1
M3:13491
April 30, 2004
ppGpp-dependent stationary phase induction of genes on Salmonella pathogenicity
island 1 (SPI1)
Miryoung Song,1,2 Hyun-Ju Kim, 1,2 Eun Young Kim, 1,2 Minsang Shin, 1,2 Hyun
Chul Lee, 1,2 Yeongjin Hong, 1,2 Joon Haeng Rhee, 1,2 Sangryeol Ryu, 3 Sangyong
Lim, 3 Hyon E. Choy1,2,4
1 Genome Research Center for Enteropathogenic Bacteria and Research Institute of
Vibrio Infection
2Department of Microbiology, Chonnam National University Medical College,
Kwangju 501-746, South Korea
3 Department of Food Science and Technology, School of Agricultural Biotechnology,
Center for Agricultural Biomaterials, Seoul National University, Seoul, 151-742, South
Korea
4 Corresponding author
Hyon E. Choy
Department of Microbiology,
Chonnam National University Medical College,
Kwangju 501-746, South Korea
Tel) +82 62 220 4137
Fax) +82 62 228 7294
e.mail) [email protected]
JBC Papers in Press. Published on May 25, 2004 as Manuscript M313491200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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We have examined expression of the genes on Salmonella pathogenicity island 1 (SPI1)
during growth under the physiologically well-defined standard growth condition of LB
medium with aeration. We found that the central regulator hilA and the genes under its
control are expressed at the onset of stationary phase. Interestingly, two-component
regulatory genes, hilC/hilD, sirA/barA ,and ompR, known to modulate expression from
the hilA promoter (hilAp) under so-called ‘inducing conditions (LB medium containing
0.3 M NaCl without aeration)’ acted under standard conditions at the stationary phase
induction level. The induction of hilAp depended not on RpoS, the stationary phase
sigma factor, but on the stringent signal molecule, ppGpp. In the ppGpp null mutant
background, hilAp showed absolutely no activity. The stationary phase induction of
hilAp required spoT but not relA. Consistent with this requirement, hilAp was also
induced by carbon source deprivation, which is known to transiently elevate ppGpp
mediated by spoT function. The observation that amino acid starvation elicited by
addition of serine hydroxamate did not induce hilAp in a RelA+ SpoT+ strain suggested
that in addition to ppGpp some other alteration accompanying entry into stationary
phase might be necessary for induction. It is speculated that during the course of
infection, Salmonella encounters various stressful environments that are sensed and
translated to the intracellular signal, ppGpp, that allows expression of Salmoenella
virulence genes including SPI1 genes.
[key words: Stringent response, ppGpp, Transcription regulation, Salmonella
pathogenicity island 1 genes]
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Infection with Salmonella enterica serovar Typhimurium can cause a systemic,
typhoid-like disease in mice. Following ingestion, bacteria can colonize the intestinal
tract, penetrate the intestinal epithelium, and access systemic sites such as the spleen and
liver through lymphatic and blood circulation (1). Passage of the bacteria through the
intestinal lining is initiated by bacterial invasion into enterocytes and M cells (1, 2, 3, 4).
The invasion is mediated by a bacterial type III secretion system (TTSS) encoded by
genes on Salmonella pathogenicity island 1 (SPI1) (5). The TTSS translocates
bacterial effector proteins, also encoded on SPI, into the host cell cytosol to reorganize
the cytoskeleton, resulting in membrane ruffling and eventual bacterial uptake (6).
Expression of the SPI1 secretion system and of its secreted effectors is coordinately
regulated by HilA encoded on SPI1, a member of the OmpR/ToxR family of
transcriptional regulators (7). The genes on SPI1 regulated by HilA include invF and
sicA (8, 9, 10). InvF, a member of the AraC/XylS family of transcriptional regulators,
in conjunction with SicA, a TTSS chaperone, take part in the coordinated regulation of
SPI1 encoded genes.
Regulation of hilA expression has been studied extensively because of its
central role in invasion gene activation. Environmental signals, like oxygen
concentration and osmolarity, and the growth state of bacteria, have been shown to
influence the expression of hilA and the secretion of invasion-associated proteins (11,
12). Thus, most studies have been carried out using bacteria grown under so called
inducing conditions, namely high osmolarity and low oxygen conditions (LB containing
0.3 M NaCl without aeration). Studies of bacteria grown under these conditions have
so far revealed that hilA expression is regulated by a complex array of regulatory
systems including hilC/sirC/sprA (13, 14, 15), hilD (15), sirA/barA (16, 17), fis (18, 19),
csrAB (16, 20), envZ/ompR (21), phoB, fadD, and fliZ (7), hha (22), and H-NS and HU
(19). Two of these genes, hilC and hilD, encode AraC-like transcriptional activators
that activate hilA transcription by binding upstream of the hilA promoter DNA (15, 23,
24). Members of the phosphorylated response regulator superfamily involved in hilA
expression include sirA/barA, envZ/ompR, phoR/phoB, and phoP/phoQ (7, 17, 21, 12).
However, none of these regulatory systems has been shown to directly relay
environmental signals to hilA expression.
Enteric bacteria elicit stringent control of ribosome production during the
transition from exponential growth to stationary phase (25, 26). The effector molecule
of the stringent control modulation is the alarmone guanosine tetraphosphate, ppGpp
(27, 28). The ppGpp is synthesized by two synthetases, PSI and PSII, encoded by relA
and spoT genes, respectively. These two enzymes respond differently to
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environmental conditions. PSI is activated during amino acid starvation but is largely
inactive during exponential growth; in contrast, PSII is mostly inactive during amino
acid starvation, but is active during exponential growth to determine basal levels and to
response to certain environmental stress including deprivation of carbon or energy (29,
30, 31, 32, 33, 34). Accumulation of ppGpp during the exponential phase of growth
results in the reduction of stable RNA synthesis and the activation of certain mRNA
synthesis.
In this study, we examined expression of SPI1 genes including hilA under
physiologically well defined standard growth conditions (LB with vigorous aeration)
and observed that these genes were induced at the onset of the stationary phase. This
stationary phase induction was, however, not dependent on stationary phase specific ,38, but on the stringent signal molecule ppGpp. Most interestingly we found the
stationary phase hilAp induction depended not on RelA but on SpoT function. This
suggests an unusual inducing role of the SpoT protein distinct from its ability to
produce the stringent signal molecule ppGpp. We conclude that Salmonella virulence
genes, including SPI1 genes, are expressed under stressed conditions in a ppGpp-
dependent manner.
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Materials and Methods
Strains and plasmids
The Salmonella strains, derived from 14028s, and plasmids used in this study are listed
in Table 1. All bacterial strains were constructed by P22HT int transduction as
described previously (35). The hilC::kan strain was constructed following the method
developed by Datsenko and Wanner (36). The hilC carrying kan in the place of its
ORF was generated by PCR amplification using a pair of 60-nt primers that included
40-nt homology extensions and 20-nt priming sequences with pKD13 as a template: 5’
primer
(TTCAATGAATAAATCAGTTGAGGCCATTAGCAATAATCACGTGTAGGCTGGA
GCTGCTTC) and 3’ primer
(CTAATCCATTTATTAATGGAAATTTGTTCGGCTGTTGAAGATTCCGGGGATCC
GTCGACC) (hilC sequences are underlined).
The 1.4-kbp PCR products were purified and transformed into bacteria carrying a Red
helper plasmid (pKD46) by electroporation. The electrocompetent cells were grown in
LB broth with ampicillin and L-arabinose (1 mM) at 30 C to an OD600 of 0.5. The
mutants were confirmed by PCR using original and common test primers: k1
(CAGTCATAGCCGAATAGCCT) and kt (CGGCCACAGTCGATGAATCC) for kan.
Growth conditions
Except when indicated otherwise, cultures were grown in LB medium (Difco
Laboratories) containing 1% NaCl with vigorous aeration at 37℃. For solid support
medium, 1.5% granulated agar (Difco Laboratories) was included. MacConkey
lactose, Nutrient Broth and Brain Heart Infusion (BHI) media were purchased from
Difco Laboratories. Antibiotics were from Sigma Chemical. When present,
antibiotics were added at the following concentrations: ampicillin, 50 g/ml;
chloramphenicol, 15 g/ml; tetracycline, 15 g/ml. X-gal (Sigma) was used at 20
g/ml. The carbon source starvation experiment was carried out in LB+0.1% glucose
with alpha-methyl glucoside ( -MG, Sigma). Amino acid starvation experiment was
carried out as described in Shand et al. (37) using DL-serine hydroxamate (Sigma) in
NB supplemented with serine (0.75 mM).
Analysis of culture supernatant
Cultures were grown overnight in 5 ml of LB broth with antibiotics and vigorous
aeration, and then harvested. Bacteria were pelleted at 8,000 g for 15 min, and
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supernatants were immediately transferred to clean tubes. The supernatants were
filtered through a 0.45 m pore size syringe filter (Sartorius), and proteins were
precipitated with cold trichloroacetic acid (TCA) at a final concentration of 10%. The
proteins were collected by centrifugation at 8,000 g at 4 C and resuspended in 1 ml
cold acetone. These mixtures were centrifuged for 10 min at 8,000 rpm at 4 C, and
pellets were resuspended in 20 l of 1 PBS. The protein sample buffer containing -
mercaptoethanol was added to the samples, the samples were boiled for 5 min, and
proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) (7.5%).
Proteins were visualized with silver stain (38).
-galactosidase assays
-galactosidase assays were performed as described by Miller (39), using cells
permeabilized with Koch’s lysis solution (40). -galactosidase specific activity was
expressed as Miller units (A420/min/A600/ml x1000). To measure -galactosidase levels
in bacteria at different stages of growth, fresh overnight cultures were diluted 1:50 into
LB or the media condition described in the text and grown at 37℃ until the cultures
reached the stationary phase. Samples were taken for enzyme assay at regular time
intervals. Each strain was assayed in triplicate and average enzyme activities were
plotted as a function of time.
Primer extension analysis
Total RNA was isolated from Salmonella grown statically using Trizol reagent (Life
Technologies, Inc). To study hilA and hisG transcriptions, the primers with 5’-
TAATAATATTGTTATAACTAACTGTGATTA -3’, complementary to +134 to -+114 of
the transcripton start site of hilA, and 5’-
ACTGGAAGATCTGAATGTCTTCCAGCACAC-3’, complementary to +124 to +95 of
the transcription start site of hisG, were used. 32P-labeled primers (50,000 cpm) were
co-precipitated with 30 µg of total RNA. Primer extension reactions were performed
as described by Shin et al. (41).
Invasion assay
The assays were performed essentially as previously described (42). Monolayers for
bacterial invasion were prepared by seeding 5X105 HEp-2 cells into each well of 24-
well plates. The HEp-2 cells were grown in DMEM (GibcoBRL) +10% fetal bovine
serum (GibcoBRL) at 37℃ with 5% CO2. Salmonellae prepared as described in the
text were added to HEp-2 cells at a ratio of 10:1, and the mixture was incubated at 37℃
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under 5% CO2 for 30 min. Infected cells were washed three times with phosphate-
buffered saline (PBS, PH 7.4) and DMEM containing gentamicin (5 mg/ml, Sigma) was
added, and the mixture was incubated for an additional 60 min. Intracellular bacteria
were harvested by extraction with lysis buffer (0.05% triton X-100 in PBS, PH 7.4), and
replica plated for colony counting on BHI agar plates.
Results
Growth phase-dependent invasiveness of bacteria grown under standard conditions
The ability of Salmonellae to invade cultured nonphagocytic cells has been correlated
with the expression of SPI1 encoded genes (43). In an attempt to investigate the
regulation of invasion genes under a physiologically well-defined standard growth
conditions, we determined the invasiveness of bacteria grown under standard conditions
(LB with aeration). In this experiment, overnight culture of bacteria grown in LB was
diluted 40-fold in the same media or a high salt media (LB+0.3 M NaCl) and grown
with or without aeration, respectively. The high salt media without aeration was
considered the ‘inducing condition’ for the expression of SPI1 genes (11, 12). Fig. 1A
shows Salmonellae growth under the two conditions. Under the standard condition,
bacteria grew rapidly and reached the stationary phase in about 4 hrs. Under the
inducing condition, the culture entered into the stationary phase at a much lower A600,
<1 A600. Bacteria were sampled from the middle of the exponential phase (~2 hrs) and
the early (~4 hrs) and late (~12 hrs) stationary phases grown under the two conditions,
and the invasiveness of each growth phase culture was determined using HEp-2 cells.
In this experiment, the number of bacteria from various growth phases was adjusted to
MOI=10: bacteria (5x106) and host cells (5x105). Fig. 1B shows the actual number of
intracellular bacteria that survived gentamicin treatment (10 g/ml), recovered from the
host cells following an 1 hr incubation of bacteria and host cells. When grown under
the standard condition (filled bars), the early stationary phase bacteria were found to be
about 10~20-fold more invasive than the exponential phase and the late stationary phase
bacteria. By contrast, the invasiveness of bacteria grown under the inducing condition
showed a different pattern; the early stationary phase culture was about 3-fold more
invasive than the exponential phase culture, but slightly less invasive than the late
stationary culture (open bars). The maximum invasion was obtained with the early
stationary culture grown under the standard condition. It was thought that the loss of
invasiveness with the late stationary culture grown under standard condition was due to
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destruction of TTSS by continuous agitation of the culture. Thus, although the
inducing condition might closely represent the intestinal milieu (11, 12), bacteria grown
under the standard condition were used in the subsequent experiments to identify the
factor(s) conferring the maximum invasiveness at the early stationary phase.
Next, we determined the presence of secreted effector proteins encoded within
SPI1 (38) in the cultures at different time grown under the standard condition (see Fig.
1C). Total supernatants of cultures at different phases were collected, precipitated with
TCA, and analyzed on 7.5% SDS-PAGE gel (Fig. 1C). The volume of supernatant
was proportionally adjusted to the number of bacteria at each growth phase. The
secreted effector proteins, namely SipA (89 Kd), SipB (67 Kd), SigD (62 Kd), and SipC
(42 Kd), were detected only in the supernatant of cultures entering the stationary phase
(4 hrs) and thereafter (6 hrs). Thus, Salmonellae at the entry of the stationary phase
were most invasive because SPI1 encoded genes, including those constituting the TTSS
apparatus and effector proteins, were expressed exclusively at this growth phase under
the standard condition (see below).
Growth phase-dependent expression from the promoters in SPI1
We analyzed the activity of the promoters driving expression of the genes involved in
Salmonella invasion of host cells encoded on SPI1, namely hilA, invF and sicA
promoters (hilAp, invFp and sicAp in short), during growth under the standard condition.
To determine activity of these promoters, S. typhimurium strains carrying lacZY genes
transcriptionally fused to individual promoters on the chromosome (SMR2063, Fig. 2A)
or on a plasmid in 14028s strain background (Fig. 3) were used. Bacteria were taken
at regular time intervals and -galactosidase activity representing activity of each
promoter during the course of growth was determined. Fig. 2 shows the hilAp activity
determined under the standard and inducing growth conditions. Under the inducing
condition, hilAp activity was about the same throughout the exponential and stationary
phases. By contrast, hilAp activity under the standard growth condition was induced
~30-fold when the culture entered the stationary phase. hilAp activity under the
standard condition was ~10-fold less during the exponential phase but ~3.5-fold more at
the entry of the stationary phase compared with the activities under the inducing
condition. This result, thus, accounts for the different invasiveness of the cultures
grown under the two conditions; the central regulator hilAp is selectively expressed at
the onset of the stationary phase under the standard condition but is maintained at more
or less the same level irrespective of the growth phase under the inducing condition.
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To further verify the hilAp induction under the standard growth condition, the hilAp
specific transcript was monitored during the course of growth by the primer extension
assay (Fig. 2B). The hilAp specific RNA was detected at 3 hrs as culture entered the
stationary phase, peaked at 5 hrs, and disappeared at 7 hrs.
Subsequently, invF and sicA as well as hilA promoters on the pRS415 plasmid
(44) were determined during growth under the standard condition (Fig. 3). The
episomal hilAp activity (3A) showed a similar pattern of induction at the onset of the
stationary phase but the magnitude of induction was significantly reduced to ~4-fold.
The reduction was ascribed to the huge increase in the basal level activity at the
exponential phase, as if a repressor acting at the exponential phase was titrated out by
the episomal hilAp DNA. The invFp and sicAp activities were determined using the
strain carrying the individual promoters fused to lacZYA on the pRS415. Both invFp
(3C) and sicAp (3B) were increased more than 50-fold as the culture entered the
stationary phase. The extension in the activation of downstream activators, invF and
sicA, as compared with the upstream activator, hilA, might represent a magnification of
physiological response in cascade regulation. Taken together, these results clearly
establish that the early stationary phase bacteria grown under the standard condition are
most invasive due to the selective expression of the central activator, hilA, presumably
thereby downstream activators invF and sicA under its control, and thereby those
encoding TTSS and the effectors.
Regulation of stationary phase induction of hilA expression
We then set out to establish the molecular mechanism underlying the stationary phase
induction of hilA under the standard growth condition. Stationary phase induction of
gene expression in enteric bacteria is due at least partly to the stationary phase sigma
factor 38, the rpoS gene product (45). The heat shock sigma factor ( 24), the rpoE
product, has also recently been shown to be strongly induced upon entry of Salmonella
into the stationary phase (46, 47). We determined the chromosomal hilAp activity in
the RpoS- mutant background and found the stationary phase activation pattern was
even greater than in the wild type (WT) (Fig. 4). In the RpoE- mutant background, no
difference was observed. Thus, the stationary phase induction of hilAp is apparently
independent of rpoS or rpoE.
Subsequently, we examined the regulation of hilA by those two-component
regulatory systems known to activate hilA under the inducing condition, namely
hilC/hilD, sirA/barA, and envZ/ompR (7, 48) (Fig. 5). Under the standard growth
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condition, hilAp activity in the HilC- mutant in the exponential phase was equivalent to
that in the WT but was not induced at the entry of the stationary phase. On the other
hand, hilAp activity in the HilD- mutant in the exponential phase was ~10-fold lower
than that in the WT, and the activity remained reduced throughout the course of growth.
The hilAp activity in either the SirA- or BarA- mutant in the exponential phase was not
much different than that in the WT but was only partially induced at the entry of the
stationary phase. The hilAp activity was virtually undetected in the OmpR- mutant
throughout the growth period. However, in the EnvZ- mutant strain, hilAp was
induced at the entry of the stationary phase, although ~2.5-fold less than in the WT.
The differences in hilAp induction in OmpR- and EnvZ- suggest that OmpR could be
phosphorylated by a protein(s) other than EnvZ (49), if phosphorylated OmpR induces
hilAp activity. Nevertheless, defect in the two-component regulatory systems resulted
in a failure to induce hilAp at the entry of the stationary phase under the standard
condition. It is speculated that these activators might respond to a certain signal at the
entry of the stationary phase.
ppGpp-dependent induction of hilA and SPI1 genes
In an attempt to identify the global regulatory system responsible for the stationary
phase induction of hilAp, we examined hilAp activity in a strain lacking ppGpp, the
effector molecule of the stringent response (28). ppGpp is produced and maintained
by PSI and PSII, the respective relA and spoT gene products. We examined the hilAp
activity in the relA or relA spoT strain, which lack PSI or both PSI and PSII,
respectively (Fig. 6). Growth of the mutants did not differ much from the WT strain
under the standard growth condition in LB. We observed that in the relA mutant
strain, hilAp activity was indistinguishable from that in the WT strain. However, hilAp
activity was completely silent throughout the growth phase in the relA spoT mutant
strain lacking ppGpp. These observations suggest that hilAp induction at the entry of
the stationary phase is mediated by ppGpp, which is synthesized primarily by SpoT
activity. To further verify the route of ppGpp synthesis leading to hilAp induction,
hilAp activity was determined during carbon source starvation, which is known to
elevate ppGpp in a SpoT-dependent manner (50, 51, 52, 34). Carbon starvation was
elicited by the addition of 2.5% -methyl glucoside ( -MG), a competitive inhibitor of
glucose uptake, into LB containing 0.1% glucose (Fig. 7). The addition of -MG only
slightly reduced the growth rate: generation time shifted from ~30 min to ~40 min for
all three WT, relA, and relA spoT strains. Fig. 7A shows a representative growth
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curve for all three strains. The basal hilAp activity levels prior to -MG addition in the
strains fell within a 2-fold range: WT > relA > relA spoT in order (Fig. 7B). Upon
addition of -MG, hilAp activity increased drastically in the WT and relA strains but
not in the relA spoT strain. We also examined the hilAp activity during amino acid
starvation that elevates ppGpp levels in a RelA-dependent manner (37) (Fig. 8). The
condition of amino acid starvation was elicited by the addition of 2 mM serine
hydroxamate (SerHX) to a culture grown in Nutrient Broth supplemented with 0.75 mM
serine. Both WT and relA spoT strains grew with more or less the same generation
time (~30 min) prior to the addition of SerHX (Fig. 8A). The addition of SerHX in the
middle of exponential phase of growth immediately reduced the growth rate for WT
strain (top panel). By contrast, the cell mass (A600) of relA spoT (bottom panel)
increased at the same rate for some period (~ 1 hr) as prior to the addition of SerHX and
then ceased (data not shown). It has been shown under this growth condition, the
addition of SerHX drastically increased the ppGpp level in RelA-dependent manner,
~10-fold (37). Under this condition, we first determined an amino acid histidine
biosynthesis operon promoter (hisGp), a classical promoter known to respond in parallel
with the change in ppGpp level (62, 37, 63). The promoter activity was determined by
measuring the transcripts. The addition of SerHX increased hisGp activity in WT
(~30-fold) within 5 min but not in relA spoT mutant strain (Fig. 8B, top). Under the
same condition, hilAp activity remained unchanged by the addition of SerHX in either
WT or relA spoT strain (Fig. 8B, bottom). These results confirm that induction of
hilAp, and thereby those genes under its control at the entry of the stationary phase,
results from the elevation of ppGpp levels but in a SpoT-dependent manner.
Lastly, we evaluated the WT and relA spoT strains for their abilities to invade
HEp-2 cells to access the in vivo role of ppGpp in Salmonella virulence (Table 2). The
early stationary phase bacteria were used in this assay. The analysis revealed that
invasion by the relA spoT strain was less than 1% of the level of invasion by the WT
bacteria. Thus, the lack of hilA expression in the relA spoT strain, and thereby the
lack of expression of those genes under its control, including SPI1 encoded TTSS and
effector proteins, caused an apparent reduction in invasiveness.
Discussion
Stationary phase induction of hilA under the standard growth condition
In this study, we reported that hilA and therefore those genes under its control are
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expressed under the standard growth condition at the onset of the stationary phase based
on the following observations: 1) invasiveness culminated at the early stationary phase
culture (Fig. 1); 2) some representative secreted proteins encoded in SPI1 were detected
in the supernatant from early stationary phase cultures but not in supernatant from
exponential phase cultures (Fig. 1); 3) hilAp and those promoters under its control,
sicAp and invFp, were induced at the onset of the stationary phase (Fig. 2 and 3).
Similarly, early stationary phase bacteria grown under the standard condition are
reportedly most cytotoxic to cultured mammalian cells (53). The primer extension
analysis revealed that hilAp was transitionally expressed during transition from the
exponential phase to the stationary phase, demonstrating a pattern of growth phase-
dependent expression. The induction of hilA was, however, independent of the
stationary phase or the heat shock , 24, which have been implicated in
Salmonella pathogenesis in animals and are induced at the entry of the stationary phase
(45, 46, 47) (Fig 4). Therefore, hilAp seems be induced in response to an unidentified
environmental signal built up as culture enters the stationary phase.
We have observed that hilAp induction on a multicopy plasmid was lower than
that on the chromosome (~4-fold vs ~30-fold), largely due to a huge increase in hilAp
activity at the exponential phase (~1000-fold). Therefore, the stationary phase
induction of hilAp activity could be, at least in part, ascribed to removal of repressor
acting during the exponential phase. In this case, the hypothetical repressor might be
titrated out by episomal hilAp DNA, resulting in the elevation of hilA activity during the
exponential phase. Since a plethora of regulatory systems has been proposed to
regulate hilAp (7), the titratable factors should include an activator(s) as well as a
repressor(s).
Interestingly, it was noted that the two-component regulatory systems known to
activate hilAp under the inducing condition acted at the level of its induction at the entry
of the stationary phase under the standard growth condition (Fig. 5). Amongst the
regulatory components, hilC/hilD has been shown to exert its regulatory effect by
directly binding to a site upstream of hilAp (15, 23, 54). We observed under the
standard growth condition that hilAp activity remained at the basal level in the HilC- or
HilD- mutant background, although the defect was more severe in the HilD- mutant.
In fact, hilAp activity was reduced ~10-fold in the HilD- mutant background even
during the exponential phase. This result suggests that the mechanism of hilAp
activation by hilC/hilD might be different depending on whether the cells are in the
exponential growth phase or at the entry into the stationary phase. We obtained similar
results with the strains lacking the two-component regulatory systems reported to up-
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regulate hilAp activity under the inducing condition, barA/sirA and envZ/ompR (55, 16,
20, 48). Under the standard growth condition, hilAp in the BarA- or SirA- mutant was
defective at the level of its induction in stationary phase but not in exponential phase.
The hilAp activity in the OmpR- mutant was virtually undetected while that in the
EnvZ- was almost normally induced, although the peak activity was ~2.5-fold less than
that in the WT. It is well established that EnvZ phosphorylates OmpR upon sensing an
increase in osmolarity in the environment (49). Under the standard condition, if the
phosphorylated OmpR was responsible for hilAp induction, the phosphorylation must
occur through a route(s) other than EnvZ. In fact, an EnvZ-independent mechanism of
OmpR phosphorylation has been postulated (56, 57). OmpR might take part in hilAp
induction at the onset of the stationary phase by sensing an unknown environmental
signal through a currently unknown sensor. In this case, OmpR is unlikely to be
responding to a change in media osmolarity since we did not detect any noticeable
change in the media osmolarity as the culture entered stationary phase (data not shown).
Further study is required to elucidate the underlying mechanism of hilA induction and
its regulation by two-component regulatory systems under the standard growth
condition.
Implication of ppGpp in hilAp induction
Since establishing hilAp induction at the entry of the stationary phase under the
standard growth condition, we searched for the global regulatory signal responsible for
the induction and found ppGpp. hilAp activity remained at the basal level in
relA spoT strain while normally induced in relA strain (Fig. 6). SPI1 encoded sicA
and invF promoters also remained at their basal levels in the relA spoT mutant strain
(data not shown). Consistently, the relA spoT strain showed reduced invasiveness
by more than 100-fold, as determined in vitro assay using HEp-2 cells (Table 2). RelA
is known to sense an imbalance or lack of amino acid supply and to synthesize ppGpp,
resulting in the reduction of stable RNA synthesis, the phenomenon known as “stringent
response” (29, 26, 28). Alternatively, the basal level of ppGpp during balanced growth
is regulated by spoT, which carries both ppGpp synthetase (PSII) and hydrolase (58, 34).
Therefore, the basal level of ppGpp depends on the balance of two activities. Some
conditions, including carbon and energy starvation have been shown to result in
accumulation of ppGpp in a SpoT-dependent manner (59, 60, 34). Under the standard
laboratory growth conditions, the transition from the exponential phase to the stationary
phase presumably represents a stressed condition. It has been reported recently that
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changes in the proteome pattern of E. coli entering the stationary phase were
significantly different between WT and relA1 spoT mutant, which had a proteome
pattern that appeared to be locked in the exponential growth mode (61). We speculate
that the transitional stress at the entry of stationary phase must be sensed and translated
to ppGpp synthesis primarily by SpoT function.
Most interestingly, it was observed that hilAp was induced during exponential
phase of growth by the carbon source starvation known to elevate ppGpp level in a
SpoT-dependent manner (50, 51, 52, 34) but not by the amino acid starvation known to
elevate ppGpp level in a RelA-dependent manner (37) (Fig. 7 and 8). Whereas, the
amino acid histidine biosynthesis operon promoter (hisGp), a classical promoter
positively regulated by ppGpp (62, 37, 63), responded in parallel with the RelA-
dependent increase in ppGpp level in this study (Fig 8). It has been reported that the
amino acid starvation elicited by the addition of SerHX causes an immediate increase in
ppGpp level in RelA-dependent manner: 28.7 pmol/A650 to 1,042 pmol/A650 (37).
The carbon source deprivation could also increase ppGpp concentration up to ~500
pmol/A650 in WT bacteria (51). However, it must be noted that hilAp was induced
normally in relA strain following the carbon source starvation in which ppGpp pool
was measured to be increased only a few-fold, a little more than 100 pmol/A650 (34).
Thus, regulation of gene expression following carbon source starvation and amino acid
starvation seem to be mechanistically different. Likewise, gene induction at the entry
of the stationary phase and that observed during the classical stringent response
following amino acid deprivation must also be different. In addition to ppGpp, some
other alteration accompanying entry into the stationary phase may be necessary for gene
induction, including hilAp activation. The physiological consequence of the two
routes of ppGpp elevation, the RelA- and SpoT-dependent mechanisms, remains
unknown.
During the course of animal infection, Salmonella bacteria encounter diverse
environments in the intestinal lumen and inside various host cells. Thus, it is
imperative that Salmonellae must be able to sense and respond to changing
environments in order to survive (64). We speculate that environmental stress is
sensed and translated to the intracellular signal ppGpp that enables expression of
various Salmonella virulence genes including those encoded on SPI1 that are required
for the invasion of host cells and induction of macrophage apoptosis (1, 65).
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Acknowledgement
This work was supported by Korea Health 21 R & D (01-PJ10-PG6-01GM02-002) by
the Ministry of Health and Welfare, Republic of Korea. We thank C. Lee (Boston) and
K. Tedin (Berlin) for providing important Salmonella strains.
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Figure legends
Fig.1. Host cell (Hep-2) invasion by the bacteria (14028s) grown in the inducing
condition or standard growth condition (A & B) and the expression of representative
secreted TTSS components (C). A. Bacterial growth (A600) under the inducing
condition (open circles) and under the standard condition (closed circles). B. The
actual number of intracellular gentamicin resistant bacteria after incubation of Hep-2
cells and bacterial cultures at different growth phases. Closed bars represent
invasiveness of bacteria grown under the standard condition and open bars represent
invasiveness of bacteria grown under the inducing condition. C. Proteins excreted into
media during Salmonella growth under the standard condition, on 7.5% SDS PAGE gel.
The secreted proteins, SipA (89 Kd), SipB (67 Kd), SigD (62 Kd), and SipC (42 Kd),
were identified by their sizes as described in Hong and Miller (38). Protein markers
(BioRad) are shown in the first lane.
Fig. 2. Expression of hilAp::lacZY during Salmonella growth under the inducing
(triangles) or standard condition (circles) (A). The curves with open symbols represent
growth (A600) and curves with closed symbols represent chromosomal hilAp activity as
determined by -galactosidase assay (Miller units). B. Expression of hilA under the
standard growth condition as determined by primer extension analysis (left panel).
Thirty micrograms of total Salmonella RNA, extracted at each time point during growth,
was co-precipitated and annealed with end-labeled hilA primer. Reactions were
performed as described under "Materials and Methods." The products were resolved
on a 6% sequencing gel. Right panel shows DNA sequencing ladder of the region
around hilA transcription initiation site. Arrow indicates the first nucleotide of the hilA
transcript, T with circle.
Fig. 3. Expression from hilAp (pMS009, A), sicAp (pMS011, B) and invFp (pMS010,
C) on the transcription fusion plasmid pRS415 during Salmonella growth under the
standard growth condition. The curves with open symbols represent growth (A600) and
curves with closed symbols represent the activity of each promoter activity as
determined by the -galactosidase assay (Miller units).
Fig. 4. Chromosomal hilAp activity in the WT (SMR2063, closed circles), RpoS-
(SMR2065, closed triangles), and RpoE- (SMR2090, closed squares) background during
growth under the standard condition. Dotted curve with open circles shows
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representative growth (A600). hilAp::lacZY activity was as determined by the -
galactosidase assay (Miller units).
Fig. 5. Chromosomal hilAp activity in WT (SMR2063, closed circles), SirA-
(SMR2094, open triangles), BarA-(SMR2095, closed triangles), OmpR- (SMR2064,
open squares), EnvZ- (SMR2084, closed squares), HilC- (SMR2106, open reverse
triangles), and HilD- (SMR2099, closed reverse triangles). Dotted curve with open
circles represents growth (A600). hilAp::lacZY activity was as determined by -
galactosidase assay (Miller units). Note that hilAp activity in OmpR- and HilD- was
virtually undetected (open squares and closed reverse triangles overlapped at the
bottom).
Fig. 6. Chromosomal hilAp activity in WT (SMR2063, circles), relA SHJ2070,
triangles and relA spoT (SHJ2057, squares) backgrounds. The curves with open
symbols represent growth (A600) and curves with closed symbols represent hilAp
activity as determined by -galactosidase assay (Miller units).
Fig. 7. Chromosomal hilAp activity in WT (SMR2063, circles),
relA SHJ triangles and relA spoT (SHJ2057, squares) backgrounds during
carbon source deprivation elicited by the addition of -MG during exponential growth.
-MG was added at time = 0 (arrow). A. A representative growth curve (A600) before
and after the addition of -MG. The growth pattern for WT, relA and relA spoT
were indistinguishable. Closed and open circles represent A600 with and without -MG
addition, respectively. B. hilAp activity as determined by -galactosidase assay (Miller
units): closed and open symbols represent the hilAp activities with and without -MG
addition, respectively.
Fig. 8. hilAp and hisGp activities determined by primer extension analysis in WT
(SMR2110)and relA spoT (SMR2112) background during amino acid starvation
elicited by the addition of SerHX into cultures grown in NB supplemented with 0.75
mM serine. SerHX was added at time 0 (arrow) when A600 was 0.15 and 0.3 for WT
and relA spoT, respectively. A. Growth (A600) of WT (top) and relA spoT
(bottom) strains as function of time. Closed and open symbols represent the
measurements with and without SerHX addition, respectively. B. hisGp (top) and
hilAp (bottom) activities determined by primer extension assay in WT and
relA spoT strains.
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Table 1. Strains and plasmids
Strains Description Reference or source
S. typhimurium
14028s Wild type
SCH2006 rpoS::Amp, Ampr This work
SMR2063 hilA080::Tn5lacZY, Tetr This work
SMR2064 ompR43::MudJ, hilA080::Tn5lacZY, Kanr, Tetr This work
SMR2065 rpoS::Ap, hilA080::Tn5lacZY, Ampr, Tetr This work
SMR2084 envZ1005::MudP, hilA080::Tn5lacZY, Camr, Tetr This work
SMR2090 rpoE::cat, hilA080::Tn5lacZY, Camr, Tetr This work
SMR2094 sirA::kan, , hilA080::Tn5lacZY, Kanr, Tetr This work
SMR2095 barA::kan, hilA080::Tn5lacZY, Kanr, Tetr This work
SMR2099 hilD::kan-1, hilA080::Tn5lacZY, Kanr, Tetr This work
SMR2106 hilC::kan, hilA080::Tn5lacZY, Kanr, Tetr This work
SMR2110 hisO1242hisD9953::MudA, Ampr This work
SMR2112 spoT::cat, relA::kan, hisO1242hisD9953::MudA, Ampr, Camr,
Kanr
This work
SHJ2037 spoT::cat, relA::kan, Camr, Kanr This work
SHJ2057 spoT::cat, relA::kan, hilA080::Tn5lacZY, Camr, Kanr, Tetr This work
SHJ2070 relA::kan, hilA080::Tn5lacZY, Kanr, Tetr This work
EE658 SL1344, hilA080::Tn5lacZY, Tetr 12
EE715 SL1344, sirA::kan, Kanr 15
EE731 SL1344, barA::kan, Kanr 15
JF2757 UK1, ompR43::MudJ, Kanr (lacZ-) 66
KT2184 LT2, relA71::kan, Kanr 67
KT2192 LT2, relA71::kan, spoT281::cat, Kanr, Camr 67
LM399 SL1344 , hilD::kan-1, Kanr 15
SF799 LT2, envZ1005:: MudP, Camr 68
TF951 14028s, rpoE::cat, Camr 47
TT11082 LT2, hisO1242hisD9953::MudA, Ampr 37
Plasmids
pRS415 LacZ fusion vector, Ampr 44
pMS009 pRS415 containing –138 to +84 of hilA This work
pMS010 pRS415 containing –300 to +130 of invF This work
pMS011 pRS415 containing –200 to +70 of sicA This work
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Table 2. Invasiveness of WT and relA spoT strains in Hep-2 cells
Strain WT relA spoT
Invasiveness (%) 100 <1
The invasiveness was determined as described in the Materials and Methods.
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Hong, Joon Haeng Rhee, Sangryeol Ryu, Sangyong Lim and Hyon E. ChoyMiryoung Song, Hyun-Ju Kim, Eun Young Kim, Minsang Shin, Hyun Chul Lee, Yeongjin
island 1 (SPI1)ppGpp-dependent stationary phase induction of genes on salmonella pathogenicity
published online May 25, 2004J. Biol. Chem.
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