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Inhibition of bacterial DNA replication by zincmobilization during nitrosative stressJeffrey 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 ofMicrobiology, 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

hostpathogen interactions. Inhibition of DNA replication during

nitrosative stress is invariably accompanied by zinc mobilization,

implicating DNA-binding zinc metalloproteins as critical targets ofNO-related antimicrobial activity.

N itric oxide (NO) mediates a diverse variety of signalingfunctions in eukaryotic cells via modification of cysteinethiols and transition metal centers (1). NO biosynthesis has beendemonstrated to play important and complex roles in infection(2), including the limitation of microbial proliferation withinhost cells (3). Although multiple potential microbial targets ofnitrosylation have been identified (4), the specific mechanis-tic basis of NO-related antimicrobial activity has not beenestablished.

In a murine Salmonella enterica serovar Typhimurium modelof systemic infection, genetic or biochemical abrogation of NO

production enhances bacterial proliferation within host macro-phages (3) and increases host lethality (5). Although NO canexhibit synergistic bactericidal activity in combination with re-active oxygen species (ROS) in vitro (6), NO by itself appearsprincipally to mediate ROS-independent bacteriostatic actionsin vitro (6) and during murine salmonellosis in vivo (3, 5). Thepresent study examines the consequences of nitrosative stress onSalmonella in vitro in an effort to understand the mechanism by

which host-derived nitrogen ox ides inhibit bacterial replication.

Materials and Methods

Bacterial Strains and Plasmids. S. enterica serovar TyphimuriumATCC 14028s or its isogenic derivatives were used and grownin 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 ef fect onrecD. S. enterica recD::MudCm was isolated from a MudCmlibrary and was confirmed by sequencing of the transposoninsertion site. Escherichia coli NO56 sulA:lacZ was obtainedfrom D. Mount (University of Arizona, Tucson) (10). Amutation in the RuvC resolvase was constructed by the methoddescribed (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 fragmentshowed 83% identity at the nucleotide level to the E. coli ruvCgene. Interruption of the ruvC gene was confirmed by South-ern blot analysis by using a digoxigenin-labeled (DIG HighPrime, Roche Molecular Biochemicals) PCR-amplified ruvC

gene fragment as a probe (data not shown). JS1A03 wascombined with recBC::Tn10 by the method described (11) tocreate strain JS1A09. A mutation in the gene encoding theRecG helicase was constructed by using linear transformationand lambda red-dependent recombination as described (12) byusing primers 5-CGCGGCAAGTATGGCGCAGAGAT-GATCCATCCGGAATATCGCCTGCAGGGCGATCT-CAGCACGGGGAGAGCCTGAGCAAA-3 and 5-TCCAG-CAGGTCTGACTCTTCAATCAGCGTACAGACCCAG-TAGGCCTGACGGCCTTCGGTTAAGCCACTGGAGCA-CCTCAA-3, creating strain JS1A06, and combined withrecBC::Tn10 to create strain JS1A07. The S. enterica ruvC gene was cloned w ith the primers 5-CGGTGAGATCTCTGA-

TGAGGTGGCGGCGAC-3 and 5-CCCCTCAACGCGA-GGCTGAGGGAG-3 into the pSC101-derived plasmidpSX34 Cmr (S. Y. Xu, unpublished observation). The pSX34-ruvC construct was electroporated into JS1A09 to make strainJS1A10. Complementation was assessed by testing candidatestrains for susceptibility to UV irradiation and assessment ofDNA fragmentation by pulsed-field gel electrophoresis (datanot 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 wasthen adjusted to 7.5 with NaOH (Sigma). SpermineNONOate(SPERNO) was obtained f rom 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) byadding 15 l of NO donor compound to a paper disk overlyinga lawn of 106 bacteria, and incubating plates overnight at 37C.-Galactosidase activity of E. coli NO56 sulA::lacZ was mea-sured after treatment with 0500 M SPERNO according tothe 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-folddilutions were plated on individual LB agar plates and incubatedovernight at 37C for enumeration of c olony-forming units (cfu).

Pulsed-Field Gel Electrophoresis. Protocol and buffers wereadapted from the methods of Birren and Lai (15, 16). Bacteria

were grown anaerobically overnight in M9 with 0.2% glucose byusing a BBL GasPak (Becton Dickinson). Cells were diluted to

OD600 0.5 in a 1-ml volume, pelleted, resuspended in 100 lof cell suspension buffer (10 mM Tris, pH 7.220 mM NaCl50mM EDTA), and mixed with an equal volume of molten 2%InCert agarose (FMC) at 40C to create a plug containing intactbacteria. Plugs were distributed into individual tubes containing3 ml of M9 minimal medium with 0.2% glucose. GSNO wasadded to a final concentration of 500 M, and the samples wereincubated in a 37C water bath for 2 h. Untreated plugs wereused as a control. After incubation, plugs were placed in 3 ml oflysis buffer (10 mM Tris, pH 7.250 mM NaCl0.2% Na deoxy-

Abbreviations: GSNO, S-nitrosoglutathione; SPERNO, spermine NONOate.

To whom correspondence should be addressed. E-mail: fcfang@washington.edu.

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by homologous recombination (24) after base excision by DNAglycosylases (25). However, these experimental conditions do

not represent simple nitrosative stress, because ONOO

is apotent oxidizing agent and oxidative DNA alterations such as8-oxoguanine are produced (25). The nitrosative stress wehave induced in Salmonella by using GSNO does not requirethe presence of oxygen (6) and differs fundamentally from theantimicrobial actions of NO gas under aerobic conditions,

which are substantially more pronounced for recA mutant thanfor recBC mutant bacteria (24). In contrast, the toxicity ofGSNO is substantially greater for recBC mutant than for recAmutant Salmonella. The limited release of NO from GSNOand the ability of GSNO to enter cells (6) and facilitatenitrosylation of intracellular targets may account for reducedformation of genotoxic nitrogen oxides that could otherwisemask important actions of NO at replication forks. The criticalRecA-independent role of RecBCD in both GSNO resistanceand Salmonella virulence indicates that nitrosative stress-

related DNA damage can occur by a mechanism distinct fromthe direct modification of DNA by hydrogen peroxide (26, 27),

UV irradiation (28), or N2O3 and ONOO

(24) that is repairedby homologous recombination.The requirement for recBC (but not recA) in resistance to

GSNO is best rationalized by a mechanism of indirect DNAdamage 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 recombinationpathway, the free double-strand end can be processed into aRecA-usable form by RecBCD to permit fork reassembly.

Alternatively, in the degradative pathway, the stalled fork canbe stabilized by the removal of the double-strand end by theRecBCD nuclease in the absence of RecA, thereby displacingthe RuvAB proteins from the arrested fork and preventingsubsequent DNA cleavage by the RuvC endonuclease. Eithera recBC mutation or a combination ofrecA and recD mutationseliminates both the recombination and degradative repair

Fig. 4. Zinc mobilization correlates with cell filamentation

during nitrosative stress. Bacteria from the edge of a zone of

GSNOinhibitionwere observedby 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).

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. RuvCmay resolve the RuvAB-bound Holliday junction to create a double-strandbreak, whichcan be repairedonly 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.)

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