MOLECULAR PATHOGENESIS

crossm

Cooperative Roles of Nitric Oxide-Metabolizing Enzymes

To Counteract Nitrosative Stress in Enterohemorrhagic
Escherichia coli
Takeshi Shimizu,a Akio Matsumoto,b Masatoshi Nodaa

a
Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, Chiba, Japan
b Department of Aging Pharmacology, School of Medicine, Toho University, Tokyo, Japan

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) has at least three enzymes,

NorV, Hmp, and Hcp, that act independently to lower the toxicity of nitric oxide
(NO), a potent antimicrobial molecule. This study aimed to reveal the cooperative
roles of these defensive enzymes in EHEC against nitrosative stress. Under anaerobic
conditions, combined deletion of all three enzymes significantly increased the NO
sensitivity of EHEC determined by the growth at late stationary phase; however, the
expression of norV restored the NO resistance of EHEC. On the other hand, the
growth of Δhmp mutant EHEC was inhibited after early stationary phase, indicating
that NorV and Hmp play a cooperative role in anaerobic growth. Under microaerobic
conditions, the growth of Δhmp mutant EHEC was inhibited by NO, indicating that
Hmp is the enzyme that protects cells from NO stress under microaerobic condi-
tions. When EHEC cells were exposed to a lower concentration of NO, the NO level
in bacterial cells of Δhcp mutant EHEC was higher than those of the other EHEC mu-
tants, suggesting that Hcp is effective at regulating NO levels only at a low concen-
tration. These findings of a low level of NO in bacterial cells with hcp indicate that
the NO consumption activity of Hcp was suppressed by Hmp at a low range of
NO concentrations. Taken together, these results show that the cooperative ef-
fects of NO-metabolizing enzymes are regulated by the range of NO concentra-
tions to which the EHEC cells are exposed.

KEYWORDS NO reductase, Shiga toxin, enterohemorrhagic Escherichia coli, nitric

oxide

E nterohemorrhagic Escherichia coli (EHEC) is classified into multiple serotypes,

among which O157:H7 is the serotype most commonly linked to epidemic and
sporadic disease in humans throughout Japan, North America, and parts of Europe.
Citation Shimizu T, Matsumoto A, Noda M.
2019. Cooperative roles of nitric oxide-
metabolizing enzymes to counteract
EHEC infections are a primary cause of hemorrhagic colitis and hemolytic-uremic nitrosative stress in enterohemorrhagic
Escherichia coli. Infect Immun 87:e00334-19.
syndrome (HUS) (1–3). The pathogenesis of EHEC infections is associated with the https://doi.org/10.1128/IAI.00334-19.
production of Shiga toxins (Stxs) that are identical to the Shiga toxin produced by Editor Shelley M. Payne, The University of
Shigella dysenteriae type I (4). Stxs produced by EHEC include Stx1 and Stx2, and the Texas at Austin
nucleotide sequences of these two Stxs are ⬃56% identical (5). Copyright © 2019 American Society for
Microbiology. All Rights Reserved.
In 2006, a high frequency of HUS was seen following an outbreak of the EHEC O157
Address correspondence to Takeshi Shimizu,
TW14359 strain caused by consumption of contaminated spinach (6). The percentage tshimizu@faculty.chiba-u.jp.
(15%) of TW14359 cases that developed HUS was significantly higher than the per- Received 27 April 2019
centage (4.1%) of all EHEC O157 cases that developed HUS (7). Comparison of the Returned for modification 20 May 2019
genome sequences between TW14359 and other EHEC O157 strains that caused lower Accepted 8 June 2019
Accepted manuscript posted online 17
frequencies of HUS identified the intact nitric oxide (NO) reductase gene norV as a
June 2019
candidate gene for virulence (8). There are two types of norV genes in EHEC O157, an Published 21 August 2019
intact norV and a deleted norV gene (9–11). The product of deleted norV is an internal

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Shimizu et al. Infection and Immunity

68-amino-acid deletion in the norV coding sequence and did not exhibit NO reductase
activity (9, 10). Moreover, intact norV-type EHEC showed better survival in macrophages
than deleted norV-type EHEC (10). An evolutionary analysis revealed that intact norV in
EHEC O157 was strictly correlated with subgroup C cluster 1, and deleted norV was
correlated with subgroup C clusters 2 and 3, but cluster 2 is a minor group (11). EHEC
O157 subgroup C cluster 1 and cluster 2 usually produce only Stx2, while almost all the
members of EHEC O157 subgroup C cluster 3 produce both Stx1 and Stx2 (11).
Epidemiological studies have shown that EHEC O157 strains that produce only Stx2 are
more commonly associated with severe human disease than EHEC O157 strains that
produce both Stx1 and Stx2 (12–14). Therefore, the intact norV gene is a direct virulence
determinant of EHEC O157, even though, with the exception of serotype O157, no EHEC
strains carrying the deleted norV gene have been obtained (11). This fact indicates that
reducing NO in bacterial cells is important for the growth and survival of EHEC O157 in
the intestinal tract during EHEC infection (11).
NO is a water-soluble reactive nitrogen species. It is rapidly oxidized by molecular
oxygen but is more stable under anaerobic conditions. It is potentially toxic in biolog-
ical systems because of its ability to react with a variety of cellular molecules, especially
thiol groups and transition metal centers in proteins (15). NO is an important compo-
nent of the innate immune system against infection, and it is synthesized at high
concentrations (⬃10⫺7 M to 10⫺6 M) by the inducible NO synthases (iNOSs) in
phagocytic cells (15–18). On the other hand, lower concentrations of NO (⬃10⫺9 M to
10⫺7 M) are produced by certain bacteria, either as a by-product of nitrite reduction to
ammonia or as an intermediate of denitrification (19, 20). In fact, the physiological
concentrations of NO within the gut are thought to be within the range of ⬃10⫺7 M
to 10⫺6 M (21, 22). Therefore, bacteria can potentially be exposed to various concen-
trations of NO produced by host cells and endogenously generated NO.
Bacteria have evolved specific sensor proteins to survive against nitrosative stress;
these proteins detect the NO molecule and switch on the expression of enzymes that
rapidly detoxify NO before it reaches lethal levels. NO sensor proteins are iron-
containing transcriptional factors that have evolved roles in the cellular response to NO
(23). NO interacts with the heme, iron-sulfur clusters, and iron present in NO sensor
proteins to form iron-nitrosyl complexes, which likely leads to protein conformational
changes and/or changes in DNA affinity that modulate gene expression in response to
the presence of NO. In Escherichia coli, two direct NO-sensing proteins, the transcrip-
tional activator NorR and the transcriptional repressor NsrR, appear to exclusively sense
NO (24–28). NorR is a ␴54-dependent transcriptional activator that activates the tran-
scription of the norVW operon in E. coli in response to NO. NorR senses NO directly
through a mononuclear nonheme iron center. On the other hand, NsrR contains an
iron-sulfur cluster and regulates the expression of several genes in response to NO. NsrR
binds to the promoter region upstream of its target gene and represses transcription.
NsrR controls the expressions of the flavohemoglobin Hmp and the hybrid cluster
protein Hcp in response to NO in E. coli (26, 27, 29).
NO detoxification pathways appear to be widespread among bacteria to defend
against NO. Enteric bacteria have developed multiple mechanisms for protecting
themselves from NO. Three main methods for detoxifying NO have been identified in
E. coli (30–32). The NorV flavorubredoxin and its redox partner protein NorW NADH:
flavorubredoxin oxidoreductase constitute an electron transfer chain, with NADH acting
as the primary electron donor (33). NorV and NorW exhibit NADH-dependent NO
reductase activity, which is considered to be responsible for the detoxification of NO
under anaerobic conditions (33). NorV and NorW are encoded in a bicistronic unit,
which is under the regulation of NorR. NorV is composed of three structural domains,
the flavodiiron structural core, a flavin mononucleotide (FMN)-binding flavodoxin-like
module, and a C-terminal rubredoxin module, while NorW contains a flavin adenine
dinucleotide (FAD) moiety (33, 34).
The Hmp flavohemoglobin in E. coli catalyzes both a rapid reaction of heme-bound
O2 with NO to form nitrate under aerobic conditions and a slower reduction of

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Cooperative Roles of NO-Metabolizing Enzymes in EHEC Infection and Immunity

heme-bound NO to form N2O under anaerobic conditions (35, 36). Hmp is made of an
N-terminal globin domain and a C-terminal FAD- and NAD-binding domain (37, 38). The
heme in the globin domain is covalently linked to the polypeptide chain through a
proximal histidine residue (His85) in addition to the conservation of the typical globin
fold, and a phenylalanine residue (Phe43) appears to be necessary to keep the heme in
the correct orientation within the pocket (37, 38). In the FAD- and NAD-binding domain
of Hmp, the aromatic ring of the conserved residue Phe390 is packed against the
isoalloxazine central ring of FAD (37, 38).
Hcp is a high-affinity NO reductase that is the major enzyme for reducing NO to N2O
under physiologically relevant conditions (32). Recently, the Stamler group demon-
strated that Hcp is an essential enzyme required for S-nitrosylation (39). Hcp contains
two types of iron-sulfur clusters unique in biological systems: a [4Fe-4S] cubane cluster
and a novel type of hybrid [4Fe-2S-2O] cluster (40). The hcp gene is closely linked to a
gene for an NADH-dependent Hcp oxidoreductase, Hcr, which serves as the electron
donor for the Hcp in the presence of NADH (40). Hcr contains FAD and a [2Fe-2S] cluster
as cofactors (40) and protects Hcp against nitrosylation by NO (32).
In this study, we clarified the cooperative roles of these NO-metabolizing enzymes,
NorV, Hmp, and Hcp, in reducing the NO level and protecting against the growth of
EHEC bacterial cells in response to nitrosative stress.

RESULTS
Expressions of NO-metabolizing enzyme genes in EHEC in response to NO.
EHEC contains three NO-metabolizing enzymes, NorV, Hmp, and Hcp. To determine the
expressions of norV, hmp, and hcp and, thus, the levels of the corresponding enzymes
in EHEC following exposure to NO, we constructed EHEC reporter strains using a
chromosome-plasmid hybrid bioluminescent reporter system (41). Three single-copy
chromosomal norV::luxE fusion, hmp::luxE fusion, and hcp::luxE fusion strains were
constructed, and these mutant strains were transformed with the LuxCDAB expression
plasmid pluxCDAB3 (41). The levels of luminescence in EHEC reporters depended on
the level of luxE expression from each promoter. To determine the effect of the
NO-induced expressions of norV, hmp, and hcp on growth conditions, EHEC cells were
cultured in LB broth at 37°C for 6 h (early stationary phase) under various growth
conditions, and specific luminescence was determined by a luminometer as a measure
of the expression of the corresponding gene, followed by normalization by colony
counting. Since the concentration of NO in culture following treatment with 200 ␮M
diethylenetriamine NONOate (DETA/NO) under anaerobic conditions was 12 ␮M (42),
the concentration of NO in culture after treatment with 100 ␮M DETA/NO under
anaerobic conditions might be no more than about 10⫺6 M, which corresponds to the
physiological concentration of NO in the gut (21, 22). In the presence of 100 ␮M
DETA/NO, the expression levels of norV, hmp, and hcp were increased ⬃160- to
200-fold, ⬃16- to 53-fold, and ⬃133- to 163-fold, respectively, under anaerobic growth
and aerobic static growth conditions (Fig. 1A to C). However, under aerobic growth
conditions with shaking, the expressions of norV and hcp were not increased, although
the expression of hmp was increased (Fig. 1A to C). It was previously reported that the
hcp promoter is not NO inducible under aerobic conditions (43). Therefore, these results
indicated that the culture under aerobic growth with shaking constituted aerobic
conditions and that the cultures under both anaerobic growth and aerobic static
growth were oxygen limited. To further determine the differences between anaerobic
growth and aerobic static growth, we investigated bacterial growth of wild-type, ΔnorV
mutant, Δhmp mutant, and Δhcp mutant EHEC strains following treatment with an NO
donor. Under aerobic static growth conditions, Δhmp mutant EHEC displayed a reduc-
tion of growth 18 h after treatment with 200 ␮M DETA/NO, although the growth levels
of wild-type, ΔnorV mutant, Δhmp mutant, and Δhcp mutant EHEC strains were not
inhibited under anaerobic growth conditions (Fig. 1D and E). Since NO detoxification by
Hmp occurs most effectively via O2-dependent NO dioxygenation (36), and NO reduc-
tase activity of NorV is exhibited in the absence of an O2 molecule (44), these results

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Shimizu et al. Infection and Immunity

(A) (B) (C)

100 norV 100 hmp 100 hcp
norV expression ( RLU / cfu)

hmp expression ( RLU / cfu)

10-1 10-1 10-1

hcp expression ( RLU / cfu)

10-2 10-2 10-2

10-3 10-3 10-3

10-4 10-4 10-4

10-5 10-5 10-5

DETA/NO DETA/NO DETA/NO
(PM) 0 100 0 100 0 100 (PM) 0 100 0 100 0 100 (PM) 0 100 0 100 0 100

Growth Anaerobic Aerobic Aerobic Growth Anaerobic Aerobic Aerobic Growth Anaerobic Aerobic Aerobic
conditions static static shaking conditions static static shaking conditions static static shaking
Fold Fold Fold
induction 204 ± 6 158 ± 12 0.8 ± 0.03 induction 53 ± 3 16 ± 3 5.6 ± 0.2 induction 133 ± 11 163 ± 22 1.2 ± 0.1

(D) Anaerobic (E) Aerobic

static static
200 PM DETA/NO 200 PM DETA/NO
108 108
Number of bacteria (cfu / 100 Pl)

Number of bacteria (cfu / 100 Pl)

107 107

106 106

'norV * 'norV *
'hmp * 'hmp *
'hcp * 'hcp *

FIG 1 Effect of growth conditions on gene expressions of NO-metabolizing enzymes and bacterial growth in EHEC treated with an NO donor.
EHEC cells grown overnight were diluted 1:100 with LB medium–10 mM HEPES (pH 7.0) with or without DETA NONOate (DETA/NO) and grown
at 37°C for 6 h for gene expression or 18 h for bacterial growth under anaerobic growth, aerobic static growth, or aerobic growth with shaking
at 220 rpm. Relative light units (RLU) and the number of bacteria were measured by a luminometer and bacterial plate counts (CFU), respectively.
Data are the means ⫾ standard deviations of values from three experiments. (A) norV reporter, 100 ␮M DETA/NO; (B) hmp reporter, 100 ␮M
DETA/NO; (C) hcp reporter, 100 ␮M DETA/NO; (D) anaerobic growth, 200 ␮M DETA/NO; (E) aerobic static growth, 200 ␮M DETA/NO.

indicate that the culture under aerobic static growth and anaerobic static growth
constituted microaerobic and anaerobic conditions, respectively.
EHEC enters the gastrointestinal tract during infection. While the lumen of the
intestinal tract is anaerobic, there is a zone of lower oxygenation adjacent to the
mucosal surface that is generated by diffusion from the microvillus capillary network
(45). Moreover, oxygen levels modulate pathogen virulence during infection (46).
Therefore, we analyzed the roles of NO-metabolizing enzymes in EHEC under anaerobic
and microaerobic conditions. EHEC reporters were cultured in LB broth at 37°C for 6 h
and 18 h (late stationary phase) under anaerobic and microaerobic conditions. In this

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Cooperative Roles of NO-Metabolizing Enzymes in EHEC Infection and Immunity

(A) (B) (C) (D)

norV hmp hcp espA
15 * * 15 * * 500 * * 15

100

Fold induction (espA)

Fold induction (norV)

Fold induction (hmp)

Fold induction (hcp)

10 10 10

Anaerobic 10

5 5 5
1

0 0 0.1 0
Nitrite - + - + Nitrite - + - + Nitrite - + - + Nitrite - +
6h 18 h 6h 18 h 6h 18 h 18 h

(E) norV (F) hmp (G) hcp (H) espA

* * * * * *
15 15 500 15

100

Fold induction (espA)

Fold induction (norV)

Fold induction (hmp)

Fold induction (hcp)

10 10 10

Microaerobic 10

5 5 5
1

0 0 0.1 0
Nitrite - + - + Nitrite - + - + Nitrite - + - + Nitrite - +
6h 18 h 6h 18 h 6h 18 h 18 h

FIG 2 Effect of nitrite on gene expressions of NO-metabolizing enzymes in EHEC. EHEC reporters for norV, hmp, or hcp grown overnight were diluted 1:100
with LB medium–10 mM HEPES (pH 7.0) with or without 3 mM nitrite and grown statically at 37°C for 6 h or 18 h under anaerobic and microaerobic conditions.
RLU and the number of bacteria were measured by a luminometer and bacterial plate counts (CFU), respectively. The results are expressed relative to the values
obtained for EHEC reporters without nitrite treatment after 6 h. Data are the means ⫾ standard deviations of values from three experiments. An asterisk
indicates a significant difference (P ⬍ 0.01). (A) Anaerobic, norV; (B) anaerobic, hmp; (C) anaerobic, hcp; (D) anaerobic, espA; (E) microaerobic, norV; (F)
microaerobic, hmp; (G) microaerobic, hcp; (H) microaerobic, espA.

study, nitrite was used as a source of NO (40, 41). In the presence of 3 mM nitrite, the
expression levels of norV, hmp, and hcp were increased ⬃7- to 11-fold, ⬃10- to 12-fold,
and ⬃180- to 290-fold under anaerobic conditions, respectively (Fig. 2A to C). Under
microaerobic conditions, the expression levels of norV, hmp, and hcp in the presence of
3 mM nitrite were increased ⬃4- to 5-fold, ⬃10- to 13-fold, and ⬃300- to 400-fold,
respectively (Fig. 2E to G). The expression of espA in the presence of 3 mM nitrite was
not increased under anaerobic and microaerobic conditions (Fig. 2D and H). The
product of espA is one of the type III secreted proteins in EHEC (47). These results
indicated that among these three genes, induction of hcp is most sensitive to NO under
anaerobic and microaerobic conditions.
To further determine the expressions of norV, hmp, and hcp in EHEC following
exposure to NO, we treated the EHEC reporter strains with various concentrations of the
NO donor DETA/NO for 18 h. At DETA/NO concentrations as low as 0.1 ␮M, the
expression level of hcp was significantly higher than those of norV and hmp under both
anaerobic and microaerobic conditions, indicating that the expression of hcp has a
lower threshold for a response to NO than the expressions of norV and hmp (Fig. 3A and
B). The expression of espA following treatment with 100 ␮M DETA/NO was not in-
creased under anaerobic or microaerobic conditions (Fig. 3A and B). Under anaerobic

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Shimizu et al. Infection and Immunity

(A) Anaerobic (B) Microaerobic

300
norV 300 norV
hmp hmp
100 hcp 100 hcp
espA espA
Fold Induction

Fold Induction
10 10

*
*

1 1

0.1 0.1
0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100

DETA/NO (PM) DETA/NO (PM)

FIG 3 Measurement of the NO responsiveness of NO-metabolizing enzyme genes in EHEC. EHEC reporters for norV, hmp, or hcp grown overnight were diluted
1:100 with LB medium–10 mM HEPES (pH 7.0) containing various concentrations of DETA/NO and grown statically at 37°C for 18 h under anaerobic (A) and
microaerobic (B) conditions. RLU and the number of bacteria were measured by a luminometer and bacterial plate counts (CFU), respectively. The results are
expressed relative to the values obtained for EHEC reporters without DETA/NO treatment. Data are the means ⫾ standard deviations of values from three
experiments. An asterisk indicates a significant difference (P ⬍ 0.01).

and microaerobic conditions, the expression levels of norV, hmp, and hcp following
treatment with 10 ␮M DETA/NO were increased ⬃7- to 8-fold, ⬃5- to 6-fold, and ⬃140-
to 200-fold, respectively (Fig. 3A and B). These results were similar to those in the
presence of 3 mM nitrite.
Role of NO-responsive transcription factors in expressions of NO-metabolizing
enzyme genes and growth protection in EHEC in response to NO. NorR is a
␴54-dependent transcriptional activator that regulates the expression of norV in E. coli
(48). In addition, there is direct evidence for NsrR binding at sites upstream of hmp and
hcp, and these are known to be NsrR targets (29, 43, 49). Fnr-binding sites have also
been predicted in the promoter regions of hmp and hcp (31). Thus, to determine
whether the expressions of these NO-metabolizing enzyme genes depend on the
response of NorR, NsrR, and/or Fnr to NO in EHEC, we constructed ΔnorR mutant, ΔnsrR
mutant, and Δfnr mutant EHEC reporters and determined their specific luminescences
under anaerobic and microaerobic conditions. As expected, the expression of norV in
the ΔnorR mutant EHEC reporter was not increased in response to NO under either
anaerobic or microaerobic conditions after 18 h (Fig. 4A and D). However, the ΔnorR
mutant norV reporter could not be complemented by a plasmid clone containing the
norR gene (data not shown). This was consistent with a previous report (50). These
results indicate that the transcriptional activator NorR in EHEC positively regulates the
expression of norV in response to NO under anaerobic and microaerobic conditions. In
addition, the expressions of norV in the ΔnsrR mutant and Δfnr mutant EHEC reporters
were slightly and largely increased in response to NO under anaerobic and microaero-
bic conditions after 18 h, respectively (Fig. 4A and D). The ΔnsrR mutant and Δfnr
mutant norV reporters could be complemented by plasmid clones containing the nsrR
gene and the fnr gene, respectively (see Fig. S1 in the supplemental material).
The expressions of hmp in the wild-type, ΔnorR mutant, and Δfnr mutant EHEC
reporters were increased in response to NO under anaerobic and microaerobic condi-
tions after 18 h (Fig. 4B and E). However, the expression level of hmp in the ΔnsrR
mutant EHEC reporter was constantly higher than the hmp expression levels in the
other strains in both the presence and absence of NO donor treatment and under both
anaerobic and aerobic conditions (Fig. 4B and E). The ΔnsrR mutant hmp reporter could
be complemented by a plasmid clone containing the nsrR gene (Fig. S1). These facts

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Cooperative Roles of NO-Metabolizing Enzymes in EHEC Infection and Immunity

(A) (B) (C)

200 400
500

100
Wild 100
100 norR
nsrR
: fnr

Fold Induction (hcp)

Fold Induction (hmp)
Fold Induction (norV)

10
10
Anaerobic 10
Wild
norR
nsrR
: fnr
1 1
1 Wild
norR
nsrR
: fnr

0.1 0.1
0.1 0 50 100 0.001 0.01 0.1 1 10
0 50 100
DETA/NO (PM) DETA/NO (PM) DETA/NO (PM)

(D) (E) (F)

200 400
1000

100
100

100

Fold Induction (hcp)

Fold Induction (hmp)
Fold Induction (norV)

10
10

Microaerobic 10

Wild
1 1
norR
1 Wild
Wild nsrR
norR
norR : fnr
nsrR
nsrR : fnr
: fnr
0.1
0.1 0.1 0.001 0.01 0.1 1 10
0 50 100 150 200 0 50 100 150 200
DETA/NO (PM)
DETA/NO (PM) DETA/NO (PM)

FIG 4 Measurement of the NO responsiveness of NO-metabolizing enzyme genes in NO-sensing transcription factor gene-deficient EHEC. EHEC reporters for
norV, hmp, or hcp grown overnight were diluted 1:100 with LB medium–10 mM HEPES (pH 7.0) containing various concentrations of DETA/NO and grown
statically at 37°C for 18 h under anaerobic and microaerobic conditions. RLU and the number of bacteria were measured by a luminometer and bacterial
plate counts (CFU), respectively. The results are expressed relative to the values obtained for the wild-type EHEC reporter without DETA/NO treatment. Data
are the means ⫾ standard deviations of values from three experiments. (A) Anaerobic, norV; (B) anaerobic, hmp; (C) anaerobic, hcp; (D) microaerobic, norV;
(E) microaerobic, hmp; (F) microaerobic, hcp.

indicated that the transcriptional repressor NsrR in EHEC repressed the expression of
hmp and was inactivated by exposure to NO, resulting in the derepression of hmp.
The expressions of hcp in the wild-type and ΔnorR mutant EHEC reporters were
increased in response to NO under anaerobic and microaerobic conditions after 18 h
(Fig. 4C and F). However, the expression level of hcp in the ΔnsrR mutant EHEC reporter
was constantly higher than those in the other strains either with or without NO donor
treatment and under both anaerobic and microaerobic conditions. In contrast, the
expression of hcp in the Δfnr mutant EHEC reporter remained at the basal level either
with or without NO donor treatment and under both anaerobic and microaerobic
conditions (Fig. 4C and F). The ΔnsrR mutant and Δfnr mutant hcp reporters could be
complemented by plasmid clones containing the nsrR gene and the fnr gene, respec-
tively (Fig. S1). These results indicated that the transcriptional repressor NsrR in EHEC
repressed the expression of hcp and was inactivated by exposure to NO, resulting in the
derepression of hcp, and that Fnr was essential for the expression of hcp under
anaerobic and microaerobic conditions.
To further examine the roles of NorR, NsrR, and Fnr in EHEC under NO stress
conditions, we investigated growth inhibitions of the wild-type, ΔnorR mutant, ΔnsrR
mutant, and Δfnr mutant EHEC strains following treatment with an NO donor. Under
anaerobic conditions, ΔnorR mutant EHEC displayed a reduction of growth 18 h after
treatment with 400 ␮M DETA/NO, although the growth levels of the wild-type, ΔnsrR
mutant, and Δfnr mutant EHEC strains were not inhibited (Fig. S2). Under microaerobic
conditions, the growth levels of the wild-type, ΔnorR mutant, ΔnsrR mutant, and Δfnr
mutant EHEC strains were not inhibited by 400 ␮M DETA/NO after 18 h (Fig. S2). These
results suggested that NorR was essential for anaerobic growth at a very high concen-
tration of NO (400 ␮M DETA/NO).
Role of NO-metabolizing enzymes in protecting the growth of EHEC in re-
sponse to NO. To examine the role of NO-metabolizing enzymes in EHEC under NO
stress conditions, we constructed NO-metabolizing enzyme gene-deficient mutant

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Shimizu et al. Infection and Immunity

(A) (B) (C) (D)

Anaerobic, 100 PM DETA/NO, 18 h Anaerobic, 100 PM DETA/NO, 6 h Microaerobic, 200 PM DETA/NO, 18 h Microaerobic, 200 PM DETA/NO, 6 h
* * * * * * *
* *
108 108 108 108
Number of bacteria (cfu / 100 Pl)

Number of bacteria (cfu / 100 Pl)

Number of bacteria (cfu / 100 Pl)

Number of bacteria (cfu / 100 Pl)

107 107 107 107

106 106 106 106

105 105 105 105

104 104 104 104

'norV * * * * 'norV * * * * 'norV * * * * 'norV * * * *

'hmp * * * * 'hmp * * * * 'hmp * * * * 'hmp * * * *
'hcp * * * * 'hcp * * * * 'hcp * * * * 'hcp * * * *

(E) (F) (G) (H)

Anaerobic, 100 PM DETA/NO, 18 h Anaerobic, 100 PM DETA/NO, 6 h Microaerobic, 200 PM DETA/NO, 18 h Microaerobic, 200 PM DETA/NO, 6 h
* * * * * *
*
101
* 101
101
101

100 100
100 100
NO level (RLU / cfu)

NO level (RLU / cfu)

NO level (RLU / cfu)

NO level (RLU / cfu)

10-1 10-1 10-1
10-1

10-2 10-2 10-2 10-2

10-3 10-3 10-3 10-3

'norV * * * * 'norV 'norV * * * * 'norV

* * * * * * * *
'hmp * * * * 'hmp 'hmp * * * * 'hmp
* * * * * * * *
'hcp * * * * 'hcp 'hcp * * * * 'hcp
* * * * * * * *

FIG 5 Effect of NO-metabolizing enzymes on bacterial growth (A to D) and NO levels (E to H) in bacterial cells treated with a high concentration of an NO donor.
EHEC NO reporters grown overnight were diluted 1:100 with LB medium–10 mM HEPES (pH 7.0) containing 100 ␮M or 200 ␮M DETA/NO and grown statically
at 37°C for 6 h or 18 h under anaerobic and aerobic conditions. RLU and the number of bacteria were measured by a luminometer and bacterial plate counts
(CFU), respectively. Data are the means ⫾ standard deviations of values from three experiments. An asterisk indicates a significant difference (P ⬍ 0.01). (A and
E) Anaerobic, 100 ␮M DETA/NO, 18 h; (B and F) anaerobic, 100 ␮M DETA/NO, 6 h; (C and G) microaerobic, 200 ␮M DETA/NO, 18 h; (D and H) microaerobic,
200 ␮M DETA/NO, 6 h. Closed bars, shaded bars, hatched bars, and open bars indicate wild-type strains, single mutant strains, double mutant strains, and triple
mutant strains, respectively.

strains and investigated growth inhibition following treatment with an NO donor under
anaerobic and microaerobic conditions. The ΔnorV mutant, Δhmp mutant, and Δhmp
mutant strains could be complemented by plasmid clones containing the norV gene,
the hmp gene, and the hcp gene, respectively (Fig. S3). Under anaerobic conditions, the
growth of Δhmp Δhcp mutant EHEC 18 h after treatment with 100 ␮M DETA/NO was
not inhibited in comparison to the growth of ΔnorV Δhmp Δhcp mutant EHEC (Fig. 5A),
indicating that NorV plays an important role in anaerobic growth in the presence of a
high concentration of NO. However, although norV was expressed at 6 h (Fig. 2A), the
growth levels of the Δhmp mutant and Δhmp Δhcp mutant EHEC strains were inhibited
6 h after treatment with 100 ␮M DETA/NO under anaerobic conditions (Fig. 5B). These
results indicated that NorV and Hmp play a cooperative role for anaerobic growth
under a high concentration of NO.
Under microaerobic conditions, Δhmp mutant EHEC displayed a reduction of mi-
croaerobic growth in response to 200 ␮M DETA/NO after 6 h and 18 h (Fig. 5C and D).
The growth of ΔnorV Δhcp mutant EHEC was not inhibited by 200 ␮M DETA/NO after
6 h and 18 h (Fig. 5C and D). These results indicated that Hmp was important for
protection of bacterial growth under microaerobic conditions in the presence of a high
concentration of NO.
Role of NO-metabolizing enzymes in the regulation of NO toxicity in EHEC
bacterial cells. EHEC strains that expressed NO-metabolizing enzymes were previously
reported to produce a lower level of NO than the corresponding mutant EHEC strains

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Cooperative Roles of NO-Metabolizing Enzymes in EHEC Infection and Immunity

(10, 42). To confirm this, here we determined the NO levels in bacterial cells treated with
an NO donor under anaerobic and aerobic conditions. As an NO reporter system, we
used the NO reporter plasmid pRPL3, whose luxCDABE expression is dependent on
NorR activation of the promoter of norV in response to NO in bacterial cells (10). Under
anaerobic conditions, the specific luminescence (relative light units [RLU]/CFU) of the
wild-type EHEC NO reporter was much lower than that of the ΔnorV Δhmp Δhcp mutant
EHEC NO reporter 6 h and 18 h following treatment with 100 ␮M DETA/NO, suggesting
that NO-metabolizing enzymes in EHEC detoxify NO to yield a low concentration of NO
in bacterial cells (Fig. 5E and F). Among the EHEC strains expressing only a single
NO-metabolizing enzyme gene, the specific luminescence of the Δhmp Δhcp mutant
EHEC NO reporter was lower than those of the ΔnorV Δhcp mutant and ΔnorV Δhmp
mutant EHEC NO reporters 18 h after treatment with 100 ␮M DETA/NO under anaerobic
conditions, suggesting that NorV in EHEC was important for NO detoxification at 18 h
under anaerobic conditions at a high concentration of NO (Fig. 5E). However, the
specific luminescences of the Δhmp mutant and Δhmp Δhcp mutant EHEC NO reporters
at 6 h were higher than those 18 h after treatment with 100 ␮M DETA/NO, suggesting
that NorV in EHEC could not detoxify NO in bacterial cells under anaerobic conditions
at 6 h in the presence of a high concentration of NO (Fig. 5E and F). This result is
consistent with that for growth inhibition following treatment with the same concen-
tration of DETA/NO. On the other hand, under microaerobic conditions, the specific
luminescence of the ΔnorV Δhcp mutant EHEC NO reporter was lower than those of
the Δhmp Δhcp mutant and ΔnorV Δhmp mutant EHEC NO reporters 6 h and 18 h
after treatment with 200 ␮M DETA/NO, suggesting that Hmp in EHEC is important
for NO detoxification under microaerobic conditions at high concentrations of NO
(Fig. 5G and H).
Because much lower concentrations of NO were required to induce hcp expression
than to induce the expressions of norV and hmp (Fig. 3A and B), we next examined the
role of NO-metabolizing enzymes at lower concentrations of NO under anaerobic and
microaerobic conditions. Under both anaerobic and microaerobic conditions, the
growth of ΔnorV Δhcp mutant EHEC was similar to that of ΔnorV Δhmp Δhcp mutant
EHEC and lower than those of the Δhmp Δhcp mutant and ΔnorV Δhmp mutant EHEC
strains following treatment with 10 ␮M DETA/NO or in the presence of 3 mM nitrite (Fig.
6A to D), indicating that NorV and Hcp protect EHEC from growth inhibition at low
concentrations of NO under anaerobic and microaerobic conditions.
To further examine the roles of NorV and Hcp in EHEC at low concentrations of NO,
we investigated the NO levels in bacterial cells at low concentrations of NO under
anaerobic and microaerobic conditions. The specific luminescence of the ΔnorV Δhmp
mutant EHEC NO reporter was lower than those of the Δhmp Δhcp mutant and ΔnorV
Δhcp mutant EHEC NO reporters treated with 10 ␮M DETA/NO or in the presence of
3 mM nitrite under anaerobic and microaerobic conditions (Fig. 6E to H). These results
indicate that Hcp in EHEC is the major enzyme responsible for the reduction of NO in
the presence of a low concentration of NO under anaerobic and microaerobic condi-
tions.
Hmp suppresses the NO reductase activity of Hcp at a low concentration of NO.
Unexpectedly, the specific luminescences of the Δhmp mutant and ΔnorV Δhmp
mutant EHEC NO reporters were lower than that of the wild-type EHEC NO reporter
treated with 10 ␮M DETA/NO or in the presence of 3 mM nitrite under anaerobic and
microaerobic conditions (Fig. 6E to H). The levels of specific luminescence of the Δhmp
mutant and ΔnorV Δhmp mutant EHEC NO reporters following treatment with 10 ␮M
DETA/NO or in the presence of 3 mM nitrite were similar to those of the wild-type EHEC
NO reporter without NO donor or nitrite treatment (data not shown). However, the level
of specific luminescence of the ΔnorV mutant EHEC NO reporter was similar to that of
the wild-type EHEC NO reporter (Fig. 6E to H). This result indicated that the NO
reductase activity of Hcp in EHEC was suppressed by Hmp at a low concentration of NO
under anaerobic and microaerobic conditions. Thus, to further examine the effect of
Hmp on NO reductase activity of Hcp at a low concentration of NO, we constructed

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Shimizu et al. Infection and Immunity

(A) Anaerobic, 10 PM DETA/NO (B) Anaerobic, 3 mM nitrite (C) Microaerobic, 10 PM DETA/NO (D) Microaerobic, 3 mM nitrite
* * * *
* * * *
* * * *
* * * *
* * * * * * * *
9 9 9 9
Number of bacteria ( 107 cfu / 100 Pl)

Number of bacteria ( 107 cfu / 100 Pl)

Number of bacteria ( 107 cfu / 100 Pl)

Number of bacteria ( 107 cfu / 100 Pl)

8 8 8 8

7 7 7 7

6 6 6 6

5 5 5 5

4 4 4 4

3 3 3 3

2 2 2 2

1 1 1 1

0 0 0 0

'norV * * * * 'norV * * * * 'norV * * * * 'norV * * * *

'hmp * * * * 'hmp * * * * 'hmp * * * * 'hmp * * * *
'hcp * * * * 'hcp * * * * 'hcp * * * * 'hcp * * * *

(E) (F) (G) (H)

Anaerobic, 10 PM DETA/NO Anaerobic, 3 mM nitrite Microaerobic, 10 PM DETA/NO Microaerobic, 3 mM nitrite
* * * * * * * *
* * * * 100 * * * *
100 100 100

10-1
10-1 10-1 10-1
NO level (RLU / cfu)

NO level (RLU / cfu)

NO level (RLU / cfu)

NO level (RLU / cfu)

10-2
10-2 10-2 10-2

-3
10-3 10
10-3 10-3

10-4 10-4
10-4 10-4

10-5 10-5 10-5 10-5

10-6 10-6 10-6

10-6

'norV * * * * 'norV * * * * 'norV 'norV * * * *

* * * *
'hmp * * * * 'hmp * * * * 'hmp 'hmp * * * *
* * * *
'hcp * * * * 'hcp * * * * 'hcp 'hcp * * * *
* * * *

FIG 6 Effect of NO-metabolizing enzymes on bacterial growth (A to D) and NO levels (E to H) in bacterial cells treated with a low concentration of an NO donor
or in the presence of nitrite. EHEC NO reporters grown overnight were diluted 1:100 with LB medium–10 mM HEPES (pH 7.0) containing 10 ␮M DETA/NO or
3 mM nitrite and grown statically at 37°C for 18 h under anaerobic and microaerobic conditions. RLU and the number of bacteria were measured by a
luminometer and bacterial plate counts (CFU), respectively. Data are the means ⫾ standard deviations of values from three experiments. An asterisk indicates
a significant difference (P ⬍ 0.01). (A and E) Anaerobic, 10 ␮M DETA/NO; (B and F) anaerobic, 3 mM nitrite; (C and G) microaerobic, 10 ␮M DETA/NO; (D and
H) microaerobic, 3 mM nitrite. Closed bars, shaded bars, hatched bars, and open bars indicate wild-type strains, single mutant strains, double mutant strains,
and triple mutant strains, respectively.

deleted and point-mutated hmp-expressing EHEC strains (Fig. 7). The aromatic side
chain of Phe43 in the globin domain binds nearly parallel to the heme plane, and Hmp
is stabilized by a stacking interaction of the isoalloxazine central ring of FAD with
Phe390 in the FAD- and NAD-binding domain (37, 38). These deleted and point-

Hmp Globin domain FAD- and NAD-binding domain

396

Hmp305
305

Hmp100
100

HmpF43A F43A

HmpF390A F390A

FIG 7 Structure of intact and mutant Hmp. Hmp is made of an N-terminal globin domain and a
C-terminal FAD- and NAD-binding domain.

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Cooperative Roles of NO-Metabolizing Enzymes in EHEC Infection and Immunity

(A) (B) (C) (D) (E)

Microaerobic, 200 PM DETA/NO Anaerobic, 10 PM DETA/NO Microaerobic, 10 PM DETA/NO Anaerobic, 10 PM DETA/NO Microaerobic, 10 PM DETA/NO
*
108 *
*
9
* 9 9 9

Number of bacteria ( 107 cfu / 100 Pl)

Number of bacteria ( 107 cfu / 100 Pl)

Number of bacteria ( 107 cfu / 100 Pl)

Number of bacteria ( 107 cfu / 100 Pl)

Number of bacteria (cfu / 100 Pl)

8 8 8 8

107
7 7 7 7

6 6 6 6

106 5 5 5 5

4 4 4 4

3 3 3 3
105
2 2 2 2

1 1 1 1

104 0 0 0 0
hmp - + 305 100 F43AF390A hmp - + 305 100 F43AF390A + hmp - + 305 100 F43AF390A + 'norV * * * * 'norV * * * *
'hcp * * * * * * 'hcp 'hcp 'hmp * * 'hmp * *
* * 'hcp
'norV * * * * * * 'norV * 'norV * 'hcp * * * *
* * * * * * * * * * * * 'hcr 'hcr
* * * * * * * *

(F) (G) (H) (I) (J)

Microaerobic, 200 PM DETA/NO Anaerobic, 10 PM DETA/NO Microaerobic, 10 PM DETA/NO Anaerobic, 10 PM DETA/NO Microaerobic, 10 PM DETA/NO
100 * 100 * 100 *
* * *
100 * 100 * *
* * *
* * 10-1 *
10-1
NO level (RLU / cfu)

NO level (RLU / cfu)

NO level (RLU / cfu)

NO level (RLU / cfu)

10-1 10-1

NO level (RLU / cfu)

10 10-2 10-2

10-3 10-3
10-2 10-2

1 10-4 10-4

10-3 10-3
10-5 10-5

0.1 10-6 10-6

10-4 10-4
hmp - + 305 100 F43AF390A hmp - + + hmp - + 305 100 F43AF390A +
305 100 F43AF390A 'norV * * * * 'norV *
'hcp * * * * * * 'hcp 'hcp * 'hmp 'hmp
* * *
* * * * *
'norV * * * * * * 'norV * 'norV * * * * * * 'hcp 'hcp
* * * * * * * * * * *
'hcr * * * * 'hcr * * * *

FIG 8 Effect of Hmp on bacterial growth (A to E) and NO levels in bacterial cells (F to J) following treatment with an NO donor. EHEC NO reporters grown
overnight were diluted 1:100 with LB medium–10 mM HEPES (pH 7.0) containing 10 ␮M or 200 ␮M DETA/NO and grown statically at 37°C for 18 h under
anaerobic and microaerobic conditions. RLU and the number of bacteria were measured by a luminometer and bacterial plate counts (CFU), respectively. Data
are the means ⫾ standard deviations of values from three experiments. An asterisk indicates a significant difference (P ⬍ 0.01). (A and F) Microaerobic, 200
␮M DETA/NO; (B, D, G, and I) anaerobic, 10 ␮M DETA/NO; (C, E, H, and J) microaerobic, 10 ␮M DETA/NO.

mutated hmp-expressing Δhmp ΔnorV Δhcp mutant EHEC strains display lower growth
and higher NO levels than ΔnorV Δhcp mutant EHEC following treatment with 200 ␮M
DETA/NO under microaerobic conditions, indicating that mutated Hmp did not exhibit
NO consumption activity (Fig. 8A and F). The specific luminescences of the deleted-
hmp-expressing Δhmp ΔnorV mutant and point-mutated-hmp-expressing Δhmp ΔnorV
mutant EHEC NO reporters were lower than that of the ΔnorV mutant EHEC NO reporter
(Fig. 8B and G). These results indicated that the NO reductase activity of Hcp in EHEC
was suppressed by the NO consumption activity of Hmp in the presence of a low
concentration of NO, although the NO consumption activity of Hmp in ΔnorV Δhcp
mutant EHEC could not be detected following treatment with 10 ␮M DETA/NO under
anaerobic and microaerobic conditions (Fig. 6E and G).
Hcp may accept electrons from NADH via Hcr (40). Thus, to determine whether Hcr
played a role in the suppression of NO reductase activity for Hmp under a low
concentration of NO, we measured NO levels in bacterial cells following treatment with
10 ␮M DETA/NO under anaerobic and microaerobic conditions using Δhcr mutant
EHEC. The specific luminescence of the ΔnorV Δhmp Δhcr mutant EHEC NO reporter was
lower than that of the ΔnorV Δhmp Δhcp Δhcr mutant EHEC NO reporter (Fig. 8I and J),
suggesting that there is another route of electron transfer to Hcp that is independent
of Hcr. The specific luminescence of the ΔnorV Δhmp Δhcr mutant EHEC NO reporter
was lower than that of the ΔnorV Δhcr mutant EHEC NO reporter under microaerobic
conditions (Fig. 8J). However, under anaerobic conditions, the level of specific lumi-
nescence of the ΔnorV Δhmp Δhcr mutant EHEC NO reporter was similar to that of the
ΔnorV Δhcr mutant EHEC NO reporter (Fig. 8I). This result at least indicated that the NO
reductase activity of Hcp in EHEC was suppressed by Hmp in an Hcr-independent

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Shimizu et al. Infection and Immunity

Fnr NorR
Anaerobic: High concentration of NO
NorV Microaerobic: Low concentration of NO

norV
Fnr NorR
binding binding
site site

norR

Fnr NsrR
Anaerobic: High concentration of NO
Hmp Microaerobic: High concentration of NO

hmp
Fnr NsrR
binding binding
site site

Fnr NsrR
Anaerobic: Low concentration of NO
Hcp Microaerobic: Low concentration of NO

hcp
Fnr NsrR
binding binding
site site

FIG 9 Gene regulation and function of NorV, Hmp, and Hcp in EHEC. Schematic (not to scale) of upstream regions and structures of the norV, hmp, and hcp
genes in EHEC. Regulatory proteins and their corresponding cis-acting elements are indicated. The NorR-binding site in the upstream region of norV has been
experimentally verified (48). The NsrR-binding sites in the upstream regions of hmp and hcp have been experimentally verified (29). The Fnr-binding sites in
the upstream regions of hmp and hcp have been experimentally verified (43, 51). The Fnr-binding site completely overlaps the NsrR-binding site in the hmp
promoter. NorV plays a role at a high concentration of NO under anaerobic conditions. Hmp plays a role at a high concentration of NO under anaerobic and
microaerobic conditions. Hcp plays a role at a low concentration of NO under anaerobic and microaerobic conditions.

manner in the presence of a low concentration of NO under microaerobic conditions.

However, we did not obtain any evidence of a direct interaction between the Hmp
protein and the Hcp protein by a pulldown assay (data not shown).

DISCUSSION
The data presented in this paper indicate that the NO-induced expression of hcp has a
low threshold relative to those of norV and hmp under anaerobic and microaerobic
conditions. Moreover, Hcp effectively decreased the NO concentration in bacterial cells at
a low concentration of NO under anaerobic and microaerobic conditions. Therefore, the
regulation of transcription of hcp in response to a low concentration of NO and the high
affinity of Hcp for NO might enable Hcp to reduce NO to N2O under normal physiological
conditions when the concentration of NO in bacterial cells is too low for NorV and Hmp to
be expressed or effective. These results are consistent with a previous report (32).
The expressions of hcp and hmp in EHEC were regulated by NsrR (Fig. 4B, C, E, and
F), and the promoter regions of both hcp and hmp have NsrR-binding sites in E. coli (Fig.
9) (29, 31). However, the NsrR-regulated expression of hcp was significantly greater than
the NsrR-regulated expression of hmp in EHEC. NsrR-binding sites in E. coli fall into two

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Cooperative Roles of NO-Metabolizing Enzymes in EHEC Infection and Immunity

classes: those comprising two copies of an 11-bp inverted repeat with 1-bp spacing and
those which have a single copy of an 11-bp element (29). Although the NsrR-binding
sites of both hcp and hmp were of the former type, the first and second NsrR-binding
sites in hcp satisfied the consensus sequence of the NsrR-binding motif (29). On the
other hand, the second NsrR-binding site in hmp was far from satisfying this sequence
(29). Therefore, one reason for the differential expression between hcp and hmp in
response to NO may have been differences in the sequences of the NsrR-binding sites
between hcp and hmp.
In addition to NsrR-binding sites, hmp and hcp in E. coli were preceded by a binding
site of Fnr (Fig. 9) (31, 51). Both hmp and hcp were consititutively expressed at higher
levels in the ΔnsrR mutant (Fig. 4B, C, E, and F). Increased expression of hcp, but not
hmp, was strongly dependent on Fnr. This indicates that the hcp promoter was
activated by Fnr and repressed by NsrR. However, in the case of the hmp promoter,
repression by NsrR was the dominant regulatory mechanism in this experiment,
although there is some evidence that repression by Fnr also played a role, as reported
previously (52, 53). Under our growth conditions, hmp might not have been signifi-
cantly derepressed in the fnr mutant grown in LB medium, which masked the indirect
effect of NsrR. These different effects of the fnr mutant on the expression of hmp may
be due to the choice of growth medium (26). At least in part, the consequence is that
the absence of an effect or derepression of the hmp promoter in an fnr mutant can be
explained by an indirect effect acting through NsrR (26). However, it was previously
reported that there is an Fnr-binding site in the hmp promoter that can be bound by
Fnr in vitro (Fig. 9) (51). Since the Fnr-binding site completely overlaps the NsrR-binding
site in the hmp promoter (Fig. 9) (49), Fnr might not bind to the hmp promoter when
NsrR binds to the hmp promoter in vivo. On the other hand, the Fnr-binding site in the
hcp promoter does not overlap the NsrR-binding site (Fig. 9) (31).
The expression of norV in the fnr-deficient reporter was largely increased in response
to NO. This was consistent with a previous report in which an Δfnr mutant of E. coli
caused an increase in norV-norW transcription without NO treatment (54). Analysis of
the region upstream of norV shows that this regulation may be achieved by the binding
of Fnr to the Fnr-binding site within the norR coding region (Fig. 9) (31, 54).
Macrophages are important components of the innate immune response. One of
the antimicrobial systems of macrophages is the iNOS pathway, which is responsible for
the generation of NO (16, 17, 55). The concentration of NO that accumulates in bacterial
cells within macrophages and during nitrite reduction has not been reported. However,
in a previous study, we revealed that the specific luminescences (RLU/CFU) of the
deleted norV-type EHEC NO reporter within macrophages were ⬃5 ⫻ 10⫺1 to 100 (42),
which correspond to the specific luminescences (RLU/CFU) of the norV-deficient EHEC
NO reporter after treatment with more than 100 ␮M and 200 ␮M DETA/NO under
anaerobic and aerobic conditions, respectively (Fig. 5E to H). In addition, since the
maximum steady-state concentration of NO in LB medium following treatment with
200 ␮M DETA/NO under anaerobic conditions was 12 ␮M (42), the steady-state con-
centration of NO after treatment with 100 ␮M DETA/NO under anaerobic conditions
might be no higher than 10⫺6 M. On the other hand, in the presence of 3 mM nitrite
under anaerobic and aerobic conditions, the specific luminescences (RLU/CFU) of the
wild-type EHEC NO reporter were ⬃5 ⫻ 10⫺5 to 10⫺4, which correspond to treatment
with 10 ␮M DETA/NO under anaerobic and aerobic conditions (Fig. 6E to H). Therefore,
these results suggested that the concentrations of NO generated from the NO donor in
this study corresponded to the NO concentrations under physiological conditions.
In EHEC O157, the intact norV-type EHEC strains are more virulent than the deleted
norV-type EHEC strains (10). In this study, we show that NorV plays an important role for
anaerobic growth in the presence of high concentrations of NO. Since the lumen of the
intestinal tract is anaerobic, this result suggests that intact norV in EHEC O157 would be
involved in determining clinical outcomes. On the other hand, although the hmp deletion
type of E. coli was not observed, we revealed that Hmp was also involved in the protection
of anaerobic growth under high concentrations of NO. When oxygen is available, Hmp

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Shimizu et al. Infection and Immunity

TABLE 1 Bacterial strains

Strain Relevant characteristics Reference or source
K47 Enterohemorrhagic Escherichia coli O157:H7; stx2, intact norV type 10
K47(⫺2)S3 K47; deletion of stx2 This study
K47(⫺2)VE1 K47; deletion of stx2, insertion of luxE downstream of the norV promoter This study
K47(⫺2nor)VE1 K47; deletion of stx2 and norR, insertion of luxE downstream of the norV promoter This study
K47(⫺2nsr)VE1 K47; deletion of stx2 and nsrR, insertion of luxE downstream of the norV promoter This study
K47(⫺2fnr)VE1 K47; deletion of stx2 and fnr, insertion of luxE downstream of the norV promoter This study
K47(⫺2)hmpE1 K47; deletion of stx2, insertion of luxE downstream of the hmp promoter This study
K47(⫺2nor)hmpE1 K47; deletion of stx2 and norR, insertion of luxE downstream of the hmp promoter This study
K47(⫺2nsr)hmpE1 K47; deletion of stx2 and nsrR, insertion of luxE downstream of the hmp promoter This study
K47(⫺2fnr)hmpE3 K47; deletion of stx2 and fnr, insertion of luxE downstream of the hmp promoter This study
K47(⫺2)hcpE3 K47; deletion of stx2, insertion of luxE downstream of the hcp promoter This study
K47(⫺2nor)hcpE1 K47; deletion of stx2 and norR, insertion of luxE downstream of the hcp promoter This study
K47(⫺2nsr)hcpE1 K47; deletion of stx2 and nsrR, insertion of luxE downstream of the hcp promoter This study
K47(⫺2fnr)hcpE1 K47; deletion of stx2 and fnr, insertion of luxE downstream of the hcp promoter This study
K47(⫺2)espE1 K47; deletion of stx2, insertion of luxE downstream of the espA promoter This study
K47(⫺2nor)SK1 K47; deletion of stx2 and norR This study
K47(⫺2nsr)SK6 K47; deletion of stx2 and nsrR This study
K47(⫺2fnr)SK1 K47; deletion of stx2 and fnr This study
K47(⫺2V)SC4 K47; deletion of stx2 and norV This study
K47(⫺2H)SC2 K47; deletion of stx2 and hmp This study
K47(⫺2C)SK5 K47; deletion of stx2 and hcp-hcr This study
K47(⫺2VH)KSC2 K47; deletion of stx2, norV, and hmp This study
K47(⫺2VC)SCK1 K47; deletion of stx2, norV, and hcp-hcr This study
K47(⫺2HC)SCK1 K47; deletion of stx2, hmp, and hcp-hcr This study
K47(⫺2VHC)SCK3 K47; deletion of stx2, norV, hmp, and hcp-hcr This study
K47(⫺2VC)H100 K47; deletion of stx2, norV, and hcp-hcr, replacement of mutant hmp(1–300) This study
K47(⫺2V)H100 K47; deletion of stx2 and norV, replacement of mutant hmp(1–300) This study
K47(⫺2VC)H305 K47; deletion of stx2, norV, and hcp-hcr, replacement of mutant hmp(1–915) This study
K47(⫺2V)H305 K47; deletion of stx2 and norV, replacement of mutant hmp(1–915) This study
K47(⫺2HVC)hR43A1 K47; deletion of stx2, norV, and hcp-hcr, replacement of mutant hmp(R43A) This study
K47(⫺2HV)hR43A1 K47; deletion of stx2, norV, and hcp-hcr, replacement of mutant hmp(R43A) This study
K47(⫺2HVC)hR390A1 K47; deletion of stx2, norV, and hcp-hcr, replacement of mutant hmp(R390A) This study
K47(⫺2HV)hR390A1 K47; deletion of stx2, norV, and hcp-hcr, replacement of mutant hmp(R390A) This study
K47(⫺2VR)SK2 K47; deletion of stx2, norV, and hcr This study
K47(⫺2VHR)SCK1 K47; deletion of stx2, norV, hmp, and hcr This study
BL21(DE3) E. coli B; F⫺ dcm ompT hsdS(rB⫺ mB⫺) gal ␭ (DE3) Nippon Gene
DH5␣(␭pir) recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 deoR (lacZYA-argF)U169 ␭pir⫹ 58

protects bacteria against NO stress by oxidizing NO to nitrate (35, 56). However, under
anaerobic conditions, Hmp also catalyzes a slower reduction of NO, followed by the
formation of N2O (35). Therefore, this result indicated that the NO reductase activity of Hmp
was important for anaerobic growth in the presence of high concentrations of NO.
Finally, we show that Hcp and Hmp played cooperative roles for NO consumption
in EHEC. Although Hmp alone was not effective at decreasing the NO levels in bacterial
cells in the presence of a low concentration of NO, Hmp partially repressed the NO
reductase activity of Hcp under anaerobic and aerobic conditions. We revealed that the
NO consumption activity of Hmp was necessary for the repression of the NO reductase
activity of Hcp at low concentrations of NO. However, NorV, which has NO consumption
activity, could not repress the NO consumption activity of Hcp. Since the concerted
action of NsrR-dependent Hcp for NO consumption and NsrR-dependent YtfE for the
repair of damaged iron centers would maintain the cytoplasmic concentration of NO in
the nanomolar range (57), repression of the NO reductase activity of Hcp by Hmp might
be important for the coordinate regulation of Hcp and Hmp synthesis by NsrR following
exposure to NO. The mechanism by which the NO reductase activity of Hcp is partially
repressed in the presence of Hmp under a low concentration of NO remains unknown.
However, our results may at least suggest that this mechanism does not interfere with
either the interaction between Hcp and Hcr or the transfer of electrons from Hcr to Hcp,
since Hmp also partially represses the NO reductase activity of Hcp in hcr-deficient
EHEC. Furture studies will be required to explain how Hmp represses the NO reductase
activity of Hcp at low concentrations of NO.

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Cooperative Roles of NO-Metabolizing Enzymes in EHEC Infection and Immunity

TABLE 2 Plasmidsa
Plasmid Relevant characteristic(s) Reference or source
pTVm2 NorV-His expression plasmid; Ampr; Ptrc 10
pThmp1 Hmp-His expression plasmid; Ampr; Ptrc This study
pBhcp1 Hcp expression plasmid; Cmr; PBAD This study
pBnorR2 NorR expression plasmid; Cmr; PBAD This study
pBnsrR2 NsrR expression plasmid; Cmr; PBAD This study
pBfnr1 Fnr expression plasmid; Cmr; PBAD This study
pTrcHis 2C Expression plasmid; Ampr; Ptrc Invitrogen
pBAD33 Expression plasmid; p15-ori PBAD; Cmr National BioResource Project (NIG) (Japan)
pRPL3 NO reporter plasmid; Ampr 10
phmpC10 Suicide plasmid; hmp and loxP-flanked Cmr cassette This study
phmp100C2 Suicide plasmid; deleted hmp(1–300) and loxP-flanked Cmr cassette This study
phmp305C15 Suicide plasmid; deleted hmp(1–915) and loxP-flanked Cmr cassette This study
phmpCF43A1 Suicide plasmid; mutated hmp(F43A) and loxP-flanked Cmr cassette This study
phmpCF390A1 Suicide plasmid; mutated hmp(F390A) and loxP-flanked Cmr cassette This study
pRed/ET(amp) Red/ET expression plasmid; Ampr Gene Bridges GmbH
pFRT-Kan Suicide plasmid; FRT-flanked PGK-gb2-neo cassette Gene Bridges GmbH
ploxCm1 Suicide plasmid; loxP-flanked Cmr cassette 41
ploxSm11 Suicide plasmid; loxP-flanked Smr cassette This study
pAH144 Source of Smr cassette National BioResource Project
pCreA1 Thermosensitive Cre expression plasmid; Ampr 41
pFT-A Thermosensitive FLP expression plasmid; Ampr National BioResource Project
pLCE19 Suicide plasmid; luxE and loxP-flanked Cmr cassette 41
pluxCDAB3 LuxCDAB expression plasmid; Ampr; Plac 41
aAmpr, ampicillin resistant; Cmr, chloramphenicol resistant; Smr, streptomycin resistant.

MATERIALS AND METHODS

Bacterial strains, plasmids, and oligonucleotides. The bacterial strains and plasmids used in this
study are listed in Tables 1 and 2. The oligonucleotides used for this study are listed in Table S1 in the
supplemental material.
Culture conditions. EHEC strains grown overnight were diluted 1:100 with LB broth containing
10 mM HEPES (pH 7.0). For anaerobic conditions, EHEC strains were grown statically at 37°C using an
Anaero Pack-Anaero 5% system (Mitsubishi Gas Chemical Company, Tokyo, Japan). For microaerobic
conditions, EHEC strains were grown statically at 37°C in an aerobic culture. For aerobic conditions, EHEC
strains were grown with shaking at 220 rpm at 37°C in an aerobic culture.
Gene reporter assay, growth inhibition assay, and NO reporter assay. For the gene reporter
assay, mutant EHEC strains containing a single-copy chromosomal target gene-luxE fusion construct were
transformed using the LuxCDAB expression plasmid pluxCDAB3 (41). For the growth inhibition assay and
NO reporter assay, mutant EHEC strains were transformed using the NO reporter plasmid pRPL3 (10).
Relative light units (RLU) and the number of bacteria were measured by using a Glomax 20/20
luminometer (Promega, Madison, WI) and bacterial plate counts (CFU), respectively.
Reagents and media. NO was generated by an NO donor, namely, diethylenetriamine NONOate
(DETA/NO) (Cayman Chemical Company, Ann Arbor, MI). The half-life of DETA/NO is 20 h at 37°C in 0.1 M
phosphate buffer (pH 7.4). NaNO2 was purchased from Wako (Tokyo, Japan).
Plasmid construction. To construct plasmids expressing Hmp, hmp was amplified by PCR from the
genomic DNA of the EHEC K47 strain using the primer set P1032-P1033 (Table S1). A DNA fragment was
cleaved with NcoI and HindIII and then inserted into these sites in pTrcHis 2C to yield pThmp1 expressing
His-tagged Hmp (Table 2 and Table S2). To construct plasmids expressing Hcp-Hcr, NorR, NsrR, and Fnr,
the hcp, norR, nsrR, and fnr genes were amplified by PCR from the genomic DNA of the EHEC EDL933
strain using the primer sets P1345-P1346, P1339-P1340, P1341-P1342, and P1343-P1344, respectively
(Table S1). Each DNA fragment was cleaved with SacI and SalI and then inserted into these sites in
pBAD33 to yield pBhcp1 expressing Hcp-Hcr, pBnorR2 expressing NorR, pBnsrR2 expressing NsrR, and
pBfnr1 expressing Fnr, respectively (Table 2 and Table S2). To construct a template plasmid for
homologous recombination using a Red/ET recombination system (Gene Bridges GmbH, Heidelberg,
Germany), a DNA fragment containing a streptomycin-resistant (Smr) gene was obtained by PCR from the
plasmid DNA of pAH144 using the primer set P730-P731 (Table S1) and cloned into a DNA fragment that
was amplified by PCR using the primer set P736-P737 (Table S1) from plasmid DNA of ploxCm1 (41) to
yield ploxSm11 (Table 2 and Table S2). To construct template plasmids for hmp mutation, DNA fragments
containing intact and deletion hmp genes were amplified by PCR from the genomic DNA of K47 using
the primer sets P1296-P1297, P1296-P1298, and P1296-P1299 (Tables S1 and S2). These fragments were
cloned by the ligation of a NotI fragment into ploxCm1 to yield the template hmp plasmids phmpC10,
phmp100C2, and phmp305C15 (Table 2 and Table S2). In addition, to construct template plasmids for
hmp point mutation, a PrimeSTAR mutagenesis kit (TaKaRa Bio Inc., Shiga, Japan) was used with the
plasmid DNA of phmpC10 as a template and primer sets P1310-P1311 and P1312-P1313 (Table S1) to
yield the template hmp plasmids phmpCF43A1 and phmpCF390A1, respectively (Table 2 and Table S2).
These plasmids were confirmed using restriction digestion and DNA sequencing.

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Shimizu et al. Infection and Immunity

Strain construction. Mutant strains were derivatives of EHEC O157:H7 K47 and were produced using
a Red/ET recombination system (Table 1 and Table S3). Briefly, for construction of the deletion and
replacement mutants, PCR primers containing 50 bp immediately upstream and downstream, respec-
tively, of the target locus were used to amplify the DNA fragment containing the FLP recombination
target (FRT)-flanked PGK-gb2-neo (kanamycin-resistant [Kanr]) cassette, a loxP-flanked chloramphenicol-
resistant (Cmr) cassette, or a loxP-flanked Smr cassette found in the appropriate plasmids (Table S3).
For construction of reporter strains, PCR primers containing 50 bp immediately upstream and
downstream, respectively, of the target locus were used to amplify the DNA fragment containing the
promoterless RBS-luxE gene and loxP-flanked Cmr cassette in the pLCE19 plasmid (Table S3). The
resulting linear PCR product was electroporated with the appropriate parent strain previously transformed
with plasmid pRed/ET(amp) (Table S3). Recombinants containing appropriate antibiotic-resistant elements in
place of the target locus were selected on appropriate antibiotic plates and confirmed by PCR. Deletions of
antibiotic-resistant elements were accomplished by transformation with plasmid pCreA1 or pFT-A, resulting
in antibiotic-sensitive mutants that were verified by PCR. The plasmids pRed/ET(amp), pCreA1, and pFT-A are
temperature sensitive and were cured by overnight growth at 37°C.
Statistics. An unpaired t test was used to determine significant differences between two treatment
groups. One-way analysis of variance (ANOVA) with a Student-Newman-Keuls multiple-comparison test
was used to analyze significant differences among three or more groups.

SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/IAI
.00334-19.
SUPPLEMENTAL FILE 1, PDF file, 1 MB.
SUPPLEMENTAL FILE 2, PDF file, 0.1 MB.

ACKNOWLEDGMENTS
We thank Kanako Hirano for technical assistance.
This research was supported by JSPS KAKENHI grant number 16K08771 and AMED
grant number JP18jk021007.

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