Inhibition of bacterial DNA replication by zinc mobilization during nitrosative stress

Jeffrey M. Schapiro; Stephen J. Libby; Ferric C. Fang

doi:10.1073/pnas.1033133100

Communicated by Roy Curtiss, Washington University, St. Louis, MO, May 23, 2003 (received for review January 6, 2003)

Abstract

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 depends 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.

Nitric 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 mechanistic 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 macrophages (3) and increases host lethality (5). Although NO can exhibit synergistic bactericidal activity in combination with reactive 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′-TGACCCCGGCTCGCGTATTACCGG-3′ and 5′-CCCCGCGCCAGATTGAGTCGGCTC-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 Southern blot analysis by using a digoxigenin-labeled (DIG High Prime, Roche Molecular Biochemicals) PCR-amplified ruvC 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′-CGCGGCAAGTATGGCGCAGAGATGATCCATCCGGAATATCGCCTGCAGGGCGATCTCAGCACGGGGAGAGCCTGAGCAAA-3′ and 5′-TCCAGCAGGTCTGACTCTTCAATCAGCGTACAGACCCAGTAGGCCTGACGGCCTTCGGTTAAGCCACTGGAGCACCTCAA-3′, creating strain JS1A06, and combined with recBC::Tn10 to create strain JS1A07. The S. enterica ruvC gene was cloned with the primers 5′-CGGTGAGATCTCTGATGAGGTGGCGGCGAC-3′ and 5′-CCCCTCAACGCGAGGCTGAGGGAG-3′ into the pSC101-derived plasmid pSX34 Cm^r (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 glutathione (Sigma) and NaNO₂ (Sigma) in 1 M HCl. The pH was then adjusted to 7.5 with NaOH (Sigma). Spermine/NONOate (SPER/NO) was obtained from Alexis Biochemicals (San Diego). A 500 mM stock solution was prepared with 0.01 M NaOH. Disk diffusion susceptibility assay was performed (6, 9) by adding 15 μl of NO donor compound to a paper disk overlying a lawn of 10⁶ bacteria, and incubating plates overnight at 37°C. β-Galactosidase activity of E. coli NO56 sulA::lacZ was measured after treatment with 0–500 μM SPER/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 μl of cell suspension buffer (10 mM Tris, pH 7.2/20 mM NaCl/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 μM, 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 7.2/50 mM NaCl/0.2% Na deoxycholate/0.5% sarcosyl/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 8.0/1% sarcosyl/1 mg/ml proteinase 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 8.0/50 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/cm and 120^o 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 Documentation System (Bio-Rad).

Mouse Virulence Assay. Six to 8 week-old C57BL/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 μl of 500 mM stock solution) or ciprofloxacin (5 μg) 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 μl of PBS and incubated at 37°C for 30 min with 5 μl 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/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 METAMORPH 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 associated with induction of the SOS regulatory network, a coordinated genetic response to DNA replication arrest (17). SulA expressed during the SOS response reversibly inhibits cell division via interactions with the FtsZ protein (18). Exposure to NO released from SPER/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).

Fig. 1.

Nitrosative stress induces the SOS response and exerts effects on DNA repair-deficient bacteria distinct from hydrogen peroxide or UV irradation. β-Galactosidase activity (A) was measured after treatment of E. coli NO56 sulA::lacZ with increasing concentrations of the NO donor SPER/NO. Susceptibility 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)orthe 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 ≤0.00001%).

Nitrosative Stress Causes DNA Damage via Replication Arrest. Genetic loci involved in the recombinational or degradative repair of arrested DNA replication forks (recA, recBC, recD) or in the secondary damage associated with replication arrest (ruvC, recG) were examined for their effects on susceptibility to nitrosative stress. S. enterica lacking recBC was found to have dramatically 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 peroxide (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 homologous recombinational repair in the recBC mutant strain was considered (20). However, no evidence of a suppressor mutation was observed in either the recBC/ruvC or recBC/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 into the recBC/ruvC mutant strain complemented the ruvC mutation by restoring GSNO susceptibility (data not shown).

Nitrosative Stress Causes Double-Strand DNA Breakage by a RuvC/ RecG-Dependent Process. The formation of large, linear chromosomal fragments produced by double-stranded DNA breakage can be detected by pulsed-field gel electrophoresis (21). Nitrosative 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 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/ruvC mutant in comparison to a recBC/recG or WT strain, which is not enhanced by GSNO. Most notably, the protective phenotypes of recBC and recA/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).

Fig. 2.

Chromosomal fragmentation occurs during nitrosative stress. Bacteria were untreated (-) or incubated with 500 μM 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/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.

Attenuated Virulence of recBC Mutant S. enterica Requires a Functional 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/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.

Fig. 3.

A recG mutation restores virulence to recBC mutant Salmonella. Mortality of C57BL/6, gp91phox^-/-, and iNOS^-/- mice after i.p. inoculation of ≈500 cfu of mutant or WT Salmonella is shown over time. A recBC/recG mutant is fully virulent despite its deficiency in homologous recombination and impaired ability to repair direct DNA damage.

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 simultaneous observation of inhibited and noninhibited cells. Bacterial cells with normal morphology and filamentous cells were readily distinguishable by differential interference contrast microscopy (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 filamentous 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 concentrations of GSNO (6). Zinc mobilization was not found to be inevitably associated with stalled replication or cell filamentation 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. 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 contrast (A) and fluorescence microscopy (B, ×600) after the addition 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).

Discussion

Phagocytic cells can inhibit the growth of ingested microbes by subjecting them to nitrosative stress via the enzymatic production 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 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· 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 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 proteins. Mutations in recA alone have little effect on GSNO susceptibility, because RecBCD exonuclease-mediated degradation 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 exonuclease 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 formation at sites of replication fork collapse (32), whereas the RuvC endonuclease mediates the actual strand breakage (33).

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.)

One specific prediction of this model is the RuvABC-dependent formation of double-stranded DNA breaks in bacteria under conditions 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 integrity to a recBC mutant strain. Enhanced levels of chromosomal fragmentation in untreated recBC mutant bacteria (Fig. 2) presumably reflect the need for replication fork repair, even under routine in vitro conditions (34).

It must be concluded that repair of arrested replication forks is more critical for cell viability during nitrosative stress than homologous recombination per se. Mutations in ruvC or recG restore genomic integrity to recBC mutant bacteria, despite the persistent recombination deficiency and hydrogen peroxide/UV hypersusceptibility of these strains (Fig. 1 B and C). Although nitrogen oxides are capable of direct DNA modification (35–37), our observations indicate that an important mechanism of nitrosative stress-related DNA damage in intact bacterial cells is the inhibition of DNA replication with subsequent strand breakage, mediated by RuvC in a process facilitated by the repair proteins RecG and RuvAB.

In considering the mechanism by which nitrosative stress might inhibit bacterial DNA replication, it should be recalled that nitrogen oxides have been shown to target cysteine thiols and metal centers in diverse biological systems (38–40). Cysteine-containing zinc-finger motifs found within many DNA-binding proteins are highly susceptible to S-nitrosylation (Fig. 6). Mobilization of zinc has been shown to reversibly inhibit the transcription factors LAC9, Sp1, and EGR-1 in eukaryotic cells (41, 42), and the DNA repair enzymes DNA ligase and O⁶-methylguanine DNA methyltransferase in E. coli (43, 44). We therefore propose that zinc metalloproteins involved in DNA replication and the restarting of collapsed replication forks (e.g., PriA, DnaG, DnaJ) might be targeted during nitrosative stress. In particular, mutations in priA and dnaG have been shown to result in SOS induction, cell filamentation (45), and enhanced susceptibility to RuvC-mediated double-stranded DNA breakage (46–48). The invariable association of cell filamentation during nitrosative stress with zinc mobilization (Fig. 4B) provides strong support for this hypothesis.

Fig. 6.

Reversible mobilization of zinc after S-nitrosylation of a zinc-finger domain. C, cysteine; H, histidine; Zn, zinc ion.

The greater attenuation of a S. enterica recBC mutant compared with a recA mutant in mouse virulence studies (23) can be better accounted for by RecBC-dependent replication fork repair than by double-stranded break repair, which requires both RecBC and RecA. Remarkably, a recG mutation can restore full virulence to a recBC mutant Salmonella strain after i.p. inoculation of C57BL/6, iNOS^-/-, or gp91phox^-/- mice (Fig. 3), despite a persistent deficiency in homologous recombination and an impaired ability to repair direct DNA damage. This indicates that DNA damage associated with replication arrest not only is seen under in vitro conditions, but also occurs during host–pathogen interactions in vivo. The virulence of a recG/recBC Salmonella mutant is comprehensible if host defenses are considered to inhibit DNA replication rather than damage DNA directly (Fig. 5). In the absence of RecG, fork reversal during DNA replication arrest is prevented, thereby averting RuvABC-mediated double-stranded DNA breakage and obviating the need for RecBCD rescue. The restoration of attenuated virulence by the addition of a recG mutation to recBC mutant Salmonella is also observed in iNOS^-/- mice, suggesting that host-derived mediators, in addition to NO, are able to target microbial DNA replication during infection. Reactive oxygen species are likely candidates for this role, because they have been shown to inhibit bacterial DNA replication in vitro (49) and mobilize zinc from zinc fingers (50). Microbial DNA replication therefore appears to be a critical final common pathway targeted by multiple host defenses.

In summary, NO is an essential antimicrobial mediator produced by host phagocytic cells. After exposure to nitrogen oxides, bacterial replication appears to be inhibited by zinc release from metalloproteins involved in DNA synthesis. Interactions between nitrogen oxides and thiol-stabilized zinc-containing DNA-binding proteins are involved in both the regulation of eukaryotic gene expression (51) and the inhibition of invading prokaryotic microbes, underscoring the universality of S-nitrosylation as a redox signaling mechanism.

Acknowledgments

We are grateful to D. Mount for strain NO56, S. Sandler for helpful discussions, and A. Vazquez-Torres and J. Stamler for critical review of the manuscript before submission. Support for this work was provided by the National Institutes of Health.

Footnotes

↵§ To whom correspondence should be addressed. E-mail: fcfang{at}washington.edu.
Abbreviations: GSNO, S-nitrosoglutathione; SPER/NO, spermine NONOate.

Received January 6, 2003.

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