JOURNAL OF BACTERIOLOGY, May 2008, p. 3444–3455
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 10
An Ortholog of OxyR in Legionella pneumophila Is Expressed
Postexponentially and Negatively Regulates the Alkyl
Hydroperoxide Reductase (ahpC2D) Operon?
Jason J. LeBlanc,1,2Ann Karen C. Brassinga,4Fanny Ewann,4
Ross J. Davidson,1,2,3and Paul S. Hoffman2,3,4,5*
Department of Pathology and Laboratory Medicine, Queen Elizabeth II Health Sciences Center, Halifax,
Nova Scotia, Canada1; Department of Microbiology and Immunology2and Medicine,3Faculty of
Medicine, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada; and Department of
Medicine, Division of Infectious Diseases and International Health4and Department of
Microbiology,5University of Virginia School of Medicine,
Charlottesville, Virginia 22908-1340
Received 28 January 2008/Accepted 11 March 2008
Legionella pneumophila expresses two peroxide-scavenging alkyl hydroperoxide reductase systems (AhpC1 and
expressed AhpC1 system is compensated for by increased expression of the exponentially expressed AhpC2D
system. In this study, we used an acrylamide capture of DNA-bound complexes (ACDC) technique and mass
spectrometry to identify proteins that bind to the promoter region of the ahpC2D operon. The major protein
captured was an ortholog of OxyR (OxyRLp). Genetic studies indicated that oxyRLpwas an essential gene expressed
postexponentially and only partially complemented an Escherichia coli oxyR mutant (GS077). Gel shift assays
confirmed specific binding of OxyRLpto ahpC2D promoter sequences, but not to promoters of ahpC1 or oxyRLp;
however, OxyRLpweakly bound to E. coli OxyR-regulated promoters (katG, oxyR, and ahpCF). DNase I protection
studies showed that the OxyRLpbinding motif spanned the promoter and transcriptional start sequences of ahpC2
and that the protected region was unchanged by treatments with reducing agents or hydrogen peroxide (H2O2).
Moreover, the OxyRLp(pBADLpoxyR)-mediated repression of an ahpC2-gfp reporter construct in E. coli GS077 (the
mutations have locked OxyRLpin an active DNA-binding conformation, which has permitted a divergence of
function from a regulator of oxidative stress to a cell cycle regulator, perhaps controlling gene expression during
Legionella pneumophila is found in aquatic and moist soil
environments as an intracellular parasite of protozoa (12, 14,
15). Legionnaires’ disease results from aerosol transmission of
bacteria from the environment to susceptible humans. In nat-
ural hosts (and some mammalian cell lines), L. pneumophila
displays a dimorphic lifestyle in which vegetative replicating
bacilli differentiate into metabolically dormant cyst-like plank-
tonic forms (17). Analogous to the small-cell variants of Cox-
iella burnetii or the elementary bodies of Chlamydia spp., these
highly resilient cyst-like forms of L. pneumophila enable the
organism to survive extracellularly for extended periods of
time in a highly infectious state. The cyst form likely contrib-
utes to the noted resilience of L. pneumophila to the actions of
oxidizing agents (chlorine), chloramines, and biocides, used to
control infection of man-made aquatic systems (17). Several
lines of evidence suggest that antioxidant enzymes play crucial
roles during the early stages of infection of protozoa or human
phagocytes (3, 6, 8, 19, 27) and may also be important for
environmental persistence (25).
We previously characterized two peroxide-scavenging alkyl
hydroperoxide reductase systems of L. pneumophila: AhpC1,
similar to the AhpC-thioredoxin system of Helicobacter pylori
(30, 49), and AhpC2D, similar to the AhpC AhpD system
found in mycobacteria (30, 42). These studies demonstrated
that both systems contributed to efficient scavenging of H2O2
and organic peroxides and that at least one system was re-
quired for viability. Moreover, these genes were inversely ex-
pressed during the growth cycle; ahpC2D induced early in
exponential phase and repressed postexponentially, concomi-
tant with induction of ahpC1. Finally, a compensatory increase
in expression of ahpC2D was observed in an ahpC1 mutant,
suggesting that the ahpC2D operon might be upregulated in
response to changes in cellular redox status or by increases in
intracellular peroxides. However, previous studies of the anti-
oxidant defense genes of L. pneumophila (sodB, sodC, katA,
katB, ahpC1, or ahpC2D) had concluded that none of these
genes were activated in response to oxidative stress (4, 5, 30,
39, 43). In contrast, these studies revealed that all of these
antioxidant defense genes, with the exception of sodB (consti-
tutive), were growth stage regulated.
The emerging pattern of growth stage-regulated antioxidant
* Corresponding author. Mailing address: Division of Infectious
Diseases and International Health, Room 2146, MR-4 Bldg., 409 Lane
Road, University of Virginia Health Systems, Charlottesville, VA
22908-1340. Phone: (434) 924-2893. Fax: (434) 924-0075. E-mail:
?Published ahead of print on 21 March 2008.
defense genes in L. pneumophila together with the postexpo-
nential expression of transmission (virulence) traits is consis-
tent with the dimorphic lifestyle of this organism (17). In sup-
port of this view, microarray analyses comparing sessile and
planktonic cells of L. pneumophila under biofilm conditions
revealed ahpC2 and ahpD to be the most highly induced genes
in sessile cells (25). Related studies have similarly shown that
expression of the ahpC2D operon increases significantly during
intracellular growth in protozoa (8). Despite these findings,
little is known of the regulatory systems controlling ahpC2D
expression or other antioxidant defense genes in L. pneumo-
phila. With the exception of an OxyR homolog (described
herein), typical regulators of oxidative stress defenses (such as
SoxRS, OhrR, and PerR) are absent in the L. pneumophila
genome. Growth phase-dependent regulation of genes associ-
ated with virulence and transmission traits has been attributed
in part to global regulators, such as the stationary-phase sigma
factor (RpoS) (2, 20), the carbon storage regulator (CsrA) (13,
33), and the LetAS, CpxRA, and PmrBA two-component reg-
ulatory systems (13, 16, 22, 32, 52); however, the predicted
hierarchal regulatory cascade is incomplete, and none of these
regulators has been linked to oxidative stress defenses.
Here we report the use of an acrylamide capture of DNA-
bound complexes (ACDC) technique (35) and tandem mass
spectrometry of ahpC2D promoter-bound proteins to identify
a previously uncharacterized putative regulator of oxidative
stress in L. pneumophila, designated OxyRLp. These studies
will show that while oxyRLpwas able to partly complement an
oxyR mutant of Escherichia coli and purified OxyRLpexhibited
weak binding to OxyR-regulated promoters of E. coli by gel
shift experiments, the protein was no longer redox active and
its repression of ahpC2D could not be reversed by H2O2. Fi-
nally, the apparent essentiality of oxyRLptogether with its post-
ahpC2D, suggests regulatory roles that are unrelated to oxida-
with repression of
MATERIALS AND METHODS
Bacterial strains and growth conditions. Bacterial strains and plasmids used in
this study are listed in Table 1 and oligonucleotides in Table 2. L. pneumophila
strains were grown aerobically at 37°C on buffered charcoal yeast extract agar or
in buffered yeast extract (BYE) broth, supplemented with ?-ketoglutaric acid (1
mg/ml), ferric pyrophosphate (250 ?g/ml), L-cysteine (40 ?g/ml), thymidine (100
?g/ml), and antibiotics when required. E. coli strains DH5?, MG1655, and
GS077 were grown at 37°C on Luria-Bertani (LB) agar or in LB broth supple-
mented with the appropriate antibiotics. For some studies, E. coli GS077 was
grown at 37°C under anaerobic conditions using the BD GasPak EZ (Becton
Dickinson, Oakville, Ontario) and AnaeroGen anaerobic atmosphere generation
system (Oxoid, Ltd., Nepean, Ontario). Antibiotics (Sigma-Aldrich Canada Ltd.,
Oakville, Ontario) were added to media at the following concentrations, when
appropriate: streptomycin (100 ?g/ml), kanamycin (40 ?g/ml), chloramphenicol
(20 ?g/ml), and ampicillin (100 ?g/ml). All strains were stored at ?85°C in
nutrient broth containing 10% dimethyl sulfoxide.
Standard molecular techniques. Preparation of DNA, restriction enzyme di-
gestions, and other molecular techniques have been described elsewhere (40).
Plasmid isolation was performed with the QIAprep spin miniprep kit (Qiagen
Inc., Mississauga, Ontario) or using a standard alkaline lysis method (40). Puri-
fication of DNA fragments from agarose gels for subcloning was carried out with
a QIAquick gel purification kit (Qiagen). PCR was performed under conditions
described by LeBlanc et al. (30) using oligonucleotides synthesized by Integrated
DNA Technologies, Inc. (Coralville, IA). DNA and protein sequencing was
carried out by the DalGen Microbial Genetics Center (Halifax, Nova Scotia,
TABLE 1. Strains, genotypes, and plasmids used in this study
Bacterial strain, genotype,
Relevant property(ies)Source or reference
BL21 Codon Plus
F??80dlacZ?M15 ?(lacZYA-argF)U169 endA1 recA1 hsdR17 deoR thi-1 supE44 gyrA96 relA1
F?ompT hsdS (rB
?) dcm?Tetrgal? (DE3) endA Hte ?argU ileY leuW Camr?
Philadelphia-1 derivative, rpsL hsdR thyA (Smr) mutant
Lp02 ahpC1::kan mutant
Lp02 ahpC2D::kan mutant
Lp02 strain MB 379::kan
Lp02 strain MB 414::kan
Lp02 integration host factor double mutant (lacking both ? and ? subunits); KanrGmr
IPTG-inducible T7 expression vector
pET29b-derived overexpression vector for His6-OxyRLp
RSF1010 ori, promoterless gfpmut3 td?i (Ampr)
PahpC2region (?199 to ?27 bp upstream of ahpC2) cloned upstream of gfpmut3 of pBH6119
PahpC2region (?179 to ?27 bp upstream of ahpC2) cloned upstream of gfpmut3 of pBH6119
PahpC2region (?156 to ?27 bp upstream of ahpC2) cloned upstream of gfpmut3 of pBH6119
PahpC2region (?132 to ?27 bp upstream of ahpC2) cloned upstream of gfpmut3 of pBH6119
PahpC2region (?109 to ?27 bp upstream of ahpC2) cloned upstream of gfpmut3 of pBH6119
PahpC2region (?91 to ?27 bp upstream of ahpC2) cloned upstream of gfpmut3 of pBH6119
PahpC2region (?66 to ?27 bp upstream of ahpC2) cloned upstream of gfpmut3 of pBH6119
oxyR promoter region cloned upstream of gfpmut3 of pBH6119
Arabinose-inducible expression vector (Ampr)
pBAD22-derived expression vector for E. coli oxyR
pBAD22-derived expression vector for L. pneumophila oxyR
VOL. 190, 2008 OxyRLpREGULATION OF ahpC2D IN LEGIONELLA3445
Canada). Protein concentrations were estimated using a Bio-Rad protein assay
(Bio-Rad, Hercules, CA).
ACDC. To identify transcription factors that bind to the promoter region of
ahpC2D (PahpC2), an ACDC was performed according to a modified version of
the methods described by Nelson et al. (35). Briefly, PCRs were performed using
forward primers containing 5? Acrydite (Ac) moiety, Ac-FC2PROM, and un-
modified reverse primer RC2PROM. Amplicons were purified using a MinElute
gel extraction kit (Qiagen). Protein lysates were obtained as follows: bacteria
were harvested (4,800 ? g, 6 min, 4°C) from a 500-ml, 24-h culture of L.
pneumophila. The pellet was washed with cold 50 mM Tris-HCl, pH 7.5, and
resuspended in buffer consisting of 50 mM Tris-HCl, pH 7.5; 1 mM EDTA, pH
8.0; 50 mM NaCl; 10% glycerol with 0.1 mM phenylmethylsulfonyl fluoride
(PMSF); and 1 mM dithiothreitol (DTT). Cells were lysed using a French press
and subjected to ultracentrifugation at 100,000 ? g. Protein lysates were ali-
quoted and stored at ?85°C. Before use, protein lysates were diluted in a buffer
consisting of 100 mM Tris-HCl, pH 7.5, and 10 mM MgCl2and treated for 15
min at room temperature with 100 mM of acetyl phosphate (phosphorylate
DNA-binding proteins). Binding reactions for the ACDC assay were performed
in a 25-?l volume and consisted of 0 (no DNA for control), 2, 4 or 8 ?g of
Ac-DNA, 1 ?g of protein lysate, 7.5 ?l buffer D (20 mM HEPES, pH 7.8; 100
mM KCl; 10% glycerol; and 1 mM DTT), 1 ?g poly(dI-dC), and 3.5 ?l of 30%
acrylamide. Reactions were incubated for 15 min at room temperature before
addition of 1.25 ?l of 5% ammonium persulfate and 1.25 ?l of diluted
N,N,N?,N??-tetramethylethylenediamine (1:30 in buffer D). The reactions were
immediately loaded into the wells of a precast nondenaturing 5% polyacrylamide
gel (0.5? Tris-borate-EDTA [TBE], Bio-Rad Laboratories). After polymeriza-
tion (approximately 5 min), electrophoresis was performed in 0.5? TBE for 1 h
at 125 V to remove nonspecific proteins. Each well was excised from the gel,
transferred to a microtube containing 100 ?l of 10% sodium dodecyl sulfate
(SDS) sample buffer (NEB) with 5% ?-mercaptoethanol and heated for 3 min at
94°C to remove captured proteins. After a brief centrifugation, 50-?l aliquots
were loaded onto a precast 4 to 15% Tris-HCl polyacrylamide gel (Bio-Rad
Laboratories) and subjected to SDS-polyacrylamide gel electrophoresis (SDS-
PAGE). After silver staining, proteins distinct from the no-DNA controls were
excised and submitted for mass spectrometry analysis (DalGen Microbial
Cloning and overexpression of OxyRLpin E. coli GS077. For complementation
of the E. coli strain GS077 (oxyR::kan) (generously donated by Gisela Storz,
National Institutes of Health, Bethesda, MD), E. coli strain K-12 oxyR (oxyREc)
or oxyRLpwas expressed using the arabinose-inducible expression systems
of pBAD22 (18). Amplicons were generated from PCRs using primer pairs
FECOXYRPBAD and RECOXYRPBAD for oxyREcand FLPOXYRPBAD and
RLPOXYRPBAD for oxyRLp, digested with EcoRI and HindIII and subcloned
into pBAD22. The resulting constructs (pBADEcoxyR and pBADLpoxyR) were
transformed into E. coli DH5? and verified by PCR and DNA sequencing.
Plasmids were extracted and transformed into electrocompetent E. coli GS077.
Ampicillin-resistant GS077 transformants obtained anaerobically were con-
firmed by PCR analysis and termed GS077 pBADEcoxyR and GS077 pBADL-
poxyR. The pBAD22 empty vector was transformed into wild-type MG1655 and
GS077 strains, verified by PCR analysis using primers for the ampicillin resis-
tance cassette FBLA and RBLA, and termed MG1655 pBAD22 and GS077
Disk diffusion assays. Strains were grown under anaerobic conditions to mid-
exponential phase (optical density at 620 nm [OD620] of 0.4), and expression of
oxyREcor oxyRLpfrom respective pBAD22-derived plasmids was induced with
various concentrations of arabinose (0, 0.02, and 0.2%) or repressed using 0.2%
glucose. Samples were analyzed by SDS-PAGE to confirm the expression of
OxyREcand OxyRLp. Cells corresponding to 0.1 OD620were inoculated in 4 ml
of prewarmed LB 0.75% top agar, mixed by inversion, and poured onto LB
media with proper antibiotics and in the presence of arabinose or glucose. Once
solidified, 10 ?l of 30% H2O2was placed on sterile one-fourth-inch antibiotic
disks in the center of the plate. Following overnight incubation at 37°C under
anaerobic conditions, the diameters of the zones of inhibition were measured
and reported for three independent experiments.
Overproduction and purification of OxyRLp. The primer pair FLPOXYRPET
and RLPOXYRPET was used to generate an amplicon containing NdeI and
XhoI restriction sites, respectively. Following digestion, the NdeI-XhoI fragment
was subcloned into the corresponding sites of pET29b, generating pETLpoxyR.
The in-frame fusion of L. pneumophila oxyR with the C-terminal hexameric
histidine (His6) tag was confirmed by DNA sequencing. For overexpression, an
overnight culture of E. coli BL21(DE3) Codon Plus harboring pETLpoxyR was
inoculated 1% (vol/vol) into 200 ml of fresh LB broth and grown to an OD620of
0.4. Isopropyl-?-D-thiogalactopyranoside was added to a final concentration of 1
mM, and incubation was continued for another 60 min at 37°C. Bacteria were
then harvested by centrifugation (5,800 ? g for 6 min), resuspended (5 ml/g [wet
weight]) in suspension buffer (20 mM Tris-HCl, pH 7.9; 500 mM NaCl; 5 mM
imidazole; and 1 mM PMSF), and lysed on ice by sonication (six bursts of 10 s at
200 W). Following clarification by centrifugation (10,000 ? g for 15 min, 4°C),
the His6-OxyRLpwas purified by nickel interaction chromatography as previously
described (41). Samples taken from each step of purification were analyzed by
SDS-PAGE, and the purified protein fractions were pooled, aliquoted, and
stored at ?85°C. A single band was excised and submitted for mass spectrometry
analysis. Purified OxyRLpprotein used in this study is considered to be wholly
EMSA. For electrophoretic mobility shift assay (EMSA), amplicons spanning
the promoter regions of ahpC1, ahpC2, and oxyR (PahpC1, PahpC2, and PoxyR) were
generated by PCR amplification using primers sets FC1PROM and RC1PROM,
FC2PROM and RC2PROM, and FLPOXYRPROM and RLPOXYRPROM,
respectively. For binding assays to the E. coli ahpCF, katG, and oxyREcpromot-
ers, primer pairs FECAHPCPROM and RECAHPCPROM, FECKATGPROM
and RECKATGPROM, and FECOXYRPROM and RECOXYRPROM were
used to generate respective amplicons. Fifty nanograms of the DNA probes was
mixed with various amounts of His6-OxyRLp(0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250,
and 500 ng) in a 20-?l reaction mixture containing 20 mM Tris-HCl, pH 8.0; 5
mM MgCl; 20 mM KCl; 50 ?g bovine serum albumin; 20% glycerol; 1 mM
TABLE 2. Oligonucleotide primers used in this study
3446LEBLANC ET AL. J. BACTERIOL.
PMSF; and 0 or 200 mM DTT (Promega, Madison, WI) for oxidizing or reducing
conditions, respectively. After 30 min of incubation at room temperature, pro-
tein-DNA complexes were separated by electrophoresis for approximately 1 h at
125 V on a 5% nondenaturing polyacrylamide gel in 0.5? TBE buffer. Gels were
then soaked in 10,000-fold diluted Sybr green I nucleic acid stain (Invitrogen, La
Jolla, CA) and washed twice in distilled water, and DNA was visualized using a
Typhoon 9410 system (GE Healthcare) under blue light excitation at 488 nm and
using a 520-nm emission filter. Densitometry was performed using ImageQuant
version 5.2 (Molecular Dynamics, Sunnyvale, CA).
Mapping of PahpC2. An EMSA was performed as described above using 500 ng
of His6-OxyRLpand 50 ng of variously sized 5?-end deletion DNA fragments
generated by PCR using a common reverse primer, RC2PROM, with different
forward primers: FC2PROM, FC2PROM179, FC2PROM156, FC2PROM132,
FC2PROM109, FC2PROM91, and FC2PROM66. All promoter fragments were
also subcloned into pBH6119 and electroporated into L. pneumophila Lp02 and
the ahpC1::kan mutant (30) to evaluate promoter activity using the green fluo-
rescence protein (GFP) reporter assay.
DNase I footprinting. DNase I protection experiments were performed using
a 226-bp DNA fragment of PahpC2generated from PCRs using primer pair
FC2PROM and RC2PROM (containing EcoRI and BamHI restriction sites,
respectively). The digested DNA (EcoRI for the top strand and BamHI for the
bottom strand) was then treated with calf intestinal phosphatase (New England
BioLabs, Ipswich, MA), and reactions were purified using a QIAquick gel ex-
traction kit (Qiagen). End-labeled DNA was prepared using [?-32P]dATP and
Ready-To-Go T4 polynucleotide kinase (GE Healthcare) as described by the
manufacturer and purified with a QIAquick nucleotide removal kit (Qiagen).
Binding reactions were performed by incubating approximately 20,000 cpm of
labeled probe with 10 ?g of OxyRLpprotein in an assay buffer consisting of 20
mM Tris-HCl, pH 8.0; 10 mM MgCl2; 100 mM KCl; 1 mM CaCl2; 10% glycerol;
0.05 mg/ml bovine serum albumin; 4 ?g/ml poly(dI-dC); and H2O2(0 to 50 mM)
for oxidizing or 200 mM DTT for reducing conditions. In cases where peroxi-
dation abolished DNA binding, replicates were treated with 200 mM DTT to
restore DNA binding activity. After 20 min of incubation, 0.5 to 1 U of DNase
I (Sigma) was added. The reaction was continued for 90 s before 100 ?l of a stop
solution (200 mM EDTA, pH 8.0; 5 M ammonium acetate; and 100 ?g of salmon
sperm DNA) was added. DNA was ethanol precipitated following phenol-chlo-
roform extraction. Dry pellets were mixed with a formamide loading dye and
loaded onto a 6% sequencing gel. The DNA ladder was performed by cycle
sequencing using an ?-35S-dATP label according to the SequiTherm Excel II
DNA sequencing kit (Epicenter Technologies).
Primer extension. Total RNA was obtained from exponentially grown L.
pneumophila by an automated Maxwell-16 protocol (Promega), and primer ex-
tension reactions were performed as follows. Briefly, 2 pmol of [?-32P]ATP
end-labeled RC2PROM primer was added to 25 ?g total RNA and allowed to
anneal by dropping the temperature from 80°C to 41°C over a period of 30 min.
cDNA extension products were generated at 41°C for 1 h in a reaction mixture
containing 1 ?l of 200 U/?l SuperScript reverse transcriptase (Invitrogen) and
subsequently treated with RNase A (Sigma) to eliminate residual RNA. Samples
were loaded onto a 6% polyacrylamide urea sequencing gel along with the
corresponding DNA sequence ladder obtained by cycle sequencing using an
?-35S-dATP label with the appropriate primer in accordance with the Sequi-
Therm Excel II DNA sequencing kit protocol.
GFP reporter assay. A GFP-transcriptional fusion of PoxyRwas constructed
using primer pair FLPOXYRPROM and RLPOXYRPROM and cloned into the
EcoRI and BamHI sites of pBH6119 (21). Plasmid constructs were first trans-
formed into E. coli DH5? strains, and correct orientation was determined by
PCR using the respective combination of FLPOXYRPROM and RGFP primers.
The pBH6119 plasmid or its derivatives were electroporated into wild-type,
ahpC1, ahpC2D, rpoS, letA, or himAB mutants of L. pneumophila. Transformants
capable of growth without thymidine were verified for respective plasmids by
PCR analysis using primer pairs FLPOXYRPROM and RLPOXYRPROM or
FBLA and RBLA for the empty vector controls. Fluorometric detection of
samples taken from BYE-grown bacteria was performed as described previously
(30). Values are expressed as relative fluorescence units (RFU) per OD620and
represent triplicate values obtained from three independent experiments.
OxyRLpregulation of ahpC2-gfp in E. coli GS077. DNA fragments containing
gfp (no promoter) or PahpC2gfpwere excised with EcoRI and PstI from plasmids
pBH6119 and pC2gfp, respectively, and were subcloned into pMMB206. The
resulting constructs, pgfp and pahpC2gfp, respectively, were electroporated into
either GS077 pBAD22 or GS077 pBADLpoxyR. Ampicillin- and chloramphen-
icol-resistant transformants were confirmed by PCR analysis and subjected to the
GFP reporter assay (30). Overnight cultures, grown at 37°C in LB broth under
anaerobic conditions, were inoculated 1% into fresh media until an OD620of 0.4
was obtained. Then, glucose or arabinose was added to a final concentration of
0.02%, and cultures were incubated for an additional hour. In some cases, strains
were then challenged for 30 min with 100 ?M of H2O2. Fluorometric detection
of samples taken from LB-grown bacteria was performed as described previously
(30). Values are expressed as RFU per OD620and represent triplicate values
obtained from three independent experiments.
Acrylamide capture of ahpC2D DNA-bound complexes. We
employed the ACDC technique (Fig. 1) to screen for transcrip-
tion factors that bound the promoter region of ahpC2D. The
principle of this method relies on capture of DNA-binding
proteins from L. pneumophila lysates by interaction with
ahpC2D promoter DNA sequences immobilized in the poly-
acrylamide matrix by covalent linkage to its 5? Ac moiety.
Proteins that do not bind the Ac-DNA can be removed by
standard nondenaturing electrophoresis. Following SDS-
PAGE, silver staining, and mass spectrometry analysis of pro-
teins captured in the wells of the native acrylamide gel, pro-
teins able to bind target DNA sequences can be identified.
Using increasing concentrations of Ac-labeled ahpC2D pro-
moter DNA (Ac-PahpC2) in the ACDC assay (Fig. 1 inset), a
distinct protein was captured by the immobilized Ac-DNA,
whereas no bands could be seen in the absence of Ac-DNA.
Peptides identified by tandem mass spectroscopy analysis cor-
responded to 181 of 296 amino acids (61% sequence coverage)
of a previously uncharacterized protein annotated as the hy-
drogen peroxide-inducible gene activator, OxyR, of L. pneu-
mophila Philadelphia-1. This protein, designated OxyRLp,
shared 45 to 50% identity and 63 to 69% similarity to well-
characterized OxyR regulators of Xanthomonas campestris, E.
coli, Salmonella enterica serovar Typhimurium, Haemophilus
influenzae, and C. burnetii by BLAST analysis.
The identification of OxyRLpas a putative regulator of
ahpC2D was not unexpected, since OxyR mediates oxidative
stress induction of alkyl hydroperoxide reductase and catalase
genes in many organisms (1, 26, 31, 36). Moreover, in those
organisms expressing the ahpC2D operon, including close rel-
ative C. burnetii, oxyR is usually located immediately upstream
and shares promoter and regulatory sequences. However, the
oxyRLpgene was not located upstream of ahpC2D, but rather
shares a divergent promoter region with a gene annotated as a
major facilitator transporter, and downstream is a gene of
unknown function oriented in the opposite direction. Our at-
tempts to knock out oxyRLpby allelic replacement with a drug
resistance cassette (kanamycin) by methods previously used to
obtain ahpC1 and ahpC2D mutants (30) proved unsuccessful.
Since catalase supplementation to media enabled the recovery
of oxyR mutants in Pseudomonas aeruginosa (23), buffered
charcoal yeast extract agar was supplemented with catalase
(10,000 units); however, no oxyRLpmutants were recovered.
The apparent essentiality of oxyRLpwas unexpected, since the
genomic organization was uncomplicated by overlapping reg-
ulatory elements or downstream polarity effects. Possible func-
tional divergence was considered, since previous studies had
indicated that genes typically regulated in response to oxidative
stress in other bacteria (ahpC1, ahpC2D, katA, katB, sodB, and
sodC) were not similarly regulated in L. pneumophila (4, 5, 30,
38, 39, 43). Rather, most of these genes exhibited growth
stage-dependent expression (4, 5, 30, 43).
VOL. 190, 2008OxyRLpREGULATION OF ahpC2D IN LEGIONELLA 3447
Complementation of E. coli oxyR mutant. Functional
complementation was assessed by cloning (pBAD22) and ex-
pressing the oxyRLpgene in E. coli oxyR mutant strain GS077.
Studies were conducted under anaerobic conditions because
this strain is highly susceptible to peroxide killing (compared
with wild-type strain MG1655) due to a loss of OxyR regula-
tory functions. Under inducing conditions (arabinose) or re-
pressing conditions (glucose), the presence of pBAD22 did not
alter peroxide susceptibility (Table 3). Complementation of
GS077 with pBADEcoxyR fully restored peroxide resistance to
wild-type E. coli MG1655 levels under inducing conditions, but
not under repressing conditions. In contrast, overexpression of
pBADLpoxyR partially rescued the peroxide sensitivity of E.
coli GS077, suggesting that OxyRLprecognizes OxyR-regu-
lated promoters. The noted partial complementation by
OxyRLpmight be due to DNA sequence differences in OxyR
binding motifs between these organisms.
Interaction of OxyRLpwith the promoter region of ahpC2.
To both confirm ACDC results and extend functional studies,
Ni2?-nitrilotriacetic acid-purified OxyRLp
from pETLpoxyR) was assessed by EMSA for DNA-binding
specificity to the promoter regions (PahpC2, PahpC1, and PoxyR)
of these genes. As seen in Fig. 2A, OxyRLpbound to a 226-bp
DNA fragment containing PahpC2(?199 to ?27 relative to the
ahpC2 coding sequence). This is evident in the sample con-
taining 500 ng of His6-OxyRLp, where 84% of the DNA probe
was in the bound state. No differences in binding were ob-
served under oxidizing (no added reducing agent) or reducing
conditions (200 mM DTT) (see Fig. 2B). Similar experiments
were also carried out with PahpC1and PoxyR; however, no DNA
binding was observed (data not shown). EMSAs were also
performed with amplicons containing promoter regions of
known members of the E. coli OxyR regulon (ahpCF, katG,
and oxyR) under oxidizing conditions. As seen in Fig. 2C,
FIG. 1. Acrylamide capture of proteins interacting with PahpC2. The ACDC assay was performed by adding 1 ?g of protein (cell extract)
prepared from BYE-grown L. pneumophila to various amounts of Ac-DNA (0, 2, 4, and 8 ?g). Nonspecific proteins were removed by nondena-
turing electrophoresis on a 0.5? TBE-5% polyacrylamide gel. Captured proteins (inset) were excised from wells, heated in SDS sample buffer
containing ?-mercaptoethanol, and following SDS-PAGE, visualized by silver staining. dsDNA, double-stranded DNA; APS, ammonium persul-
fate; TEMED, N,N,N?,N??-tetramethylethylenediamine.
TABLE 3. Zones of inhibition (mm) in a disk diffusion assay using
10 ?l of 3% H2O2
Zones of inhibition (mm) with:
Glucose (%) Arabinose (%)b
0 0.2 0.020.2
21 ? 1
21 ? 1
38 ? 2
39 ? 1
37 ? 2
39 ? 1
23 ? 1
22 ? 1
40 ? 1
41 ? 1
40 ? 1
39 ? 1
21 ? 1
22 ? 1
38 ? 1
38 ? 1
19* ? 1
29* ? 2
21 ? 1
22 ? 1
37 ? 2
38 ? 1
18* ? 1
29* ? 2
aTriplicate experiments were performed under anaerobic conditions on Muel-
ler-Hinton agar without antibiotics (†) or supplemented with 100 ?g/ml ampi-
cillin (‡), with 40 ?g/ml kanamycin (§), or with 100 ?g/ml ampicillin and 40 ?g/ml
b*, significance (P ? 0.01) was determined in relation to strain GS077.
3448LEBLANC ET AL. J. BACTERIOL.
OxyRLpweakly bound these promoters and only at the highest
concentrations of protein used (500 ng). The EMSA results
indicate that the partial complementation of an E. coli oxyR
mutant by oxyRLpmight be due to a combination of OxyRLp
overexpression and partial activation of OxyR-regulated pro-
moters in GS077.
Mapping of the OxyR binding site within PahpC2. To identify
upstream DNA sequences necessary for ahpC2 expression,
PCR-generated 5?-end deletion fragments of PahpC2were
cloned upstream of gfpmut3, encoding GFP. The promoter
fragments shared a common 3? end at ?27 (relative to the
ahpC2 initiation codon) and a variable 5? end ranging from
?199 to ?66 (Fig. 3A). The ability of PahpC2fragments to
express GFP was assessed in the ahpC1 mutant (Fig. 3B), since
ahpC2 levels are increased in this mutant (30). The 5?-end
deletions of PahpC2from base pairs ?179 to ?109 showed
transcriptional activity similar to that of the previously studied
full-length fragment (?199 to ?27) (30). However, PahpC2
deletions of ?91 or ?66 no longer expressed GFP. It should be
noted that similar results were obtained in the wild-type strain,
albeit at a lower expression level (data not shown). In a parallel
series of experiments, mobility shift assays were performed
with various PahpC2fragments (Fig. 3C). Deletion of nucleo-
tides ?199 to ?109 had no effect on OxyRLpbinding (Fig. 3C,
lanes 2 to 5). However, further deletion of nucleotides ?91 to
?66 (which had ablated transcription) did not completely abol-
ish DNA binding, suggesting that OxyRLpbinding motif(s)
remained. An alignment of DNA sequences for the ?105 to
?55 region of PahpC2for the three genome sequences for L.
pneumophila together with sequences from OxyR-regulated
genes from E. coli and other species reveals some conservation
with the E. coli OxyR consensus (Fig. 3D). Taken together,
these findings suggest that the OxyRLp-protected region con-
tains an OxyR motif that might include promoter sequences.
DNase I footprinting and primer extension. To further de-
lineate the specific DNA binding sequence of PahpC2recog-
nized by OxyRLp, the untreated and reduced (200 mM DTT)
forms of OxyR (OxyRLp-oxand OxyRLp-red, respectively) were
used in a footprinting assay. OxyRLp-redand OxyRLp-oxshowed
similar DNA footprints (Fig. 4A and B) and confirmed that the
FIG. 2. Interaction between OxyRLpand PahpC2. (A) Fifty ng of a 226-bp PahpC2fragment spanning the region ?199 to ?27 (relative to the L.
pneumophila ahpC2 initiation codon) was incubated in the presence of various amounts of His6-OxyRLp. Lanes: 1, low-mass ladder (Invitrogen);
2, DNA without protein; 3 to 10, DNA in the presence of 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, or 500 ng of His6-OxyRLp, respectively. Arrows indicate
bands representing the DNA probe (P) and mobility shift (S). Densitometry analysis reported as a percentage of DNA probe (P [%]) or shift (S
[%]) relative to the total intensity of each lane. (B) Mobility shift assay performed in the presence of 25 ng PahpC2with OxyRLpunder reducing
(200 mM DTT, OxyRLp-red) or oxidizing (no DTT, OxyRLp-ox) conditions. (C) Mobility shift assay performed in the presence of 50 ng each of E.
coli PahpCF, PkatG, and PoxyRin the presence (?) or absence (?) of 500 ng His6-OxyRLpunder nonreducing conditions.
VOL. 190, 2008OxyRLpREGULATION OF ahpC2D IN LEGIONELLA 3449
protected region included the ?91 and ?66 regions. Primer
extension analysis (Fig. 4C) revealed that the transcription
start site as well as the ?10 hexamer was located within the
OxyRLp-protected region (Fig. 4D). The location of the pro-
moter region and transcription start site within the OxyRLp-
protected region is unusual, since in E. coli, reduced OxyR
binds upstream of the promoter region of OxyR-regulated
genes and represses transcription by overlapping the ?35 hex-
amer (9, 31, 34, 36, 45, 46). In the case of PahpC2, the ?35
hexamer is outside of the OxyRLp-protected region, suggesting
a different regulatory mechanism from that reported for
OxyREc(46, 47). However, if OxyRLpwas not fully oxidized
during the purification process, as is typical for other OxyR
proteins (47, 48), then perhaps further oxidation with H2O2
might reveal more typical redox-mediated DNA-binding dif-
To test this possibility, we treated OxyRLpwith a range of
peroxide concentrations (10 ?M to 50 mM) and determined
that 1 mM completely abolished DNA binding (Fig. 4A, lane
4). While not shown, treatment of OxyRLpwith 500 ?M H2O2
reduced DNA binding by 50% but did not alter the protected
area relative to untreated OxyRLpcontrols. To distinguish
between reversible and irreversible oxidation of OxyRLp, we
followed 1 mM H2O2treatment with 200 mM DTT treatment
FIG. 3. Promoter deletion analysis of the OxyRLpbinding site within PahpC2. (A) Schematic representation of the 5?-end deletions of PahpC2
cloned upstream of promoter-less gfp. (B) Fluorescence detected from ahpC1::kan mutants harboring plasmids and PahpC2promoter DNA-GFP
constructs. (C) Mobility shift assay performed with the 5?-end deletions of PahpC2. Each lane contained 50 ng of the DNA fragments incubated with
(right panel) or without (left panel) 500 ng of His6-OxyRLp. For both gels, lanes are as follows (symbols as shown in panel B): 1, low-mass ladder
(Invitrogen); 2, PahpC2fragment ?199 to ?27 (f); 3, PahpC2fragment ?179 to ?27 (?); 4, PahpC2fragment ?156 to ?27 (Œ); 5, PahpC2fragment
?132 to ?27 (*); 6, PahpC2fragment ?109 to ?27 (E); 7, PahpC2fragment ?91 to ?27 (●); 8, PahpC2fragment ?66 to ?27 (‚); and 9, no-DNA
control (}). DNA fragments are annotated relative to the ahpC2 initiation codon. (D) DNA alignments of the ahpC2 upstream region (?115 to
?55) from L. pneumophila Philadelphia-1, L. pneumophila Paris, and L. pneumophila Lens; ahpC from Xanthomonas campestris (Xc) and P.
aeruginosa (Pa); and ahpC, katG, oxyR, and oxyS from E. coli. The consensus sequence from E. coli is provided, and the matching motifs are
3450LEBLANC ET AL. J. BACTERIOL.
to reduce the disulfide bonds, and as seen in Fig. 4A, lane 5,
subsequent reduction could not restore DNA binding activity.
These studies indicate that the OxyRLpfootprint is unchanged
by treatments with H2O2at concentrations that should be
sufficient to fully oxidize the protein.
OxyRLprepresses ahpC2-gfp expression. Since the OxyRLp
footprint for ahpC2 appears to block the binding of the RNA
polymerase complex, we sought to confirm that OxyRLpwas
indeed a repressor. We first introduced pahpC2gfp into E. coli
GS077 (the oxyR mutant) and noted constitutive expression of
GFP (Fig. 5). We then introduced pBADLpoxyR and pBAD22
vector controls into GS077 strains harboring pahpC2gfp and
pgfp controls and induced each with either arabinose or glu-
cose. As seen in Fig. 5, arabinose-dependent induction of
FIG. 4. OxyRLpbinding of PahpC2and ahpC2 transcript start site. Shaded bars indicate OxyRLpDNA footprints for bottom strand (end labeled
at BamHI) (A) and top strand (end labeled at EcoRI) (B). OxyRLpbinding to PahpC2occurred under the following conditions: lanes 1 and 6, no
protein control reactions; lane 2, oxidizing conditions (no DTT); lane 3, 200 mM DTT; lanes 4, 1 mM H2O2; lane 5, treated with 1 mM H2O2and
then with 200 mM DTT to recover DNA-binding activity. In panels A and C, asterisk denotes ahpC2 transcript start site as indicated by alignment
of primer extension product with the corresponding DNA sequence ladder. (D) Schematic of PahpC2. Arrows indicate FC2PROM and RC2PROM
primers, open boxes denote OxyRLpsites as determined by DNase I protection assays shown in panels A and B, boldface indicates open reading
frames of ahpC2 and lpg2351, and the ribosome-binding site (RBS) for ahpC2 is underlined. The transcription start site is indicated by an asterisk,
and the ?10 sequence is indicated in bold. The numbers in parentheses depict the numbering and sites of deletions shown in Fig. 3.
VOL. 190, 2008OxyRLpREGULATION OF ahpC2D IN LEGIONELLA3451
OxyRLp from pBADLpoxyR repressed fluorescence from
pahpC2gfp, consistent with our hypothesis that binding of
OxyRLpto the promoter of ahpC2-gfp blocked transcription.
Moreover, there was no change in ahpC2-gfp expression fol-
lowing challenge with 100 ?M H2O2(oxidative stress), further
evidence that OxyRLpis not a redox-active regulator.
Cell cycle expression of oxyRLp. To determine the temporal
expression of oxyR during in vitro growth, a GFP reporter assay
was performed. Figure 6 shows that the expression of oxyRLpis
growth cycle-dependent, increasing during mid- and postexpo-
nential phases and decreasing slightly during late-stationary
phase. Similar trends were observed in peroxide-sensitive
ahpC1 and ahpC2D mutants. In an attempt to identify possible
regulators of oxyR expression, the poxyRgfp reporter construct
was transformed into mutant strains lacking known regulators
of L. pneumophila virulence, rpoS, letA, and himAB (encoding
integration host factor). No differences in oxyR expression
were observed in any of the mutants analyzed (Fig. 6), indi-
cating that the postexponential expression of OxyRLpis not
activated by RpoS or the LetA/S regulatory cascade.
Our laboratory has characterized two alkyl hydroperoxide
reductases (AhpC1 and AhpC2D) that are expressed at differ-
ent stages of bacterial growth and provide essential peroxidatic
functions in L. pneumophila. Loss of AhpC1 function results in
increased expression of ahpC2D, suggesting a compensatory
response to cellular oxidative stress (30). Using an ACDC
assay, we identified OxyRLpas a regulatory factor controlling
expression of the ahpC2D operon. OxyRLpbound to ahpC2D
promoter sequences but did not bind to the promoter regions
of ahpC1 or itself (autoregulation). Members of the OxyR
family are well-characterized, redox-active transcriptional ac-
tivators of genes associated with defense against oxidative
stress. In contrast, none of the antioxidant defense genes
(sodB, sodC, katA, katB, or ahpC1 and ahpC2D) of L. pneu-
mophila appear to be activated in response to oxidative stress
(4, 5, 30, 39, 43). Moreover, the expression of most of these
genes, including oxyRLp, is aligned with growth stage, increas-
ing dramatically around mid-exponential phase and peaking in
early stationary phase, consistent with the view that oxidative
stress is more acute for stationary-phase bacteria (4, 39). By
demonstrating that OxyRLp repressed transcription from
ahpC2D, we have partly resolved the basis for the reciprocal
gene expression noted between ahpC1 and ahpC2D. Studies
are in progress to identify regulatory mechanisms associated
with postexponential activation of ahpC1 and oxyR.
OxyRLpappears to be functionally different from other
OxyR proteins that have been shown to function as redox-
active repressors (47, 48). Here we present several lines of
evidence to suggest that OxyRLpmay no longer function as a
global regulator of antioxidant defenses: (i) oxyRLpexpression
is growth stage dependent, (ii) OxyRLpfunction is apparently
essential, (iii) DNA-binding properties of OxyRLpare not re-
dox dependent, (iv) OxyRLpcontains key amino acid substitu-
tions that likely ablate disulfide-bond formation or conforma-
tional changes that are required for activation, and (v) H2O2
treatment did not increase ahpC2-gfp levels in L. pneumophila
or in an E. coli oxyR mutant (GS077) expressing oxyRLp. How-
ever, these findings do not exclude the possibility that OxyRLp
retains some DNA sequence recognition for OxyR-regulated
FIG. 5. Regulation of ahpC2-gfp in E. coli GS077 by OxyRLp.
Strains were grown in LB broth and subjected to repressing (glucose)
or inducing (arabinose) conditions. Treatment with 100 ?M H2O2is
indicated where appropriate. RFUs were normalized to 1.0 OD620
unit. Results are the means ? standard deviations of three indepen-
FIG. 6. Cell cycle-dependent oxyR expression in L. pneumophila.
(A) GFP reporter assays and (B) bacterial growth curves for PoxyRwere
performed in BYE broth, and fluorescence was determined for sam-
ples taken every 3 h, following the growth of the wild type (}) or the
ahpC2D (●), rpoS (‚), letA (Œ), and himAB (encoding integration host
factor) (E) mutant strains. RFUs were normalized to 1.0 OD620unit.
Results are the means ? standard deviations of three independent
3452LEBLANC ET AL. J. BACTERIOL.
promoters of E. coli, as indicated by complementation and
EMSA studies. Our studies indicate that OxyRLpmay be
locked in an activated DNA-binding conformation (29), inde-
pendent of further activation by peroxide but sufficient to ac-
tivate the antioxidant defense system of E. coli.
In E. coli, OxyR binds DNA as a dimer of dimers under both
reducing and oxidizing conditions, yet only the latter promotes
expression of its target genes (47). Treatment of E. coli OxyR
with millimolar amounts of DTT was shown to promote the
binding of OxyR to contact sites that blocked ?35 sequences of
OxyR-regulated promoters, whereas peroxide oxidation of
OxyR led to a conformational change in the protein that con-
tracted the DNA footprint and permitted strong induction of
these promoters (44, 46, 47). In the case of L. pneumophila,
treatment of OxyRLpwith micromolar concentrations of H2O2
did not alter the footprint, which suggested either that OxyRLp
was fully oxidized during protein purification or that DNA
binding was independent of oxidation state. While millimolar
concentrations of H2O2indeed abolished DNA binding, the
result is not considered physiologically relevant since biological
activity was not recoverable by treatments with 200 mM DTT.
In this regard, functional studies have shown that the redox-
active thiols (C199 and C208) of OxyREcspontaneously oxidize
to form disulfide bonds (redox potential of ?185 mV) during
protein purification (1, 50). In some organisms, such as Deino-
coccus radiodurans, oxidation of a single (peroxidatic) cysteine
residue of OxyR is sufficient to promote activation (10). Thus,
regardless of whether a single cysteine becomes oxidized or
even a disulfide bond is formed, our studies suggest that the
redox-sensing domain of OxyRLpno longer signals conforma-
tional changes that promote activation.
Members of the LysR family (which includes OxyR) use a
ligand-binding conformational change mechanism to mediate
DNA binding (11). Mutation-based studies have identified key
amino acids required for the function of OxyR. For example,
E. coli OxyR H125I and H218D mutations resulted in wild-
type DNA binding but a loss of redox-mediated regulation
(11). These amino acid substitutions occur naturally in the
OxyRLpproteins from all four strains of L. pneumophila whose
genomes have been sequenced (125 and 218) (Fig. 7). While
the two redox-active cysteine residues involved in OxyR acti-
vation (Cys199 and Cys208) are conserved in OxyRLp, there is
substantial amino acid sequence divergence in the activation
domain that might affect redox activity. For example, a key
arginine at position 220 in OxyREc(Fig. 7) that is predicted to
interact with catalytic cysteines and peroxide (28) is changed to
FIG. 7. Clustal W alignment of selected OxyR amino acid sequences. The alignments are of L. pneumophila Philadelphia-1 (LpnOxyR
[OxyRLp]), Xanthomonas campestris (XanOxyR [OxyRXc]), E. coli (EcoliOxyR [OxyREc]), and C. burnetii (CbOxyR [OxyRCb]). The DNA binding
helix-turn-helix (HTH) is shaded, and relevant amino acid positions are numbered and bolded.
VOL. 190, 2008 OxyRLpREGULATION OF ahpC2D IN LEGIONELLA 3453
threonine in OxyRLpproteins. Interestingly, in the OxyR pro-
teins of close relative C. burnetii and in Francisella tularensis,
Arg220 is replaced by lysine, which might suggest a more
traditional function in these organisms. Further comparisons
revealed amino acid sequence variation within the helix-turn-
helix region that might account for differences in the DNA-
binding specificity noted between OxyRLpand OxyREcin
complementation experiments and EMSAs. We suggest that
the loss of the redox-sensing function of OxyRLphas occurred
naturally through specific amino acid substitutions that have
been demonstrated through empirical studies to alter disulfide
bond formation and conformational changes required for ac-
In contrast to other bacteria, such as F. tularensis, C. burnetii,
and Streptomyces species, in which oxyR is located upstream
and divergently expressed from the ahpC2D system, the oxyRLp
gene is located elsewhere in the chromosome. It is conceivable
that the present oxyRLpwas acquired laterally, perhaps con-
comitantly with the deletion or loss of an ancestral upstream
ortholog, perhaps a necessary event associated with the evolu-
tion of a dimorphic lifestyle.
It seems unlikely that the only function for OxyRLpis to
repress ahpC2D during the growth transition to stationary
phase and into cyst morphogenesis. While downregulation of
ahpC2D is consistent with metabolic changes and a shift to the
NADPH-thioredoxin AhpC1 system that occur during the
transition to stationary phase, ahpC2D deletion mutants are
both viable and infectious (30). Moreover, high-level ahpC2D
expression, noted with the ahpC1 deletion mutant, also sug-
gested that the postexponential presence of AhpC2D was not
toxic. Thus, OxyRLpmust provide other essential regulatory
functions to L. pneumophila, since this mutant could not be
obtained by methods routinely used to generate mutants
(30). We used the pattern search option of the L. pneumophila
Paris and Lens genome website (http://genolist.pasteur.fr
/LegioList/) to screen for additional genes, using motif patterns
deduced from comparisons with either the E. coli or L. pneu-
mophila OxyR binding motif. While these motifs are relatively
degenerate, binding sites were observed upstream of approxi-
mately 50 genes, including the dps homolog, which has recently
been found to prevent Fenton-mediated DNA damage by se-
questering iron (37). Others included promoters of genes en-
coding efflux pumps (that exclude redox-cycling compounds
and organic solvents); metal ion transporters (which regulate
rates of Fenton chemistry); components of the respiratory
chain, such as cytochrome oxidases, DNA repair, or modifica-
tion enzymes; known virulence factors, such as the zinc met-
alloproteinase (shown to inhibit the oxidative burst); and sub-
strates of the Dot/Icm type IVB secretion system (SidG, SdhB,
and SdeA). Finally, OxyR binding motifs were also found up-
stream of genes encoding numerous transcriptional regulators,
like Fur, FleQ, FleN, and other members of the LysR family,
suggesting that there could be cross talk with other regulatory
pathways. Interestingly, unlike in E. coli, fur is an essential gene
in L. pneumophila (24). Further studies will be required to
dissect additional regulatory functions associated with essenti-
Our studies predict the participation of additional regulatory
elements in controlling the expression of ahpC2D, since re-
pression of ahpC2D by OxyRLpdoes not fully explain the
upregulation of ahpC2D in an ahpC1 mutant or the postexpo-
nential activation of ahpC1 (30). In L. pneumophila, transition
from exponential phase to stationary phase/cyst differentiation
is controlled in part by postexponential regulators RpoS, LetA,
and HimAB (2, 13, 20, 22, 32, 33, 52). However, the oxyRLp-gfp
gene expression studies of rpoS, letA, and himAB mutants were
indistinguishable from those of the wild type. While not di-
rectly examined, carbon storage regulator CsrA, an important
repressor of stationary-phase genes during exponential phase
(13, 33), may not be involved in regulating oxyRLp, since CsrA
should have repressed postexponential expression of oxyRLpin
letA and rpoS mutants. Future studies will employ the ACDC
strategy to capture putative regulatory factors associated with
the control of oxyRLpexpression.
In summary, we have shown that OxyRLprepresses ahpC2D
expression and that oxyRLpis induced postexponentially. Our
studies further establish that OxyRLpno longer functions as an
oxidative response regulator in L. pneumophila, which is con-
sistent with previous observations that L. pneumophila mounts
no response to oxidative stress and that these bacteria are
rather resistant to oxidative damage, even in phagocytic cells
(30). We propose that OxyRLphas been adapted from an
oxidative stress response regulator to a growth stage-specific
regulator of genes mediating the transition from vegetative to
resilient cyst-like transmissible forms.
We thank Gisela Storz and Michele Swanson for providing bacterial
strains and plasmids.
This work was supported by CIHR grant MOP 14443 and NIH grant
AI066058 to P.S.H. and a CIHR postdoctoral fellowship to A.K.C.B.
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