Molecular Microbiology (1999) 31(6), 1759–1773
Environmental regulation of Salmonella pathogenicity
island 2 gene expression
Jörg Deiwick, Thomas Nikolaus, Sezgin Erdogan and
Michael Hensel*
Lehrstuhl für Bakteriologie, Max von Pettenkofer-Institut
für Hygiene und Medizinische Mikrobiologie,
Pettenkoferstr. 9a, D-80336 Munich, Germany.
Summary
The enteric pathogen Salmonella typhimurium co-ordinates the expression of virulence determinants in
response to environmental cues from the host organism. S. typhimurium possesses Salmonella pathogenicity island 2 (SPI2), a large virulence locus
encoding a type III secretion system for virulence
determinants required for systemic infections and
accumulation inside host cells. We have generated
transcriptional fusions to SPI2 genes to analyse
expression and used antibodies against recombinant
SPI2 proteins to monitor levels of SPI2 proteins under
various conditions. Here, we demonstrate that SPI2
gene expression is induced by Mg2þ deprivation and
phosphate starvation. These conditions are likely to
represent the environmental cues encountered by S.
typhimurium inside the phagosome of infected host
cells. The induction of SPI2 gene expression is modulated by the global regulatory system PhoPQ and is
dependent on SsrAB, a two-component regulatory
system encoded by SPI2.
Introduction
The enteric pathogen Salmonella spp. is one of the leading causes of gastrointestinal infections ranging from
mild, self-limiting inflammation of the intestinal mucosa to
typhoid fever, a life-threatening systemic infection. During
the complex multistage pathogenesis, Salmonella spp.
have to respond to a variety of environmental signals
(such as deprivation of nutrients), stress conditions
(osmolarity, temperature) and the antimicrobial defence
mechanisms of the host (Foster and Spector, 1995; Slauch
et al ., 1997). To successfully colonize the host organism
and to avoid clearance by the host immune system, a
large number of general stress response systems as well
Received 28 September, 1998; revised 7 December, 1998; accepted
10 December, 1998. *For correspondence. E-mail hensel@m3401.
mpk.med.uni-muenchen.de; Tel. (þ49) 89 5160 5248; Fax (þ49) 89
5160 5223.
䊚 1999 Blackwell Science Ltd
as specific virulence factors are required (Groisman and
Ochman, 1997).
The expression of these sets of genes is co-ordinately
regulated in a temporal and spatial fashion (Mekalanos,
1992). Often, the expression of virulence genes is regulated by two-component regulatory systems composed
of the membrane-bound sensor and a transcriptional regulator of gene expression (Parkinson and Kofoid, 1992).
Although some of the two-component regulatory systems
are shared between non-pathogenic bacteria like Escherichia coli K-12 and pathogens like S. typhimurium, additional systems can be found in the pathogen. The
two-component regulatory system PhoPQ is present in
both species, but represents an important global regulator
of Salmonella virulence. The expression of more than 40
loci is under the control of PhoPQ (Soncini et al., 1996),
and it has been demonstrated that mutants defective in
PhoP or PhoQ, and also a mutant exhibiting a hyperactive
state of the PhoQ kinase, are dramatically attenuated for
virulence (Miller et al., 1989; Miller and Mekalanos, 1990).
Garcia Vescovi et al. (1996) demonstrated that the PhoPQ
system senses the concentration of the divalent cations
Mg2þ and Ca2þ. PhoPQ has been proposed to be a
regulatory system for genes which allows Salmonella to
survive and replicate within macrophages (Miller, 1991;
Garcia Vescovi et al., 1994).
An important virulence trait of S. typhimurium is the
ability to invade host cells (Galan, 1996). The invasion
phenotype is determined by a large cluster of genes present in Salmonella pathogenicity island 1 (SPI1), encoding
a complex type III secretion system for virulence determinants (Mills et al., 1995). Expression of SPI1 genes is
regulated in a co-ordinated fashion in response to environmental signals (Bajaj et al., 1996).
A large cluster of virulence genes located within Salmonella pathogenicity island 2 (SPI2) has been identified
(Hensel et al., 1995; Ochman et al., 1996; Shea et al.,
1996). It has been shown that this locus encodes a second
type III secretion system (Hensel et al., 1997a; 1997b),
secreted effector proteins and their specific chaperones
(Cirillo et al., 1998; Hensel et al., 1998), as well as a
two-component regulatory system (Ochman et al., 1996;
Shea et al., 1996). Inactivation of these genes by transposon insertions or introduction of antibiotic resistance
cassettes results in a dramatic attenuation of virulence in
the model system of murine salmonellosis. In vitro analyses showed that SPI2 mutations cause pleiotropic
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J. Deiwick, T. Nikolaus, S. Erdogan and M. Hensel
effects, including effects on the expression of SPI1 genes
(Deiwick et al., 1998). It has been suggested that SPI2 is
important for the survival of Salmonella inside macrophages because SPI2 mutant strains fail to accumulate
in macrophages (Ochman et al., 1996; Cirillo et al., 1998;
Hensel et al., 1998). Recently, Valdivia and Falkow (1997)
reported that a green fluorescent protein (GFP) reporter
gene fusion to a promoter of SPI2 was highly induced
inside macrophages and epithelial cells. However, the
chemical or physical nature of the signals inducing the
gene expression of SPI2 was not investigated.
In this study, we set out to characterize the environmental cues affecting the expression of SPI2 genes.
Furthermore, the modulation of SPI2 gene expression by
regulatory systems in S. typhimurium was analysed. Our
data allowed us to define signals inducing SPI2 expression
in vitro and indicate that conditions encountered by the
pathogen inside the intracellular compartment of the host
cell specifically induce the expression of SPI2 genes.
Results
Generation of reporter gene fusions to SPI2 promoters
and antibodies against recombinant SPI2 proteins
For the analysis of the expression of SPI2 virulence genes,
transcriptional reporter gene fusions of SPI2 genes to firefly
luciferase (luc ) were generated (Fig. 1). To avoid artefacts by increased copy numbers of promoter elements,
fusions were integrated into the S. typhimurium chromosome using luc suicide vectors (Gunn and Miller, 1996).
As a second approach, the amount of proteins encoded
by SPI2 genes was monitored using antibodies raised
against recombinant SPI2 proteins rSsaP and rSscA.
SsaP is encoded by a gene of the ssaK /U operon, encoding structural components of the type III secretion system
(Hensel et al., 1997b). In contrast to other components of
the SPI2 type III secretion system, SsaP has no significant
similarity to components of other type III secretion systems, including the SPI1 secretion system of S. typhimurium. SscA is encoded by the third gene of a cluster
of nine genes encoding putative effector proteins of SPI2
and their chaperones, and has low sequence similarity
to specific chaperones of other type III secretion systems
(Hensel et al., 1998). Detection with antisera raised against
rSsaP and rSscA allowed us to monitor protein levels of
different classes of SPI2 proteins (Fig. 2B).
Effect of environmental parameters on expression of
SPI2 genes
The amounts of SPI2 proteins SsaP and SscA under various growth conditions were monitored by Western blot
analysis (Fig. 2B), and the expression was analysed
Fig. 1. Genetic organization of SPI2, position of reporter gene fusions, plasmids and mutations used in this study.
A. Genes encoding the type III secretion system apparatus (ssa, open arrows), putative effector proteins (sse, filled arrows), specific
chaperones (ssc, shaded arrows) and the two-component regulatory system (ssr, hatched arrows) of SPI2 are indicated. The position of
transcriptional fusions to luc of strains MvP127, MvP131 and MvP266, as well as the mTn5 insertion of mutant strain P8G12 is shown.
B. The positions of restriction sites (B, Bam HI; E, Eco RI; H, Hin dIII; K, Kpn I; P, Pst I; Sa, Sal I; Sm, Sma I; Sp, Sph I; V, Eco RV), and the
position of inserts of plasmids used in this study is indicated.
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SPI2 gene expression 1761
Fig. 2. Expression of SPI2 genes and levels of SPI2 proteins under various growth conditions. S. typhimurium wild-type strain and strain
MvP131 harbouring a ssaB ::luc fusion were grown overnight in various media. In detail, the following conditions were used: BHI, brain–heart
infusion broth; E salts, Vogel–Bonner minimal medium containing 38 mM glycerol and 0.1% casamino acids; NCE, non-citrate Vogel–Bonner
minimal medium containing 1 mM MgCl2 , 38 mM glycerol and 0.1% casamino acids; M9, M9 minimal medium containing 0.2% glucose (Miller,
1992); ISM, intracellular salt medium (Headley and Payne, 1990); RPMI, RPMI-1640; PCN, MOPS-buffered minimal medium (Neidhardt et al.,
1974); -N, -P, -C, star vation conditions for phosphate, carbon, and nitrogen according to Spector and Cubitt (1992); PCN anaerobic, medium
according to Stewart and Parales (1988); PCN -P 8 ␮M Mg2þ, PCN medium containing 8 ␮M Mg2þ and 113 ␮M phosphate; N salts (100 mM
Bis/Tris-HCl, pH 7.0, 5 mM KCl, 7.5 mM (NH4 )2SO4 , 0.5 mM K2SO4 , 1 mM KH2PO4 , 38 mM glycerol, 0.1% casamino acids) containing 10 mM
MgCl2 or 8 ␮M MgCl2 as indicated.
A. The Luc activities of lysates of strain MvP131 were determined. Error bars indicate standard deviation from mean.
B. Lysates of the S. typhimurium wild type were subjected to SDS–PAGE, protein was transferred onto nitrocellulose membranes, and SsaP
and SscA were detected using antibodies raised against rSsaP and rSscA respectively. Equal amounts of cells as adjusted by OD at 600 nm
(OD600 ) were analysed. The positions of SsaP and SscA are indicated. We observed that SscA appears as diffuse double bands in Western
analyses. Total cell fractions of IPTG-induced cultures of E. coli BL21(DE3) harbouring pET21(þ) (negative control), p5-4-T7 (T7 control for
SscA, 50-fold diluted), and p7-26-T7 (T7 control for SsaP, 500-fold diluted) were analysed.
using single-copy luc reporter gene fusions to SPI2 genes
(Fig. 2A). No significant expression of SPI2 genes was
detectable after growth in LB, tryptic soy broth, or tissue
culture media such as RPMI-1640 and DMEM, either
with or without the addition of 5% fetal calf serum. This
observation is in accordance with previous work of Valdivia and Falkow (1997), who observed that expression of
a plasmid-borne fusion of the reporter gene GFP to
ssaH was highly induced in S. typhimurium residing in
䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1759–1773
the intracellular compartment of infected host cells but
repressed during growth in LB or tissue culture media.
Expression of ssaP was detected in cells grown in certain
batches of BHI (brain–heart infusion) medium, however
use of other batches did not induce SPI2 gene expression.
In minimal media of various formulations, only low levels of
SPI2 gene expression were detected (Fig. 2A). To simulate conditions experienced by S. typhimurium during
intracellular episodes, such as the low pH of the
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J. Deiwick, T. Nikolaus, S. Erdogan and M. Hensel
Fig. 3. Dependence of SPI2 gene expression
on Mg2þ concentration. S. typhimurium
strains MvP127 (sseA ::luc, open columns)
and MvP131 (ssaB ::luc, hatched columns)
were grown for 16 h at 37⬚C with aeration in
N salts minimal medium buffered by 100 mM
Tris-HCl, pH 7.4 (A) or N salts minimal medium
buffered by 100 mM Bis/ Tris-HCl, pH 7.0
(B) which contained various concentrations of
MgCl2 as indicated. Luc activities of bacterial
lysates were determined. The final OD600
(circles), as well as the final pH of the
medium (triangles) of cultures of MvP131
after growth for 16 h are indicated.
phagolysosomal compartment (Garcia del Portillo et al.,
1992; Rathman et al., 1996), the effect of exposure of bacteria to media at various pHs on SPI2 gene expression
was assayed. During growth in LB, no effects of lowered
pH on SPI2 gene expression were observed, whereas a
two- to fivefold increase in expression was observed if
the pH of E or NCE minimal medium was lowered from
7.0 to 5.0 (data not shown). However, only low rates of
expression were observed.
Bacteria inside the phagolysosomal compartment of
host cells also experience deprivations of nutrients and
a low concentration of divalent cations (Garcia del Portillo
et al., 1992). The minimal media formulation of Neidhardt
et al. (1974) was used to simulate effects of starvation for
phosphate, carbon or nitrogen. High levels of expression
were only observed after starvation for phosphate
(Fig. 2). Next, we analysed the effect of various concentrations of Mg2þ or Ca2þ on SPI2 gene expression
(Fig. 3). Although expression of SPI2 genes was very
low after growth in minimal media containing high amounts
of Mg2þ (10 mM) or Ca2þ (2 mM), reporter gene fusions to
SPI2 genes were highly induced after growth in minimal
media containing Mg2þ or Ca2þ at a minimal concentration
of 8 ␮M. This effect of divalent cation concentration on
SPI2 gene expression was observed in various minimal
media such as N, M9, E or NCE, with or without additional
nitrogen sources (casamino acids) and with carbon sources
such as glycerol, ethanol or glucose (data not shown). As
the sole exposure to acidic pH did not induce SPI2 gene
expression, the combined exposure of bacteria to limiting
concentrations of Mg2þ and to acidic pH was analysed.
However, no additive effect of both conditions on SPI2
gene expression was observed.
We also analysed the effects of other stress conditions
known to activate the expression of Salmonella virulence
genes according to Abshire and Neidhardt (1993). The
addition of a cationic peptide (2.4 ␮g ml¹1 polymyxin B)
or oxidizing agents (195 ␮M paraquat; 125 ␮M or 300 ␮M
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SPI2 gene expression 1763
H2O2 ) did not induce the expression of SPI2 genes (data
not shown). Increasing the osmolarity of medium with
minimal amounts of Mg2þ resulted in the reduction of
SPI2 gene expression (Table 1). Changes in osmolarity
resulting from various concentrations of divalent cations
Table 1. Expression of ssaB in media of various osmolarities.
Growth conditions
(% NaCl)
Relative Luc
activity ⫾ SD
0
1
2
3
816 141 ⫾ 228 305
808 144 ⫾ 241 541
505 080 ⫾ 188 106
62 663 ⫾ 15 842
Strain MvP131 harbouring a ssaB ::luc fusion was grown in N salts
minimal medium containing 8 ␮M Mg2þ and various concentrations
of NaCl as indicated, and Luc activities ware assayed.
had no effect on SPI2 expression. A reduced osmolarity
did not result in increased expression of the ssaB ::luc
fusion, however evaluation of media of very low osmolarity
was limited by the buffer component required (data not
shown). Western blot analysis revealed that the levels of
proteins encoded by SPI2 genes are not affected by the
growth temperature of cultures.
Effects of divalent cations on SPI2 gene expression
To analyse the effect of divalent cations in more detail,
expression of reporter fusions was assayed after growth
in minimal media containing various concentrations of
divalent cations (Fig. 3). The effect of the Mg2þ concentration on SPI2 gene expression was analysed using the
Tris-buffered N salts medium formulation as described
previously (Hmiel et al., 1986; Garcia Vescovi et al.,
1996). SPI2 gene expression was highly induced in media
containing less than 30 ␮M Mg2þ, but not induced in
Fig. 4. Effect of divalent cation concentration on SPI2 gene
expression and SPI2 protein levels.
A. Induction of SPI2 gene expression by withdrawal of Mg2þ
according to Garcia Vescovi et al. (1996). Cultures of MvP131
(ssaB ::luc, open symbols) and MvP127 (sseA ::luc, filled symbols)
were grown in N salts minimal media containing 2 mM CaCl2 and
8 ␮M MgCl2 . At OD600 of about 0.5, EGTA was added to a final
concentration of 6 mM and growth of the cultures was continued.
Aliquots of the cultures were sampled at various time points and
Luc activities of cultures after EGTA addition (circles) and control
cultures without the addition of EGTA (triangles) were determined.
In the presence of 10 mM Mg2þ, the addition of 6 mM EGTA did not
induce SPI2 gene expression (data not shown).
B. S. typhimurium strains MvP131 (ssaB ::luc, open symbols) and
MvP266 (ssaJ ::luc, filled symbols) were grown to mid-log phase
(OD600 of about 0.5) in N salts minimal medium containing 1 mM
MgCl2 . Bacteria were recovered by centrifugation, washed three
times in minimal medium containing 8 ␮M MgCl2 and resuspended
either in medium containing 8 ␮M MgCl2 (circles) or 10 mM MgCl2
(triangles). Growth at 37⬚C was continued and aliquots of the
cultures were withdrawn at the time points indicated and subjected
to analysis of luciferase activity.
C. S. typhimurium wild type was grown and washed as described
in (B), and cultivation was continued in media containing Mg2þ
concentrations as indicated. Culture aliquots were withdrawn at the
time points indicated, and total protein of equal amounts of bacterial
cells as adjusted by OD600 were separated by SDS–PAGE,
transferred onto nitrocellulose membranes, and SscA and SsaP
was detected as described in Fig. 2.
䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1759–1773
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J. Deiwick, T. Nikolaus, S. Erdogan and M. Hensel
media with higher concentrations of Mg2þ. A similar effect
was observed if Mg2þ was replaced by Ca2þ; however,
expression of reporter fusions to ssaB and sseA was
observed at concentrations equal to or below 100 ␮M
Ca2þ (data not shown). We observed that the pH of this
medium dropped below 5 after growth of cultures for
14 –16 h. Additional experiments were performed with
Bis/ Tris-buffered N salts medium to ensure medium pH
was constant over the entire period of growth (Fig. 3B).
Under these conditions, highest induction of SPI2
expression was observed at about 50 ␮M Mg2þ (Fig.
3B). In contrast, analysis of a reporter gene fusion to
mgtB, a previously defined PhoPQ-regulated gene (Hmiel
et al., 1989), indicated highest induction at 8 ␮M Mg2þ,
without effects of the buffer capacity of the medium (data
not shown).
Further experiments were performed to determine
whether the expression of SPI2 genes could be induced
by withdrawal of divalent cations. (Fig. 4). After growth
in minimal medium containing 8 ␮M Mg2þ and 2 mM
Ca2þ, the expression of reporter gene fusions to SPI2
genes was induced after chelation of Ca2þ by addition of
EGTA (ethylene glycol-bis[␤-aminoethylether]-N,N,N ⬘,N ⬘tetraacetic acid) (Fig. 4A). A similar result was obtained
after growth of cultures in minimal media containing
1 mM Mg2þ, washing of cells, and shifting cultures to a
medium containing 8 ␮M Mg2þ (Fig. 4B). This effect on
SPI2 gene expression was observed for reporter gene
fusions to ssaB and ssaJ (Fig. 4B), and correlated with
the levels of SsaP and SscA detected in Western blots
(Fig. 4C).
Effects of starvation for phosphate on SPI2 gene
expression
The effect of phosphate starvation on SPI2 expression
was further investigated by growing cultures in media
containing saturating or limiting amounts of phosphate
and various concentrations of Mg2þ, and subsequent analysis of the expression of a ssaB ::luc fusion (Fig. 5). In the
presence of high amounts of phosphate, this fusion was
expressed in media with low Mg2þ concentrations. In contrast, under phosphate starvation conditions, the ssaB ::luc fusion was highly induced, but the expression was
not dependent on Mg2þ deprivation. This observation
suggests that phosphate starvation may represent a
further environmental cue stimulating the expression of
SPI2 genes.
Identification of the transcriptional regulator SsrB
The partial sequence of the gene of the sensor component
SsrA (or SpiR) of a two-component regulatory system in
SPI2 has been reported previously (Ochman et al., 1996).
Fig. 5. Effect of phosphate starvation on SPI2 gene expression.
Strain MvP131 was grown in minimal medium according to O’Neal
et al. (1994) containing 25 mM or 110 ␮M PO43¹ and various
concentrations of Mg2þ (filled circles, 8 ␮M Mg2þ; open circles
100 ␮M Mg2þ; triangles, 10 mM Mg2þ ) as indicated. Cultures were
grown at 37⬚C with aeration, samples were withdrawn at various
time points, and the Luc activity of bacterial lysates was
determined.
As part of our effort to characterize the SPI2 locus, cloning
and sequence analysis of DNA downstream of the ssrA
gene was performed. The insert of a clone of a ␭ library
of S. typhimurium genomic DNA harboured the 3⬘ end of
ssrA as well as the region downstream of ssrA . The
ssrA gene encodes a protein of 920 amino-acid residues
and a theoretical molecular weight of 103.6 kDa. Sequence
analysis revealed an open reading frame with coding
capacity for a 24.3 kDa protein downstream of ssrA . The
predicted protein shares significant sequence similarity
with a family of transcriptional activators such as DegU
of Bacillus subtilis, NarL and UvrY of E. coli, SirA of S.
typhimurium and BvgA of Bordetella pertussis (Fig. 6A).
Therefore, it is likely that the protein acts as the regulatory
component of the ssr system and the gene was designated ssrB. The size of proteins identified after expression
of ssrAB under control of the T7 promoter was in accordance with the predicted molecular weight (Fig. 6B).
Further characterization of the SPI2 locus indicated that
ORF 242 and ORF 319 downstream of ssrB are not
required for S. typhimurium virulence (Hensel et al.,
1999).
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SPI2 gene expression 1765
Fig. 6. Alignment of SsrB of S. typhimurium
to various bacterial transcriptional regulators.
A. The deduced amino-acid sequence of SsrB
was aligned to SirA of S. typhimurium
(Swiss-Prot accession number P96058), UvrY
of E. coli (P07027), DegU of Bacillus subtilis
(P13800), NarL of E. coli (P10957) and BvgA
of Bordetella pertussis (P16574) using the
ClustalW module of MacVector version 6.0.
Similar residues are boxed, identical residues
are indicated by shading.
B. Expression of ssrAB under control of the
T7 promoter according to Studier et al.
(1990). E. coli BL21(DE3) harbouring
pWSK29 (lane 1) or pssrAB (lane 2) were
grown to mid-log phase, 1 mM IPTG and
20 ␮Ci [ 35S]-methionine/cysteine (Promix,
Amersham) were added and labelling was
performed for 5 min. Bacteria were pelleted,
boiled for 5 min in SDS–PAGE sample buffer,
and aliquots corresponding to 100 ␮l culture
were analysed by SDS–PAGE on 12% gels
and subsequent autoradiography.
Effects of SsrAB, PhoPQ and further regulatory
systems on SPI2
We next analysed the effect of mutations in the ssrAB
system on the levels of SPI2 proteins. Characterization
of the mTn5 insertion of mutant strain P8G12 initially
identified by signature-tagged mutagenesis (Hensel et
al., 1995) indicated that ssrB was inactivated in this mutant
strain. Virulence properties and effects on SPI2 gene
expression of a mutant strain in which ssrB had been inactivated by insertion of a aphT gene cassette lacking transcriptional terminators were similar to those of mutant
strain P8G12, indicating that the phenotype of the latter
is not the result of polar effects on downstream genes
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(data not shown). Analysis of luc fusions in the strain
backgrounds of mutants P3F4 (ssrA ::mTn5 ; Hensel et
al., 1998) and P8G12 (ssrB ::mTn5 ; Hensel et al., 1998;
Fig. 9) resulted in a strong reduction of the expression
of SPI2 genes. Furthermore, very low levels of SscA
were detected in the ssrB ¹ background after growth in
media containing limiting amounts of Mg2þ or phosphate
(Figs 7 and 8). Complementation of SPI2 expression in
mutant strain P8G12 was possible by introduction of plasmid pssrAB harbouring ssrAB (data not shown). Presence
of pssrAB resulted in decreased effects of environmental
signals on the amounts of SPI2 proteins present in the
cell (Fig. 8). High levels of SsaP and SscA were observed
in the wild type and ssrB ¹ mutant strain harbouring
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J. Deiwick, T. Nikolaus, S. Erdogan and M. Hensel
Fig. 7. Role of SsrAB and PhoPQ. S. typhimurium wild type (wild
type), strain P8G12 harbouring a mTn5 insertion in ssrB (ssrB ¹ ),
and strains CS022 ( pho-24 ) and CS015 ( phoP ¹ ) were analysed.
Bacterial cultures were grown in minimal medium containing 10 mM
MgCl2 to mid-log phase (OD600 about 0.5), pelleted, washed three
times in minimal medium with 8 ␮M MgCl2 , and resuspended in
fresh minimal media containing 8 ␮M or 10 mM MgCl2 as indicated.
Cultures were incubated at 37⬚C and samples were withdrawn at
various time points as indicated. Total protein from equal amounts
of bacterial cells as adjusted by OD600 was separated by
SDS–PAGE, transferred onto membranes and SscA was detected
using antibodies raised against the recombinant protein.
pssrAB in the presence of high and low amounts of Mg2þ,
however effects of phosphate starvation were not fully
restored in P8G12 [pssrAB] (Fig. 8).
The induction of SPI2 gene expression in minimal media
containing micromolar amounts of Mg2þ or Ca2þ is similar
to the characteristics of a subset of genes whose expression is controlled by the global regulatory system PhoPQ
(Miller et al., 1989; Garcia Vescovi et al., 1996; Soncini
et al., 1996). These loci are referred to as ‘PhoP-activated
genes’ or pag. To determine whether SPI2 represents a
pag locus or whether exposure of S. typhimurium to conditions of low cation concentrations is sensed by a regulatory system independent from PhoPQ, luc fusions to
SPI2 genes were moved into strains CS015 ( phoP¹ )
and CS022 ( pho-24) by P22-mediated transduction. It
was observed before that phoP¹ strains show growth
defects in minimal media with limiting concentrations of
divalent cations (Miller and Mekalanos, 1990). To analyse
the effect of the PhoPQ system on SPI2 expression under
similar conditions, cells were grown to mid-log phase in
media with 10 mM Mg2þ, washed, then transferred to
media containing 8 ␮M or 10 mM Mg2þ. Under these
assay conditions, all strains had similar growth rates for
up to 2.5 h after transfer to low-Mg2þ media. After this
time point, no further growth of CS015 was observed.
Levels of SscA were analysed at various time points
after the shift using antibodies raised against rSscA
(Fig. 7). In the wild type and pho-24 background, the
expression of sscA was induced after shift of cultures to
media containing 8 ␮M Mg2þ. However, levels of SscA
were highly reduced in the phoP¹ strain. This observation
was confirmed by analysis of the expression of reporter
gene fusions in the genetic background of wild-type,
pho-24 and phoP¹ strains. After shifting cultures to minimal media containing 8 ␮M Mg2þ, no expression of the
luc fusions to SPI2 genes was observed in the phoP¹
background (data not shown).
The role of other regulatory systems affecting Salmonella
virulence gene expression on SPI2 gene expression was
analysed. Reporter gene fusions were transduced in strains
harbouring mutations in hilA , the local transcriptional
activator of SPI1 gene expression (Bajaj et al., 1995;
1996), sirA , a regulator affecting the expression of SPI1
(Johnston et al., 1996), and rpoS, encoding an alternative
sigma factor important for the regulation of spv genes of
the Salmonella virulence plasmid (Swords et al., 1997;
Wilmes Riesenberg et al., 1997). Expression of luc fusions
to SPI2 genes was assayed after growth in minimal media
containing various concentrations of MgCl2 . The induction
of SPI2 gene expression was similar in the wild-type background and in strain backgrounds with mutations in hilA,
sirA and rpoS (data not shown). Under the in vitro conditions defined in this study, SPI2 gene expression is not
affected by these regulators.
Regulation of SPI1 and SPI2 gene expression
We studied the expression of genes for the SPI1 and SPI2
type III secretion systems under conditions previously
described to be inducing for SPI1 genes (Bajaj et al.,
1996), and defined here to be inducing for SPI2 genes
(Fig. 9). Growth in media with high osmolarity and O2
limitation induced SPI1 gene expression, but not SPI2
gene expression. As previously shown (Bajaj et al., 1995;
1996), our experiments confirm that the expression of
SPI1 genes is dependent on the function of the local
Fig. 8. Effect of overexpression of ssrAB. S. typhimurium wild type
and mutant strain P8G12 (ssrB ¹ ) either harbouring plasmid
pssrAB or without a plasmid were grown for 16 h in various media
as described in the legend of Fig. 2. Total protein of equal amounts
of cells as adjusted by OD600 was subjected to SDS–PAGE,
transferred onto membranes and SsaP and SscA were detected.
Strains harbouring the vector pWSK29 had protein levels identical
to strains without a plasmid (data not shown).
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SPI2 gene expression 1767
uptake of bacteria was inhibited by the addition of
cytochalasin D (data not shown).
Discussion
Induction of SPI2 gene expression by environmental
signals
Fig. 9. Inverse regulation of expression of genes in SPI1 and SPI2.
The expression of reporter gene fusions in SPI2 (ssaB ::luc, open
bars) and SPI1 (sipC ::lacZ) were analysed in the background of S.
typhimurium wild-type strain, and ssrB and hilA mutant strains.
Strains were grown under conditions previously described as highly
inducing for SPI1 gene expression (Bajaj et al., 1996) or defined
here as inducing for SPI2 gene expression. Cultures were grown
for about 16 h in LB containing 1% NaCl for induction of SPI1, or in
N salts minimal medium containing 8 ␮M Mg2þ for the induction of
SPI2. The Luc activities of lysates of strains harbouring the
ssaB ::luc fusion (open columns) and ␤-galactosidase activities of
strains harbouring the sipC ::lacZ fusion (solid columns) were
determined.
transcriptional activator HilA. However, under these conditions only low levels of expression of sipC were detected in
the ssrB ¹ background, indicating that SsrB is required for
the expression of sipC. The effect of mutations in ssrB and
other SPI2 genes on the expression of SPI1 genes has
been reported in detail (Deiwick et al., 1998). The molecular basis of these effects of SPI2 mutations has not yet
been elucidated. In contrast, under conditions inducing
SPI2 gene expression, no significant expression of SPI1
genes was observed. Inactivation of HilA had no effect
on the expression of the ssaB ::luc fusion.
SPI2 plays a pivotal role in the virulence of S. typhimurium
in the model of murine salmonellosis (Ochman et al., 1996;
Shea et al., 1996), and it has been shown that SPI2 contributes to the intracellular proliferation of S. typhimurium
within infected host cells (Cirillo et al., 1998; Hensel et
al., 1998). Recently, a fusion of a SPI2 gene to GFP was
identified which was highly induced in bacteria inside
macrophages (Valdivia and Falkow, 1997), indicating
that the presence within an intracellular compartment
specifically induces the expression of SPI2 genes. We
identified environmental signals inducing SPI2 gene
expression by analysing SPI2 gene expression under in
vitro conditions. These experiments indicated that the
exposure of S. typhimurium to media with limiting concentrations of divalent cations was highly inducing for
the expression of various SPI2 genes. Furthermore, starvation of S. typhimurium for phosphate resulted in the
induction of the expression of SPI2 genes. Similarities
between protein patterns of S. typhimurium grown under
phosphate starvation in vitro and bacteria exposed to
the intracellular environment were observed using proteome analysis (Abshire and Neidhardt, 1993). Recently,
a GFP fusion to a gene encoding a phosphate transport
SPI2 gene expression of intracellular Salmonella
To investigate whether in vitro conditions that induce SPI2
reflect the conditions encountered by S. typhimurium
during infection of host cells, the expression of reporter
gene fusions by bacteria localized within macrophages
was analysed (Fig. 10). Intracellular location of S. typhimurium resulted in a strong induction of the reporter
gene fusion ssaB ::luc and ssaJ ::luc. No significant expression was observed in the ssrB ::mTn5 background, indicating that induction of SPI2 gene expression within host cells
is dependent on the function of SsrB. These observations
are in keeping with the results of Valdivia and Falkow
(1997) and Cirillo et al. (1998), who observed that GFP
fusions to SPI2 genes are specifically induced by intracellular bacteria. We did not observe expression of luc
fusions to SPI2 genes under conditions in which the
䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1759–1773
Fig. 10. Expression of SPI2 genes within macrophages. Cells of
the murine macrophage-like line J774.A1 were infected at a
multiplicity of infection of 10 bacteria per macrophage with MvP131
(ssaB ::luc, filled circles), MvP266 (ssaJ ::luc, open circles) and
MvP244 (ssaB ::luc in a ssrB ¹ background, triangles). Extracellular
bacteria were killed by the addition of gentamicin. At the various
time points indicated, macrophages were lysed, and intracellular cfu
and Luc activity were determined as described in Experimental
procedures. The number of bacteria recovered for strains MvP244
and MvP266 was plotted (dotted lines). Luc activities were
expressed as relative Luc activity per bacterial cell.
1768
J. Deiwick, T. Nikolaus, S. Erdogan and M. Hensel
protein has been identified as intracellularly induced (Valdivia and Falkow, 1997). These observations suggest that
phosphate limitation is an important environmental cue
within intracellular compartments. Our in vitro analyses
did not identify further inducing SPI2 gene expression. It
has been suggested that exposure to acidic pH is an
important signal for the induction of S. typhimurium virulence genes (Alpuche Aranda et al., 1992; Rathman et
al., 1996). However, the environmental stimulus ‘acidic
pH’ did not significantly induce SPI2 expression. These
observations may indicate that the acidification of the
phagosome is a prerequisite for subsequent changes in
the phagosomal milieu that affects gene expression of
intracellular Salmonella, or that there are subsets of
genes whose expression is induced by low pH, and others
whose expression is induced by other signals such as
nutrient starvation or low concentrations of divalent
cations.
Role of two-component regulatory systems in SPI2
gene expression
The expression of SPI2 virulence genes is dependent on
the function of the two-component regulatory system
SsrAB, encoded by SPI2. Inactivation of SsrA or SsrB
resulted in a strong attenuation of virulence (Shea et al.,
1996; Valdivia and Falkow, 1997). SPI2 gene expression
was not induced in the ssrB¹ background in vitro (this
study) or in intracellular bacteria (Valdivia and Falkow,
1997). The induction of reporter gene fusions in intracellular bacteria indicated that environmental signals received
and transmitted by SsrAB are present during growth in
vitro as well as within infected host cells.
A strong correlation between the expression of various
SPI2 genes and the amount of Mg2þ present in the growth
medium was observed. Furthermore, only very low levels
of SPI2 proteins were detected in the phoP¹ background.
However, levels of SPI2 proteins were dependent on the
Mg2þ concentration of the growth medium in the background of the pho-24 mutation. The observation that the
pho-24 allele did not result in a constitutive expression of
SPI2 genes may indicate that SPI2 gene expression is
not directly regulated by but modulated via the PhoPQ system. The effect of PhoPQ on the expression of SPI2 genes
described here is in contrast to the observations of Valdivia and Falkow (1997), who reported that the intramacrophage induction of a GFP fusion to a SPI2 promoter
was independent of PhoP. Possible explanations of this
difference may be the type of reporter fusion used (singlecopy chromosomal fusions in this study compared with
plasmid-encoded fusions) or the existence of signals
specific for the intracellular environment that compensate
the PhoP¹ phenotype. It is worth noting that a phoP¹
background results in attenuation of virulence (Miller et
al., 1989), defects in intramacrophage accumulation (Miller
et al., 1989; Hensel et al., 1998), as well as in growth
defects in minimal media containing limiting amounts of
Mg2þ (Garcia Vescovi et al., 1996; Soncini et al., 1996).
An alternative explanation to the modulation of SPI2
expression via PhoPQ is a direct sensing of divalent cation
concentrations by SsrA, or the sensing of changes to the
cell envelope under conditions of Mg2þ or phosphate
starvation. Further work has to be carried out to identify
the signal integrated by the SsrAB system.
Inverse regulation of SPI1 and SPI2
Two structurally similar type III secretion systems for
virulence determinants are present in S. typhimurium.
This observation raised the question of how the secretion
of target proteins can be directed via either of the secretion
systems. Our results indicate that the expression of the
type III secretion systems of SPI1 and SPI2 is induced
by different environmental conditions. It has been demonstrated that certain SPI1 genes are PhoPQ repressed
(Pegues et al., 1995), and it was assumed that expression
of SPI1 genes is not required after entry into cells or penetration of the epithelial layer. In contrast, the expression of
SPI2 genes is positively modulated by PhoPQ and
induced by the intracellular location of bacteria. We have
not been able to define media or growth conditions
under which the expression of both type III secretion
systems of SPI1 and SPI2 is induced. The comparison
of in vitro conditions activating SPI1 and SPI2 gene
expression indicates that environmental conditions that
result in the simultaneous induction of both SPI are not
likely to occur in vivo.
Co-ordinated regulation of SPI2 gene expression
Our data indicate that SPI2 represents a virulence locus
whose expression is co-ordinately regulated and activated
in a specific phase of the pathogenesis. S. typhimurium
possesses sensory systems to detect stress conditions
and situations of nutritional deprivation present in the
vacuolar environment of a host cell. Previous reports indicate that nutritional deprivation within the vacuole results
in the induction of various stress response systems and
high-affinity uptake systems for ions and nutrients (Buchmeier and Heffron, 1990; Garcia del Portillo et al., 1992;
Burns-Keliher et al., 1998). Mutants in PhoP or the transport systems MgtCB are unable to proliferate within the
intracellular environment of infected macrophages. These
mutants have been rescued by addition of high amounts
of extracellular Mg2þ (Blanc-Potard and Groisman, 1997),
however the physiological significance of these observations is controversial (Moncrief and Maguire, 1998).
We suggest that the environmental stimuli ‘low-Mg2þ
䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1759–1773
SPI2 gene expression 1769
concentration’ and ‘phosphate starvation’ signal to
Salmonella that it is within a vacuole of the host cell.
One of the specific responses is the induction of SPI2
gene expression. Proteins translocated by the SPI2encoded type III secretion system may then influence
the intracellular trafficking of the Salmonella -containing
vacuole and contribute to the survival and accumulation
of Salmonella inside the host cell. The regulation by
environmental signals reported here further support the
role of SPI2 during intracellular life.
The regulatory cascade postulated in this work
also reflects an important event in the evolution of the
Salmonella virulence. ssrAB, the local regulatory system
for the expression of SPI2 genes, was probably introduced
together with genes encoding the type III secretion system
of SPI2 in the same event of horizontal gene transfer.
However, the bacterial cell gained control over this
virulence factor by modulation of SPI2 expression by the
PhoPQ system. A similar proposal has been made for
the pmr locus (Gunn and Miller, 1996; Gunn et al., 1996),
and for the mgtCB genes within SPI3 which are also
under the control of PhoPQ (Blanc-Potard and Groisman,
1997). Our results extend the role of the PhoPQ system as
a global regulator of Salmonella virulence.
There are several questions arising from the environmental regulation of SPI2 reported here. Further work is
needed to reveal how SPI2 gene expression is modulated
by PhoPQ, and how the regulatory hierarchy of SPI2
genes is organized. Because the two-component regulatory
system SsrAB is absolutely required for the expression of
SPI2 genes, identification of the signal acting on the
sensory component SsrA will be crucial for the understanding of SPI2 function and intracellular pathogenesis
of S. typhimurium.
Table 2. Bacterial strains, phages and plasmids used in this study.
Strain, phage or plasmid
Description
Reference/ source
E. coli strains
DH5␣
CC118 ␭ pir
S17-1 ␭ pir
BL21(DE3)
M15
See reference
␭ pir lysogen
␭ pir lysogen
See reference
pREP
Gibco-BRL
de Lorenzo and Timmis (1994)
de Lorenzo and Timmis (1994)
Novagen
Qiagen
S. typhimurium strains
NCTC12023
LT2a
CS015
CS022
EE638
P8G12
MvP127
MvP131
MvP239
MvP244
MvP266
Wild type
Wild type
phoP -102::Tn10d -Cm, PhoP¹
pho-24
sipC ::lacZY
ssrB ::mTn5
sseA ::luc
ssaB ::luc
EE638 in NCTC12023
ssaB ::luc ssrB ::mTn5
ssaJ ::luc
NCTC
SGSC
Miller et al. (1989)
Miller and Mekalanos (1990)
Hueck et al. (1995)
Shea et al. (1996)
Hensel et al. (1998)
This study
This study
This study
This study
Phages
␭1, 2, 5, 7
Clones from a library of S. typhimurium genomic DNA in ␭1059
Shea et al. (1996)
Plasmids
pUC18
pSKþ, pKSþ
pWSK29
pT7-Blue
pET21(þ)
pLB02
pGPL01
pQE30
pQE32
p1-4
p2-2
p5-2
p5-4
p7-26
p5-4-T7
p7-26-T7
pssrAB
pJD11
pJD12
Ampr, high-copy vector
Ampr, high-copy vector
Ampr, low-copy vector
Ampr, high-copy vector
Ampr, T7 expression vector
Ampr, luc suicide vector
Ampr, luc suicide vector
Ampr, His tag expression vector
Ampr, His tag expression vector
7 kb Pst I fragment of ␭1 in pT7-Blue
5.7 kb Bam HI fragment of ␭2 in pUC18
5.7 kb Eco RI fragment of ␭5 in pKSþ
5.5 kb Hin dIII fragment of ␭5 in pSKþ
7 kb Nsi I fragment of ␭7 in pSKþ
Ampr, p5-4 insert in pET21(þ)
Ampr, p7-26 insert in pET21(þ)
5.9 kb Kpn I/Pvu I fragment of SPI2 in pWSK29
ssaP in pQE30
sscA in pQE32
Gibco-BRL
Stratagene
Wang and Kushner (1991)
Novagene
Novagene
Gunn and Miller (1996)
Gunn and Miller (1996)
Qiagen
Qiagen
This study
This study
Hensel et al. (1998)
Hensel et al. (1998)
This study
This study
This study
This study
This study
This study
䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1759–1773
1770
J. Deiwick, T. Nikolaus, S. Erdogan and M. Hensel
Experimental procedures
Bacterial strains and growth conditions
Strains used in this study are listed in Table 2. E. coli and S.
typhimurium were routinely cultivated in Luria broth (LB) or on
LB agar (Miller, 1992). Minimal media were used as follows:
M9, E salts (Vogel-Bonner minimal salts); NCE salts (citratefree minimal salts) (Maloy et al., 1996) containing 1 mM
MgCl2 ; and intracellular salt medium (ISM) (Headley and
Payne, 1990). N salts minimal medium containing 0.1%
casamino acids (Difco) and 38 mM glycerol (Hmiel et al.,
1986) with various concentrations of divalent cations added
as indicated. For buffering of N salts medium over the entire
time of cultural growth, 100 mM Tris-HCl (pH 7.4) used in the
original formulation was replaced by 100 mM MOPS-NaOH
(pH 7.0) or 100 mM Bis/Tris-HCl (pH 7.0). For the analysis of
starvation conditions, MOPS-buffered salts minimal medium
(Neidhardt et al., 1974) was used with modifications for anaerobic culture conditions (Stewart and Parales, 1988) and carbon, nitrogen or phosphate starvation (Spector and Cubitt,
1992). The basal MOPS salts containing micronutrients,
15 mM NH4Cl, and 0.4% glucose has been used with 25 mM
K2 HPO4 /KH2 PO4 (pH 7.4) for high-phosphate and 0.113 mM
K2HPO4 /KH2PO4 for phosphate starvation conditions (O’Neal
et al., 1994). Other growth media used were brain–heart infusion medium (BHI, Difco), RPMI-1640 and DMEM. Routinely,
bacterial cultures of 3 ml volume were grown aerobically in
glass test tubes in a roller drum (New Brunswick) with agitation at 50 r.p.m. Antibiotics were used in the following concentrations: 50 ␮g ml¹1 carbenicillin, 50 ␮g ml¹1 kanamycin, 50 ␮g
ml¹1 chloramphenicol, 100 ␮g ml¹1 nalidixic acid, 20 ␮g ml¹1
tetracycline.
The pho-24 allele of strain CS022 and its derivatives was
confirmed by streaking on LB agar containing 40 ␮g ml¹1
X-phosphate and a selection of deep-blue colonies (Belden
and Miller, 1994).
For the generation of recombinant SsaP (rSsaP), a 374 bp
PCR fragment was generated using primers SsaP-For (5⬘CTCGGATCCAAAGTTGAGGGAAG-3⬘) and SsaP-Rev (5⬘GTGAAAGCTTCATTCGCTATTCTTA-3⬘); and for rSscA, a
475 bp PCR fragment was generated using SsaC-For
(5⬘-CTCGGATCCCGACCCTACAACAG-3⬘) and SsaC-Rev
(5⬘-GTGAAAGCTTAGCTCCTGTCAGAAAG-3⬘) introducing
Bam HI and Hin dIII sites as indicated by underlining. After
digestion with Bam HI and Hin dIII, fragments were ligated to
pQE30 and pQE32 vectors to generate pJD11 and pJD12 for
the expression of rSsaP and rSscA, respectively, introducing
N-terminal His-tag fusions.
Recombinant SPI2 proteins, generation of antibodies
and Western blotting
Recombinant proteins rSsaP and rSscA were expressed
according to standard protocols (Qiagen), and purified by
metal-chelating chromatography using ‘Hi-trap’ columns
according to the manufacturer (Pharmacia). Antigens were
administered to rabbits by subcutaneous injection of purified
protein (about 1 mg) and emulsified with complete or incomplete Freund’s adjuvant for initial or booster immunisations
respectively (Harlow and Lane, 1988). Antibody titres were
checked by Western blots using the purified antigen and
total cell fractions of E. coli expression SPI2 genes under
the control of the T7 promoter.
For Western blot analysis, total bacterial cells were resuspended in SDS–PAGE sample buffer (Schägger and von
Jagow, 1987), boiled for 5 min and protein was separated
on 12% SDS–PAGE gels using the tricine buffer system
(Schägger and von Jagow, 1987). Protein was transferred
into nitrocellulose membranes (Schleicher and Schuell)
using a semidry blotting apparatus (Bio-Rad) with a buffer
system according to Kyhle-Andersen (1984). Detection of
antibodies was performed using the ECL system according
to the manufacturer’s protocol (Amersham).
DNA biochemistry
DNA manipulations were performed according to standard
procedures (Sambrook et al., 1989). For the sequencing and
cloning of ssrB and the 3⬘ end of ssrA, a Pst I fragment of a
clone ␭1 (Shea et al., 1996) harbouring a portion of SPI2
was subcloned to generate p1-4. Sequence analysis was
performed by a primer-walking strategy using the dye
terminator chemistry on an ABI 373 sequencer. The DNA
sequence is available under EMBL accession number Z95891.
Plasmids for expression of SPI2 genes under control of the
T7 promoter were generated by subcloning the insert of plasmids p5-4 and p7-26 in pET21(þ) to generate p5-4-T7 and
p7-26-T7 respectively. E. coli BL21(p5-4-T7) and BL21(p726-T7) expressed sscA and ssaP, respectively, after induction by IPTG. A plasmid for complementation and expression
of ssrAB under control of the T7 promoter was constructed by
subcloning a 3.7 kb Bam HI/Kpn I fragment of p2-2 into p1-4.
The resulting construct was digested by Pvu I, blunt ended
by treatment with the Klenow fragment of DNA polymerase
I, followed by digestion with Kpn I. A 5.9 kb fragment was
recovered and ligated to Kpn I/Eco RV-digested pWSK29 to
generate pssrAB.
Generation of reporter gene fusions
Fusions of the reporter gene firefly luciferase to various genes
in SPI2 were obtained using the suicide vectors pLB02 and
pGPL01 (Gunn and Miller, 1996), kindly provided by Drs
Gunn and Miller (Seattle). For the generation of a fusion to
ssaB, a 831 bp Eco RV fragment of p2-2 was subcloned in
Eco RV-digested pSKþ. For the generation of a transcriptional fusion to sseA , a 1060 bp Sma I/Hin cII fragment of
p5-4 was subcloned in pSKþ. The inserts of the resulting
constructs were recovered as Eco RI/Kpn I fragments and
ligated with Eco RI/Kpn I-digested reporter vectors pGPL01
and pLB02. For the generation of a transcriptional fusion to
ssaJ, a 3 kb Sma I/Kpn I fragment of p5-2 was directly subcloned in pGPL01 and pLB02 and maintained in E. coli
CC118 ␭ pir. Constructs with transcriptional fusions of SPI2
genes to luc were then integrated into the chromosome of
S. typhimurium by mating between E. coli S17-1 ␭ pir harbouring the respective construct and a spontaneous mutant
of S. typhimurium resistant to 100 ␮g ml¹1 nalidixic acid, and
then selection for exconjugants resistant to carbenicillin and
nalidixic acid. The targeted integration into the chromosome
䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1759–1773
SPI2 gene expression 1771
of S. typhimurium was confirmed by Southern analysis.
Fusions were then moved into a mouse-passaged strain of
S. typhimurium 12023 by P22 transduction according to
standard procedures (Maloy et al., 1996).
Assay of reporter genes
␤-Galactosidase activities of reporter gene fusions were
determined according to standard procedures (Miller, 1992).
Luciferase activities of bacterial cultures were determined
using the Promega (Heidelberg) luciferase assay kit or custom-made reagents accordingly. Briefly, bacteria were pelleted
by centrifugation for 5 min at 20 000 × g at 4⬚C and resuspended
in lysis buffer (100 mM KH2PO4 – KOH, pH 7.8, 2 mM EDTA,
1% Triton X-100, 5 mg ml¹1 bovine serum albumin, 1 mM
DTT, 5 mg ml¹1 lysozyme). Lysates were incubated for 15 min
at room temperature with repeated agitation and subjected to
a freeze–thaw cycle. Aliquots of the lysates (25 ␮l) were transferred into microtitre plates (Microfluor, Dynatech) and immediately assayed after addition of 50 ␮l of luciferase reagent
[20 mM Tricine-HCl, pH 7.8, 1.07 mM (MgCO3 )4Mg(OH)2 ,
100 ␮M EDTA, 33.3 mM DTT, 270 ␮M Li3-coenzyme A,
470 ␮M D(¹)-luciferin, 530 ␮M Mg-ATP] for photon emission
using the TriLux MicroBeta luminometer (Wallac). All assays
were carried out in triplicate and repeated on independent
occasions. Luc activities of equal amounts of cells as adjusted
by OD 600 are shown.
Cell culture and infection assays
The macrophage-like cell line J774.A1 was maintained in
DMEM with 10% fetal calf serum at 37⬚C in an atmosphere
containing 5% CO2 . J774.A1 cells were seeded at ⬇ 5.0 × 105
cells per well in 24-well tissue culture dishes and incubated
overnight. Bacterial strains were grown in LB broth to stationary phase and washed three times in PBS, opsonized for
20 min in 10% heat-inactivated normal mouse serum. Bacteria were added to the cell monolayer at a multiplicity of
infection (m.o.i.) of 10 : 1 and centrifuged for 5 min at 300 × g.
After incubation for 20 min at 37⬚C, the infected cells were
washed three times with prewarmed PBS and incubated
with media containing 12 ␮g ml¹1 gentamicin for 90 min to
kill extracellular bacteria. The infected monolayers were
either lysed or further incubated for 20 h in the presence of
6 ␮g ml¹1 gentamicin. The cells were lysed by addition of
1 ml 0.1% Triton X-100 in PBS per well. For determination of
colony-forming units (cfu), aliquots were plated on Mueller–
Hinton plates with a WASP spiral plater (Meintrup) at
appropriate dilutions.
The luciferase activity of intracellular bacteria was determined as described above. Briefly, the lysates from the gentamicin protection assay were centrifuged at 4⬚C for 5 min at
15 000 × g. After aspirating the supernatant, the bacterial pellets were resuspended in 100 ␮l of luciferase lysis buffer for
15 min at RT and frozen immediately at ¹20⬚C. The frozen
samples were thawed and 25 ␮l used for the Luc assay.
The luciferase activity per recovered bacteria was calculated.
Acknowledgements
This work was supported by DFG grant 1964/2 and the BMBF
䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 1759–1773
grant 01KI 9609 ‘Molekulare Pathogenese der Salmonellosen’. We would like to thank Drs S. I. Miller, J. Gunn, C. J.
Hueck, and M. E. Maguire for providing bacterial strains and
plasmids. Critical reading of the manuscript by Drs W.-D.
Hardt and D. W. Holden is acknowledged. We are indebted
to Drs E. Riedel and J. Heesemann for generous support of
this work and stimulating discussions.
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