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Apu L mu an o Sino hizobium edii HH103 is
symbio ically de ec i e and al e ed in i s
lipopolysaccha ide
Ana M. Buendı´a-Cla e ı´a,
1
Ahmed Moussaid,
1
F. Ja ie Olle o,
1
Jose
´M. Vina dell,
1
An onio To es,
1
Ja ie Mo eno,
2
An onio M. Gil-Se ano,
3
Miguel A. Rod ı´guez-Ca ajal,
3
Pila Teje o-Ma eo,
3
Jan L. Pea ,
4
Nicholas J. B ewin
4
and Jose
´E. Ruiz-Sainz
1
Co espondence
Jose
´E. Ruiz-Sainz
[email p o ec ed]
1
Depa amen o de Mic obiologı´a, Facul ad de Biologı´a, Uni e sidad de Se illa, Apdo 1095,
41080 Se illa, Spain
2
Depa amen o de Biologı´a Celula , Facul ad de Biologı´a, Uni e sidad de Se illa, A . Reina
Me cedes s/n, 41012 Se illa, Spain
3
Depa amen o de Quı´mica O ga
´nica, Facul ad de Quı´mica, Uni e sidad de Se illa, Apdo 553,
41071 Se illa, Spain
4
Depa men o Molecula Mic obiology, John Innes Cen e, No wich NR4 7UH, UK
Recei ed 5 No embe 2002
Re ised 13 Ma ch 2003
Accep ed 18 Ma ch 2003
The pleio opic pheno ype o an auxo ophic pu L mu an (SVQ295) o Sino hizobium edii HH103
has been in es iga ed. SVQ295 o ms colonies ha a e anslucen , p oduce mo e slime and
abso b less Congo ed han hose o wild- ype s ain HH103. SVQ295 did no g ow in minimal
medium unless he cul u e was supplemen ed wi h hiamin and adenine o wi h hiamin and
AICA- iboside (5-aminoimidazole-4-ca boxamide 1-b-D- ibo u anoside), an in e media e o pu ine
biosyn hesis. Bac e ial cul u es supplemen ed wi h AICA- iboside o adenine eached he same
cul u e densi y, al hough he doubling ime o SVQ295 cul u es con aining AICA- iboside was
clea ly longe . S. edii SVQ295 induced pseudonodules on Glycine max and ailed o nodula e
six di e en legumes. On Glycy hiza u alensis, howe e , nodules showing ni ogenase ac i i y and
con aining in ec ed plan cells we e o med. SVQ295 showed au o-agglu ina ion when g own
in liquid TY medium and i s lipopolysaccha ide (LPS) elec opho e ic p o ile di e ed om ha o i s
pa en al s ain HH103-1. In addi ion, ou monoclonal an ibodies ha ecognize he LPS o S. edii
HH103 ailed o ecognize he LPS p oduced by SVQ295. In con as ,
1
H-NMR spec a o
K-an igen capsula polysaccha ides (KPS) p oduced by SVQ295 and he wild- ype s ain HH103
we e simila . Co-inocula ion o soybean plan s wi h SVQ295 and SVQ116 (a nodA mu an
de i a i e o HH103) p oduced ni ogen- ixing nodules ha we e only occupied by SVQ116.
INTRODUCTION
The in e ac ion be ween hizobia and legumes o o m
ni ogen- ixing oo nodules in ol es se e al communica-
ion s eps o coo dina e gene exp ession and nodule
de elopmen . The ini ial exchange o signals ac i a es
hizobial nod gene exp ession by plan -p oduced la onoid
compounds. The nod gene p oduc s a e esponsible o he
p oduc ion o hos -speci ic lipooligosaccha ide signal
molecules ha cause oo hai cu ling and nodule me is em
ini ia ion (Fishe & Long, 1992; Day e al., 2000).
Unlike he bac e ial signals speci ied by nod genes, signal
molecules ha may be equi ed o ini ia e and main ain
in ec ion h ead de elopmen a e poo ly unde s ood.
S udies wi h hizobial mu an s de ec i e in exopolysac-
cha ide, lipopolysaccha ide (LPS) o K-an igen capsula
polysaccha ides (KPS) indica e ha hese polysaccha ides
a e impo an o in ec ion o a a ie y o legumes (Becke
&Pu
¨hle , 1998; Kannenbe g e al., 1998; Kannenbe g &
Ca lson, 2001). In e es ingly, Sino hizobium edii and
S. melilo i p oduce s uc u ally conse ed LPS bu s ain-
speci ic KPS, indica ing ha he conse ed LPS lack he
Abb e ia ions: AICA- iboside, 5-aminoimidazole-4-ca boxamide 1-b-D-
ibo u anoside; AICAR, 5-aminoimidazole-4-ca boxamide ibonucleo ide;
KPS, K-an igen capsula polysaccha ide; LCO, lipo-chi in oligosaccha ide.
The GenBank accession numbe o he sequence de e mined in his
wo k is AF275718.
0002-6099 G2003 SGM P in ed in G ea B i ain 1807
Mic obiology (2003), 149, 1807–1818 DOI 10.1099/mic.0.26099-0
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s uc u al in o ma ion necessa y o in luence hos speci ici y
while he a iable KPS may a ec s ain–cul i a in e -
ac ions (Reuhs e al., 1998). Mo eo e , s udies using a panel
o an i-S. melilo i monoclonal an ibodies ha ecognizes
LPS o KPS ha e e ealed changes o hese polysaccha ides
du ing symbiosis (Reuhs e al., 1999).
The pheno ypes o di e en Rhizobium auxo ophs ha e also
p o ided in e es ing clues o he physiology o nodule
de elopmen . By analysing he symbio ic beha iou o
auxo ophic mu an s, i has been demons a ed ha some
me abolic pa hways o Rhizobium bac e ia a e impo an o
an e icien symbio ic in e ac ion (Pain 1979; Noel e al.,
1988). In some legume–Rhizobium associa ions, he hos
plan can supply he auxo ophic symbion wi h he equi ed
nu ien (s) (Djo dje ic e al., 1988). Howe e , he success o
his physiological complemen a ion will depend on he hos
plan , he hizobial s ain, and he na u e o he auxo ophy
(Be inge e al., 1980). One in iguing ype o inding is ha
in some biosyn he ic pa hways, in e media es, a he han
endp oduc s, may be equi ed o he bac e ia o elici nodule
de elopmen o in ec ion. Fo example, s udies wi h
S. melilo i yp ophan auxo ophs sugges ha an h anila e
syn hesis is impo an o bac e oid de elopmen symbiosis
wi h al al a (Ba somian e al., 1992).
One o he mos uni e sally dele e ious symbio ic de ec s
is pu ine auxo ophy. Pu ine auxo ophs o mos hizobial
species a e unable o nodula e hei hos plan s e ec i ely
(Pankhu s & Schwinghame , 1974; Dena ie
´e al., 1976;
Pain, 1979). S. melilo i pu ine auxo ophs ha e been epo ed
o induce ine ec i e nodules on al al a (Dena ie
´e al., 1976).
Se e al pu ine auxo ophs o Rhizobium leguminosa um b .
iciae we e desc ibed as nonin ec i e o e en being unable o
induce nodule o ma ion (Pain, 1979). Simila ly, a pu ine
auxo oph o he b oad-hos - ange Rhizobium sp. NGR234
elici ed oo hai cu ling and nodule me is em ini ia ion on
Mac op ilium a opu pu eum, bu no in ec ion h eads we e
obse ed (Djo dje ic e al., 1988). On soybean, S. edii
pu ine auxo ophs induced pseudonodules ha did no
con ain bac e ia (Kim e al., 1988).
Pu ine auxo ophs (Pu
2
)o R. e li CFN42 elici ed pseudo-
nodules on bean plan s (Noel e al., 1988). These mu an s
caused oo hai cu ling and nodule me is em ini ia ion bu
did no elici in ec ion h eads. Supplemen a ion o he oo
medium wi h 0?1 mM pu ine, o pu ine nucleosides, did
no enhance nodula ion abili y o R. e li pu mu an s.
Howe e , he addi ion o 0?1 mM 5-aminoimidazole-4-
ca boxamide 1-b-D- ibo u anoside (AICA- iboside, an
in e media e in he biosyn he ic pu ine pa hway) o he
plan nu i i e solu ion signi ican ly enhanced nodule
de elopmen (Noel e al., 1988; Newman e al., 1994).
Nodules induced by he mu an s in he p esence o AICA-
iboside esembled hose o med by he wild- ype, bu we e
unpigmen ed and lacked ni ogenase ac i i y. Examina ion
o hese nodules by ligh mic oscopy e ealed he p esence o
in ec ion h eads illed wi h bac e ia, bu in ec ed plan cells
we e no obse ed. Time-cou se expe imen s showed ha
he addi ion o AICA- iboside o Phaseolus ulga is oo s
wi hin he i s 6 days a e inocula ion wi h a R. e li pu
mu an allowed he o ma ion o ascula bundles in he
nodule co ex. Beyond his ime, AICA- iboside supple-
men a ion p oduced nodules ha con ained hei ascula
bundles in a cen al posi ion (Newman e al., 1992). AICA-
iboside also p omo es Rhizobium leguminosa um b . iciae
and Sino hizobium edii pu mu an s o he o ma ion o
oo nodules in Pisum sa i um and Glycine max, espec i ely
(Newman e al., 1994). All hese esul s ha e led o he
hypo hesis ha hizobia migh con e AICA iboside in o
AICAR (5-aminoimidazole-4-ca boxamide ibonucleo ide)
o ini ia e and/o sus ain in ec ion h ead de elopmen ,
possibly by using his subs ance in he p oduc ion o a
signal molecule.
We epo he e he isola ion and cha ac e iza ion o a pu L
mu an (SVQ295) o S. edii HH103 ha equi es he
addi ion o adenine and hiamin o g ow on minimal
medium and o ms pseudonodules on Glycine max oo s.
We demons a e ha he symbio ic pheno ype o SVQ295
a ies depending on he legume assayed. In Mac o yloma
axylla e no nodules we e o med, while in Glycy hiza
u alensis, nodules con aining in ec ed cells we e obse ed.
Fu he mo e, he LPS p o ile o mu an SVQ295 is di e en
om ha shown by i s wild- ype pa en HH103, and
monoclonal an ibodies aised agains he HH103 LPS ailed
o ecognize he LPS o SVQ295.
METHODS
S ains, plasmids and media. The bac e ial s ains and plasmids
used in his wo k a e lis ed in Table 1. Sino hizobium s ains we e
g own in TY medium (Be inge , 1974), yeas manni ol (YM)
medium (Vincen , 1970), B
2
medium (Spaink e al., 1992) o MMB
(Be gensen, 1961) a 28 uC. To sc een o auxo ophic s ains he
minimal medium (MMB) was solidi ied wi h aga ose a 1?5 % (w/ ).
Esche ichia coli was cul u ed in Lu ia–Be ani (LB) medium
(Mania is e al., 1982) a 37 uC. When equi ed, he media we e sup-
plemen ed wi h he app op ia e an ibio ics as desc ibed by Lam abe
e al. (1999). When equi ed, MMB medium was supplemen ed wi h
adenine (12 mgml
21
), AICA- iboside (0?1o 0?5 mM) and/o
hiamin (0?8mgml
21
).
Gene ic manipula ions. Plasmids we e ans e ed by conjuga ion
as desc ibed by Simon (1984). Random ansposon Tn5-Mob mu a-
genesis o Sino hizobium edii s ain HH103-1 was ca ied ou by
using he suicide plasmid pSUP5011 as desc ibed by Simon (1984).
T ansconjugan s we e selec ed on TY medium con aining kanamycin
and s ep omycin. Single colonies wi h al e ed mo phology on solid
TY o YM media we e es ed o auxo ophy by using a modi ica ion
o he Holliday es (Be inge e al., 1984).
To al genomic DNA and plasmid isola ions we e ca ied ou as
desc ibed by Mania is e al. (1982). DNA manipula ion, including
es ic ion analyses, liga ion, bac e ial ans o ma ion and elec o-
pho esis we e pe o med acco ding o he gene al p o ocols o
Mania is e al. (1982).
The suicide hizobial plasmid pSUP202 (Simon e al., 1983) was used
o clone he hizobial DNA ha is adjacen o he Tn5-Mob inse ion in
mu an S. edii SVQ295. B ie ly, plasmid pSUP202 was ans e ed
om E. coli S17-1 o mu an SVQ295, and Tc
R
ansconjugan s we e
1808 Mic obiology 149
A. M. Buendı´a-Cla e ı´a and o he s
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selec ed. Since his plasmid canno eplica e in S. edii, he only
possible inhe i ance o pSUP202 is by homologous ecombina ion
be ween he Mob si e o ansposon Tn5-Mob and he Mob egion o
plasmid pSUP202. This ecombina ion will lead o he in eg a ion o
plasmid pSUP202 inside ansposon Tn5-Mob. Plasmid pSUP202 has a
unique EcoRI si e (inside he Cm
R
gene) and his enzyme does no cu
he Tn5-Mob ansposon. Hence, EcoRI diges ion o o al DNA isola ed
om SVQ295 : : pSUP202 gene a es an EcoRI agmen ha con ains
he e acycline- esis ance gene, he O iV o pSUP202, he Mob si e,
pa o he ansposon including he IS50R and a agmen o hizo-
bial DNA adjacen o he inse ed ansposon. EcoRI-diges ed
SVQ295 : : pSUP202 o al DNA was subjec ed o sel -liga ion and
E. coli HB101 Tc
R
ans o man s we e selec ed.
An oligonucleo ide (pdad2: 59-AGATTTAGCCCAGTCG-39) was
designed om he IS50R DNA sequence o ansposon Tn5-Mob
and used as a p ime o sequence he hizobial DNA ha is adjacen o
he igh a m o Tn5-Mob in mu an s ain SVQ295. DNA sequence
was ca ied ou by he dideoxy chain- e mina ion me hod (Sange e al.,
1977). Compu e analysis o he sequences was pe o med by using he
GCG p og am (Uni e si y o Wisconsin; De e eux e al., 1984).
Plan assays. The nodula ion abili y o s ain SVQ295 was es ed
on Glycine max (L.) Me . c . Williams, Cajanus cajan (L.) Millsp.,
Mac op ilium a opu pu eum (Moc. & Sesse
´ex DC) U b., Vigna
adia a (L.) Wilczek, Indigo e a inc o ia (L.), Mac o yloma axilla e
(E. Mey.) Ve dc., Albizia lebbek (L.) Ben h., Kumme owia s ipulacea
(Maxim.) Makino, Neono onia wigh ii (A n.) Lackey and Glycy hiza
u alensis (L.) as desc ibed by Buendı
´a-Cla e ı
´ae al. (1989).
Ge mina ed seeds we e ans e ed o Leona d ja s con aining s e i-
lized e miculi e supplemen ed wi h Fa
˚h aeus nu ien solu ion
(Vincen , 1970). When equi ed, he Fa
˚h aeus plan nu i i e solu-
ion was supplemen ed wi h adenine a 12 mgml
21
, hiamin a
0?8mgml
21
and AICA- iboside a 0?1o 0?5 mM. Plan s we e
g own o a leas 5 weeks wi h a 16 h pho ope iod a 25 uC in he
ligh and 18 uC in he da k. Ni ogenase ac i i y o soybean (Glycine
max) nodules was de e mined by ace ylene educ ion assays as
p e iously epo ed (Buendı
´a-Cla e ı
´ae al., 1986). Plan ops
we e d ied a 70 uC o 48 h and weighed. Bac e ia we e isola ed
om su ace-s e ilized nodules as desc ibed by Buendı
´a-Cla e ı
´a
e al. (1986).
In double-inocula ion expe imen s, soybean plan s c . Williams we e
inocula ed wi h 3610
8
bac e ia pe plan o each co-inoculan ,
SVQ116 (Ri
Km
, p o o ophic) and SVQ295 (S
Km
, Ade
2
Thi
2
).
Plan s we e ha es ed 82 days a e co-inocula ion. To de e mina e
nodule occupancy, isola es om soybean nodules we e es ed o
an ibio ic esis ance and auxo ophic ma ke s.
Radiolabelling o lipo-chi in oligosaccha ide (LCO). Bac e ia
we e g own in 5 ml B
2
medium in he p esence o he induce
na ingenin (3?6mM) and N-ace yl[
14
C]glucosamine. The
14
C-labelled
LCOs we e analysed by TLC as desc ibed by Spaink e al. (1992).
SDS-PAGE analysis o lipopolysaccha ide. Bac e ial cul u es
we e g own on solid TY medium o on solid MMB medium supple-
men ed wi h adenine (o AICA- iboside) and hiamin. Bac e ial cells
we e washed in 0?9 % NaCl and pelle ed by cen i uga ion. The bac-
e ial pelle was esuspended and lysed by hea ing a 100 uCin
125 ml 60 mM T is/HCl (pH 6?8) con aining 2 % (w/ ) SDS and
1 mM EDTA o 5 min and hen dilu ed o 1 ml wi h he same
bu e wi hou SDS. The bac e ial c ude ex ac was ea ed wi h
RNase, DNase and p o einase K as desc ibed by Ko
¨pling e al.
(1993). The elec opho esis o c ude bac e ial ex ac s was
pe o med on a 16?5 % (w/ ) polyac ylamide gel wi h he T icine
bu e sys em desc ibed by Lesse e al. (1990). Fo he de ec ion o
LPS, gels we e s ained wi h sil e as desc ibed by Ki elbe ge &
Hilbink (1993).
Monoclonal an ibodies (mAbs) and immunoblo ing.
P oduc ion o mAbs agains ee-li ing cul u es o HH103 o
Table 1. Bac e ial s ains and plasmids
S ain o plasmid De i a ion and ele an p ope ies Sou ce o e e ence
Sino hizobium edii
HH103-1 HH103 S
R
Buendı
´a-Cla e ı
´ae al. (1989)
SVQ295 HH103-1 pu L ::Tn5-Mob This wo k
SVQ269 HH103 Ri
R
Madinabei ia e al. (2002)
SVQ526 Pu ine auxo ophic mu an o
SVQ269 by Tn5-Mob mu agenesis
This wo k
SVQ116 SVQ269 nodA ::Tn5-B20 Buendı
´a-Cla e ı
´ae al. (1996)
Rhizobium e li
CE3 De i a i e o CFN42, S
R
Noel e al. (1984)
CE382 CE3 pu L ::Tn5Newman e al. (1995)
Esche ichia coli
HB101 Res ic ion-minus, ecA backg ound, S
R
Boye & Roulland-Dussoix (1969)
Plasmids
pBluesc ip Cloning ec o , Ap
R
S a agene
pSUP202 pBR325 con aining he Mob egion subcloned
in Sau3A, Ap
R
Cm
R
Tc
R
Simon e al. (1983)
pSUP5011 Suicide plasmid ca ying he Tn5-Mob ansposon Simon (1984)
pMUS375 pSUP202 con aining a 11 kb EcoRI agmen o SVQ295 DNA
adjacen o he IS50R om Tn5-Mob
This wo k
pMUS509 Cosmid pLAFR1 ca ying pu L o S. edii HH103 This wo k
pCOS110 Cosmid pLAFR1 ca ying pu Y,pu Q and pu L o R. e li CE3 Newman e al. (1994)
pJN382A pCOS110 pu L ::Tn5Newman e al. (1994)
h p://mic.sgmjou nals.o g 1809
pu L mu an o S. edii
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nodule-de i ed bac e oids ollowed p e iously published p ocedu es
(Lucas e al., 1996). Spleen cells de i ed om immunized male a s
(Line LOU/IAP) we e used o he myeloma line IR983F. Fo immuno-
blo ing expe imen s, mAbs ha ecognize HH103 LPS we e
de i ed om ou cul u e supe na an s. NB3-4.72 and NB3-4.79
(class IgG 2c) we e wo clones de i ed om he same hyb idoma
line o igina ing om a a immunized wi h a p epa a ion o HH103
bac e oids, NB6-228.22 (Class IgG 2b) was also ob ained ollowing
immuniza ion wi h a bac e oid p epa a ion, while NB7-242.33
(Class IgG 2c) was ob ained ollowing immuniza ion wi h ee-li ing
cul u es o HH103.
Polyac ylamide gels we e elec oblo ed on o ni ocellulose shee s,
using a Bio-Rad Semi-d y ansblo appa a us a 20 V o 1 h a oom
empe a u e. T ans e bu e was 48 mM T is/HCl, 39 mM glycine,
con aining 20 % ( / ) me hanol and 0?0375 % (w/ ) SDS (pH 8?0).
Wes e n-blo ed shee s we e immunos ained wi h he app op ia e
mAb, using as he seconda y an ibody a goa an i- a IgG conjuga ed o
alkaline phospha ase.
NMR analyses o KPS. KPS we e isola ed om S. edii HH103
and i s mu an SVQ295 as desc ibed by Gil-Se ano e al. (1999).
The samples con aining KPS we e deu e ium-exchanged se e al
imes by eeze-d ying om
2
H
2
O and hen examined in solu ion
(4 mg ml
21
)in99?98 %
2
H
2
O. Spec a we e eco ded a 303 K on a
B uke AMX 500 spec ome e ope a ing a 500?13 MHz. Chemical
shi s a e gi en in p.p.m., using he
2
H
2
O signal (4?75 p.p.m.)
as e e ence.
Mic oscopic s udies. Small agmen s o nodules and pseudo-
nodules we e ixed in 4 % ( / ) glu a aldehyde p epa ed in 0?1M
cacodyla e bu e , pH 7?2, o 3 h a 4 uC and pos - ixed in 1 %
OsO
4
o 2 h a 4 uC. Samples we e dehyd a ed in an ace one se ies
and embedded in Epon (epoxy embedding medium). Toluidine-
blue-s ained semi- hin sec ions (0?5mm hick) we e iewed in a
Lei z (A is oplan) ligh mic oscope.
RESULTS
Isola ion o Tn5-Mob mu an s showing al e ed
colony mo phology
S. edii HH103-1 was mu agenized wi h Tn5-Mob (Simon,
1984) and 80 000 Nm
R
ansconjugan s we e sc eened o
al e a ions in colony mo phology. One mu an , called
SVQ295, was u he in es iga ed. SVQ295 ha bou s only
one copy o Tn5-Mob, as indica ed by hyb idiza ion
expe imen s using a 0?9kbPs I in e nal agmen o he
kanamycin- esis ance gene o Tn5-Mob as a p obe (da a no
shown). I o ms colonies ha a e anslucen , p oduce
mo e slime and abso b less Congo ed han hose o wild-
ype s ain HH103-1. SVQ295 au o-agglu ina es when
g own in liquid TY, an indica ion ha his mu an migh
be al e ed in i s LPS, as epo ed o R.leguminosa um LPS
mu an s (P ie e , 1989). PAGE showed ha he LPS I and
LPS II egions o SVQ295 cul u es g own on solid TY
medium con ain bands ha mig a e as e han hose o i s
pa en al s ain HH103-1 (Fig. 1A, lanes 2 and 1 espec-
i ely). On he o he hand, he
1
H-NMR spec a ob ained
o he KPS om S. edii HH103 and om i s mu an
de i a i e SVQ295 appea o be e y simila (Fig. 2). The
LCO and plasmid p o iles o mu an s ain SVQ295 we e
also iden ical o hose o i s pa en al s ain HH103-1 (da a
no shown).
Mu an SVQ295 does no g ow in minimal medium (MMB)
unless he bac e ial cul u e is supplemen ed wi h hiamin
and adenine o wi h hiamin and AICA- iboside, an
in e media e o pu ine biosyn hesis.
Al hough he doubling ime o SVQ295 cul u es g own in
Fig. 1. LPS p o iles o s ains g own in TY medium, unless o he wise indica ed. (A) Lane 1, HH103-1; lane 2, SVQ295; lane
3, SVQ295(pMUS509); lane 4, SVQ295(pJN382A); lane 5, SVQ295; lane 6, SVQ295(pCOS110). (B) Lane 1, HH103-1;
lane 2, SVQ295; lane 3, SVQ295 (MMB medium supplemen ed wi h 0?5 mM AICA- iboside and hiamin); lane 4, SVQ295
(MMB medium supplemen ed wi h 0?1 mM AICA- iboside and hiamin); lane 5, SVQ295 (MMB medium supplemen ed wi h
adenine and hiamin); lane 6, SVQ295 (TY medium supplemen ed wi h 0?5 mM AICA- iboside); lane 7, SVQ295 (TY medium
supplemen ed wi h 0?1 mM AICA- iboside). (C) Lane 1, SVQ295; lane 2, SVQ526; lane 3, HH103-1.
1810 Mic obiology 149
A. M. Buendı´a-Cla e ı´a and o he s
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he p esence o AICA- iboside is longe (app ox. 9 h) han
ha in he p esence o adenine (app ox. 5 h), bo h bac e ial
cul u es (wi h AICA- iboside o adenine) eached he same
OD
660
a e 6 days o incuba ion (5610
8
bac e ia ml
21
).
Inocula ion o MMB medium supplemen ed wi h adenine
o AICA- iboside wi h SVQ295 p e iously g own in he
p esence o AICA- iboside showed again ha bac e ia g ew
as e in he p esence o adenine han in he p esence o
AICA- iboside (da a no shown). Mu an SVQ295 and i s
pa en al s ain HH103-1 showed he same doubling ime
(135 min) in comple e TY medium. The p esence o
adenine o AICA- iboside in SVQ295 cul u es did no
es o e he wild- ype LPS elec opho e ic p o ile (Fig. 1B).
Iden i ica ion o he mu a ed gene in SVQ295
Plasmid pSUP202 was used o clone he SVQ295 DNA
egion ha is adjacen o he IS50R o ansposon Tn5-Mob.
Following he s a egy desc ibed in Me hods, a plasmid
(pMUS375) ha con ained abou 16 kb o he SVQ295
DNA ha is adjacen o he IS50R o ansposon Tn5-Mob
was isola ed. A XhoI agmen o pMUS375 ha con ains
485 bp o he IS50R and abou 4 kb o SVQ295 DNA was
subcloned in o plasmid pBluesc ip . By using p ime pdad2,
a DNA agmen con aining he las 120 bp o he IS50R and
500 bp o he adjacen hizobial DNA was sequenced.
Compu e analysis o his sequence indica es homology
wi h he pu L gene o S. melilo i, which encodes a
59-phospho ibosyl o mylglycinamidine syn hase (FGAR
amido ans e ase, EC 6.3.5.3). This enzyme ca alyses he
s ep om FGAR (59-phospho ibosyl-N- o mylglycinamide)
o FGAM (59-phospho ibosyl-N- o mylglycinamidine) in
he biosyn he ic pu ine pa hway and i is essen ial o
biosyn hesis o bo h adenine and hiamin. These esul s a e
in acco dance wi h he auxo ophic pheno ype exhibi ed by
mu an SVQ295.
Symbio ic p ope ies o mu an SVQ295
SVQ295 induces Fix
2
pseudonodules on soybean c .
Williams. Mic oscopic analysis showed ha plan cells o
hese pseudonodules we e de oid o bac e ia. Nodula ion
assays we e ca ied ou o in es iga e whe he he addi ion o
hiamin and/o adenine o AICA- iboside o he plan
nu ien solu ion es o ed soybean nodula ion abili y o
mu an SVQ295. Adenine, hiamin, o bo h oge he , did
no enhance he symbio ic p ope ies o mu an s ain
SVQ295 whe eas 0?1o 0?5 mM AICA- iboside enabled
SVQ295 o o m in ec ed nodules ha did no ix ni ogen
and we e smalle han hose o med by HH103. Bac e ia
isola ed om hese nodules had he app op ia e an ibio ic
esis ance (S
R
Nm
R
) and we e auxo ophic o adenine
and hiamin.
The symbio ic p ope ies o SVQ295 we e also s udied in
o he leguminous plan s ha es ablish ni ogen- ixing
symbioses wi h he wild- ype s ain HH103-1. Mu an
SVQ295 ailed o nodula e Cajanus cajan,Indigo e a
inc o ia,Mac o yloma axilla e,Albizia lebbek,Kumme owia
s ipulacea and Neono onia wigh ii. I o med small ine ec i e
nodules on Mac op ilium a opu pu eum. Al hough in ec ed
nodule cells we e obse ed (Fig. 3D, E), bac e ia we e no
eco e ed om hese nodules. In con as , SVQ295 was able
o o m ni ogen- ixing nodules on Glycy hiza u alensis
(Fig. 3A) as demons a ed by ace ylene educ ion assays.
G. u alensis nodules con ained in ec ed plan cells wi h a la ge
cen ally posi ioned acuole (Fig. 3B, C) and bac e ia isola ed
om hese nodules showed he ma ke s o SVQ295.
Isola ion and cha ac e iza ion o he pu L gene
o S. edii HH103-1
Fo unknown easons, i is o en di icul o isola e cosmid
clones ha complemen HH103 mu an s by conjuga ional
ans e o he HH103 genomic lib a y o he ecipien
mu an . Because o his di icul y, an HH103 gene lib a y,
cons uc ed in cosmid pLAFR1, was ans e ed o R. e li
CE382, a de i a i e o CE3 con aining Tn5in pu L
(Newman e al., 1995). CE382 Tc
R
Km
R
ansconjugan s
we e selec ed on MMB aga and a cosmid named pMUS509
was isola ed om one o hem. Cosmid pMUS509 was
ans e ed (by ans o ma ion) o E. coli S17-1 and om
his dono s ain i was ein oduced (by conjuga ion) in o
R. e li CE382. T ansconjugan s ca ying pMUS509 did no
equi e he p esence o adenine and hiamin o g ow on
MMB medium and we e able o o m ni ogen- ixing
nodules on Phaseolus ulga is.
54321
d (p.p.m.)
Fig. 2.
1
H-NMR spec a (500 MHz) ob ained o he capsula
polysaccha ide om S. edii HH103 (A) and SVQ295 (B).
h p://mic.sgmjou nals.o g 1811
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Cosmids pMUS509, pCOS110 and pJN382A we e ans-
e ed o S. edii SVQ295. Cosmid pCOS110 ca ies he
pu YQL egion o R. e li and pJN382A is a de i a i e o
pCOS110 ca ying Tn5in pu L. SVQ295 ansconjugan s
ca ying pMUS509 o pCOS110 we e able o g ow on MMB,
demons a ing ha he R. e li pu L gene can complemen he
pu L mu a ion o S. edii HH103-1. SVQ295 ca ying
pMUS509, pCOS110 o pJN382A was es ed o nodula ion
abili y on soybean c . Williams. Plan s we e g own in he
p esence o absence o hiamin. Soybean plan s inocula ed
wi h SVQ295(pCOS110) o SVQ295(pMUS509) induced
ni ogen- ixing nodules, while hose inocula ed wi h
SVQ295(pJN382A) only o med Fix
2
pseudonodules.
These esul s demons a ed ha he mu a ion on he pu L
gene is esponsible o he symbio ic-de ec i e pheno ype o
mu an SVQ295. The p esence o hiamin in he plan
nu ien solu ion did no ha e any signi ican e ec on
bac e ial nodula ion.
A3?2kb BamHI agmen o cosmid pMUS509, which
posi i ely hyb idized o a p obe con aining 0?5 kb o he
SVQ295 DNA ha is adjacen o he ansposon IS50Rin
plasmid pMUS375, was subcloned in o pBluesc ip . F om
he esul an plasmid a KpnI–BamHI 2870 bp agmen was
sequenced (accession numbe AF275718). By ma ching his
sequence wi h ha ob ained om he pa ial sequencing o
pMUS375 using he IS50R p ime pdad2, i was possible o
posi ion he ansposon a nucleo ide 832 o he inal
2870 bp sequence.
The sequenced agmen con ains one ORF wi h a high
p obabili y o encoding p o ein, as indica ed by he Tes code
algo i hm. This ORF encodes a p o ein ha is homologous
o se e al Pu L p o eins o di e en o ganisms such as
S. melilo i,Meso hizobium lo i and Ag obac e ium
ume aciens (Table 2). pu L begins a posi ion 461 and
ex ends o 2129 bp, encoding a deduced polypep ide o
Fig. 3. Nodula ion pheno ype o SVQ295.
(A) Mo phology o nodules induced by
SVQ295 on Glycy hiza u alensis. Ba , 1 cm.
(B–E) Ligh mic oscopy o sec ions o
nodules induced on G. u alensis by
HH103-1 (B) and SVQ295 (C), and on
Mac op ilium a opu pu eum by HH103-1
(D) and SVQ295 (E). Magni ica ion: 636in
(B–D); 166in (E).
1812 Mic obiology 149
A. M. Buendı´a-Cla e ı´a and o he s
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743 amino acids wi h a p edic ed size o 79?1 kDa. The ORF
is p eceded by a pu a i e ibosome-binding si e, GAAGGA,
a posi ions 219/214. The sequence AGCGGCCGAACC-
GGCT a posi ions 2229/2214 o he pu a i e ATG s a
codon o he S. edii pu L gene shows simila i ies o he
Pu Box epo ed in Lac ococcus lac is (Kils up e al., 1998).
In addi ion, he sequence CGCAAAGCGCCCCCTGTTTGT-
GTTGCGGTTC a posi ions 290/259 om he ATG codon
esembles he E. coli Pu Box (GGCAAACGGTTTCGTC) as
de ined by Sampei & Mizobuchi (1989). DNA sequences
ups eam and downs eam o he iden i ied ORF do no
show clea homology o any known gene. The abundance o
s op codons in hese wo egions indica es ha hey should
no con ain any coding egion.
Se ological s udies on he LPS p oduced by
S. edii SVQ295
PAGE showed ha SVQ295 ca ying cosmid pMUS509
has an LPS p o ile ha is indis inguishable om ha o
HH103-1 (Fig. 4A, lanes 3 and 1 espec i ely).
Fou mAbs aised agains ee-li ing cul u es o HH103
(NB7-242.33) o nodule-de i ed bac e oids (NB3-4.72;
NB3-4.79 and NB6-228.22) and ha ecognize he same
pa e n o bands ha a e isible in sil e -s ained LPS gels o
S. edii HH103 we e es ed o hei eac i i y agains he
LPS o mu an SVQ295. Fig. 4 shows ha he ou mAbs
eac ed wi h he HH103-1 LPS bands co esponding o he
LPS I and LPS II egions (lane 1 o panels B–E) bu ailed o
ecognize any LPS band in mu an s ain SVQ295 (lane 2 o
panels B–E). SVQ295 ca ying pMUS509 o pCOS110
p oduced LPS ha we e ully ecognized by all he assayed
mAbs (lanes 3 and 4 o panels B–E).
Co-inocula ion expe imen s
We in es iga ed he e ec o co-inocula ing soybean plan s
wi h SVQ295 and he S. edii HH103 nodA mu an
SVQ116, which canno p oduce nodula ion ac o s
(LCOs) (Buendı
´a-Cla e ı
´ae al., 1996). Hence, he ino-
culum mix u e was composed o a mu an (SVQ295) ha
p oduces LCOs bu is unable o in ec soybean oo s and
ano he one (SVQ116) ha should be in ec i e bu is o ally
unable o igge nodule de elopmen . Soybean plan s
g owing in a plan nu i i e solu ion con aining adenine
we e also inocula ed wi h SVQ295 o wi h SVQ295 and
SVQ116.
As expec ed, single inocula ion wi h SVQ295 only induced
pseudonodules on soybean. In his expe imen , howe e ,
SVQ295 was able o induce non-ni ogen- ixing nodules on
soybean when adenine was added o he plan nu i i e
solu ion. Soybean plan s inocula ed wi h bo h mu an s (in
he p esence o absence o adenine) o med a mix u e o
pseudonodules and ue ni ogen- ixing nodules (Table 3).
No colonies we e o med when nodules we e di ec ly
c ushed on TY medium con aining s ep omycin (ch omo-
somal ma ke o SVQ295). All bac e ial isola es (900
colonies es ed) om nodules c ushed on TY medium
showed he ma ke s o SVQ116 (Ri
,Km
, S
s
and
p o o ophy). These esul s demons a e ha , ega dless
o he p esence o absence o adenine, only he p o o ophic
LCO
2
mu an SVQ116 was able o pene a e in o he
plan cells.
DISCUSSION
P e ious epo s ha e clea ly shown ha pu ine auxo ophic
mu an s o (Sino)Rhizobium a e symbio ically de ec i e,
Table 2. Compa ison o HH103 Pu L p o ein wi h Pu L p o eins om di e en o ganisms.
O ganism Iden i y (%) Simila i y (%) Amino acids o e lapping Leng h o Pu L p o ein Accession no.
Sino hizobium melilo i 92 96 743 743 Q92PH7
Meso hizobium lo i 81 90 739 743 Q98NN7
Ag obac e ium ume aciens 86 91 769 775 AAL42846
Zymomonas mobilis 61 73 745 734 AAF23789*
Bacillus sub ilis 48 61 725 742 AAA22679*
Synechocys is sp. 48 61 741 777 BAA16646
Lac obacillus lac is 46 61 731 739 AAD12626
S ep omyces coelicolo 42 59 729 752 CAB56359
Mycobac e ium ube culosis 42 56 748 754 P54876
Campylobac e jejuni 44 62 701 728 CAB73212
Lac obacillus casei 43 58 744 741 P35852
Me hanococcus jannaschii 41 56 737 733 Q58660
Esche ichia coli 25 41 771 1295 P15254
Salmonella yphimu ium 24 40 770 1295 AAB08888
Vib io chole ae 25 41 667 1297 AAF94031
Pseudomonas ae uginosa 24 40 685 1298 AAG07150
*De ined in he p o ein lib a y as p o eins encoded by he pu Q gene.
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o ming Fix
2
pseudonodules ha show cen ally loca ed
ascula bundles (Pankhu s & Schwinghame 1974;
Dena ie
´e al., 1976; Pain, 1979; Noel e al., 1984, 1988;
Newman e al., 1992, 1995; Djo dje ic e al., 1996).
A possible explana ion o he linking o pu ine auxo ophy
and symbio ic de iciency is ha , du ing nodule o ma ion,
adenine migh ac as a p ecu so o cy okinins, which a e
N-6 subs i u ed de i a i es o adenine (Pankhu s &
Schwinghame , 1974; Pain, 1979). This possibili y does
no exclude ha adenine auxo ophs migh su e o he
al e a ions ha , o any eason, could p e en no mal
nodule de elopmen . In ac , pu ine auxo ophs usually
exhibi pleio opic pheno ypes ha indica e he occu ence
o mul iple al e a ions o he bac e ial physiology. Fo
ins ance, R. e li pu ine auxo ophs exp essed, in ee-li ing
Table 3. Co-inocula ion o soybean plan s c . Williams wi h S. edii mu an s SVQ116 and SVQ295 in he p esence o
absence o adenine
Inoculan (s) Nodula ion* Appea ance o plan sDMa ke s o he nodule isola esd
SVQ116 Nod
2
Yellow –
SVQ295 Pseudonodules Yellow –
SVQ295 +ade§ Pseudonodules and small nodules (8) Yellow S
Km
, Ade
2
Thi
2
(28)
SVQ116 +SVQ295 Pseudonodules and nodules (41) G een Ri
Km
S
s
, Ade
+
Thi
+
(50)
SVQ116 +SVQ295 +ade§ Pseudonodules and nodules (43) G een Ri
Km
S
s
, Ade
+
Thi
+
(40)
HH103-1 Fix
+
(160) G een S
Km
s
, Ade
+
Thi
+
(4)
*Six plan s we e used o each ea men . The numbe s in pa en heses indica e he mean numbe o nodules pe soybean plan . Plan s we e
analysed 82 days a e inocula ion.
DNi ogenase ac i i y o g een soybean plan s was con i med by ace ylene educ ion assay.
dThe numbe s in pa en heses indica e he numbe o nodules analysed. F om each nodule 10 colonies we e checked o an ibio ic esis ance and
auxo ophy ma ke s.
§Fa
˚h aeus nu i i e solu ion was supplemen ed wi h 0?8mg adenine ml
21
.
Fig. 4. Sil e s aining (A) and immunos aining (B–E) o LPS p o iles om S. edii s ains using he mAbs NB3-4.72 (B),
NB3-4.79 (C), NB-6-228.22 (D) and NB7-242.33 (E). Lanes: 1, HH103-1; 2, SVQ295; 3, SVQ295(pMUS509); 4,
SVQ295(pCOS110).
1814 Mic obiology 149
A. M. Buendı´a-Cla e ı´a and o he s
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condi ions, highe le els o he ixNOQP ope on, which
codes o he symbio ic cy och ome e minal oxidase ccb
3
(Sobe on e al., 1997). Mo eo e , Rhizobium sp. NGR234
pu Y and pu F mu an s exhibi ed mul iple di e ences in
gene exp ession when compa ed o he pa en al wild- ype
s ain (Gue ei o e al., 1998).
In his in es iga ion we ha e s udied an auxo ophic mu an
o S. edii HH103 ha was isola ed in a sc een o he
appea ance o al e a ions in colony mo phology. Mu an
SVQ295 equi es adenine and hiamin o g ow h in
minimal media, which is in ag eemen wi h he ac ha
he eac ion ca alysed by he Pu L enzyme occu s be o e
AICAR in he biosyn he ic pa hway o pu ines (Neuha d &
Nygaa d, 1987). In addi ion, SVQ295 can also g ow in
minimal media i he supplemen a ion wi h adenine is
eplaced by he pu ine p ecu so AICA- iboside. Al hough
SVQ295 cul u es g own in he p esence o 0?5 mM AICA-
iboside showed a longe doubling ime han ha obse ed
in MMB medium wi h adenine, he inal op ical densi y was
he same in bo h bac e ial cul u es (wi h AICA- iboside o
adenine). Fully g own SVQ295 cul u es in he p esence o
AICA- iboside did no con ain ade
+
e e an s, since
bac e ial subcul u es ailed o g ow in MMB medium
de oid o adenine o AICA- iboside. Hence, we conclude
ha AICA- iboside can indeed ac as a pu ine sou ce o pu
mu an s. P e ious epo s showed ha pu mu an s o
S. edii HH303 and R. leguminosa um b . iciae 128C53
g ew poo ly 18 h a e he addi ion o AICA- iboside
(Newman e al., 1994). Ou esul s also suppo his
obse a ion, since an ini ial popula ion o app oxima ely
4610
5
bac e ia ml
21
in MMB supplemen ed wi h AICA-
iboside did no show any inc ease a e 5 h incuba ion and
only inc eased o 7610
5
bac e ia ml
21
a e 18 h. Hence,
he la ency pe iod exhibi ed by SVQ295 cul u es in he
p esence o AICA- iboside and hei long doubling ime
may explain he poo g ow h o hese pu mu an s du ing
hei i s hou s o incuba ion in he p esence o AICA-
iboside. Howe e , a e 18 h incuba ion, a pu F mu an o
R. e li did no show poo g ow h in minimal medium
supplemen ed wi h AICA- iboside (Newman e al., 1994).
Mu an SVQ295 ailed o nodula e six di e en legumes
(see Resul s), and o med pseudonodules on G. max.On
Mac op ilium a opu pu eum, small ine ec i e nodules we e
o med. Bac e ial elease in M. a opu pu eum nodules was
only obse ed in a ew clus e ed plan cells (Fig. 3E). In
Glycy hiza u alensis, howe e , bac e ial elease was much
mo e ex ended h ough he nodule issue and ni ogenase
ac i i y was de ec ed (Fig. 3C). This a iable symbio ic
pheno ype o SVQ295 migh be due o he di e en ial abili y
o each legume hos in supplying pu ine, o pu ine
p ecu so s, o he bac e ia du ing he nodula ion p ocess.
The ac ha G. u alensis o ms inde e mina e nodules
(Fig. 3A) is no ele an o he abili y o SVQ295 o in ec
nodule cells, since Albizia lebbek also o ms inde e mina e
nodules (wi h he pa en al s ain HH103) bu ails o
nodula e wi h he pu L mu an .
P e ious epo s ha e clea ly shown ha he addi ion o
AICA- iboside o he plan nu i i e solu ion p omo es
nodula ion abili y o di e en hizobial pu mu an s
(Newman e al., 1994, 1995; Djo dje ic e al., 1996). In
addi ion o suppo ing hese obse a ions ou esul s also
show ha , in long- e m expe imen s (82 days), he addi ion
o adenine can also p omo e he o ma ion o small soybean
nodules by mu an SVQ295. This la e nodula ion appea s o
be he consequence o he exogenous addi ion o adenine o
he plan oo medium because, in he absence o adenine,
soybean plan s inocula ed wi h SVQ295 only o med
pseudonodules (Table 3).
Mu an SVQ295 ca ies a ansposon Tn5-Mob inse ion
be ween nucleo ides 831 and 832 o he sequenced DNA
agmen , which co esponds o he 59end o a 2690 bp ORF
showing homology o he pu L gene o S. melilo i. This ORF
would encode a pu a i e polypep ide o 743 amino acids,
clea ly sho e han he E. coli Pu L p o ein (1295 amino
acids). The assump ion ha SVQ295 is mu a ed in i s pu L
gene is suppo ed by he ac ha he mu an was
complemen ed by cosmid pCOS110 (i ca ies R. e li
pu YQL genes), bu no by cosmid pJN382A (a de i a i e
o pCOS110 ca ying a Tn5inse ion in he pu L gene).
In E. coli, he pu L gene encodes a 59-phospho ibosyl-
o mylglycinamidine syn hase (FGAR amido ans e ase)
ha ca alyses he con e sion o 59-phospho ibosyl-N-
o mylglycinamide in o 59-phospho ibosyl-N- o myl-
glycinamidine (Sampei & Mizobuchi, 1989). In Bacillus
sub ilis and Lac obacillus casei, howe e , his enzyme is
o med by he p oduc s o wo di e en genes, pu Q and
pu L (Ebbole & Zalkin, 1989; Pel onen & Ma
¨n sa
¨la
¨, 1999).
The Pu L enzyme in E. coli has h ee di e en domains, I, II
and III, which ha e been assigned as po en ial ATPase,
iosephospha e-isome ase-like isome ase, and glu aminase,
espec i ely (Sampei & Mizobuchi, 1989). The E. coli Pu L
domain III aligns wi h he B. sub ilis and L. casei Pu Q
p o ein, while domains I and II align wi h he Pu L p o ein
o B. sub ilis,L. casei and S. edii HH103. This indica es ha
S. edii HH103 migh also need a leas wo di e en
genes o he o ma ion o a unc ional FGAR amido ans-
e ase. O he bac e ia, such as Me hanococcus jannaschii,
Mycobac e ium ube culosis and Synechocys is sp. also appea
o ha e a mul i-subuni o m o he FGAR amido ans e ase
enzyme (Bul e al., 1996; Jackson e al., 1996; Kaneko
e al., 1996). In all hese cases, in which he enzyme appea s
o be composed o di e en subuni s, he ORF de ined as
pu L encodes a polypep ide o 705–777 amino acids, which
is clea ly sho e han he FGAR amido ans e ase
(1295 amino acids) encoded by he pu L gene o E. coli
(Table 2).
The L. casei and B. sub ilis pu Q and pu L genes belong o an
ope on composed o a clus e o o he pu ine genes, while
he E. coli pu L gene cons i u es a single ansc ip ional uni
which is independen o o he pu ine genes. The pu L
o S. edii HH103 also appea s o cons i u e a single
ansc ip ional uni , since pu a i e ORFs we e no ound
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