RESEARCH ARTICLE
Summa y.T ansposon Tn5-Mob mu agenesis allowed he selec ion o a Sino hizobium edii HH103 mu an de i a i e
(SVQ 292) ha equi es he p esence o u acil o g ow in minimal media. The mu a ed gene, py F, codes o an o o idine-5´-
monophospha e deca boxylase (EC 4.1.1.23). Mu an SVQ 292 and i s pa en al p o o ophic mu an HH103 showed simila
Nod- ac o and lipopolysaccha ide p o iles. The symbio ic p ope ies o mu an SVQ 292 we e se e ely impai ed wi h all
legumes es ed. Mu an SVQ 292 o med small ine ec i e nodules on Cajanus cajan and abno mal nodules (pseudonodules)
unable o ix ni ogen on Glycine max (soybean), Mac op i lium a opu pu eum, Indigo e a inc o ia, and Desmodium cana-
dense. I also did no induce any mac oscopic esponse in Mac o yloma axilla e oo s. The symbio ic capaci y o SVQ 292 wi h
soybean was no enhanced by he addi ion o u acil o he plan nu i i e solu ion. [In Mic obiol 2007; 10(3):169-176]
Key wo ds: Sino hizobium edii HH103 · Mac o yloma axilla e ·py F · symbio ic de ec s · soybean · legumes
In oduc ion
In he symbio ic ela ionship ha is es ablished be ween hi-
zobia and hei hos legumes, a complex se o molecula
in e ac ions akes place du ing he de elopmen and main e-
nance o ni ogen- ixing nodules on he plan oo s. Inside
he nodules, hizobia p o ide he plan wi h an assimilable
sou ce o ni ogen. In e u n, he legume supplies compounds
ha can be used by he bac e ia as sou ces o ca bon and
ene gy [14]. Analyses o he symbio ic p ope ies o aux-
o ophic hizobial mu an s a e one way o iden i y some o
he me abolic unc ions ha a e necessa y o es ablish a suc-
cess ul symbio ic ela ionship.
Symbio ically-de ec i e auxo ophic mu an s ha e been
isola ed om se e al as -g owing s ains o hizobia, and
hei e ec s in he nodula ion p ocess a e highly a iable
[ e iewed in 14]. Auxo ophic mu an s o leucine, his idine,
o ni hine, aspa ic acid, o cys eine induce ine ec i e ni o-
gen- ixing nodules. Howe e , he nodules induced by
Sino hizobium melilo i mu an s a ec ed in glu amine
biosyn hesis a e ni ogen- ixing, which indica es ha his
amino acid is no essen ial o symbio ic ni ogen ixa ion in
al al a plan s. Fu he mo e, S. melilo i auxo ophic mu an s
o glycine a e mo e e ec i e in ni ogen ixa ion han hei
pa en al p o o ophic s ain.
INTERNATIONAL MICROBIOLOGY (2007) 10:169-176
DOI: 10.2436/20.1501.01.24 ISSN: 1139-6709 www.im.mic obios.o g
*Co esponding au ho : A.M. Buendía-Cla e ía
Depa amen o de Mic obiología, Facul ad de Biología
Uni e sidad de Se illa
A . Reina Me cedes, 6
41012 Se illa, Spain
Tel. +34-954557121. Fax +34-954557830
E-mail: [email p o ec ed]
§These wo au ho s con ibu ed equally o his wo k.
Juan C. C espo-Ri as,1§ Isabel Ma ga e ,1§ F ancisco Pé ez-Mon año,1
F ancisco J. López-Baena,1José M. Vina dell,1F ancisco J. Olle o,1
Ja ie Mo eno,2José E. Ruiz-Sainz,1Ana M. Buendía-Cla e ía1*
1Depa men o Mic obiology, Facul y o Biology, Uni e si y o Se illa, Se illa, Spain. 2Depa men o Cellula Biology,
Facul y o Biology, Uni e si y o Se illa, Se illa, Spain
Recei ed 9 Ap il 2007 · Accep ed 26 July 2007
A
py F
auxo ophic mu an o
Sino hizobium
edii
HH103 impai ed in i s symbio ic
in e ac ions wi h soybean and o he legumes
170 INT. MICROBIOL. Vol. 10, 2007
Pu ine auxo ophs (pu mu an s) o mos hizobia species
a e unable o e ec i ely nodula e hei hos plan s [7,12,14].
While adenine auxo ophs o Rhizobium leguminosa um b .
iciae a e unable o nodula e pea plan s, hose o S. melilo i
o m nodules on al al a ha ail o ix ni ogen [14]. Apu ine
auxo ophic mu an o 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 in ec ion h eads (inwa d- acing ubula
s uc u es h ough which hizobia pene a e he oo ) we e
no obse ed [14]. A pu L mu an o S. edii HH103 induces
pseudonodules on soybean oo s bu i is s ill able o induce
a ew ni ogen- ixing nodules on Glycy hiza u alensis [7].
In con as , a pu F mu an o R. leguminosa um b . i olii L1
has been desc ibed o e ain he capaci y o induce ully
de eloped ni ogen- ixing nodules on i s legume hos [24].
A py imidine auxo ophic R. e li mu an , unable o syn-
hesize u acil, induces small ine ec i e pseudonodules on
Phaseolus ulga is (bean). Simila ly, u acil auxo ophic
de i a i es o S. melilo i 2001 and S. edii HH303 only
induce pseudonodules on al al a and soybean, espec i ely
[14]. S. melilo i Rmd201 py imidine auxo ophic mu an s
a ec ed in ca bamoyl phospa e syn he ase (ca ) and in
py imidine biosyn hesis (py ) induce whi e ine ec i e nod-
ules on al al a plan s. The nodules induced by py mu an s
a e mo e ad anced han hose induced by ca mu an s [22].
In some cases, hizobia equi e a pa icula in e media e
o a biosyn he ic pa hway (bu no he end p oduc s) o es ab-
lish success ul symbio ic in e ac ions. S udies wi h yp o-
phan auxo ophic mu an s o S. melilo i sugges ed ha
an h anila e syn hesis, a he han yp ophan, is equi ed o
bac e oid de elopmen in al al a nodules [14]. In o ma ion
abou he impo ance o he di e en me abolic pa hways o
he Sino hizobium edii symbio ic capaci y is sca ce. In e -
es ingly, S. edii s ains show a b oad hos - ange o nodula-
ion, a cha ac e is ic ha acili a es s udies o he impac o
any pa icula mu a ion on he bac e ial symbio ic capaci y
wi h legumes in which de e mined o unde e mined nodules
a e o med [8]. In his wo k, we cha ac e ized gene ically and
symbio ically a u acil auxo ophic mu an o S. edii HH103
a ec ed in he py F gene.
Ma e ial and me hods
S ains, media, and gene ic manipula ions. 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 [2], yeas manni ol (YM) [23], B– [20], o MMB [1] min-
imal medium a 28ºC. Auxo ophic s ains we e pla ed on MMB solidi ied
wi h 1.5% (w/ ) aga ose. Esche ichia coli was cul u ed in Lu ia-Be ani
(LB) medium [16] a 37ºC. When equi ed, he media we e supplemen ed
wi h he app op ia e an ibio ics, as desc ibed by Lam abe e al. [10], o wi h
u acil a 12 µg/ml.
Plasmids we e ans e ed by conjuga ion as desc ibed by Simon [17].
Tn5-Mob mu agenesis o S. edii s ain SVQ269 was ca ied ou using he
suicide plasmid pSUP5011 [17]. Sino hizobium plasmid p o iles we e
ob ained using he Ekha d me hod, as desc ibed by Olle o e al. [13]. Those
subs ances equi ed by he auxo ophic mu an SVQ292 o g ow in minimal
media we e iden i ied as desc ibed by Be inge e al. [3]. DNA was manip-
ula ed acco ding o he gene al p o ocols o Samb ook e al. [16]. The p ime
pdad2 (5′AGATTTAGCCCAGTCG 3′), loca ed inside he IS50R o ans-
poson Tn5-Mob, was used as a p ime o sequence he hizobial DNA adja-
cen o he igh si e o he inse ed ansposon in mu an s ain SVQ292.
The UWGCG p og am was used o basic DNA sequence analysis and
assembly. The NCBI ORF-Finde [h p://www.ncbi.nlm.nih.go /go /
go .h ml] and BLAST [h p://www.ncbi.nlm.nih.go /BLAST/] p og ams
CRESPO-RIVAS ET AL
Table 1. Bac e ial s ains and plasmids used in his wo k
S ains De i a ion and ele an p ope ies Sou ce o e e ence
Sino hizobium edii
SVQ269 HH103 Ri [6]
SVQ292 SVQ269 py F::Tn5-Mob This wo k
Esche ichia coli
HB101 Res ic ion-minus, ecA backg ound, S [4]
Plasmids
pBluesc ip Cloning ec o , Ap S a agene®
pSUP202 pBR325 con aining he Mob egion subcloned in Sau3A, Ap ,
Cm , Tc
[18]
pSUP5011 Suicide plasmid ca ying he Tn5-Mob ansposon [19]
pMUS390 pSUP202 de i a i e plasmid ha bo ing abou 0.5 kb o SVQ292
DNA adjacen o igh o Tn5-Mob ansposon This wo k
pMUS344 Cosmid pLAFR1 ca ying he py F gene o S. edii HH103 This wo k
171
INT. MICROBIOL. Vol.10, 2007
we e used o open eading ame (ORF) iden i ica ion and homology
sea ches, espec i ely. The Clus alW and Box-shade p og ams we e used o
align pep ide sequences. Nucleo ide and/o amino acid sequence da a a e o
be ound a he EMBL da abase as accession no. AF311321.
In i o isola ion o SVQ292 mu a ion. The mu a ed gene in
SVQ292 was cloned ollowing he me hod desc ibed by Madinabei ia e al.
[11] and was used o cons uc plasmid pMUS390. This plasmid ha bo s
abou 0.5 kb o he SVQ292 DNA adjacen o he IS50R o he inse ed
ansposon.
Plan assays. Nodula ion assays on Glycine max (L.) Me . c . Williams,
Cajanus cajan (L.) Millsp., Desmodium canadense (L.) DC, Mac op ilium
a opu pu eum (Moc.& Sessé ex DC) U b, Indigo e a inc o ia (L.), and
Mac o yloma axilla e (E. Mey) Ve dc, we e ca ied ou as desc ibed by
Buendía-Cla e ía e al. [6]. Ge mina ed seeds we e ans e ed o Leona d
ja s con aining s e ilized e miculi e supplemen ed wi h Fåh aeus nu ien
solu ion [23]. Each Leona d ja con ained wo plan s. Each plan was inocu-
la ed wi h abou 108bac e ia and hen g own o 40 days wi h a 16 h-pho-
ope iod a 25ºC in he ligh and 18ºC in he da k [8]. Ni ogen ixa ion was
de e mined by ace ylene educ ion assay [5]. Plan ops we e d ied a 80ºC
o 48 h and weigh ed. Bac e ia we e isola ed om su ace-s e ilized nod-
ules as p e iously epo ed [10]. When equi ed, he Fåh aeus plan nu i i e
solu ion was supplemen ed wi h u acil a 12 µg/ml.
Lipo-chi in oligosaccha ide (LCO) de ec ion. Bac e ia we e
g own in 5 ml o B–medium in he p esence o he induce na ingenin (3.6
µM) and N-ace yl[14C] glucosamine. The 14C-labeled LCO we e analyzed by
e e se hin-laye ch oma og aphy, as desc ibed by Spaink e al.[20].
Lipopolysaccha ide SDS/PAGE analysis. To in es iga e lipopo-
lysaccha ide (LPS) p o iles, bac e ial cul u es we e g own on solid TY
medium. Bac e ial cells we e washed in 0.9% NaCl and pelle ed by cen i u-
ga ion. T ea men s applied o bac e ial pelle , elec opho esis o c ude bac-
e ia ex ac s, and he sil e -s aining p ocedu es we e done as desc ibed by
Buendía-Cla e ía e al.[7].
Mic oscopic s udies. Nodules and pseudonodules we e ixed in 4%
( / ) glu a aldehyde p epa ed in 0.1 M cacodyla e bu e , pH 7.2, o 3 h a
4°C and pos - ixed in 1% OsO4( / ) o 2 h a 4°C. 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 (0.5 µm hick) we e iewed in a
Lei z (A is oplan) ligh mic oscope.
Resul s
Isola ion and cha ac e iza ion o a
S. edii
HH103 Tn
5
-Mob auxo ophic mu an .S. edii
SVQ269 (= HH103 Ri ) was subjec ed o Tn5-Mob mu age-
nesis using plasmid pSUP5011, a e which ansconjugan
colonies we e selec ed on TY supplemen ed wi h i ampicin
and kanamycin. In o de o selec auxo ophic mu an s,
ansconjugan s we e eplica-pla ed on MMB solidi ied wi h
aga ose. One o he colonies ha ailed o g ow in MMB was
selec ed o u he in es iga ion (auxo ophic mu an
SVQ292). SVQ292 was able o g ow in MMB i he minimal
medium was supplemen ed wi h u acil.
In o de o iden i y and subclone he gene mu a ed in
SVQ292, plasmid pMUS390 was ob ained as desc ibed in
Ma e ial and Me hods. This plasmid ha bo s abou 0.5 kb o
he hizobial DNA adjacen o he IS50R o he Tn5-Mob
inse ed in mu an SVQ292. An EcoRI/XhoI 0.9-kb agmen
o pMUS390 was cloned in o pBluesc ip and abou 450 bp
we e sequenced by using p ime pdad2. Compu e analysis
o his sequence indica ed homology wi h he py F gene o S.
melilo i, which encodes an o o idine-5´-monophospha e
deca boxylase (EC 4.1.1.23). This enzyme ca alyzes he inal
s ep in py imidine biosyn hesis, he con e sion o o o idine-
5´-monophospha e (OMP) o u idine-5´-monophospha e.
S. FREDII PYRIMIDINE MUTANT
In . Mic obiol.
Fig. 1. Toluidine blue-s ained semi hin sec ions o pseudonodule induced by he py F mu an SVQ292 (A) and by he pa en al s ain SVQ269 (B) on soy-
bean Glycine max c . Williams. The a ows ma k nodule cells ull o bac e oids. (Magni ica ion, ca. 16×)
172 INT. MICROBIOL. Vol. 10, 2007
SVQ292 only induced pseudonodules on soybean c .
Williams ha did no ix ni ogen. Mic oscopic analysis
showed ha plan cells o hese pseudonodules we e de oid
o bac e ia, while many cells o nodules induced by he
pa en al s ain SVQ269 we e illed o hizobia (Fig. 1A,B).
This symbio ic de ec was used o cons uc a genomic
lib a y o he wild- ype s ain HH103 (in cosmid pLAFR1)
and hen o isola e he wild- ype py F gene. The genomic
lib a y was ans e ed “en masse” o mu an SVQ292, and
he e acycline- esis an ansconjugan s we e used o inocu-
la e soybean c . Williams plan s. Soybean oo s de eloped
many pseudonodules and a ew ni ogen- ixing nodules. All
bac e ia e-isola ed om he e ec i e nodules we e Tc (p es-
ence o cosmid pLAFR1), Nm , and Km (p esence o Tn5-
Mob) and con ained he same cosmid. This cosmid, named
pMUS344, was isola ed, in oduced in o E. coli HB101, and
hen e- ans e ed o he py F mu an SVQ292. S ain
SVQ292 (pMUS344) was able o g ow in MMB wi hou
u acil and o med no mal ni ogen- ixing nodules on soy-
bean.
A 1.5-kb Ps I agmen o cosmid pMUS344, which pos-
i i ely hyb idized o he 0.9-kb EcoRI/XhoI agmen o plas-
mid pMUS390, was subcloned in o pBluesc ip . The 1511 bp
sequence appea s in he EMBL nucleo ide da a lib a y as
accession numbe AF311321. By ma ching his sequence
wi h ha ob ained by pa ial sequencing o pMUS390 using
he IS50R p ime pdad2, i was possible o loca e he inse -
ion o he ansposon Tn5-Mob a nucleo ide 955 o he
1511-bp sequence.
The sequenced agmen comp ised one comple e and
wo pa ial ORFs, each wi h he same pola i y and wi h a
high p obabili y o encoding p o eins, as indica ed by he
Tes code algo i hm. The le side o he sequenced agmen
con ained he 3´ agmen o a pu a i e ORF ha is 87.5%
iden ical o smc00413, a pu a i e S. melilo i gene o unknown
unc ion (GenBank accession no. AL591783). The pu a i e
p o ein encoded by his HH103 ORF was 66% iden ical o
p o ein AGR_C_57 o Ag obac e ium ume aciens C58
(NP_353329) and con ained he conse ed domain GpmB,
which is cha ac e is ic o uc ose 2,6 biphospha ases (EC
3.1.3.46).
The second ORF was 85% iden ical o he py F gene o
S. melilo i. This ORF began a posi ion 397 and ex ended o
702 bp, encoding a deduced polypep ide o 234 amino acids
wi h a p edic ed molecula mass o 24.5 kDa. In mu an
SVQ292, he Tn5-Mob ansposon was inse ed 558 nucle-
o ides a e he s a codon o he py F gene. A posi ion
–79/–66 he e is a pu a i e GGN10GC mo i (GGcaa c c
GC), desc ibed as a p omo e [21]. The pe cen ages o iden-
i y be ween he S. edii HH103 Py F p o ein and hose o di -
e en hizobia and o he g am-nega i e bac e ia a e shown in
Table 2. In addi ion, alignmen o he Py F p o eins o S. edii
HH103 and di e en hizobial s ains is shown in Fig. 2.
The inal 175 nucleo ides (posi ions 1323–1511) co e-
sponded o he 5´ egion o a pu a i e ORF ha is 82% iden-
ical o he S. melilo i gene smc00411, which encodes a hypo-
he ical p o ein o 95 amino acids. In he S. edii sequence,
he in e genic egion be ween py F and sm00411 spans 221
CRESPO-RIVAS ET AL
Table 2. Compa ison o HH103 Py F p o ein wi h Py F p o eins om di e en bac e ia
Bac e ial s ains Iden i y (%) Simila i y (%) Leng h o he Py F
p o ein (amino acids) Amino-acid o e lap Accession numbe
Sino hizobium melilo i 86 94 234 233 CAC41769
Sino hizobium medicae 83 91 232 231 ABR62357
Meso hizobium sp. BNC1 79 89 249 223 ZP_00194064
Rhizobium leguminosa um b . iciae 79 89 235 228 CAK05821
Rhizobium e li 78 89 235 228 ABC89136
Ag obac e ium ume aciens 77 88 255 225 AAK86113
Meso hizobium lo i 74 87 237 225 BAB51336
B ucella suis 71 85 238 227 AAN31018
B ucella meli ensis 70 85 259 227 AAL53180
Ba onella bacilli o mis 68 84 235 225 Q44843
B ady hizobium japonicum 54 72 237 229 CAB91880
Vib io chole ae 40 60 231 215 AAF95059
Pseudomonas ae uginosa 38 57 232 231 CAA46564
Esche ichia coli 36 55 245 227 NP_415797
173
INT. MICROBIOL. Vol.10, 2007
nucleo ides, ins ead o he 106 nucleo ides p esen in he co -
esponding egion o S. melilo i.
Symbio ic p ope ies o mu an SVQ292 on
se e al legumes. S. edii HH103 is conside ed a b oad
hos - ange nodula ion s ain. Thus, we s udied whe he he
mu a ion in py F could a ec he symbio ic capaci y o HH103
wi h di e en legumes, as i did o soybean. Mu an SVQ292
o med pseudonodules wi h Glycine max c . Williams, M. a o-
pu pu eum, I. inc o ia, and D. canadense, and ine ec i e
nodules wi h C. cajan. Inocula ion o M. axilla e wi h mu an
SVQ292 did no induce any mac oscopic oo esponse.
Plan es s we e ca ied ou o de e mine whe he he p es-
ence o u acil in he plan nu i i e solu ion would comple-
S. FREDII PYRIMIDINE MUTANT
In . Mic obiol.
Fig. 2. Alignmen o he Py F p o eins o di e en hizobia. S , Sino hizobium edii; Sm, S. melilo i; Sme, S. medicae; Msp, Meso hizobium sp.; Rl ,
Rhizobium leguminosa um b . iciae; Re, Rhizobium e li; A , Ag obac e ium ume aciens; Ml, Meso hizobium lo i.

174 INT. MICROBIOL. Vol. 10, 2007 CRESPO-RIVAS ET AL
men he symbio ic de ec s o mu an SVQ292. The p esence
o u acil did no enhance he symbio ic capaci y o SVQ292
wi h any o he legumes es ed. Since SVQ292 was symbio -
ically de ec i e, we decided o in es iga e whe he his
mu an was al e ed ei he in symbio ic signals (such as LCO
and LPS) o in i s plasmid p o ile. The LCO and LPS p o iles
p oduced by SVQ292 we e simila o hose obse ed o he
pa en al s ain SVQ269, as demons a ed by hin-laye ch o-
ma og aphy and SDS-PAGE, espec i ely (da a no shown).
Howe e , plasmid aga ose elec opho esis showed ha ,
appa en ly, SVQ292 lacked a c yp ic plasmid o abou 85 MDa
(Fig. 3).
Discussion
Pu ine and py imidine auxo ophic hizobial mu an s a e usu-
ally unable o nodula e hei hos plan s o o induce ni ogen-
ixing nodules [7,14]. Vinee ha e al. [22] ob ained di e en
py imidine auxo ophs o S. melilo i ha induce whi e ine -
ec i e nodules on al al a plan s. These epo s sugges ha
he exp ession o some pu ine and py imidine biosyn he ic
genes is essen ial o an e ec i e symbiosis o hizobia wi h
hos plan s.
We cloned and sequenced a 1.5-kb Ps I DNA agmen
ha con ained he py F gene o S. edii HH103. This DNA
agmen ha bo ed h ee ORFs (5´ sm00413-py F-smc00411
3´) wi h he same o ganiza ion as he co esponding ORFs o
S. melilo i. The only di e ence de ec ed was in he in e genic
egion be ween py F and smc00411. In S. melilo i, his egion
spans 106 nucleo ides. O hese, only he six immedia ely
a e py F and he 54 p eceding smc00411 a e conse ed in
HH103. Mo eo e , be ween hese wo conse ed agmen s,
HH103 ha bo s 161 addi ional nucleo ides wi hou homolo-
gy o any known DNA sequence. In M. lo i, a gene homolo-
gous o smc00411 is loca ed 17 nucleo ides a e he end o
py F. Howe e , he m l7505 gene (NC_002678), which is
homologous o smc00413, is a a dis ance o 3000 kb om
he o he wo genes.
In his wo k, we ound ha s ain SVQ292, a py F mu an
o S. edii HH103 ha equi es u acil o g ow, induced Fix–
pseudonodules on plan s—including Glycine max c .
Williams and e en Mac o yloma axilla e— ha we e e ec-
i ely nodula ed by he pa en al s ain. These esul s sugges -
ed ha in S. edii HH103 he py F gene, which encodes o o-
idine-5´-monophospha e deca boxylase, is essen ial o he
es ablishmen o an e ec i e symbiosis be ween he bac e i-
um and i s hos s. The symbio ic impai men wi h M. axilla e
should occu a he e y ea ly s ages o nodula ion, since nei-
he pseodunodules no any o he mac oscopic oo espons-
es o he p esence o SVQ292 we e obse ed. Pseudonodules
induced by SVQ292 on Williams soybean we e de oid o
bac e ia (Fig. 1A). Simila ly, py imidine auxo ophs o S.
melilo i 2011 induced whi e ine ec i e nodules in al al a
plan s. The al al a nodules induced by S. melilo i py E/py F
mu an s a e mo e de eloped han hose induced by a py C
mu an , and he la e a e mo e ad anced han hose induced
by ca mu an s [22]. In he py imidine biosyn he ic pa hway,
enzymes encoded by ca genes ac ea lie han Py enzymes.
All hese indings indica ed ha he py imidine biosyn he ic
pa hway, o a pa icula in e media e, is essen ial o nodule
mo phogenesis. Also, he impai ed nodule de elopmen
caused by he auxo ophy a ies acco ding o he posi ion a
which he biosyn he ic pa hway is blocked.
The symbio ic de ec o SVQ292 was no e e ed by he
addi ion o u acil o he nu i i e plan solu ion. Simila
esul s we e desc ibed o py imidine auxo ophic mu an s o
R. e li CFN42, S. edii HH303, and S. melilo i Rmd2, in
which he addi ion o he plan nu ien medium o u acil o
any o he in e media e in he biosyn he ic py imidine pa h-
way does no es o e he symbio ic de ec s [14,22]. Howe e ,
in di e en hizobial pu ine auxo ophs, he addi ion o
AICA- iboside (5-aminoimidazole-4-ca boxamide 1-β-D-
ibo u anoside, 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
enhances nodule de elopmen [7,12,14]. This is also he case
In . Mic obiol.
Fig. 3. Plasmid p o iles o S. edii SVQ269 and i s py F de i a i e SVQ292.
The indica ed molecula masses o he plasmids a e app oxima e.
175
INT. MICROBIOL. Vol.10, 2007
o o he auxo ophic hizobial mu an s ha induce ine ec i e
nodules bu a e able o e ec i ely nodula e hei hos plan s
i he auxo ophic equi emen is added o he plan nu i i e
solu ion. Fo example, his idine auxo ophs o B ady hi-
zobium japonicum and S. melilo i and a ibo la in- equi ing
mu an o R. leguminosa um induce ni ogen- ixing nodules
on hei hos plan s only when he nu i i e plan solu ion is
supplemen ed wi h he equi ed g ow h ac o [14].
SVQ292 lacked a c yp ic plasmid o abou 85 MDa (Fig.
3) bu his was no he cause o he mu an ’s symbio ic de ec
because SVQ292 ha bo ing cosmid pMUS344, which con-
ained he wild- ype py F gene, induced ni ogen- ixing nod-
ules on soybean. These esul s sugges ed ha he c yp ic
plasmid cu ed in mu an SVQ292 is no essen ial o he
es ablishmen o an e ec i e symbiosis be ween S. edii
HH103 and soybean.
Nodula ion ac o s (LCO) and hizobial su ace polysac-
cha ides ac as signal molecules in he nodula ion p ocess.
Rhizobial mu an s ha do no p oduce LCO a e o ally
unable o ini ia e nodule o ma ion [10,20]. Many hizobial
mu an s a ec ed in he p oduc ion o bac e ial su ace poly-
saccha ides o m ine ec i e pseudonodules ha a e de oid
o bac e ia [14]. Since a leucine auxo ophic mu an o S.
melilo i is unable o p oduce LCO [15] and a pu ine aux-
o ophic mu an o S. edii has an al e ed LPS p o ile [7], we
in es iga ed whe he mu an SVQ292 p oduced no mal LCO
and LPS p o iles. Indeed, he he LCO and LPS p o iles p o-
duced by SVQ292 we e simila o hose p oduced by i s
wild- ype pa en al s ain HH103. Thus, he symbio ic impai -
men o SVQ292 was no appa en ly due o al e a ions in
hese wo symbio ic signals.
Al hough he possible symbio ic ole o amino acids,
pu ines, and py imidines is no clea , a ecen epo p o id-
ed new insigh s indica ing ha pu ines and py imidines, o
any in e media y o hei biosyn he ic pa hways, play an
impo an ole in igge ing nodule o ma ion [9]. Pho osyn-
he ic B ady hizobium sp. s ain ORS278 does no equi e
LCO p oduc ion o i s symbio ic in e ac ion wi h he legume
Aeschynomene sensi i a. Howe e , di e en mu an s a ec -
ed in he biosyn hesis o pu ines and a s ain ORS278 py F
mu an only o med a ew pseudonodules on his legume.
Thus, pu ines and py imidines a e essen ial o he o ma ion
o nodules, ega dless o whe he he mechanism o nodule
o ganogenesis equi es he p esence o bac e ial nodula ion
ac o s.
Acknowledgemen s. This wo k was suppo ed by he Spanish
Minis y o Educa ion and Science g an s BIO2005-08691-C02-02 and
AGL2006-13758-C05-03/AGR. We a e g a e ul o Ahmed Moussaid, Rocío
Gu ié ez, Ángeles Hidalgo, and Remedios Ga cía-Na a o o echnical
assis ance.
Re e ences
1. Be gensen FJ (1961) The g ow h o Rhizobium in syn he ic media. Aus
J Biol Sci 14:349-360
2. Be inge JE (1974) R ac o ans e in Rhizobium leguminosa um. J
Gen Mic obiol 84:188-198
3. Be inge JE, Ruiz-Sainz JE, Johns on AWB (1984) Me hods o he
gene ic manipula ion o Rhizobium. In: G ainge JM, Lynch JM (eds)
Mic obiological me hods o en i onmen al bio echnology. Academic
P ess, New Yo k, pp 79-94
4. Boye HW, Roulland-Dossoix D (1969) Complemen a ion analysis o
he es ic ion and modi ica ion o DNA in Esche ichia coli. J Mol Biol
41:459-472
5. Buendía-Cla e ía AM, Ruiz-Sainz JE, Cubo-Sánchez T, Pé ez Sil a J
(1986) S udies o symbio ic plasmids in Rhizobium i olii and as -
g owing bac e ia ha nodula e soybean. J Appl Bac e iol 17:155-160
6. Buendía-Cla e ía AM, Chambe M, Ruiz-Sainz JE (1989) A compa a-
i e s udy o he physiological cha ac e is ics, plasmid con en and sym-
bio ic p ope ies o di e en Rhizobium edii in Eu opean soils. Sys
Appl Bac e iol 17:55-160
7. Buendía-Cla e ía AM, Moussaid A, Olle o FJ, e al. (2003) A pu L
mu an o Sino hizobium edii HH103 is symbio ically de ec i e and
al e ed in i s lipopolysaccha ide. Mic obiology 149:1807-1818
8. de Ly a MCCP, López-Baena FJ, Madinabei ia N, e al. (2006)
Inac i a ion o he Sino hizobium edii HH103 hcJ gene abolishes
nodula ion ou e p o eins (Nops) sec e ion and dec eases symbio ic
capaci y wi h soybean. In Mic obiol 9:125-133
9. Gi aud E, Moulin L, Vallene D, e al. (2007) Legumes symbioses:
absence o nod genes in pho osyn he ic b ady hizobial. Science
316:1307-1312
10. Lam abe Y, Bellogín RA, Cubo T, e al. (1999) Mu a ion in GDP- ucose
syn hesis genes o Sino hizobium edii al e s Nod ac o s and signi i-
can ly dec eases compe i i eness o nodula e soybeans. Mol Plan -
Mic obe In e ac 12:207-217
11. Madinabei ia N, Bellogín RA, Buendía-Cla e ía A, e al. (2002) Sino-
hizobium edii HH103 has a unca ed nolO gene due o a –1 ame-
shi mu a ion ha is conse ed among o he geog aphically dis an S.
edii s ains. Mol Plan -Mic obe In e ac 15:150-159
12. Newman JD, Roso i z MJ, Noel KD (1995) Requi emen o hizo-
bial p oduc ion o 5-aminoimidazole-4-ca boxamide ibonucleo ide
(AICAR) o in ec ion o bean. Mol Plan -Mic obe In e ac 8:407-414
13. Olle o FJ, Espuny MR, Bellogín RA (1989) Mobiliza ion o he symbi-
o ic plasmid om a s ain o Rhizobium sp. (Hedysa um co ona ium).
Sys em Appl Mic obiol 11:217-222
14. Randhawa GS, Hassani R (2002) Role o hizobial biosyn he ic pa h-
ways o amino acids, nucleo ide bases and i amins in symbiosis. Indian
J Exp Biol 40:755-764
15. Sanjuán-Pinilla JM, Muñoz S, Nogales J, Oli a es J, Sanjuán J (2002)
In ol emen o he Sino hizobium melilo i leuA gene in ac i a ion o
nodula ion genes by NodD1 and lu eolin. A ch Mic obiol 178:36-44.
16. Samb ook J, F i sch EF, Mania is T (1989) Molecula cloning. A labo-
a o y manual, 2nd edn. Cold Sp ing Ha bo Lab P ess, Cold Sp ing
Ha bo , NY
17. Simon R (1984) High equency mobiliza ion o g am-nega i e bac e -
ial eplicons by he in i o cons uc ed Tn5-Mob ansposon. Mol Gen
Gene 196:413-420
18. Simon R, P ie e U, Pühle A (1983) A b oad hos ange mobiliza ion
sys em o in i o gene ic enginee ing: T ansposon mu agenesis in g am
nega i e bac e ia. Bio/Technology 1:784-791
S. FREDII PYRIMIDINE MUTANT
176 INT. MICROBIOL. Vol. 10, 2007
19. Simon R, Quand J, Klipp W (1989) New de i a i es o ansposon Tn5
sui able o mobiliza ion o eplicons, gene a ion o ope on usions, and
induc ion o genes in g am-nega i e bac e ia. Gene 80:161-169
20. Spaink HP, Aa s A, S acey G, Bloembe g GV, Lug enbe g BJJ,
Kennedy EP (1992) De ec ion and sepa a ion o Rhizobium and
B ady hizobium Nod me aboli es using hin-laye ch oma og aphy. Mol
Plan -Mic obe In e ac 5:72-80
21. S ych U, Wohl a h S, Winkle UK (1994) O o idine-5´-monophospha e
deca boxylase om Pseudomonas ae uginosa PAO1: cloning, o e exp es-
sion, and enzyme cha ac e iza ion. Cu Mic obiol 29: 353-359
22. Vinee ha KE, Vij N, P asad CK, Hassani R, Randhawa GS (2001) Ul a-
s uc u al s udies on nodules induced by py imidine auxo ophs o Sino-
hizobium melilo i. Indian J Exp Biol 39:371-377
23. Vincen JM (1970) The modi ied Fåh aeus slide echnique. In: Vincen
JM (ed) A manual o he p ac ical s udy o oo nodule bac e ia.
Appendix III. Blackwell Scien i ic Pub, Ox o d, pp 144-145
24. Wo land S, Gue ei o N, Yip L, Djo dje ic MA, Djo dje ic SP,
Weinman J, Rol e BG (1999) Rhizobium pu ine auxo ophs, pe u bed
in nodula ion, ha e mul iple changes in p o ein syn hesis. Aus J Plan
Physiol 26:511-519
CRESPO-RIVAS ET AL