Microbiology (2014), 160, 733–741 DOI 10.1099/mic.0.

071381-0

OmpR phosphorylation regulates ompS1

Correspondence
Miguel Ángel De la Cruz
miguel_angel_81@live.com
expression by differentially controlling the use of
promoters
Mario Alberto Flores-Valdez,13 Marcos Fernández-Mora,23
Miguel Ángel Ares,3 Jorge A. Girón,4 Edmundo Calva2
and Miguel Ángel De la Cruz2,3
1
Centro de Investigación y Asistencia en Tecnologı́a y Diseño del Estado de Jalisco A. C.,
Normalistas 800, Colinas de la Normal, Guadalajara 44270, Jalisco, Mexico
2
Departamento de Microbiologı́a Molecular, Instituto de Biotecnologı́a, Universidad Nacional
Autónoma de México, Av. Universidad 2001, Cuernavaca, Morelos 62210, Mexico
3
Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, Hospital de Pediatrı́a,
Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Mexico
4
Department of Molecular Genetics and Microbiology, Emerging Pathogens Institute,
University of Florida, Gainesville, FL, USA

Received 16 July 2013
Accepted 15 January 2014
The Salmonella enterica ompS1 gene encodes a quiescent porin that belongs to the OmpC/
OmpF family. In the present work we analysed the regulatory effects of OmpR phosphorylation on
ompS1 expression. We found that in vivo, OmpR in its phosphorylated form (OmpR-P) was
important in the regulation of the two ompS1 promoters: OmpR-P activated the P1 promoter and
repressed the P2 promoter in an EnvZ-dependent manner; expression occurs from the P2
promoter in an ompR mutant. In vitro, OmpR-P had a higher DNA-binding-affinity to the ompS1
promoter region than OmpR and OmpRD55A, showing an affinity even higher than that of
equivalent DNA regions in the 59-upstream regulatory sequence of the major porin-encoding
genes ompC and ompF. By analysing different environmental conditions, we found that glucose
and glycerol enhanced ompS1 expression in the wild-type strain. Interestingly the stimulation by
glycerol was OmpR-dependent while the effect of glucose was still observed in the absence of
OmpR. Acetyl phosphate produced by the AckA-Pta pathway did not influence ompS1 regulation.
These data indicate the important role of the phosphorylation in the activity of OmpR on the
differential regulation of both ompS1 promoters and its impact on the pathogenesis.

INTRODUCTION regulatory sequences of the ompC and ompF promoters

(Forst et al., 1989; Delgado et al., 1993; Head et al., 1998).
Salmonella enterica serovar Typhi (S. Typhi) is the
aetiological agent of typhoid fever in humans (Pang et al., The S. enterica ompS1 gene codes for the OmpS1 quiescent
1998). S. Typhi synthesizes three major outer-membrane porin that belongs to the OmpC/OmpF super family
proteins, OmpC, OmpF and OmpA. OmpC and OmpF are (Fernández-Mora et al., 1995). OmpS1 appears to have a
under the control of the EnvZ-OmpR two-component role in swarming motility, biofilm formation and virulence
system encoded by the ompB locus (Puente et al., 1991) that in mice (Toguchi et al., 2000; Mireles et al., 2001; Rodrı́guez-
responds to several environmental stimuli, among which Morales et al., 2006). Expression of the ompS1 gene is mainly
osmolarity and pH have been the most studied (Russo & dependent on two overlapping promoters, P1 and P2
Silhavy, 1991; Bang et al., 2000). EnvZ senses an envir- (Oropeza et al., 1999; Flores-Valdez et al., 2003; De la Cruz
onmental signal, which modulates OmpR function by a et al., 2007; Fig. 1a). OmpR activates P1, which is located in
mechanism involving phosphorylation and dephosphoryla- the ompS1 upstream regulatory region and possesses six
tion (Forst et al., 1989; Forst & Roberts, 1994; Mizuno & OmpR-binding sites, and thus in the absence of OmpR there
Mizushima, 1990; Pratt et al., 1996). OmpR is phos- is no P1 activity. The P2 promoter is located in the
phorylated at the aspartate 55 residue to produce OmpR- overlapping OmpR 235 and 210 boxes and does not
P, an event that enhances its ability to bind to the upstream require OmpR for activation; instead P2 is only active in the
absence of OmpR (Fig. 1a). A third promoter with low
3These authors contributed equally to this work. expression was described by using RNA from plasmid
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M. A. Flores-Valdez and others

(a) OmpR-binding box III

(b) 2
ompS1

Bits
pRO88 1

P2
OmpR-binding box II OmpR-binding box I
0

10
11
12
13
14
15
16
17
18
1
2
3
4
5
6
7
8
9
5′ 3′

–35 –10 –35 2

ompC
P1

Bits
1

–10 +1 0

1
2
3
4
5
6
7
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9
10
11
12
13
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5′ 3′
(c) 30 000
25 000 (d)
(U mg–1 protein)
β-Galactosidase

20 000
15 000
10 000 1 2 3 4
5000 a-OmpR 1. IMSS-1
2. IMSS-40
0
3. IMSS-40 pFM2000
a-DnaK 4. IMSS-40 pFVD55A
Vector pFM2000 pFVD55A Vector pFM2000 pFVD55A

IMSS-1 IMSS-40 IMSS-1 IMSS-40

(wt) (ompR) (wt) (ompR)
pSCZ10 (ompC-lacZ) pRO88 (ompS1-lacZ)

(e)
+1 P2

pRO88

CTAG 1 2 3 4

P2 ompS1

P1 ompS1

1. IMSS-1
2. IMSS-40 (ompR)

+1 P1 3. IMSS-40 pFM2000
4. IMSS-40 pFVD55A

Fig. 1. OmpR-P differentially regulates both ompS1 promoters. (a) Nucleotide sequence of the ompS1 promoter region. Start-
points and ”10 and ”35 boxes for both promoters are shown. OmpR-binding boxes (I”II) overlap with the P2 promoter. The
sequence of the promoter region from the pRO88 plasmid is shown. (b) Logo motif analysis (Crooks et al., 2004) using the six
and three OmpR-binding sites for ompS1 and ompC promoter regions, respectively. (c) b-Galactosidase activity of ompC-lacZ
(pSCZ10) and ompS1-lacZ (pRO88) gene reporter fusions in S. Typhi wild-type (IMSS-1) and a null ompR mutant (IMSS-40)

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complemented with OmpR wt (pFM2000) and OmpR D55A (pFVD55A). The data represent means±SD of three independent
experiments. (d) Western blotting experiments showing OmpR levels in different backgrounds: wt, ompR, ompR (pFM2000)
and ompR (pFVD55A). DnaK is shown as a loading control. (e) Primer extension analysis of the ompS1 P1 and P2 promoters
from the pRO88 in S. Typhi wild-type and in the null ompR mutant complemented with OmpR wt (pFM2000) and OmpR D55A
(pFVD55A). The ompA transcript was included as a loading control.
fusions (Oropeza et al., 1999); however, this promoter has not
been detected from chromosomal RNA (Flores-Valdez et al.,
2003; De la Cruz et al., 2007). OmpR dimerizes and recognizes
around 18–20 nt (approximately 10 nt recognized by each
OmpR molecule; Yoshida et al., 2006). In a similar manner to
the ompC promoter, the OmpR-binding sites in the ompS1
promoter showed conservation of AT-rich sequences (nt 1–7)
and a CATNT motif (nt 12–16; Fig. 1b). Sequences upstream
of position 288 are required for negative regulation, and
sequences downstream are required for positive regulation
(Oropeza et al., 1999; Flores-Valdez et al., 2003). OmpR-
binding boxes I–II and part of III were identified in this
positive regulation zone (Oropeza et al., 1999; Fig. 1a). Thus,
OmpR acts as a modulator of ompS1 expression by selectively
binding to the P1 or P2 promoters (De la Cruz et al., 2007). In
contrast to OmpC and OmpF, no osmoregulation was seen
for OmpS1 (Oropeza et al., 1999). However, the role of
OmpR-P in ompS1 regulation remains unclear.
In addition to OmpR, H-NS and StpA nucleoid-binding
proteins negatively regulated ompS1 expression by binding
to P1 and P2 (Flores-Valdez et al., 2003; De la Cruz et al.,
2007). Interestingly, we recently reported that DNA static
curvature favours the repressive effect of both nucleoid
proteins on ompS1 expression (De la Cruz et al., 2009).
LeuO, a LysR-type regulator, antagonizes the repression
mediated by H-NS and StpA on the S. Typhi ompS1 gene
by displacement of these proteins (De la Cruz et al., 2007).
The aim of this study was to elucidate the role of the P1
and P2 promoters in the regulation of ompS1 expression
mediated by OmpR-P.
METHODS
Bacterial strains, plasmids, and recombinant DNA techniques.
Bacterial strains and plasmids used in this study are listed in Table 1.
DNA manipulations were performed according to standard protocols
(Sambrook & Russell, 2001). Oligonucleotides used for amplification
by PCR were provided by the Oligonucleotide Synthesis Facility at the
Instituto de Biotecnologı́a, Universidad Nacional Autónoma de
México, and are listed in Table 2. PCRs were performed with Taq
DNA polymerase (Invitrogen), according to the manufacturer’s
instructions. The one-step mutagenesis procedure described by
Datsenko & Wanner (2000) for bacterial chromosomal genes was
used to generate gene deletions and replacements for antibiotic
resistance markers. The S. Typhi ompR D55A allele was subcloned
from a BlueScript KS vector (Tran et al., 2000) by using XbaI and
HindIII, and ligated to pMPM-A3 (Mayer, 1995) digested with the
same enzymes generating pFVD55A.
Bacterial culture and b-galactosidase assays. The culture
conditions and microplate protein and b-galactosidase assays were
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OmpR phosphorylation controls both ompS1 promoters
as previously described (Flores-Valdez et al., 2003). Acid pH (5.2),
high osmolarity (0.3M NaCl) or oxidative stress (5 mM H2O2
60 min) was tested in Nutrient Broth (NB) or M9 medium
containing glucose or glycerol to a final concentration of 0.4 %.
Western blotting. Whole-cell extracts of S. Typhi strains grown in
NB cultures at 37 uC were subjected to SDS-PAGE (12 % poly-
acrylamide) and blotted onto nitrocellulose membranes (Millipore) as
previously described (De la Cruz et al., 2007). After blocking with
non-fat milk, the membranes were incubated with a primary antibody
against OmpR (1 : 2000). Goat anti-rabbit (Biomeda) conjugated to
horseradish peroxidase (1 : 10 000) was used as a secondary antibody,
and the reactions were visualized with chemiluminescence reagents
(PerkinElmer).
Primer extension analysis. Total RNA was isolated from samples of
cultures grown in NB at 37 uC and collected at mid-exponential phase.
Five micrograms of total RNA (for ompA) or 25 mg of total RNA (for
ompS1), isolated using a commercial kit (RNeasy; Qiagen), were
denatured at 90 uC for 3 min and then slowly cooled to 50 uC. The
RNA was annealed with [c-32P]ATP-labelled ompA-PE 59-
TTTGCGCCTCGTTATCATCCAA-39 (annealing from position +3
to 224 with respect to the translational start site) or primer ompS1-PE
59-TTGCTGCGCCTGCCACTAATAAC-39 (annealing from +57 to
+35 with respect to the translational start site). Primers were extended
with Moloney murine leukaemia virus reverse transcriptase at 37 uC for
2 h, and the extended products were collected with a Microcon-30
microconcentrator (Amicon) and analysed by electrophoresis in urea–
8 % polyacrylamide gels alongside sequencing ladders.
OmpR purification. OmpR was overexpressed and purified from E.
coli BL21 (Novagen) carrying pQR254 that encodes a His6x-OmpR
fusion protein as previously described (Oropeza et al., 1999). This
plasmid was used as a template to generate pQRD55A by inverse PCR
as was previously described (Oropeza & Calva, 2009). The primers
used are listed in Table 2.
Phosphorylation of OmpR by acetyl phosphate. OmpR protein
was phosphorylated using 25 mM acetyl phosphate (Sigma) as
previously described (Kenney et al., 1995).
Fluorescence anisotropy. Equilibrium binding measurements were
performed using fluorescence anisotropy (Head et al., 1998). OmpR
was labelled at the amino terminus using an amine fluorescein
labelling kit (Panvera). The labelled protein was purified using a series
of three 5 ml desalting columns (Amersham Biosciences). Fluorescent
OmpR was excited at 490 nm, and emission was measured at 530 nm
in a Beacon fluorometer (Panvera). The samples were incubated in
the fluorometer for 30 s, and five measurements were taken at 10 s
intervals after each protein addition. A typical binding experiment
lasted approximately 30 min. Each point plotted in the figures
represents the mean of at least three independent measurements. The
change in anisotropy, where (A2Ao)/Ao represents the difference in
anisotropy in the presence of protein minus the anisotropy in the
absence of protein divided by the anisotropy in the absence of
protein, was plotted as a function of the total protein concentration.
The results from the binding curves were fitted by non-linear least-
squares regression as described previously (Head et al., 1998). The
735
M. A. Flores-Valdez and others

Table 1. Bacterial strains and plasmids

Strain or plasmid Genotype and/or relevant markers Reference

E. coli
MC4100 F9 araD139 D (argF-lac)U169 rpsL150 relA1 flb5301 deoC1 ptsF25 rbsR Casadaban (1976)
BL21(DE3) F2 ompT gal (dcm) (lon) hsdSB(rB2 mB2) metBL21(DE3) Novagen

S. enterica
IMSS-1 Salmonella enterica serovar Typhi 9.12, d, Vi serotype; Mexican reference clinical strain Puente et al. (1987)
IMSS-40 IMSS-1 DompR : : Kmr Fernández-Mora et al. (2004)
IMSS-81 IMSS-1 DompB : : Kmr Martı́nez-Flores et al. (1999)
IMSS-45 IMSS-1 D(ackA-pta) : : Kmr This work
IMSS-85 IMSS-81 D(ackA-pta) : : Cmr This work

Plasmids
pRO310 pMC1871-derived plasmid, containing a translational fusion of the ompS1 59 regulatory Oropeza et al. (1999)
region, up to 310 to 27 bp upstream of the transcriptional start point, to the lacZ
reporter gene
pRO88 pMC1871-derived plasmid, containing a translational fusion of the ompS1 59 regulatory Oropeza et al. (1999)
region, up to 88 to 27 bp upstream of the transcriptional start point, to the lacZ
reporter gene
pSCZ10 pMC1871-derived plasmid, containing a translational fusion of the ompC 59 regulatory Martı́nez-Flores et al. (1999)
region, 1450 bp upstream of the transcriptional star point and the first codon, to the
lacZ reporter gene
pFM2000 pACYC184 carrying the S. Typhi ompR gene; p15A Fernández-Mora et al. (2004)
pFVD55A pMPM-A3 carrying the S. Typhi ompR gene with a point mutation in the Asp-55 This work
(D55A); p15A
pQR254 pQE32 carrying the S. Typhi ompR gene with His6 in the N-terminal Oropeza et al. (1999)
pQRD55A pQR254 carrying the S. Typhi ompR gene with a point mutation in the Asp-55 (D55A) This work

apparent dissociation constants reported in the text represent the
mean.
RESULTS
OmpR phosphorylation is key for the differential
regulation of the P1 and P2 ompS1 promoters
In order to test the role of OmpR phosphorylation on
ompS1 expression, two plasmids coding for OmpR wild-
type (pFM2000) or for OmpR mutated in the D55
(aspartate 55) phosphorylation site (pFVD55A) were used.
D55A OmpR protein is unable to become phosphorylated
and thus has a decreased affinity for DNA (Delgado et al.,
1993; Head et al., 1998).
As a control of OmpR function, the expression of the
major OmpR-dependent porin gene ompC was assessed by
using the pSCZ10 fusion, which contains the ompC
regulatory region fused to lacZ (Martı́nez-Flores et al.,
1999). The expression of ompC was measured in two S.
Typhi backgrounds, wild-type (IMSS-1) and an ompR null
mutant (IMSS-40). In IMSS-40, ompC expression was
abolished, which is in agreement with the reported
dependency of ompC expression on OmpR-mediated
regulation (Hall & Silhavy, 1981). Introduction of an
ompR wild-type allele (pFM2000) into the mutant resulted

Table 2. Oligonucleotides used in this work

Oligonucleotide Sequence Reference

ackA-H1P1 GTA TCA TAA ATA GGT ACT TCC ATG TCG AGT AAG TTA GTA CTG GTT TGT AGG This work
CTG GAG CTG CTT C
pta-H2P2 ATC CGG CAT TAG CTT TTA CTG TTA CTG CTG CTG TTG AGA AGC CTG CAT ATG This work
AAT ATC CTC CTT AGT TCC
ackA-5 ATA AAT GTC AGG GCT ATC ATG CGC This work
pta-3 AAC GCG TCT TAT CCG GCC TAC AAC GG This work
ompR-D55A-59 CAT CTC ATG GTA CTG GCT TTA ATG CTG CCA GGT GAA GAT This work
ompR-D55A-39 CAT CTT CAC CTG GCA GCA TTA AAG CCA GTA CCA TGA GAT This work

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in restoration of ompC expression, as expected. Conversely,
the mutated ompR allele coding for a phosphorylation-
deficient OmpR (pFVD55A) did not restore ompC
expression (Fig. 1c), strongly showing a central role for
OmpR phosphorylation on ompC expression. To deter-
mine the OmpR levels produced in the plasmids, we
performed Western blotting experiments detecting OmpR
protein in both the wild-type and the ompR mutant
complemented with pFM2000 and pFVD55A. OmpR wt
and OmpRD55A proteins expressed in trans showed
similar levels compared to the wild-type strain (Fig. 1d).
The role of OmpR-P in regulating ompS1 expression was
further examined by using the pRO88 fusion, which attains
the maximum level of expression and contains the two and
half OmpR-binding sites that are required for activation of
P1 (Fig. 1a, b; Oropeza et al., 1999; Flores-Valdez et al.,
2003; De la Cruz et al., 2007). As reported previously, in an
ompR null mutant, ompS1 expression was diminished to
half of the value observed in the wild-type strain (Fig. 1c).
When this mutant was complemented with pFM2000,
ompS1 expression reached wild-type-like levels as expected
(Fig. 1c). Moreover, upon introduction of pFVD55A the
level of ompS1 expression remained similar to that of the
ompR mutant, at half of the wild-type levels (Fig. 1c),
consistent with the notion that OmpR (D55A) is non-
functional and thus neither binds to DNA nor activates P1,
allowing for expression of ompS1 from P2. In agreement
with these results, primer extension experiments showed
that in the wild-type strain ompS1 expression started from
the P1 promoter, while in the null ompR mutant expression
was started from P2 (Fig. 1e). In the ompR mutant
transformed with pFM2000, ompS1 activity was restored,
indicating it was P1-dependent, but when complemented
with pFVD55A, ompS1 activity was P2-dependent (Fig. 1e).
Therefore, transcriptional activity from P2 was observed
only in the absence of OmpR or when OmpR was not
phosphorylated at D55. Our data demonstrate that OmpR-
P positively regulates the P1 promoter and negatively
affects P2.
OmpR-P has a higher DNA-binding affinity than
OmpR for the ompS1 promoter region
The differential expression of both ompS1 P1 and P2
promoters demonstrated that the phosphorylation status of
OmpR determines which promoter is to be used for
initiation of transcription of this gene. In order to
determine the relevance of OmpR phosphorylation status
to ompS1 regulation, we compared the DNA-binding
affinity of OmpR, OmpR-P and OmpRD55A for the
ompS1 promoter region. We phosphorylated OmpR in
vitro by incubation with acetyl phosphate, and the
proportion of OmpR-P obtained was in the range of 92–
97 % as assessed by HPLC (data not shown). Next, the
equilibrium binding of OmpR and OmpR-P to the ompS1
promoter region was compared by fluorescence anisotropy
experiments. The apparent dissociation constant (Kd) for
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OmpR phosphorylation controls both ompS1 promoters
OmpR binding to OmpR-boxes I-II and the half of III on
ompS1 was 157.3 nM (Fig. 2a, b). Strikingly, OmpR-P
showed a 72-fold increase in DNA-binding affinity, with a
Kd of 2.2 nM (Fig. 2a). To corroborate the relevance of the
phosphorylation site, we purified the OmpRD55A protein.
The Kd for OmpRD55A on ompS1 was 121.2 nM, very
similar to non-phosphorylated OmpR (Fig. 2a). These
compelling data indicate that OmpR-P displays a high
affinity for the ompS1 promoter region activating P1
promoter and repressing P2 (Figs 1 and 2).
EnvZ is required for the differential regulation of
both P1 and P2 promoters of ompS1
Under certain conditions, acetyl phosphate promotes
EnvZ-independent OmpR phosphorylation (Hsing &
Silhavy, 1997; Matsubara & Mizuno, 1999; Bang et al.,
2000; Heyde et al., 2000). In order to define whether
phosphorylation of OmpR mediated by EnvZ was the main
source of transcriptionally active OmpR-P for regulating
ompS1 expression, we monitored gene expression in several
genetic backgrounds, including an ompB (ompR-envZ)
background (IMSS-81; Martı́nez-Flores et al., 1999). In an
ompB mutant, ompS1 expression reached levels similar to
those observed in the single ompR mutant (Fig. 3a).
Interestingly, when pFM2000 (OmpR wt) was introduced
into the ompB mutant, ompS1 expression remained as if no
OmpR-P was produced (Fig. 3a). We further demonstrated
by primer extension experiments the participation of EnvZ
in producing OmpR-P for ompS1 differential promoter
use. In an ompB mutant, ompS1 transcription initiated
from P2 and when this mutant was transformed with
pFM2000 or pFVD55A, P2 remained as an active promoter
(Fig. 3b). Production of acetyl phosphate in the cell is
mediated by the acetate kinase (AckA) and phosphotrasce-
tylase (Pta) enzymes (Wanner & Wilmes-Riesenberg, 1992;
McCleary & Stock, 1994; Wolfe, 2005). To discard the
effect of the acetyl phosphate produced in the cell on
OmpR phosphorylation, we analysed ompS1 transcription
in the absence of the AckA-Pta pathway. No differences
were observed in both ackA-pta and ompB ackA-pta
compared to the wild-type and ompB backgrounds (Fig.
4b). These results strongly indicate that EnvZ is the main
kinase involved in the production of phosphorylated
OmpR for ompS1 expression, since in the absence of
EnvZ such expression was P2-dependent, while the OmpR-
P dependent P1-promoter was unable to become activated
at least to detectable levels.
Glucose and glycerol stimulate ompS1 expression
To analyse the ompS1 expression under different envir-
onmental conditions, we monitored pRO88 fusion both in
the wild-type strain and in the ompR mutant. The conditions
used were pH, osmolarity, oxidative stress and carbon
source. No differences were found in acid pH (5.2), high
osmolarity (0.3 M NaCl) or oxidative stress (5 mM H2O2
for 60 min, Fig. 4a) in agreement with previous reports
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M. A. Flores-Valdez and others

(a) 0.25 0.25

0.20 0.20
(A-Ao)/Ao

(A-Ao)/Ao
0.15 0.15
Kd=2.2 nM
0.10 Kd=157.3 nM 0.10

0.05 0.05

0 0
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 0.1 0.2 0.3 0.4 0.5

[OmpR] mM [OmpR-P] mM

0.25

0.20
(A-Ao)/Ao

0.15
Kd=121.2 nM
0.10

0.05

0
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
[OmpR-D55A] mM
(b)
5′ –3′

Fig. 2. Binding of OmpR and OmpR-P to the ompS1 promoter region. (a) DNA-binding curves of OmpR, OmpR-P and
OmpRD55A to ompS1 OmpR-binding boxes I–II, and half of III, contained in a 54 bp oligonucleotide. The fractional change in
anisotropy is plotted against OmpR or OmpR-P concentration. Ao is the anisotropy value of the oligonucleotide in the absence
of the protein and A is the measured anisotropy value at each protein concentration. The apparent dissociation constants
calculated from these curves were 2.2 nM for OmpR-P, 157.3 nM for OmpR and 121.2 for OmpRD55A. (b) Sequence of the
oligonucleotide used in this study.

(Oropeza et al., 1999). However, when S. Typhi harbouring
the pRO88 plasmid was grown in M9 medium with glucose
or glycerol, ompS1 transcription was enhanced (Fig. 4a).
Interestingly, this stimulation was differential: induction by
glycerol and glucose was dependent and independent of
OmpR, respectively. By evaluating the role of the AckA-Pta
pathway in stimulation by glucose and glycerol in ompS1
expression, we measured pRO88 levels in wild-type, ompR,
ompB, ackA-pta and ompB ackA-pta backgrounds. pRO88
levels were similar between the wild-type strain and the
ackA-pta single mutant (Fig. 4b). Similarly, DompB D(ackA-
pta) and DompB had a similar phenotype (Fig. 4b). Acetyl
phosphate produced in the cell did not influence the ompS1
expression stimulated by glycerol and glucose.
DISCUSSION
The expression of ompS1 is dependent on two promoters,
P1 and P2. OmpR differentially regulates both promoters
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in its phosphorylated state, acting as a modulator of ompS1
expression and selectively choosing one or the other
promoter (Fig. 1). OmpR-P regulates P1 and P2 positively
and negatively, respectively (Fig. 1b). In contrast to the
regulation of both ompC and ompF porin genes, whose
expression is strictly OmpR-P-dependent, ompS1 expres-
sion can be independent of OmpR-P; even though OmpR-
P had a much higher affinity to the ompS1 promoter region
than OmpR and OmpRD55A (72-fold; Fig. 2). Similar
fluorescence anisotropy experiments performed by other
workers showed that OmpR-P had a 32-fold higher affinity
for ompF than OmpR, in contrast to a threefold affinity
displayed for ompC (Head et al., 1998).
Moreover, acetyl phosphate is a phospho-donor in the cell,
which can phosphorylate response regulators including
OmpR (Hsing & Silhavy, 1997; Matsubara & Mizuno,
1999; Bang et al., 2000; Heyde et al., 2000). Nevertheless,
we observed that ompS1 expression from the P1 promoter,
under our culture conditions (NB), was completely
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(b)
+1 P2

pRO88

CTAG 1 2 3 4
(a) 35 000 P2 ompS1
30 000
(U mg–1 protein)
β-Galactosidase

25 000
20 000
15 000
10 000
5000
0
Vector pFM2000 pFVD55A Vector pFM2000 pFVD55A
P1 ompS1
IMSS-1 IMSS-40 IMSS-81
(wt) (ompR) (ompR-envZ)
1. IMSS-1
pRO88 (ompS1-lacZ) 2. IMSS-81 (ompB)
+1 P1 3. IMSS-81 pFM2000
4. IMSS-81 pFVD55A

Fig. 3. EnvZ is necessary for OmpR-P-mediated ompS1 regulation. (a) b-Galactosidase activity of the ompS1-lacZ (pRO88)
gene reporter fusion in S. Typhi wild-type (IMSS-1), a null ompR mutant (IMSS-40) and in an ompB (ompR-envZ) mutant
(IMSS-81) complemented with OmpR wt (pFM2000) or the OmpR D55A variant mutated in the phosphorylation site
(pFVD55A). The data represent means±SD of three independent experiments. (b) Primer extension analysis of the ompS1 P1
and P2 promoters from pRO88 in S. Typhi wild-type, the null ompR mutant and in the ompB mutant complemented with OmpR
wt (pFM2000) or OmpR D55A (pFVD55A). The ompA transcript was included as a loading control.

(a) NB (b)
60 000 NB
β-Galactosidase (U mg–1 protein)

NBS
β-Galactosidase (U mg–1 protein)

50 000 NBS
50 000
NBH NBGlu
40 000
40 000
NBGlu NBGly
30 000
30 000
NBGly
20 000 20 000
NBA
10 000 10 000

0 0
IMSS-1 IMSS-40 IMSS-1 IMSS-40 IMSS-81 IMSS-45 IMSS-85
(wt) (ompR) (wt) (ompR) (ompB) (ackA-pta) (ompB
ackA-pta)

Fig. 4. Environmental conditions and ompS1 regulation. (a) b-Galactosidase activity of the ompS1-lacZ (pRO88) gene reporter fusion
in S. Typhi wild-type (IMSS-1) and a null ompR in low osmolarity (NB), high osmolarity (NBS), oxidative stress (NBH), acid pH (NBA)
and the presence of glucose (NBGlu) or glycerol (NBGly). (b) b-Galactosidase activity of the ompS1-lacZ (pRO88) gene reporter
fusion in S. Typhi wild-type (IMSS-1), ompR, ompB (ompR-envZ), ackA-pta and ompB ackA-pta in low osmolarity (NB), high osmolarity
(NBS) and the presence of glucose (NBGlu) or glycerol (NBGly). The data represent means±SD of three independent experiments.

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M. A. Flores-Valdez and others
dependent on EnvZ indicating a lack of cross-talk or the
involvement of acetyl phosphate (AckA-Pta pathway) (Figs
3 and 4). Therefore, in the absence of EnvZ or in the
presence of OmpR D55A, which results in the absence of
OmpR-P, ompS1 expression was P2-dependent (Fig. 3b).
While for the P1 promoter OmpR-P is a transcriptional
activator, it acts as a repressor for the P2 promoter (Fig.
1b). A repressor effect of OmpR-P has been reported for
flagella and invasin in Escherichia coli and Yersinia entero-
colitica, respectively (Shin & Park, 1995; Brzostek et al.,
2007). The work presented here indicates that the P2
promoter can act as a transcriptional support for P1 where,
in the absence of OmpR-P, S. Typhi uses the second
promoter, in contrast with ompC and ompF that are strictly
OmpR-P-dependent. Presumably, discriminating between
both ompS1 promoters will depend on the response of
Salmonella to the conditions in different ecological niches.
In this manner, glycerol and glucose differentially regulated
ompS1 expression: induction by glycerol and glucose was
dependent and independent of OmpR, respectively (Fig. 4).
These results suggest specific functions for both ompS1
promoters in responding to different environmental signals.
Mechanistic details about how both glucose and glycerol
stimulated ompS1 expression are being studied. OmpR-P, in
addition to regulating the porin repertoire (De la Cruz &
Calva, 2010), activates the expression of Salmonella
pathogenicity island 2 (SPI-2) via ssrAB, conferring
Salmonella enterica with the ability to replicate and survive
within the macrophage (Lee et al., 2000). Hence, ompS1
would have a selective advantage to be expressed under
various environments found in the host. In this respect,
ompS1 has been previously implicated in virulence in the
mouse (Lawley et al., 2006; Rodrı́guez-Morales et al., 2006).
ACKNOWLEDGEMENTS
We thank Dr Linda Kenney for assistance with the fluorescence
anisotropy assays; Dr Ann Stock for anti-OmpR antibody; Dr Vı́ctor H.
Bustamante and Dr José Luis Puente for helpful discussions; Dr Verónica
I. Martinez and Dr Alejandra Vázquez for excellent technical assistance. E.
C. was supported by a grant from the CONACYT, Mexico (No. 179946).
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Edited by: J. Stülke
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