JOURNAL OF BACTERIOLOGY,
0021-9193/98/$04.0010Oc . 1998, p. 5231–5234 Vol. 180, No. 19
Copy igh © 1998, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
Cell Di ision Inhibi ion in Salmonella yphimu ium His idine-
Cons i u i e S ains: an sI-Like De ec in he P esence
o Wild-Type Penicillin-Binding P o ein 3 Le els
DAVID A. CANO,
1
CHAKIB MOUSLIM,
1
JUAN A. AYALA,
2
FRANCISCO GARCI
´A-DEL PORTILLO,
2
AND JOSEP CASADESU
´S
1
*
Depa amen o de Gene´ ica, Facul ad de Biologı´a, Uni e sidad de Se illa, Se ille 41080,
1
and Cen o de Biologı´a
Molecula Se e o Ochoa, CSIC-Uni e sidad Au o´noma de Mad id, Can oblanco, Mad id 28049,
2
Spain
Recei ed 6 Ap il 1998/Accep ed 11 July 1998
His idine-cons i u i e (His
c
) s ains o Salmonella yphimu ium unde go cell di ision inhibi ion in he p es-
ence o high concen a ions o a me abolizable ca bon sou ce. Filamen s o med by His
c
s ains show con-
s ic ions and con ain e enly spaced nucleoids, sugges ing a de ec in sep um o ma ion. Inhibi o s o peni-
cillin-binding p o ein 3 (PBP3) induce a ilamen a ion pa e n iden ical o ha o His
c
s ains. Howe e , he
His
c
sep a ion de ec is caused nei he by educed PBP3 syn hesis no by educed PBP3 ac i i y. G oss
modi ica ions o pep idoglycan composi ion a e also uled ou . D-Cyclose ine, an inhibi o o he soluble
pa hway p oducing pep idoglycan p ecu so s, causes pheno ypic supp ession o ilamen a ion, sugges ing ha
he sep a ion de ec o His
c
s ains may be caused by sca ci y o PBP3 subs a e.
When his idine-cons i u i e (His
c
) mu an s o Salmonella
yphimu ium we e i s isola ed, he au ho s no ed ha high
le els o his idine biosyn he ic enzymes caused w inkled colony
mo phology on 2% glucose pla es (25). W inkledness e lec s
cell ilamen a ion (12, 19), which is igge ed by o e p oduc-
ion o hisH and hisF gene p oduc s (5, 9, 19). A simila e-
sponse has been desc ibed o Esche ichia coli (11). HisH and
HisF a e subuni s o he he e odime ic imidazole-glyce ol-
phospha e syn hase (1, 34), which ca alyzes he o ma ion o
imidazole-glyce ol-phospha e (IGP) wi h elease o he pu ine
p ecu so AICAR (5-aminoimidazole-4-ca boxamide ibonu-
cleo ide) (17, 26). Howe e , di ision inhibi ion does no e-
qui e me abolic low h ough he his idine biosyn he ic pa h-
way, sugges ing ha HisH and HisF igge ilamen a ion
h ough an ac i i y un ela ed o IGP syn hesis (10, 19). The
in ol emen o AICAR has been also uled ou (10, 11). In
bo h S. yphimu ium and E. coli, he cell di ision de ec o His
c
s ains is un ela ed o he SOS esponse and does no in ol e
he cell di ision inhibi o SulA (11, 12). We show below ha
he cell di ision de ec o S. yphimu ium His
c
s ains is a block
in sep um o ma ion, as p oposed by F andsen and D’A i (11).
We also desc ibe he unexpec ed inding ha s ains ha o e -
p oduce IGP syn hase con ain wild- ype le els o ac i e peni-
cillin-binding p o ein 3 (PBP3). These con adic o y da a a e
en a i ely econciled by he abili y o D-cyclose ine o supp ess
ilamen a ion in His
c
mu an s. The la e obse a ion sugges s
ha HisHF o e p oduc ion may cause a sho age in PBP3
subs a e.
Filamen o ma ion by His
c
s ains. Mid-exponen ial-phase
cul u es o s ains LT2 (hisO
1
) and TR6753 (hisO1242 [5, 16])
we e obse ed unde he mic oscope by using Hi aga’s luo-
phase combined me hod, a p ocedu e ha pe mi s he simul-
aneous obse a ion o nucleoids and cells (15). Nucleoid
s aining was achie ed wi h DAPI (49,6-diamino-2-phenylin-
dole). The His
c
s ain o med long ilamen s which con ained
e enly spaced nucleoids, indica ing ha hei di ision de ec is
un ela ed o DNA syn hesis o ch omosome pa i ion (Fig. 1).
Mo eo e , he p esence o blun cons ic ions indica es ha
he di ision block lies beyond he s age o F sZ ac ion (8). The
ilamen s a e simila in mo phology and leng h o hose o med
by sI and sA mu an s o E. coli (8). The sI gene encodes
PBP3, an essen ial cell di ision p o ein in ol ed in sep um
o ma ion (27). F sA is a memb ane-bound p o ein ha in e -
ac s wi h PBP3 (22, 31). A di e ence is ha sI and sA
mu an s a e condi ional ( he mosensi i e) le hals unable o
o m colonies unde es ic i e condi ions (3, 18), while he
ilamen s p oduced by His
c
s ains o S. yphimu ium in he
p esence o 2% glucose gi e ise o colonies which a e dis-
inc ly w inkled (9, 19, 25).
An ibio ics ha inhibi PBP3, such as az eonam and azlocil-
lin, p oduce phenocopies o sI mu an s in E. coli (29). Based
on his p eceden , we in es iga ed whe he PBP3 inhibi o s
we e able o ep oduce he ilamen a ion pheno ype o His
c
s ains. Addi ion o az eonam (1 mg/li e ) o a cul u e o
s ain LT2 igge ed ilamen o ma ion (Fig. 1C), and he
ilamen s we e iden ical in mo phology and leng h o hose
o med by he His
c
s ain TR6753 (Fig. 1A). The same e ec
was induced by azlocillin (da a no shown). A he concen a-
ions used, hese an ibio ics inhibi speci ically PBP3 (21).
These obse a ions sugges ha His
c
s ains o S. yphimu ium
beha e as sI mu an s.
Addi ional e idence agains an F sA-like de ec was p o-
ided by he ailu e o a plasmid ca ying he E. coli sA gene
o elie e cell di ision inhibi ion when in oduced in s ain
TR6753. I His
c
s ains we e sA-like, a plasmid-bo ne sA
gene should es o e he F sA/F sZ a io (6), he eby causing a
ce ain deg ee o supp ession. Howe e , he ac ual esul was
ha pMFV26, an sA
1
plasmid p o ided by Miguel Vicen e
(CIB-CSIC, Mad id, Spain), ailed o elie e sep a ion inhibi-
ion. The conclusion ha His
c
s ains beha e as sI ( a he
han sA) mu an s ecei es u he suppo om he abili y o
D-cyclose ine o supp ess ilamen a ion (see below).
HisHF o e p oduc ion does no cause educed syn hesis o
PBP3. The le els o PBP3 p oduced by HisO
1
and HisO
c
s ains we e compa ed by using en elope ex ac s om mid-
* 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 ille 41080, Spain. Phone: 34 95 455 7105. Fax: 34 95 455 7104.
E-mail: [email p o ec ed].
5231
exponen ial-phase cul u es (op ical densi y a 600 nm, 0.5 o
0.6) in E medium con aining 2% glucose (33). En elope p o-
eins we e ac iona ed by sodium dodecyl sul a e (SDS)-poly-
ac ylamide gel elec opho esis (8% ac ylamide) and de ec ed
by immunoblo ing agains a polyclonal an i-PBP3 se um (21).
The le els o PBP3 p o ein a e simila in HisO
1
and HisO
c
s ains (Fig. 2); densi ome ic analysis (no shown) con i med
he absence o di e ences. Thus, he cell di ision de ec asso-
cia ed wi h IGP syn hase o e p oduc ion is no caused by e-
duced syn hesis o PBP3. An addi ional obse a ion is ha he
elec opho e ic mobili ies o PBP3 a e simila in HisO
1
and
HisO
c
s ains, he eby elimina ing he occu ence o g oss
s uc u al changes in he p o ein.
HisHF o e p oduc ion does no cause educed ac i i y o
PBP3. As an indica o o he unc ionali y o PBP3 in mem-
b ane ex ac s om HisO
1
and HisO
c
s ains, we analyzed
hei capaci ies o bind co alen ly
3
H-labelled benzylpenicillin
(2). Cul u es we e p epa ed as desc ibed abo e. P o ein sep-
a a ion was pe o med on an SDS-polyac ylamide gel (2). Ra-
dioac i i y was de ec ed by luo og aphy (28, 30). P o ein
quan i a ion was pe o med by he me hod o B ad o d (4). No
di e ences in penicillin binding we e ound (Fig. 3 and den-
si ome ic da a no shown). A side obse a ion is ha he e-
maining high-molecula -weigh PBPs we e also una ec ed
(Fig. 3).
Pep idoglycan composi ion o His
c
s ains. To in es iga e
he possibili y ha His
c
s ains migh syn hesize an abno mal
cell wall, we p epa ed pep idoglycan ex ac s om s ains LT2
and TR6753 g own in E medium con aining 2% glucose. Con-
cen a ed exponen ial cul u es con aining app oxima ely 10
11
bac e ial cells we e cooled o 4°C, cen i uged a 12,000 3g o
15 min, and esuspended in 3 ml o phospha e-bu e ed saline,
pH 7.4. The suspension was mixed 1:1 ( ol/ ol) wi h a boiling
solu ion o 8% SDS (24). The SDS-insoluble ma e ial was
washed in dis illed wa e (13). Pep idoglycan was diges ed wi h
Cellosyl mu amidase (20 mg/ml) (Hoechs , Somme ille, N.J.);
his ea men yields mu opep ides o low molecula weigh
(23). The eac ion was s opped in a boiling ba h o 5 min.
FIG. 1. Mic oscopic pho og aphs o cells and ilamen s p epa ed wi h Hi a-
ga’s luo-phase combined me hod (15). (A) Filamen s o med by he His
c
s ain
TR6753 g own in E medium con aining 2% glucose. (B) An isogenic His
1
s ain
(LT2) g own unde he same condi ions does no o m ilamen s. (C) Addi ion o
az eonam o a cul u e o s ain LT2 in E medium induces ilamen s iden ical o
hose o med by a His
c
s ain in high-glucose medium (compa e panels A and C).
Ba , 5 mm.
FIG. 2. Immunode ec ion o PBP3 om he wild ype (lanes A and C) and
he His
c
s ain TR6753 (lanes B and D). Lanes A and B con ain memb ane
esicles co esponding o 80 mg o p o ein in a inal eac ion olume o 20 ml.
Lanes C and D con ain memb ane esicles co esponding o 120 mg o p o ein in
a inal olume o 20 ml.
FIG. 3. Binding o
3
H-labelled benzylpenicillin o cell en elopes om expo-
nen ially g owing cells o he His
1
s ain LT2 (lane I) and he His
c
s ain TR6753
(lane II). PBPs a e numbe ed on he igh by s anda d nomencla u e (27).
5232 NOTES J. BACTERIOL.
Insoluble ma e ial was emo ed by cen i uga ion (1,000 3g,
10 min). Pep idoglycan composi ion was de e mined by high-
pe o mance liquid ch oma og aphy analysis (13, 14). The main
conclusions a e ha (i) abno mal pep idoglycan mu opep ides
we e no ound and (ii) g oss di e ences in pep idoglycan
composi ion o o ganiza ion we e no ound be ween HisO
1
and HisO
c
s ains (Table 1). These expe imen s do no ule ou
he o ma ion o an abno mal PBP3 subs a e bu ce ainly ex-
clude he possibili y ha any unusual subs a e is inco po a ed
in o g owing pep idoglycan chains. This absence o di e ences
does no elimina e he possibili y ha His
c
s ains may ha e a
de ec in he eac ion ca alyzed by PBP3; pep idoglycan om
s ain LT2 ea ed wi h he PBP3 inhibi o az eonam showed
also s anda d composi ion (Table 1). In ac , a well-known and
su p ising ea u e o cell wall syn hesis is ha he global pep-
idoglycan composi ion emains unal e ed unde condi ions
ha cause majo changes in cell shape (20).
D-Cyclose ine causes pheno ypic supp ession o he cell di-
ision de ec o His
c
s ains. To explo e he possibili y ha he
sep a ion de ec o His
c
s ains migh be caused by lowe ed
le els o PBP3 subs a e, we in es iga ed whe he D-cyclose ine
was able o cause pheno ypic supp ession o ilamen a ion in a
His
c
mu an . D-Cyclose ine educes he numbe o pen apep-
ide side chains in pep idoglycan and inc eases he numbe o
ipep ides (21), an e ec caused by inhibi ion o D-alanine-D-
alanine ligase and alanine acemase (32). The esul ing imbal-
ance inc eases sep a ion a he expense o elonga ion, gi ing
ise o ounded cells in he wild ype and es o ing cell di ision
in F sI
2
mu an s (3).
His
c
s ains o med w inkled colonies on bo h g een pla es
and E pla es con aining 2% glucose. In he p esence o D-cy-
close ine (20 mg/li e ), he colonies o med by he HisO
c
s ain
TR6753 and hose o med by he wild ype we e iden ical: smoo h
and small. Fo mic oscopic obse a ion o cul u es, s ains LT2
and TR6753 we e g own in E medium con aining 2% glucose
and 15 mg o D-cyclose ine pe li e . Exponen ial cul u es we e
ans e ed o aga -coa ed slides and pho og aphed unde
phase-con as op ics (7). S ain LT2 o med ounded cells
(da a no shown). S ain TR6753 unde wen nea ly comple e
supp ession o ilamen a ion and o med od-shaped cells (Fig.
4). Because hese esul s s ongly esemble hose ob ained wi h
sI mu an s o E. coli (3), we hypo hesize ha D-cyclose ine
may supp ess he sep a ion de ec o His
c
s ains by inc easing
PBP3 subs a e. I his iew is co ec , he cell di ision de ec o
His
c
s ains may be en a i ely e o mula ed as a sho age o
ipep ide side chains, he p oposed subs a e o he sep al
machine y (21).
This s udy was suppo ed by g an PB93-649 om he Di eccio´n
Gene al de In es igacio´n Cien ı´ icayTe´cnica o he Go e nmen o
Spain and g an 97-109-96 om he Comunidad de Mad id.
We hank Miguel Vicen e o he gi o pMFV26 and Bo is Ma-
gasanik, Dick D’A i, and Molly Schmid o help ul discussions. The
assis ance o Glo ia Chaco´n, Ana Mo eno, Jose´Co´ doba, and Luis
Romanco is also app ecia ed.
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ec o His
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s ains. (A) S ain TR6753 g own in E medium con ains only ila-
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TABLE 1. Mu opep ide composi ion o pep idoglycan om His
1
and His
c
s ains and om a His
1
s ain ea ed wi h az eonam
Sample
Rela i e abundance (mol%)
a
C oss-
linkage
(%)
Monome s Dime s T ime s Lpp
D-D L-D
LT2 (His
1
) 65.2 27.7 2.9 4.12 9.25 38.8
TR6753 (His
c
) 66.9 26.4 3.2 3.2 9.0 36.3
LT2 (wi h az eonam) 66.0 26.2 3.0 4.6 8.2 38.5
a
Mu opep ides a e g ouped acco ding o s uc u al simila i ies (13). D-D a e
dime ic mu opep ides c oss-linked by a D-D pep ide b idge; L-D a e dime ic
mu opep ides c oss-linked by an L-D pep ide b idge. T ime s a e ime ic mu-
opep ides c oss-linked by D-D pep ide b idges. Lpp a e mu opep ides bound o
he C- e minal dipep ide o B aun’s lipop o ein (13, 14).
VOL. 180, 1998 NOTES 5233
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