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
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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.
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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 .
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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-
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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.
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