Molecular Microbiology (2014) 91(6), 1057–1069 ■
12500
Regulation of Salmonella enterica pathogenicity island 1
(SPI-1) by the LysR-type regulator LeuO
Elena Espinosa and Josep Casadesús*
Departamento de Genética, Facultad de Biología,
Universidad de Sevilla, Apartado 1095, Sevilla
E-41080, Spain.
Summary
LeuO is a quiescent LysR-type regulator belonging to
the H-NS regulon. Activation of leuO transcription
represses expression of pathogenicity island 1 (SPI-1)
in Salmonella enterica serovar Typhimurium and
inhibits invasion of epithelial cells. Loss of HilE sup-
presses LeuO-mediated downregulation of SPI-1. Acti-
vation of leuO transcription reduces the level of HilD
protein, and loss of HilE restores the wild type HilD
level. Hence, LeuO-mediated downregulation of SPI-1
may involve inhibition of HilD activity by HilE, a view
consistent with the fact that HilE is a HilD inhibitor. In
vivo analyses using β-galactosidase fusions indicate
that LeuO activates hilE transcription. In vitro analyses
by slot blotting, electrophoretic mobility shift analysis
and DNase I footprinting show that LeuO binds the hilE
promoter region. Although residual SPI-1 repression
by LeuO is observed in the absence of HilE, the LeuO-
HilE-HilD ‘pathway’ appears to be the major mecha-
nism. Because both leuO and SPI-1 are repressed by
H-NS, activation of leuO transcription may provide a
backup mechanism for SPI-1 repression under condi-
tions that impair H-NS-mediated silencing.
Introduction
LysR-type transcriptional regulators (LTTRs) are members
of a large family of prokaryotic proteins involved in diverse
cellular functions including transport, response to oxidative
stress, nitrogen fixation, biofilm formation and bacterial
virulence (Henikoff et al., 1988; Maddocks and Oyston,
2008; Momany and Neidle, 2012). LTTRs are typically
300–350 amino acids long and contain an N-terminal
helix–turn–helix DNA binding motif (Maddocks and
Oyston, 2008). Some LTTRs act in combination with
Accepted 18 December, 2013. *For correspondence. E-mail
casadesus@us.es; Tel. (+34) 95542 0881; Fax (+34) 95455 7104.
© 2013 John Wiley & Sons Ltd
doi:10.1111/mmi.
First published online 13 January 2014
ligands that modulate their DNA binding ability upon
binding to a C-terminal domain (Zaim and Kierzek, 2003).
LTTRs can be either activators or repressors of transcrip-
tion (Maddocks and Oyston, 2008).
LeuO is a LTTR originally identified in Salmonella
enterica (Hertzberg et al., 1980; Haughn et al., 1986), and
later found in other genera of gramnegative bacteria
including Escherichia, Shigella, Vibrio and Yersinia
(Maddocks and Oyston, 2008; Stratmann et al., 2008).
Many genes activated by LeuO are repressed by H-NS,
and for this reason LeuO is often viewed as a transcrip-
tional antagonist of H-NS (Chen and Wu, 2005; Chen et al.,
2005; Hernandez-Lucas et al., 2008; Stratmann et al.,
2008; Shimada et al., 2009; 2011; Gallego-Hernandez
et al., 2012) and of its paralogue StpA (De la Cruz et al.,
2007; Stratmann et al., 2012). Antagonism may involve
competition with H-NS for binding to DNA (De la Cruz et al.,
2007; Shimada et al., 2011) or formation of a barrier that
hinders H-NS polymerization (Chen and Wu, 2005; Chen
et al., 2005). In other genes, however, LeuO has been
shown to repress transcription (Shi and Bennett, 1995;
Repoila and Gottesman, 2001). In the last few years,
identification of LeuO binding sites in the genomes of
E. coli and Salmonella has defined DNA motifs bound by
LeuO, and high throughput analysis of gene expression
has increased the number of loci ascribed to the LeuO
regulon (Shimada et al., 2011; Dillon et al., 2012;
Hernandez-Lucas and Calva, 2012).
Expression of the leuO gene is low during standard
laboratory growth as a consequence of H-NS-mediated
repression (Klauck et al., 1997). It is thus conceivable that
activation of LeuO synthesis under specific circumstances
can permit expression of genes that are usually repressed
by H-NS and its paralogues (Dorman, 2007; Hernandez-
Lucas and Calva, 2012). Such circumstances remain
largely unknown, although leuO expression in E. coli is
known to be moderately higher in minimal low phosphate
medium than in LB, and also in stationary phase compared
with exponential growth (Fang et al., 2000; Majumder
et al., 2001).
Examples of bacterial functions under LeuO control
include biofilm formation in Vibrio cholerae (Moorthy and
Watnick, 2005) and E. coli (Shimada et al., 2011), and
1058 E. Espinosa and J. Casadesús ■
synthesis of E. coli fimbriae (Shimada et al., 2011). In
Salmonella enterica serovar Typhi, LeuO activates the
synthesis of quiescent porins OmpS1 and OmpS2
(Fernandez-Mora et al., 2004; Rodriguez-Morales et al.,
2006; De la Cruz et al., 2007; Hernandez-Lucas et al.,
2008). In Salmonella enterica serovar Typhimurium,
OmpS1 and OmpS2 are required for virulence in
the mouse model of typhoid (Lawley et al., 2006;
Rodriguez-Morales et al., 2006). LeuO was also found in a
screen for Salmonella virulence factors using Caenorhab-
ditis elegans as host (Tenor et al., 2004) and in a screen for
Salmonella genes required for long-term infection of mice
(Lawley et al., 2006). Lack of LeuO causes attenuation in
the mouse model of typhoid (Rodriguez-Morales et al.,
2006).
In this study, we show that LeuO downregulates the
expression of a major virulence determinant of Salmo-
nella enterica serovar Typhimurium, the gene cluster
known as Salmonella pathogenicity island 1 (SPI-1)
(Galan and Curtiss, 1989). SPI-1 encodes a type 3
secretion system and secreted effectors that promote
invasion of the intestinal epithelium by interacting with
proteins inside epithelial cells (Galan and Curtiss, 1989).
SPI-1 genes are organized in seven or more transcrip-
tional units under the control of the SPI-1-encoded tran-
scription factors HilA, HilC, HilD and InvF (Lostroh and
Lee, 2001) and of RtsA, a transcription factor encoded
outside SPI-1 (Ellermeier and Slauch, 2003). SPI-1
expression is controlled by additional regulators located
outside the island such as the Fur and BarA/SirA acti-
vators (Fortune et al., 2006; Ellermeier and Slauch,
2008) and the HilE repressor (Fahlen et al., 2000).
Under conditions that permit Salmonella invasion of epi-
thelial cells, SPI-1 expression is bistable (Sturm et al.,
2010; Bailly-Bechet et al., 2011).
Our study was inspired by previous findings indicating
that SPI-1 contains a high density of LeuO binding sites,
and that deletion of leuO in serovar Typhimurium
increases expression of the SPI-1 gene sopA (Dillon
et al., 2012). Indeed, we show that LeuO represses tran-
scription of SPI-1 and inhibits epithelial cell invasion.
However, we provide evidence that LeuO mainly exerts
SPI-1 repression by an indirect mechanism, hitherto
unsuspected: LeuO activates transcription of the hilE
gene, which is located outside SPI-1 and encodes a
SPI-1 repressor (Fahlen et al., 2000; Baxter et al., 2003).
Because LeuO is a quiescent regulator under laboratory
conditions (Hernandez-Lucas and Calva, 2012), the sig-
nificance of LeuO-mediated SPI-1 repression in the Sal-
monella lifestyle remains to be established. However, the
fact that both leuO and SPI-1 are repressed by H-NS
(Klauck et al., 1997; Lucchini et al., 2006; Navarre et al.,
2006) suggests that LeuO might provide a backup
mechanism for SPI-1 repression. Such a mechanism
© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 1057–1069
might be relevant under circumstances that impair SPI-1
silencing by H-NS.
Results
Activation of leuO transcription represses SPI-1
To confirm previous evidence indicating that SPI-1 might
be repressed by LeuO (Dillon et al., 2012), we compared
the expression of selected SPI-1 genes in the presence
and in the absence of LeuO. Activation of leuO transcrip-
tion was achieved by insertion of a T-POP element
upstream of the leuO coding sequence (Fig. S1). Tran-
scription of leuO was activated by addition of autoclaved
chlortetracycline (Rappleye and Roth, 1997; Lee et al.,
2007; Dillon et al., 2012). This experimental design was
chosen for the following reasons: (i) In E. coli, leuO expres-
sion increases in stationary cultures; in S. enterica,
however, increase of leuO expression under such condi-
tions is small (Fig. S2); (ii) We discarded the use of an
Hns− mutant because hns mutations are detrimental in
S. enterica ser. Typhimurium, unless accompanied by an
rpoS mutation as in strain LT2 (Wilmes-Riesenberg et al.,
1997); (iii) The combination of hns and leuO mutations
strongly impairs S. enterica viability, and normal growth
may require the acquisition of suppressor mutations of
unknown nature (data not shown). Use of T-POP to drive
leuO transcription does not cause viability problems, and
has two additional advantages: (i) the transcription rate can
be controlled by using different concentrations of tetracy-
cline or chlortetracycline (Lee et al., 2007); (ii) in the strain
engineered for this study (SV6141), T-POP prevents tran-
scription from the native leuO promoter, thus avoiding a
feedback loop of autogenous activation (Fang and Wu,
1998a,b) that might yield undesirably high levels of LeuO.
Under the conditions employed in this study, the level of
leuO expression was similar to that of an Hns− mutant
(Fig. S3).
Expression of SPI-1 was monitored by measuring the
β-galactosidase activity of lac fusions in six genes: hilA,
hilC, hilD and invF, which encode transcriptional regulators
of SPI-1 (Altier, 2005; Jones, 2005; Ellermeier and Slauch,
2007); invH, which encodes a component of the SPI-1
secretion apparatus (Ellermeier and Slauch, 2007); and
rtsA, a transcriptional regulator of SPI-1 encoded outside
SPI-1 (Ellermeier and Slauch, 2003; 2007; Jones, 2005).
Activation of leuO transcription reduced the expression of
all lac fusions (Fig. 1). As controls, strains carrying the
same lac fusions and a T-POP insertion unlinked to leuO
(zzz:T-POP) were used. In four control strains, the
β-galactosidase activities were similar to that of the wild
type, in one strain was slightly higher, and in another strain
was lower (Fig. S4). This experiment ruled out the possi-
bility of an artefact caused by either T-POP or chlortetra-
cycline. We thus concluded that LeuO does repress SPI-1.

LeuO downregulates SPI-1 expression via hilE and hilD
Because SPI-1 expression is responsive to multiple
regulators (Ellermeier and Slauch, 2007), we devised a
genetic screen to ascertain whether a single cell function
might transmit LeuO-mediated regulation to SPI-1. For
this purpose, Tn10dCm mutagenesis was performed in
a strain that carried the T-POP leuO construct and a
hilC::lac translational fusion (SV6844). When leuO tran-
scription is activated, strain SV6844 is Lac− and forms
white colonies on plates containing Xgal. In the screen,
Lac+ (blue) colonies were sought among the Cmr isolates
generated by Tn10Cm insertion. One such isolate was
purified and re-constructed by P22 HT transduction to
confirm that the Tn10dCm insertion suppressed hilC::lac
downregulation by LeuO. The isolate was propagated as
strain SV7036.
Amplification by semi-random PCR and sequencing of
the Tn10dCm boundaries indicated that the Tn10dCm
element of SV7036 had disrupted the hilE gene. Use of a
HilE− null mutant constructed ad hoc (strain SV5586) pro-
vided independent evidence that lack of HilE suppressed
hilC::lac downregulation by LeuO.
Single cell analysis of gene expression by flow cytometry
(Fig. 2) confirmed that HilE plays a role in LeuO-mediated
repression of SPI-1: (i) activation of leuO expression
decreased the activity of a sipB::GFP fusion, abolishing
bistable SPI-1 expression; (ii) lack of LeuO restored the
wild type pattern of sipB::GFP expression, indicating that
SPI-1 downregulation was caused by LeuO indeed; (iii) a
hilE null mutation increased sipB::GFP expression and
reduced the size of the SPI-1(OFF) subpopulation, in
agreement with the role of HilE as a SPI-1 repressor
(Baxter et al., 2003); (iv) activation of leuO expression in a
HilE− background yielded a small subpopulation of sipB-
::GFP(OFF) cells; and (v) absence of both LeuO and HilE
restored the wild type pattern of sipB::GFP expression.
Altogether, these observations suggest that activation of
LeuO expression downregulates SPI-1 by both HilE-
© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 1057–1069
Regulation of SPI-1 by LeuO 1059
Fig. 1. Regulation of SPI-1 expression by
LeuO. β-Galactosidase activity of hilD::lac,
hilC::lac, invF::lac, rtsA::lac, hilA::lac and
invH::lac fusions in the presence and in the
absence of LeuO. Data are averages and
standard deviations from > 3 independent
experiments.
dependent and HilE-independent mechanisms. However,
activation of leuO transcription in a HilE+ background yields
a homogeneous population of (SPI-1)OFF cells while acti-
vation of leuO transcription in the absence of HilE yields
a small subpopulation of SPI-1(OFF) cells. Hence,
HilE-dependent downregulation seems to be the major
‘pathway’ of SPI-1 repression by LeuO.
Because HilE is a negative regulator of hilD (Baxter
et al., 2003), a tentative interpretation for the occurrence of
HilE-dependent SPI-1 repression was that LeuO might
increase the HilE level, which in turn might inhibit HilD
activity. Western blot analysis revealed that activation of
leuO transcription decreased the HilD level, and the
decrease was suppressed in a ΔleuO background (Fig. 3).
We also observed that a hilE null mutation increased the
HilD levels in the presence and in the absence of LeuO
(Fig. 3), in accordance with two well known facts: the
inhibition of HilD activity by HilE (Baxter et al., 2003) and
the occurrence of autogenous activation of hilD transcrip-
tion (Ellermeier et al., 2005). These experiments support
the view that downregulation of SPI-1 by LeuO may involve
HilE-mediated inhibition of HilD activity.
The involvement of HilD in LeuO-mediated SPI-1 repres-
sion was further investigated by epistasis analysis. This
kind of analysis takes advantage of two well known traits of
SPI-1 expression. One is redundancy of certain transcrip-
tion factors involved in SPI-1 control (Altier, 2005; Jones,
2005; Ellermeier and Slauch, 2007). Another is that lack of
a single transcription factor does not completely abolish
expression of certain SPI-1 transcriptional units (Ellermeier
et al., 2005). We thus designed experiments to test the
effect of a hilD null mutation on the expression of rtsA::lac
and hilC::lac fusions in the presence and in the absence of
LeuO. If HilD was required for SPI-1 repression by LeuO,
we reasoned, downregulation of SPI-1 by LeuO should not
be observed in the absence of HilD. Results shown in
Fig. 4 did not completely fulfil this prediction: moderate
downregulation of SPI-1 by LeuO was observed in a HilD−
background. Furthermore, even though a hilD mutation
1060 E. Espinosa and J. Casadesús ■

Fig. 2. Flow cytometry analysis of SPI-1 expression. Effect of a hilE null mutation on sipB::GFP expression in the presence and in the
absence of LeuO (left column). Data were collected for 40 000 events per sample. In the histograms presented, the cell numbers have been
normalized to 100. Data are also represented by a dot plot (right column).

© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 1057–1069

Fig. 3. Effect of a hilE mutation on the levels of HilD protein in the
presence and in the absence of LeuO. DnaK was used as loading
control in Western blots.
was epistatic over hilE, moderate repression of SPI-1
by LeuO was still observed in a HilD− HilE− background
(Fig. 4). Hence, LeuO seems to repress SPI-1 by a major
‘pathway’ involving HilD and HilE, and also by minor, HilD-
and HilE-independent mechanisms. This conclusion is
coherent with the detection of a subpopulation of SPI-
1(OFF) cells when LeuO expression was activated in a
HilE− background (Fig. 2).
LeuO activates hilE transcription
A tentative model for LeuO-mediated downregulation of
SPI-1 via HilE and HilD is that LeuO may activate hilE
expression, and that HilE-mediated inhibition of HilD
activity (Baxter et al., 2003) may contribute to SPI-1
downregulation. To test whether LeuO is an activator of
hilE expression, a hilE::lacZ translational fusion was con-
structed on the Salmonella chromosome. Comparison of
β-galactosidase activities in the presence and in the
absence of LeuO (strains SV7327 and SV7328) indicated
that hilE expression is activated by LeuO (Fig. 5A).
Transcription of hilE is known to be driven by three
promoters (Lim et al., 2007). To identify the hilE promot-
er(s) under LeuO control, the P1, P2 and P3 promoters
were cloned on the promoter-probe vector pIC552 (Macian
et al., 1994) to generate transcriptional lac fusions. A con-
struct contained the three promoters (pIZ1997), and the
© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 1057–1069
Regulation of SPI-1 by LeuO 1061
others contained individual promoters P1 (pIZ1998), P2
(pIZ1999) and P3 (pIZ2000). Diagrams of the constructs
are shown in Fig. 5B. Measurements of β-galactosidase
activities of the plasmid-borne lac fusions showed that
LeuO regulates expression of the P1, P2 and P3 hilE
promoters in an independent manner (Fig. 4C). However,
LeuO-dependent regulation was found to be stronger
when the three promoters were present (Fig. 5C).
Binding of LeuO to the hilE promoter region
To test whether LeuO is able to bind the hilE promoter
region, a slot blot binding assay was performed. A 449 bp
DNA fragment containing the P1, P2 and P3 hilE promot-
ers was incubated with increasing concentrations of LeuO
protein. Binding was unambiguously detected (Fig. 6A).
Quantitative analysis of binding (Fig. 6B) indicated that,
under the conditions of the assay, LeuO bound the hilE
DNA fragment with an approximate Kd of 0.37 μM. As a
negative control, a binding assay with the rtsA promoter
was performed, and LeuO was unable to bind the DNA
fragment (data not shown).
Binding of LeuO to the hilE promoter region was also
tested by electrophoretic mobility shift assays. DNA frag-
ments that contained the entire promoter region or the
individual P1, P2 and P3 promoters were used. LeuO
binding was observed upstream of P2 and P3 but not
upstream of P1 (Fig. 6C). This observation is consistent
with two facts: (i) the weak level of P1-driven hilE tran-
scription detected with the promoter-probe plasmid
(Fig. 5C); (ii) the existence of putative LeuO binding sites
upstream of P2 and P3 but not upstream of P1 (Fig. S5).
Further evidence of LeuO binding to the hilE promoter
region was obtained by DNA footprinting using the cus-
tomary 449 bp DNA fragment as target. Protection from
DNase I digestion was observed in the presence of LeuO
(Fig. 6D). Protection was stronger at a region that over-
laps the P3 promoter. Because LTTRs can act at a dis-
tance (Maddocks and Oyston, 2008; Momany and Neidle,
2012), LeuO binding at the P3 region may be sufficient to
permit regulation of the downstream promoter P2 (and
Fig. 4. Epistasis analysis of SPI-1
expression. Effect of LeuO expression on the
β-galactosidase activity of rtsA::lac and
hilC::lac translational fusions in HilD− and
HilD− HilE− backgrounds. Data are averages
and standard deviations from > 3 independent
experiments.
1062 E. Espinosa and J. Casadesús ■
perhaps P1). It is also possible that lower affinity binding
to P2 may boost transcription.
Activation of leuO transcription inhibits
epithelial cell invasion
During infection of animals, Salmonella Typhimurium
invades epithelial cells using the SPI-1 type 3 secretion
system. The observation that LeuO downregulates SPI
expression thus raised the question of whether activation
of hilE transcription by LeuO might inhibit epithelial cell
invasion. To test this possibility, assays of epithelial cell
invasion were performed in vitro, and the invasion rates of
T-POP leuO and T-POP ΔleuO strains were compared. As
a control, a SPI-1 deletion mutant was included in the
assays. Activation of leuO transcription decreased inva-
sion > 100-fold, and lack of LeuO restored the wild type
invasion rate (Fig. 7A). A HilE− mutant was more invasive
than the wild type (Fig. 7B), an observation coherent with
the role of HilE as a negative regulator of SPI-1 (Fahlen
et al., 2000; Baxter et al., 2003). In the absence of HilE,
activation of leuO transcription reduced epithelial cell inva-
sion three- to fourfold (Fig. 7B), thereby providing further
evidence for HilE-independent downregulation of SPI-1 by
LeuO. We thus conclude that LeuO inhibits invasion of
epithelial cells by Salmonella enterica serovar Typhimu-
rium, and that the main inhibition ‘pathway’ requires HilE.
These conclusions are in agreement with the ability of
LeuO to activate hilE transcription (Fig. 5).
© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 1057–1069
Fig. 5. Regulation of hilE expression by
LeuO.
A. β-Galactosidase activity of an hilE::lac
translational fusion in the presence and in the
absence of LeuO.
B. Diagram of the hilE promoter region, drawn
using information from (Lim et al., 2007). The
DNA fragments cloned on pIC552 are
represented below. The −505 to −56 fragment
contains the three hilE promoters (plasmid
pIZ1997). The −159 to −56 fragment contains
the P1 promoter (pIZ1998). The −334 to −161
fragment contains the P2 promoter (pIZ1999).
The −505 to −336 fragment contains the P3
promoter (pIZ2000).
C. β-Galactosidase activities of pIZ1997,
pIZ1998, pIZ1999 and pIZ2000 in the
presence and in the absence of LeuO. The
pIC552 vector was included as control. Data
are averages and standard deviations from
> 3 independent experiments.
Discussion
The existence of LeuO has been known for more than two
decades (Hertzberg et al., 1980), and its recognition as a
member of the LysR family was published fifteen years ago
(Henikoff et al., 1988). Studies in a variety of gramnegative
bacteria have shown that LeuO regulates transcription of
genes involved in diverse physiological processes, either
as an activator or as a repressor (Shi and Bennett, 1995;
Repoila and Gottesman, 2001; Chen and Wu, 2005;
Stratmann et al., 2008; 2012; Shimada et al., 2009; 2011;
Dillon et al., 2012; Hernandez-Lucas and Calva, 2012). In
serovars Typhi and Typhimurium of Salmonella enterica
LeuO has been shown to regulate virulence-related
genes (Fernandez-Mora et al., 2004; Tenor et al., 2004;
Rodriguez-Morales et al., 2006; Hernandez-Lucas et al.,
2008). Given these antecedents, the identification of LeuO
binding sites in Salmonella pathogenicity island 1 (Dillon
et al., 2012), combined with the observation that deletion of
leuO increased expression of the SPI-1 gene sopA (Dillon
et al., 2012), suggested that LeuO might regulate SPI-1, a
major determinant of Salmonella virulence (Galan and
Curtiss, 1989). Multiple controls adjust SPI-1 expression
to conditions that permit invasion of epithelial cells
(Altier, 2005; Jones, 2005; Ellermeier and Slauch, 2007).
Because activation of SPI-1 expression requires relief from
H-NS-mediated silencing (Lucchini et al., 2006; Navarre
et al., 2006) and LeuO acts often as an H-NS antagonist
(Hernandez-Lucas et al., 2008; Shimada et al., 2009;
 Regulation of SPI-1 by LeuO 1063

Fig. 6. Binding of LeuO-His6x to the hilE promoter region.

A. Slot blot binding assay. A 449 bp DNA fragment containing the P1, P2 and P3 hilE promoters was incubated with increasing concentrations
of LeuO-His6x.
B. Quantification of the binding assay. The fluorescence of the DNA bound to the membrane was represented versus the concentration of
LeuO-His6x protein.
C. Electrophoretic mobility shift assays of LeuOxHis6x binding to the hilE promoter region. PhilE designates a DNA fragment containing the
entire hilE promoter region, while P1, P2 and P3 designate DNA fragments containing individual hilE promoters. A DNA fragment containing
the S. enterica gene STM4318 was used as control. The concentrations of LeuO-His6x are indicated above each lane.
D. DNase I footprinting of LeuOxHis6x binding to the hilE promoter region using end-labelled linear DNA fragments. The size and quantity of
6-FAM-labelled digestion products were measured using a capillary electrophoresis DNA sequencing instrument.

Fig. 7. Inhibition of S. enterica invasion by LeuO.

A. Invasion of HeLa cells by the wild type (WT), a strain carrying a SPI-1 deletion (ΔSPI-1), a strain with leuO transcription driven by T-POP
(T-POP-leuO), and a leuO deletion mutant (T-POP ΔleuO).
B. Invasion of HeLa cells by the wild type (WT), a strain carrying a SPI-1 deletion (ΔSPI-1), a HilE− null mutant, (ΔhilE), a HilE− null mutant
with leuO transcription driven by T-POP (T-POP-leuO ΔhilE), and a strain lacking both LeuO and HilE (T-POP ΔleuO ΔhilE). The invasion rate
of the wild type was 0.1–0.3%, and was normalized to 1. Data are averages and standard deviations from > 6 independent experiments.
Because of the disparate invasion rates of the strains under study, different scales are used in each graph.

© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 1057–1069
1064 E. Espinosa and J. Casadesús ■
2011; Stratmann et al., 2012), the possibility that LeuO
might repress SPI-1 was intriguing.
LeuO is a quiescent LTTR under standard laboratory
conditions because leuO transcription is repressed by
H-NS (Klauck et al., 1997). To study LeuO-dependent
regulation of SPI-1 in S. enterica serovar Typhimurium,
transcription of the leuO gene was freed from H-NS
repression by introducing a T-POP element upstream of
leuO (Fig. S2). Activation of leuO transcription was found
to downregulate genes belonging to independent tran-
scriptional units within SPI-1, as well as the SPI-1-
controlled rtsA gene located outside SPI-1 (Fig. 1). These
experiments confirmed that LeuO represses SPI-1
expression. A consequence of SPI-1 repression by LeuO
is reduced invasion of epithelial cells in vitro (Fig. 7).
A genetic screen for loss-of-function mutations that
restored SPI-1 expression in the presence of LeuO pro-
vided evidence that the hilE gene is necessary for LeuO-
mediated repression of SPI-1, an hypothesis confirmed
upon directed construction of a HilE− mutant. The hilE
gene is located outside SPI-1, and encodes a repressor of
SPI-1 expression (Baxter et al., 2003). HilE inactivates
the transcriptional activator HilD (Baxter et al., 2003).
HilD inactivation disrupts a positive feedback loop for
hilD autogenous activation and causes SPI-1 repression
(Ellermeier et al., 2005). We thus considered that LeuO
might activate hilE transcription, and that HilE might
repress SPI-1 via HilD inactivation.
The existence of a LeuO-HilE-HilD ‘pathway’ of SPI-1
repression in S. enterica serovar Typhimurium is sup-
ported by several lines of evidence: (i) lack of HilE relieves
LeuO-mediated repression of SPI-1 (Fig. 2); (ii) activation
of leuO transcription decreases the level of HilD protein
(Fig. 3); (iii) the HilD protein decrease caused by LeuO is
suppressed by a hilE mutation (Fig. 3); and (iv) LeuO
activates transcription of hilE (Fig. 5).
In the absence of HilE, however, LeuO remains able to
downregulate expression of SPI-1 (Fig. 4) and to reduce
epithelial cell invasion (Fig. 7). HilD is likewise dispensa-
ble for HilE-independent downregulation of SPI-1 by
LeuO (Fig. 4). However, HilE-independent SPI-1 repres-
sion appears to be weak in comparison with the HilE-
dependent ‘pathway’ (Figs 2 and 4). This conclusion is in
agreement with the observation that activation of leuO
transcription decreases epithelial cell invasion > 100-fold
in the presence of HilE and only three- to fourfold in the
absence of HilE (Fig. 7).
Our ignorance of natural conditions that permit leuO
expression advises against interpretation of the physiologi-
cal significance of SPI-1 regulation by LeuO. However, a
conceivable scenario is that LeuO might either backup
or relieve H-NS repression of certain loci, in a fashion
reminiscent of the HN-S-repressed VirT-VirB regulatory
cascade in Shigella (Tobe et al., 1993). Because HilE is a
© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 1057–1069
SPI-1 repressor, a tentative model is that activation of hilE
transcription by LeuO might boost SPI-1 repression,
perhaps under conditions in which H-NS fails to do so.
Such hypothetical conditions can be expected to activate
leuO expression because leuO transcription is also
repressed by H-NS (Klauck et al., 1997). The existence of
a subordinate machinery to secure SPI-1 silencing might
have selective value because SPI-1 expression causes
growth retardation (Sturm et al., 2010). Tight repression of
SPI-1 might have also selective value in environments
different from the animal intestine. For instance, coloniza-
tion of plants by Salmonella is more efficient in the absence
of SPI-1 components, perhaps because the presence of
the SPI-1 secretion apparatus in the Salmonella envelope
triggers a defence response by the plant (Iniguez et al.,
2005).
The view that LeuO may back up or replace H-NS to
silence SPI-1 is consistent with the existence of overlap-
ping controls that contribute to silencing of horizontally
acquired genes. For instance, the E. coli nucleoid-
associated proteins Hha and YdgT enhance silencing of
foreign genes by H-NS (Vivero et al., 2008; Banos et al.,
2009). Interestingly, YdgT and Hha appear to be redun-
dant, and YdgT has been proposed to act as a backup
molecule (Paytubi et al., 2004). The intricacy of accommo-
dating horizontally acquired genes in the host regulatory
network (Ochman et al., 2000; Lercher and Pal, 2008;
Price et al., 2008) may confer adaptive value to redundant
control.
Experimental procedures
Bacterial strains, media and culture conditions
Strains of S. enterica serovar Typhimurium (henceforth abbre-
viated as S. enterica) used in this study are listed in Table 1,
and derive from ATCC 14028. An exception is SV6829 which
derives from SL1344. Strain SV6413 carries a hilD::lac
transcriptional fusion downstream from the hilD stop codon,
constructed in such a way that the strain remains HilD+
(Lopez-Garrido and Casadesus, 2012). The remaining chro-
mosomal lac fusions are translational. E. coli DH5∝ [endA1
hsdR17 supE44 thi1 recA1 gyrA96 relA1 ΔlacU189 (Φ80
lacZΔM15)] (Hanahan, 1983) was used as the host of plas-
mids. E. coli BL21 [F− dcm ompT hsdS(rB− mB−) gal [malB+]K-
12(λS)] is a product of Stratagene, La Jolla, CA.
Transductional crosses using phage P22 HT 105/1 int201
(Schmieger, 1972) were used for S. enterica strain construc-
tion operations involving chromosomal markers, and for
transfer of small plasmids between S. enterica strains. The
transduction protocol has been described elsewhere (Garzon
et al., 1995). To obtain phage-free isolates, transductants
were purified by streaking on green plates (Chan et al., 1972),
using methyl blue (Sigma-Aldrich, St Louis, MO) instead of
aniline blue. Phage sensitivity was tested by cross-streaking
with the clear-plaque mutant P22 H5. Strain SV6141 was
constructed by transducing the T-POP insertion of SV7424
 Regulation of SPI-1 by LeuO 1065

Table 1. Strain list. (Dillon et al., 2012) to ATCC 14208. Tagging of proteins with 3×
FLAG or HA epitopes (strains SV6929 and SV7313) was
Strain Genotype performed as described by Lionello Bossi and co-workers
(Uzzau et al., 2001). Luria-Bertani (LB) broth was used as
SV5282a hilA::lacZ
standard liquid medium. Solid LB contained agar at 1.5%.
SV5295a invF::lacZ
SV5301a invH::lacZ Antibiotics were used at the concentrations described previ-
SV5384a hilC::lacZ ously (Torreblanca et al., 1999). The indicator for monitoring
SV5386a ΔhilD hilC::lacZ β-galactosidase activity in plate tests was 5-bromo-4-chloro-
SV5546 ΔhilD rtsA::lacZ 3-indolyl-β-D-galactopyranoside (‘Xgal’, Sigma Chemical Co.,
SV5584a rtsA::lacZ 40 μg ml−1). To activate leuO transcription, chlortetracycline
SV5586 hilE::Km
(5 μg ml−1) was added to the culture.
SV6055b SPI-1::Km
SV6141 T-POP-leuO
SV6142 T-POP-ΔleuO Construction of a T-POP ΔleuO mutant (SV6142)
SV6413e hilD930::lacZ
SV6829c leuO::3xFLAG Disruption and replacement of leuO with a chloramphenicol
SV6841 T-POP-leuO hilA::lacZ
resistance cassette was performed by lambda Red recom-
SV6842 T-POP-ΔleuO hilA::lacZ
SV6844 T-POP-leuO hilC::lacZ bineering (Datsenko and Wanner, 2000). Briefly, the Cmr
SV6845 T-POP-ΔleuO hilC::lacZ resistance cassette from plasmid pKD3 was PCR-amplified
SV6847 T-POP-leuO invF::lacZ with primers leuOP1 and leuOP2 (Table S1). The PCR
SV6848 T-POP-ΔleuO invF::lacZ product was used to transform an ATCC 14028 derivative
SV6850 T-POP-leuO hilD930::lacZ carrying the Red recombinase plasmid pKD46. Transfor-
SV6851 T-POP-ΔleuO hilD930::lacZ
mants were selected on LB with chloramphenicol.
SV6853 T-POP-leuO rtsA::lacZ
SV6854 T-POP-ΔleuO rtsA::lacZ
SV7034 T-POP-leuO invH::lacZ Construction of a sipB::GFP fusion (strain SV7975)
SV7035 T-POP-ΔleuO invH::lacZ
SV7036 T-pop leuO hilE::Tnd10dCm hilC::lacZ A fragment containing the promoterless green fluorescent
SV7037 T-POP-leuO ΔhilE protein (gfp) and the chloramphenicol resistance cassette
SV7038 T-POP-ΔleuO ΔhilE
SV7044 T-POP-leuO ΔhilD hilC::lacZ
was amplified from the pZEP07 plasmid using primers sipB-
SV7045 T-POP-ΔleuO ΔhilD hilC::lacZ GFP+-For and sipB-GFP+-Rev (Table S1). The 5′ regions of
SV7048 T-POP-leuO ΔhilD rtsA::lacZ these primers are homologous to the 3′ untranslated region of
SV7049 T-POP-ΔleuO ΔhilD rtsA::lacZ the S. enterica sipB gene, so that the fusion is formed down-
SV7312 T-POP-leuO hilD::HA stream of the sipB stop codon. The construct was integrated
SV7313 T-POP-ΔleuO hilD::HA into the chromosome of S. enterica using the Lambda Red
SV7315 T-POP-leuO ΔhilE hilD::HA
SV7316 T-POP-ΔleuO ΔhilE hilD::HA
recombination system.
SV7317 ΔhilD ΔhilE rtsA::lacZ
SV7318 T-POP-leuO ΔhilD ΔhilE rtsA::lacZ Construction of a hilE mutant (SV5586)
SV7319 T-POP-ΔleuO ΔhilD ΔhilE rtsA::lacZ
SV7320 ΔhilD ΔhilE hilC::lacZ Disruption and replacement of hilE with a Kmr cassette was
SV7321 T-POP-leuO ΔhilD ΔhilE hilC::lacZ
SV7322 T-POP-ΔleuO ΔhilD ΔhilE hilC::lacZ
performed by lambda Red recombineering (Datsenko and
SV7327 T-POP-leuO hilE::lacZ Wanner, 2000). The Kmr resistance cassette of plasmid
SV7328 T-POP-ΔleuO hilE::lacZ pKD13 was PCR-amplified with primers hilEPS1 and hilEPS4
SV7741 T-POP leuO/pIC552 (Table S1). The PCR product was used to transform the
SV7742 T-POP ΔleuO/pIC552 wild-type strain carrying pKD46. Transformants were
SV7743 T-POP leuO/pIZ1997 selected on LB plates with kanamycin. The antibiotic resist-
SV7744 T-POP ΔleuO/pIZ1997
SV7745 T-POP leuO/pIZ1998
ance casette was excised by recombination with plasmid
SV7746 T-POP ΔleuO/pIZ1998 pCP20 (Datsenko and Wanner, 2000). For the construction of
SV7747 T-POP leuO/pIZ1999 a translational hilE::lac fusion on the Salmonella chromo-
SV7748 T-POP ΔleuO/pIZ1999 some (strain SV7327), FRT sites generated by excision of
SV7749 T-POP leuO/pIZ2000 Kmr cassette were used to integrate plasmid pCE40
SV7750 T-POP ΔleuO/pIZ2000 (Ellermeier et al., 2002).
SV7975d sipB::GFP
SV7976 ΔhilE sipB::GFP
SV7977 T-POP leuO sipB::GFP Screen for suppressors of SPI-1 downregulation in a
SV7978 T-POP ΔleuO sipB::GFP
LeuO-constitutive strain
SV7979 ΔhilE T-POP leuO sipB::GFP
SV7980 ΔhilE T-POP ΔleuO sipB::GFP
Strain SV6842 (hilC::lac T-POP-leuO) was transduced with a
a. Constructed by J. López-Garrido. Tn10dCm pool, and transductants were selected on LB
b. Constructed by F. Baisón-Olmo. plates supplemented with chloramphenicol and Xgal. Inde-
c. SL1344 derivative. pendent Lac+ transductants were sought and purified on
d. Constructed by I. Cota and S. B. Hernández. green plates. To characterize Tn10dCm insertions, a semi-
e. Strain described by (Lopez-Garrido and Casadesus, 2012). random, two step PCR protocol (Chun et al., 1997) was used
to amplify genomic regions adjacent to the Tn10dCm

© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 1057–1069
1066 E. Espinosa and J. Casadesús ■
element. The reactions were carried out as described else-
where (Baison-Olmo et al., 2012), using primers listed in
Table S1. Amplification products were sequenced using
primers ST-PCR-Cm2-Int and st1. The Tn10dCm insertion
had occurred at a TGCT/GCCA motif in the hilE coding
sequence.
Plasmids
Plasmid pZEP07 (Hautefort et al., 2003) was used to
construct a sipB::GFP fusion. Plasmid pIZ1871 is a pET21a
derivative (Novagen EMD4 Biosciences, Darmstadt,
Germany) containing leuO-His6x (Dillon et al., 2012). Plas-
mids carrying transcriptional lacZ fusions are derivatives of
the promoter-probe vector pIC552 (Macian et al., 1994). To
construct plasmid-borne lac fusions on pIC552, DNA from
strain ATCC 14028 was used as template for PCR amplifica-
tion with primers listed in Table S1. The amplified fragments
were digested with BglII and XhoI and ligated with BglII/XhoI-
digested pIC552. The DNA fragments from the hilE promoter
region carried by pIC552 derivatives are as follows: pIZ1997,
−505 to −56; pIZ1998, −159 to −56; pIZ1999, −334 to −161;
and pIZ2000, −505 to −336.
β-Galactosidase assays
Bacterial cultures were grown in LB + chlortetracycline until
stationary phase (OD600 ≤ 2). Levels of β-galactosidase activ-
ity were assayed using the CHCl3-sodium dodecyl sulphate
permeabilization procedure (Miller, 1972).
Western immunoblot assays
Whole cell protein extracts were prepared from bacterial cul-
tures in LB broth containing chlortetracycline. Cultures were
grown at 37°C until stationary phase (OD600 ∼ 2). Bacterial
cells contained in 1 ml of culture were collected by centrifu-
gation (16 000 g, 2 min, 4°C) and suspended in 50 μl of
Laemmli sample buffer [1.3% sodium dodecyl sulphate, 10%
(v/v) glycerol, 50 mM tris(hydroxymethyl)aminomethane-HCl
(Tris-HCl), 1.8% β-mercaptoethanol, 0.02% bromophenol
blue, pH 6.8]. Proteins were resolved by Tris-Tricine-PAGE
using 12% gels. Conditions for protein transfer have been
described elsewhere (Balbontin et al., 2006). Primary anti-
bodies were anti-FLAG M2 monoclonal antibody (1:5000,
Sigma Chemical Co, St Louis, MO) and anti-DnaK monoclo-
nal antibody (1:5000, MBL International, MA). Goat anti-
mouse horseradish peroxidase-conjugated antibody (1:5000,
Bio-Rad, Hercules, CA) was used as secondary antibody.
Proteins recognized by the antibodies were visualized by
chemiluminiscence using luciferin–luminol.
Flow cytometry
Bacterial cultures were grown at 37°C in LB + chlortetracy-
cline until stationary phase (OD600 ∼ 2). Cells were washed
and re-suspended in PBS to a final concentration of
5 × 106 cells ml−1. Data acquisition and analysis were per-
formed using a Cytomics FC500-MPL cytometer (Beckman
Coulter, Brea, CA). Approximately 5 × 106 cells were ana-
lysed for GFP expression. Data were collected for 40 000
© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 1057–1069
events per sample, and were analysed with CXP and FlowJo
8.7 Software.
Quantitative reverse transcriptase PCR
RNA was extracted from stationary phase cultures
(OD600 ∼ 2). Cells were centrifuged at 13 000 rpm at 4°C and
resuspended in 100 μl of lysozyme (Sigma Chemical)
(3 mg ml−1). RNA was extracted using Trizol (Invitrogen,
Carlsbad, CA), according to manufacturer’s instructions. The
quality and quantity of the extracted RNA was determined
using a ND-1000 spectrophotometer (Nanodrop Technolo-
gies, Wilmington, DE). To diminish genomic DNA contamina-
tion and to obtain cDNA, 1 μg RNA was treated with the
Quantitec Reverse Transcription Kit (Quiagen, Venlo, The
Netherlands). Quantitative RT-PCR reactions were per-
formed in LightCycler 480 II (Roche). Each reaction was
carried out in a total volume of 10 μl on a 480-well optical
reaction plate (Roche, Basel, Switzerland) containing 5 μl
SYBR, 0.5 DYE II (Takara Bio, Otsu, Japan), 4 μl cDNA (1/50)
and two gene-specific primers at a final concentration of
0.2 mM each. Real-time cycling conditions were as follows:
(i) 95°C for 10 min; (ii) 40 cycles at 95°C for 15 s, 60°C for
1 min. A non-template control was included for each primer
set. Melting curve analysis verified that each reaction con-
tained a single PCR product. Gene expression levels were
normalized to transcripts of gmk that served as an internal
control. Gene-specific primers were designed with PRIMER3
software (http://primer3.sourceforge.net) and are listed in
Table S1.
Purification of LeuO protein
For LeuO purification, plasmid pIZ1871 was used (Dillon et al.,
2012). E. coli BL21/pIZ1871 was grown in LB broth, and
expression of leuO was induced with 1 mM isopropylβ-D-thio-
galactopyranoside (IPTG). After 4 h induction, cells were cen-
trifuged and resuspended in lysis buffer [20 mM Tris, 300 mM
NaCl, 10 mM imidazole, 1 mM phenylmethylsulphonyl fluoride
(PMSF), 5 μl ml−1 protein inhibitor cocktail (Sigma-Aldrich, St
Louis, MO)], and lysed by sonication. The suspension was
centrifuged at 10 000 rpm for 30 min. LeuO-His6x was purified
by nickel affinity chromatography (GE Healthcare). The
column was washed once with 5 ml of washing buffer (20 mM
Tris, 300 mM NaCl, 50 mM imidazole), and twice with the
same buffer containing 75 mM and 100 mM of imidazole.
Protein elution was performed with 4 ml of elution buffer
(20 mM Tris, 300 mM NaCl, 300 mM imidazole). Imidazole
was removed and the protein was concentrated by washing
with storage buffer (20 mM Tris, 300 mM NaCl, 10% glycerol)
and centrifuged using Amicon® Ultra centrifugal filters. LeuO-
His6x protein was stored at −80°C.
Slot blot and gel mobility shift assays of LeuO binding
DNA probes labelled with 6-caroxyfluorescein (FAM) were
prepared by PCR amplification using primer pairs listed in
Table S1. PCR products were purified with the Wizard® SV
Clean-Up-System (Promega, Madison, WI). Ten nanograms
of FAM-labelled probe were used for slot blot assays, and
25 ng for electrophoretic gel mobility shift assays. In both

types of experiments, the FAM-labelled probes were incu-
bated at room temperature for 30 min with increasing con-
centrations of LeuO-6xHis in a final volume of 20 μl. The binding
buffer L10× contained 20 mM HEPES, 100 mM KCl, 2 mM
MgCl2, 0,1 mM EDTA and 20% glycerol (De la Cruz et al.,
2007). For slot blot binding assays, protein-DNA complexes
were diluted 1:10 with PBS and blotted onto nitrocellulose
filters (AmershamTM HybondTM-ECL, GE Healthcare) using a
PR 600 Slot Blot Manifold (Hoefer Scientific Instruments, San
Francisco, CA) connected to a portable vacuum/pressure
pump (Merck Millipore, Darmstadt, Germany). Wells were
then washed five times with PBS and membranes were air-
dried. DNA fragments were visualized with a FLA-5100
Imaging system (Fujifilm, Tokyo, Japan). For gel shift assays,
protein-DNA complexes were subjected to electrophoresis at
4°C in a 6% non-denaturing acrylamide : bisacrylamide
(29:1) gel in 0.5 Tris-borate-EDTA buffer.
DNase I footprinting of LeuO binding to
PCR-amplified DNA
A PhilE DNA probe labelled with 6-caroxyfluorescein (FAM)
was prepared by PCR amplification using primers listed in
Table S1. DNase I footprinting was performed as described
elsewhere (Cameron and Dorman, 2012) with minor modifi-
cations. DNase I footprinting reactions were performed in
15 μl reaction volumes containing 1× LeuO binding buffer
(2 mM HEPES, 10 mM KCl, 0.2 mM MgCl2, 0.01 mM EDTA
and 2% glycerol), 0.01 mM DTT, 100 ng μl−1 bovine serum
albumin (BSA), 50 nM bait DNA, and 4 μM LeuO-His6x. LeuO-
DNA binding was allowed to equilibrate at room temperature
for 30 min. One μl (0.05 units) of DNase I (Roche) was then
added, mixed gently, and incubated at room temperature for
5 min. Reactions were stopped by addition of 2 μl EDTA
(100 mM) followed by vigorous vortexing and heat denatura-
tion at 95°C for 10 min. Digestion products were desalted
using MicroSpin G-25 columns (GE Healthcare, Sevilla,
Spain), and were analysed on an ABI 3730 DNA Analyzer
along with GeneScan 500-LIZ size standards (Applied Bio-
systems, Alcobendas, Spain).
Invasion assays in HeLa epithelial cells
HeLa human epithelial cells (ATCC CCL2) were grown in
Dulbecco’s modified Eagle medium (DMEM) containing 10%
fetal calf serum and 1 mM glutamine (Life Technologies, Prat
de Llobregat, Spain). HeLa cells were seeded in 24-well
plates (Costar, Corning, New York, NY) the day before infec-
tion, and were grown at 37°C with 5% CO2. Bacteria were
grown overnight in LB broth containing NaCl 0.3 M and chlo-
rtetracycline 5 μg ml−1 at 37°C without shaking. An aliquot
from the bacterial culture was added to HeLa cells to reach a
multiplicity of infection of 50 bacteria per eukaryotic cell.
Bacterial dilutions were prepared in DMEM. The colony-
forming units of the strains in the input were enumerated by
plating a dilution series of the inoculum, using the appropriate
antibiotic to distinguish the strains. Thirty minutes after inva-
sion, the cells were washed twice with phosphate-buffered
saline (PBS) and incubated for 90 min in fresh DMEM con-
taining 100 μg ml−1 gentamicin. Numbers of viable intracellu-
© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 1057–1069
Regulation of SPI-1 by LeuO 1067
lar of bacteria were obtained after lysis of infected cells with
1% Triton X-100 and plating on appropriate media. Strain
discrimination was performed as for the input. Infections were
carried out in triplicate. The invasion rate was defined as the
number of bacteria in the output (intracellular bacteria recov-
ered after 2 h of infection) divided by the number of bacteria
in the input (initial inoculum).
Acknowledgements
This study was supported by grants BIO2010–15023 and
CSD2008-00013 from the Spanish Ministry of Economy and
Competitivity (MINECO) and the European Regional Fund.
We are grateful to Charles Dorman, Shane Dillon, Francisco
Ramos-Morales and Elena Cardenal-Muñoz for discussions,
to Javier López-Garrido, Fernando Baisón-Olmo, Ignacio
Cota, and Sara B. Hernández for strain constructions, to
María Antonia Sánchez-Romero for advice in flow cytometry
experiments, and to Modesto Carballo, Laura Navarro, and
Cristina Reyes of the Servicio de Biología, Centro de Inves-
tigación, Tecnología e Innovación de la Universidad de
Sevilla (CITIUS) for help with experiments performed at the
facility.
References
Altier, C. (2005) Genetic and environmental control of salmo-
nella invasion. J Microbiol 43 (Spec. No.): 85–92.
Bailly-Bechet, M., Benecke, A., Hardt, W.D., Lanza, V.,
Sturm, A., and Zecchina, R. (2011) An externally modu-
lated, noise-driven switch for the regulation of SPI1 in
Salmonella enterica serovar Typhimurium. J Math Biol 63:
637–662.
Baison-Olmo, F., Cardenal-Munoz, E., and Ramos-Morales,
F. (2012) PipB2 is a substrate of the Salmonella patho-
genicity island 1-encoded type III secretion system.
Biochem Biophys Res Commun 423: 240–246.
Balbontin, R., Rowley, G., Pucciarelli, M.G., Lopez-Garrido,
J., Wormstone, Y., Lucchini, S., et al. (2006) DNA adenine
methylation regulates virulence gene expression in Salmo-
nella enterica serovar Typhimurium. J Bacteriol 188: 8160–
8168.
Banos, R.C., Vivero, A., Aznar, S., Garcia, J., Pons, M.,
Madrid, C., and Juarez, A. (2009) Differential regulation of
horizontally acquired and core genome genes by the bac-
terial modulator H-NS. PLoS Genet 5: e1000513.
Baxter, M.A., Fahlen, T.F., Wilson, R.L., and Jones, B.D.
(2003) HilE interacts with HilD and negatively regulates
hilA transcription and expression of the Salmonella
enterica serovar Typhimurium invasive phenotype. Infect
Immun 71: 1295–1305.
Cameron, A.D., and Dorman, C.J. (2012) A fundamental
regulatory mechanism operating through OmpR and DNA
topology controls expression of Salmonella pathogenicity
islands SPI-1 and SPI-2. PLoS Genet 8: e1002615.
Chan, R.K., Botstein, D., Watanabe, T., and Ogata, Y. (1972)
Specialized transduction of tetracycline by phage P22 in
Salmonella typhimurium. II. Properties of a high frequency
transducing lysate. Virology 50: 883–898.
Chen, C.C., and Wu, H.Y. (2005) LeuO protein delimits the
1068 E. Espinosa and J. Casadesús ■
transcriptionally active and repressive domains on the bac-
terial chromosome. J Biol Chem 280: 15111–15121.
Chen, C.C., Chou, M.Y., Huang, C.H., Majumder, A., and Wu,
H.Y. (2005) A cis-spreading nucleoprotein filament is
responsible for the gene silencing activity found in the
promoter relay mechanism. J Biol Chem 280: 5101–5112.
Chun, K.T., Edenberg, H.J., Kelley, M.R., and Goebl, M.G.
(1997) Rapid amplification of uncharacterized transposon-
tagged DNA sequences from genomic DNA. Yeast 13:
233–240.
Datsenko, K.A., and Wanner, B.L. (2000) One-step inactiva-
tion of chromosomal genes in Escherichia coli K-12 using
PCR products. Proc Natl Acad Sci USA 90: 6640–6645.
De la Cruz, M.A., Fernandez-Mora, M., Guadarrama, C.,
Flores-Valdez, M.A., Bustamante, V.H., Vazquez, A., and
Calva, E. (2007) LeuO antagonizes H-NS and StpA-
dependent repression in Salmonella enterica ompS1. Mol
Microbiol 66: 727–743.
Dillon, S.C., Espinosa, E., Hokamp, K., Ussery, D.W.,
Casadesus, J., and Dorman, C.J. (2012) LeuO is a global
regulator of gene expression in Salmonella enterica
serovar Typhimurium. Mol Microbiol 85: 1072–1089.
Dorman, C.J. (2007) H-NS, the genome sentinel. Nat Rev
Microbiol 5: 157–161.
Ellermeier, C.D., and Slauch, J.M. (2003) RtsA and RtsB
coordinately regulate expression of the invasion and flagel-
lar genes in Salmonella enterica serovar Typhimurium. J
Bacteriol 185: 5096–5108.
Ellermeier, C.D., Janakiram, A., and Slauch, J.M. (2002)
Construction of targeted single copy lac fusions using
lambda Red and FLP-mediated site-specific recombination
in bacteria. Gene 290: 153–161.
Ellermeier, C.D., Ellermeier, J.R., and Slauch, J.M. (2005)
HilD, HilC and RtsA constitute a feed forward loop that
controls expression of the SPI1 type three secretion
system regulator hilA in Salmonella enterica serovar Typh-
imurium. Mol Microbiol 57: 691–705.
Ellermeier, J.R., and Slauch, J.M. (2007) Adaptation to the
host environment: regulation of the SPI1 type III secretion
system in Salmonella enterica serovar Typhimurium. Curr
Opin Microbiol 10: 24–29.
Ellermeier, J.R., and Slauch, J.M. (2008) Fur regulates
expression of the Salmonella pathogenicity island 1 type III
secretion system through HilD. J Bacteriol 190: 476–486.
Fahlen, T.F., Mathur, N., and Jones, B.D. (2000) Identification
and characterization of mutants with increased expression
of hilA, the invasion gene transcriptional activator of Sal-
monella typhimurium. FEMS Immunol Med Microbiol 28:
25–35.
Fang, M., and Wu, H.Y. (1998a) A promoter relay mechanism
for sequential gene activation. J Bacteriol 180: 626–633.
Fang, M., and Wu, H.Y. (1998b) Suppression of leu-500
mutation in topA+ Salmonella typhimurium strains. The pro-
moter relay at work. J Biol Chem 273: 29929–29934.
Fang, M., Majumder, A., Tsai, K.J., and Wu, H.Y. (2000)
ppGpp-dependent leuO expression in bacteria under
stress. Biochem Biophys Res Commun 276: 64–70.
Fernandez-Mora, M., Puente, J.L., and Calva, E. (2004)
OmpR and LeuO positively regulate the Salmonella
enterica serovar Typhi ompS2 porin gene. J Bacteriol 186:
2909–2920.
© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 1057–1069
Fortune, D.R., Suyemoto, M., and Altier, C. (2006) Identifica-
tion of CsrC and characterization of its role in epithelial cell
invasion in Salmonella enterica serovar Typhimurium.
Infect Immun 74: 331–339.
Galan, J.E., and Curtiss, R., 3rd (1989) Cloning and molecu-
lar characterization of genes whose products allow Salmo-
nella typhimurium to penetrate tissue culture cells. Proc
Natl Acad Sci USA 86: 6383–6387.
Gallego-Hernandez, A.L., Hernandez-Lucas, I., De la Cruz,
M.A., Olvera, L., Morett, E., Medina-Aparicio, L., et al.
(2012) Transcriptional regulation of the assT-dsbL-dsbI
gene cluster in Salmonella enterica serovar Typhi IMSS-1
depends on LeuO, H-NS, and specific growth conditions. J
Bacteriol 194: 2254–2264.
Garzon, A., Cano, D.A., and Casadesus, J. (1995) Role of Erf
recombinase in P22-mediated plasmid transduction.
Genetics 140: 427–434.
Hanahan, D. (1983) Studies on transformation of Escherichia
coli with plasmids. J Mol Biol 166: 557–580.
Haughn, G.W., Wessler, S.R., Gemmill, R.M., and Calvo,
J.M. (1986) High A+T content conserved in DNA
sequences upstream of leuABCD in Escherichia coli and
Salmonella typhimurium. J Bacteriol 166: 1113–1117.
Hautefort, I., Proenca, M.J., and Hinton, J.C. (2003) Single-
copy green fluorescent protein gene fusions allow accurate
measurement of Salmonella gene expression in vitro and
during infection of mammalian cells. Appl Environ Microbiol
69: 7480–7491.
Henikoff, S., Haughn, G.W., Calvo, J.M., and Wallace, J.C.
(1988) A large family of bacterial activator proteins. Proc
Natl Acad Sci USA 85: 6602–6606.
Hernandez-Lucas, I., and Calva, E. (2012) The coming of age
of the LeuO regulator. Mol Microbiol 85: 1026–1028.
Hernandez-Lucas, I., Gallego-Hernandez, A.L., Encarnacion,
S., Fernandez-Mora, M., Martinez-Batallar, A.G., Salgado,
H., et al. (2008) The LysR-type transcriptional regulator
LeuO controls expression of several genes in Salmonella
enterica serovar Typhi. J Bacteriol 190: 1658–1670.
Hertzberg, K.M., Gemmill, R., Jones, J., and Calvo, J.M.
(1980) Cloning of an EcoRI-generated fragment of the
leucine operon of Salmonella typhimurium. Gene 8: 135–
152.
Iniguez, A.L., Dong, Y., Carter, H.D., Ahmer, B.M., Stone,
J.M., and Triplett, E.W. (2005) Regulation of enteric endo-
phytic bacterial colonization by plant defenses. Mol Plant
Microbe Interact 18: 169–178.
Jones, B.D. (2005) Salmonella invasion gene regulation: a
story of environmental awareness. J Microbiol 43 (Spec.
No.): 110–117.
Klauck, E., Bohringer, J., and Hengge-Aronis, R. (1997) The
LysR-like regulator LeuO in Escherichia coli is involved in
the translational regulation of rpoS by affecting the expres-
sion of the small regulatory DsrA-RNA. Mol Microbiol 25:
559–569.
Lawley, T.D., Chan, K., Thompson, L.J., Kim, C.C., Govoni,
G.R., and Monack, D.M. (2006) Genome-wide screen for
Salmonella genes required for long-term systemic infection
of the mouse. PLoS Pathog 2: e11.
Lee, C., Wozniak, C., Karlinsey, J.E., and Hughes, K.T.
(2007) Genomic screening for regulatory genes using the
T-POP transposon. Methods Enzymol 421: 159–167.

Lercher, M.J., and Pal, C. (2008) Integration of horizontally
transferred genes into regulatory interaction networks
takes many million years. Mol Biol Evol 25: 559–567.
Lim, S., Yun, J., Yoon, H., Park, C., Kim, B., Jeon, B., et al.
(2007) Mlc regulation of Salmonella pathogenicity island I
gene expression via hilE repression. Nucleic Acids Res 35:
1822–1832.
Lopez-Garrido, J., and Casadesus, J. (2012) Crosstalk
between virulence loci: regulation of Salmonella enterica
pathogenicity island 1 (SPI-1) by products of the std fim-
brial operon. PLoS ONE 7: e30499.
Lostroh, C.P., and Lee, C.A. (2001) The Salmonella patho-
genicity island-1 type III secretion system. Microbes Infect
3: 1281–1291.
Lucchini, S., Rowley, G., Goldberg, M.D., Hurd, D., Harrison,
M., and Hinton, J.C. (2006) H-NS mediates the silencing of
laterally acquired genes in bacteria. PLoS Pathog 2: e81.
Macian, F., Perez-Roger, I., and Armengod, M.E. (1994) An
improved vector system for constructing transcriptional
lacZ fusions: analysis of regulation of the dnaA, dnaN, recF
and gyrB genes of Escherichia coli. Gene 145: 17–24.
Maddocks, S.E., and Oyston, P.C. (2008) Structure and func-
tion of the LysR-type transcriptional regulator (LTTR) family
proteins. Microbiology 154: 3609–3623.
Majumder, A., Fang, M., Tsai, K.J., Ueguchi, C., Mizuno, T.,
and Wu, H.Y. (2001) LeuO expression in response to star-
vation for branched-chain amino acids. J Biol Chem 276:
19046–19051.
Miller, J.H. (1972) Experiments in Molecular Genetics. Cold
Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Momany, C., and Neidle, E.L. (2012) Defying stereotypes:
the elusive search for a universal model of LysR-type regu-
lation. Mol Microbiol 83: 453–456.
Moorthy, S., and Watnick, P.I. (2005) Identification of novel
stage-specific genetic requirements through whole
genome transcription profiling of Vibrio cholerae biofilm
development. Mol Microbiol 57: 1623–1635.
Navarre, W.W., Porwollik, S., Wang, Y., McClelland, M.,
Rosen, H., Libby, S.J., and Fang, F.C. (2006) Selective
silencing of foreign DNA with low GC content by the H-NS
protein in Salmonella. Science 313: 236–238.
Ochman, H., Lawrence, J.G., and Groisman, E.A. (2000)
Lateral gene transfer and the nature of bacterial innovation.
Nature 405: 299–304.
Paytubi, S., Madrid, C., Forns, N., Nieto, J.M., Balsalobre, C.,
Uhlin, B.E., and Juarez, A. (2004) YdgT, the Hha paralogue
in Escherichia coli, forms heteromeric complexes with
H-NS and StpA. Mol Microbiol 54: 251–263.
Price, M.N., Dehal, P.S., and Arkin, A.P. (2008) Horizontal
gene transfer and the evolution of transcriptional regulation
in Escherichia coli. Genome Biol 9: R4.
Rappleye, C.A., and Roth, J.R. (1997) A Tn10 derivative
(T-POP) for isolation of insertions with conditional
(tetracycline-dependent) phenotypes. J Bacteriol 179:
5827–5834.
Repoila, F., and Gottesman, S. (2001) Signal transduction
cascade for regulation of RpoS: temperature regulation of
DsrA. J Bacteriol 183: 4012–4023.
Rodriguez-Morales, O., Fernandez-Mora, M., Hernandez-
Lucas, I., Vazquez, A., Puente, J.L., and Calva, E. (2006)
Salmonella enterica serovar Typhimurium ompS1 and
© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 1057–1069
Regulation of SPI-1 by LeuO 1069
ompS2 mutants are attenuated for virulence in mice. Infect
Immun 74: 1398–1402.
Schmieger, H. (1972) Phage P22 mutants with increased or
decreased transducing abilities. Mol Gen Genet 119:
75–88.
Shi, X., and Bennett, G.N. (1995) Effects of multicopy LeuO
on the expression of the acid-inducible lysine decarboxy-
lase gene in Escherichia coli. J Bacteriol 177: 810–814.
Shimada, T., Yamamoto, K., and Ishihama, A. (2009) Involve-
ment of the leucine response transcription factor LeuO in
regulation of the genes for sulfa drug efflux. J Bacteriol
191: 4562–4571.
Shimada, T., Bridier, A., Briandet, R., and Ishihama, A. (2011)
Novel roles of LeuO in transcription regulation of E. coli
genome: antagonistic interplay with the universal silencer
H-NS. Mol Microbiol 82: 378–397.
Stratmann, T., Madhusudan, S., and Schnetz, K. (2008)
Regulation of the yjjQ-bglJ operon, encoding LuxR-type
transcription factors, and the divergent yjjP gene by H-NS
and LeuO. J Bacteriol 190: 926–935.
Stratmann, T., Pul, U., Wurm, R., Wagner, R., and Schnetz,
K. (2012) RcsB-BglJ activates the Escherichia coli leuO
gene, encoding an H-NS antagonist and pleiotropic regu-
lator of virulence determinants. Mol Microbiol 83: 1109–
1123.
Sturm, A., Heinemann, M., Arnoldini, M., Benecke, A.,
Ackermann, M., Benz, M., et al. (2010) The cost of viru-
lence: retarded growth of Salmonella Typhimurium cells
expressing type III secretion system 1. PLoS Pathog 7:
e1002143.
Tenor, J.L., McCormick, B.A., Ausubel, F.M., and Aballay, A.
(2004) Caenorhabditis elegans-based screen identifies
Salmonella virulence factors required for conserved host-
pathogen interactions. Curr Biol 14: 1018–1024.
Tobe, T., Yoshikawa, M., Mizuno, T., and Sasakawa, C.
(1993) Transcriptional control of the invasion regulatory
gene virB of Shigella flexneri: activation by virF and repres-
sion by H-NS. J Bacteriol 175: 6142–6149.
Torreblanca, J., Marques, S., and Casadesus, J. (1999) Syn-
thesis of FinP RNA by plasmids F and pSLT is regulated by
DNA adenine methylation. Genetics 152: 31–45.
Uzzau, S., Figueroa-Bossi, N., Rubino, S., and Bossi, L.
(2001) Epitope tagging of chromosomal genes in Salmo-
nella. Proc Natl Acad Sci USA 98: 15264–15269.
Vivero, A., Banos, R.C., Mariscotti, J.F., Oliveros, J.C.,
Garcia-del Portillo, F., Juarez, A., and Madrid, C. (2008)
Modulation of horizontally acquired genes by the Hha-
YdgT proteins in Salmonella enterica serovar Typhimu-
rium. J Bacteriol 190: 1152–1156.
Wilmes-Riesenberg, M.R., Foster, J.W., and Curtiss, R., 3rd
(1997) An altered rpoS allele contributes to the avirulence
of Salmonella typhimurium LT2. Infect Immun 65: 203–210.
Zaim, J., and Kierzek, A.M. (2003) The structure of full-length
LysR-type transcriptional regulators. Modeling of the full-
length OxyR transcription factor dimer. Nucleic Acids Res
31: 1444–1454.
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