458

Journal of Health Science, 57(6) 458–471 (2011)— Review —SEp22, Salmonella Dps, a Key Molecule Bearing Both
Pathogenicity and Resistance to Environmental Stresses
in Salmonella
Fumio Amano∗
Laboratory of Biodefense & Regulation, Osaka University of Pharmaceutical Sciences, 4–20–1 Nasahara, Takatsuki, Osaka 569–1094,
Japan(Received August 26, 2011)

We isolated and characterized a pathogenicity-related protein in Salmonella Enteritidis (SE) from poultry farms,
and designated it as SEp22, which was identified later as Salmonella Dps, a DNA-binding protein from starved
cells. Expression of SEp22 was regulated by bacterial growth in Luria-Bertani (LB) medium, showing reduced
expression in the logarithmic phase but increased expression from the late logarithmic to stationary phases, which
was linked to the expression of σS both in protein as well as mRNA levels. In addition, induction of SEp22 required
nutritional factors in LB or casamino acids during overnight incubation in M9 minimal medium, or even throughshort-term exposure to H2 O2 for 5–15 min. Induction of SEp22 was also remarkable after sudden addition of H2 O2and exposure of the bacteria to the drying protocol. The roles of SEp22 in the pathogenicity of Salmonella inmice, the survival of Salmonella exposed to H2 O2 , or that under dry-stress were proved not only by the difference
among Salmonella clones from environmental isolates with different levels of SEp22 expression, but also by sep22gene-depleted mutants that originated from SECl#15-1, a wild type virulent strain with high levels of SEp22.
These results suggest that SEp22 is a key molecule that is responsible for pathogenesis as well as environmental
stress-resistance. In this review, the diverse roles of SEp22/Dps in Salmonella are also described, suggesting theimportance of this protein in the bacterial stress responses in infection and survival, as well as in the regulation ofbacterial growth through aerobic metabolism. Recent progress in SEp22/Dps research at the molecular levels is
also discussed.
Key words —— Salmonella Enteritidis, Dps, pathogenicity, resistance to environmental stresses, H2 O2 -resistance,
dry-resistance

INTRODUCTION
Salmonella is one of the most causative bacteria
of food-borne diseases in Japan1) and other countries,2) and is detected not only in food but also innatural environments including river water, soil or
air-borne dust. Among Salmonella spp., Salmonella
enterica serover Enteritidis (S. Enteritidis; SE) has
been the most dominant species detected, and is frequently found in contaminated foods such as eggs,
chicken meat, and even vegetables including cabbages, tomatoes, and pimentos. It has also been the∗

To whom correspondence should be addressed: Laboratory of
Biodefense & Regulation, Osaka University of Pharmaceutical Sciences, 4–20–1 Nasahara, Takatsuki, Osaka 569–1094,
Japan. Tel.: +81-72-690-1054; Fax: +81-72-690-1054; E-mail:amano@gly.oups.ac.jp

cause of mass food-poisoning in Japan and U.S.A.,
which might be because SE shows rather strong virulence in humans among Salmonella species thatcause food-poisoning. Poultry is an importantsource for spreading SE, not only thorough food but
also through environmental contamination.3) Epidemiological studies have revealed that the major
vehicles of SE outbreaks in humans have included
contaminated poultry meat and eggs.4) In order tocontrol the spread of SE to foods, it is of a great
importance to understand the characteristics of SE
that allow it to survive in natural environments under various conditions, and also to understand themechanisms underlying the spread of SE in environments as well as in clinical cases.
It is also important to remind ourselves thatthe virulence and pathogenicity of Salmonella areC 2011 The Pharmaceutical Society of Japan

459

No. 6

Fig. 1. Salmonella-macrophage Interaction (a Model)
Table 1. Expression of SEp22 and invA, Pathogenicity in Mice, and Resistance to H2 O2 and to the
Drying Protocol in SE Environmental Isolates
Clone
SECl#4-1
SECl#7-1
SECl#14-1
SECl#15-1
SECl#16-1
SECl#23-1
SECl#26-1
SECl#28-1
SECl#40∗

SEp22a)
±
+++
±
+++
±
+++
++
−
+++

invAb)
+
+
+
+
+
+
−
+
+

pathogenicityc)
−
+
−
+
−
+
−
−
+

H2 O2 -resistd)
−
+
−
+
−
+
N/A
−
+

dry-resiste)
±
+++
±
+++
±
+++
N/A
−
+++

∗ A clinical isolate from a patient of sporadic diarrhea. The other bacterial clones in this table are environmental isolates from poultry farms in Japan. a) SEp22 was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting. b) invA was determined by RT-PCR.
c) Pathogenicity of the bacteria was estimated by lethality after oral administration of 1 × 108 cfu of eachbacterial clone to BALB/c mice. d) H2 O2 -resistance was assayed by colony-formation of each bacterium after
exposure of 0.1–10 mM H2 O2 . e) Dry-resistance was assayed by the drying protocol. In this table, the extents of each phenotype are relatively expressed as negative (−), sometimes positive (±), positive (+), strongly
positive (++), and very strongly positive (+++). N/A means “not assayed.”tightly linked to its behavior in the host after infection: As a food-poisoning bacterium, Salmonella
usually invades from gut epithelium through M
cells in the neighboring Peyer’s patches, or throughintestinal epithelial cells directly, where polymorphonuclear leukocytes (PMNs) and macrophages
phagocytose them.5) The macrophages play major roles in elimination of these Salmonella fromthe host by exerting bactericidal activity including
phagocytosis, secretion of lysozyme or other lysosomal enzymes, and the production of reactive oxygen species (ROIs) such as superoxide anion (O2 ·− ),hydrogen peroxide (H2 O2 ), hydroxyl radical (OH·),
singlet oxygen (1 O2 ), nitric oxide (NO·), or peroxynitrite (ONOO− ).6) Among them, H2 O2 plays central roles in bactericidal activity, because it readily

penetrates through bacterial membranes and is converted to OH· in the presence of Fe2+ by the Fenton
reaction.
To cope with these attacks and effector
molecules produced by macrophages, Salmonella
has a counter system to attack host cells (Fig. 1); oneof the most important systems is the Type III secretion system (T3SS) which is composed of a series ofmolecules necessary for injection of virulence factors through a needle-like structure. Among them,invA encodes an inner membrane protein that forms
the apparatus of T3SS.7)
So far, the virulence of bacteria has been estimated by the presence of invA, spv8) or enterotoxin genes. However, as shown in Table 1, thepathogenicity of Salmonella clones is not entirely460

Vol. 57 (2011)

Fig. 2. Expression of SEp22 in SE Clones

consistent with the presence of invA. Therefore,
invA is a necessary factor for acquisition of virulence that is required to attack the host. Apparently,
another defense factor for protection of the bacteria
from certain effector molecules which are derived
from the immune phagocyte system like ROIs, isimportant. This is an adequate factor that is closely
linked to the pathogenicity of the bacteria during
infection of the host. Therefore it is important toconsider both the offensive and defensive factorsof Salmonella when estimating the pathogenicity inthe host.
In our studies searching for the factors andmechanisms underlying acquisition of pathogenicity in the mice by environmental SE clones, werecognized one protein band of MW of approximately 22 kDa, that was evident in all of the cell
extracts of the virulent clones but not in the avirulent clone [Fig. 2(a) Coomassie brilliant blue (CBB)
stain]. This experiment lead me to discover SEp22,
which will be described in the following section.

ISOLATION AND CHARACTERIZATION
OF SEp22 PROTEIN
Isolation of SEp22
More than 30 SE clones, isolated from poultry
farms (CAF Laboratories; Fukuyama, Japan), wereindividually cultured in 100 ml of Luria-Bertani(LB) medium in 300-ml Erlenmeyer flasks at 37◦ C
overnight, with shaking at 150 strokes min−1 . The
cells were harvested by centrifugation at 4120 × g
for 20 min at 4◦ C, washed with ice-cold phosphatebuffered saline (PBS), and disrupted with 0.1 g ofglass beads (Sigma) in a cell beater (BIO 101 Savant, Fast PrepTM FP120) and centrifuged at 40300

× g for 5 min at 4◦ C. The resultant supernatant wascollected as bacterial cell extract, which was analyzed by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE, Terai et al.).9) As
shown in Fig. 2(a), SECl#4-1, #14-1, #16-1, and#28-1 showed only faint bands of CBB-staining
around 22 kDa, while SECl#7-1, #15-1, and #231 as well as SECl#40, a clinical isolate strain,
all showed clear bands in the corresponding MW.
We designated this protein band as SEp22, andthen tried to purify it using a large scale culture of SECl#15-1, because infection of these SE
strains into BALB/c mice orally resulted in two distinct classifications; i.e., a strongly virulent group(SECl#7-1, #15-1, and #23-1 as well as SECl#40),
and a completely avirulent group (SECl#4-1, #14-1,#16-1, and #28-1), which showed good correlation
with the expression of SEp22.
One liter culture of SECl#15-1 in LB medium
in an Erlenmeyer flask at 37◦ C overnight yielded asufficient number of stationary phase cells, whichwere washed with PBS, disrupted, and the resultant
cell extract was mixed with ammonium sulfate solution to 50% saturation. SEp22 was not precipitatedby 50% saturation but most of the other cell proteins were precipitated and separated from SEp22.
Dialysis of the supernatants of the 50% ammonium
sulfate saturation against 20 mM Tris-HCl, pH 7.5,
resulted in precipitation of SEp22 aggregates to almost homogeneity.
Characterization of SEp22
We analyzed the sequences of N-terminal 22
amino acids of SEp22, and performed cloning of
the sep22 gene covering full length coding of nucleotide sequences corresponding to 167 aminoacids (Fig. 3). The sep22 gene was shown tobe similar to Escherichia coli (E. coli) dps/pexB,
which was first reported by Almiron et al.,9) and
we subsequently showed10) sep22 to be identicalto Salmonella dps.11) SEp22 contains a proposed
DNA-binding domain at the N-terminal (5 K-XX-K-X-K) as E. coli DNA-binding proteins fromstarved cells (Dps) does. In addition, a characteristic aggregate formation of SEp22 during dialysis of the 50% ammonium sulfate supernatantfraction suggested an automatic crystallization of
SEp22 under low salt concentration. In fact, analysis of the crystal structure of SEp22, based on
our research, has been reported,12) showing a dodecameric shell with 23 symmetry, together withthe ferroxidase center of a single iron ion per pro-

461

No. 6

Fig. 3. Amino Acid Sequence of SEp22

Fig. 5. Iron Entry, Oxidation, Mineralization and Reutilizationin SEp22/Dps

Fig. 4. Crystal Structure of SEp22
Stereoview of the dimer (a) and dodecamer (b) structure of SEp22
is shown.12)

tomer (Fig. 4). These results show that SEp22 is actually Salmonella Dps, which has a structure similar
to that of other bacterial species such as E. coli,13)
Halobacterium salinarum14) and so on.

SEp22/Dps AS AN IRON-BINDING,FERRITIN-LIKE PROTEIN
Because SEp22 was assigned to Salmonella
Dps, which has been identified as a ferritin-like protein partly because of its ferroxidase activity, or
more specifically, its ability to oxidize bound ferrous ions to the ferric state,15) extensive study has

The pathway of iron movement in Dps is composed of: (1) entering of Fe2+ into the dodecamer through the N-terminal pores, (2) binding of Fe2+ to the ferroxidase site, (3) oxidization of Fe2+ to Fe3+ ,(4) move of Fe3+ to the nucleation site, (5) starting of mineralization
with the formation of FeOOH at the nucleation site, (6) neutralization of iron by reducing FeOOH back to Fe2+ , and (7) transport of
Fe2+ back to cytosol through N-terminal pores. Alternatively, (i) Fe2+flows to the cavity where it might also get oxidized on the surface ofthe growing mineral, or (ii) iron is transported out of the protein via
the C-terminal pores. (Transferred and modified from the model by
Haikarainen et al.17) )been done on whether SEp22 has iron-binding ability at the ferroxidase center (FOC) inside the dodecamer structure of this ferritin-like protein, Dps.16)
X-ray diffraction analysis of the SEp22 crystals
without or with iron-soaking revealed that iron ion
was bound to the FOCs between the acidic side
chains of D78 and E82 of one protomer and the basic side chains of H51, K48 and H63 of the otherprotomer in the iron-soaked SEp22 crystals.12) Dpslike proteins, including SEp22, are known to be
widely distributed among bacterial species, and all
Dps-like proteins have common characteristic three-462Vol. 57 (2011)

Fig. 6. Inhibition of Hydroxyl Radical Formation by the Fenton Reaction in Dps Ferroxidase CenterIn the cells, ferrous ion (Fe2+ ) is oxidized by hydrogen peroxide (H2 O2 ) at the molar ratio of 1 : 1 through the Fenton reaction and hydroxyl radical(OH ·) is produced as shown in (1). However, in the presence of Dps, ferrous ion is incorporated into Dps, then bound to the ferroxidase center andsubsequently reacted with hydrogen peroxide at the molar ratio of 2 : 1, forming ferric peroxide complex (FeOOH-P) at the binding site (P), which
inhibit hydroxyl radical formation as shown in (2). (Transferred and modified from the model by Calhoun and Kwon16) ).

dimensional architecture of spherical dodecamers
with a hallow central cavity where FOC exists,
containing the several conserved core amino acids(H51, H63, D78, E82 and W52 in case of E. coli) in
the neighborhood.17)
As for the processes of iron uptake, binding andoxidation in SEp22/Dps, the 23 symmetry of the
SEp22/Dps dodecamer (Fig. 4) creates 4 threefold
axes through the protein leading to 8 trimeric interfaces, formed at the end of the C- or N-termini of themonomers creating channels, which connect the exterior of the dodecamer to the internal cavity of thisprotein.16, 17) The pathway is composed of iron entry
inside the dodecamer, binding of iron to the ferroxidase center, oxidation of iron at the ferroxidase center, nucleation, mineralization, iron re-utilization by
reduction to ferrous iron, and transport back to cytosol (Fig. 5).17)
Dps was reported to bind and chelate iron ion
and thus inhibit production of highly toxic hydroxyl
radical (OH·) by the Fenton reaction, as shown in
Fig. 6. This inhibition proceeds at the FOC of Dps,
which prefers H2 O2 to O2 as an oxidization agent
for ferrous iron, harboring catalase-like activity to
Dps. In this sense, Dps is not comparable to ferritin,in which ferrous iron is oxidized preferentially notby H2 O2 but by O2 .16, 18)

SEp22/Dps AS A DNA-BINDING
PROTEIN
Because the finding of Dps from E. coli starved
cells by Almiron et al.9) was accompanied by characterization of this protein as a DNA-binding protein, subsequent studies concerning Dps in otherlaboratories as well as their own one have focusedon both the structure and function of the DNADps complex. Intensive structural research, especially using with X-ray crystallography, has revealed a highly conserved structural fold of proteins
within the Dps protein family isolated from different organisms.12, 16, 17, 19–25) However, most of thesestudies mainly showed structural features common
to other ferritins characterized in bacteria, including the paradigmatic ferritin (FtnA) and the hemecontaining bacterioferritin (Bfr), which are composed of at least 12 subunits folded into four-helix
bundles and assembled into spherical protein shells
as Dps is.13, 26) E. coli Dps has a very compact andstable structure with protrusion of the highly flexible and lysine-rich N-terminus (Fig. 3) of each ofits monomers,17) which has been believed to be responsible for Dps-DNA co-crystallization as well
as Dps self-aggregation in solution.26) The DNAbinding ability of Dps was initially examined by addition of Dps to supercoiled or linear plasmid DNA,
which resulted in very stable Dps-DNA complex
formation.9) Although heating of Dps at 65◦ C priorto addition of DNA failed to form a Dps-DNA complex, the Dps-DNA complex itself is, once formed,
stable against heating at 100◦ C, deoxyribonuclease(DNase) I digestion, and treating with 5% Triton
X-100 or 5% Brij-58, but is dissociated with 30%
formamide or 2% SDS. The binding of E. coli Dps
to DNA showed no apparent sequence specificity,
because Dps-bound DNA reveals no clear footprint
after DNase I digestion.9)
N-terminal deletion mutants of Dps revealed
the importance of N-terminus lysine residue multiplicity in the formation of large Dps-DNA complexes: wild-type Dps, carrying three positively
charged lysine residues at its N-terminus, forms
large Dps-DNA complexes in vitro at physiolog-

463

No. 6

ical pH, while Dps mutants, possessing only oneof the three terminal lysine residues, are also able
to bind DNA but are unable to sequester and condense it. However, mutants lacking all three terminal lysine residues are unable to bind or condense DNA.26) In addition, the binding of Dps to
DNA in E. coli cells was also examined by electron microscopy and the results revealed DNA-Dps
co-crystallization in the Dps-overexpressed cells aswell as the starved ones.27) This highly organizedco-crystallization process is produced with closed
super-coiled plasmids, linear double-stranded DNA,as well as single-stranded RNA molecules in vitro,and the DNA-Dps co-crystallization proceeds very
rapidly within seconds, by addition of DNA to Dps
solution.27) Because immediate sequestration of vital macromolecules like DNA in intracellular crystalline assemblies might be an effective and efficient
way of protection of the molecule,27) Dps seems
no doubt to play an important role in biodefense
mechanisms in bacterial survival and environmental stress responses. Furthermore, Dps-DNA complex formation has been shown to be involved in
the architecture of bacterial nucleoids by atomic
force microscopy,26, 28) providing further proof that
protein-protein interactions play a major role ingeneric DNA protection29) and reduction of mutation frequency.30)
However, DNA does not seem to bind directly
to the surface of Dps, because the spherical surfaceof this protein has no apparent DNA-binding motifs
and is negatively charged, which might repel binding of negatively charged DNA.13) Frenkiel-Krispin
et al.29) proposed that DNA-Dps complex formation
relies on ion bridges formed by Mg2+ . The similarity between packing of Dps dodecamers in protein crystals and that within Dps-DNA complexes
suggest a close relationship between Dps selfaggregation and DNA condensation.26) Dps selfaggregation forms a crystal lattice, creating a lineof lysine-rich N-termini with holes derived from 3
adjacent Dps molecules,13) where DNA might bethreaded through. Wolf et al.27) also suggested asimilar mechanism of DNA threading through the
Dps crystalline structure. The important role of the
N-termini in Dps-DNA interaction is also supported
by studies on Dps family members that do not possess positive residues at N-termini, such as Listeria
innocua ferritin, Bacillus anthracis Dlp, and Helicobacter pylori HP-NAP, showing no DNA binding
ability.31–33)

REGULATION OF SEp22/Dps
EXPRESSION
The first finding of SEp22 was based on
the different expression of stationary phase proteins among SE clones from environmental isolates which were pre-incubated in LB medium
overnight.10) Because SEp22 was proved to be
Salmonella Dps10) as described in the previous section, the expression and induction of SEp22 and thatof Dps should occur similarly, if the experimental
conditions are controlled. Almiron et al.9) also described the induction of Dps in E. coli cells during
incubation in LB medium at the stationary phase but
not at the mid-exponential phase. However, subsequent studies on E. coli Dps and related Dps seemed
to have paid much more attention to the expressionof Dps in starved cells cultured in minimal medium
like M639, 16) as well as the molecular structure of
Dps and regulation of gene expression.16) It remains
largely unknown whether nutrient starvation in M63
medium, which means depletion of glucose, induces
the same signals for induction of SEp22/Dps during
culture in LB medium or not. To address this question, we used M9 minimal medium containing 0.4%
glucose, in which SE did not induce SEp22 after
incubation overnight even when the cells reached
the stationary phase. However, addition of 10%(v/v) LB medium to M9 medium turned out to induce SEp22 during overnight culture. A similar effect on SEp22 induction was obtained by additionof casamino acids, although the effect was dosedependent.34) These results imply the involvementof unknown regulatory mechanisms concerning nutrient factors and their depleting effects.
Expression of SEp22 during culture in LB
medium was growth phase-dependent, regulated at
the transcriptional level, and dependently on σs expression (Fig. 7).10) It has also been shown that
Dps expression is highly dependent on the growthphase: In the stationary phase, σs , the stationary
phase-specific sigma factor, controls the expressionof dps mRNA,35) as was the case of sep22 in SE.10)
In the dps promoter, consensus sequences for thehistone-like integration factor (IHF) are present, andheterodimeric IHF protein was shown to be required
for the induction of σs -dependent dps mRNA in
the starved cells.35) In the exponentially growing
cells, H2 O2 induces OxyR activation,36) leading to
the activation of another sigma factor, σ70 , thatbinds to dps promoter and stimulates gene expres-464

Fig. 7. Expression of SEp22 in the Course of Cell Growth in
LB Medium(A) Growth curve of SECl#15-1 in LB medium. Bacteria numbers
are shown as CFU ml−1 (•) and OD550 (). An overnight culture of
SECl#15-1 in the stationary phase was diluted and incubated at 37◦ C,
with shaking at 150 strokes min−1 (1st cycle). On the second day, dilution and culture of the bacterium was repeated (2nd cycle). (B) Expression of (a) SEp22, (b) σ38 (σS ) and (c) σ70 proteins in SECl#15-1
in the growth phases during culture (hr). Western blot analysis was
performed with specific antibodies against SEp22, σ38 , or σ70 . Panels(a), (b) and (c) correspond to the proteins with MW of 19 kDa (SEp22),
38 kDa (σ38 ) and 70 kDa (σ70 ), respectively. (C) Expression of SEp22
mRNA (upper panel) and 16S rRNA (lower panel). Equal amounts
of total RNA (1.5 µg) were loaded in each lane and northern blots of
SEp22 mRNA are shown in the upper panel. In the lower panel, theEtBr-stained 16S rRNA bands are shown. [Cited from Ref. 8) withmodification.]Vol. 57 (2011)

sion via σ70 -RNA polymerase.37) This pathway isredox-sensitive,36) and thus could be activated bysuch reagents and chemicals that activate peroxideformation, such as antibiotics.38)
Recently, down-regulation of Dps expression
was reported: selective repression of dps promotor was found in E. coli by Fis and H-NS throughinhibition of transcription of dps gene.38) Fis represses by trapping RNA polymerase containing
σ70 at the promoter, while H-NS functions by displacing RNA polymerase containing σ70 , but not
RNA polymerase containing (σS ) σ38 . Because Fislevels are high in exponentially growing cells but
decrease to near zero when the cells enter stationary
phase, down-regulation of Dps expression might be
explained by the functions of Fis in exponentiallygrowing cells. On the contrary, the repression by
H-NS can be overcome by binding of σ38 that also
recognizes dps promoter. These results also suggest mechanisms by which Dps-dependent condensation of bacterial chromosome occurs in the courseof cell growth from exponentially growing phase tostationary phase.16, 39)
Accumulation of Dps during stationary phase
was suggested to be due to the post-transcriptional
help of ClpAP: ClpAP is an ATP-dependent protease with molecular chaperon activity, and it increased Dps levels through indirectly maintaining
the ongoing translation of dps mRNA in the prolonged stationary phase, although it did not digest Dps protein in this period.40) Another effect ofClpAP on translation of Dps was suggested to be
due to proteolytic degradation of translational repressor of Dps during starvation of the cells, which
resulted in accumulation of Dps.
In addition to the translational regulation,
SEp22/Dps levels are post-translationally regulated
by proteolysis. During re-incubation in fresh LB
medium of SE which were pre-cultured overnight tothe stationary phase, SEp22 decreased rapidly withtime and reached a non-detectable level at the exponentially growing phase.10) This decrease of SEp22
was dependent on cell density, and incubation time
and temperature, and was sensitive to sodium azide(data not shown).10) These results imply the presence of energy-dependent proteolysis of SEp22 during the exponential growth phase. Similar results
were reported concerning Dps degradation: Dps
was shown to be degraded directly by ClpXP and
ClpAP during exponential growth,41) and ClpXPmediated degradation of Dps occurred upon re-entry

No. 6

into the logarithmic phase.40) Recent studies haverevealed that Dps was a target of N-end rule degradation in E. coli,40) and that ClpS played a role as
an adapter protein for ClpAP-mediated degradationof Dps.42) The N-rule degradation pathway, a major pathway of protein degradation in E. coli, requires the primary structure of target proteins attheir N-terminal residues.43) ClpS targets a truncated form of Dps (Dps6-167) and interacts directlywith Leu6 at the N-terminal (Fig. 3) to recruit theprotein for ClpAP protease.41) On the other hand,
ClpXP recognizes the N-terminal tail of Dps for
degradation, and thus N-terminal cleavage of Dps
decreases ClpXP-mediated turnover.44)
Furthermore, recent studies by Hanna et al.
showed the presence,45) purification and cloning46)
of glycosylated form of Dps in Sarmonella Typhimurium (S. Typhimurium). It is a kind of posttranslational modification and probably a new typeof regulation of Dps, although the physiologicalmeaning and importance of glycosylation of Dps remain largely unknown.
Taken together, Dps expression is regulated
at the transcriptional, translational, and posttranslational levels through a variety of regulators.16)

SEp22/Dps AS A
PATHOGENICITY-RELATED FACTOR
Salmonella is one example of intracellular parasitism that invades, survives and multiplies in thephagocytic vacuoles of macrophages, which allows
this bacterium to persist in the host and sometimes
to cause latent infections.47, 48) In these processesof Salmonella infection, the mononuclear phagocyte systems, especially macrophages, plays major roles in exerting bactericidal activity toward
Salmonella (Fig. 1). Among the effector molecules,
ROIs show the central, and the strongest activity tokill the bacterium in the early stages of the infection. As SEp22/Dps is rapidly induced by exposureof H2 O2 ,9, 10, 16) it seems feasible to consider that
SEp22/Dps plays some of the key roles in acquiring resistance to immune attack from the host. In
fact, Halsey et al.11) showed the oxidative stress resistance and virulence in mice was endowed to S.
Typhimurium by Dps, showing that a dps gene deletion mutant clone of Salmonella was highly susceptible to H2 O2 , which was restored in the revertant

465

clone with pBAD-dps transformation, and that thedps gene deletion mutant showed much reduced virulence to mice than the wild type Salmonella after
intraperitoneal infection. Similar results were obtained in our laboratory with SE clones.10)
Induction of dps gene expression in S. Typhimurium was examined after infection to themacrophages in vitro: Infection of wild type,virulent strain of S. Typhimurium to J774A.1
macrophage-like cells resulted in sudden elevation of dps gene expression at 2 hr after the infection, and a similar increase in spv genes, essential genes of virulence for establishment of asystemic infection of Salmonella, was also observed.49) These results seem to suggest a very important response of Salmonella during infection into
macrophages (Fig. 1): Dps is not an offense factor of Salmonella but rather a defense factor of thebacterium, as described in the previous sections, toprotect Salmonella from harmful ROIs produced bymacrophages and neutrophils. On the other hand,
spv is an offense factor and is closely related to
the virulent plasmid expression.50) The expressionof both the dps and spv genes, which is regulated
under the control of RpoS (σS ), seems to inducea co-ordinated response of Salmonella of the host,
implying these genes are needed for survival andgrowth in macrophages. In this sense, Dps is apathogenicity-related, or even pathogenic, factor,
although it does not seem proper to call it a virulence factor.
Studies on the host factor concerning the induction of dps gene expression in S. Typhimurium
during infection to macrophages were performed
using Toll-like receptor 4 (TLR4)- and NADPH
oxidase-deficient mice.51) Compared with TLR4deficient mice, bone marrow-derived macrophages
in the presence of TLR4 resulted in the inductionof dps gene in S. Typhimurium. In addition, thedps gene-deletion mutant of S. Typhimurium failed
to increase the bacterial number to about 1/100of the wild type clone at 3 days after administration of these bacteria to C57BL/6 mice. Similar
results were obtained from the experiments using
gp91Phox -deficient mice, i.e., NADPH oxidasedeficient mice, and wild type as well as dps genedeletion mutant of S. Typhimurium. These results
show that intracellular survival of S. Typhimuriumin normal macrophages requires Dps expression
in the bacterium, and suggest that a TLR4signaling cascade(s) links NADPH oxidase activity
in macrophages to induce fully bactericidal activity.

466

Vol. 57 (2011)

REGULATORY ROLES OF SEp22/Dps
IN ENVIRONMENTAL
STRESS-RESISTANCE OF Salmonella
Although extensive studies have revealed thatDps not only functions as an iron-binding ferritinlike protein,17) but also works as a DNA-binding
protein,16) or more accurately, a DNA-guard protein. Because iron is an essential metal for mostof cells in both prokaryotic and eukaryotic organisms, it is very important for these cells to manage
the various effects of redox changes of iron and of
the resultant ROIs.16) One of the most remarkablechanges that cause Dps expression is nutritional deprivation and oxidative stress, as described in theprevious section. However, it remains largely unknown whether Dps has a pleiotropic effect in theregulation of gene and/or protein expression during
times of starvation or environmental stresses such asoxidative stresses.
To address this question, we analyzed environmental isolates of SE, with various levels of
SEp22/Dps, and examined whether these clones arepathogenic, or resistant to H2 O2 , heat-shock, ultraviolet (UV)-irradiation, or other chemical stresses.
We found dry-resistance was closely linked to the
SEp22/Dps level,52) showing that the clones expressing high levels of SEp22/Dps were resistantto the drying protocol53) but those with little or extremely low levels were sensitive to the dry-stress(Fig. 8). In addition, all of the sep22-deletion mutants that originated from the wild type, virulent
strain, showed high susceptibility toward the drystress (Fig. 9). Induction of SEp22 expression during the drying procedure in the wild type strain
also supported the importance and involvement of
SEp22 in the acquisition of the dry-resistance.52)
Dry-resistance is one of the remarkable characteristics of Salmonella in an environment, and is recognized as an important point for food-safety control.54) It is interesting that lactoferrin, which is
known as an iron-binding protein in mammals,55)
functions not only as an iron-supplier but also as a
bio-protective material against infection, and shows
strong inhibitory effects toward the acquisition of
dry-resistance by various Salmonella species.56) Although the mechanisms underlying the inhibitory
effects of lactoferrin remain largely unknown, it
may be due to the iron-binding property of lactoferrin that competes with the iron-capturing activityof SEp22/Dps.12)

Fig. 8. Dry-resistance of SE Clones with Varied Expression
Levels of SEp22/Dps52)

Fig. 9. Dry-resistance of sep22-deletion Mutants52)

Another way to determine the regulatory rolesof Dps in the stress responses is to investigatechanges in protein expression patterns, showing a
dramatic difference in the proteomes of the parental
and dps-deleted mutant strains, and also to examine the pleiotropic phenotype of mutants lacking
a functional dps gene, as discussed by Calhoun
and Kwon.16) Dps has a DNA-binding property, although the sequence-specificity of DNA has not

467

No. 6

Fig. 10. SEp22/Dps Induction by Internal and Environmental Stresses in Salmonella

been reported.9) However, the binding of Dps to
DNA should have some effect on cell functions andgene expression. Proteome analysis seems to provide good clues for understanding the regulatory
roles of Dps, using the sets of parental and mutant strains grown under various conditions, suchas exponentially growing and stationary phases,9)or stress-treated and non-treated states.57) In addition, DNA microarray analysis seems to be useful
to monitor changes at the mRNA levels between
wild type and dps-deletion mutants under the various conditions as described above. Anyway, if theuseful clue, either protein or gene, becomes apparent, possible targets of regulation will be identified, leading to a breakthrough in future studies of
SEp22/Dps in biology and pathology, as well as application of the findings to microbial regulation in
many fields including food-safety, infection control,
and agriculture.58)
However, it is possible, as pointed out by Calhoun and Kwon,16) that Dps does not have any
special role in the pleiotropic regulation of othergenes or phenotypes, but instead merely binds andenvirons DNA at the stationary phase or understarved conditions for DNA protection and survival.15, 30, 59, 60) It is worth conducting studies on
Dps to determine the regulatory role of Dps, because Dps may be considered as a histone-like protein showing many similarities with the eukaryotic
histone.61, 62) Histones are well known not as a stillplayer surrounding DNA but as an active moleculethat undergoes covalent modifications in response tocell behavior, and also make DNA accessible or inaccessible to transcriptional machinery, finally leading to regulation of gene expression.63)

SEp22/Dps AS A COMMONREGULATOR OF Salmonella SURVIVAL
IN THE NATURAL ENVIRONMENT AND
INFECTION: CONCLUDING REMARKSAND FUTURE PERSPECTIVES
SEp22 was initially discovered as apathogenicity-related protein of SE, and wasthen assigned as Salmonella Dps.10) As shownin this review and others,16, 17) Dps is a protein
normally associated with stress resistance during
stationary phase, as originally found and describedby Almiron et al.9) However, Dps is also recognized
as a H2 O2 -resistant protein during exposure to oxidative stress in the exponential growth phase.35)
Because expression of SEp22/Dps is regulated
primarily at the transcriptional level through OxyR
regulon and σS , it seems rational toassume that
there are common pathways and regulators for the
induction of Dps under different culture conditions
and exposure to stresses which activate thesetranscriptional factors.
As shown in Fig. 10, Salmonella and other bacteria exist under stressful conditions, both environmentally and internally: environmental stresses include ROIs, UV, radiation, heat, dryness, nutritional

468

starvation, and other stresses such as chemicals andantibiotics. During infection of a host, effector
molecules from the host immune phagocyte system, such as ROIs, acidic pH in phagolysosomes,
lysosomal acid hydrolases, and lysozymes (Fig. 1),
also become stresses for the invading pathogens.
Salmonella copes with most of these stresses fromthe environment by inducing SEp22/Dps, which
protects DNA not only by detoxifying iron by chelation and subsequent trapping ferroxide in the center of Dps to avoid hydroxyl radical formation, butalso binding and sequestration of DNA to stabilize it.16) On the other hand, SEp22/Dps is also induced by internal, i.e., intracellular stresses, including the growth phase, respiration as well as cell division, which have close relationships to oxygen radical production or chromosomal rearrangement. Because Dps is a major DNA-binding protein in stationary phase cells and also in stress-exposed cells,
the expression of Dps is under σS control. As described in the previous section, down-regulation of
Dps expression by H-NS and Fis is closely linked to
the growth phases.
It is interesting to summarize the circumstances
surrounding the bacterium in Fig. 10 with regard
to SEp22/Dps, because both environmental stresses
and internal stresses are factors common to the induction of (sometimes the reduction of) SEp22/Dps,and because SEp22/Dps combats these stresses
in order to survive and grow. In this sense,
SEp22/Dps is a key molecule that plays essential
roles in both the environmental stress-resistance andpathogenicity of Salmonella. Our recent study suggests that accumulation of dry-resistant Salmonella
clones in poultry farms is accompanied increase in
SEp22 expression in these cells through repeateddry procedure. Furthermore, maintaining viability in the VNC (viable but non-culturable) state ofSalmonella, obtained during prolonged incubationin M9 minimal medium, is also linked to SEp22 expression. Future studies on SEp22/Dps should examine the molecular information of SEp22/Dps andthe phenotypes of sep22/dps gene-deletion mutants,
and perform careful observation and detailed analysis of natural environmental isolates with various
levels of SEp22/Dps proteins.
Acknowledgements The author wishes to express grateful thanks to the following collaborators: Drs. S. Terai and K. Fujimori, Ms. A. Tamura,
Ms. E. Nishio, Ms. N. Minami, Mr. M. Tanda,
Mr. Y. Morishige, and Mr. R. Kobayashi of Os-

Vol. 57 (2011)

aka University of Pharmaceutical Sciences; Drs.
S. Igimi and A. Okutani of the National Instituteof Health Sciences; Dr. M. Yamasaki of the Institute of Microbial Chemistry; Ms. N. Saitoh of
the National Institute of Infectious Diseases; Drs.
T. Ekawa, and H. Ohta of the CAF Laboratories;
and Dr. Y. Katayama of the Tokyo University ofAgriculture & Technology. The author also thanks
CAF Laboratories (Fukuyama, Japan) for providing S. Enteritidis, S. Johanesburg, and S. Infantis; Dr. A. Nakamura (National Institute of Infectious Diseases) for providing S. Typhimurium and
S. Oranienburg; Dr. J. Kudaka (Okinawa Prefectural
Institute of Health and Environment) for providing
S. Weltevreden, and Rokko Butter Co. LTD (Kobe,
Japan) for providing lactoferrin and apolactoferrin.
This work was supported by grants (KH51050,
KH51043) from the Japan Health Sciences Foundation, by grants-in-aid (15590117, 21590141) fromthe programs Promotion of Science and Grants-inAid for High Technology Research of the Ministryof Education, Culture, Sports, Science and Technology in Japan.
The author also wishes to thank Dr. A.
Nakayama of Kyoto University, for suggesting thisreview be written.

REFERENCES
1) National Institute of Infectious Diseases and Tuberculosis and Infectious Disases Control Division,
Ministry of Health, Labor and Welfare, Japan (2006)
Salmonellosis. Infectious Agents Surveillance Report, 27, 191–192.
2) Center for Disease Control and Prevention (CDC)(2009) Multistate outbreaks of Salmonella infections associated with live poultry-United States.
2007. Morb. Mortal. Wkl. Rep., 58, 25–29.
3) Allen-Vercoe, E., Dibb-Fuller, M., Thorns, C. and
Woodward, M. (1997) SEF17 fimbriae are essential
for convoluted colonial morphology of S. enteritidis.
FEMS Microbiol. Lett., 153, 33–42.
4) Centers for Disease Control and Prevention (1996)
Outbreak of Salmonella serotype enteritidis infection associated with consumption of raw shell eggs
— United States. 1994–1995. MMWR Morb. Mortal. Wkly. Rep., 45, 737–742.
5) Tam, M. A., Rydström, A., Sundquist, M. and Wick,
M. J. (2008) Early cellular responses to Salmonella
infection: dendritic cells, monocytes, and more. Immunol. Rev., 225, 140–162.

No. 6

6) Vazquez-Torres, A. and Fang, F. C. (2001) Oxygendependent anti-Salmonella activity of macrophages
Trends Microbiol., 9, 29–33.
7) Lilic, M, Quezada, C. M. and Stebbins, C. E. (2010)
A conserved domain in type III secretion links thecytoplasmic domain of InvA to elements of the basal
body. Acta Crystallogr. Sect. D Biol. Crystallogr.,
66, 709–713.
8) Hueck, C. J. (1998) Type III protein secretion systems in bacterial pathogens of animals and plants.
Microbiol. Mol. Biol. Rev, 62, 379–433.
9) Almiron, M., Link, A. J., Furlong, D. and Kolter, R.
(1992) A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli.
Genes Dev., 6, 2646–2654.
10) Terai, S., Yamasaki, M., Igimi, S. and Amano,
F. (2005) Expression and degradation of SEp22,
a pathogenicity-related protein of Salmonella Dps,
in Salmonella Enteritidis isolated from the poultry
farms in Japan. Biosci. Microflora, 24, 113–118.
11) Halsey, T. A., Vazquez-Torres, A., Gravdahl, D. J.,
Fang, F. C. and Libby, S. J. (2004) The ferritinlike Dps protein is required for Salmonella enterica
Serovar Typhimurium oxidative stress resistancesand virulence. Infect. Immun., 72, 1155–1158.
12) Miyamoto, T., Asahina, Y., Miyazaki, S., Shimizu,
H., Ohto, U., Noguchi, S. and Satow, Y. (2011)
Structures of the SEp22 dodecamer, a Dps-likeprotein from Salmonella enterica subsp. enterica
serovar Enteritidis. Acta Crystallogr. Sect. F, Struct.
Biol. Cryst. Commun., 1, 17–22.
13) Grant, R. A., Filman, D. J., Finkel, S. E., Kolter, R.
and Hogle, J. M. (1998) The crystal structure of Dps,a ferritin homolog that binds and protects DNA. Nat.
Struct. Biol., 5, 294–303.
14) Zeth, K., Offermann, S., Essen, L. O. andOesterhelt, D. (2004) Iron-oxo clusters biomineralizing on protein surfaces: structural analysis of
Halobacterium salinarum DpsA in its low- andhigh-iron states. Proc. Natl. Acad. Sci. U.S.A., 101,
13780–13785.
15) Nair, S. and Finkel, S. E. (2004) Dps protects cells
against multiple stresses during stationary phase. J.
Bacteriol., 186, 4192–4198.
16) Calhoun, L. N. and Kwon, Y. M. (2010) Structure,function and regulation of the DNA-binding protein
Dps and its role in acid and oxidative stress resistance in Escherichia coli: a review. J. Appl. Microbiol., 110, 375–386.
17) Haikarainen, T. and Papageorgiou, A. C. (2010)
Dps-like proteins: structural and functional insights
into a versatile protein family. Cell. Mol. Life Sci.,
67, 341–351.

46918) Zhao, G., Arosio, P. and Chasteen, N. D. (2006)
Iron(II) and hydrogen peroxide detoxification by human H-chain ferritin. An EPR spin-trapping study.
Biochemistry, 45, 3429–3436.
19) Ren, B., Tibbelin, G., Kajino, T., Asami, O. and
Ladenstein, R. (2003) The multi-layered structureof Dps with a novel di-nuclear ferroxidase center.
J. Mol. Biol., 329, 467–477.
20) Kauko, A., Haataja, S., Pulliainen, A. T., Finne, J.
and Papageorgiou, A. C. (2004) Crystal structure ofStreptococcus suis Dps-like peroxide resistance protein Dpr: implications for iron incorporation. J. Mol.
Biol., 338, 547–557.
21) Bhattacharyya, G. and Grove, A. (2007) The Nterminal extensions of Deinococcus radiodurans
Dps-1 mediate DNA major grove interactions aswell as assembly of the dodecamer. J. Biol. Chem.,
282, 11921–11930.
22) Gauss, G. H., Benas, P., Wiedenheft, B., Young, M.,
Douglas, T. and Lawrence, C. M. (2006) Structureof the Dps-like protein from Sulfolobus solfataricus
reveals a bacterioferritin-like dimetal binding site
within a Dps-like dodecameric assembly. Biochemistry, 45, 10815–10827.
23) Romao, C. V., Mitchell, E. P. and McSweeney, S.
(2006) The Crystal structure of Deinococcus radiodurans Dps protein (DR2263) reveals the presenceof a novel metal centre in the N-terminus. J. Biol.
Inorg. Chem., 11, 891–902.
24) Thumiger, A., Polenghi, A., Papinutto, E.,
Battistutta, R., Montecucco, C. and Zanotti, G.
(2006) Crystal structure of antigen TpF1from Treponema pallidum. Proteins, 62, 827–830.
25) Pesek, J., Buchler, R., Albrecht, R., Boland, W. andZeth, K. (2011) Structure and mechanism of irontranslocation by a Dps protein from Microbacteriumarborescens. J. Biol. Chem., 286, 34872–34882.
26) Ceci, P., Cellai, S., Falvo, E., Rivetti, C., Rossi, G. L.
and Chiancone, E. (2004) DNA condensation andself-aggregation of Escherichia coli Dps are coupled phenomena related to the properties of the Nterminus. Nucleic Acids Res., 32, 5935–5944.
27) Wolf, S. G., Frenkiel, D., Arad, T., Finkel, S. E.,
Kolter, R. and Minsky, A. (1999) DNA protection by
stress-induced biocrystallization. Nature, 400, 83–85.
28) Kim, J., Yoshimura, S. H., Hizume, K., Ohniwa,
R. L., Ishihama, A. and Takayasu, K. (2004) Functional structural units of the Escherichia coli nucleoid revealed by atomic force microscopy. Nucleic
Acids Res., 32, 1982–1992.
29) Frenkiel-Krispin, D., Levin-Zaidman, S., Shimoni,
E. and Wolf, S. G. (2001) Regulated phase transi-

470

tions of bacterial chromatin: a non-enzymatic pathway for generic DNA protection. EMBO J., 20,
1184–1191.
30) Martinez, A. and Kolter, R. (1997) Protection of
DNA during oxidative stress by the nonspecificDNA-binding protein Dps. J. Bacteriol., 179, 5188–
5194.
31) Bozzi, M., Mignogna, G., Stefanini, S., Barra, D.,Longhi, C., Valenti, P. and Chiancone, E. (1997) A
novel non-heme iron-binding ferritin related to the
DNA-binding proteins of the Dps family in Listeria
innocua. J. Biol. Chem., 272, 3259–3265.
32) Papinutto, E., Dundon, W. G., Pitulis, N.,
Battistutta, R., Montecucco, C. and Zanotti, G.
(2002) Structure of two iron-binding proteins from
Bacillus anthracis. J. Biol. Chem., 277, 15093–
15098.
33) Zanotti, G., Papinutto, E., Dundon, W., Battistutta,
R., Seveso, M., Giudice, G., Rappuoli, R. andMontecucco, C. (2002) Structure of the neutrophilactivating protein from Helicobacter pylori. J. Mol.
Biol., 323, 125–130.
34) Terai, S., Yasuda, M. and Amano, F. (2006) Regulation of SEp22 expression in Salmonella enterica subsp. enterica Serovar Enteritidis by culture
medium. Microbes Environ., 21, 36–43.
35) Altuvia, S., Almiron, M., Huisman, G., Kolter, R.
and Storz, G. (1994) The dps promoter is activatedby OxyR during growth and by IHF and σS in stationary phase. Mol. Microbiol., 13, 265–272.
36) Tartaglia, L. A., Storz, G. and Ames, B. N.
(1989) Identification and molecular analysis of
oxyR-regulated promoters important for the bacterial adaptation to oxidative stress. J. Mol. Biol., 210,
709–719.
37) Schnetz, K. (2008) Fine-tuned growth phase controlof dps, encoding a DNA protection protein, by FIS
and H-NS. Mol. Microbiol., 68, 1345–1347.
38) Calhoun, L. N. and Kwon, Y. M. (2011) The
ferritin-like protein Dps protects Salmonella enterica serotype Enteritidis from the Fenton-mediatedkilling mechanism of bactericidal antibiotics. Int. J.
Antimicrobiol. Agents, 37, 261–265.
39) Grainger, D. C., Goldberg, M. D., Lee, D. J. andBusby, S. J. (2008) Selective repression by Fis and
H-NS at the Escherichia coli dps promoter. Mol. Microbiol., 68, 1366–1377.
40) Schmidt, R., Zahn, R., Bukau, B. and Mogk, A.
(2009) ClpS is the recognition component for Escherichia coli substrates of the N-end rule degradation pathway. Mol. Microbiol., 72, 506–517.
41) Stephani, K., Weichart, D. and Hengge, R. (2003)
Dynamic control of Dps protein levels by ClpXP

Vol. 57 (2011)

and ClpAP proteases in Escherichia coli. Mol. Microbiol., 49, 1605–1614.
42) Ninnis, R. L., Spall, S. K., Talbo, G. H., Truscott,
K. N. and Dougan, D. A. (2009) Modificationof PATase by L/F-transferase generates a ClpSdependent N-end rule substrate in Escherichia coli.
EMBO J., 28, 1732–1744.
43) Mogk, A., Schmidt, R. and Bukau, B. (2007) The Nend rule pathway for regulated proteolysis: prokaryotic and eukaryotic strategies. Trends Cell Biol., 17,
165–172.
44) Flynn, J. M., Neher, S. B., Kim, Y. I., Sauer, R. T.
and Baker, T. A. (2003) Proteomic discovery of cellular substrates of the ClpXP protease reveals fiveclasses of ClpX-recognition signals. Mol. Cell, 11,
671–683.
45) Hanna, E. S., Roque-Barreira, M. C., Bemardes,
E. S., Panunto-Castelo, A., Sousa, M. V., Almeida,
I. C. and Brocchi, M. (2007) Evidence for glycosylation on a DNA-binding protein of Salmonella enterica. Microb. Cell Fact., 6, 11.
46) Hanna, E. S., Roque-Barreira, M. C., Mendes,
G. M., Soares, S. G. and Brocchi, M. (2008)
Cloning, expression and purification of a glycosylated form of the DNA-binding protein Dps from
Salmonella enterica Typhimurium. Protein Expr.
Purif., 59, 197–202.
47) Chappell, L., Kaiser, P., Barrow, P., Jones, M. A.,
Johnston, C. and Wigley, P. (2009) The immunobiology of avian systemic salmonellosis. Vet. Immunol.
Immunopathol., 128, 53–59.
48) Mittrücker, H.-W. and Kaufmann, S. H. E. (2000)
Immune response to infection with Salmonella typhimurium in mice. J. Leukoc. Biol., 67, 457–463.
49) Marshall, D. G., Bowe, F., Hale, C., Dougan, G. andDorman, C. (2000) DNA topology and adaptation of
Salmonella typhimurium to an intracellular environment. Philos. Trans. R. Soc. Lond. B Biol. Sci., 355,
565–574.
50) Gulig, P. A., Danbara, H., Guiney, D. G., Lax, A. J.,
Norel, F. and Rhen, M. (1993) Molecular analysisof spv virulence genes of the Salmonella virulence
plasmids. Mol. Microbiol., 7, 825–830.
51) Wright, J. A., Totemeyer, D., Hautefort, I.,
Appia-Ayme, C., Alston, M., Daniro, V., Paterson,
G. K., Mastoeni, P., Menager, N., Rolfe, M.,
Thompson, A., Ugrinovic, S., Sait, L., Humphrey,T., Northen, H., Peters, S., Maskell, D. J., Hinton,
J. C. D. and Bryant, C. E. (2009) Multiple redundant stress resistance mechanisms are inducedin Salmonella enterica serovar Typhimurium in response to alteration of the intracellular environment
via TLR4 signaling. Microbiology, 155, 2919–2929.

No. 652) Tamura, A., Yamasaki, M., Okutani, A., Igimi, S.,
Saitoh, N., Ekawa, T., Ohta, H., Katayama, Y. andAmano, F. (2009) Dry-resistance of Salmonella enterica subsp. enterica Serovar Enteritidis is regulated by both SEp22, a novel pathogenicity-related
factor of Salmonella, and nutrients. Microbes Environ., 24, 121–127.
53) Amano, F., Fujimori, K., Igimi, S. and Yamasaki, M.
(2011) Regulation of dry-resistance of Salmonella
enterica subsp. enterica Serovar Enteritidis. In
Salmonella, Book 2 (Annous, B. A. and Gurtler, J.,
Eds.), Inteck, in press.
54) Baron, F., Nau, F., Guerin-Dublard, C., Gonnet, F.,
Dubois, J. J. and Gautier, M. (2003) Effect of dry
heating on the microbiological quality, functional
properties, and natural bacteriostatic ability of egg
white after reconstitution. J. Food Prot., 66, 825–
832.
55) González-Chávez, S. A., Arévalo-Gallegos, S. andRascón-Cruz, Q. (2009) Lactoferrin: structure,function and applications. Int. J. Antimicrobiol.
Agents, 33, 301.e1–301.e8.
56) Tamura, A., Nishio, E., Fujimori, K., Igimi, S. andAmano, F. (2009) Lactoferrin inhibits the acquisition of dry-resistance by Salmonella spp. Biosci. Microflora, 28, 81–88.
57) Calhoun, L. N., Liyanage, R., Lay, J. O., Jr.

471

and Kwon, Y. M. (2010) Proteomic analysis of
Salmonella enterica serovar Enteritidis following
propionate adaptation. BMC Microbiol., 10, 249.
58) Ceci, P., Ilari, A., Falvo, E. and Chiancone, E.
(2003) The Dps protein of Agrobacterium tumefaciens does not bind to DNA but protects it toward
oxidative cleavage. J. Biol. Chem., 278, 20319–
20326.
59) Zhao, G., Ceci, P., Ilari, A., Giangiacomo, L., Laue,
T. M., Chiancone, E. and Chasteen, N. D. (2002)
Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. J.
Biol. Chem., 277, 27689–27696.
60) Choi, S. H., Baumler, D. J. and Kaspar, C. W. (2000)
Contribution of dps to acid stress tolerance and oxidative stress tolerance in Escherichia coli O157:H7.
Appl. Environ. Microbiol., 66, 3911–3916.
61) Azam, T. A. and Ishihama, A. (1999) Twelve speciesof the nucleoid-associated protein from Escherichia
coli. J. Biol. Chem., 274, 33105–33113.
62) Luijsterburg, M. S., White, M. F., van Driel, R. andDame, R. T. (2008) The major architects of chromatin: architectural proteins in bacteria, archaea andeukaryotes. Crit. Rev. Biochem. Mol. Biol., 43, 393–
418.
63) Jenuwein, T. (2006) The epigenetic magic of histonelysine methylation. FEBS J., 273, 3121–3135.