JOURNAL OF BACTERIOLOGY, Sep . 1995, p. 4841–4850 Vol. 177, No. 17
0021-9193/95/$04.0010
Copy igh q1995, Ame ican Socie y o Mic obiology
Supp ession o he Pleio opic E ec s o HisH and HisF
O e p oduc ion Iden i ies Fou No el Loci on he Salmonella
yphimu ium Ch omosome: osmH,s iW,s iX, and s iY
AMANDO FLORES AND JOSEP CASADESU
´S*
Depa amen o de Gene´ ica, Uni e sidad de Se illa, Se ille 41080, Spain
Recei ed 30 Ma ch 1995/Accep ed 19 June 1995
Inse ion mu a ions ha supp ess some o all he pleio opic e ec s o HisH and HisF o e p oduc ion we e
ob ained by using ansposons Tn10dTe and Tn10dCam. All supp esso mu a ions p o ed o be ecessi e,
indica ing ha hei e ec s we e caused by loss o unc ion; hus, he supp esso s iden i y genes ha a e
necessa y o igge he pleio opic esponse when HisH and HisF a e o e p oduced. Gene ic mapping o he
supp esso mu a ions iden i ies ou no el loci on he Salmonella yphimu ium gene ic map. Mu a ions in osmH
(min 49) beha e as gene al supp esso s ha abolish all mani es a ions o he pleio opic esponse. Mu a ions
in s iY (min 83) supp ess cell di ision inhibi ion and he mosensi i i y bu no osmosensi i i y. Mu a ions ha
supp ess only cell di ision inhibi ion de ine ano he locus, s iX (min 44). A ou h no el locus, s iW (min 19),
is also in ol ed in cell di ision inhibi ion. The pheno ype o s iW mu a ions is in u n pleio opic: hey supp ess
cell di ision inhibi ion, make S. yphimu ium unable o g ow in minimal media, and cause slow g ow h and
abno mal colony and cell shape. The inabili y o s iW mu an s o g ow in minimal medium canno be elie ed
by any known nu i ional equi emen o by he use o ca bon sou ces o he han glucose. The hie a chy o
supp esso pheno ypes and he exis ence o epis a ic e ec s among supp esso mu a ions sugges a pa hway-
like model o he His
c
pleio opic esponse.
Cons i u i e exp ession o he Salmonella yphimu ium his-
idine ope on causes g ow h inhibi ion a 428C (24), w inkled
mo phology o colonies g own in ei he 2% glucose o ‘‘g een’’
pla es (28, 42), and g ow h inhibi ion on pla es con aining
h ee o i e imes he no mal concen a ion o E sal s (12, 37).
The w inkled-colony mo phology is due o cell ilamen a ion
which is also obse ed in liquid cul u es, especially i g own
wi h a high concen a ion o glucose (25, 28, 42). This ilamen-
a ion is sulA independen and un ela ed o he SOS esponse
(28).
The His
c
pleio opic esponse is caused by inc eased le els
o p o eins HisH and HisF (12, 25, 42). In he close ela i e
Esche ichia coli, a simila esponse is obse ed when HisH and
HisF a e o e p oduced (27). HisH and HisF ca alyze he e-
lease o 5-amino-4-imidazole ca boxamide iboside 59-mono-
phospha e (AICAR), which is also an in e media e in pu ine
biosyn hesis (43, 44, 53). Since AICAR is a po en ial p ecu so
o he ala mone 5-amino-4-imidazole ca boxamide iboside 59-
iphospha e (9, 46), se e al au ho s had p oposed ha in-
c eased syn hesis o AICAR migh be esponsible o he
pleio opic e ec s o his o e exp ession (11, 22). Howe e ,
AICAR does no seem o be in ol ed in he pleio opic e-
sponse (25, 26).
We ha e add essed he iden i ica ion o genes in ol ed in
he His
c
pleio opic esponse by he classical app oach o
s udying in e genic (ex e nal) supp ession (30). Mu a ions ha
supp ess some o all he mani es a ions o he pleio opic e-
sponse iden i y ou no el loci on he S. yphimu ium ch omo-
some: osmH (min 49), s iW (min 19), s iX (min 44), and s iY
(min 83). The hie a chy o supp esso pheno ypes and he
exis ence o epis a ic e ec s in ce ain supp esso combina ions
sugges ha he pleio opic esponse o His
c
mu an s in ol es
a complex ne wo k o pa hway whose ul ima e physiological
signi icance emains unknown.
MATERIALS AND METHODS
Bac e ial s ains, bac e iophages, and s ain cons uc ion. The S. yphi-
mu ium s ains used in his s udy, all de i ed om s ain LT2, a e lis ed in Table
1. S ain TR6753, ca ying he a enua o dele ion hisO1242, was used as he
s anda d His
c
s ain (12). S ain hisD3050, ob ained om J. R. Ro h (Depa -
men o Biology, Uni e si y o U ah, Sal Lake Ci y), ca ies a comple e dele ion
o he his idine ope on. T ansduc ional c osses using phage P22 HT 105/1 in 201
(44a, 49) we e used o s ain cons uc ion; he ansducing phage will be hence-
o h e e ed as P22 HT. Phage sensi i i y was es ed by c oss-s eaking wi h he
clea -plaque mu an P22 H5. To ob ain phage- ee isola es, ansduc an s we e
pu i ied by s eaking on g een indica o pla es.
Plasmids and ansposons. The episome F9128 p o
1
lac
1
zz -1836::Tn10dCam
was desc ibed by Ellio and Ro h (23). pNK972 (Amp
) is a pBR333 de i a i e
ca ying he IS10 ansposase gene unde he con ol o a ac p omo e (41).
pNK2880 con ains he IS10 npA(a s1 a s2) gene exp essed om a ac p omo e ;
his plasmid con ains a lacI
q
allele and con e s ampicillin esis ance (35). The
a s1 and a s2 mu a ions con e elaxed speci ici y o Tn10 ansposase (5).
Tn10dTe , Tn10dCam, and Tn10dKan a e dele ion de i a i es o ansposon
Tn10 ha do no ca y ansposi ion unc ions (52). The E. coli episome F9128
p o
1
lac
1
zz -1831::Tn10dTe and he S. yphimu ium episome F9152 nad
1
zz -1833::Tn10dKan we e bo h ob ained om J. R. Ro h. Mud1-8(Amp Lac) (32)
and MudI1734(Kan Lac) (13) a e ansposi ion-de icien Mu de i a i es ha
gene a e ope on usions upon inse ion. MudI1734(Kan Lac) (13) was enamed
MudJ by Hughes and Ro h (34).
Media, chemicals, and cul u e condi ions. The E medium o Vogel and Bon-
ne (51) supplemen ed wi h 0.2% glucose was used as minimal medium. In
ce ain expe imen s, glucose was eplaced by 0.2% uma a e. High-sal (33E)
pla es con ained h ee imes he no mal concen a ion o E sal s (12). NCE
medium is E medium wi hou ci a e. The ich medium was nu ien b o h (NB;
8 g/li e ; Di co) wi h added NaCl (5 g/li e ). Solid media con ained aga a a inal
concen a ion o 1.5%. An ibio ics we e p epa ed and used as desc ibed by
Maloy (38). Fo he selec ion o Te
s
de i a i es o Te
s ains, we used he
medium o Bochne e al. (10) as modi ied by Maloy and Nunn (39). G een
indica o pla es we e p epa ed as desc ibed by Chan e al. (14) excep ha
me hyl blue (Sigma) subs i u ed o aniline blue. Nu i ional supplemen s and
auxanog aphy pools we e p epa ed as desc ibed by Da is e al. (19). Casamino
Acids we e used a concen a ions o 0.2, 0.4, and 0.6%. Excep when speci ically
indica ed, cul u es we e incuba ed a 378C. Liquid cul u es we e g own in a
Gallenkamp o bi al incuba o a 160 pm.
* 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: 345-455-7105. Fax: 345-455-7104. Elec-
onic mail add ess: [email p o ec ed].
4841
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Mu agenesis wi h Tn10dCam and isola ion o sal - esis an de i a i es o a
His
c
s ain. We used a a ian o he nonhomologous ansduc ion p ocedu e
desc ibed by Ellio and Ro h (23). A lysa e g own on s ain SV2056 was used o
ansduce s ain SV2057, selec ing Cam
Amp
ansduc an s. T ansducing mix-
u es we e made on NB pla es and p eincuba ed o 4 o6ha 378C be o e
eplica pla ing o NB supplemen ed wi h chlo amphenicol and ampicillin. Use o
a ecipien ca ying he dele ion hisD3050 p e en ed he inse ion o he mini-
Tn10 in he his idine ope on. Cam
Amp
ansduc an s we e eplica pla ed mo e
han h ee imes o NB-chlo amphenicol pla es con aining EGTA (10 mM); he
la e was added o p e en ein ec ion ( hus allowing he isola ion o phage- ee
TABLE 1. S. yphimu ium s ains used
S ain Geno ype Re e ence o sou ce
a
LT2 Wild ype Labo a o y s ock
PP1002 cya-1091::Tn10 pB223 SGSC
b
SA4247 a oA554::Tn10 SGSC
SMS409 aspC409::Tn10 SGSC
SV2056 hisD3050/F9128 lac
1
p o
1
zz -1836::Tn10dCam
SV2057 hisD3050/pNK972
SV2058 hisO1242/F9128 lac
1
p o
1
zz -1831::Tn10dTe
SV2059 hisO1242/pNK2880
SV2060 hisO1242 osmH1::Tn10dCam
SV2061 hisO1242 osmH2::Tn10dCam
SV2064 hisO1242 osmH3::Tn10dTe
SV2068 hisO1242 s iW1::Tn10dTe
SV2069 hisO1242 s iX1::Tn10dTe
SV2070 hisO1242 s iX2::Tn10dTe
SV2071 hisO1242 s iY1::Tn10dTe
SV2072 hisO1242 s iX3::Tn10dTe
SV2073 hisO1242 s iW2::Tn10dTe
SV2074 hisO1242 s iY2::Tn10dTe
SV2082 hisO1242 osmH4::Tn10dTe
SV2090 hisO
1
hisC9968::MudJ
SV2091 hisO1242 hisC9968::MudJ
SV2092 hisO
1
hisC9968::MudJ osmH1::Tn10dCam
SV2093 hisO1242 hisC9968::MudJ osmH1::Tn10dCam
SV2129 hisO1242 osmH3::Tn10dCam s iX2::Tn10dTe
SV2130 hisO1242 osmH3::Tn10dCam s iY1::Tn10dTe
SV2131 hisO1242 osmH3::Tn10dCam s iW1::Tn10dTe
SV2132 hisO1242 s iW1::Tn10dTe s iX2::Tn10dKan
SV2133 hisO1242 s iW1::Tn10dTe s iY1::Tn10dKan
SV2134 hisO1242 s iX2::Tn10dKan s iY1::Tn10dTe
SV2135 hisO1242 osmH3::Tn10dCam s iW1::Tn10dTe s iY1::Tn10dKan
SV2136 hisO1242 osmH3::Tn10dCam s iW1::Tn10dTe s iX2::Tn10dKan
SV2137 hisO
1
hisIE9969::Mud1-8
SV2138 hisO1242 hisIE9969::Mud1-8
SV2139 hisO
1
hisIE9969::Mud1-8 osmH1::Tn10dTe
SV2140 hisO1242 hisIE9969::Mud1-8 osmH1::Tn10dTe
SV2150 hisO1242 DUP [hisH9962 pMudPpcysA1586]
c
SV2155 hisO1242 DUP [pu E2154 pMudPppu B1879]
c
TR6753 hisO1242 12
TT2242 a oD5 hisW1824 pu F145 psL me G319 zee-78::Tn10 SGSC
TT7294 zea-1032::Tn10 SGSC
TT10423 p oAB47/F9128 p o
1
lac
1
zz -1836::Tn10dTe J. Ro h
TT10425 nadA56/F9152 nad
1
zz -1833::Tn10dKan J. Ro h
TT10604 p oAB47/F9128 p o
1
lac
1
zz -1836::Tn10dCam J. Ro h
TT13736 eu -153::MudJ SGSC
TT15222 leuA414
2
m
1
els2 h -458::MudQ N. Benson
TT15225 leuA414
2
m
1
els2 py A2413::MudP N. Benson
TT15228 leuA414
2
m
1
els2 nadC220::MudP N. Benson
TT15233 leuA414
2
m
1
els2 pu E2155::MudP N. Benson
TT15234 leuA414
2
m
1
els2 pu E2155::MudQ N. Benson
TT15251 leuA414
2
m
1
els2 hisH9950::MudP N. Benson
TT15252 leuA414
2
m
1
els2 hisH9950::MudP N. Benson
TT15252 leuA414
2
m
1
els2 a oC566::MudP N. Benson
TT15253 leuA414
2
m
1
els2 cysA1586::MudQ N. Benson
TT15259 leuA414
2
m
1
els2 nadB226::MudP N. Benson
TT15260 leuA414
2
m
1
els2 nadB226::MudQ N. Benson
TT15262 leuA414
2
m
1
els2 cysHIJ1574::MudQ N. Benson
TT15269 leuA414
2
m
1
els2 il A2648::MudP N. Benson
TT15274 leuA414
2
m
1
els2 pu D1874::MudP N. Benson
TT17163 leuA414
2
m
1
els2 hisH9962::MudP N. Benson
a
Omi ed o s ains i s desc ibed in his s udy.
b
SGSC, Salmonella Gene ic S ock Cen e, Uni e si y o Calga y, Calga y, Albe a, Canada.
c
Nomencla u e o duplica ions (DUP) ollows he ules desc ibed in e e ences 15 and 33.
4842 FLORES AND CASADESU
´S J. BACTERIOL.
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de i a i es). Pools o 1,000 o 2,000 Cam
colonies we e made and lysed wi h
phage P22 HT. The pools we e hen used o ansduce s ain TR6753; Cam
ansduc an s we e selec ed on 33E pla es. The o al numbe o Cam
isola es
pooled was a ound 60,000. To gua an ee independence, only one Cam
, sal -
esis an isola e pe pool was u he s udied.
Mu agenesis wi h Tn10dTe . A lysa e g own on s ain SV2058 was used o
ansduce s ain SV2059; he la e con ains plasmid pNK2880 (35) o pe mi
ans complemen a ion o he de ec i e Tn10dTe elemen by ATS ansposase.
T ansducing mix u es we e di ec ly made on NB pla es and p eincuba ed o 4 h
a 378C. The cul u es we e hen eplica pla ed o NB pla es supplemen ed wi h
e acycline and o app op ia e media o de ec supp ession o he mu a ion
hisO1242:33E pla es o selec sal - esis an de i a i es, E pla es incuba ed a
428C o selec he mo esis an isola es, and g een pla es o de ec he p esence o
non ilamen ing (smoo h) colonies. The o al numbe o Te
isola es sco ed (all
o independen o igin) was a ound 110,000. Candida es we e made phage ee on
g een pla es and used o ansduce TR6753. This second ansduc ion allowed
he isola ion o plasmid- ee Te
de i a i es.
T ansposon subs i u ions. Lysa es g own on s ains TT10604, TT10423, and
TT10425 we e used o ansposon subs i u ions. These s ains ca y F-p ime
plasmids con aining Tn10dCam, Tn10dTe , and Tn10dKan (Table 1). The lysa es
we e i adia ed wi h UV ligh , using a 15-W Syl ania lamp a a dis ance o 30 cm
du ing 30 s; i adia ion o he phage suspensions can be expec ed o inc ease
ecombina ion in he ecipien s (45). T ansduc ions selec ing he incoming
ma ke (Te
, Cam
,o Kan
) we e ca ied ou . T ansduc an s we e hen sco ed
o loss o he esiden ma ke (Te
o Cam
). These p ocedu es allowed us o
ob ain allele a ian s agged wi h di e en an ibio ic esis ance genes. S ains
ca ying allele a ian s a e no included in Table 1; he allele a ian s a ailable
a e lis ed in Table 2.
Rapid mapping wi h Mud-P22 p ophages. We ollowed he p ocedu e de-
sc ibed by Benson and Goldman (6), using 67 ‘‘locked-in’’ Mud-P22 p ophages.
In addi ion o he 54-lysogen collec ion desc ibed in e e ence 6, we used 13
addi ional Mud-P22 lysogens (s ains TT15222, TT15225, TT15228, TT15233,
TT15234, TT15251, TT15252, TT15253, TT15259, TT15260, TT15262, TT15274,
and TT17163, all ob ained om Nick Benson, Depa men o Biology, Uni e si y
o U ah). These addi ional lysogens (bu no he o iginal collec ion desc ibed in
e e ence 6) a e included in Table 1.
Co ansduc ional mapping. Upon ansduc ion wi h phage P22 HT, ansduc-
an s we e eplica pla ed o sui able pla es o sco e ansduc ion o unselec ed
ma ke s. Compa ible ma ke combina ions we e made possible by using di e en
e sions (Te
, Kan
,o Cam
) o he alleles o be mapped. Co ansduc ion
equencies a e a e ages o mo e han ou independen c osses, sco ing a leas
300 ansduc an s om each c oss. The ela i e o de o ma ke s was de e mined
by h ee- ac o c osses. Co ansduc ion equencies below 1% we e no aken
in o accoun (38). Table 1 con ains he o iginal s ains ca ying ma ke s co ans-
ducible wi h he loci s udied (TT7294, TT15253, SA4247, SMS409, TT2242,
PP1002, and TT15269) bu no hei de i a i es cons uc ed by adding one o he
alleles o be mapped; such cons uc ions we e equi ed o pe o m ce ain h ee-
ac o c osses. S ains used in unsuccess ul co ansduc ional mapping a emp s
a e also omi ed in Table 1.
Complemen a ion analysis. Complemen a ion was achie ed by he cons uc-
ion o me odiploid s ains (2, 15, 31). Duplica ions wi h p ede e mined end-
poin s we e ob ained by using a s a egy adap ed om e e ence 33.
b-Galac osidase assays. Le els o b-galac osidase we e assayed as desc ibed
by Mille (40), using he CHCl
3
-sodium dodecyl sul a e pe meabiliza ion p oce-
du e. To measu e his ope on exp ession by using ansc ip ional lac usions a
hisC and hisIE, cul u es we e g own in E medium con aining 0.2% glucose and
0.1 M his idine; o e nigh cul u es we e dilu ed in o esh medium o each an
op ical densi y a 540 nm (OD
540
) o 0.4 o 0.6.
Osmosensi i i y assays in liquid medium. All assays we e ca ied ou a 378C
wi h shaking. Cul u es we e p eadap ed o .8 h in E medium con aining 0.3 M
NaCl; he cul u es we e hen dilu ed o ob ain an ini ial OD
540
o 0.05. G ow h
was moni o ed by nephelome ic measu emen s wi h a Spec onic 200 spec o-
pho ome e . Osmop o ec ion was achie ed in he p esence o 0.5 mM p oline
(17).
The mosensi i i y assays. All assays we e ca ied ou in E medium. A e
o e nigh p eincuba ion a 308C, he cul u es we e dilu ed o an OD
540
o 0.05
and ans e ed o a shaking wa e ba h p ewa med a 428C. G ow h was mon-
i o ed by nephelome ic measu emen s as desc ibed abo e.
UV su i al assays. O e nigh cul u es made in NB we e dilu ed 1/10 in he
same medium. When he cul u es eached an OD o 0.5, he cells we e ha es ed
and esuspended in E bu e (E medium wi hou glucose). Fi e-millili e aliquo s
we e ans e ed o s e ile, emp y pe i dishes. I adia ion was achie ed by
opening he pla es unde a 15-W Syl ania UV lamp a a dis ance o 30 cm in he
absence o dayligh illumina ion. Cell suspensions we e s i ed du ing i adia ion.
A e se ial dilu ion in oil-co e ed ubes, i adia ed cul u es we e pla ed on NB.
Mic oscopic obse a ion o cul u es. Aliquo s o exponen ially g owing cells in
E medium con aining 2% glucose we e ex ac ed and ans e ed o slides coa ed
wi h a hin laye o aga (21). Cells we e pho og aphed wi h a Zeiss Ul apho
mic oscope unde phase-con as op ics, using a Kodak Pan ilm. Pho og aphs
we e de eloped wi h Kodak Technidol liquid de elope .
RESULTS
Supp ession o sal sensi i i y by inse ion o mini-Tn10
elemen s. A e mu agenesis wi h ei he Tn10dTe o Tn10d
Cam, sal - esis an de i a i es o he His
c
s ain TR6753 we e
isola ed on 33E pla es con aining ei he e acycline
o chlo amphenicol. Sal - esis an isola es appea ed a a e-
quency o 1 pe ;3,000 inse s es ed. To be classi ied as
supp esso mu a ions, he inse ions we e equi ed o pass he
ollowing es s.
(i) A e mo e han wo ounds o pu i ica ion, he isola es
ca ying pu a i e supp esso s we e lysed wi h phage P22 HT;
he lysa es we e hen used o ansduce he pa en al s ain
TR6753, selec ing ei he Te
o Cam
. An ibio ic- esis an
ansduc an s we e eplica pla ed o 33E pla es. Comple e
(100%) linkage be ween he ansposon-encoded an ibio ic e-
sis ance and he supp esso pheno ype (sal esis ance) was
aken as e idence ha supp ession was caused by he inse ion
mu a ion.
(ii) The lysa es desc ibed abo e we e also used o ansduce
s ain hisD3050. His
1
ansduc an s we e eplica pla ed o 33
E pla es; sal sensi i i y indica ed inhe i ance o he mu a ion
hisO1242.
(iii) To con i m ha he supp esso pheno ype was caused by
he ansposon inse ion (and no by ano he mu a ion loca ed
elsewhe e in he genome), econs uc ion expe imen s we e
ca ied ou . Fi s , he ansposon inse ion was ansduced o
s ain hisD3050, selec ing he ansposon-encoded an ibio ic
esis ance. The esul ing ansduc an s we e hen ansduced
o His
1
wi h a lysa e g own on he His
c
s ain TR6753. The
p esence o he supp esso was con i med because all o he
TABLE 2. Loci in ol ed in he pleio opic e ec s o HisH and HisF o e exp ession
Locus Map loca ion
(min) Cha ac e is ics o mu an s O iginal alleles ully
cha ac e ized O he allele e sions
a ailable
a
osmH 49 Supp ession o osmosensi i i y, he mosensi-
i i y, and cell di ision inhibi ion osmH1::Tn10dCam Te
osmH2::Tn10dCam Te
osmH3::Tn10dTe Kan
, Cam
osmH4::Tn10dTe None
s iW 19 Supp ession o cell di ision inhibi ion; in-
abili y o g ow in minimal medium s iW1::Tn10dTe Kan
s iW2::Tn10dTe Kan
s iX 44 Supp ession o cell di ision inhibi ion only s iX1::Tn10dTe Kan
s iX2::Tn10dTe Kan
s iX3::Tn10dTe Kan
s iY 83 Supp ession o cell di ision inhibi ion and
he mosensi i i y s iY1::Tn10dTe Kan
s iY2::Tn10dTe Kan
a
No included in he s ain lis .
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His
1
Te
o His
1
Cam
ansduc an s we e sal esis an . As a
con ol, when he same lysa es we e used o ansduce s ain
hisD3050, all o he ansduc an s we e sal sensi i e.
Ele en (se en Cam
and ou Te
) sal - esis an isola es o
independen o igin we e used o u he s udy. All p o ed o
be he mo esis an on E pla es incuba ed a 428C, sugges ing
ha osmosensi i i y and he mosensi i i y had been simul a-
neously supp essed by a single inse ion mu a ion. Mo eo e ,
hese sal - esis an isola es we e smoo h on g een pla es (al-
hough hei colony mo phology was sligh ly di e en om
ha o he pa en al s ain: compa e Fig. 1B and C). Mic o-
scopical obse a ion o sal - esis an isola es g own in E liquid
cul u es con aining 2% glucose con i med ha he inse ions
s udied we e able o supp ess cell di ision inhibi ion, al hough
sho ilamen s we e s ill o med (see Fig. 4C).
Supp ession o he mosensi i i y by inse ions o Tn10dTe .
To asce ain whe he supp ession o he mosensi i i y was al-
ways associa ed wi h high-sal esis ance, he mo esis an de-
i a i es o s ain TR6753 we e isola ed by mu agenesis wi h
Tn10dTe and pla ing on E medium a 428C (p ewa med).
The mo esis an isola es appea ed a a equency o 1 pe
4,000 Te
inse s. All ( ou o ou , independen ) p o ed o be
sal esis an , he eby con i ming ha osmosensi i i y and he -
mosensi i i y had been simul aneously supp essed. These iso-
la es we e smoo h on g een pla es, like he sal - esis an iso-
la es desc ibed abo e.
Inse ions ha supp ess osmosensi i i y, he mosensi i i y,
and cell di ision inhibi ion a e alleles o a single locus. To
asce ain whe he he supp esso alleles belonged o one o
mo e loci, ecombina ion es s we e ca ied ou by ansduc-
ion and eplica p in ing. To assay e e y allele combina ion,
ansposon subs i u ions we e pe o med. Two inse ions we e
conside ed allelic whene e he esiden allele was excluded
om he ecombinan s (e.g., when .99% o he Te
ansduc-
an s we e no longe Cam
). These es s indica ed ha all
gene al supp esso s ha abolish sal sensi i i y, he mosensi-
i i y, and cell di ision inhibi ion a e alleles o a single locus.
Hence o h, his locus will be called osmH ( o osmosensi i i y
caused by HisH and HisF o e p oduc ion). Fu he wo k was
ca ied ou wi h he ollowing alleles: osmH1::Tn10dCam,
osmH2::Tn10dCam, and osmH3::Tn10dTe (isola ed as sal e-
sis an ) and osmH4::Tn10dTe (isola ed as he mo esis an ).
Supp ession o cell di ision inhibi ion by Tn10dTe inse -
ions. Fo he de ec ion o inse ions supp essing cell di ision
inhibi ion, Tn10dTe inse s we e ob ained on s ain TR6753.
Te
ansduc an s we e eplica pla ed o g een pla es supple-
men ed wi h e acycline, o gi e 300 o 500 colonies pe pla e.
The pla es we e sco ed o he p esence o smoo h colonies;
hese appea ed a a equency o 1 pe ;2,000 Te
isola es.
The ini ial sample included 47 independen smoo h isola es; 14
p o ed o be P22 esis an and we e disca ded. The emaining
pu a i e supp esso -con aining isola es we e subjec ed o es s
simila o hose desc ibed abo e, o co ela e he p esence o
he inse ion wi h he supp esso pheno ype and o p o e ha
he pa en al s ain s ill con ained he mu a ion hisO1242.
None o he smoo h isola es o med ilamen s in E liquid
medium con aining 2% glucose. Howe e , hese isola es be-
longed o h ee classes, on he basis o cell and colony mo -
phology, nu i ional equi emen s, and he abili y o supp ess
o he mani es a ions o he pleio opic esponse.
Class I isola es o m small, smoo h, da k colonies on g een
pla es (Fig. 1F). They a e unable o g ow on minimal medium
(ei he E o NCE). This inabili y o g ow in minimal medium
hampe s he s udy o he mosensi i i y and osmosensi i i y,
because HisO
c
s ains g own in ich medium a e osmo esis an
and he mo esis an (da a no shown). I inse ions o his class
a e ans e ed o he wild- ype s ain LT2, he inabili y o
g ow in liquid medium and he abno mal colony shape a e
likewise obse ed. Thus, class I inse ions cause hese de ec s
on hei own, i espec i e o he gene ic backg ound o he
s ain (His
1
o HisO
c
). The g ow h de ec o S iW
2
mu an s is
no elie ed (i) wi h any single componen o an auxanog aphy
pool, (ii) wi h any pai wise combina ion o he nu ien s o he
auxanog aphy es o Da is e al. (19), (iii) wi h any concen-
a ion o Casamino Acids (0.2, 0.4, o 0.6%), o (i ) wi h
uma a e as a ca bon sou ce al e na i e o glucose.
Class II isola es o m la ge, smoo h, ligh colonies on g een
pla es (Fig. 1E). They a e p o o ophic and he mo esis an
bu osmosensi i e.
Class III isola es o m la ge, smoo h, ligh colonies on g een
pla es (Fig. 1D). They a e p o o ophic, he mosensi i e, and
osmosensi i e.
Supp esso s o cell di ision inhibi ion a e alleles o h ee
loci. Tn10dTe inse ions supp essing cell di ision inhibi ion
we e eplaced wi h Tn10dKan; allelic inse ions we e de ec ed
by ansduc ional ecombina ion as desc ibed abo e. These
es s de ined h ee g oups o inse ions, each co esponding o
one o he classes desc ibed abo e (which, in u n, a e no
A
F
E
DC
B
FIG. 1. Colonies o S. yphimu ium HisO
1
, HisO
c
, and supp esso -
con aining de i a i es o he la e , all g own on g een pla es. (A) TR6753
(hisO1242); (B) LT2 (hisO
1
); (C) SV2064 (hisO1242 osmH3::Tn10dTe ); (D)
SV2069 (hisO1242 s iX1::Tn10dTe ); (E) SV2071 (hisO1242 s iY1::Tn10dTe );
(F) SV2068 (hisO1242 s iW1::Tn10dTe ).
4844 FLORES AND CASADESU
´S J. BACTERIOL.
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allelic wi h osmH inse ions). Thus, each class o inse ions
de ines a dis inc locus. These loci will hence o h be called
s iW (class I), s iY (class II), and s iX (class III). The o iginal
collec ion con ained 10 s iW alleles, 16 s iY alleles, and 7 s iX
alleles; ep esen a i e alleles o each class a e lis ed in Table 2.
Gene ic mapping o supp esso s. Fo mapping, all Tn10
inse ions cha ac e ized as supp esso s o one o mo e mani-
es a ions o he pleio opic esponse we e ans e ed o s ain
LT2; he esul ing s ains a e no included in Table 1.
(i) Mapping o osmH alleles. T ansduc ions wi h he
locked-in lysa e collec ion gene a ed pa ches o Te
s
ansduc-
an s when s ains TT15258 and TT15253 we e used as dono s.
The Mud-P22 p ophages o hese s ains map a min 50 and 49,
espec i ely. Lysa es om TT15258 a e packaged clockwise;
he packaging di ec ion o TT15253 is unknown. Co ansduc-
ional mapping con i med ha osmH is 1% linked o cysA and
5% linked o zej-3271::Tn10. In u n, cysA and zej-3271::Tn10
a e 92% linked. These esul s indica e ha osmH lies on min
49; he ma ke o de is osmH-zej-3271::Tn10-cysA.
(ii) Mapping o s iW alleles. Dense pa ches o Te
s
ansduc-
an s we e ob ained wi h locked-in lysa es o s ain TT15240,
whose Mud-P22 p ophage maps a min 21.5 and packages
coun e clockwise. Co ansduc ional mapping indica ed ha
s iW is 95% co ansducible wi h a oA and 7% co ansducible
wi h aspC. The linkage be ween a oA and aspC is 3%. Thus,
s iW maps on min 19, and he ma ke o de is a oA-s iW-aspC.
(iii) Mapping o s iX alleles. Te
s
ansduc an s we e ob-
ained wi h lysa es om TT15249 and TT17163, which package
clockwise om min 40.5 and 42, espec i ely. Co ansduc ional
mapping indica ed ha s iX is 11% linked o zee-78::Tn10 and
3% linked o me G. In u n, zee-78::Tn10 and me G a e 77%
co ansducible. Thus, s iX maps on min 44, and he ma ke
o de is s iX-zee-78::Tn10-me G.
(i ) Mapping o s iY alleles. Lysa es om TT15268 and
TT15269, mapping a min 83 and packaging clockwise and
coun e clockwise, espec i ely, ga e hick pa ches o Te
ansduc an s. A lysa e om TT15271 (min 84, coun e clock-
wise) ga e a ain ansduc ion pa ch. These da a indica ed ha
he s iY locus maps a ound min 83 on he S. yphimu ium
ch omosome. Co ansduc ional mapping con i med ha s iY is
25% linked o il A and 72% linked o cya. The linkage be ween
il A and cya is 6%; hus, s iX lies be ween il A and cya on min
83.
E ec o osmoly es on he g ow h o a His
c
s ain and i s
mu an de i a i es ca ying supp esso mu a ions. The sal -
esis an pheno ype o supp esso -con aining de i a i es o
TR6753 is no obse ed only on 33E pla es. Wi h o he
osmoly es es ed (NaCl, D-xylose, and D-so bi ol), clea di e -
ences we e obse ed be ween he pa en al His
c
s ain and i s
osmH de i a i es.
(i) When osmosensi i i y was assayed on NCE pla es, he
ollowing MICs we e ound o he His
c
s ain: 0.3 M NaCl, 0.6
MD-xylose, and 1 M D-so bi ol. To p e en he use o D-xylose
and D-so bi ol as ca bon sou ces (29), he pla es con ained
0.2% glucose. The HisO
1
s ain and he His
c
s ains ca ying
an osmH supp esso s ill g ew (slowly) on 1 M NaCl, .1M
D-xylose, and .1MD-so bi ol.
(ii) G ow h cu es ob ained in he p esence o 0.3 M NaCl
a e shown in Fig. 2A. G ow h o he HisO
c
s ain was se e ely
inhibi ed, while he g ow h cu es o hisO1242 osmH de i a-
i es we e nea ly iden ical o ha o he HisO
1
s ain. Thus,
mini-Tn10 inse ions a osmH comple ely supp ess he osmo-
sensi i i y o His-cons i u i e s ains. In Fig. 2A, one o he
s ains shown ca ied an osmH allele (osmH4::Tn10dCam) iso-
la ed as a he mo esis an supp esso , while ano he s ain
ca ied he allele osmH3::Tn10dTe , isola ed as osmo esis an ;
bo h cu es a e iden ical. Osmosensi i i y was no supp essed
by s iX and s iY alleles. Because HisO
c
s ains a e no osmo-
sensi i e in ich media (da a no shown), s iW alleles could no
be es ed o supp ession o osmosensi i i y. As an in e nal
con ol, we es ed he abili y o he HisO
c
s ain TR6753 and
i s de i a i es ca ying osmH,s iX,o s iY alleles o g ow unde
low-osmola i y condi ions (E liquid medium). All g ow h
cu es we e simila o ha o he wild- ype (da a no shown).
These expe imen s con i med ha osmH alleles speci ically
supp ess he inabili y o HisO
c
s ains o g ow in high-osmo-
la i y media.
(iii) Osmosensi i i y o he HisO
c
s ain and i s de i a i es
con aining s iX and s iY alleles was elie ed by he p esence o
p oline 1 mM (Fig. 2B). The alle ia ing e ec o p oline on he
osmosensi i i y o HisO
c
s ains has been p e iously epo ed
by Csonka (18).
G ow h o he His
c
s ain and i s supp esso -con aining
de i a i es a 42&C. The HisO
c
s ain and i s de i a i es ca -
ying osmH,s iX,o s iY alleles g ew no mally in E medium a
308C, and hei g ow h cu es we e simila o ha o an iso-
genic HisO
1
s ain (da a no shown). In con as , g ow h o he
HisO
c
s ain was se e ely inhibi ed a 428C, while i s de i a-
i es con aining osmH o s iY alleles we e he mo esis an (Fig.
3). HisO
c
S iX
2
s ains g ew poo ly, con i ming ha s iX mu-
a ions do no supp ess he mosensi i i y (Fig. 3). Addi ion o
p oline o E medium did no elie e he he mosensi i e phe-
FIG. 2. G ow h cu es o S. yphimu ium HisO
1
, HisO
c
, and supp esso -
con aining de i a i es o he la e , all g own in 33E liquid medium (A) and in
33E liquid medium supplemen ed wi h 1 mM p oline (B). E, LT2 (hisO
1
); F,
TR6753 (hisO1242); Ç, SV2082 (hisO1242 osmH4::Tn10dTe ); h, SV2064
(hisO1242 osmH3::Tn10dTe ); å, SV2069 (hisO1242 s iX1::Tn10dTe ); ■,
SV2071 (hisO1242 s iY1::Tn10dTe ).
VOL. 177, 1995 PLEIOTROPIC EFFECTS OF HisH AND HisF OVERPRODUCTION 4845
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no ype o he HisO
c
s ain and i s HisO
c
S iX
2
de i a i es
(da a no shown). Thus, he mosensi i i y and osmosensi i i y
a e wo independen mani es a ions o he pleio opic pheno-
ype (and no wo ea u es o he same phenomenon).
Supp ession by osmH alleles is un ela ed o changes in
exp ession o he his idine ope on. All pheno ypes o he pleio-
opic esponse a e elie ed o abolished by osmH mu a ions.
Thus, he e ec o osmH mu a ions is o mally simila o ha
o cis-ac ing mu a ions (e.g., p
1
p omo e mu a ions) ha e-
duce his ansc ip ion (12). OsmH
2
mu a ions canno be ac i e
in cis because hey a e unlinked o he his ope on and ecessi e
(see below). Al hough he possibili y ha a ecessi e mu a ion
could lowe his ansc ip ion seemed a p io i unlikely because
ans-ac ing posi i e egula o s o he p
1
p omo e a e no
known o exis (53), we used ansc ip ional b-galac osidase
usions o measu e his ansc ip ion le els in osmH mu an s.
Supp esso osmH alleles we e in oduced in isogenic hisO
1
and hisO1242 s ains ha con ained a MudJ inse ion in hisC.
While he p esence o he a enua o dele ion hisO1242 caused
a 10- o 15- old di e ence in he b-galac osidase ac i i y o he
lac usion hisC9968::MudJ, no signi ican di e ence was ob-
se ed when he s ains con ained an osmH supp esso (Table
3). These esul s indica e ha osmH mu a ions do no lowe
ansc ip ion om he p
1
p omo e .
Howe e , HisH and HisF a e also p oduced om ansc ip s
o igina ing a he in e nal p
2
p omo e (48). The weakness o
his p omo e allows supp ession o he pleio opic e ec s o
HisH and HisF o e exp ession by pola mu a ions ups eam p
2
(12), indica ing ha high le els o HisH and HisF p oduc s a e
no ob ained in he absence o cons i u i e exp ession om p
1
.
Howe e , p ocessed mRNA species de i ed om he main his
ansc ip a e known o ha e inc eased s abili y (1). Thus mu-
a ions a ec ing he p oduc ion and/o he s abili y o p o-
cessed mRNAs con aining he dis al pa o he ope on can be
expec ed o a ec HisH and HisF le els. As a consequence,
hese (hypo he ical) mu a ions migh supp ess he pleio opic
esponse. Since all p ocessed mRNA species known include
hisIE (1), we in es iga ed he e ec o osmH mu a ions on he
ac i i y o hisIE9969::Mud1-8 lac usions. The esul s (Table 3)
indica e ha osmH mu a ions do no a ec hisIE exp ession
le els. Thus, he possibili y ha osmH mu a ions mimic sup-
p ession by lowe ing HisH and HisF exp ession seems unlikely.
E ec o supp esso mu a ions on cell di ision. S ains LT2
(HisO
1
), TR6753 [HisO
c
], SV2064 [HisO
c
OsmH
2
], SV2069
[HisO
c
S iX
2
], and SV2071 [HisO
c
S iY
2
] we e g own in E
medium con aining 2% glucose; s ain SV2068 [HisO
c
S iW
2
]
was g own in NB con aining 2% glucose. Pho og aphs ob-
ained om la e-exponen ial-phase cul u es a e shown in Fig.
4. Di ision o he pa en al HisO
c
s ain is se e ely inhibi ed.
Filamen a ion is no obse ed in i s supp esso -con aining de-
i a i es, al hough sho ilamen s seem o coexis wi h no mal
cells in s ain SV2064 [HisO
c
OsmH
2
]. None o he s i mu an s
o ms ilamen s, bu all show abno mal cell mo phology; lysis
migh be occu ing in he s ains con aining s iX and s iY sup-
p esso s, while HisO
c
S iW
2
cells a e small and da k.
Supp ession o cell di ision inhibi ion in HisO
c
s ains does
no cause UV sensi i i y. To explo e he possibili y ha mu a-
ions ha supp ess HisO
c
di ision inhibi ion can a ec genes
ela ed o he SOS pa hway, we de e mined he UV sensi i i y
o s ains ca ying osmH,s iX,s iW, and s iY inse ion mu a-
ions. No di e ences we e obse ed be ween he supp esso -
con aining isola es and he pa en al His
c
s ain TR6753; in
u n, he UV sensi i i y o TR6753 was simila o ha o he
pa en al s ain LT2 (da a no shown). These esul s ag ee wi h
ea lie obse a ions indica ing ha he di ision inhibi ion pa h-
way igge ed by HisH and HisF o e exp ession is un ela ed o
he SOS esponse (28).
Complemen a ion o supp esso alleles and dominance
s udies. Al hough mos inse ion mu a ions can be expec ed o
be ecessi e (7, 36), Tn10 ou wa d p omo e s can also ac i a e
adjacen genes (16, 50). Thus, we ca ied ou complemen a ion
es s o asce ain whe he he supp esso mu a ions s udied a e
dominan o ecessi e.
(i) Fo complemen a ion analysis o s iW mu an s, we con-
s uc ed a duplica ion wi h endpoin s a min 12 and 25, using a
s a egy adap ed om ha o Hughes and Ro h (33). Two
s ains, each ca ying a Mud-P22 p ophage in he same o ien-
a ion in pu E (TT15235) and pu B (TT15241), we e lysed wi h
P22 HT. The lysa es we e mixed o ansduce s ain TR6753,
selec ing Cam
on minimal pla es. P o o ophic Cam
ans-
duc an s can be expec ed o con ain a duplica ion o he pu E-
pu B egion, wi h an hyb id locked-in Mud-P22 p ophage in
he middle. This assump ion was con i med because seg ega-
ion in he absence o an ibio ic selec ion yielded only Cam
s
p o o ophs (see e e ence 33). One isola e ca ying one such
Mud-held duplica ions be ween min 12 and 25 was p opaga ed
as s ain SV2155. This s ain was ough on g een pla es and
showed all he addi ional mani es a ions o he pleio opic
esponse.
To examine whe he s iW mu a ions a e dominan o eces-
si e, a s iW::Tn10dTe allele was in oduced in o s ain SV2155
by P22 HT ansduc ion, selec ing Te
. To main ain he du-
plica ion, chlo amphenicol was also added o he selec i e
pla es. The esul ing Cam
Te
ansduc an s con ain one copy
o he s iW
1
allele and one s iW::Tn10dTe inse ion. All Te
FIG. 3. G ow h cu es o S. yphimu ium HisO
1
, HisO
c
, and supp esso -
con aining de i a i es o he la e , all g own in E liquid medium a 428C. E, LT2
(hisO
1
); F, TR6753 (hisO1242); Ç, SV2082 (hisO1242 osmH4::Tn10dTe ); h,
SV2064 (hisO1242 osmH3::Tn10dTe ); å, SV2069 (hisO1242 s iX1::Tn10dTe );
■, SV2071 (hisO1242 s iY1::Tn10dTe ).
TABLE 3. Ac i i ies o ansc ip ional hisC::lac and hisIE::lac
usions in OsmH
1
and OsmH
2
backg ounds
S ain Geno ype b-Galac osi-
dase ac i i y
(Mille uni s)
SV2090 hisO
1
hisC9968::MudJ 493
SV2091 hisO1242 hisC9968::MudJ 5,953
SV2092 hisO
1
hisC9968::MudJ osmH1::Tn10dTe 445
SV2093 hisO1242 hisC9968:MudJ osmH1::Tn10dTe 6,028
SV2137 hisO
1
hisIE9969:Mud1-8 52
SV2138 hisO1242 hisIE9969:Mud1-8 494
SV2139 hisO
1
hisIE9969::Mud1-8 osmH1::Tn10dTe 55
SV2140 hisO1242 hisIE9969::Mud1-8 osmH1::Tn10dTe 471
4846 FLORES AND CASADESU
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FIG. 4. Phase-con as mic og aphs o la e-exponen ial-phase cul u es o S. yphimu ium HisO
1
, HisO
c
, and supp esso -con aining de i a i es o he la e , all
g own in liquid E medium con aining 2% glucose. (A) TR6753 (hisO1242); (B) LT2 (hisO
1
); (C) SV2064 (hisO1242 osmH3::Tn10dTe ); (D) SV2069 (hisO1242
s iX1::Tn10dTe ); (E) SV2071 (hisO1242 s iY1::Tn10dTe ); (F) SV2068 (hisO1242 s iW1::Tn10dTe ).
VOL. 177, 1995 PLEIOTROPIC EFFECTS OF HisH AND HisF OVERPRODUCTION 4847
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Cam
ansduc an s we e ough and ligh g een, indica ing ha
s iW mu a ions a e ecessi e. The p esence o bo h s iW
1
and
s iW alleles in he s ain was con i med by seg ega ion: upon
nonselec i e s eaking on g een pla es, Cam
s
isola es we e
ob ained. These belonged o wo ypes: ough, ligh g een, Te
s
and smoo h, da k, Te
.
(ii) Fo complemen a ion analysis o s iX mu an s, we con-
s uc ed a Mud-held duplica ion wi h endpoin s in hisH (min
42) and cysA (min 50). The esul ing s ain (SV2150) was hen
used as he ecipien o a s iX::Tn10dTe allele. All he Te
ansduc an s we e ough, indica ing ha s iX mu a ions a e
ecessi e. The me odiploid na u e o hese ansduc an s was
con i med by seg ega ion as desc ibed abo e.
(iii) Complemen a ion analysis o osmH and s iY mu an s
was analyzed wi h a sligh ly di e en s a egy. Since osmH is
linked o cysA, bo h he supp esso and a linked cysA::MudP
inse ion can be co ansduced o a His
c
ecipien (TR6753). Si-
mul aneous selec ion o p o o ophy and he wo incoming
ma ke s allows he isola ion o a e ansduc an s in which he
ansduced agmen has ecombined wi h one copy o a p e-
exis ing duplica ion. The OsmH
1
/OsmH
2
me odiploids ob-
ained p o ed o be sal sensi i e, he mosensi i e, and ough
on g een pla es, indica ing ha osmH mu a ions a e ecessi e.
Seg ega ion o he me odiploids p o ided an in e nal con ol
o he expe imen .
Since s iY is linked o il A, an analogous p ocedu e was used
o ob ain S iY
1
/S iY
2
me odiploids: he HisO
c
s ain TR6753
was ansduced wi h a P22 HT lysa e g own on a dono ca y-
ing an s iY supp esso and a linked il A::MudQ inse ion, se-
lec ing Il
1
Cam
Te
ansduc an s. The S iY
1
/S iY
2
me o-
diploids ob ained o med w inkled colonies on g een pla es
(and ilamen s in liquid medium) and we e he mosensi i e,
indica ing ha s iY mu a ions a e ecessi e.
Combina ions o supp esso mu a ions. Taking ad an age
o he allele a ian s ha bo ing di e en mini-Tn10 inse ions
(Tn10dTe , Tn10dCam, and Tn10dKan), we ca ied ou ans-
duc ional c osses o cons uc His
c
s ains con aining mo e
han one supp esso mu a ion. The ele an conclusions om
hese expe imen s a e as ollows.
(i) All allele combina ions a e iable.
(ii) The hie a chy o pheno ypes de ined by single supp es-
so s emains unal e ed in double- and iple-supp esso mu-
an s; o ins ance, osmH s iX,osmH s iY, and osmH s iX s iY
s ains a e osmo esis an and he mo esis an and do no o m
ilamen s. In u n, s iX s iY mu an s a e he mo esis an and do
no o m ilamen s.
(iii) The cell shape o osmH s iX and osmH s iY mu an s
esembles ha o osmH s ains (compa e Fig. 5 wi h Fig. 4C).
Thus, he cell mo phology con e ed by osmH alleles is epi-
s a ic o e hose o s iY and s iX. The same conclusion applies
o colonial mo phology (da a no shown).
(i ) When s iY and s iX alleles a e combined, bo h he colony
shape and he cell mo phology o he double-supp esso mu-
an s emain like hose o he pa en al s ains, which a e in u n
ha d o dis inguish (Fig. 1 and 4D and E). These esul s do no
cla i y whe he he pheno ypes con e ed by s iX and s iY mu-
a ions a e epis a ic o addi i e. Howe e , he ac ha s iX s iY
mu an s a e he mo esis an indica es ha s iX mu a ions a e
no epis a ic o e s iY.
Al oge he , hese obse a ions sugges a model in which he
pleio opic esponse in ol es he hie a chical exp ession o
osmH,s iY, and s iX (Fig. 6). The obse a ion ha s iX s iY
mu an s a e he mo esis an places s iX below s iY (and no in
a pa allel posi ion).
The exis ence o s iW supp esso s complica es he si ua ion.
When combined wi h osmH,s iX,o s iY alleles, he esul ing
double- and iple-supp esso mu an s do no g ow in minimal
medium and show he colony and cell shape ypical o S iW
2
mu an s. Thus, he s iW e ec s on g ow h pa e n, cell shape,
and colony mo phology a e epis a ic o e hose o osmH,s iX,
and s iY. The g ow h de ec caused by s iW alleles does no
allow es ing HisO
c
OsmH
2
S iW
2
and HisO
c
S iX
2
S iW
2
s ains o supp ession o he mosensi i i y and osmosensi i -
i y; hus, he consequences (i any) o combining s iW alleles
wi h osmH and s iY canno be analyzed.
DISCUSSION
This epo desc ibes inse ion mu a ions ha supp ess one
o mo e mani es a ions o he His
c
pleio opic esponse. All o
he supp esso mu a ions a e ecessi e; he e o e, hey iden i y
genes ha igge he pleio opic esponse when HisH and
HisF a e o e p oduced. Wi h he excep ion o s iW alleles,
FIG. 5. Phase-con as mic og aph o a la e-exponen ial-phase cul u e o
s ain SV2129 (hisO1242 osmH3::Tn10dCam s iX2::Tn10dTe ) g own in liquid E
medium con aining 2% glucose.
FIG. 6. A pa hway-like model o he His
c
pleio opic esponse, based on he
hie a chy o supp esso mu a ions and on he epis a ic e ec s obse ed among
ce ain supp esso pheno ypes.
4848 FLORES AND CASADESU
´S J. BACTERIOL.
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none o he supp esso s s udied con e s a ecognizable pheno-
ype o he wild ype.
Inse ions in osmH (min 49) supp ess osmosensi i i y, he -
mosensi i i y, and cell di ision inhibi ion. Inse ions in s iY
(min 83) supp ess he mosensi i i y and cell di ision inhibi ion
(bu no osmosensi i i y). Inse ions in s iX (min 44) supp ess
cell di ision inhibi ion only. The gene al supp ession exe ed
by osmH inse ions migh indica e ha he pleio opic e-
sponse igge ed by HisH and HisF o e exp ession has a single
cellula a ge , he p oduc o he osmH gene. Howe e , he
exis ence o pa ial supp esso s, he hie a chy o hei pheno-
ypes, and he exis ence o epis a ic e ec s among he supp es-
so s sugges ha he pleio opic esponse is a pa hway which
can be in e up ed a di e en s ages (Fig. 6).
The exis ence o a ou h ype o supp esso , s iW, adds
u he complexi y o he pic u e. Ce ain pheno ypes o s iW
mu a ions a e epis a ic o e hose o osmH,s iY, and s iX,
sugges ing ha s iW migh be placed abo e osmH in he en-
a i e pa hway o Fig. 6. Al e na i ely, s iW migh supp ess
ilamen a ion by a mechanism un ela ed o he pa hway de-
ined by osmH,s iY, and s iX. The la e explana ion seems
mo e easonable because he dis inc pheno ypes con e ed by
s iW mu a ions (biza e mo phology o cells and colonies, in-
abili y o g ow in minimal medium) a e likewise obse ed in a
HisO
1
backg ound. On his a ionale, s iW has been placed
ou side he osmH-s iY-s iX pa hway d awn in Fig. 6.
None o he supp esso s s udied causes UV sensi i i y; hus,
he co esponding wild- ype genes mus be in ol ed in an SOS-
independen p ocess o ilamen o ma ion. This assump ion
ag ees wi h he p e ious obse a ion ha he pleio opic e -
ec s o HisH and HisF o e p oduc ion a e SOS un ela ed and
sulA independen (28). In E. coli, SOS-independen di ision
inhibi o s a e encoded by he minB ope on (8, 20). The map
posi ions o he osmH,s iW,s iX, and s iY loci on he S. yphi-
mu ium ch omosome a e all di e en om ha o he E. coli
minB ope on (4). Thus, he cellula unc ions ha cause ila-
men a ion upon HisH and HisF o e p oduc ion seem o iden-
i y no el loci in ol ed in an SOS-independen p ocess o cell
di ision inhibi ion. The uniqueness o he His
c
pleio opic e-
sponse (and hus o he sco es used o supp esso isola ion)
may explain why no el genes pu a i ely ela ed o cell di ision
ha e been de ec ed in ou hun s.
The lis o supp esso s o he His
c
pleio opic esponse de-
sc ibed in his epo may be incomple e, i s because all o he
supp esso s desc ibed a e inse ion mu a ions, and hus any
essen ial genes ha migh be in ol ed in he His
c
esponse
would be o e looked in ou hun s, and second because use o
ATS ansposase can be expec ed o b oaden Tn10 a ge spec-
i ici y, bu a ge p e e ences mus s ill exis (5). This may
explain why one ype o mu a ion which has been p e iously
shown o supp ess he pleio opic esponse o His
c
mu an s
(en B [3]) has no been ound among he supp esso s de-
sc ibed in his wo k.
ACKNOWLEDGMENTS
This s udy was suppo ed by g an s PB90-0898 and PB93-649 om
he DGICYT o he Go e nmen o Spain. Addi ional unds we e
ob ained om he Regional Go e nmen o Andalusia (Jun a de An-
dalucı´a, Spain).
We hank Miguel Vicen e o ad ice and Da id A. Cano o helping
in mapping expe imen s. Cell pic u es we e aken by Manuel Sa´nchez
and Pila Palacios. We acknowledge he e icien and gene ous help
ecei ed om And ew Hessel and Kenne h Sande son, Salmonella
Gene ic S ock Cen e , Uni e si y o Calga y, Calga y, Albe a, Can-
ada. S ains we e also p o ided by Nick Benson and John Ro h. We
a e also g a e ul o And e´s Ga zo´n, Molly Schmid, Alan Campbell,
Richa d D’A i, Diana Downs, John Ro h, Lionello Bossi, Ra ael Mal-
donado, and Juan Aguila o help ul sugges ions and o Edua do
San e o and And e´s Aguile a o c i ical eading o he manusc ip .
The echnical assis ance o Asuncio´n Blasco, Ana Mo eno, Jose´Co´ -
doba, and Luis Romanco is also app ecia ed.
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VOL. 177, 1995 PLEIOTROPIC EFFECTS OF HisH AND HisF OVERPRODUCTION 4849
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