one possible mechanism
suggesting a role for the native 3O-C12-HSL in mediating native for the impact of oxidative stress on LasR as a transcriptional
LBD folding and perhaps blocking disulfide bond formation. activator is through reduction in DNA binding affinity. Our
The yield of soluble protein also decreased with shorter AHLs, next aim was to catalogue the biochemical transformations
and no soluble protein was produced with C6-HSL or C4-HSL. explaining this observed change in binding affinity.
CD results also indicate that the secondary structure and ther-
The relevance of Cys79 in connecting oxidative stress to
mal stability of LasR LBD are impacted by changes to acyl chain LasR-DNA binding affinity was measured using mutant LasR
length (Fig. 2D). Our results join previous findings suggesting C79S in similar EMSA experiments. The addition of H2O2 also
that autoinducer acyl side chain length is particularly important reduced DNA binding affinity for this mutant (Fig. 3A), sug-
in dictating the stimulation of LasR activity (36) and recent gesting that Cys79 was not completely responsible for the
work cataloging hydrogen-bonding interactions in the LasR observed reduction in binding affinity. This somewhat unex-
LBD critical for LasR activation and inhibition (37, 38).
pected finding led us to investigate the three additional cys-
We also investigated the impact of Cys79 mutagenesis on teines found outside the ligand binding domain of LasR, as
LBD structure by measuring the thermal stability of LBD detailed below.
mutants C79S (Tm ϭ 55.1 °C) and C79A (Tm ϭ 55.7 °C), find-
Interestingly, mutagenesis of Cys79 does have a significant
ing that replacement of Cys79 does lower thermal stability for impact on DNA binding affinity based on results from quanti-
these mutants expressed in the presence of native 3O-C12-HSL tative EMSA. Replacement of Cys79 with serine increased the
compared with wild-type LBD (Tm ϭ 59.4 °C). Expression and apparent equilibrium dissociation constant (Kd) from 0.400 Ϯ
purification of C79A LBD in the presence of non-native C10- 0.086 ␮M to 48.4 Ϯ 8.0 ␮M (Fig. 4). The Kd for C79A LasR also
HSL gave still lower thermal stability (Tm ϭ 43.8 °C), a finding increased to 4.89 Ϯ 0.84 ␮M. Although Cys79 may not represent
that parallels the diminished stability observed for wild-type the thiol that is mediating oxidative stress response in native
LBD in the presence of shortened ligands (Fig. 2D). Taken LasR, it does appear to be important for DNA binding, likely by
together, our experiments with the LasR LBD suggest that promoting correct protein folding and dimerization in
Cys79 is important for protein folding and ligand recognition, response to native autoinducer binding.
and may form a disulfide-linked dimer in the presence of oxi-
DNA Binding Domain Cys201 and Cys203 Respond to Oxida-
dative stress. Additional experiments with full-length LasR tive Stress—Based on the EMSA experiments reported above, it
were required to probe how Cys79 oxidation might be linked to seemed likely that one or more of the three cysteines within the
the function of LasR as a transcriptional activator.
LasR DNA binding domain (Fig. 1A) might be linked to the
Oxidative Stress Impacts LasR-DNA Binding Affinity—The observed change in DNA-binding affinity in the presence of
full-length LasR protein with a C-terminal His6 tag was ex- oxidant. We generated a construct representing only the LasR
pressed and purified in the presence of 3O-C12-HSL, yielding DBD (residues 175–240) to explore this portion of the protein
soluble protein. EMSA was used to measure the interaction more closely. Expression and purification of this domain with
between LasR and one of its known target sequences, the lasB C-terminal His6 tag yielded a soluble DNA-binding protein.
operator 1 (OP1) site (39). Addition of H2O2 to binding reac- Although this domain alone was sufficient to bind DNA, the Kd
tions reduced LasR-DNA binding, particularly when oxidant (23.8 Ϯ 3.6 ␮M) was again increased over that observed for
was added to LasR prior to the addition of DNA (Fig. 3A). Bind- full-length LasR (Fig. 4).
ing was recovered when excess DTT was added to the reaction,
Addition of H2O2 to DNA binding reactions containing LasR
indicating that the impact of H2O2 was thiol-specific and DBD yielded similar results to that seen with full-length LasR
11780 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 291•NUMBER 22•MAY 27, 2016

Oxidative Stress and Quorum Sensing Regulator LasR
Da calculated). Similar experiments with LasR C79S yielded
results that also supported the formation of a Cys201-Cys203
disulfide bond. Here, treatment of the protein with H2O2
followed by iodoacetamide gave a mass equal to that pre-
dicted for one alkylated cysteine and one disulfide bond
(27,860.8 Da observed and 27,861.7 Da calculated), whereas a
water control experiment in this case gave a mass indicative of
alkylation of all three thiols (27,980.4 Da observed and 27,977.7
Da calculated). Taken together, these results suggest that
Cys201 and Cys203 form a disulfide bond under these oxidative
conditions, whereas Cys188 is not similarly oxidized, remaining
reactive toward iodoacetamide. To further characterize the
possible Cys201-Cys203 disulfide bond, LasR C79S/C188S that
had been treated with H2O2 and iodoacetamide was digested
with Glu-C protease. The expected mass for the disulfide-
linked fragment containing Cys201 and Cys203 was discerned
ϩ
((MϩH) observed 965.40, calculated 965.42). LC-MS/MS
FIGURE 4. Quantitative EMSA comparing equilibrium binding affinity for
wild-type LasR (●), LasR C79S (f), LasR C79A (Ⅺ), and LasR DBD (E). The
apparent Kd for each was determined by fitting data to the Langmuir equa-
tion as described in Ref. 27. Error bars denote standard deviation for triplicate
data.
fragmentation analysis confirmed the identity of this peptide
fragment (Fig. 5).
LasR Reporter Assay Results Parallel in Vitro Experiments—
We expanded our in vitro characterization of LasR by perform-
ing parallel experiments using an E. coli-based reporter assay.
(Fig. 3B), confirming that one or more of the cysteines within The known plasmid pKDT17 (21) includes the P. aeruginosa
this domain mediates LasR oxidative response. Reacting the lasR gene and a lasB::lacZ fusion, allowing monitoring of LasR
DBD with iodoacetamide prior to EMSA experiments resulted activity as reflected by its binding to the lasB promoter, activat-
in a triply alkylated protein (8874.4 Da observed, 8873.14 Da ing production of an elastase-␤-galactosidase fusion protein
calculated) that no longer bound DNA (Fig. 3B). These results with resultant enzyme activity detected in a Miller assay. Here,
indicate that cysteines in the DBD are important for DNA bind- LasR and its target lasB promoter are the same as components
ing affinity and that chemical modification of these thiols, of our EMSA experiments, but this cell-based assay allowed us
either through oxidation or alkylation, has a decided effect on to interrogate our findings in a different type of system. E. coli
DNA binding.
(pKDT17) was cultured under a variety of conditions, testing
To inspect each of the three DBD cysteines in turn, each was the impact of 3O-C12-HSL and H2O2 on lasB induction medi-
mutated to alanine in full-length LasR. C188A yielded a protein ated by LasR. Inclusion of AHL ligand was important for detec-
that weakly bound DNA at 6 ␮M, forming complexes that tion of robust ␤-galactosidase activity as anticipated, and the
appeared to dissociate during electrophoresis. C201A and addition of 100 ␮M H2O2 decreased observed ␤-galactosidase
C203A expression resulted in proteins that gave no discernible activity (Fig. 6A). These results are consistent with our findings
DNA binding at 3 ␮M compared with completely saturated that H2O2 impacts the DNA binding affinity of LasR. Mutagen-
wild-type LasR binding observed at ϳ1 ␮M (Fig. 4). Generation esis of pKDT17 to convert each LasR cysteine to serine allowed
of C201S and C203S mutants also gave proteins with no detect- inspection of the importance of each for LasR activity in this
able DNA binding even at 7.6 ␮M. The mutation of Cys201 or assay (Fig. 6B). The activity of all of the mutants was reduced
Cys203 consistently resulted in poor protein overexpression compared with wild-type LasR, but C201S and C203S both
yields as well, at most one-third of the yield observed for wild- showed especially low activity, similar to the wild-type con-
type LasR and Cys79 or Cys188 mutants (typically 3 mg/liter). struct assayed in the absence of AHL ligand. Again, these results
Based on these experiments that highlight the importance of are comparable with our findings with purified LasR mutants,
proximal Cys201 and Cys203, we hypothesized that a disulfide particularly highlighting the essential nature of Cys201 and
bond between these two residues might represent a redox Cys203 for functional LasR. Because of the fact that Cys201 and
switch impacting the DNA binding affinity of wild-type LasR.
Cys203 mutagenesis resulted in LasR with negligible activity
Cys201 and Cys203, seemingly important for LasR oxidative under normal growth conditions, further studies to test the
sensing, were probed further through a series of mass spec- impact of oxidative stress on these mutants were not likely to be
trometry experiments designed to assess the possibility of disul- illustrative because their level of activity is already so low.
fide bond formation. LasR C79S/C188S, retaining only Cys201
Modeling LasR DBD Structure—Although the structure of
and Cys203, was treated with H2O2, followed by excess iodoacet- the LasR DBD is not known because of reported difficulties with
amide to cap any free thiols. LC-MS analysis revealed only the full-length LasR protein solubility (22), we generated and eval-
mass anticipated for LasR C79S/C188S with one disulfide bond uated a homology model of this domain using the Phyre2 pro-
(27,788.8 Da observed and 27,788.6 Da calculated). As a control tein fold recognition server (40) to consider the placement of
experiment, treatment of LasR C79S/C188S with water fol- this disulfide bond. The model, shown in Fig. 7A, illustrates the
lowed by iodoacetamide yielded the mass expected for alkyla- bond between Cys201 and Cys203 and predicts its position
tion of the two free thiols (27,906.2 Da observed and 27,904.6 between the two helices of the helix-turn-helix motif. These
MAY 27, 2016•VOLUME 291•NUMBER 22
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Oxidative Stress and Quorum Sensing Regulator LasR
A
y8 y7 y6
y4
y2 y1
197I S V I C N C S E205
b1 b2 b3 b4 b5 b6 b7 b8
B
+
b9
+
y6
+
y7
+
b8
+
b7
+
y5
+
b4
+
y8
+
b3
FIGURE 5. Mass spectrometric mapping of the Cys201-Cys203 disulfide bond in LasR. A, graphical fragment map correlating the relevant peptide sequence
with the observed fragmentation ions. B, representative MS/MS fragmentation of the ion at m/z 965.40 corresponding to the disulfide-linked Glu-C peptide
fragment 197–205.
FIGURE 6. Miller assay to detect the activity of ␤-galactosidase produced by the E. coli lasR lasB::lacZ reporter gene system. A, impact of adding H2O2
and 20 ␮M AHL (3O-C12-HSL) to cell cultures. Error bars denote standard deviation for triplicate data. B, impact of the indicated lasR gene mutations reflected
in the reporter assay. Cultures were grown in the presence of 20 ␮M 3O-C12-HSL except where noted. Error bars denote standard deviation for triplicate data.
helices correlate to “scaffold” helix ␣8 and “recognition” helix tion of these thiols also disrupts DNA binding. Combining LasR
␣9 in sequence alignment with TraR, a LuxR-type regulator and target DNA prior to the addition of H2O2 reduces the
from Agrobacterium tumefaciens whose dimeric crystal struc- impact of the oxidant, also supporting the possibility that one
ture bound to both autoinducer and target DNA has been or both of these cysteines interact(s) closely with DNA. We
solved (41). To roughly approximate the position of Cys201 and anticipate that future structural studies will permit further
Cys203 when the DBD associates with DNA, we superimposed understanding of the roles of Cys201 and Cys203 in target site
the LasR DBD with one of the helix-turn-helix motifs in the recognition.
TraR crystal structure (Fig. 7B). It is conceivable that the rela-
Discussion
tive orientation of these helices could be impacted by the for-
mation of a Cys201-Cys203 disulfide bond, altering DNA binding
The QS regulator LasR is of interest because of its role in
affinity. It is also likely that one or both of these cysteines is/are controlling P. aeruginosa response to increased population
important for DNA recognition or proper protein folding density, linking autoinducer concentration to up-regulation of
because conversion of either to alanine or serine yields a protein processes that lead to heightened bacterial virulence. Inhibition
that is poorly expressed and no longer binds DNA, and alkyla- of LasR, therefore, represents an attractive antivirulence strat-
11782 JOURNAL OF BIOLOGICAL CHEMISTRY
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Oxidative Stress and Quorum Sensing Regulator LasR
A
B
Cys201
Cys203
Cys188
FIGURE 7. Modeling the LasR DBD. A, homology model of DBD generated using the Phyre2 web server (40). The three DBD cysteines are shown, including the
Cys201-Cys203 disulfide bond. B, overlay of the DBD homology model (purple) with the TraR DBD (right monomer, shown in gray) within the crystal structure (41)
of TraR bound to its autoinducer and target DNA.
egy, and additional understanding of LasR function and activity ing affinity is still reduced for these mutant proteins under oxi-
should enable more effective inhibition. The possibility that dizing conditions.
LasR responds to oxidative stress in addition to its known func-
Although our results do not suggest that LasR Cys79 is acces-
tion as a QS regulator is intriguing, and we have worked to sible or redox-sensitive in folded LasR, others have noted the
characterize this possible additional LasR function at the possibility that conserved LBD cysteines might mediate the oxi-
molecular level. Our work expands on earlier studies that dem- dative stress response in other LuxR-type regulator proteins.
onstrated a link between oxidative stress and LasR QS efficacy Recent efforts to catalogue solo luxR genes indicate that
in P. aeruginosa (15). Here we developed in vitro assays with many of the LuxR proteins identified feature multiple con-
purified LasR and individual LasR domains to enable us to served cysteines, particular in their LBDs, and it has been
probe the biochemical mechanism that may permit LasR to act postulated that these cysteines, including Cys79 in solo LuxR
as a redox sensor.
proteins, are positioned well for disulfide bond formation
Systematic inspection of the four cysteines within LasR finds and possible redox regulation (42). Based on prior reports
that Cys79 is important for protein folding and DNA binding (15) and our studies, it is certainly conceivable that LasR
but does not suggest a likely mechanism for this residue to act as Cys79 has an important role in vivo as a reactive thiol, par-
an oxidative sensor in native, ligand-bound LasR. Although ticularly in poorly folded LasR, and continued work will
native LBD with 3O-C12-HSL bound does not react with DTNB enable a full understanding of this residue.
or H2O2, denatured LBD does form a disulfide-linked dimer
Our experiments with the LasR DBD indicate that oxidative
when exposed to oxidative stress, indicating that Cys79 is a reac- stress does impact the DNA binding affinity of this domain
tive thiol that appears to be inaccessible when native autoin- under in vitro reaction conditions similar to those used by oth-
ducer is bound. Experiments with non-native AHLs with ers in characterizing oxidation-sensitive DNA binding proteins
shorter acyl chains further demonstrate that the absence of (18–20, 28–30). To further explore this result, we employed a
native autoinducer may permit LasR to adopt a conformation cell-based reporter assay that also indicated a reduction in LasR
that more readily forms a disulfide dimer. Cys79 is conserved in activity in the presence of H2O2. These findings intersect with
some LasR homologs that similarly recognize 3O-C12-HSL (Fig. prior work describing the influence of oxidative stress on LasR
8), underscoring our finding that this residue aids in specific activity in P. aeruginosa assays (15). Although this previous
ligand recognition. We note that mutating Cys79 significantly report focused on Cys79, their results indicating a reduction in
disrupts the enhancement in binding affinity offered by the wild-type LasR activity in the presence of H2O2 did not delin-
properly folded, dimeric LasR ligand binding domain because eate which of the four LasR cysteines is critical for this response
the observed DNA binding affinity for the DBD alone only dif- and did not consider any of the DBD cysteines. Based on our
fers by a factor of two from that measured for LasR C79S, 2 studies, we now propose that the formation of a disulfide bond
orders of magnitude greater than that for wild-type LasR. It is between Cys201 and Cys203 might regulate that stress response.
interesting that the measured DNA binding affinity is so similar Because mutagenesis of Cys201 or Cys203 yields inactive LasR in
for LasR completely lacking the ligand binding domain (DBD both EMSA and cell-based reporter assays, it is difficult to com-
alone) and LasR with a single ligand binding domain mutation pletely characterize their response to oxidative stress or inves-
(C79S), and these findings highlight the importance of Cys79. tigate a direct link between disulfide bond formation and
Mutagenesis of Cys79 to serine or alanine does not abolish sen- reduced quorum sensing in P. aeruginosa because complemen-
sitivity of LasR to oxidative stress, though, because DNA bind- tation studies with these mutants in a LasR deletion strain are
MAY 27, 2016•VOLUME 291•NUMBER 22
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Oxidative Stress and Quorum Sensing Regulator LasR
|P25084|LasR
|P12746|LuxR
|B1VD86|BraR
|Q3Y8H6|PpuR
|Q9AH79|MupR
|A5WYC9|TraR
|P07026|SdiA
|B6E4Z5|RhlR
|D3W065|CviR
1
1
1
1
1
1
1
1
1
MALVDG----------------------FLELERSSGKLEWS-AILQKMASDLGFSKILFGLL---PKDSQDYENAFIVGNYPAAWREHYD
MKNINA-------DDTY------RI---INKIKACRSNNDIN-QCLSDMTKMVHCEYYLLAII---YPHSMVKSDISILDNYPKKWRQYYD
MSPIL----------------------------AASDETQWF-DAVAGLAGSWGFSQVLLAIL---PRPGMRLEDAYVRSNYASSWRQAYT
MTLLVM-------DE-------------LLRLSEMERFEDWL-AHLKILTRKLGYSNFLIGLK---PAPTDANQQVLIYSDYPDAWRTRYD
ML---------------------------EDILMCETLEAWS-KVLFALAKEYGFSSVLFGVK---PTISTPFSSSTIISNYSRSWRSIYD
MQHW---------------------LDKLTDLAAIEGDECILKTGLADIAEHYGFTGYAY-------L-HIQHRHITAVTNYHREWQSTYF
MQDKD-FFSWR--RT---------M---LLRFQRMETAEEVY-HEIELQAQQLEYDYYSLCVR---HPVPFTRPKVAFYTNYPEAWVSYYQ
MRNDGGFLLWW--DG---------L---RSEMQPIHDSQGVF-AVLEKEVRRLGFDYYAYGVR---HTIPFTRPKTEVHGTYPKAWLERYQ
MVISKPIN-ARPLPAGLTASQQWTLLEWIHMAGHIETENEL-KAFLDQVLSQAPSERLLLALGRLNNQNQIQRLERVLNVSYPSDWLDQYM
|P25084|LasR
|P12746|LuxR
|B1VD86|BraR
|Q3Y8H6|PpuR
|Q9AH79|MupR
|A5WYC9|TraR
|P07026|SdiA
|B6E4Z5|RhlR
|D3W065|CviR
70 ARVDPTVSHCTQSVLPIFWEP--SIY---QTRKQHEFFEEASAAGLVYGLTMPLHGARGELG-ALSLSVEAENR-AEANRFMESVLPTLWM
76 IKYDPIVDYSNSNHSPINWNIFENNA---VNKKSPNVIKEAKTSGLITGFSFPIHTANNGFG-MLSFAHSEKDNYIDSLF--LHACMNIPL
64 AYIDPTVSHCTKHSSPLVWSP--DLF---ATTPQKSMYEEASAHGLRAGVTLPIHGPRQEAG-MLCFVNDSNPN-DKFWQHIDTALPNLVL
72 AAVDPVVQHCLNANRPLLWDR--DNY---RRPSESEFFEEAAAHGLQQGLALPLHGPRGEAG-MLCLKPFESGPRATATM--IESLPMATL
65 YEVDPVVFHCLNSSLPLVWTK--ENF---INKPEQQLYEAASASGVCSGLCMPIHGPRGEFG-MLNFITDEKSP-ADVIR--SKGLPQFAL
67 VALDPVVKRARSRKHIFTWSG--EQERPSLSRDERSFYARAADFGIRSGITIPIRTANGSMS-MFTLASDKPVI--DLDR--EIDAVAAAS
77 LAIDPVLNPENFSQGHLMWND--DLF---S--EAQPLWEAARAHGLRRGVTQYLMLPNRALG-FLSFSRCSARE-IPILS--DELQLKMQL
78 GAVDPAILNGLRSSEMVVWSD--SLF---D--QSRMLWNEARDWGLCVGATLPIRAPNNLLS-VLSVARDQQNI-SSFER--EEIRLRLRC
94 AQHDPILRIHL-GQGPVMWEE--RFN-RAKGAEEKRFIAEATQNGMGSGITFSAASERNNIGSILSIAGREPGR--NAA-----LVAMLNC
|P25084|LasR
|P12746|LuxR
|B1VD86|BraR
|Q3Y8H6|PpuR
|Q9AH79|MupR
|A5WYC9|TraR
|P07026|SdiA
|B6E4Z5|RhlR
|D3W065|CviR
158 ALQSGAGLA-F--EHPVSKPVVLTSREKEVLQWCAIGKTSWEISVICNCSEANVNFHMGNIRRKFGVTSRRVAAIMAVNLGLIT-L-----
165 LVDNYRKIN-I---ANNKSNNDLTKREKECLAWACEGKSSWDISKILGCSERTVTFHLTNAQMKLNTTNRCQSISKAILTGAIDCPYFK-N
152 VIDTSQRHL-S--AHAQTLIPKLTPRERECLKWTALGKSTWEISHILNCSEAVVNFHMKNIRAKFGVNSRRAAAVIATQLGLIDPG-----
159 AMEGMLK---A--RIECSTPVHLTSREKEVLQWSAAGKTTWEISMILSCTTSAIDFHFKNIRRKFQVSSRQMAVLKAIQQKSITP------
151 LIESFQKIQ-SAAEKEVEKPPKLTARELECLKWVAAGKTSWEISRILSCAEVTVNYHVTNFMRKFNAESRQHAVISGIRSGLVTP------
155 IHARISYL-RT--TPTAEDAAWLDPKEASYLRWIAVGMTMEEIADVEGVKYNSVRVKLREAMKRFDVHSKAHMIALAIRRKLI--------
161 SLMALMRLN-D--EIVMTPEMNFSKREKEILRWTAEGKTSAEIAMILSISENTVNFHQKNMQKKINAPNKTQVACYAAATGLI--------
162 LTQKLTDLE-H--PMLMSNPVCLSHREREILQWTADGKSSGEIAIILSISESTVNFHHKNIQKKFDAPNKTLAATYAAALGLI--------
178 LHQAAIRVANL--PPASPSNMPLSQREYDIFHWMSRGKTNWEIATILDISERTVKFHVANVIRKLNANNRTHAIVLGMHLAMPPST-VANE
FIGURE8. MultiplesequencealignmentofLasRanditshomologs. ProteinsequencesshownincludeP. aeruginosaLasR(UniProtP25084), VibriofischeriLuxR
(UniProt P12746), Burkholderia kururiensis BraR (UniProt B1VD86), Pseudomonas putida PpuR (UniProt Q348H6), Pseudomonas fluorescens MupR (UniProt
Q9AH79), A. tumefaciens TraR (UniProt A5WYC9), E. coli SdiA (UniProt P07026), P. aeruginosa RhlR (UniProt B6E4Z5), and Chromobacterium violaceum CviR
(UniProt D3W065). The positions of the four LasR cysteines are indicated (stars). Cys79 is shared by BraR (47), PpuR (48), and MupR (49), all believed to recognize
3O-C12-HSL. This is not the autoinducer for the other proteins shown. With LuxR, these proteins also share Cys203. Interestingly, there is no sequence homology
to Cys201 even in proteins that share Cys203, suggesting that the potential for a disulfide bond at that position is unique to LasR among known regulator
sequences. It is possible that Cys201 in its reduced state may be an important hydrophobic amino acid, based on the conserved leucine at this position in most
homologous sequences and the known hydrophobic nature of free cysteines (50).
likely to show deficient LasR-dependent quorum sensing with H2O2 (45), and other recent reports provide increased evidence
or without oxidative stress.
for the close connection between QS and central metabolism in
There is additional evidence that Cys203 is important for P. aeruginosa (46). Although it will be interesting to know pre-
LasR function. Although we find that both Cys201 and Cys203 cisely how oxidative sensing by LasR might serve P. aeruginosa,
are essential for LasR activity, Cys203 appears to be more highly it is perhaps more valuable to have learned more about this
conserved based on comparison with homologous sequences protein at the chemical level, noting multiple critical cysteines
(Fig. 8) and is even shared by LuxR. Furthermore, one LasR that may prove to be useful targets in working toward effective
mutation observed in two different studies of clinical P. aerugi- treatment of this pathogen.
nosa isolates is C203R, correlated with loss of LasR function as
measured by elastase activity (3, 6). These clinical findings fur- Author Contributions—K. A. T. cloned and expressed LasR and
ther support an important role for Cys203. It is not surprising
LBD. P. K., A. N. A., J. M. R., and T. L. S. prepared and characterized
the LasR, DBD, and LBD variants. R. L. T. synthesized and charac-
terized AHLs. E. G. S. and T. L. S. performed the LasR reporter
assays. T. L. S. supervised the project and wrote the paper with input
and approval from all authors.
that the formation of a disulfide dimer directly involving an
essential DNA contact cysteine could have a significant impact
on DNA binding affinity. Similar findings with transcription
factor p53 have recently been reported, as Cys275, critical for
p53-DNA binding affinity, is believed to form a disulfide bond
with Cys277 upon oxidation, leading to a decrease in binding
affinity (43).
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Oxidative Stress and Quorum Sensing Regulator LasR
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11786 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 291•NUMBER 22•MAY 27, 2016

Molecular Insights into the Impact of Oxidative Stress on the Quorum-Sensing
Regulator Protein LasR
Prapti Kafle, Amanda N. Amoh, Jocelyn M. Reaves, Emma G. Suneby, Kathryn A.
Tutunjian, Reed L. Tyson and Tanya L. Schneider
J. Biol. Chem. 2016, 291:11776-11786.
doi: 10.1074/jbc.M116.719351 originally published online April 6, 2016
Access the most updated version of this article at doi: 10.1074/jbc.M116.719351
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