JOURNAL OF BACTERIOLOGY, Oct. 2007, p. 6957–6967
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 189, No. 19
Anaerobic Regulation of Shigella flexneri Virulence: ArcA Regulates
fur and Iron Acquisition Genes?†
Megan L. Boulette and Shelley M. Payne*
Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, The University of Texas at
Austin, 1 University Station A5000, Austin, Texas 78712
Received 22 April 2007/Accepted 16 July 2007
Invasion and plaque formation in epithelial monolayers are routinely used to assess the virulence of Shigella
flexneri, a causative agent of dysentery. A modified plaque assay was developed to identify factors contributing
to the virulence of S. flexneri under the anaerobic conditions present in the colon. This assay demonstrated the
importance of the ferrous iron transport system Feo, as well as the global transcription factors Fur, ArcA, and
Fnr, for Shigella plaque formation in anoxic environments. Transcriptional analyses of S. flexneri iron transport
genes indicated that anaerobic conditions activated feoABC while repressing genes encoding two other iron
transport systems, the ABC transporter Sit and the Iuc/Iut aerobactin siderophore synthesis and transport
system. The anaerobic transcription factors ArcA and Fnr activated expression of feoABC, while ArcA re-
pressed iucABCD iutA. Transcription of fur, encoding the iron-responsive transcriptional repressor of bacterial
iron acquisition, was also repressed anaerobically in an ArcA-dependent manner.
Shigella flexneri is a pathogenic, gram-negative bacterium
that causes dysentery in humans. During infection of the colon,
shigellae gain access to the basolateral surface of the epithe-
lium via M cells. Upon ingestion by macrophages, shigellae
promote apoptosis of these cells and are released to subse-
quently invade intestinal epithelial cells. S. flexneri multiplies
within the cytoplasm of the epithelial cells and spreads directly
to adjacent cells, thus delaying detection by immune cells (re-
viewed in references 22, 44, and 46). Because shigellosis is
restricted to higher primates, Shigella pathogenesis is routinely
investigated with cultured human epithelial cells. S. flexneri
forms plaques in confluent monolayers, and the size and num-
ber of these plaques correlate with virulence, as plaque forma-
tion requires that the bacteria invade, grow intracellularly, and
spread directly to adjacent epithelial cells (43).
Shigella, like other pathogenic bacteria, has multiple mech-
anisms for coping with the iron-restricted environment of the
host. The genome of S. flexneri 2a encodes at least four iron
acquisition systems (23, 65). The aerobactin operon encodes
the biosynthesis (iucABCD) and transport (iutA) of the hydrox-
amate siderophore aerobactin. This siderophore transports
ferric iron (Fe3?), the predominant form of iron under aerobic
conditions at neutral pH. Additionally, S. flexneri expresses the
fhu genes for transport of the fungal siderophore ferrichrome.
S. flexneri also encodes two iron acquisition systems, Feo and
Sit, which are predicted to transport ferrous iron (Fe2?), the
more abundant form of iron in anaerobic environments. The
Shigella SitABCD system has similarity to the Salmonella en-
terica serovar Typhimurium Sit system, which primarily trans-
ports manganese (3). The S. flexneri Sit system has been shown
to function in iron transport (50, 52), and the S. flexneri sit
genes were up-regulated in the intracellular environment (31,
51). A sit feo iuc mutant did not grow in the absence of exog-
enously supplied siderophore or form plaques in epithelial cell
monolayers (52), indicating that there are no other iron trans-
port systems in strain SA100.
Iron is essential for growth, yet free iron can be toxic to
cellular components. Therefore, the expression of iron acqui-
sition genes is regulated in response to the intracellular iron
concentration. Under iron-replete conditions, the transcription
factor Fur binds iron and Fe-Fur represses the expression of
iron transport genes (11). Fe-Fur also represses ryhB, which
encodes a small RNA that promotes degradation of transcripts
for iron storage, oxidative metabolism, and stress proteins (35–
38). Iron availability influences the transcription of fur as well
as the activity of the Fur protein. Fe-Fur is an autorepressor,
reducing fur expression in response to iron (9, 10, 19, 53). fur
expression is also reduced in strains with mutations in cya,
encoding adenylate cyclase, and crp, encoding the cyclic AMP
receptor protein, suggesting that the source of cellular carbon
impacts iron uptake (10). fur transcription is also activated by
OxyR and SoxS, which are redox regulators activated by oxi-
dative stress (68). The increased level of Fur scavenges un-
bound iron to prevent cell-damaging radical formation as well
as turning off iron acquisition. Bacteria also regulate specific iron
transporters in response to the oxygen availability. Fnr and ArcA
are the primary redox regulators responsible for the activation or
repression of genes associated with the transition to anaerobiosis
(17), and Fnr has been shown to stimulate transcription of
feoABC in Escherichia coli under anoxic conditions (24). The
expression of genes encoding the Sit system in S. enterica se-
rovar Typhimurium decreases anaerobically (20). This anaer-
obic repression was not due to ArcA and Fnr but rather to the
availability of the redox metals iron and manganese, which
bind to the transcription factors Fur and MntR (20).
Anaerobiosis has been shown to influence the persistence
and virulence of enteric pathogens such as E. coli (12, 21),
* Corresponding author. Mailing address: The University of Texas
at Austin, Section of Molecular Genetics and Microbiology, 1 Univer-
sity Station A5000, Austin, TX 78712. Phone: (512) 471-9258. Fax:
(512) 232-5841. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jb
?Published ahead of print on 27 July 2007.
Salmonella spp. (6, 27, 60), Vibrio cholerae (2, 58), and Yersinia
enterocolitica (57). Because S. flexneri infects the colon, which
is an oxygen-limited environment, studies were undertaken to
determine whether anaerobiosis and the anaerobic transcrip-
tion factors ArcA and Fnr affect S. flexneri iron metabolism and
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. Bacterial strains and plas-
mids are described in Table 1. E. coli strains were grown in Luria-Bertani (LB)
broth or on LB agar (16). S. flexneri strains were grown in RPMI (RPMI 1640
[Invitrogen] with L-glutamine, without phenol red [Gibco; Invitrogen], and buff-
ered with 100 mM HEPES) supplemented with 2.5 ?M FeSO4where indicated
or on tryptic soy broth agar plus 0.01% Congo red dye (Congo red agar) at 37°C.
Antibiotics were used at the following concentrations (per milliliter): 125 ?g of
carbenicillin (Car), 200 ?g of streptomycin (Str), 20 ?g of kanamycin (Kan), and
7.5 ?g of chloramphenicol (Cam).
For green fluorescent protein (GFP) reporter and real-time reverse transcrip-
tion (RT)-PCR assays, S. flexneri strains were plated on Congo red agar with
appropriate antibiotics and grown overnight at 37°C. Isolated colonies were used
to inoculate 2 ml RPMI with 2.5 ?M FeSO4and appropriate antibiotics for
plasmid maintenance. After overnight growth at 37°C, cultures were diluted to an
optical density at 600 nm (OD600) of 0.05 into 2 ml RPMI containing antibiotics
and grown aerobically at 37°C for 2 h. These were subcultured to an OD600of
0.03 into the same medium and grown to exponential phase either aerobically
with vigorous shaking or anaerobically in Oxoid AnaeroJars with AnaeroGen
TABLE 1. Strains and plasmids
Strain or plasmidCharacteristicsReference or sourcea
E. coli strains
Donor strain for triparental conjugation
Helper strain for triparental conjugation
R. Meyer, UTA
S. flexneri strains
Wild-type S. flexneri serotype 2a
SA100 Crb?arcA::kan fnr::cam
SA100 feoB::dhfr iucD::Tn5
SM100 feoB::dhfr sitA::cam
SA100 iucD::Tn5 sitA::cam
SM100 feoB::dhfr iucD::Tn5 sitA::cam
S. Seliger, UTA
Plasmids for mutant construction
and gene expression
Single- and inducible-copy vector
Sucrose-selectable suicide vector for allelic exchange
PCR cloning vector
Source of cam cassette
IPTG-inducible gene expression vector
pDRIVE with SA100 fnr region
pDRIVEfnr with cam cassette disruption
pCVD442N with fnr::cam of pDRIVEfnr::cam
pQE-2 with arcA coding region inserted into BseRI
and HindIII sites
pCC1 with IPTG-inducible arcA and lacI regions from
pQE-2 with fur coding region inserted into BseRI and
pMBarcAccQE This study
Plasmids for GFP reporter assays
gfp reporter vector
pLR29 with sitA promoter
pLR29 with iutA promoter
L. Runyen-Janecky and
E. Gonzales, UTA
pLR29 with altered feoA promoter
pLR29 with feoA promoter
pLR29 with altered fur promoter
pLR29 with fur promoter
pLR29 with lldP promoter
pLR29 with minC promoter
pLR29 with narG promoter
aUTA, University of Texas—Austin.
6958BOULETTE AND PAYNEJ. BACTERIOL.
sachets and Anaerobic Indicators (Oxoid Ltd., Hampshire, England), as indi-
cated. All data are the average of at least three independent experiments.
Tissue culture and plaque assays. Henle cells (Intestine 407 cells; American
Type Culture Collection, Manassas, VA) were grown in minimal essential me-
dium (MEM) or RPMI 1640 (Invitrogen) supplemented with 10% Bacto tryp-
tose phosphate broth (Difco; Becton Dickinson Company), 2 mM glutamine,
MEM nonessential amino acid solution (Invitrogen Corporation), and 10% fetal
bovine serum (Invitrogen) in a 5% CO2atmosphere at 37°C. Plaque assays were
performed as described previously (52), except that the medium was supple-
mented with 100 mM HEPES (pH 7.5) and agarose was omitted from the
overlay. For the anaerobic plaque assays, the plates were incubated in the BD
BBL GasPack pouch anaerobic system (Becton, Dickinson, and Company) for
the duration of the experiment.
Construction of mutants. The S. flexneri arcA::kan mutant was constructed by
bacteriophage P1 transduction (56) from the E. coli arcA::kan mutant ECL5331
(30). Insertional inactivation of the arcA gene in MBF200 was confirmed by PCR
using primers flanking the arcA gene, IWDArcAOut1 and IWDArcAOut2.
The S. flexneri fnr strain was made by allelic exchange. The fnr region was
amplified from SA100 by PCR using primers MWDFnrUS1 and MWDFnrDS1,
digested with PstI and SalI, and ligated with the pDRIVE vector. A cam cassette
was excised from pMTLcam using SmaI and was ligated into pDRIVEfnr di-
gested with BclI and made blunt with the Klenow fragment of DNA polymerase
I (New England Biolabs, Ipswich, MA). fnr::cam was then excised from
pDRIVEfnr::cam using SmaI and ligated into SmaI-digested pCVD442N. The
resulting plasmid, pCVD442fnr::cam, was then mated into SM100 by triparental
conjugation. Primary integrants were selected by growth in the presence of Car,
Str, and Cam and verified with primers MWFFnrDS1 and FNRintFor. The
fnr::cam mutant was isolated by growth in the presence of sucrose and Cam and
was confirmed by PCR using primer pair FNRintFor and FNRintRev and primer
pair MWFnrUS1 and MWFFnrDS1. MBF100 (SA100 fnr::cam) and MBF300
(SA100 arcA::kan fnr::cam) were obtained by P1 transduction of fnr::cam from
SM100 fnr::cam to SA100 and MBF200, respectively, and verified by PCR. All
primer sequences are listed in Table S1 in the supplemental material.
Construction of plasmids for gene expression. A single-copy, IPTG (isopropyl-
?-D-thiogalactopyranoside)-inducible arcA vector was engineered by first cloning
the arcA gene under the inducible T5 promoter of plasmid pQE-2 (QIAGEN).
SA100 genomic DNA was used as a template for PCR using the primers MBarc
AforQE and MBarcArevQE (see Table S1 in the supplemental material), and
the fragment was cloned into pQE-2 digested with BseRI and HindIII to gen-
erate pMBarcAQE. The FspI fragment, including the lacI and T5-arcA genes,
was excised from pMBarcAQE and ligated into the blunt-cloning-ready pCC1
vector (Epicenter Biotechnologies, Madison, WI), resulting in pMBarcAccQE,
which was verified by DNA sequencing.
A plasmid for IPTG-inducible expression of fur was constructed by PCR
amplification of the fur gene from SA100 DNA using primers MBfurForQE and
MBfurRevQE (see Table S1 in the supplemental material). The PCR product
and pQE-2 vector were digested with BseRI and HindIII and ligated, and the
resulting pMBfurQE plasmid was confirmed by DNA sequence analysis.
Microarray analysis. Microarrays were printed and postprocessed as described
previously (45). Wild-type S. flexneri batch cultures were grown aerobically in RPMI
medium without added iron to mid-logarithmic phase in a BIOFLO 110 Fer-
mentor/Bioreactor (New Brunswick Scientific, Edison, NJ) to maintain constant
pH, dissolved oxygen concentration, temperature, and agitation. A portion of the
culture was removed to obtain the aerobically grown bacteria. The dissolved
oxygen concentration was then depleted in the remaining culture by the addition
of nitrogen gas, and 15 min after depletion, the anaerobic bacteria were isolated.
RNA was purified with RNeasy Midi kits (QIAGEN). Reverse transcription of
RNA to generate amino allyl-dUTP-incorporated cDNA, Cy3 and Cy5 coupling,
probe generation, and array hybridization were performed as described previ-
ously (45), with RNA derived from aerobically grown bacteria labeled with Cy3
and from anaerobically grown strains labeled with Cy5. Microarrays were
scanned by the Genepix array scanner 4000A (Axon Instruments, Union City,
CA). Preliminary analysis of microarrays was performed with Genepix 5.0 soft-
ware, and normalization of microarray data was carried out by the Longhorn
Array Database, an open-source, MIAME-compliant implementation of the
Stanford Microarray Database (28). Normalized data were filtered so that spots
with a regression correlation of lower than 0.6 and those that were in areas of
high background were excluded. Additionally, genes that did not exhibit greater
than a twofold difference in expression in at least two arrays and those that
showed inconsistent patterns of induction or repression were excluded from
GFP reporter assays. gfp transcriptional fusions (Table 1) were constructed by
PCR amplification of the promoter of each gene (primers are listed in Table S1
in the supplemental material) and ligation into the promoterless gfp vector
pLR29. To measure GFP, overnight cultures were diluted to an OD600of 0.05
into RPMI with Car and 100 mM HEPES and grown aerobically at 37°C for 2 h.
These were subcultured to an OD600of 0.03 into the same medium and grown for
2 h either aerobically with vigorous shaking or anaerobically in Oxoid AnaeroJars
with AnaeroGen and Anaerobic Indicators (Oxoid Ltd., Hampshire, England).
The cultures were diluted to an OD600of 0.08, and fluorescence was measured
in a VersaFluor fluorometer (Bio-Rad Laboratories, Hercules, CA). The instru-
ment was blanked using the parent strains with the pLR29 control plasmid, and
the range was set to 15,000 relative fluorescence units (RFU) with the constitu-
tive pMBminC vector, which expresses gfp constitutively. The data are shown as
relative expression levels, with the culture giving maximal expression set at 100%.
The average RFU/OD600of each transcriptional fusion was normalized to the
RFU/OD600of the pMBminC vector in the same strain. Results are the average
of three independent experiments. The plasmids pMBnarG and pMBlldP served
as positive controls for Fnr and ArcA regulation, respectively.
Real-time RT-PCR. S. flexneri strains SA100/pCC1, MBF200/pCC1, and
MBF200/pMBarcAccQE were grown as for the GFP reporter assays, except that
1 ?M IPTG was added to the final subculture medium to induce ArcA expres-
sion; anti-ArcA (generously provided by P. Silverman ) immunoblots showed
that this amount of IPTG induced wild-type levels of ArcA. RNA was isolated on
RNeasy Mini columns (QIAGEN) following the addition of 1/5 volume of 95%
ethanol–5% phenol (vol/vol) to logarithmically growing, anaerobic cultures.
RNA was DNase treated (DNase I; QIAGEN) on the RNeasy column and again
after elution with amplification-grade DNase I (Invitrogen) according to the
manufacturers’ instructions. cDNA was generated from approximately 5 ?g of
each RNA sample with the High Capacity cDNA Archive kit (Applied Biosys-
tems). Real-time RT-PCR mixtures in a total volume of 25 ?l contained 1?
Power SYBR green PCR Master Mix (Applied Biosystems), 800 nM concentra-
tions of the indicated primers, and 1/200 of the cDNA reaction mixture. fur
cDNA was detected with primers MBfurRT1 and MBfurRT2. rrsA cDNA was
detected with primers RrsA.for and RrsA.rev. Real-time RT-PCR and analyses
were carried out with an Applied Biosystems 7300 Real Time PCR System and
software. Standard curves for each primer set were generated by using cDNA
obtained from 10-fold dilutions of SA100 RNA, and the amount of cDNA in
each sample was extrapolated from the standard curve. The relative amounts of
fur cDNA were normalized by dividing the values by the relative amounts of rrsA
control cDNA in each sample.
Antibody supershift assays. The promoter-gfp fusion plasmids served as tem-
plates for PCRs to generate probes for the promoter regions, using primers listed
in Table S1 in the supplemental material. After gel extraction of the PCR
fragments, the DNA was digested with XmaI for probes generated with the
pLR29EMSAfor 5? primer, which cuts just upstream of the BamHI restriction
enzyme site in the pLR29 vector, while the iuc probe was digested with XbaI. The
probes were gel purified, and the ends were filled in using Klenow fragment
(New England Biolabs, Ipswich, MA) and a mixture of nucleotides for cold
probes. dCTP was replaced with [?-32P]dCTP (Perkin Elmer, Boston, MA) for
radiolabeled probes. Unincorporated nucleotides were removed with Micro Bio-
Spin P-30 Tris chromatography columns (Bio-Rad Laboratories), and all probes
were phenol-chloroform extracted and ethanol precipitated (1). Radioactivity
was measured by liquid scintillation counting, and probe concentrations were
determined by measuring the absorbance.
Cell extracts from MBF200/pCC1 and MBF200/pMBarcAccQE were prepared
by the method of Tardat and Touati (61), with the exception of growth condi-
tions. S. flexneri cultures were grown overnight in HEPES-buffered RPMI with
2.5 ?M FeSO4and antibiotics, subcultured at 1:100 into the same medium
without added iron, and grown aerobically at 37°C to mid-logarithmic phase. The
cultures were then diluted into the same medium containing 1 ?M IPTG to
induce arcA expression, and the cultures were grown for 2 h under anaerobic
The antibody supershift assays were performed essentially as described by
Ausubel et al. (1). Binding buffer consisted of 10 mM Tris-HCl (pH 7.4), 10%
glycerol, 10 mM CaCl2, 100 mM KCl, 1 mM EDTA, 5 ?g/ml bovine serum
albumin, 1 mM dithiothreitol, and 1 ?g poly(dI-dC) in a reaction volume of 30
?l, also containing 5 ?g of crude protein extract, 1 ?l of antiserum diluted
1/1,000, and approximately 1 ng of labeled probe. Fragments were separated by
electrophoresis in a 5% polyacrylamide–Tris-borate-EDTA gel, and radioactive
bands were visualized with a Bio-Rad Molecular Imager FX after overnight
exposure of the dried gel to a phosphor screen (Bio-Rad Laboratories). Fluoro-
phore band intensity was analyzed with Quantity One software (Bio-Rad Labo-
VOL. 189, 2007ArcA REGULATION OF SHIGELLA fur AND IRON TRANSPORT6959
Roles of iron transport systems in aerobic and anaerobic
plaque formation. The plaque assay, a standard measure of S.
flexneri virulence, is performed under aerobic conditions,
which may not allow assessment of factors influenced by the
anaerobic environment of the colon. Therefore, we developed
a modified plaque assay in which infected Henle cell monolay-
ers were incubated anaerobically. As shown in Fig. 1, wild-type
S. flexneri formed plaques under both aerobic and anaerobic
conditions, whereas the avirulent Crb?strain was unable to
form plaques under either condition. S. flexneri mutants defec-
tive in one or more iron transport systems (29, 52) were also
tested in this assay (Fig. 1), since we had previously shown that
the S. flexneri iuc, feo, and sit iron transport systems contribute
to iron acquisition in cultured cells. A mutant lacking all three
of these systems, which is defective in aerobic plaque forma-
tion (52), also failed to form plaques anaerobically (Fig. 1),
while all strains expressing at least two of these three iron
transport systems formed plaques in either environment (data
not shown). The strain with only the Sit transporter was also
able to form plaques under both conditions, although under
anaerobic conditions the plaque size was reduced compared to
that of the wild type (feo iuc mutant [Fig. 1]). The iuc sit double
mutant, in which only the Feo system is functional, formed
plaques under anaerobic but not aerobic conditions. This is
consistent with the ferrous iron ligand for the Feo system being
enriched under anaerobic conditions. In contrast, the feo sit
double mutant, which expressed only the aerobactin system,
formed plaques solely under aerobic conditions, as might be
expected for a ferric iron transporter. This effect of oxygen
availability on the function of these iron transport systems
suggested that the regulation of their genes could also be
influenced by oxygen.
Regulation of iron acquisition genes by oxygen availability.
Because plaque formation was influenced by the availability of
oxygen, microarray analysis was used to screen S. flexneri iron
transport genes or other virulence genes for the effect of oxy-
gen on expression. Wild-type cultures grown aerobically were
compared with those subjected to oxygen depletion. Genes
previously reported to be influenced in E. coli by oxygen avail-
ability or by the anaerobic transcription factor ArcA or Fnr
(25, 30, 54, 55) showed the expected regulation in S. flexneri
(Table 2). Representative genes induced aerobically included
the TCA cycle loci acnA, mdh, and lpd-ace, the F1FoATPase
atpCD GAHF genes, the ndk/nrd aerobic ribonucleotide reduc-
tases, and genes encoding Fe-S cluster biogenesis and oxidative
stress proteins. Under anaerobic conditions, the genes encod-
ing nitrate reductase (narGHJI) and fermentation genes (adhE
and pflB) were elevated (Table 2). This indicates that the
conditions used for analysis of S. flexneri were appropriate for
detecting anaerobically induced or repressed genes. The tran-
scription of several iron acquisition genes also responded to
oxygen availability. The feo genes were induced anaerobically
(Table 2). Conversely, the sit and iuc transcripts were more
abundant aerobically (Table 2). dps, which has been shown to
bind ferrous iron (4), was also elevated anaerobically (Table 2).
The regulatory effects detected by microarray were further
analyzed with GFP reporter fusion assays (Fig. 2). Transcrip-
tional gfp reporter fusions in the vector pLR29 were made
using promoters upstream of the feoABC, sitABCD, and
iucABCD iutA operons. Strains of wild-type S. flexneri contain-
ing these reporter fusions were grown aerobically or anaero-
bically to compare the relative fluorescence. The reporter data
confirmed that the iuc and sit promoters were more active
aerobically than anaerobically, while the feo promoter was
stronger anaerobically (Fig. 2).
feo is induced by ArcA and Fnr, while ArcA represses iuc. To
determine whether the transcription factors ArcA and Fnr
have a role in the regulation of iron acquisition genes in re-
sponse to oxygen availability, arcA and fnr mutants of S. flexneri
FIG. 1. Plaque formation by S. flexneri under aerobic and anaerobic conditions. Henle cell monolayers were infected with 104wild-type S.
flexneri (SA100), avirulent Crb?mutant (SA101), feoB iucD mutant (SA192), feoB sitA mutant (SM191), iucD sitA mutant (SA167), or feoB iucD
sitA mutant (SM193) cells. The plates were incubated for 2 days in medium containing gentamicin under either aerobic (top) or anaerobic (bottom)
conditions and stained to visualize plaque formation.
6960 BOULETTE AND PAYNEJ. BACTERIOL.
TABLE 2. Transcriptional changes of selected genes in response to oxygen availability in S. flexneri
Transcripts elevated aerobically
O2-regulated metabolic genes
Aerobic ribonucleotide reductase
Aerobic ribonucleotide reductase
Aerobic ribonucleotide reductase
O2-regulated stress response genes
Fe-S cluster formation
Fe-S cluster formation
Fe-S cluster formation
Fe-S cluster formation
Fe-S cluster formation
Fe-S cluster formation
Fe-S cluster formation
Fe-S cluster formation
Oxidative stress response
Oxidative stress response
O2-regulated iron transport genes
Fe and Mn acquisition
Fe and Mn acquisition
Fe and Mn acquisition
Fe and Mn acquisition
Transcripts elevated anaerobically
O2-regulated metabolic genes
Pyruvate formate lyase
O2-regulated stress response genes
Oxidative stress, iron-binding protein
O2-regulated iron transport genes
Ferrous iron transport
Ferrous iron transport
Ferrous iron transport
aMicroarrays were use to determine the transcriptional profiles of cells switched to anaerobic conditions after aerobic growth. Genes known to be oxygen regulated
in E. coli and those that may be involved in plaque formation were analyzed. The range, average, and 1 standard deviation (SD) for the indicated gene spot on six arrays
are shown. Data are shown as fold change for the anaerobic sample compared to the aerobic sample. *, only two of the arrays had spots that passed the filters; thus,
the standard deviation is not shown.
VOL. 189, 2007ArcA REGULATION OF SHIGELLA fur AND IRON TRANSPORT 6961
were constructed and the relative expression from the gfp re-
porter fusions was determined in these strains. The activity of
iuc promoter fusions was the same in wild-type and fnr strains
(data not shown) but was slightly elevated in the arcA mutant
(Fig. 3A). This suggests that ArcA represses iuc, and a putative
ArcA box was found in the promoter sequence (Fig. 3B). In
contrast, the activity of the feo promoter was approximately
half of the wild-type level in the arcA and fnr single mutants
and one-fifth of the wild-type level in the arcA fnr double
mutant (Fig. 4A), indicating that the anaerobic induction of feo
transcription in the wild-type strain is due to Fnr and ArcA and
that their effects are additive.
The Fnr regulation of feo transcription was expected since
induction of the feo promoter under anaerobiosis was shown
previously to be Fnr dependent in E. coli. The S. flexneri feo
promoter sequence is identical to that of E. coli feo, including
a site with near identity to the Fnr consensus sequence
(TTGAT[n4]ATCAA) (24). However, ArcA regulation of the
feo promoter has not been shown previously. Although con-
sensus sequences for the ArcA recognition site have been
reported in the literature, the optimal ArcA binding sequence
is not known (13, 14, 30, 32, 39, 40). Using parameters from
one of these predictions (14) and adding sequences from newly
identified ArcA binding sites, we generated an ArcA consensus
sequence (Fig. 5) and used this sequence to identify a putative
ArcA binding sequence in the feo promoter (Fig. 4B). To
FIG. 2. Effect of O2on expression of gfp fused to iron transport
gene promoters. Wild-type S. flexneri containing plasmids carrying iron
acquisition gene promoters fused to gfp were grown to mid-log phase
in the presence or absence of oxygen, and relative fluorescence was
measured. For each promoter, the condition with the highest fluores-
cence value was set at 100%. Experiments were performed in tripli-
cate, and error bars represent 1 standard deviation.
FIG. 3. Effect of anaerobiosis on expression of the iuc promoter.
(A) Cultures were grown anaerobically to mid-log phase, and relative
gfp expression from the iuc promoter of pEG6 in SA101 (WT) and
MBF200W (arcA) was determined. The highest relative fluorescence
value was set at 100%. Experiments were performed in triplicate, and
error bars represent 1 standard deviation. (B) The S. flexneri iucABCD
iutA chromosomal region is depicted, showing the relative positions of
the Fur box, Shine-Dalgarno sequence (SD), and putative ArcA box.
The putative ArcA box sequence in iuc is shown below the map, with
bases matching the ArcA box consensus sequence in black and bases
not matching the consensus in gray. Lowercase letters indicate bases
not conserved in the ArcA box weight matrix.
FIG. 4. Anaerobic expression of native and altered feo promoters.
(A) Cultures were grown anaerobically to mid-log phase, and relative
gfp expression from the feo (pMBfeo) and feoAlt(pMBfeoAlt) promoter
fusions in SA101 (WT), MBF100W (fnr), MBF200W (arcA), and
MBF300W (arcA fnr) was determined. The highest relative fluores-
cence value was set at 100%. Experiments were performed in tripli-
cate, and error bars represent 1 standard deviation. (B) The S. flexneri
feoABC chromosomal region shows the relative positions of the Fur
box, Shine-Dalgarno sequence (SD), and putative ArcA box and Fnr
box. The sequences resembling the putative ArcA box in the native and
altered feo promoters are indicated below, with bases matching those
of the putative ArcA box consensus sequence in black, bases not
matching the consensus in gray, and bases changed in the altered
promoter underlined. Lowercase letters indicate bases not conserved
in the ArcA box weight matrix.
FIG. 5. Predicted ArcA regulatory motif. Sequences of ArcA-reg-
ulated promoters were entered into the SeSiMCMC interface (http:
//favorov.imb.ac.ru/SeSiMCMC/), and the algorithm reported a con-
served weight matrix for ArcA sequence recognition. The sequence
logo was obtained by entering the weighted matrix derived from a
multiple sequence alignment into the interface at http://weblogo
6962 BOULETTE AND PAYNE J. BACTERIOL.
determine whether this site is important for ArcA regulation of
feo, several bases were changed to the least-common bases
found at those positions in the weight matrix of the ArcA box
consensus sequence. The activity of the altered feo promoter
(feoAlt) was similar in wild-type and arcA strains (Fig. 4A),
indicating that ArcA did not stimulate expression from the
altered feo promoter. This confirms that ArcA stimulates feo
transcription and that the sequences changed in the feoAlt
promoter are required for stimulation. The activity of the al-
tered feo promoter in the fnr mutant was reduced to approxi-
mately half its level in the wild-type strain (Fig. 4A), which
indicates that Fnr binding was not disrupted by the base
changes. This is consistent with the sequence prediction that
the Arc and Fnr binding sites do not overlap (Fig. 4B).
The sit promoter fusion was also examined for ArcA regu-
lation. The average relative gfp expression of the reporter fu-
sion in the arcA mutant grown under aerobic conditions
(0.98 ? 0.01) was the same as under anaerobic conditions
(0.98 ? 0.2), indicating that the higher level of expression seen
under aerobic conditions in the wild type (Fig. 2) required
ArcA. However, no sequence matching the ArcA consensus
was identified in the sit promoter region, suggesting that an
effect of ArcA on sit expression might not be direct.
ArcA represses transcription of fur. Since several iron ac-
quisition genes were regulated in response to oxygen availabil-
ity (Table 2), the effect of ArcA and Fnr on transcription of fur,
which encodes the regulator of iron transport genes, was de-
termined. Real-time RT-PCR was performed with RNA iso-
lated from anaerobically grown wild-type S. flexneri and the
arcA, fnr, and arcA fnr mutants. While there was no difference
in the level of fur mRNA between the wild type and the fnr
mutant (data not shown), there was a significant increase of
the fur transcript in strains lacking arcA (Fig. 6A), suggesting that
fur transcription was repressed in an ArcA-dependent manner.
fur repression was restored in the arcA strain by inducing arcA
expression from a plasmid (Fig. 6A).
To confirm that ArcA regulates fur promoter activity, a
gfp reporter fusion to the fur promoter was constructed and
the relative fluorescence of gfp was determined in the wild-
type and arcA mutant strains. Relative to the wild type, the
level of GFP was elevated in the arcA mutant (Fig. 6B),
indicating that this promoter was negatively regulated by
ArcA. Several bases matching the ArcA box consensus se-
quence in the fur promoter were then changed to the least-
common bases occurring at those positions in the weight
matrix derived from ArcA boxes of known ArcA-regulated
promoters (furAlt[Fig. 5 and Fig. 6C]). The activity of the
altered fur promoter was higher than that of the native fur
promoter in the wild-type ArcA?strain, and the difference
between the amount of GFP in the arcA strain relative to the
wild type was significantly decreased (Fig. 6B). These results
were consistent with ArcA repression of fur transcription
and implicated the altered bases in contributing to ArcA
binding at this promoter.
FIG. 6. ArcA-dependent repression of fur transcription under anaerobic conditions. (A) S. flexneri SA100 (WT) with pCC1 (vector) and
MBF200 (arcA) with pCC1 (vector) or pMBarcAccQE (pArcA) were grown to mid-log phase anaerobically with 1 ?M IPTG. The level of fur
mRNA was determined by RT-PCR. Experiments were performed in triplicate, and error bars represent 1 standard deviation. (B) The relative gfp
expression levels obtained from fur (pMBfur) and furAlt(pMBfurAlt) promoters in SA101 (WT) and MBF200W (arcA) are shown with the highest
relative fluorescence value set at 100%. Experiments were performed in triplicate, and error bars represent 1 standard deviation. (C) The S. flexneri
fur chromosomal region shows the relative positions of promoter elements involved in fur regulation, including the putative ArcA boxes, OxyR box,
Crp box, and Fur box. The two putative ArcA boxes are indicated by the arrows, and their sequences are shown below. The bases in the native
and altered fur promoters matching those of the putative ArcA box consensus sequence are shown in black. Bases not matching the consensus are
in gray, and bases changed in the altered promoter are underlined. Lowercase letters indicate bases not conserved in the ArcA box weight matrix.
VOL. 189, 2007ArcA REGULATION OF SHIGELLA fur AND IRON TRANSPORT 6963
ArcA binds the iuc, feo, and fur promoters. The presence of
putative ArcA binding sites in the fur, iuc, and feo promoters
suggested that the observed regulation of these genes by oxy-
gen availability was mediated directly by ArcA. To detect bind-
ing of ArcA to the feo, iuc, and fur promoters, anti-ArcA
antibody supershift assays were performed (Fig. 7). The lld
(lct) promoter, which contains a known ArcA binding site (32),
was used as a positive control. Incubation of the lld, iuc, feo, or
fur promoter with an extract containing ArcA and anti-ArcA
antibody slowed electrophoretic mobility (Fig. 7). Incubation
of the probes with extract from the arcA mutant did not affect
mobility of the radiolabeled probe (Fig. 7). The altered feo and
fur probes were also tested, and as predicted from the gfp
expression results, the altered probes showed reduced anti-
ArcA-dependent supershifting (Fig. 7; compare feo with feoAlt
and fur with furAlt). Some binding of ArcA to the altered
probes was still observed, particularly with furAlt. The amount
of probe in the supershifted bands was quantified by measuring
the radioactivity; the mean counts in the supershifted region
were 3,910 for the native feo, compared with 1,511 for feoAlt,
and 1,918 for fur, compared with 1,607 for furAlt. The relatively
small effect of changing the fur promoter may reflect the fact
that there are regions with homology to the consensus ArcA
box on both DNA strands of the fur probe. The base changes
introduced alter each site differently, and there may be residual
ArcA binding to one or both sequences on the altered fur
probe. ArcA did not bind the sit probe, as indicated by the lack
of supershifting (Fig. 7), which was in agreement with our
failure to find an ArcA box sequence in the promoter. These
data demonstrate that ArcA directly binds the feo, iuc, and fur
promoters and that the altered bases of feo are within the
region required for DNA recognition by ArcA.
S. flexneri transcription factors ArcA, Fnr, and Fur impact
plaque formation in Henle cell monolayers. The expression of
iron acquisition genes is regulated by ArcA, Fnr, and Fur, and
since iron uptake is required for plaque formation by S. flex-
neri, we assessed the importance of these regulators in aerobic
and anaerobic plaque assays. The arcA and fnr single mutants
formed plaques under anaerobic conditions (Fig. 8A). In the
absence of oxygen, these mutants also formed plaques but
there was a slight reduction in plaque size (Fig. 8A). These
data indicate that ArcA and Fnr exhibit a partial redundancy in
the ability to regulate one or more of the steps required for
plaque formation. In contrast, the arcA fnr double mutant was
able to form plaques only in the presence of oxygen (Fig. 8A).
Additionally, the double mutant formed plaques when the
infected Henle cells were incubated aerobically for 2 days
following the usual period of anaerobic incubation (data not
shown). This indicates that under anaerobic conditions, the
double mutant could invade and remain viable inside epithelial
cells but was defective in intracellular growth or cell-to-cell
spread. Since these transcription factors influence iron acqui-
sition, the defective anaerobic plaque formation by arcA fnr
mutants may reflect reduced feo expression and aberrant ex-
pression of fur.
FIG. 7. ArcA binds feo, fur, and iuc promoters. S. flexneri protein
extracts prepared from strain MBF200 (arcA) containing either pCC1
vector (ArcA?) or pMBarcAccQE (ArcA?) were incubated with the
indicated radiolabeled probes and anti-ArcA antibody. Samples were
electrophoresed on a 5% polyacrylamide gel. Positions of probes (?)
and supershifted bands (brackets) are indicated.
FIG. 8. Fur and the anaerobic regulators ArcA and Fnr are important for plaque formation by S. flexneri. Henle cell monolayers were infected
with 104CFU of the indicated S. flexneri strain. The plates were incubated for 2 days in medium containing gentamicin under either aerobic (A) or
anaerobic (B) conditions and stained to visualize plaque formation. (A) SA100 (WT) and mutants MBF100 (fnr), MBF200 (arcA), and MBF300
(arcA fnr). (B) SM100 (WT), SM1301 (fur), or SM100/pMBfurQE (WT/pfur) incubated with or without 50 ?M IPTG to induce fur expression.
6964 BOULETTE AND PAYNE J. BACTERIOL.
The effect of altered fur expression on the ability of S. flex-
neri to form plaques was then investigated. An S. flexneri fur
mutant and the wild-type strain expressing fur from an IPTG-
inducible plasmid were assayed for aerobic and anaerobic
plaque formation in the Henle cell model of infection. Over-
expression of fur prevented plaque formation, while a mutation
in fur reduced the number of plaques formed (Fig. 8B). The
effects of altered expression of fur on plaque formation oc-
curred in both aerobic and anaerobic environments, indicating
that proper regulation of fur is critical for the virulence of S.
flexneri regardless of oxygen availability.
Studies on the growth of Shigella spp. in response to condi-
tions encountered within the host or in model systems have
shed light on genes required for initiating and sustaining in-
fection. S. flexneri virulence is typically assayed in epithelial cell
monolayers to determine its proficiency in adherence, invasion,
intracellular growth, and intercellular spread (43). Because
tissue culture models are typically performed under aerobic
conditions, while the lumen of the colon is considered to be
anaerobic, we developed a plaque assay model of S. flexneri
pathogenesis to measure S. flexneri plaque formation in an
anaerobic environment. This model of infection may be of
value in assessing mutants of S. flexneri and other enteric
pathogens, since it measures aspects of virulence under condi-
tions more like those encountered in vivo.
The anaerobic plaque assay demonstrated that the Feo sys-
tem is an important iron transporter for anaerobic iron acqui-
sition by S. flexneri in epithelial cells. The lack of ability of the
Feo system to sustain aerobic plaque formation likely reflects
its reduced expression when oxygen is abundant. The Feo iron
transport system has recently been shown to be important for
intracellular survival or virulence of a number of pathogenic
bacteria, including Legionella pneumophila (5, 48, 54), Campy-
lobacter jejuni (41), Helicobacter pylori (63), and S. enterica
serovar Typhimurium (3, 62).
Not all environments encountered by S. flexneri have limited
oxygen availability; therefore, aerobically expressed iron trans-
port systems may also be important for the virulence of S.
flexneri. Under aerobic conditions, the Sit system was sufficient
for wild-type plaque formation. The genes of the Sit system are
widespread among enteroinvasive pathogens (52). Further, the
S. flexneri sit genes have been shown to be derepressed upon
entry into epithelial cells (51). Thus, this system may be im-
portant during intracellular replication. In addition to its role
in iron acquisition, the Sit system is able to transport manga-
nese (26), which correlates with increased survival of S. flexneri
in macrophages and under oxidative stress conditions (50).
Thus, the prevalence of the sit genes among enteroinvasive
pathogens may reflect its role in both iron and manganese
The strain that expressed only the aerobactin system, which
transports Fe3?, formed small plaques aerobically and failed to
form plaques anaerobically. This is in agreement with our
previous findings demonstrating that the aerobactin locus is
repressed intracellularly (18). This suggests that the sid-
erophore is less critical than other mechanisms of iron trans-
port for the intracellular growth of S. flexneri. The aerobactin
genes have been shown to be important for the growth of S.
flexneri within extracellular tissues in a ligated ileal loop model
of infection, however (29, 42). Thus, expression of siderophore
iron acquisition systems is likely related to their role during
certain aerobic, extracellular stages of pathogenesis.
The availability of oxygen is expected to influence iron ac-
quisition in several ways. The amount of available ferric and
ferrous iron is affected by oxygen, with the ferric form predom-
inating when oxygen is present and ferrous iron more abundant
in anaerobic environments. Additionally, the activity of en-
zymes such as the oxygen-requiring lysine/ornithine N-mono-
oxygenase (IucD), which is necessary for aerobactin biosynthe-
sis, would also be reduced in the absence of molecular oxygen
(64). Consistent with these expectations, the promoters of the
iucABCD iutA operon and fur were repressed anaerobically by
ArcA, while the feoABC promoter, previously shown to be
induced by Fnr in E. coli, was induced by both Fnr and ArcA
in S. flexneri. Although expression of the sit operon was ele-
vated aerobically, its promoter did not appear to be directly
regulated by ArcA or Fnr. A study of transcriptional regulation
of the Salmonella sit locus also demonstrated that its expres-
sion increased under aerobic conditions independently of
ArcA and Fnr; both Fur and MntR contributed to the reduc-
tion in sit transcription under anaerobic conditions (20).
Our data agree with studies indicating regulation of fur and
iron acquisition genes by anaerobiosis and ArcA in E. coli (7,
25, 30, 54, 55). Liu and De Wulf (30) identified putative ArcA
boxes upstream of the E. coli feo and fur operons, and the fur
transcript, but not the feo transcript, was derepressed in the
arcA mutant. Our studies provide direct evidence that ArcA
represses fur and confirm ArcA involvement in the regulation
of feo and siderophore biosynthesis and transport genes. These
data support a role for oxygen availability as a general signal
for regulation of iron acquisition.
Since anoxic conditions and the anaerobic regulators ArcA
and Fnr impact the virulence of several enteric pathogens, it
was not surprising that mutations in these genes affected an-
aerobic plaque formation. However, the transition to anaero-
biosis did not appear to regulate the expression of the viru-
lence genes found on the Shigella virulence plasmid (Table 2
and data not shown). Because ArcA and Fnr are pleiotropic
transcription factors with overlapping functions, it is impossi-
ble to attribute the lack of plaque formation by the arcA fnr
double mutant to one specific pathway. ArcA and Fnr, how-
ever, both regulate iron acquisition on more than one level,
and given the importance of iron acquisition in S. flexneri
virulence, aberrant iron uptake in the arcA fnr double mutant
is likely a contributing factor in the loss of plaque formation.
Factors in addition to oxygen deprivation activate ArcA (15,
33, 34, 49). Reduced quinones accumulate in the membrane
when the respiratory dehydrogenases, reductases, or electron
carriers are inactive due to an inadequate supply of cofactors,
including iron, or a lack of substrate availability. These quinols
lead to autophosphorylation of the ArcB sensor, which acti-
vates ArcA. ArcA helps the cell conserve energy and acquire
ATP through substrate-level phosphorylation by down-regulat-
ing TCA cycle and aerobic respiratory enzymes and inducing
fermentation genes and anaerobic respiratory complexes.
ArcA may also help the cell conserve energy by derepressing
the Feo ferrous iron transporter while repressing synthesis of
VOL. 189, 2007ArcA REGULATION OF SHIGELLA fur AND IRON TRANSPORT 6965
the more energetically expensive siderophore system. It is also
likely that ArcA repression of fur leads to an increase in RyhB,
which helps regulate iron storage and cellular metabolism (36).
Interestingly, many of the targets of RyhB overlap those of
ArcA and Fnr, and so an increase in RyhB may be a mecha-
nism for additional fine-tuning of metabolic pathways during
the depletion of oxygen as well as of iron.
We thank Ian J. Whitney for technical assistance during mutant
construction, Philip M. Silverman for generously providing anti-ArcA
and anti-goat control antiserum, Peter De Wulf for supplying the E.
coli arcA strain ECL5331, and Elizabeth E. Wyckoff for thorough
review and editing of the manuscript.
This work was funded by grant AI16935 from the National Institutes
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