Role of the RecBCD recombination pathway in Salmonella virulence

JOURNAL OF BACTERIOLOGY, 0021-9193/02/$04.00⫹0 DOI: 10.1128/JB.184.2.592–595.2002 Jan. 2002, p. 592–595 Vol. 184, No. 2 Copy igh © 2002, Ame ican Socie y o Mic obiology. All Righ s Rese ed. Role o he RecBCD Recombina ion Pa hway in Salmonella Vi ulence Da id A. Cano, 1 M. G aciela Puccia elli, 2 F ancisco Ga cı´a-del Po illo, 2 and Josep Casadesu´s 1 * Depa amen o de Gene´ ica, Uni e sidad de Se illa, Se ille 41080, 1 and Depa amen o de Bio ecnologı´a Mic obiana, Cen o Nacional de Bio ecnologı´a, C.S.I.C., Campus de Can oblanco, Mad id 28049, 2 Spain Recei ed 10 Sep embe 2001/Accep ed 24 Oc obe 2001 Mu an s o Salmonella en e ica lacking he RecBC unc ion a e a i ulen in mice and unable o g ow inside mac ophages (N. A. Buchmeie , C. J. Lipps, M. Y. H. So, and F. He on, Mol. Mic obiol. 7:933–936, 1993). The i ulence- ela ed de ec s o RecBC ⴚ mu an s a e no supp essed by sbcB and sbcCD mu a ions, indica ing ha ac i a ion o he RecF ecombina ion pa hway canno eplace he i ulence- ela ed unc ion(s) o RecBCD. Func ions o he RecF pa hway such as RecJ and RecF a e no equi ed o i ulence. Since he RecBCD pa hway, bu no he RecF pa hway, is known o pa icipa e in he epai o double-s and b eaks p oduced du ing DNA eplica ion, we p opose ha sys emic in ec ion by S. en e ica may equi e RecBCD-media ed ecombina ional epai o p ime DNA eplica ion inside phagocy es. Mu an s lacking bo h RecD and RecJ a e also a enua ed in mice and a e unable o p oli e a e in mac ophages, sugges ing ha exonucleases V and IX p o ide al e na i e unc ions o RecBCD-media ed ecombina ional epai du ing Salmonella in ec ion. Salmonella en e ica is a g am-nega i e bac e ial pa hogen ha causes gas oin es inal diso de s and sys emic in ec ions in humans and li es ock animals (29). A hallma k o Salmonella pa hogenesis is he capaci y o he bac e ium o su i e and p oli e a e wi hin phagocy ic cells (29). In i o and in i o s udies ha e p o ided e idence ha bac e ial g ow h inside mac ophages and neu ophils is equi ed o sys emic in ec ion (3, 8, 18, 25, 31). Phagocy ic cells syn hesize DNA-damaging agen s such as ni ic oxide and oxygen adicals (37) as mam- malian de ense mechanisms agains pa hogens; hence, Salmo- nella mus ace he a ack o compounds ha challenge ge- nome in eg i y (21, 33, 37). Ac i e unc ions and mechanisms ha p o ec Salmonella om oxida i e s ess inside phagocy es ha e been desc ibed ecen ly (2, 7, 38). In addi ion, almos a decade ago Buchmeie e al. showed ha mu an s o S. en e ica lacking RecA o RecBC unc ions a e a i ulen in he mu ine yphoid model and highly sensi i e o oxida i e compounds syn hesized by mac ophages, sugges ing ha ecombina ion is equi ed o he epai o oxida i e DNA damage (4). This iew is suppo ed by he ecen epo ha Esche ichia coli uses ecombina ional epai o su i al o exposu e o ni ic oxide (35). Like E. coli and o he bac e ial species, S. en e ica can pe - o m ecombina ion be ween homologous DNA molecules by means o wo main p ocesses o pa hways, RecBCD and RecF (20). Each pa hway has dis inc DNA subs a e p e e ences and equi es a speci ic se o ecombina ion unc ions (20). In his s udy, we ha e examined he abili y o S. en e ica ecom- bina ion mu an s o cause sys emic in ec ion in mice and o p oli e a e inside mac ophages. The s ains used lack ecom- bina ion unc ions o he RecBCD pa hway and/o he RecF pa hway. Ou da a indica e ha he RecBCD enzyme is he key unc ion equi ed o bo h sys emic in ec ion and g ow h inside mac ophages. Supp esso mu a ions ha es o e ecombina- ion p o iciency in RecBC ⫺ mu an s by ac i a ing he RecF pa hway do no es o e i ulence, indica ing ha a p ocess di e en om s anda d ecombina ion is a ec ed. Because he RecF pa hway canno unc ionally eplace RecBCD-media ed ecombina ion o he epai o double-s and DNA b eaks (15, 36), he inabili y o RecBC ⫺ SbcB ⫺ and RecBC ⫺ SbcB ⫺ SbcCD ⫺ mu an s o S. en e ica o g ow inside mac ophages may esul om a es ed ch omosome eplica ion upon DNA damage. We also show ha he unc ions o he RecF pa hway a e no equi ed o i ulence; howe e , mu an s lacking bo h RecD and RecJ a e a i ulen , sugges ing ha exonuclease IX (RecJ) can subs i u e o he exonuclease ac i i y o RecBCD. Tes s o i ulence o S. en e ica se o a Typhimu ium e- combina ion mu an s in BALB/c mice. To iden i y ecombina- ion unc ions equi ed o i ulence in he mu ine yphoid model, BALB/c mice we e challenged wi h ecombina ion-de- icien s ains o S. en e ica se o a Typhimu ium (Table 1). In pa allel, in ec ions we e pe o med wi h he i ulen s ain SV1445, a His ⫹ de i a i e o he mouse i ulen s ain SL1344 (12). The inse ion zeb-6312::Tn10dTc ca ied by s ains SV4189 and SV4190 is a Tn10dTc inse ion linked o he sbcB locus and has no e ec on i ulence (da a no shown). The 50% le hal doses (LD 50 ) shown in Table 2 suppo he ollowing conclusions. (i) Among he single mu an s, he mos a enu- a ed is he RecBC ⫺ mu an , which has an o al LD 50 mo e han 10 4 - old highe han ha o he pa en al s ain. Ac ually, he RecBC ⫺ mu an was unable o cause he dea h o any animal, e en a he highe doses used (7.2 ⫻10 9 CFU). We also obse ed ha s ain SV4191 ( ecA1) was able o kill mice a doses 10 o 100 imes lowe han hose o SV4188 ( ecB497::MudJ) (da a no shown). The di e ence obse ed be ween he RecA ⫺ and RecBC ⫺ mu an s may e lec he pleio opy o ecBC mu a ions; i.e., hey impai g ow h in s an- da d cul u e media and cause a ious kinds o DNA epai de ec s (1, 16, 19, 22). RecF ⫺ , RecJ ⫺ , and RecF ⫺ RecJ ⫺ s ains we e all i ulen , sugges ing ha , aside om he RecA unc ion, unc ions o he RecF pa hway o ecombina ion a e no equi ed o sys emic in ec ion. LD 50 ials by in ape i o- neal inocula ion con i med ha RecA ⫺ and RecBC ⫺ s ains * Co esponding au ho . Mailing add ess: Depa amen o de Ge- ne´ ica, Facul ad de Biologı´a, Uni e sidad de Se illa, Apa ado 1095, Se illa 41080, Spain. Phone: 34 95 455 7105. Fax: 34 95 45 7104. E-mail: [email p o ec ed]. 592 on July 24, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om a e he only a i ulen s ains among he single mu an s es ed (da a no shown). (ii) RecA ⫺ RecF ⫺ and RecA ⫺ RecJ ⫺ mu an s we e a en- ua ed a le els simila o ha o a RecA ⫺ s ain, con i ming ha RecF and RecJ unc ions a e no equi ed o i ulence, ega dless o he p esence o RecA. (iii) A RecBC ⫺ RecF ⫺ mu an showed a enua ion a a le el simila o ha o a RecBC ⫺ s ain, he eby sugges ing ha he only ecombina ion pa hway equi ed o sys emic in ec ion is ha o RecBCD. A co olla y is ha he RecF pa hway canno p o ide he ecombina ion unc ions equi ed o i ulence, no e en when he RecBCD pa hway is absen . LD 50 ials wi h RecBC ⫺ RecJ ⫺ mu an s we e no easible because o hei low iabili y (11). (i ) Unexpec edly, a RecD ⫺ RecJ ⫺ mu an u ned ou o be a i ulen . This esul sugges s ha ei he exonuclease V o IX is equi ed o sys emic in ec ion and hence ha RecJ can pa icipa e in RecBCD-media ed ecombina ional epai in he absence o he RecD subuni . P e ious obse a ions ha e sugges ed ha RecD and RecJ p o ide al e na i e unc ions o ecombina ional epai o DNA damage in bo h Salmonella (11, 23, 27) and E. coli (39). Func ion edundancy may hus explain why RecD ⫺ and RecJ ⫺ single mu an s a e i ulen (Table 2; see also e e ence 40). E ec o supp esso s o ecB and ecC mu a ions on he i ulence o S. en e ica.Mu a ions ha supp ess he ecombi- na ion de ec o RecBC ⫺ mu an s wi hou es o ing he ac i - i ies o he RecB and RecBC enzymes ha e been cha ac e ized o bo h E. coli and Salmonella (1, 16, 19). In E. coli,sbc mu a ions a e known o ac i a e he RecF and RecE pa hways o ecombina ion (19, 20). The p ophage-associa ed ecE gene (13) does no exis in Salmonella. Mu a ions in he sbcB locus o S. en e ica supp ess he ecombina ion de iciency and he UV sensi i i y o RecBC ⫺ mu an s by ac i a ing he RecF pa hway (1). I combined wi h mu a ions in ano he locus, sbcCD, supp ession o mi omycin C sensi i i y is also obse ed (1). RecBC ⫺ SbcB ⫺ SbcCD ⫺ mu an s also egain he abili y o g ow no mally in s anda d media (1). Wi h hese ac s in mind, we examined he abili y o sbc mu a ions o supp ess he i - ulence de ec o RecBC ⫺ mu an s o S. en e ica. Bo h RecBC ⫺ SbcB ⫺ and RecBC ⫺ SbcB ⫺ SbcCD ⫺ s ains emained a i ulen by he o al and in ape i onal ou es (Table 3). The a i ulence o he RecBC ⫺ SbcB ⫺ SbcCD ⫺ mu an is specially signi ican , because hese s ains a e ecombina ion p o icien in ansduc- ional es s, a e esis an o bo h UV and mi omycin C, and do no exhibi g ow h de ec s (da a no shown; see also e e ence 1). The inabili y o sbc supp esso s o es o e i ulence in RecBC ⫺ mu an s con i ms he idea ha he ole o RecBC in Salmonella i ulence canno be pe o med by unc ions o he RecF pa hway. The hypo he ical na u e o his ole is discussed below. Abili y o ecombina ion mu an s o S. en e ica o p oli e a e in mac ophages. G ow h o Salmonella inside phagocy es is a s ic equi emen o sys emic in ec ion (8). Fu he mo e, a co ela ion be ween he a i ulence o S. en e ica RecA ⫺ and RecBC ⫺ mu an s and hei impai ed abili y o epai DNA was es ablished in he seminal s udy o Buchmeie e al.: RecA ⫺ and RecBC ⫺ mu an s can g ow wi hin mac ophages ha do no syn hesize eac i e oxygen compounds (4). As expec ed, we also ound a s aigh co ela ion be ween he i ulence o S. en e ica ecombina ion mu an s and hei abili y o p oli e a e TABLE 1. S ain lis a S ain Geno ype SV1445.................Wild ype SV4188................. ecB497::MudJ SV4189................. ecB497::MudJ sbcB21 zeb-6312::Tn10dTc SV4190................. ecB497::MudJ sbcB21 sbcCD51 zeb-6312::Tn10dTc SV4191................. ecA1 SV4212................. ecD543::Tn10dCm SV4213................. ecF522::Tn5 SV4192................. ecJ504::MudJ SV4363................. ecD543::Tn10dCm ecF522::Tn5 SV4364................. ecD543::Tn10dCm ecJ504::MudJ SV4496................. ecA1 ecF522::Tn5 SV4497................. ecA1 ecJ504::MudJ SV4498................. ecB503::Tn10 ecF522::Tn5 SV4499................. ecF522::Tn5 ecJ504::MudCm a All he ecombina ion mu a ions used we e null and had been isola ed in s ain LT2. The o igin o each mu an allele is as ollows: ecA1 and ecF522::Tn5, J. R. Ro h, Depa men o Biology, Uni e si y o U ah, Sal Lake Ci y, U ah; sbcB21, L. Bossi, Cen e de Ge´ne´ ique Mole´culai e, Cen e Na ional de la Reche che Scien i ique, Gi -su -Y e e, F ance; ecB497::MudJ, e e ence 22; ecD543::Tn10dCm, e e ence 27; ecJ504::MudJ and ecJ504::MudCm, e - e ence 23; sbcCD51, labo a o y collec ion. T ans e o he mu a ions o he mouse- i ulen s ain SV1445 was pe o med by ansduc ional c osses wi h P22 HT (34), ollowed by lysogen disposal on g een pla es (5). The ecombina ion- de icien pheno ypes o he newly cons uc ed s ains we e checked using es s desc ibed elsewhe e (11, 22, 23, 26, 27). TABLE 2. Vi ulence o S. en e ica ecombina ion mu an s in BALB/c mice S ain Rele an geno ype LD 50 (o al) a SV1445 Wild ype 1.0 ⫻10 5 SV4191 ecA1 ⬍3.0 ⫻10 8 SV4188 ecB497::MudJ ⬎7.2 ⫻10 9 SV4212 ecD543::Tn10dCm ⬍7.6 ⫻10 6 SV4213 ecF522::Tn5⬍7.4 ⫻10 6 SV4192 ecJ504::MudJ ⬍2.0 ⫻10 6 SV4496 ecA1 ecF522::Tn5⬍6.5 ⫻10 8 SV4497 ecA1 ecJ504::MudJ ⬍3.5 ⫻10 8 SV4498 ecB503::Tn10 ecF522::Tn5⬎5.0 ⫻10 9 SV4499 ecF522::Tn5 ecJ504::MudCm ⬍8.7 ⫻10 5 SV4363 ecD543::Tn10dCm ecF522::Tn5⬍6.9 ⫻10 6 SV4364 ecD543::Tn10dCm ecJ504::MudJ ⬎2.6 ⫻10 8 a Bac e ial suspensions we e p epa ed as desc ibed elsewhe e (9). The acidic pH o he s omach was bu e ed by suspending bac e ia in a 2.5% bica bona e– 0.2% lac ose solu ion p io o adminis a ion. Su i al o bac e ium-in ec ed mice was eco ded o a minimum o 4 weeks. The LD 50 was calcula ed by he me hod o Reed and Muench (30). TABLE 3. E ec o sbc supp esso mu a ions on Salmonella i ulence in BALB/c mice S ain Rele an geno ype LD 50 (o al) a LD 50 (in ape i oneal) a SV1445 Wild ype 1.0 ⫻10 5 ⬍50 SV4188 ecB497::MudJ ⬎7.2 ⫻10 9 ⬎8.0 ⫻10 3 SV4189 ecB497::MudJ sbcB21 ⬎1.5 ⫻10 8 NT b SV4190 ecB497::MudJ sbcB21 sbcCD51 ⬎1.4 ⫻10 9 ⬎2.9 ⫻10 3 a See oo no e ao Table 2. Bu e ing o bac e ial suspensions was omi ed o in ape i oneal inocula ion. b NT, no es ed. VOL. 184, 2002 NOTES 593 on July 24, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om in mac ophages. Unlike he i ulen ecombina ion mu an s, hose wi h i ulence de ec s when es ed in mice (RecA ⫺ , RecBC ⫺ , RecD ⫺ RecJ ⫺ , RecBC ⫺ SbcB ⫺ , and RecBC ⫺ SbcB ⫺ SbcCD ⫺ ) also showed de ec s o in acellula p oli - e a ion wi hin J774.A1 mouse mac ophages (Fig. 1). A no e- wo hy obse a ion is, howe e , ha sbc supp esso mu a ions we e unable o es o e in acellula p oli e a ion wi hin mac- ophages. Since sbc mu a ions es o e ecombina ion p o i- ciency by ac i a ing pa hways o he han RecBCD, ou obse - a ions indica e ha S. en e ica equi es ecombina ional epai ia he RecBCD pa hway o g ow inside phagocy es. Because o he unique abili y o he RecBCD pa hway o epai double-s and DNA b eaks (15, 17, 36), i seems logical o conclude ha S. en e ica elies on he RecBCD ecombina ion pa hway o epai his kind o DNA lesion inside mac ophages. In ac , a ecen s udy has shown he occu ence o double- s and b eaks in bac e ia g owing inside J774.A1 mac ophages (33). Wha is he ole o he RecBCD pa hway in Salmonella i ulence? A igh associa ion be ween DNA eplica ion and ecombina ion in phage T4 has long been known (28). In he las 5 yea s, he idea ha DNA eplica ion equi es ecombi- na ional epai has been ex ended o bo h bac e ia (14, 32) and Saccha omyces ce e isiae (24). Whene e a double-s and DNA b eak is p oduced, ecombina ional epai ia he RecBCD pa hway is equi ed o p ime DNA syn hesis and hence o he esump ion o ch omosome eplica ion (17, 36). Ou obse a ion ha he RecBC enzyme is equi ed o g ow h inside mac ophages co ela es wi h he ex eme sensi i i y o RecBCD ⫺ mu an s o NO (35) and wi h he occu ence o double-s and b eaks among he DNA lesions ound in E. coli cells g own inside mac ophages (33). Since he RecF pa hway canno unc ionally eplace RecBCD-media ed ecombina ion o he epai o double-s and DNA b eaks, sbcB and sbcCD supp esso mu a ions do no es o e ecombina ional epai (17). Hence, he inabili y o RecBC ⫺ SbcB ⫺ and RecBC ⫺ SbcB ⫺ SbcCD ⫺ mu an s o S. en e ica o g ow inside mac o- phages may esul om a es ed ch omosome eplica ion upon DNA damage. Ou inding ha RecD ⫺ RecJ ⫺ mu an s a e a i ulen and unable o g ow inside mac ophages seems o indica e ha , in he absence o he RecD exonuclease, ecombina ional epai ia he RecBCD pa hway can be ca ied ou wi h he pa ici- pa ion o RecJ. This si ua ion is simila o ha o Rep ⫺ mu- an s o E. coli, which equi e ei he RecD o RecJ o double- s and b eak epai (36). Fu he mo e, he e is e idence ha RecJ is in ol ed in he eco e y o eplica ion o ks upon DNA damage (6) and in o he RecBC-dependen ecombina ion p ocesses (27, 39). In summa y, we p opose ha S. en e ica uses he RecBCD ecombina ion pa hway o epai DNA double-s and b eaks p oduced du ing g ow h inside mac ophages and ha RecD and RecJ p o ide al e na i e exonuclease ac i i ies o he epai p ocess. The absence o RecBC o bo h RecD and RecJ ende s Salmonella a i ulen . A en a i e explana ion, sup- po ed by abundan li e a u e ( e iewed in e e ence 15), may be ha Salmonella needs RecBC-media ed DNA epai o p ime DNA eplica ion inside phagocy es. This wo k was suppo ed by g an s om he Di eccio´n Gene al de Ensen˜anza Supe io o he Go e nmen o Spain (PM97-0148-CO2), he Eu opean Union (QLK2-1999-00310), and he Comunidad de Ma- d id (08.2/0045.1/2000). M.G.P. is suppo ed by a pos doc o al ellow- ship om he Comunidad de Mad id. We hank Nello Bossi and John Ro h o p o iding s ains, Mike Mahan o discussions on ecombina ion, and And e´s Aguile a o c i ical eading o he manusc ip . REFERENCES 1. Benson, N. R., and J. Ro h. 1994. Supp esso s o ecB mu a ions in Salmo- nella yphimu ium. Gene ics 138:11–29. 2. Buchmeie , N., S. Bossie, C. Y. Chen, F. C. Fang, D. G. Guiney, and S. J. Libby. 1997. SlyA, a ansc ip ional egula o o Salmonella yphimu ium,is equi ed o esis ance o oxida i e s ess and is exp essed in he in acellula en i onmen o mac ophages. In ec . Immun. 65:3725–3730. 3. Buchmeie , N. A., and F. He on. 1991. Inhibi ion o phagosome-lysosome usion by Salmonella yphimu ium. In ec . Immun. 59:2232–2238. 4. Buchmeie , N. A., C. J. Lipps, M. Y. H. So, and F. He on. 1993. Recom- bina ion-de icien mu an s o Salmonella yphimu ium a e a i ulen and sen- si i e o he oxida i e bu s o mac ophages. Mol. Mic obiol. 7:933–936. 5. Chan, R. K., D. Bo s ein, T. Wa anabe, and Y. Oga a. 1972. Specialized ansduc ion o e acycline by phage P22 in Salmonella yphimu ium. II. P ope ies o a high equency ansducing lysa e. Vi ology 50:883–898. 6. Cou celle, J., and P. C. Hanawal . 1999. RecQ and RecJ p ocess blocked eplica ion o ks p io o he esump ion o eplica ion in UV-i adia ed E. coli. Mol. Gen. Gene . 262:543–551. 7. DeG oo e, M. A., U. A. Ochsne , M. U. Shiloh, C. Na han, J. M. McCo d, M. C. Dinaue , S. J. Libby, A. Vazquez-To es, Y. Xu, and F. C. Fang. 1997. Pe iplasmic supe oxide dismu ase p o ec s Salmonella om p oduc s o phagocy e NADPH-oxidase and ni ic oxide syn hase. P oc. Na l. Acad. Sci. USA 94:13997–14001. 8. Fields, P. I., R. W. Swanson, C. G. Haida is, and F. He on. 1986. Mu an s o Salmonella yphimu ium ha canno su i e wi hin he mac ophage a e a i ulen . P oc. Na l. Acad. Sci. USA 83:5189–5193. 9. Ga cı´a-del Po illo, F., M. Pucia elli, and J. Casadesu´s. 1999. DNA adenine me hylase mu an s o Salmonella yphimu ium show de ec s in p o ein sec e- ion, cell in asion and M cell cy o oxici y. P oc. Na l. Acad. Sci. USA 96: 11578–11583. 10. Ga cia-del Po illo, F., M. B. Zwick, K. Y. Leung, and B. B. Finlay. 1993. Salmonella induces he o ma ion o ilamen ous s uc u es con aining lyso- somal memb ane glycop o eins in epi helial cells. P oc. Na l. Acad. Sci. USA 90:10544–10548. 11. Ga zo´n, A., C. R. Beuzo´n, M. J. Mahan, and J. Casadesu´s. 1996. ecB ecJ mu an s o Salmonella yphimu ium a e de icien in ansduc ional ecombi- na ion, DNA epai and plasmid main enance. Mol. Gen. Gene . 270:570– 580. FIG. 1. Abili y o ecombina ion mu an s o S. en e ica o p oli e - a e in J774.A1 mac ophages. G ow h, in ec ion, and lysis o J774.A1 mouse mac ophages we e ca ied ou as desc ibed elsewhe e (9, 10). The bac e ial s ains used o in ec ion we e SV1445, SV1488, SV4189, SV4190, SV4212, SV4213, SV4192, SV4363, and SV4364. The in a- cellula p oli e a ion a e (Ip o) was calcula ed as he a io o he numbe o iable in acellula bac e ia p esen a 24 h o he numbe p esen a 2 h pos in ec ion. Da a a e means ⫾s anda d de ia ions o esul s om wo ep esen a i e expe imen s. The assay was epea ed h ee imes o each s ain. w , wild ype. 594 NOTES J. BACTERIOL. on July 24, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om 12. Hoise h, S. K., and B. A. S ocke . 1981. A oma ic-dependen Salmonella yphimu ium a e non i ulen and e ec i e as li e accines. Na u e 291:238– 239. 13. Kaise , K., and N. E. Mu ay. 1979. Physical cha ac e iza ion o he “Rac p ophage”in E. coli K-12. Mol. Gen. Gene . 175:159–174. 14. Kogoma, T., G. W. Caldwell, K. G. Ba na d, and T. Asai. 1996. The DNA eplica ion p iming p o ein, P iA, is equi ed o homologous ecombina ion and double-s and b eak epai . J. Bac e iol. 178:1258–1264. 15. Kowalczykowski, S. C. 2000. Ini ia ion o gene ic ecombina ion and ecom- bina ion-dependen eplica ion. T ends Biochem. Sci. 25:156–165. 16. Kushne , S. R., H. Nagaishi, and A. J. Cla k. 1972. Indi ec supp ession o ecB ecC mu a ions by exonuclease I de iciency. P oc. Na l. Acad. Sci. USA 69:1366–1370. 17. Kuzmino , A., and F. W. S ahl. 1999. Double-s and end epai ia he RecBC pa hway in Esche ichia coli p imes DNA eplica ion. Genes De . 13:345–356. 18. Leung, K. Y., and B. B. Finlay. 1991. In acellula eplica ion is essen ial o he i ulence o Salmonella yphimu ium. P oc. Na l. Acad. Sci. USA 88: 11470–11474. 19. Lloyd, R. G., and C. Buckman. 1985. Iden i ica ion and gene ic analysis o sbcC mu a ions in commonly used ecBC sbcB s ains o Esche ichia coli K-12. J. Bac e iol. 164:836–844. 20. Lloyd, R. G., and K. B. Low. 1996. Homologous ecombina ion, p. 2236– 2255. In F. C. Neidha d , R. Cu iss III, J. L. Ing aham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Rezniko , M. Riley, M. Schaech e , and H. E. Umba ge (ed.), Esche ichia coli and Salmonella: cellula and molecula biology, 2nd ed. ASM P ess, Washing on, D.C. 21. MacMicking, J., Q. W. Xie, and C. Na han. 1997. Ni ic oxide and mac o- phage unc ion. Annu. Re . Immunol. 15:323–350. 22. Mahan, M. J., and J. R. Ro h. 1989. The ecB and ecC genes o Salmonella yphimu ium. J. Bac e iol. 171:612–615. 23. Mahan, M. J., J. Casadesu´s, and J. R. Ro h. 1992. The Salmonella yphi- mu ium RecJ unc ion pe mi s g ow h o P22 abc phage on ecBCD ⫹ hos s. Mol. Gen. Gene . 232:470–478. 24. Malko a, A., E. L. I ano , and J. E. Habe . 1996. Double-s and b eak epai in he absence o RAD51 in yeas : a possible ole o b eak-induced DNA eplica ion. P oc. Na l. Acad. Sci. USA 93:7131–7136. 25. Ma sui, H., M. Eguchi, and Y. Kikuchi. 2000. Use o con ocal mic oscopy o de ec Salmonella yphimu ium wi hin hos cells associa ed wi h Sp -medi- a ed in acellula p oli e a ion. Mic ob. Pa hog. 29:53–59. 26. Miesel, L., and J. R. Ro h. 1994. Salmonella yphimu ium ecD mu a ions inc ease ecombina ion in a sho -sequence ansduc ion assay. J. Bac e iol. 176:4092–4103. 27. Miesel, L., and J. R. Ro h. 1996. E idence ha SbcB and RecF pa hway unc ions con ibu e o RecBCD-dependen ansduc ional ecombina ion. J. Bac e iol. 178:3146–3155. 28. Mosig, G. 1998. Recombina ion and ecombina ion-dependen DNA epli- ca ion in bac e iophage T4. Annu. Re . Gene . 32:379–413. 29. Ohl, M. E., and S. I. Mille . 2001. Salmonella: a model o bac e ial pa ho- genesis. Annu. Re . Med. 52:259–274. 30. Reed, L. J., and H. Muench. 1938. A simple me hod o es ima ing i y pe cen endpoin s. Am. J. Hyg. 27:493–497. 31. Rich e -Dahl o s, A., A. M. Buchan, and B. B. Finlay. 1997. Mu ine salmo- nellosis s udied by con ocal mic oscopy: Salmonella yphimu ium esides in acellula ly inside mac ophages and exe s a cy o oxic e ec on phagocy es in i o. J. Exp. Med. 186:569–580. 32. Sandle , S. J., H. S. Sam a, and A. J. Cla k. 1996. Di e en ial supp ession o p iA2::kan pheno ypes in Esche ichia coli K-12 by mu a ions in p iA,lexA and dnaC. Gene ics 143:5–13. 33. Schlosse -Sil e man, E., M. Elg ably-Weiss, I. Rosenshine, R. Kohen, and S. Al u ia. 2000. Cha ac e iza ion o Esche ichia coli DNA lesions gene a ed wi hin J774 mac ophages. J. Bac e iol. 182:5225–5230. 34. Schmiege , H. 1972. Phage P22 mu an s wi h inc eased o dec eased ans- ducing abili ies. Mol. Gen. Gene . 119:75–88. 35. Spek, E. J., T. L. W igh , M. S. S i , N. R. Taghizadeh, S. R. Tannenbaum, M. G. Ma inus, and B. P. Engelwa d. 2001. Recombina ional epai is c i ical o su i al o Esche ichia coli exposed o ni ic oxide. J. Bac e iol. 183:131–138. 36. Uzes , M., S. D. Eh lich, and B. Michel. 1995. Le hali y o ep ecB and ep ecC double mu an s o Esche ichia coli. Mol. Mic obiol. 17:1177–1188. 37. Vazquez-To es, A., and F. C. Fang. 2001. Oxygen-dependen an i-Salmo- nella ac i i y o mac ophages. T ends Mic obiol. 9:29–33. 38. Vazquez-To es, A., Y. Xu, J. Jones-Ca son, D. W. Holden, S. M. Lucia, M. C. Dinaue , P. Mas oeni, and F. C. Fang. 2000. Salmonella pa hogenici y island 2-dependen e asion o he phagocy e NADPH oxidase. Science 287: 1655–1658. 39. Viswana han, M., and S. T. Lo e . 1998. Single-s and DNA-speci ic exo- nucleases in E. coli: oles in epai and mu a ion a oidance. Gene ics 149: 7–16. 40. Zah , T. C., N. Buchmeie , and S. Maloy. 1999. E ec o mu S and ecD mu a ions on Salmonella i ulence. In ec . Immun. 67:6168–6172. VOL. 184, 2002 NOTES 595 on July 24, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om