Microbiology (2005), 151, 579–588
DOI 10.1099/mic.0.27520-0
Virulence attenuation in Salmonella enterica
rcsC mutants with constitutive activation of
the Rcs system
Clara B. Garcı́a-Calderón, Meritxell Garcı́a-Quintanilla, Josep Casadesús
and Francisco Ramos-Morales
Correspondence
Francisco Ramos-Morales
[email protected]
Received 27 July 2004
Revised
12 October 2004
Accepted 21 October 2004
Departamento de Genética, Facultad de Biologı́a, Universidad de Sevilla, Apartado 1095,
41080 Sevilla, Spain
Mutations in rcsC that result in constitutive colanic acid capsule synthesis were obtained in
Salmonella enterica serovar Typhimurium. Most rcsC alleles were dominant; however, recessive
rcsC alleles were also found, in agreement with the postulated double role (positive and negative)
of RcsC on the activation of the RcsB/C phosphorelay system. Salmonella rcsC mutants with
constitutive activation of the Rcs system are severely attenuated for virulence in BALB/c
mice and their degree of attenuation correlates with the level of Rcs activation. Partial relief of
attenuation by a gmm mutation indicates that capsule overproduction is one of the factors leading
to avirulence in constitutively activated rcsC mutants.
INTRODUCTION
The Rcs phosphorelay system was initially characterized in
Escherichia coli as a two-component positive regulator of
colanic acid capsule synthesis encoded by rcsC and rcsB (Brill
et al., 1988; Gottesman et al., 1985; Stout & Gottesman,
1990). The sensor protein RcsC, a hybrid histidine kinase,
and the transcriptional activator RcsB are the main components of the system, which also includes a second
transcriptional activator, RcsA (Stout et al., 1991) and an
intermediate phosphotransmitter, YojN (Takeda et al.,
2001). The Rcs system is also a negative regulator of the
flagellar master operon flhDC (Francez-Charlot et al., 2003),
whose products are necessary for the expression of genes
involved in flagellum synthesis, motility and chemotaxis.
The first environmental signal shown to strongly and
rapidly induce colanic acid synthesis was osmotic upshift
(Sledjeski & Gottesman, 1996). This effect was mediated
by the Rcs system. More recently it has been shown that
the Rcs phosphorelay can be activated when wild-type cells
are grown at 20 uC in the presence of glucose and a high
concentration of Zn2+ (Hagiwara et al., 2003). Furthermore, several genetic conditions that result in alterations
in envelope composition or integrity are known to activate
the Rcs system. For example, a mutation in the mdoH
gene involved in the biosynthesis of membrane-derived
oligosaccharides (Ebel et al., 1997) or overproduction of
the DnaJ-like transmembrane protein DjlA (Kelley &
Georgopoulos, 1997) has been shown to stimulate the
expression of genes regulated by the E. coli Rcs system. In
Abbreviations: CI, competitive index; Km, kanamycin.
0002-7520 G 2005 SGM
Printed in Great Britain
Salmonella enterica serovar Typhimurium, mutations in the
essential gene igaA (Cano et al., 2001) [also known as yrfF
or mucM (Costa & Antón, 2001)] result in mucoidy and
reduced motility (Cano et al., 2002). A recent report has
shown that viable igaA mutations also result in virulence
attenuation in the mouse model (Domı́nguez-Bernal et al.,
2004). Like the other phenotypes associated with igaA
mutations (Cano et al., 2002), attenuation is suppressed by
null mutations in rcsC, yojN or rcsB (Domı́nguez-Bernal
et al., 2004), suggesting that overactivation of the Rcs
phosphorelay causes loss of virulence. This hypothesis is
addressed in this study.
Here, we describe the isolation of Salmonella rcsC mutants
with constitutive activation of the Rcs system, and the
characterization of amino acid substitutions that lead to
different degrees of activation of the system. Virulence trials
with constitutively activated rcsC mutants provide a direct
demonstration of the link between activation of the Rcs
system and avirulence in mice. We also show that virulence
attenuation associated with activation of the RcsB/C system
is partially due to colanic acid overproduction.
METHODS
Bacterial strains, bacteriophages and strain construction.
S. enterica serovar Typhimurium strains used in this study are described in Table 1. Unless otherwise indicated, the strains derive from
the mouse-virulent strain 14028. Transductional crosses using phage
P22 HT 105/1 int201 (Schmieger, 1972) were used for strain construction operations. The transduction protocol was described by Maloy
(1990). To obtain phage-free isolates, transductants were purified by
streaking on green plates (Chan et al., 1972). Phage sensitivity was
tested by cross-streaking with the clear-plaque mutant P22 H5.
579
C. B. Garcı́a-Calderón and others
Table 1. Strains of S. enterica serovar Typhimurium used in this study
Name
14028
TH2775
SV3329
SV4379
SV4451
SV4609
SV4610
SV4756*
SV4757*
SV4758*
SV4767*
SV4948*
SV4949*
SV4952*
SV4953*
SV4954*
SV4959*
SV4960*
SV4985
Description
Source
Wild-type
LT2 flhDC5213 : : MudA
LT2 ompC396 : : Tn10
rcsA : : MudQ
rcsA : : MudJ
gmm-21 : : MudJ
flhDC5213 : : MudA
rcsC53
rcsC54
rcsC55
rcsC56
rcsC58
rcsC59
rcsC60
rcsC61
rcsC62
rcsC63
rcsC64
DUP [hisH9962* MudP*
cysA1586]
ATCC
K. Hughes, University of Washington
Laboratory stock; allele ompC from SH7241 (SGSC)
Laboratory stock
Laboratory stock
Insertion obtained from SV4407 (Cano et al., 2002)
This work; insertion obtained from TH2775
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
Duplication obtained from SV3193 (Camacho &
Casadesús, 2001)
*gmm-21 : : MudJ, flhDC5213 : : MudA, rcsB : : KmR, rcsA : : MudJ and rcsA : : MudQ derivatives of these strains
were also used where appropriate, as indicated in the text.
Media and chemicals. The culture medium for bacteria was
Motility assays. Liquid cultures were prepared in motility medium
Luria–Bertani (LB) broth. Solid media contained agar at 1?5 % final
concentration. Antibiotics were used at the following concentrations: mecillinam, 1 mg ml21; tetracycline, 20 mg ml21, kanamycin,
50 mg ml21; chloramphenicol, 20 mg ml21; ampicillin, 30 mg ml21.
Motility assays were carried out in LB prepared without yeast extract
(Gillen & Hughes, 1991). Solid motility medium contained agar at
0?25 % final concentration.
and incubated at 37 uC with shaking. At the stage of mid-exponential
growth, a sterile toothpick was soaked in the culture and used to
inoculate a motility agar plate. The plate was incubated at 37 uC.
The diameter of the bacterial growth halo was measured every hour.
Isolation of mucoid mutants in mecillinam plates. The initial
isolation of mucoid mutants was performed with mecillinam plates,
as previously described (Costa & Antón, 2001). Stationary cultures
(100 ml) of S. enterica serovar Typhimurium strain 14028 were
plated on LB agar containing 1 mg mecillinam ml21 at 37 uC. Most
mecillinam-resistant colonies were mucoid.
Construction of rcsB mutants. Disruption and replacement of
rcsB with a kanamycin (Km)-resistance gene in selected rcsC mutants
(SV4756, SV4757, SV4758 and SV4949) were performed by the
method described by Datsenko & Wanner (2000). Briefly, the Kmresistance gene from plasmid pKD4 was PCR-amplified with primers
59-ATT ATT GCC GAT GAC CAC CCG ATT GTA CTG TTC GGT
ATT CGT GTA GGC TGG AGC TGC TTC-39 and 59-AGA GAG
ATA GTT GAG CAG CGC GAT ATC ATT CTC TAC GCC CCA
TAT GAA TAT CCT CCT TAG-39. The PCR product was used to
transform rcsC mutant strains carrying the Red recombinase expression plasmid pKD46. Insertion of the Km-resistance gene was
verified by PCR with primers flanking the rcsB locus (59-AGC
GGA ATT CAG GAG GAA TAC ATG AAC AAT ATG AAC G-39
and 59-GTG AAA GCT TGT CGA CAA GCG ATT TAT TCT TTG
TCT G-39).
b-Galactosidase assays. Levels of b-galactosidase activity were
assayed for exponential cultures in LB medium as described by
Miller (1972), using the CHCl3/SDS permeabilization procedure.
580
DNA amplification with PCR and DNA sequencing. Amplifica-
tion reactions were carried out in a Perkin Elmer Gene-Amp PCR
System 2400 (Perkin Elmer Cetus). The final volume of reactions
was 50 ml, and the final concentration of MgCl2 was 1 mM. Reagents
were used at the following concentrations: dNTPs, 200 mM; primers,
1 mM; and Taq polymerase (Expand High Fidelity PCR System,
Roche Diagnostics), 1 unit per reaction. Amplification of the rcsC
coding sequence was carried out with primers 59-GTA ATG TTA
TTGCTG TGC GAG-39 and 59-TAT GTT ACC CAG CCT GAT GG39. The products of amplification were sequenced with an automated
DNA sequencer (Sistemas Genómicos) using the primers 59-GTA
ATG TTA TTG CTG TGC GAG-39, 59-CAT GAG CGG ATT ATG
AAA TAC-39, 59-TGA GTA ACG AAC TGG CGC AC-39, 59-CTG
TTG AGC AAC GCC ATT AAG-39 and 59-GAT TCC GTT GGA
AAG AGC-39. Mutations were confirmed by sequencing at least two
independent PCR products.
Mouse mixed infections. Eight-week-old female BALB/c mice
(Charles River Laboratories) were subjected to mixed infections.
Groups of three or four animals were inoculated with a 1 : 1 ratio of
the mutant and the wild-type. Bacteria were grown overnight at
37 uC in LB with shaking, diluted into fresh medium (1 : 100), and
grown to an OD600 of 0?35–0?6. Intraperitoneal inoculation was
performed with 0?2 ml physiological saline containing 105 c.f.u.
Bacteria were recovered from spleen 48 h after inoculation and c.f.u.
were enumerated on LB and on selective medium (LB with mecillinam for rcsC constitutive mutants). A competitive index (CI) for
each mutant was calculated as the ratio between the mutant and the
Microbiology 151
Constitutively activated Salmonella rcsC mutants
Fig. 1. Schematic representation of the Salmonella RcsC protein. TM1 and TM2 represent the predicted transmembrane
domains. H479 and D875 are the histidine and the aspartic residues phosphorylatable in the transmitter and the receiver
domain, respectively. The amino acid changes found in the mutants described in this work are indicated.
wild-type strain in the output (bacteria recovered from the host
after infection) divided by their ratio in the input (initial inoculum)
(Beuzón & Holden, 2001; Freter et al., 1981; Taylor et al., 1987).
In vitro CI calculation. Overnight cultures of the wild-type and
mucoid mutant strains were diluted and mixed in LB in order to get
an OD600 of about 0?05. The mixed culture was incubated in a shaker at
37 uC for 24 h; c.f.u. for both strains were enumerated on LB at the
beginning and at the end of the experiment. Strains were distinguished
by the mucoidy of the mutant colonies. A CI for each mutant was calculated as the ratio between the mutant and the wild-type strain in the
output (24 h) divided by their ratio in the input (initial mix).
RESULTS
Isolation of mucoid rcs mutants
Mucoid mutants were isolated on mecillinam plates as
described by Costa & Antón (2001). Out of 300 independent
mucoid mutants of spontaneous origin, 27 carried mutations 50 % co-transducible with ompC396 : : Tn10dTc. This
insertion is linked to rcsC, yojN and rcsB (McClelland et al.,
2001). An analogous gene arrangement is found in E. coli
(Gottesman et al., 1985). Reconstruction experiments confirmed that the mutations responsible for mucoidy and
mecillinam resistance were linked to ompC.
DNA sequence analysis of rcsC mutants
To identify mutations located in rcsC, and to characterize
them at the DNA sequence level, PCR amplification
products of the rcsC coding region from the 27 mucoid
mutants were sequenced. For 24 mutants, a mutation was
found in rcsC. The nature and position of the mutations
are indicated on the diagrammatic representation of the
predicted RcsC product in Fig. 1 and Table 2. Eleven
Table 2. Summary of features of rcsC constitutively activated mutants compared to wild-type
Values are means from representative experiments. b-Galactosidase activities were calculated for appropriate derivatives of the strains indicated. CI was calculated for representative rcsC mutants with different
degrees of activation after mouse mixed infections with the wild-type strain (mouse) or after growing
together with the wild-type strain in LB for 24 h, as described in Methods. D, dominant; R, recessive;
2, not applicable or not determined.
Strain
14028
SV4949
SV4756
SV4952
SV4767
SV4960
SV4959
SV4948
SV4954
SV4953
SV4757
SV4758
Allele
+
rcsC
rcsC59
rcsC53
rcsC60
rcsC56
rcsC64
rcsC63
rcsC58
rcsC62
rcsC61
rcsC54
rcsC55
Amino acid
substitution
2
E354K
H355R
V438F
V438I
F473I
F473Y
P484L
P484S
I531S
N877S
T903A
http://mic.sgmjournals.org
Dominance
b-Galactosidase activity
CI
(Miller units)
2
D
D
D
D
D
D
D
D
D
R
R
gmm : : lac
flhDC : : lac
Mouse
LB
4
53
253
112
218
913
786
2200
2080
955
1261
798
904
220
128
142
144
89
88
54
69
85
79
84
2
1?461023
2?061024
2
2
2
2
2
2
2
2?061026
1?861025
2
0?89
0?56
2
2
2
2
0?21
2
2
0?59
1?12
581
C. B. Garcı́a-Calderón and others
different mutations were found among the 24 rcsC mutants
sequenced. The following mutations were independently
isolated more than once: V438I, F473Y, P484L, P484S and
N877S. When the same mutation was isolated several times,
only one mutant was chosen for further study.
All the mutations were located in the putative main cytoplasmic portion of RcsC. E354K, H355R, V438F and V438I
are in the putative input domain; F473I, F473Y, P484L,
P484S and I531S in the putative histidine kinase domain;
N877S and T903A in the putative receiver domain. In silico
analysis of the predicted RcsC structure, based on its
homology with well-characterized sensors or response
regulators, has permitted predictions of the putative effect
of some of the mutations on the activity of the RcsC protein (see Discussion).
Dominance/recessivity of constitutively
activated rcsC alleles
To examine whether the rcsC mutations under study were
dominant or recessive for the mucoid phenotype, they were
introduced into a merodiploid strain carrying a chromosomal duplication of the hisH–cysA region, which encompasses centisomes 44 to 53 and includes rcsC (Camacho
& Casadesús, 2001). For dominance/recessivity analysis,
strain SV4985 was transduced with a P22 HT lysate grown
on derivatives of the rcsC mutant strains that carried the
ompC396 : : Tn10dTc mutation. Because this insertion is
50 % linked to the rcsC mutations, dominance can be
(a)
(b)
(c)
(d)
Wild-type
582
rcsC55
expected to result in about 50 % of mucoid transductants,
while recessivity will result in an absence of mucoid transductants. Most mutations were dominant: only two mutations in the putative RcsC receiver domain were found to
be recessive (Table 2). Since recessive mutations usually
correspond to loss-of-function mutations, this result
supports a negative role for this domain in the activation
of the Rcs phosphorelay (see Discussion).
Different degrees of activation of the RcsB/C
system caused by rcsC mutations as measured
by increase in wca expression
The phenotype of a representative mucoid mutant is compared with the wild-type in Fig. 2(a, b). Mucoidy is a
consequence of activation of the RcsB/C system, which
leads to an increase in colanic acid capsule synthesis
(reviewed by Gottesman, 1995). The colanic acid polysaccharide capsule biosynthetic genes (wca genes) are at
least 19 clustered genes, likely arranged in a single long
operon, in E. coli and S. enterica (Stevenson et al., 1996,
2000; Stout, 1996). Therefore, the activity of a wca : : lacZ
fusion can be used as a reliable estimation of the degree
of activation of the system in the different mutants. gmm
(also called wcaH) was used as a representative of the
whole wca cluster. Analysis of b-galactosidase activity
from exponential cultures in LB medium showed that
a gmm : : lacZ fusion was repressed in the wild-type
(<5 Miller units), while the b-galactosidase activities found
in the mutants ranged from 53 to 2200 units (Fig. 3a and
Fig. 2. Constitutively activated rcsC mutants
are mucoid and non-motile. Phenotypes of
colonies growing in LB plates for the wildtype strain (a) and one representative rcsC
mutant (b). Motility of the wild-type strain (c)
and the rcsC mutant SV4758 (d). Bacteria
were spotted onto motility agar and incubated for 6 h at 37 6C.
Microbiology 151
Constitutively activated Salmonella rcsC mutants
of inoculation. A representative experiment is shown in
Fig. 2(c, d).
Constitutive rcsC mutations decrease flhDC
expression
Since all the rcs mutants described above were non-motile,
the expression of flagellar genes in these mutants was
monitored using a transcriptional lac fusion in the flagellar
master operon flhDC. This operon encodes transcription
factors necessary to trigger the genetic cascade that controls
flagellar synthesis (Claret & Hughes, 2000; Furness et al.,
1997; Gygi et al., 1995; Macnab, 1996). The results are shown
in Fig. 3(b). As expected, expression of the flhDC fusion
was significantly decreased in all the rcsC mutants under
study. In addition, expression of flhDC negatively correlates with gmm expression. This correlation was anticipated
by the fact that the Rcs system activates wca genes but
represses flagella synthesis through flhDC.
An rcsB null mutation suppresses both
mucoidy, mecillinam resistance and motility
defects in constitutively activated rcsC mutants
Fig. 3. Effect of rcsC mutations on the expression of
gmm : : lac and flhDC : : lac fusions. (a) b-Galactosidase activities of derivatives of the indicated strains carrying a MudJ
insertion in gmm; (b) b-galactosidase activities in Miller units for
derivatives of rcsC and wild-type strains carrying a MudA insertion in flhDC. The data shown are the means and standard
deviations of at least three independent experiments. Data
are ordered according to the location of mutations. wt, Wildtype. rcsC mutants are named according to their amino acid
substitution.
Table 2). This enormous range of gmm activation (from
10-fold to 500-fold) indicated that the constitutive rcsC
alleles under study were highly heterogeneous.
Constitutively activated rcsC mutants are
non-motile
A previous study showed that igaA mutants, which are also
mucoid, have a defect in motility that can be suppressed
by null mutations in rcsC or rcsB (Cano et al., 2002). This
result suggested that activation of the RcsB/C system could
cause both mucoidy and loss of motility. Microscopic
observation of exponential cultures of the mucoid rcsC
mutants showed that all were non-motile. This result was
confirmed by comparing the wild-type and the mutant
strains in motility agar plates (see Methods). While the wildtype was able to grow, forming a halo whose diameter
increased progressively, the mutants grew only in the area
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RcsB is a downstream element in the signal transduction
pathway that leads from RcsC to transcriptional regulation
of the wca cluster (and of other genes). Hence, an rcsB null
mutation can be expected to suppress the phenotypes of
constitutively activated rcsC mutants. This prediction was
tested by replacing rcsB through transformation with a Km
resistance gene (see Methods) in the rcsC mutants. These
rcsB rcsC double mutants were non-mucoid, motile and
sensitive to mecillinam. These results confirmed that
mecillinam resistance, lack of motility and mucoidy resulted
from canonical activation of the Rcs system via RcsB.
S. enterica serovar Typhimurium rcsC mutants
with constitutive activation of the Rcs system
are avirulent in mice
A recent study has shown that viable igaA mutants are
attenuated for virulence in mice, and that attenuation
is suppressed by null mutations in rcsC, yojN or rcsB
(Domı́nguez-Bernal et al., 2004). These observations provided evidence that overactivation of the Salmonella Rcs
system causes avirulence (Domı́nguez-Bernal et al., 2004).
The rcsC mutants isolated in this work are highly suited to
test this hypothesis directly, since they affect one component
of the phosphorelay and exhibit different degrees of activation of the system. For virulence assays, BALB/c mice were
infected intraperitoneally with a mixture of the wild-type
strain and one of the mutants, and a CI was calculated for
each mutant (see Methods). All the mutants were extremely
attenuated and a correlation was found between the
individual levels of Rcs activation and their degrees of
attenuation, with CI ranging between 1?461023 and
261026 (Fig. 4 and Table 2). As expected, an rcsB mutation was able to completely suppress the attenuation of
these mutants (Fig. 4).
583
C. B. Garcı́a-Calderón and others
Fig. 4. Capsule overproduction is one factor
contributing to attenuation in constitutively
activated rcsC mutants. A competitive index
(CI) was calculated for rcsC mutants, for
rcsB rcsC, gmm rcsC and rcsA rcsC
double mutants, and for a gmm rcsA rcsC
triple mutant, after intraperitoneal infections
of BALB/c mice. rcsC mutants are ordered
from the lowest to the highest degree of
activation of the Rcs system and are named
after the changed amino acid. CI was also
calculated for an flhDC mutant (strain
SV4610) and a gmm mutant (strain
SV4609). The CIs are the means from at
least three infections. Error bars represent
the standard deviation.
To discard a potential general effect of these rcsC mutations
on bacterial growth outside the host, competition assays
were performed in LB. In vitro CI for all mutants ranged
from 0?56 to 1?12 (the only exception was SV4948, with a
CI of 0?21). Thus, the dramatic competition defect observed
in the colonization of target organs in mice cannot be
explained by the slight growth defects observed in vitro for
some strains.
A gmm mutation partially suppresses avirulence
in constitutively activated rcsC mutants
A priori, attenuation in constitutively activated rcsC mutants
could be caused by activation or repression of one or more
genes or operons regulated by the Rcs system. Two sets of
genes whose regulation by the Rcs system is well known,
both in E. coli and in S. enterica serovar Typhimurium, are
colanic acid genes (Brill et al., 1988; Cano et al., 2002), and
genes involved in flagella synthesis and in chemotaxis
(Cano et al., 2002; Francez-Charlot et al., 2003). Control of
flagella and chemotaxis is exercised through the master
operon flhDC (Macnab, 1996). Salmonella flhDC mutants
are defective in invasion of in vitro cultured epithelial cells
due to their lack of motility (Schmitt et al., 2001; Young
et al., 2000) and a role in virulence for these genes has
been shown in other bacteria (Givaudan & Lanois, 2000;
Tans-Kersten et al., 2004). However, a S. enterica serovar
Typhimurium flhDC mutant is not attenuated (Fig. 4 and
Domı́nguez-Bernal et al., 2004), indicating that lack of
motility or of expression of other genes activated by FlhDC
is not responsible for the attenuation of constitutively
activated rcsC mutants. Another possibility was that overproduction of colanic acid capsule could have a negative
effect upon virulence. To test this possibility, we measured
the CI for rcsC gmm double mutants. These are nonmucoid, since gmm is one of the structural genes necessary
for colanic acid synthesis. As shown in Fig. 4, attenuation
of the constitutively activated rcsC mutants was partially
suppressed by the gmm mutation. However, their CIs were
significantly <1 in all cases, and correlated with the degree
of activation of the system typical of each mutant, ranging
584
from 0?22 to 7?161024. Since the production of colanic
acid capsule is also dependent on the positive regulator
RcsA (Gottesman, 1995), an rcsA mutation should be able
to partially suppress the attenuation of rcsC mutants. This
prediction was confirmed by the CIs obtained by rcsA rcsC
double mutants (Fig. 4). In addition, a gmm rcsA rcsC54
mutant was no better in competition assays than the gmm
rcsC54 or the rcsA rcsC54 mutants (Fig. 4). Together these
results unambiguously suggest that overproduction of
colanic acid is one of the factors that contributes to the
avirulence of constitutively activated rcsC mutants. However,
since virulence is not fully restored, additional factors apart
from capsule overproduction must exist.
DISCUSSION
The Rcs system was initially described as a two-component
phosphorelay that controls colanic acid production in E. coli
(Brill et al., 1988; Gottesman et al., 1985). Two decades
later, this regulatory system has been shown to play roles
in other physiological processes such as the regulation of
the cell division genes ftsA and ftsZ (Carballès et al., 1999),
the modulation of swarming (Takeda et al., 2001), and the
control of flagella production (Francez-Charlot et al., 2003).
In S. enterica serovar Typhi, the RcsB/C system modulates
the expression of Vi antigen, flagellin and invasion proteins
(Arricau et al., 1998; Houng et al., 1992; Virlogeux et al.,
1995, 1996). A recent genome-wide analysis suggests that
the Rcs regulon of E. coli may include more than 40 loci
(Hagiwara et al., 2003; Oshima et al., 2002). Another study
shows that the RcsC sensor kinase is required for normal
biofilm development in E. coli and that more than 150 genes
are under Rcs control (Ferrieres & Clarke, 2003).
For a long time, the only physiological stimulus known
to activate the Rcs system in E. coli was osmotic shock
(Sledjeski & Gottesman, 1996). Growth conditions that
permit strong induction of Rcs by cold shock have recently
been described for E. coli (Hagiwara et al., 2003). In normal
laboratory growing conditions, the level of activation of the
Rcs system is very low; as a consequence, null mutations in
Microbiology 151
Constitutively activated Salmonella rcsC mutants
the components of the phosphorelay have no appreciable
effects on the phenotype. For this reason, the physiological
roles of the Rcs system have often been inferred from the
ability of null mutations in genes of the phosphorelay to
suppress mutations in other loci. For instance, in E. coli,
null mutations in mdoH lead to an increase in colanic acid
production that is suppressed by null mutations in rcsA,
rcsB or rcsC (Ebel et al., 1997). In a similar fashion, viable
igaA alleles of Salmonella enterica serovar Typhimurium
result in induction of wca genes, repression of flhDC and
avirulence, and all these phenotypes are suppressed by null
mutations in rcsB or rcsC (Cano et al., 2002; Costa & Antón,
2001; Domı́nguez-Bernal et al., 2004). This suppression
pattern has provided evidence that the S. enterica IgaA
functions as a negative regulator of the Rcs system (Cano
et al., 2002; Costa & Antón, 2001; Domı́nguez-Bernal et al.,
2004).
In this work, we describe a procedure to isolate rcsC
mutants of S. enterica with constitutive activation of the
Rcs system, followed by their characterization at the DNA
sequence level. Among 24 independent constitutively activated rcsC mutants, sequencing has revealed 11 different
amino acid substitutions, all located in the main cytoplasmic portion of the protein (Fig. 1). The mutations
causing stronger Rcs activation map inside the putative
histidine kinase domain (F473I, F473Y, P484L, P484S and
I531S) or the putative receiver domain (N877S and T903A).
Mutations in the input domain or in the histidine kinase
(transmitter) domain are dominant, while those in the
response regulatory (receiver) domain are recessive. Recessive mutations are usually loss-of-function mutations,
implying that, whereas most of our mutations increment
a positive RcsC activity (dominant, gain-of-function mutations), N877S and T903A abrogate a negative one. This is
in agreement with previous results obtained in E. coli: (i)
the rcsC137 allele (which causes constitutive colanic acid
overproduction) is recessive (Gottesman et al., 1985); (ii)
RcsC has both positive and negative regulatory effects on
the expression of the wca operon (Brill et al., 1988; Stout
& Gottesman, 1990); and (iii) the negative activity of RcsC
is localized to the receiver domain (Clarke et al., 2002). It
has been proposed that this negative activity is actually a
phosphatase activity (Clarke et al., 2002; Gottesman, 1995),
as is the case for other His-Asp phosphorelay systems
(reviewed by West & Stock, 2001). In E. coli, this putative
phosphatase activity requires the phosphorylatable Asp in
the receiver domain of RcsC (Clarke et al., 2002).
A search at the Conserved Domain Database (MarchlerBauer et al., 2003) reveals that RcsC contains two conserved
domains: the histidine kinase domain and the signal receiver
(response regulator) domain. The five mutations found in
the histidine kinase domain are located in a subdomain
called the histidine kinase A or dimerization/phosphoacceptor domain. This domain contains the phosphorylatable
His; in some instances, dimerization through this domain
has been proved to be necessary for activation (reviewed
http://mic.sgmjournals.org
by West & Stock, 2001). The three residues of this domain
that have been replaced in some of our mutants (Phe473,
Pro484 and Ile531) are present in the consensus sequence
of the domain. Pro484 is specially conserved and, interestingly, on the basis of the structure of the histidine kinase A
dimer in the E. coli protein EnvZ (Tomomori et al., 1999),
residues corresponding to Phe473 and Pro484 in RcsC are
predicted to play a role in dimerization as part of the dimer
interface.
Two different activating mutations were found in the
receiver domain of RcsC: N877S and T903A. This domain
is 38 % identical to the same domain in E. coli CheY. The
crystallographic structure of CheY reveals the presence of
five highly conserved residues in the response regulator
active site: Asp57 (the site of phosphorylation) (Sanders
et al., 1989); Asp12 and Asp13 (involved in coordination
of a Mg2+ ion essential for phosphorylation and dephosphorylation) (Stock et al., 1993); Lys109 (important for the
phosphorylation-induced conformational change) (Lukat
et al., 1991); and Thr87 (also involved in conformational
change through formation of a hydrogen bond between its
OH group and the active site acceptor) (Appleby & Bourret,
1998; Cho et al., 2000). The corresponding sites in RcsC are
Asp875, Asp831, Asp832, Lys925 and Thr903. Mutations
equivalent to T903A have been described for OmpR (T83A)
and CheY (T87A). In both cases, the substitution results in
constitutive activation, perhaps via a confomational change
which mimics the conformation of the phosphorylated
domain. It has been proposed that this substitution could
represent a universal activating substitution for response
regulators (Smith et al., 2004). This hypothesis may receive
further support from the observation that the corresponding T903A mutation also has an activating effect in RcsC.
Finally, the T87 residue in wild-type CheY and the T83
residue in OmpR have been proposed to mediate dephosphorylation of acyl phosphate (Brissette et al., 1991; Sanders
et al., 1989), since all similar proteins contain a hydroxy
amino acid residue at this position (Stock et al., 1990). This
is in agreement with the idea that T903A, a recessive
mutation, may impair a phosphatase activity of RcsC.
The second mutation in the receiver domain is located at
Asn877, a non-conserved residue in the vicinity of the active
site. In CheY, the carbonyl of the corresponding Asn59
directly chelates the active site Mg2+ in both the inactive
and the active conformations (Bellsolell et al., 1994; Lee
et al., 2001; Stock et al., 1993). According to the structure
predicted by SWISS-MODEL (Guex & Peitsch, 1997; Schwede
et al., 2003), a replacement of Asn877 by Ser increases its
contact with the site of phosphorylation, Asp875, through
hydrogen bonding. Different substitutions at the corresponding positions in VirG (N54D) (Han et al., 1992; Jin
et al., 1993; Pazour et al., 1992; Scheeren-Groot et al., 1994),
CheB (E58K) (Stewart, 1993) and CheY (N59K, N49R)
(Silversmith et al., 2001) also have an activating effect,
thereby outlining the importance of this non-conserved
residue in the activation of response regulator domains.
585
C. B. Garcı́a-Calderón and others
Two mutations in the histidine kinase domain affecting the
same residue (P484L and P484S) are of particular interest.
They have been isolated independently five and three times,
respectively, and exhibit the highest levels of activation of
the gmm : : lac fusion: over 500 times the wild-type level
(Fig. 3 and Table 2). These mutants grow more slowly
than the wild-type and the remaining constitutive rcsC
mutants (Table 2), and give rise at relatively high frequencies to non-mucoid revertants with normal growth
rate. These features can be related to previous findings
about the lethality of igaA (also called yrfF and mucM) null
mutations in Salmonella (Cano et al., 2002; Costa et al.,
2003). Lethality is suppressed by mutations in rcsC, yojN
or rcsB, suggesting that overactivation of the Rcs system
may lead either to repression of an essential gene or to
derepression of a gene whose overexpression is lethal. In
this sense, the rcsCP484L and rcsCP484S mutants might
approach the maximum level of activation of the Rcs system
still compatible with viability.
In this work, we provide direct support for the hypothesis
that overactivation of the Rcs system renders Salmonella
avirulent. First, we show that constitutively activated rcsC
mutants are attenuated in mice. Second, we describe a
nearly perfect correlation between the degree of attenuation and the level of activation of the Rcs system caused
by the different mutations (Fig. 4 and Table 2). Finally, we
show that overproduction of colanic acid capsule is a
relevant factor in the attenuation of these mutants. In all
constitutively activated rcsC mutants tested, an insertion in
gmm (one of the genes necessary for colanic acid synthesis)
or rcsA led to a partial but highly significant relief of
attenuation in mice: up to three orders of magnitude in
CI (Fig. 4). These experiments suggest that, although production of colanic acid capsule is dispensable for virulence
(see CI for strain SV4609 in Fig. 4), its overproduction
is detrimental for Salmonella infection in mice. Since
suppresion by gmm mutation is not complete, another
conclusion from these experiments is that capsule overproduction is not the only cause of attenuation of the
constitutively activated rcsC mutants. We thus postulate
the existence of additional Rcs-regulated genes involved in
Salmonella virulence.
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ACKNOWLEDGEMENTS
We are grateful to Dr Francisco Garcı́a del Portillo for helpful
discussions. This work was supported by grants QLRT-PL-00310 from
the European Union, and BIO2001-0232-C02-02 from the Ministry
of Science and Technology of Spain. F. R.-M. is an investigator of the
‘Ramón y Cajal’ programme from the Ministry of Science and
Technology of Spain. C. G.-C. and M. G.-Q. are predoctoral students
supported by grants from the Spanish Ministry of Education.
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