Inhibition of bacterial DNA replication by zinc
mobilization during nitrosative stress
Jeffrey M. Schapiro*, Stephen J. Libby†, and Ferric C. Fang*‡§
Departments of *Laboratory Medicine and ‡Microbiology, University of Washington School of Medicine, Seattle, WA 98195-7242; and †Department of
Microbiology, North Carolina State University, Raleigh, NC 27695-7615
Communicated by Roy Curtiss, Washington University, St. Louis, MO, May 23, 2003 (received for review January 6, 2003)
Phagocytic cells inhibit the growth of intracellular pathogens by
producing nitric oxide (NO). NO causes cell filamentation, induction
of the SOS response, and DNA replication arrest in the Gram-
negative bacterium Salmonella enterica. NO also induces double-
stranded chromosomal breaks in replication-arrested Salmonella
lacking a functional RecBCD exonuclease. This DNA damage de-
pends on actions of additional DNA repair proteins, the RecG
helicase, and RuvC endonuclease. Introduction of a recG mutation
restores both resistance to NO and the ability of an attenuated
recBC mutant Salmonella strain to cause lethal infection in mice,
demonstrating that bacterial DNA replication is inhibited during
host–pathogen interactions. Inhibition of DNA replication during
nitrosative stress is invariably accompanied by zinc mobilization,
implicating DNA-binding zinc metalloproteins as critical targets of
NO-related antimicrobial activity.
N itric oxide (NO) mediates a diverse variety of signaling
functions in eukaryotic cells via modification of cysteine
thiols and transition metal centers (1). NO biosynthesis has been
demonstrated to play important and complex roles in infection
(2), including the limitation of microbial proliferation within
host cells (3). Although multiple potential microbial targets of
nitrosylation have been identified (4), the specific mechanis-
tic basis of NO-related antimicrobial activity has not been
established.
In a murine Salmonella enterica serovar Typhimurium model
of systemic infection, genetic or biochemical abrogation of NO
production enhances bacterial proliferation within host macro-
phages (3) and increases host lethality (5). Although NO can
exhibit synergistic bactericidal activity in combination with re-
active oxygen species (ROS) in vitro (6), NO by itself appears
principally to mediate ROS-independent bacteriostatic actions
in vitro (6) and during murine salmonellosis in vivo (3, 5). The
present study examines the consequences of nitrosative stress on
Salmonella in vitro in an effort to understand the mechanism by
which host-derived nitrogen oxides inhibit bacterial replication.
Materials and Methods
Bacterial Strains and Plasmids. S. enterica serovar Typhimurium
ATCC 14028s or its isogenic derivatives were used and grown
in media as described (7, 8). S. enterica CL1000 (recA) and S.
enterica CL2001 (recBC) are described in ref. 9. CL2001
contains a Tn10 insertion that may also exert a polar effect on
recD. S. enterica recD::MudCm was isolated from a MudCm
library and was confirmed by sequencing of the transposon
insertion site. Escherichia coli NO56 sulA:lacZ was obtained
from D. Mount (University of Arizona, Tucson) (10). A
mutation in the RuvC resolvase was constructed by the method
described (11) by using primers 5⬘-TGACCCCGGCTCGCG-
TATTACCGG-3⬘ and 5⬘-CCCCGCGCCAGATTGAGTCG-
GCTC-3⬘ to create S. enterica ruvC mutant strain JS1A03.
Sequence analysis of the amplified S. enterica ruvC fragment
showed 83% identity at the nucleotide level to the E. coli ruvC
gene. Interruption of the ruvC gene was confirmed by South-
ern blot analysis by using a digoxigenin-labeled (DIG High
Prime, Roche Molecular Biochemicals) PCR-amplified ruvC
8496 – 8501 兩 PNAS 兩 July 8, 2003 兩 vol. 100 兩 no. 14
gene fragment as a probe (data not shown). JS1A03 was
combined with recBC::Tn10 by the method described (11) to
create strain JS1A09. A mutation in the gene encoding the
RecG helicase was constructed by using linear transformation
and lambda red-dependent recombination as described (12) by
using primers 5⬘-CGCGGCA AGTATGGCGCAGAGAT-
GATCCATCCGGA ATATCGCCTGCAGGGCGATCT-
CAGCACGGGGAGAGCCTGAGCAAA-3⬘ and 5⬘-TCCAG-
CAGGTCTGACTCT TCA ATCAGCGTACAGACCCAG-
TAGGCCTGACGGCCTTCGGTTAAGCCACTGGAGCA-
CCTCAA-3⬘, creating strain JS1A06, and combined with
recBC::Tn10 to create strain JS1A07. The S. enterica ruvC gene
was cloned with the primers 5⬘-CGGTGAGATCTCTGA-
TGAGGTGGCGGCGAC-3⬘ and 5⬘-CCCCTCAACGCGA-
GGCTGAGGGAG-3⬘ into the pSC101-derived plasmid
pSX34 Cmr (S. Y. Xu, unpublished observation). The pSX34-
ruvC construct was electroporated into JS1A09 to make strain
JS1A10. Complementation was assessed by testing candidate
strains for susceptibility to UV irradiation and assessment of
DNA fragmentation by pulsed-field gel electrophoresis (data
not shown).
Other Materials and Assays. A 500 mM stock solution of S-
nitrosoglutathione (GSNO) was prepared by dissolving gluta-
thione (Sigma) and NaNO2 (Sigma) in 1 M HCl. The pH was
then adjusted to 7.5 with NaOH (Sigma). Spermine兾NONOate
(SPER兾NO) was obtained from Alexis Biochemicals (San Di-
ego). A 500 mM stock solution was prepared with 0.01 M NaOH.
Disk diffusion susceptibility assay was performed (6, 9) by
adding 15 ␮l of NO donor compound to a paper disk overlying
a lawn of 106 bacteria, and incubating plates overnight at 37°C.
␤-Galactosidase activity of E. coli NO56 sulA::lacZ was mea-
sured after treatment with 0–500 ␮M SPER兾NO according to
the method of Miller (13). For the UV susceptibility assay, cells
were prepared (14) and placed in a UV 1800 Stratalinker
(Stratagene) for exposure to UV irradiation. Successive 10-fold
dilutions were plated on individual LB agar plates and incubated
overnight at 37°C for enumeration of colony-forming units (cfu).
Pulsed-Field Gel Electrophoresis. Protocol and buffers were
adapted from the methods of Birren and Lai (15, 16). Bacteria
were grown anaerobically overnight in M9 with 0.2% glucose by
using a BBL GasPak (Becton Dickinson). Cells were diluted to
OD600 ⫽ 0.5 in a 1-ml volume, pelleted, resuspended in 100 ␮l
of cell suspension buffer (10 mM Tris, pH 7.2兾20 mM NaCl兾50
mM EDTA), and mixed with an equal volume of molten 2%
InCert agarose (FMC) at 40°C to create a plug containing intact
bacteria. Plugs were distributed into individual tubes containing
3 ml of M9 minimal medium with 0.2% glucose. GSNO was
added to a final concentration of 500 ␮M, and the samples were
incubated in a 37°C water bath for 2 h. Untreated plugs were
used as a control. After incubation, plugs were placed in 3 ml of
lysis buffer (10 mM Tris, pH 7.2兾50 mM NaCl兾0.2% Na deoxy-
Abbreviations: GSNO, S-nitrosoglutathione; SPER兾NO, spermine NONOate.
§To whom correspondence should be addressed. E-mail: fcfang@washington.edu.
www.pnas.org兾cgi兾doi兾10.1073兾pnas.1033133100
cholate兾0.5% sarcosyl兾1 mg/ml lysozyme) and incubated at 37°C
for 2 h. Plugs were subsequently incubated in 2 ml of proteinase
K buffer (500 mM EDTA, pH 8.0兾1% sarcosyl兾1 mg/ml pro-
teinase K) and maintained at 50°C for 48 h. Plugs were washed
three times for 1 h each in 2 ml of 1⫻ wash buffer (20 mM Tris,
pH 8.0兾50 mM EDTA, pH 8.0) and once for 1 h in 0.1⫻ wash
buffer. A 2-mm slice of each plug was electrophoresed in 0.5⫻
TBE at 14°C on a CHEF DRIII apparatus (Bio-Rad) for 20 h at
a field strength of 6 V兾cm and 120o included angle in 1.1%
SeaKem LE agarose (FMC). Initial switch time was 20 sec and
final switch time was 60 sec. After staining with ethidium
bromide, the gel was visualized, and high molecular weight linear
chromosomal fragments were quantified on a Gel Documenta-
tion System (Bio-Rad).

Mouse Virulence Assay. Six to 8 week-old C57BL兾6 (The Jackson

Laboratory), and congenic iNOS⫺/⫺ or gp91phox⫺/⫺ mice were
injected i.p. with bacterial inocula of 4.0–6.9 ⫻ 102 cfu, with
inoculum size verified by quantitative plating. Three to five mice
were included in each experimental group. The mice were
monitored daily for signs of illness or death.

Detection of Zinc Mobilization. S. enterica 14028s bacteria were

obtained from the edge of a zone of inhibition surrounding
GSNO (15 ␮l of 500 mM stock solution) or ciprofloxacin (5 ␮g)
from a disk diffusion assay plate after overnight incubation. The
GSNO plate was incubated for another 8 h and additional cells
were harvested. Cells were suspended in 200 ␮l of PBS and
incubated at 37°C for 30 min with 5 ␮l of 5 mM Zinquin
[ethyl(2-methyl-8-p-toluenesulfonamido-6-quinolyloxy)acetate;
Alexis Biochemicals] then applied to an eight-well microscope
slide. The characteristic blue fluorescence of Zinquin acid兾Zn2⫹
complexes was detected by DIC and fluorescence microscopy on
a Nikon TE-200 microscope by using a CoolScan HQ camera
(Nikon). Localization and overlay were achieved with META-
MORPH software (Universal Imaging, Media, PA).

Results
Nitrosative Stress Induces the SOS Response. Previous work has
shown that the nitrosothiol GSNO causes filamentation of
bacterial cells (6), suggesting that nitrosative stress interferes
with cell division. Bacterial filamentation is frequently associ-
ated with induction of the SOS regulatory network, a coordi-
nated genetic response to DNA replication arrest (17). SulA
expressed during the SOS response reversibly inhibits cell divi-
sion via interactions with the FtsZ protein (18). Exposure to NO
released from SPER兾NO induces expression of a sulA::lacZ
fusion in a dose-dependent fashion (Fig. 1A), implicating the
SOS regulatory response in the arrest of bacterial cell division by
NO. Analogous demonstrations of SOS induction have been
obtained with GSNO and with other nitrogen oxides (19).
Nitrosative Stress Causes DNA Damage via Replication Arrest. Ge-
netic loci involved in the recombinational or degradative repair
of arrested DNA replication forks (recA, recBC, recD) or in the
Fig. 1. Nitrosative stress induces the SOS response and exerts effects on DNA
repair-deficient bacteria distinct from hydrogen peroxide or UV irradation.
␤-Galactosidase activity (A) was measured after treatment of E. coli NO56
sulA::lacZ with increasing concentrations of the NO donor SPER兾NO. Suscep-
tibility of WT and mutant Salmonella strains to oxidative and nitrosative stress
is shown in B. The zone of inhibition is a measure of susceptibility to hydrogen
peroxide (H2O2) or the S-nitrosothiol GSNO (6). Susceptibility to killing by UV
irradiation (C) parallels susceptibility to H2O2 but not to GSNO. Asterisk
indicates minimum level of detection (survival ⱕ0.00001%).
considered (20). However, no evidence of a suppressor mutation
was observed in either the recBC兾ruvC or recBC兾recG mutant
strains, which retain enhanced susceptibility to UV irradiation
(Fig. 1C) despite their resistance to GSNO. Introduction of a
functional copy of ruvC on the low-copy-number plasmid pSX34
MICROBIOLOGY

into the recBC兾ruvC mutant strain complemented the ruvC

secondary damage associated with replication arrest (ruvC,
recG) were examined for their effects on susceptibility to nitro-
sative stress. S. enterica lacking recBC was found to have dra-
matically enhanced susceptibility to GSNO, whereas S. enterica
lacking recA does not (Fig. 1B) unless a recD mutation is also
present. A mutation in ruvC or recG was found to restore WT
levels of GSNO susceptibility to a recBC mutant strain. The
effects of these mutations on susceptibility to GSNO contrast
markedly with their effects on susceptibility to hydrogen perox-
ide (Fig. 1B), which is enhanced by mutations in either recA or
recBC and not ameliorated by abrogation of ruvC or recG. The
possibility of an occult suppressor mutation restoring homolo-
gous recombinational repair in the recBC mutant strain was
mutation by restoring GSNO susceptibility (data not shown).
Nitrosative Stress Causes Double-Strand DNA Breakage by a RuvC兾
RecG-Dependent Process. The formation of large, linear chromo-
somal fragments produced by double-stranded DNA breakage
can be detected by pulsed-field gel electrophoresis (21). Nitro-
sative stress was found to induce Salmonella chromosomal
fragmentation in a process potentiated by the absence of a
functional recBC locus or by mutations in both recA and recD
(Fig. 2), but not by a mutation in recA alone. The formation of
double-strand DNA breaks was ameliorated in a recBC mutant
by the addition of mutations in ruvC or recG, implicating these

Schapiro et al. PNAS 兩 July 8, 2003 兩 vol. 100 兩 no. 14 兩 8497

Fig. 2. Chromosomal fragmentation occurs during nitrosative stress. Bacteria were untreated (⫺) or incubated with 500 ␮M GSNO (⫹) for 1 h before preparation
for pulsed-field gel electrophoresis. High-molecular-weight linear chromosomal fragments resulting from double-strand breaks can be observed in the
compression zone of the gel, most evident in recBC or recA兾recD mutant bacteria treated with GSNO. Chromosomal fragmentation in GSNO-treated recBC
mutant bacteria is restored toward untreated levels by the addition of a recG or ruvC mutation. Numbers beneath each band indicate relative signal intensity
normalized to the signal in the sample containing untreated WT bacteria.

loci in the mechanism of DNA damage. Although both ruvC and
recG mutations reduce double-stranded DNA breaks in a recBC
background, there is a modest but reproducible increase in the
level of double-stranded DNA breakage in a recBC兾ruvC mutant
in comparison to a recBC兾recG or WT strain, which is not
enhanced by GSNO. Most notably, the protective phenotypes of
recBC and recA兾recD with regard to chromosomal fragmentation
during nitrosative stress, along with the detrimental effects of
ruvC and recG, parallel their phenotypes in direct assays of
susceptibility to nitrosative stress (Fig. 1B).
duction of NO. The present study suggests a mechanism to
account for the antimicrobial actions of nitrogen oxides: the
mobilization of zinc from metalloproteins, resulting in the
inhibition of DNA replication. Earlier work has shown that
aerobically dissolved NO gas can damage DNA through the
actions of NO congeners such as N2O3 and ONOO⫺ (24, 25).
This direct DNA damage by nitrogen oxides can be repaired

Attenuated Virulence of recBC Mutant S. enterica Requires a Func-

tional recG Locus. S. enterica carrying a recBC mutation is highly
attenuated for virulence in mice (22, 23). This attenuation has
previously been attributed to the role of RecBC in double-
stranded break repair (23). However, an alternative explanation
might be the role of RecBC in preventing double-stranded
breaks after replication fork arrest. We therefore determined
whether recBC is essential for S. enterica virulence in the absence
of recG. After i.p. inoculation of C57BL兾6, iNOS⫺/⫺, and
gp91phox⫺/⫺ mice (Fig. 3), a recG mutation was found to restore
full virulence to a recBC mutant Salmonella strain, indicating
that recBC is required for Salmonella virulence only when recG
is present.

Nitrogen Oxides Mobilize Zinc from DNA-Binding Proteins. S. enterica

bacteria were obtained from the edge of a zone of inhibition
surrounding a disk impregnated with GSNO to allow the simul-
taneous observation of inhibited and noninhibited cells. Bacte-
rial cells with normal morphology and filamentous cells were
readily distinguishable by differential interference contrast mi-
croscopy (Fig. 4A). Addition of the Zn2⫹-binding fluorophore
Zinquin revealed that inhibition of bacterial replication during
nitrosative stress is invariably associated with zinc mobilization
(Fig. 4B), with free zinc found almost exclusively within fila-
mentous cells. Plates were allowed to incubate beyond several
half-lives of GSNO. Subsequent examination showed resolution
of filamentation and the disappearance of free zinc (Fig. 4C).
This observation is unlikely to result from death of filamenting
bacteria, because Salmonella is not killed, even at high concen-
trations of GSNO (6). Zinc mobilization was not found to be
inevitably associated with stalled replication or cell filamenta-
tion induced by other causes. Ciprofloxacin, which inhibits DNA
replication, produces filamentous cells that do not contain free
zinc detectable by Zinquin (Fig. 4 D and E). Zinquin-dependent
fluorescence was never observed in untreated bacteria (data not
shown). Fig. 3. A recG mutation restores virulence to recBC mutant Salmonella.
Mortality of C57BL兾6, gp91phox⫺/⫺, and iNOS⫺/⫺ mice after i.p. inoculation of
Discussion ⬇500 cfu of mutant or WT Salmonella is shown over time. A recBC兾recG
Phagocytic cells can inhibit the growth of ingested microbes by mutant is fully virulent despite its deficiency in homologous recombination
subjecting them to nitrosative stress via the enzymatic pro- and impaired ability to repair direct DNA damage.

8498 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.1033133100 Schapiro et al.

 Fig. 4. Zinc mobilization correlates with cell filamentation
during nitrosative stress. Bacteria from the edge of a zone of
GSNO inhibition were observed by differential interference con-
trast (A) and fluorescence microscopy (B, ⫻600) after the addi-
tion of the Zn2⫹-binding fluorophore Zinquin. GSNO-induced
cell filamentation is invariably associated with Zinquin-
dependent fluorescence. After an additional 8 h of incubation
(C), GSNO-treated cells no longer filament or show Zinquin-
dependent fluorescence. Ciprofloxacin-treated filamenting
bacteria (D) do not exhibit zinc mobilization when stained with
Zinquin (E).

by homologous recombination (24) after base excision by DNA
glycosylases (25). However, these experimental conditions do
not represent simple nitrosative stress, because ONOO⫺ is a
potent oxidizing agent and oxidative DNA alterations such as
8-oxoguanine are produced (25). The nitrosative stress we
have induced in Salmonella by using GSNO does not require
the presence of oxygen (6) and differs fundamentally from the
antimicrobial actions of NO gas under aerobic conditions,
which are substantially more pronounced for recA mutant than
for recBC mutant bacteria (24). In contrast, the toxicity of
GSNO is substantially greater for recBC mutant than for recA
mutant Salmonella. The limited release of NO䡠 from GSNO
and the ability of GSNO to enter cells (6) and facilitate
nitrosylation of intracellular targets may account for reduced
formation of genotoxic nitrogen oxides that could otherwise
mask important actions of NO at replication forks. The critical
RecA-independent role of RecBCD in both GSNO resistance
and Salmonella virulence indicates that nitrosative stress-
related DNA damage can occur by a mechanism distinct from
the direct modification of DNA by hydrogen peroxide (26, 27),
UV irradiation (28), or N2O3 and ONOO⫺ (24) that is repaired
by homologous recombination.
The requirement for recBC (but not recA) in resistance to
GSNO is best rationalized by a mechanism of indirect DNA
damage resulting from arrest of DNA replication (Fig. 5) (29,
30), which can be repaired by one of two possible pathways (21)
after RecG-mediated fork regression. In the recombination
pathway, the free double-strand end can be processed into a
RecA-usable form by RecBCD to permit fork reassembly.
Alternatively, in the degradative pathway, the stalled fork can
be stabilized by the removal of the double-strand end by the
RecBCD nuclease in the absence of RecA, thereby displacing
the RuvAB proteins from the arrested fork and preventing
subsequent DNA cleavage by the RuvC endonuclease. Either
a recBC mutation or a combination of recA and recD mutations
eliminates both the recombination and degradative repair

MICROBIOLOGY

Fig. 5. Model of DNA damage caused by NO-related replication arrest. Black lines indicate template strands and colored lines indicate newly synthesized
strands. NO-induced DNA replication arrest results in RecG-dependent fork reversal with subsequent Holliday junction formation. RuvAB can bind this
Holliday junction formed from the nascent strands at the arrested fork. The arrested fork can be rescued by recombination (using RecABCD or RecABCJ)
or by RecBCD-mediated degradation of the free double-strand end. RecBCD degradation continues to the replication fork, displacing RuvAB and allowing
replication to restart. RuvC may resolve the RuvAB-bound Holliday junction to create a double-strand break, which can be repaired only by recombination.
If both replication forks are inhibited, generation of double-stranded breaks by RuvC may lead to lethal chromosomal fragmentation. (Adapted from
refs. 28 and 29.)

Schapiro et al. PNAS 兩 July 8, 2003 兩 vol. 100 兩 no. 14 兩 8499

pathways (29), leaving the chromosome susceptible to double-
stranded breaks mediated by the sequential actions of RecG,
RuvAB, and RuvC, normally regarded as DNA repair pro-
teins. Mutations in recA alone have little effect on GSNO
susceptibility, because RecBCD exonuclease-mediated degra-
dation of the partially reversed fork can still prevent formation
of double-stranded DNA breaks. Similarly, mutations in recD
are tolerated during nitrosative stress because RecA and
RecBC can mediate homologous recombination with exonu-
clease activity provided by RecJ (31). However, a recBC
mutation or combination of recA and recD mutations heightens
susceptibility to nitrosative stress by simultaneously abrogating
both the recombination and degradative pathways. Nitrosative
stress-related DNA damage enhanced in the absence of recBC
can be prevented by mutations in either recG or ruvC because
the RecG helicase is responsible for Holliday junction forma-
tion at sites of replication fork collapse (32), whereas the RuvC
endonuclease mediates the actual strand breakage (33).
One specific prediction of this model is the RuvABC-dependent
formation of double-stranded DNA breaks in bacteria under con-
ditions of nitrosative stress, as observed in other instances of
replication fork arrest (21). We have been able to demonstrate that
nitrosative stress induces chromosomal fragmentation in a process
potentiated by the absence of a functional recBC locus or by a
combination of mutations in both recA and recD (Fig. 2). Moreover,
as predicted by the model in Fig. 5, a recA mutation alone has no
effect on double-stranded DNA breakage by GSNO, whereas
abrogation of RecG or RuvC activity restores chromosomal integ- Fig. 6. Reversible mobilization of zinc after S-nitrosylation of a zinc-finger
rity to a recBC mutant strain. Enhanced levels of chromosomal domain. C, cysteine; H, histidine; Zn, zinc ion.
fragmentation in untreated recBC mutant bacteria (Fig. 2) presum-
ably reflect the need for replication fork repair, even under routine
RecBC and RecA. Remarkably, a recG mutation can restore full
in vitro conditions (34).
virulence to a recBC mutant Salmonella strain after i.p. inocu-
It must be concluded that repair of arrested replication forks
lation of C57BL兾6, iNOS⫺/⫺, or gp91phox⫺/⫺ mice (Fig. 3),
is more critical for cell viability during nitrosative stress than
despite a persistent deficiency in homologous recombination and
homologous recombination per se. Mutations in ruvC or recG
restore genomic integrity to recBC mutant bacteria, despite the an impaired ability to repair direct DNA damage. This indicates
persistent recombination deficiency and hydrogen peroxide兾UV that DNA damage associated with replication arrest not only is
hypersusceptibility of these strains (Fig. 1 B and C). Although seen under in vitro conditions, but also occurs during host–
nitrogen oxides are capable of direct DNA modification (35–37), pathogen interactions in vivo. The virulence of a recG兾recBC
our observations indicate that an important mechanism of Salmonella mutant is comprehensible if host defenses are con-
nitrosative stress-related DNA damage in intact bacterial cells is sidered to inhibit DNA replication rather than damage DNA
the inhibition of DNA replication with subsequent strand break- directly (Fig. 5). In the absence of RecG, fork reversal during
age, mediated by RuvC in a process facilitated by the repair DNA replication arrest is prevented, thereby averting RuvABC-
proteins RecG and RuvAB. mediated double-stranded DNA breakage and obviating the
In considering the mechanism by which nitrosative stress need for RecBCD rescue. The restoration of attenuated viru-
might inhibit bacterial DNA replication, it should be recalled lence by the addition of a recG mutation to recBC mutant
that nitrogen oxides have been shown to target cysteine thiols Salmonella is also observed in iNOS⫺/⫺ mice, suggesting that
and metal centers in diverse biological systems (38 – 40). host-derived mediators, in addition to NO, are able to target
Cysteine-containing zinc-finger motifs found within many microbial DNA replication during infection. Reactive oxygen
DNA-binding proteins are highly susceptible to S-nitrosylation species are likely candidates for this role, because they have been
(Fig. 6). Mobilization of zinc has been shown to reversibly shown to inhibit bacterial DNA replication in vitro (49) and
inhibit the transcription factors LAC9, Sp1, and EGR-1 in mobilize zinc from zinc fingers (50). Microbial DNA replication
eukaryotic cells (41, 42), and the DNA repair enzymes DNA therefore appears to be a critical final common pathway targeted
ligase and O6-methylguanine DNA methyltransferase in E. coli by multiple host defenses.
(43, 44). We therefore propose that zinc metalloproteins In summary, NO is an essential antimicrobial mediator pro-
involved in DNA replication and the restarting of collapsed duced by host phagocytic cells. After exposure to nitrogen
replication forks (e.g., PriA, DnaG, DnaJ) might be targeted oxides, bacterial replication appears to be inhibited by zinc
during nitrosative stress. In particular, mutations in priA and release from metalloproteins involved in DNA synthesis. Inter-
dnaG have been shown to result in SOS induction, cell actions between nitrogen oxides and thiol-stabilized zinc-
filamentation (45), and enhanced susceptibility to RuvC- containing DNA-binding proteins are involved in both the
mediated double-stranded DNA breakage (46 – 48). The in- regulation of eukaryotic gene expression (51) and the inhibition
variable association of cell filamentation during nitrosative of invading prokaryotic microbes, underscoring the universality
stress with zinc mobilization (Fig. 4B) provides strong support of S-nitrosylation as a redox signaling mechanism.
for this hypothesis.
The greater attenuation of a S. enterica recBC mutant com- We are grateful to D. Mount for strain NO56, S. Sandler for helpful
pared with a recA mutant in mouse virulence studies (23) can be discussions, and A. Vazquez-Torres and J. Stamler for critical review of
better accounted for by RecBC-dependent replication fork the manuscript before submission. Support for this work was provided by
repair than by double-stranded break repair, which requires both the National Institutes of Health.

8500 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.1033133100 Schapiro et al.

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