JOURNAL OF BACTERIOLOGY, Dec. 2008, p. 8025–8032
Vol. 190, No. 24
Regulation of Cell Growth during Serum Starvation and Bacterial
Survival in Macrophages by the Bifunctional Enzyme SpoT in
Yan Ning Zhou,1William G. Coleman, Jr.,2Zhaoxu Yang,1Yi Yang,1Nathaniel Hodgson,2
Fuxiang Chen,2and Ding Jun Jin1*
National Cancer Institute—Frederick, National Institutes of Health, Frederick, Maryland1and National Institute of
Diabetes and Digestive and Kidney Diseases,2National Institutes of Health, Bethesda, Maryland
Received 12 August 2008/Accepted 25 September 2008
In Helicobacter pylori the stringent response is mediated solely by spoT. The spoT gene is known to encode
(p)ppGpp synthetase activity and is required for H. pylori survival in the stationary phase. However, neither
the hydrolase activity of the H. pylori SpoT protein nor the role of SpoT in the regulation of growth during
serum starvation and intracellular survival of H. pylori in macrophages has been determined. In this study, we
examined the effects of SpoT on these factors. Our results showed that the H. pylori spoT gene encodes a
bifunctional enzyme with both a hydrolase activity and the previously described (p)ppGpp synthetase activity,
as determined by introducing the gene into Escherichia coli relA and spoT defective strains. Also, we found that
SpoT mediates a serum starvation response, which not only restricts the growth but also maintains the helical
morphology of H. pylori. Strikingly, a spoT null mutant was able to grow to a higher density in serum-free
medium than the wild-type strain, mimicking the “relaxed” growth phenotype of an E. coli relA mutant during
amino acid starvation. Finally, SpoT was found to be important for intracellular survival in macrophages
during phagocytosis. The unique role of (p)ppGpp in cell growth during serum starvation, in the stress
response, and in the persistence of H. pylori is discussed.
Helicobacter pylori is a helical or spiral-shaped gram-neg-
ative bacterium. This gastric pathogen infects more than
one-half of the world’s population. H. pylori infection has
been linked to human gastritis, ulcers, and gastric cancer
(10, 19, 39). Gastric cancer is the fourth most common
cancer and the second leading cause of death from cancer
worldwide (38). Thus, understanding H. pylori pathogenesis
and factors that affect establishment of infection has public
The stringent response is a bacterial adaptation which affects
global gene expression during nutrient limitation and under
other stress conditions. In a well-studied Escherichia coli sys-
tem, two homologous genes, relA and spoT, are important for
the stringent response (13). RelA is a synthetase for guanosine
tetra- and pentaphosphate [(p)ppGpp], while in E. coli SpoT is
a bifunctional enzyme with both synthetase and hydrolase ac-
tivities. These two small molecular effectors bind to RNA poly-
merase (7, 14, 50) and affect global gene expression under
nutrient limitation conditions, such as amino acid starvation
(13, 17, 51). E. coli relA mutants defective in (p)ppGpp syn-
thesis have a “relaxed” phenotype and growth advantage dur-
ing amino acid starvation compared to wild-type strains (13, 17,
46, 51), while E. coli spoT mutants defective in the (p)ppGpp
hydrolase have a slow-growth phenotype (43). The relA and
spoT genes are conserved in eubacteria, implying that the func-
tions of (p)ppGpp are important for the organisms (31). The
roles of (p)ppGpp in the survival of several bacterial pathogens
during infection and transmission have been described previ-
ously (11, 20, 22, 26, 35). Many bacteria have both the relA and
spoT genes, but members of the alpha- and epsilonproteobac-
teria, including H. pylori, have only one member of the RelA/
SpoT family of proteins, SpoT (5, 20, 49, 53).
We speculated that SpoT is a potential global transcriptional
regulator for H. pylori growth and persistence. H. pylori lives
deep in the gastric mucus layer in the human stomach, where
it encounters low pH and a constantly changing environment
(44). H. pylori infection in humans persists for a lifetime unless
it is eliminated by antibiotic treatment. In order to establish an
infection and to persist in the stomach, H. pylori must over-
come the host innate immune response, including macro-
phages (1, 9, 32, 41). For optimal in vitro growth, microaero-
philic conditions (low levels of O2) and capnophilic conditions
(high levels of CO2) are used and media are supplemented
with serum (6, 12, 33). However, in vitro the viability of H.
pylori is reduced to the noncultivatable level within 7 to 10
days. The cessation of growth is accompanied by a morpho-
logical change from a helical shape to a nonhelical shape,
including a coccoid form (6). H. pylori lacks sigma S, sigma H,
and sigma E, which are typically associated with various stress
responses in many gram-negative bacteria; however, spoT is
conserved in H. pylori, implying that SpoT and (p)ppGpp are
key regulators for various stress responses in this bacterium,
including survival in macrophages.
While this study was in progress, it was reported that the H.
pylori spoT gene encodes (p)ppGpp synthetase activity and that
spoT is required for H. pylori survival in the stationary phase,
during exposure to low pH, and during aerobic shock (34, 52).
* Corresponding author. Mailing address: Transcription Control
Section, Gene Regulation and Chromosome Biology Laboratory, Na-
tional Cancer Institute—Frederick, National Institutes of Health, Fre-
derick, MD 21702. Phone: (301) 846-7684. Fax: (301) 846-1489.
?Published ahead of print on 3 October 2008.
However, the hydrolase activity of the H. pylori SpoT protein
has not been determined. Further, no “relaxed” phenotype
associated with a spoT mutant during nutrient starvation has
been reported. Moreover, the role of spoT in survival during
phagocytosis by macrophages is unknown. In this study, we
examined SpoT activity in vivo and also the effects of spoT on
cell growth under serum starvation conditions and on H. pylori
survival in macrophages. Our data provide additional evidence
that spoT is important in sensing serum limitation, in infection
persistence, and in the pathogenesis of H. pylori.
MATERIALS AND METHODS
Bacterial strains and growth conditions. All E. coli strains used are K-12
derivatives. The relA spoT double null mutant [(p)ppGpp0] and the spoT203
mutant (obtained from Mike Cashel, NIH) have been described previously (43,
55). The general bacterial techniques and media used have also been described
previously (30). H. pylori strains J99 (obtained from the American Type Culture
Collection [ATCC]), 26695 (obtained from ATCC), G27 (obtained from D. Scott
Merrell, Uniformed Services University of the Health Sciences), strain HP1061
(obtained from Paul S. Hoffman, Dalhousie University, Halifax, Nova Scotia,
Canada), and SS1 (obtained from A. Lee and J. O’Rourke, University of New
South Wales, Sydney, Australia) have been described previously (5, 23, 27, 49,
54). The G27 spoT* strain (obtained from Karen Guillemin, University of Ore-
gon) has also been described previously (34).
Unless mentioned otherwise, H. pylori cells were grown in bisulfiteless brucella
broth (BLBB) (24) supplemented with (i) Glaxo selective supplement A (20
?g/ml bacitracin, 1.07 ?g/ml nalidixic acid, 0.33 ?g/ml polymyxin B, 10 ?g/ml
vancomycin) (36) and (ii) 10% (vol/vol) fetal bovine serum (FBS; triple sterile
filtered, 0.1-?m filter) (SH30088.03HI; HyClone) (referred to below as BLBB
with 10% FBS). To make solid medium plates, Difco agar (1.7%, wt/vol) was
added to BLBB with 10% FBS described above. Kanamycin (15 ?g/ml) or
chloramphenicol (4 to 8 ?g/ml) was added when necessary. H. pylori cells were
cultivated at 37°C in a sealed jar or a humidified incubator with a microaerophilic
atmosphere (5% O2, 10% CO2, 85% N2), which was generated with an Anoxo-
mat Mark II microprocessor (www.spirabiotech.com) or was provided from a gas
H. pylori strains were maintained as frozen stocks at ?80°C. A frozen stock was
prepared by mixing equal volumes of a fresh 1-day culture (optical density at 600
nm [OD600], ?1) and BLBB supplemented with 10% (vol/vol) FBS and 50%
(vol/vol) glycerol. To ensure physiological reproducibility, experiments were
started using frozen stocks; thus, the initial cultures were prepared 3 days before
experiments were performed. On day 1, about 50 to 100 ?l of a frozen stock was
placed on a solid medium plate (60 by15 mm), which was then incubated for ?24
h. The next day, the bacteria were transferred evenly onto the entire surface of
a new solid medium plate using a moistened cotton swab, and the plate was then
incubated for ?24 h. On day 3, the fresh confluent bacterial lawn was collected
with a swab and suspended in 1 ml of BLBB without FBS in a 1.5-ml tube, and
this preparation was used as the inoculum for the starting cultures after appro-
priate dilution. In general, a starting culture (25 to 30 ml in a 225-ml cell culture
flask) with an OD600of ?0.05, which was equivalent to ?3 ? 107CFU ml?1, was
used for experiments. In each experiment, the wild-type strain and isogenic
derivatives of this strain were analyzed in parallel in the same environment (jar)
to minimize variables. Cultures were shaken gently at 60 rpm.
Bacterial growth was monitored by using three methods: (i) measurement of
the OD600of cultures using a spectrophotometer, with the growth medium
serving as the blank; (ii) determination of the number of CFU by plating serial
dilutions of cultures in duplicate on plates, which were then incubated for 3 to 6
days; and (iii) measurement of intracellular ATP levels using a bioluminescent
ATP assay kit as described previously (47) (see below).
ATP assays. ATP levels in duplicate cultures were determined using the
luciferase-based BacTiter-Glo microbial cell viability assay kit (catalog number
G8230; Promega) according to the manufacturer’s instructions; the growth me-
dium was used as a blank in these experiments. Cultures were diluted appropri-
ately to ensure that the range of measurements was linear. Light production was
measured using a microplate reader (Vector3; PerkinElmer), and the data were
expressed in relative light units. Because the wild-type and mutant strains were
always compared in the same experiment, determination of absolute ATP con-
centrations was unnecessary.
Transformation of H. pylori strains. Briefly, H. pylori cultures were prepared
as described above. On day 3, ?50 ?l of an inoculum (?108cells) was spotted
onto a fresh solid medium plate, and the plate was incubated for 3 h. Subse-
quently, ?50 ?l (?50 ng) of DNA was placed on top of the bacterial spot, and
the plate was incubated for another 3 to 5 h. Then the bacteria and DNA mixture
were transferred onto the entire surface of a fresh solid medium plate supple-
mented with the appropriate antibiotic using a moistened cotton swab. The
plates were incubated for 3 to 6 days to select for recombinants or transformants,
which were purified at least once on selective solid medium plates before frozen
stocks were prepared.
DNA techniques. Standard molecular biology techniques for DNA purifica-
tion, cloning, and PCR were performed as described previously (42). Genomic
DNA was prepared using a Wizard Genomic DNA purification kit (Promega)
according to the manufacturer’s instructions. Plasmid DNA was purified from H.
pylori as described previously (16). DNA sequencing was performed by the
National Cancer Institute Intramural DNA Sequencing MiniCore facility.
Cloning of the spoT gene from H. pylori strains. DNA fragments containing the
spoT gene from different H. pylori strains (genomic DNAs) were generated by
PCR and cloned into either the BamHI/PstI sites of vector pQE80L (Qiagen) or
the SphI/KpnI sites of the shuttle vector pHel2 (obtained from Rainer Haas )
using standard DNA techniques. The primers used for generating the DNA
fragments containing the spoT gene flanked by BamHI and PstI sites were
SpoT/BamHI F (5?-GCGGATCCATGAACGAAATTGATAAATC-3?; BamHI
site underlined) and SpoT/PstI R (5?-AAAACTGCAGTTATGATTCATAAG
CGTCAT-3?; PstI site underlined). The primers used for generating the DNA
fragments containing the spoT gene flanked by SphI and KpnI sites were SpoT-
SphI-F (5?-ACATGCATGCGATTCGCTGAGAATGTAGG-3?; SphI site un-
derlined) and SpoT-KpnI-R (5?-CGGGGTACCTTATGATTCATAAGCGT
CAT-3?; KpnI site underlined). The sequences of the primers were based on the
previously published genome sequence of H. pylori strain 26695 (49). The cloned
spoT genes were confirmed by DNA sequencing.
Construction of the ?spoT::kan mutant. A nonpolar spoT null mutation, in
which about one-half of the spoT gene encoding amino acid residues 101 to 511
of SpoT was deleted and replaced with an insertion of the nonpolar aphA-3 gene
encoding a kanamycin resistance cassette (29), was constructed in several steps.
First, a ?4.1-kb DNA fragment containing the spoT null mutation was con-
structed directly by performing three PCRs without cloning. For the first two
PCRs, H. pylori strain J99 genomic DNA was used as the template. In the first
PCR, using primer 1 (5?-AATCCCTCACTACATCCTTAAAGAAG-3?) and
primer 2 (5?-AGCCATTTATTCCTCCTAGTTAGTCACCTCACAAGGCGT
GTCTTCTACCAC-3?), a 1.5-kb DNA fragment (fragment A) was produced.
The 5? end of this DNA fragment covered the JHP715 gene sequence located
upstream of spoT (JHP712), and its 3? end had an internal spoT sequence,
followed by translational stop codons, a ribosome binding site, and the beginning
of aphA-3 in pUC18K-2 (29). In the second PCR, primer 3 (5?-GAATTGTTT
ATC-3?) and primer 4 (5?-CACGATTTCACTAGCGAGATCCTC-3?) were
used, and a 1.7-kb DNA fragment (fragment B) was produced. The 5? end
sequence of fragment B overlapped the end of the aphA-3 sequence, followed by
an extra ribosome binding site which was in frame with the remaining spoT
sequence, and the 3? end of fragment B overlapped the JHP711 gene sequence
located downstream of spoT. In the third PCR, the first two PCR products, DNA
fragments A and B, as well as pUC18K-2 DNA, were used as templates along
with primers 1 and 4, which resulted in a 4.1-kb DNA fragment that contained
the spoT null mutation. Finally, H. pylori strains were transformed with the 4.1-kb
DNA fragment, and Kanrrecombinants were selected. The spoT null mutation in
the Kanrrecombinants was verified by PCR and sequencing of the genomic
locus. The resulting spoT mutant was not able to produce (p)ppGpp when cells
were shifted from a rich growth medium to a minimal medium (data not shown),
as reported previously for another spoT mutant (34).
Analysis of bacterial morphology. Bacterial cells were fixed on glass slides by
heat, followed by Gram staining (56). The slides were examined using a Zeiss
Axiophot II microscope, and images were captured with a charge-coupled device
camera. For each condition and/or time point, more than 100 cells were scored.
Due to the limited resolution, only helical and nonhelical forms, including coc-
coid forms, were categorized.
Cell culture. Murine macrophage cell line RAW 264.7 was obtained from the
ATCC and grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitro-
gen) supplemented either with only 10% FBS or with Glaxo selective supplement
A plus 10% FBS. This cell line was cultured in a humidified incubator with 5%
CO2and 95% air at 37°C.
Invasion and intracellular survival assays. An invasion assay and an intracel-
lular viability bacterial assay were performed as described previously (18), with
some modifications. One day before invasion assays were performed, RAW 264.7
cells were seeded in 96-well tissue culture plates to obtain a concentration of ?2 ?
8026ZHOU ET AL. J. BACTERIOL.
104macrophages per well. The bacterial cells were prepared similarly to the
inoculum used for the starting cultures described above, except that DMEM
containing 10% FBS was used for the final dilution. In each independent exper-
iment, 3 to 12 wells were used for each bacterial strain tested. The monolayers
of macrophages in the wells were infected with 0.1 ml of a chilled bacterial
suspension (OD600, ?0.025; CFU ?1.5 ? 106CFU). Immediately after infection,
the plates were centrifuged at 4°C for 4 min at 600 ? g to synchronize bacterial
contact with the monolayers (4), and this was followed by three washes with cold
DMEM. After centrifugation and washing, 0.1 ml of DMEM containing 10%
FBS at 37°C was added (designated time zero), and the infected macrophage
monolayers were incubated at 37°C in a humidified incubator with 5% CO2and
95% air. After 1 h, the infected monolayers were washed once with DMEM and
then were incubated in 0.1 ml of DMEM containing 10% FBS and 100 ?g of
gentamicin/ml for 1 h to kill the extracellular bacteria but not macrophages. The
infected monolayers were then washed once with phosphate-buffered saline
(PBS) or DMEM and then either lysed (2-h time point) or incubated in 0.1 ml
of DMEM containing 10% FBS for a total of 24 h (24-h time point). To lyse the
macrophage monolayers and release H. pylori, 0.1 ml of H2O was added to each
well after it was washed with PBS or DMEM either three times (time zero) or
once (other time points). Finally, the numbers of viable bacteria (CFU) in the
macrophage lysates were determined by plating serial dilutions on solid plates as
Staining of live bacteria in infected macrophages. Live H. pylori cells within
macrophages were stained using a BacLight kit (Invitrogen) according to the
manufacturer’s instructions. Bacterial invasion and intracellular survival as-
says were performed essentially as described above, with some modifications.
Twenty-four-well tissue culture plates with clean glass coverslips (12 mm)
placed in the bottom of wells were used. About 1 ? 105RAW 264.7 macro-
phages (seeding) and 0.5 ml H. pylori cells (OD600, ?0.025; ?7.5 ? 106CFU)
in DMEM containing 10% FBS per well were used for infection. At the 2-h
(at the end of gentamicin treatment) and 24-h time points, the medium was
removed from each well by aspiration, and the wells were rinsed once with
PBS. The macrophage cells were permeabilized with 0.5 ml of saponin (1
mg/ml; freshly made in PBS) at room temperature for 10 to 15 min. After the
saponin treatment, the solution was removed very carefully (macrophages
were fragile following this treatment), and 0.25 ml of diluted BactLight
solution was added to each well to stain the viable bacteria. After incubation
at room temperature for 15 min, we removed the coverslips from the wells
and placed each coverslip on a glass slide for visualization. The slides were
examined using a Zeiss Axiophot II microscope equipped with epifluores-
cence filters. Images were captured with a charge-coupled device camera. The
images were processed with Adobe Photoshop.
RESULTS AND DISCUSSION
SpoT of H. pylori is a bifunctional enzyme with both (p)ppGpp
synthetase and hydrolase activities. H. pylori has the spoT gene
but no relA gene. This suggests that (p)ppGpp synthesis and
degradation are mediated solely by SpoT in H. pylori. We
wished to determine whether the H. pylori spoT product has
(p)ppGpp hydrolase activity in addition to its known (p)ppGpp
synthetase activity. We cloned the spoT gene from several H.
pylori strains (G27, J99, 1061, SS1, and 26695) into either the
expression vector pQE80L or the shuttle vector pHel2 (25).
The resulting spoT clones were tested to determine their abil-
ities to complement known E. coli relA and spoT mutations,
which has been used previously as a way to define the
(p)ppGpp synthetase and hydrolase activities of the products
of spoT genes from different bacteria in vivo (15, 21, 28). All
spoT clones complemented the defective functions in the E.
coli mutants (Fig. 1 and data not shown). Sequence analysis of
these clones revealed only a few minor variations in them (data
not shown); therefore, the results obtained for a representative
clone, pHPspoT (pQE80L-spoT clone from strain 1061), are
The E. coli relA spoT double null mutant was used as a host
to test the (p)ppGpp synthetase activity of H. pylori SpoT in
vivo. This E. coli double null mutant does not grow on minimal
medium due to the absence of (p)ppGpp in the cell (55). The
pHPspoT clone complemented the double null mutation, al-
lowing the host to grow on minimal medium. As a control, we
showed that the cloning vector pVector (pQE80L) did not
complement the mutation (Fig. 1A). Our results are consistent
with the results of a previous report that showed that the H.
pylori SpoT protein has (p)ppGpp synthetase activity (34).
However, in the previous study the authors observed only par-
tial complementation of the E. coli double null mutant by the
FIG. 1. H. pylori spoT exhibits both (p)ppGpp synthetase and hydrolase activities in vivo. (A) The H. pylori spoT gene complemented an E. coli
relA spoT double null mutant [(p)ppGpp0]. The H. pylori spoT gene (pHPspoT) enabled the E. coli double null mutant to grow on an M63 glucose
minimal medium plate without any amino acid supplement, but the control vector (pVector) did not grow. The plate was examined after incubation
at 37°C for 48 h. (B) H. pylori spoT gene complemented the slow-growth phenotype of the E. coli spoT203 mutant on an LB plate. The plate was
examined after incubation at 30°C for 24 h. The images were obtained using an imaging system obtained from Alpha Innotech, and they were
processed and merged with Adobe Photoshop.
VOL. 190, 2008 GROWTH CONTROL AND PHAGOCYTOSIS RESISTANCE BY ppGpp8027
H. pylori spoT construct used in their study. The differences
may be explained as follows: the spoT clones used in this study
encode the full length of SpoT (?775 amino acid residues),
whereas the spoT construct used in the previous report lacked
the last 25 amino acid residues in the C terminus of SpoT (34).
To determine if the H. pylori SpoT protein has (p)ppGpp
hydrolase activity, we took advantage of the mutant E. coli
spoT203 mutant gene, which encodes a SpoT mutant protein
defective in (p)ppGpp hydrolase activity without abolishing the
(p)ppGpp synthetase activity. The spoT203 mutation results in
a ?10-fold-higher level of (p)ppGpp in the cell and, because
this high level is toxic, a slow-growth phenotype (43). The
growth defect of the E. coli spoT203 mutant was abolished by
clones expressing the H. pylori spoT gene but not by the vector
alone (Fig. 1B). These complementation results demonstrated
that H. pylori SpoT does have both (p)ppGpp hydrolase and
synthetase activities in vivo.
The domains for the two enzyme activities associated with
SpoT are highly conserved in bacteria (31). H. pylori SpoT is
more similar to SpoT of Campylobacter jejuni (?39% identity),
a closely related bacterium, than to E. coli SpoT (?27% iden-
tity). Results of this study (see below) and other studies (34)
indicate the importance of spoT in regulation of H. pylori
growth during serum starvation and in stress responses, which
are consequences of stationary growth or phagocytosis. It is
expected that both the synthetase and hydrolase activities of
SpoT are sensitive to environmental cues. Recently, it was
reported that the SpoT-dependent stress response is linked to
fatty acid metabolism in E. coli (8). It is plausible that SpoT
behaves similarly in H. pylori. The challenge is to identify the
signals which modulate the SpoT activities and the mechanism
by which (p)ppGpp regulates gene expression in H. pylori.
A spoT null mutant is able to grow to a higher density in
serum-free medium than the wild-type strain. E. coli mutants
defective in (p)ppGpp synthesis have a “relaxed” phenotype
during amino acid starvation; therefore, we hypothesized that
a spoT mutant of H. pylori might have a growth advantage over
the wild-type strain under certain nutrient-limiting conditions.
Brucella broth supplemented with FBS has often been used for
culturing H. pylori (33). We used BLBB supplemented with
FBS to obtain optimal growth of H. pylori (24). The basis of the
requirement for serum for optimal or enhanced H. pylori
growth in vitro is unknown. Components other than bovine
serum albumin present in the serum have been suggested to be
the factor that enhances the growth of H. pylori (48). The
serum requirement for H. pylori growth prompted us to test
whether a spoT null mutation confers a growth advantage
during serum starvation.
For this study we chose the H. pylori G27 strain (54), because
this strain has been studied by several laboratories and could
serve as a prototype H. pylori strain for detailed genetic and
physiology studies. We constructed a G27 nonpolar spoT null
mutant (G27?spoT) using a method that eliminated an inter-
mediate cloning step as described in Materials and Methods.
In addition, a “complemented” or spoT* strain (G27?spoT
rdxA::Pure::HP0775) was included for comparison because
Pure-driven spoT (HP0775) expression has been reported to be
higher than wild-type spoT expression in the G27 background
(34), although the extent of the increase in SpoT expression in
this strain has not been determined. To determine whether the
spoT null mutation provides a growth advantage in serum-free
BLBB, we compared the growth of the spoT mutant with the
growth of the wild-type and spoT* strains in either BLBB with
no FBS or BLBB with 10% FBS using three different mea-
surements, OD600, intracellular ATP levels, and CFU.
After initial brief growth in serum-free BLBB, the wild-type
and spoT* strains stopped growing when the OD600reached
?0.2 at 48 h. However, the spoT mutant reproducibly contin-
ued to grow until the OD600reached ?0.8 at 48 h (Fig. 2A).
This finding is in contrast to the growth curves for cells grown
in BLBB with 10% FBS, which indicated that there were no
differences among the spoT mutant, wild-type, and spoT*
strains. Consistent with the OD600values, the peak ATP levels
were reproducibly approximately threefold higher in the spoT
mutant than in the wild-type and spoT* strains at 48 h in the
serum-free medium (Fig. 2B). The ATP level in the spoT
mutant, however, rapidly decreased after the peak was reached
and was close to the basal level at 72 h, a pattern similar to the
pattern observed for the same mutant grown in serum-supple-
mented medium. In the wild-type and spoT* strains grown in
the serum-free medium, however, the overall ATP levels were
low and remained low with no apparent peak at 48 h. In
contrast, the ATP levels were high and peaked at 48 h for both
the wild-type and spoT* strains in the serum-supplemented
medium. The ATP levels in the wild-type and spoT* strains
were ?15-fold lower in the serum-free medium than in the
serum-supplemented medium at 48 h.
Determination of the viable bacterial counts confirmed that
the number of CFU ml?1of the spoT mutant was consistently
approximately threefold higher than the numbers of CFU ml?1
of the wild-type and spoT* strains at 48 h in the serum-free
medium (Fig. 2C), in close agreement with the data for the
OD600and ATP levels. At 72 h and later time points, however,
the number of CFU ml?1was lowest for the spoT mutant, and
this was accompanied by a rapid decline in the ATP levels (Fig.
2B). These results are consistent with the report that the spoT
mutation reduced the survival rate in the stationary phase (34),
apparently independent of the presence of serum in the me-
dium. Thus, our results showed that the spoT mutant exhibits
a “relaxed” growth phenotype in a serum-free medium. To the
best of our knowledge, this is the first reported “relaxed”
phenotype associated with a spoT mutant in H. pylori.
Overall, our data suggested that one of the functions of
SpoT or (p)ppGpp in H. pylori is to limit cellular metabolic
activity and restrict cell growth during serum starvation. This
function of SpoT appears to be a general function because the
growth advantage of the spoT mutant not only is observed in
serum-free BLBB, which is a complex medium, but also is
apparent in a serum-free chemically defined Ham’s F-12 nu-
trient mixture, which has been reported to be able to support
limited growth of H. pylori (48). Our preliminary results
showed that the number of CFU ml?1of the spoT mutant was
also approximately threefold higher than the number of CFU
ml?1of the wild-type strain at 48 h in a serum-free F-12 me-
dium (data not shown). How SpoT senses serum starvation is
not known at present. It has been reported that expression of
spoT in Borrelia burgdorferi is induced during serum starvation
(15). We speculate that a spoT mutation in B. burgdorferi might
also stimulate cell grow in a serum-free medium. The mecha-
8028 ZHOU ET AL.J. BACTERIOL.
nism by which SpoT and (p)ppGpp regulate H. pylori growth in
response to serum starvation needs to be studied further.
During serum starvation H. pylori maintains helical mor-
phology due to SpoT function. We also monitored the mor-
phology of H. pylori cells grown in the serum-free medium by
using microscopy. Images of more than 100 cells at different
time points during bacterial growth were analyzed, and cell
shapes were categorized as either helical or nonhelical, includ-
ing coccoid forms. Interestingly, the morphological conversion
of wild-type cells from a helical form to a nonhelical form was
significantly delayed during serum starvation compared to the
cells grown in BLBB with 10% FBS (Fig. 3A). Almost all
(?95%) of the exponentially growing cells (up to 24 h) in
either a serum-free medium or serum-supplemented BLBB
were helical (data not shown). After 24 h, the conversion to
nonhelical cells was reproducibly faster for cells grown in se-
rum-supplemented medium than for cells grown in serum-free
medium. For example, at 48 h the values were ?31% and ?4%
for the wild-type strain grown in the serum-supplemented and
serum-free media, respectively. At 72 h, however, the values
were ?97% and ?21%, respectively. Consistent with these
results, the numbers of CFU ml?1between 48 and 72 h were
similar for the cells grown in the serum-free medium; however,
the number of CFU ml?1at 72 h was about fivefold lower than
the number of CFU ml?1at 48 h when cells were grown in
BLBB with 10% FBS (Fig. 2C). This trend was also observed
for both the spoT mutant (Fig. 3B) and the spoT* strain (Fig.
3C). Compared to the wild-type strain, however, the spoT
mutant initiated the morphological transformation prema-
turely, as reported previously (34). Most impressively, how-
FIG. 2. The spoT mutant has a growth advantage in serum-free medium compared to wild-type H. pylori. Different H. pylori strains were grown
in BLBB either in the absence (Serum-free) or in the presence (Serum-supplemented) of 10% FBS under microaerophilic conditions (5% O2, 10%
CO2, 85% N2). Bacterial growth was monitored by measuring (A) the OD600, (B) the intracellular ATP levels (expressed in relative light units
[RLU]), and (C) the number of CFU ml?1(expressed on a log scale). F, spoT mutant; Œ, wild-type strain; f, spoT* strain. The error bars indicate
the deviations from the means, and there are no error bars if the deviation from the mean was smaller than the symbol. The data are representative
of at least three independent experiments.
VOL. 190, 2008 GROWTH CONTROL AND PHAGOCYTOSIS RESISTANCE BY ppGpp8029
ever, the vast majority (?88%) of the spoT* cells, which had
higher SpoT activity than the wild-type cells, were still helical
even at 96 h in the serum-free medium (Fig. 3C). It is likely
that the higher SpoT activity in spoT* cells amplifies the func-
tion of SpoT during serum starvation.
Our results suggest that serum starvation restricts H. pylori
growth due to the SpoT function and consequently prolongs
the time that the bacterial cells are helical. Because helical H.
pylori cells are consistently found in chronically infected hosts,
it is tempting to speculate that H. pylori growth in vivo is
minimal or restricted due to nonoptimal growth conditions in
the human stomach. The majority of H. pylori cells are located
deep in the gastric mucus, close to the surface of the epithe-
lium (44). It has been suggested that tight junctions between
epithelium cells in healthy gastric tissue are unlikely to leak
significant amounts of serum, thus making the natural habitat
of H. pylori a serum-free microenvironment (47). Thus, SpoT
may play an important role in restricting cellular metabolic
activity and limiting H. pylori growth during serum starvation in
order to maintain cell vitality under these poor growth condi-
SpoT is important for intracellular survival of macrophage-
ingested H. pylori. H. pylori has developed multiple mecha-
nisms to evade elimination by host innate immune responses,
including macrophages, which may account for the persistence
of this bacterium in the host (1, 40). Once inside macrophages,
H. pylori interferes with phagosome maturation, which leads to
the formation of large phagosomes called megasomes. H. pylori
resides in these megasomes during phagocytosis (2–4). The
megasomes apparently do not fuse with the lysosomes in mac-
rophages, thus protecting the bacteria from elimination. It is
conceivable that ingested H. pylori cells following phagocytosis
by macrophages likely invoke a bacterial cell stress response as
a survival mechanism. H. pylori virulence factors, VacA, ure-
ase, and type IV secretion components have been reported
to be important for bacterial survival in macrophages (37,
40, 45, 57).
We wished to determine whether spoT plays a role in the
survival of H. pylori in macrophages. We investigated the effect
of the spoT mutation on intracellular survival by determining
the number of CFU ml?124 h after phagocytosis, as previously
described (4, 18). The number of surviving spoT mutant cells
(CFU ml?1) in macrophages after 24 h was significantly lower
than the numbers of surviving cells of the wild-type and spoT*
strains (Fig. 4).
To determine whether the spoT mutant was invasion defec-
tive, which could contribute to the apparent reduced survival,
we determined the numbers of CFU ml?1of the infected
bacteria at several time points after phagocytosis by macro-
phages. While the level of survival of the spoT mutant was
significantly lower than the levels of survival of the wild-type
and spoT* strains at 24 h after infection, the number of spoT
FIG. 3. H. pylori maintains helical morphology during serum star-
vation due to SpoT function. More than 100 cells from cultures grown
either in BLBB without FBS (black bars) or in BLBB supplemented
with 10% FBS (grey bars) at different time points were examined by
microscopy. (A) Wild-type strain. (B) spoT mutant. (C) spoT* strain.
The values are the fractions of all the cells scored which were nonhe-
lical, including coccoid forms. The error bars indicate the deviations
from the means, and there are no error bars if the deviation from the
mean was negligible. The data are representative of three independent
FIG. 4. The spoT mutant exhibits reduced survival in macrophages.
The abilities of different H. pylori strains to invade and survive in
macrophages were determined at different times postinfection and
were expressed in numbers of CFU ml?1. Black bars, spoT null mutant;
gray bars, wild-type strain; open bars, spoT* strain. The error bars
indicate the deviations from the means. The data are representative of
at least three independent experiments.
8030 ZHOU ET AL.J. BACTERIOL.
mutant cells that could be recovered from infected macro-
phages was essentially the same as the numbers of wild-type
and spoT* colonies recovered 2 h after invasion (Fig. 4). Note
that the 2-h samples were samples that were obtained after
gentamicin treatment to kill extracellular bacteria; therefore,
the CFU represented the intracellular bacteria that had in-
vaded. Thus, our data demonstrated that the ability of the spoT
mutant to invade macrophages appears to be normal, but its
ability to survive in the macrophages is defective. Consistent
with these results, microscopic imaging of live H. pylori cells
showed that while the numbers of intracellular bacteria per
macrophage were comparable for the spoT mutant, wild-type,
and spoT* strains at 2 h after infection, the numbers of spoT
mutant cells were significantly reduced after 24 h compared to
the numbers of wild-type and spoT* strain cells (data not
Our results demonstrated that SpoT or (p)ppGpp plays an
important role in bacterial evasion of elimination by macro-
phages. At present, the mechanism(s) by which SpoT or (p)p-
pGpp mediates the survival of the bacterium in macrophages is
unknown. It may be that SpoT or (p)ppGpp affects a path-
way(s) different from the pathways mediated by either VacA or
urease, because the activities of the VacA and urease proteins
in the spoT mutant were not significantly different from the
activities of these proteins in the isogenic wild-type strain (34;
unpublished results). Currently, we are studying the effects of
SpoT on the interactions between H. pylori and macrophages.
In summary, in this study we investigated the function of the
H. pylori SpoT protein in vivo and its roles in cell growth under
serum starvation conditions and in bacterial survival in mac-
rophages. Our results showed that the H. pylori spoT gene
encodes a bifunctional enzyme for the synthesis and degrada-
tion of (p)ppGpp. Also, while wild-type H. pylori maintains
helical morphology and cell growth is restricted during serum
starvation, the spoT mutant has a growth advantage over the
wild-type strain in a serum-free medium and exhibits a “re-
laxed” growth phenotype. Moreover, SpoT is critical for intra-
cellular survival in macrophages. Thus, our results demon-
strate that H. pylori SpoT or (p)ppGpp regulates cell growth in
response to serum starvation and plays a critical role in the
persistence of this bacterium.
We thank the leadership of the Gene Regulation and Chromosome
Biology Laboratory and the National Cancer Institute Center for Can-
cer Research for the support in the initiation of the H. pylori project in
D.J.J.’s laboratory, and we thank colleagues in the H. pylori research
community and in our laboratories for helpful discussions. We also
thank Scotty Merrell, Karen Guillemin, Kevin Bourzac, Rainer Haas,
Mike Cashel, and Lee-Anne Allen for providing strains, plasmids
and/or protocols. Y.N.Z. thanks Susan Gottesman for her support. We
are grateful for helpful comments on the manuscript from Don Court,
Susan Gottesman, Mikhail Kashlev, Peter McPhie, Scotty Merrell,
Herbert Tabor, and Julie Torruellas Garcia.
This research was supported by the Intramural Research Program of
the NIH National Cancer Institute Center for Cancer Research, by the
Intramural Research Program of the National Institute of Digestive
Diseases, Diabetes and Kidney Diseases, and by an intra-agency agree-
ment with the NIH National Center on Minority Health and Health
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