Download Molecular Genetics and Genomics

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Mol Gen Genet (1997) 255:131±140
ORIGINAL PAPER
B. SaÂnchez-AnduÂjar á C. Coronado
S. Philip-Hollingsworth á F. B. Dazzo á A. J. Palomares
Structure and role in symbiosis of the exoB gene
of Rhizobium leguminosarum bv trifolii
Received: 31 May 1996 / Accepted: 18 December 1996
Abstract The Rhizobium leguminosarum bv trifolii exoB
gene has been isolated by heterologous complementation of an exoB mutant of R. meliloti. We have cloned a
chromosomal DNA fragment from the R. leguminosarum bv trifolii genome that contains an open reading
frame of 981 bp showing 80% identity at the amino acid
level to the UDP-glucose 4-epimerase of R. meliloti. This
enzyme produces UDP-galactose, the donor of galactosyl residues for the lipid-linked oligosaccharide repeat
units of various heteropolysaccharides of rhizobia. An
R. leguminosarum bv trifolii exoB disruption mutant
di€ered from the wild type in the structure of both the
acidic exopolysaccharide and the lipopolysaccharide.
The acidic exopolysaccharide made by our wild-type
strain is similar to the Type 2 exopolysaccharide made
by other R. leguminosarum bv trifolii wild types. The
exopolysaccharide made by the exoB mutant lacked the
galactose residue and the substitutions attached to it.
The exoB mutant induced the development of abnormal
root nodules and was almost completely unable to invade plant cells. Our results stress the importance of
exoB in the Rhizobium-plant interaction.
Key words Rhizobium leguminosarum bv trifolii á
Trifolium subterraneum á Trifolium repens á exoB
gene á Exopolysaccharide
Communicated by E. CerdaÂ-Olmedo
B. SaÂnchez-AnduÂjar á C. Coronado á A. J. Palomares (&)
Departamento de MicrobiologõÂ a, Facultad de Farmacia,
Universidad de Sevilla, E-41012 Sevilla, Spain
e-mail [email protected]
S. Philip-Hollingsworth á F. B. Dazzo
Department of Microbiology, Michigan State University,
East Lansing, Michigan 48824, USA
F. B. Dazzo
Center for Microbial Ecology, Michigan State University,
East Lansing, Michigan 48824, USA
The nucleotide sequence data reported in this paper are available
from the EMBL data bank under accession number X96507
Introduction
The extracellular and surface polysaccharides produced
by Rhizobium constitute the interface between the bacterial cell and its environment and are involved in the
interactions leading to the establishment of an e€ective
Rhizobium-legume symbiosis (Coronado et al. 1996).
These polysaccharides include extracellular heteropolysaccharides (EPS), capsular polysaccharides, and lipopolysaccharides (LPS) of the outer membrane. Cyclic
(1±2)-b-D glucan is found in the periplasm and culture
medium of some strains.
Rhizobium meliloti synthesizes two di€erent heteropolysaccharides. EPSI is a succinoglycan necessary for
the invasion of alfalfa root nodules; EPSI-de®cient
mutants fail to form infection threads. EPSII is a galactoglucan with a completely di€erent structure; EPSIIde®cient mutants are still able to form an e€ective
symbiosis with alfalfa. Twenty-two exo genes for EPSI
synthesis have been identi®ed in R. meliloti Rm2011; 19
of them reside in a contiguous 24-kb cluster on the
second symbiotic megaplasmid (pSymb) (Leigh and
Walker 1994).
The R. meliloti exoB gene encodes a UDP-glucose 4epimerase (BuendõÂ a et al. 1991) that converts UDPglucose into UDP-galactose. Since galactose is a common component of both EPS and LPS (Diebold and
Noel 1989; Bhagwat et al. 1991), exoB mutants should
be pleiotropic and defective in the synthesis of both
polysaccharides. The symbiotic behavior of exoB mutants depends on both the Rhizobium and the legume
host species. An exoB mutant of R. meliloti induces
empty, ine€ective nodules on alfalfa (Leigh and Lee
1988). An exoB mutant of Rhizobium sp. GRH2 induces
a few empty nodules on Acacia and fails to nodulate
most herbaceous legume hosts (Lopez-Lara et al. 1995).
R. leguminosarum bv viciae exoB mutants produce only
some root hair deformation and rare, abortive infection
threads on Vicia villosa (Canter Cremers et al. 1990). In
contrast, a Rhizobium sp.(Ollero et al. 1994) exoB mu-
132
tant induces e€ective, indeterminate nodules on the host,
Hedysarum coronarium.
Strains of R. leguminosarum bv trifolii may be classi®ed into two major groups and one minor group according to the stoichiometry and the linkage site of the
non-carbohydrate substituents in EPS (Philip-Hollingsworth et al. 1989). The symbiotic behavior of some exo
mutants of R. leguminosarum bv trifolii has been described, but they have not been characterized at the
genetic level. Spontaneous and Tn5-induced exo mutants of di€erent strains of R. leguminosarum bv trifolii
induce small, white, and ine€ective nodules on clover
roots (Rolfe et al. 1980; Chakravorty et al. 1982; Derylo
et al. 1986). These nodules contain infection threads, but
few of the plant cells contain intracellular bacteria.
We have constructed an exoB disruption mutant of
R. leguminosarum bv trifolii to determine the role of this
gene in EPS structure and symbiotic behavior.
Materials and methods
Strains, plasmids, and media
Strains and plasmids used in this study are listed in Table 1.
R. meliloti strains were grown in rich medium YTA (Orosz et al.
1973) or LB medium (Ausubel et al. 1989) supplemented with
2.5 mM MgSO4 and 2.5 mM CaCl2 at 30° C. R. leguminosarum bv
trifolii strains were grown in YT medium (Beringer 1974) or de®ned
BIII agar medium (Dazzo 1982) at 30° C. For Congo red absorption, bacteria were streaked on plates of YEMCR agar
(Zevenhuizen et al. 1986). Escherichia coli strains were grown in LB
medium at 37° C. When appropriate, media were supplemented
with 200 lg/ml neomycin, 10 lg/ml nalidixic acid, 50 lg/ml kanamycin, 100 lg/ml ampicillin, 20 lg/ml gentamicin, 100 lg/ml
spectinomycin, 5 lg/ml tetracycline for Rhizobium strains and
15 lg/ml for E. coli strains or 200 lg/ml Calco¯uor white M2R
(Fluorescent Brightener 28, Sigma, St. Louis, Mo., USA).
An R. leguminosarum bv trifolii gene bank, previously constructed in E. coli HB101 (Rodriguez-QuinÄones et al. 1989), was
used for the heterologous complementation of the exoB mutant,
R. meliloti 7094. Cosmid pVK102 derivatives were transferred
conjugatively in triparental matings as described by Ditta (1986).
DNA manipulations and analysis
E. coli plasmid DNA was isolated by alkaline lysis (Ausubel et al.
1989). Rhizobium plasmid DNA was isolated according to Ish-Horowicz and Burke (1981). Bacterial genomic DNA was isolated by
selective precipitation with CTAB (hexadecyltrimethyl ammonium
bromide) as described by Ausubel et al. (1989). Plasmid characterization was performed according to the procedure of Eckhardt
(1978) as modi®ed by Rosenberg et al. (1982). DNA was digested,
electrophoresed in 0.7% agarose gels, and blotted onto nylon
membranes (Hybond-N, Amersham, Buckinghamshire, UK,
0.45 lm) according to the Southern blotting procedure. DNA
probes were prepared using the Multiprime DNA labelling system
(Amersham). Hybridizations were performed overnight at 37° C in
3 ´ SSC, 50% formamide, 25 mM NaH2PO4, 25 mM Na2HPO4,
5 ´ Denhardt's solution, containing 500 lg/ml of herring sperm
DNA (Kondorosi et al. 1982). Tn5 insertions in plasmid pBSB43
were physically mapped as described by Ditta (1986). DNA sequences were obtained from overlapping nested deletion clones
generated by exonuclease III digestion according to Heniko€
(1984). Sequencing reactions were performed using the Auto-Read
Sequencing kit (Pharmacia Biotech, Uppsala, Sweden). Sequence
data were obtained and processed using the A.L.F. DNA Sequencer
(Pharmacia Biotech) according to the manufacturer's instructions.
The nucleotide and amino acid sequences were analyzed using the
BESTFIT program of the Genetics Computer Group (GCG) program package of the University of Wisconsin (Devereux et al. 1984).
Plant nodulation
Rhizobium strains were assayed for their symbiotic phenotypes on
alfalfa (Medicago sativa cv Moapa) and clover plants (Trifolium
repens cv Huia and T. subterraneum cv Seaton Park) grown in
Leonard jars, and analyzed 4±5 weeks after inoculation, (Corbin
et al. 1982). For each strain, at least six nodules from di€erent
plants were collected. Nodules were ®xed for 2 h at 4° C with
glutaraldehyde (30 ml/l in 100 mM sodium cacodylate, pH 7.2).
After two rinses in this bu€er, the nodules were post®xed for 2 h in
OsO4 (10 g/l in 100 mM sodium cacodylate, pH 7.2). After three
further rinses, the samples were dehydrated in a graded acetone
series at 4° C. Samples were maintained for 2 h in uranyl acetate
(10 g/l in 70% acetone) before complete dehydration. Finally,
nodules were embedded in Spurr medium. Sections were examined
by combined light and transmission electron microscopy following
staining with (1 g/l) toluidine blue or lead citrate/uranyl acetate,
respectively.
Exopolysaccharide analysis
Bacteria were grown in shaker ¯asks containing 500 ml of BIII
medium at 30° C for 5 days to a density of 0.6 OD600. After removing the bacteria by centrifugation, two volumes of cold ethanol
were added to the supernatant. The EPS precipitate was collected
by centrifugation, lyophilized, redissolved in distilled water, and
dialyzed (12 000±14 000 molecular weight cuto€) against 2 l of
distilled water at 4° C with ®ve to seven changes over a 3-day period
to remove the majority of the b-1,2 glucans and other oligomers.
Finally, the EPS solution was lyophilized.
The EPS were analyzed by proton nuclear magnetic resonance
(1H-NMR) spectroscopy and combined gas-liquid chromatography/mass spectrometry (GLC/MS). For 1H-NMR analysis, 3 mg
of puri®ed EPS were dissolved in deuterium oxide (99.7%, 0.4 ml),
lyophilized, redissolved in deuterium oxide (99.99%, 0.4 ml), and
sonicated for 1 min at room temperature. Spectra were recorded at
500 MHz and 70° C with presaturation of the solvent peak at
d 4.25. For GLC/MS analysis of glycosyl compositions, EPS
samples were acid-hydrolyzed and the released sugars converted to
their corresponding alditol acetate derivatives as previously described (Hollingsworth et al. 1984). Uronic acids were identi®ed by
carboxyl reduction with sodium borodeuteride followed by GLC/
MS analysis of the alditol acetate derivatives with selected ion
monitoring for 217 a.m.u. and 219 a.m.u. as previously described
(Hollingsworth et al. 1984; Philip-Hollingsworth et al. 1989).
Electrophoresis of lipopolysaccharides
For SDS-polyacrylamide gel electrophoresis, the LPS were solubilized from proteinase K-treated cells and electrophoresed in
16.5% polyacrylamide gels as described by KoÈplin et al. (1993).
Gels were ®xed and silver-stained as described by Kittelberger and
Hilbink (1993). LPS from the smooth strain Salmonella typhimurium and the rough strains S. typhimurium TV119 and SL1181,
purchased from Sigma, were used as standards.
Results
A gene from R. leguminosarum bv trifolii homologous
to the R. meliloti exoB gene
Ten exconjugants of the exoB mutant, R. meliloti 7094,
carrying a cosmid from the R. leguminosarum bv trifolii
133
Table 1 Strains and plasmids (rantibiotic resistance, Nal nalidixic acid, Sp spectinomycin, Sm streptomycin, Tc tetracycline, Km kanamycin, Rf rifampicin, Gm gentamicin)
Strain or plasmid
Reference or source
Rhizobium strains
R. leguminosarum bv trifolii
RS800
RS12
Wild type, Nalr
exoB mutant RS800 derivative, Spr
Megias et al. (1988)
This work
R. meliloti
Rm1021
Rm7094
Rm7031
Rm7055
Rm8457
Smr, SU47 derivative
Rm1021 exoB::Tn5
Rm1021 exoA::Tn5
Rm1021 exoF::Tn5
Rm1021 exoM::Tn5
Meade et al. (1982)
Leigh et al. (1985)
Leigh et al. (1985)
Leigh et al. (1985)
Long et al. (1988)
hsdS, hsdM, pro, leu, thi, gal,
lacY, recA, Smr
recA1, endA1, gyrA96, thi,
hsdR17, supE44, relA1,
D(lac-proAB) F¢ (traD36, proAB,
lacI qZDM15)
F-, recA, hsdR, RP4-2
(Tc::Mu), (Km::Tn7)
polA1, rha, his, Nalr, Rf r
Tn5 is present at an indeterminate
chromosomal location
Boyer and Roulland-Dussoix (1969)
Escherichia coli strains
HB101
JM109
S17-1
C2110
HB101::Tn5
Plasmids
pRK2073
pVK102, gene bank
pD2
pRK404
pUT miniTn5 Sp/Sm
pJQ200SK
pBSB4
pBSB43
pBSB42
pBSB43-3
pBSB43-4
pBSB43-6
pBSB43-8
pJQ2003
``Helper'' plasmid, Spr
R. leguminosarum bv trifolii genomic DNA HindIII
fragments cloned in pVK102, Tcr
pLAFR1 derivative containing the
exoB gene of Rm1021
pRK290 derivative, Tcr
pUT derivative containing the
miniTn5 Sp/Sm element
pACYC184 derivative, Gmr
mob, sacB, lacZa
Cosmid containing exoB
gene of RS800
pRK404 derivative containing the 3.7-kb
PstI fragment DNA of RS800
pRK404 derivative containing the
2.2-kb BamHI fragment DNA of RS800
pBSB43 derivative containing a Tn5
insertion
pBSB43 derivative containing a Tn5
insertion
pBSB43 derivative containing a Tn5
insertion
pBSB43 derivative containing a Tn5
insertion
pJQ200SK derivative containing the
RS800 exoB gene disrupted by the
Sp/Sm interposon
gene bank, were examined for their capacity to form
¯uorescent colonies on Calco¯uor LB media. Physical
studies showed that all ten Rhizobium clones analyzed
were identical. The cosmid, pBSB4, which restored colony ¯uorescence to R. meliloti 7094, was chosen. This
cosmid did not complement R. meliloti exoA, exoF or
exoM mutants.
Hybridization with an internal fragment of the R.
meliloti exoB gene contained in cosmid pD2 revealed
Yanisch-Perron et al. (1985)
Simon et al. (1989)
Ditta et al. (1980)
Ditta et al. (1980)
Ditta et al. (1980)
Rodriguez-QuinÄones et al. (1989)
Leigh et al. (1985)
Ditta et al. (1985)
De Lorenzo et al. (1990)
Quandt and Hynes (1993)
This work
This work
This work
This work
This work
This work
This work
This work
that the cosmid pBSB4 contained a 3.7-kb PstI fragment
and a 2.2-kb BamHI fragment that hybridized to the
probe. These fragments were subcloned in pRK404 to
obtain plasmids pBSB43 and pBSB42, respectively.
Plasmid pBSB43 complemented R. meliloti 7094,
allowing formation of ¯uorescent colonies on Calco¯uor
LB agar and e€ective nodulation of alfalfa, but plasmid
pBSB42 did not complement any of the mutant phenotypes. These results of hybridization and complementa-
134
tion studies suggested that an R. leguminosarum bv trifolii exoB homolog, which contains a BamHI restriction
site, is located on the 3.7-kb PstI fragment.
R. leguminosarum bv trifolii RS800 harbors three
plasmids with molecular weights of about 190, 280, and
470 MDa (Rodriguez-QuinÄones et al. 1989). In order to
locate the exoB gene in the genome of RS800, a blotted
Eckhardt gel (Rosenberg et al. 1982) was hybridized
using the 3.7-kb PstI fragment as a probe. This fragment
hybridized with the second symbiotic megaplasmid of R.
meliloti 1021 and with chromosomal DNA of RS800,
but not with plasmid DNA of RS800. This con®rmed
the plasmid location of gene exoB in R. meliloti and
showed the chromosomal location of the homologous
gene of R. leguminosarum bv trifolii.
The 2-kb EcoRV-PstI fragment internal to the 3.7-kb
PstI fragment was sequenced. It contained an open
reading frame from the only ATG codon at position 210
to the TGA codon at position 1191 (Fig. 1). A potential
ribosome-binding site (AGGG) was identi®ed just 6 bp
upstream of the putative start codon. The deduced
polypeptide product was 327 amino acids in length and
had a molecular weight of 35 926 Da. This open reading
frame was identi®ed as gene exoB from its ability to
complement the exoB mutation of R. meliloti, and from
sequence comparison with exoB genes of other organisms. The exoB gene coded for a predicted protein that
showed 80.0% identity and 89.2% similarity to the deduced amino acid sequence of the UDP-glucose 4epimerase of R. meliloti. The exoB gene of R. leguminosarum bv trifolii also showed signi®cant homology
to gene sequences coding for UDP-glucose 4-epimerase
in Azospirillum brasilense (49.5% identity, 64.3% similarity) (De Troch et al. 1994) and E. coli (41.7% identity,
58.5% similarity) (Lemaire and MuÈller-Hill 1986).
nies on Calco¯uor LB agar and to nodulate alfalfa, but
the other two had lost both phenotypes. Chromosomal
exoB Tn5 insertions could not be obtained by following
the plasmid incompatibility method (Ditta 1986). A
disruption mutant of the chromosomal exoB gene was
obtained by using gene sacB (Quandt and Hynes 1993),
which renders cells unable to grow in the presence of
sucrose. The Tn5 sequence in plasmid pBSB43-4 was
excised by HpaI digestion and replaced by a 2-kb SmaI
fragment of plasmid pUTminiTn5 Sp/Sm that contains
a spectinomycin resistance gene. The 5.7-kb PstI fragment of the modi®ed plasmid was subcloned into the
PstI site of plasmid pJQ200SK that contains gene sacB.
The resulting plasmid, pJQ2003, was transferred from
E. coli S17-1 to R. leguminosarum bv trifolii RS800 by
selecting for resistance to spectinomycin and sucrose.
The resulting colonies had undergone double recombination events that were con®rmed by hybridization. The
result was the replacement of the chromosomal exoB
gene by the disrupted homolog from the plasmid. One of
these strains was designated RS12.
Mutant RS12 grew more slowly than its parental
strain, tended to ¯occulate, and formed deep red colonies on Congo red plates, whereas wild-type colonies
were pale pink. A lack of EPS seems to increase the
uptake of Congo red in other exo mutants (Breedveld
et al. 1993).
The LPS pro®le of the wild-type strain (Fig. 2, lane 4)
consisted of two major LPS species, a slow-migrating
ladder-like structure (LPSI) which could represent the
O-antigen-containing LPS, and the faster migrating
band (LPSII) that consists of lipid A and core only
(Carlson 1984). Our exoB mutant lacked the higher
molecular weight form of the LPS, and contained only
LPSII, which had a somewhat higher electrophoretic
mobility than LPSII of wild type (Fig. 2, lane 5). The
An exoB disruption mutant
of R. leguminosarum bv trifolii
Four Tn5 insertion mutants of plasmid pBSB43 were
analyzed (Fig. 1). Two of them retained the ability to
complement R. meliloti 7094 to form ¯uorescent colo-
Fig. 1 Restriction map of the 3.7-kb PstI fragment of the RS800
chromosomal DNA. Bars with open circles indicate Tn5 insertions
that give rise to ¯uorescent colonies on Calco¯uor media; insertions
indicated by closed circles do not result in ¯uorescence. The numbers
are the last digit in the name of the plasmid that bears the insertion
(Table 1). The box indicates the exoB open reading frame and the
arrow the direction of transcription. (P, PstI, Ss SspI, Pv PvuI, B
BamHI, R RsaI, Sp SphI, EV EcoRV, A AvaI, Sal I)
Fig. 2 SDS-polyacrylamide gel electrophoresis of lipopolysaccharides
(LPS) from the wild-type RS800 (lane 4), the exoB mutant strain,
RS12 (lane 5), and the same mutant with plasmid pBSB43 (lane 6).
LPS from smooth Salmonella typhimurium (lane 1), and rough
S. typhimurium TV119 (lane 2), and S. typhimurium SL1181 (lane 3)
were used as standards
135
LPS pro®le of RS12 was restored to the wild-type LPS
pro®le by complementation with plasmid pBSB43 that
contains the complete exoB gene of R. leguminosarum bv
trifolii RS800 (Fig. 2, lane 6).
Analysis of RS800 and RS12 EPS
The 1H-NMR spectrum of the EPS from the parent
strain RS800 (Fig. 3A) was consistent with a polysaccharide bearing several di€erent non-carbohydrate substitutions. The proton resonances between d 3.0±5.0
were assigned to glycosyl components, and the resonances at d 1.2, 1.4, and 2.1 were assigned to methyl
protons of 3-hydroxybutyryl, pyruvate, and acetate
group substitutions, respectively. GLC/MS analyses indicated that the EPS from this wild-type strain was an
acidic heteropolysaccharide containing glucose, glucuronic acid, and galactose residues in an approximate
glycosyl molar ratio of 4.5 : 1.2 : 1 (Fig. 4A). These 1HNMR and GLC/MS data were similar to the Type 2
EPS previously shown to be made by R. leguminosarum
bv trifolii wild-type strains ANU843, NA-30, and TA-1
(Hollingsworth et al. 1984; Philip-Hollingsworth et al.
1989). The oligosaccharide repeat unit of the Type 2
acidic EPS structure consists of an octasaccharide with
two glucuronic acid residues and two glucose residues
Fig. 3 1H-NMR spectra of isolated acidic exopolysaccharide from
Rhizobium leguminosarum bv trifolii strains RS800 (A, wild type) and
RS12 (B, exoB mutant). Resonances for pyruvate (Py), acetate (Ac),
and 3-hydroxybutyrate (3-Hb) are indicated
that constitute the tetrasaccharide main chain and a
tetrasaccharide branch that contains three internal glucose residues and a galactose residue at the non-reducing
terminus (Fig. 5). The multiplicity and splitting pattern
of the non-carbohydrate resonances in the 1H-NMR
spectrum of the EPS from strain RS800 (Fig. 3A) were
consistent with their location in the Type 2 structure
presented in Fig. 5. The single acetate group was primarily attached to O-3 (with minor migration to O-2) of
the glucuronic acid residue adjacent to glucose in the
main chain. The two pyruvate groups were attached to
the 4 and 6 positions of the terminal galactose and adjacent glucose residues of the side chain, while the 3hydroxybutyryl group was esteri®ed on the O-3 position
of the galactose residue.
From its 1H-NMR spectrum, the EPS from the exoB
mutant (Fig. 3B) was similar to that of the RS800 parent, but lacked the 3-hydroxybutyrate moiety and one of
the pyruvate groups (d 1.2, 1.4). The other pyruvate
group and the acetate group were still present in the
exoB mutant EPS. GLC/MS analysis of the EPS from
the exoB mutant (Fig. 4B) indicated the presence of
glucose and glucuronic acid, in a ratio of 4.8 : 1.3, and a
total absence of galactose.
Nodulation of clover by RS800 and RS12
Nodules induced by the RS800 wild-type strain were
typical of an e€ective symbiosis. They were pink, cylindrical, normal in size (3±4 mm in T. repens, 5±6 mm in T.
subterraneum), and developed mainly on primary roots.
In contrast, nodules induced by the RS12 mutant strain
were indicative of an ine€ective symbiosis. They lacked
the cylindrical shape and they were white, smaller, and
more numerous per plant than those of the wild type, and
frequently located at the site of secondary root emergence. The distribution of nodule development on roots
was a€ected by the mutation of the bacterial exoB gene.
In contrast to the nodules induced by the RS800 wildtype strain in T. subterraneum (Fig. 6A,B), those induced
by RS12 mutant strain were uninvaded and lacked infection threads and bacteroids. Host cells in the central
region of these nodules were smaller and contained more
starch grains than did host cells in nodules induced by
wild-type RS800. Few nodules on plants inoculated with
the RS12 mutant strain contained bacterial cells. In these
rare cases, host cells that contained bacteria were located
near the periphery of the central region (Fig. 6C,D).
Deeper sections of the same nodules (Fig. 6E,F) revealed
only uninfected host cells. These results indicated a signi®cant restriction in the ability of the exoB mutant to
invade host cells within clover nodules.
Transmission electron microscopy shows club-shaped
bacteroids of wild-type RS800 enclosed within symbiosomes of infected nodule cells (Fig. 7A). Most host cells in
the nodule central region were uninfected by the exoB
mutant, RS12, and contained an abundance of starch
granules (Fig. 7B). In the rare host cells that are invaded
136
Fig. 4 Total ion chromatogram
of alditol acetate derivatives of
glycosyl components from
exopolysaccharide of wild type,
RS800 (A) and the exoB
mutant, RS12 (B). Peak 2 is the
galactosyl derivative, whereas
peak 3 contains both the
glucosyl and glucuronosyl
derivatives, respectively. The
latter two components are
distinguished by selected ion
monitoring mass spectrometry.
Peak 1 represents a trace of the
decarboxylated product of
uronic acid
by the bacterial mutant, bacteria were released from infection threads but were not maintained in symbiosomes
(Fig. 7C,D). Instead, the released bacteria were in direct
contact with host cytosol, which itself appeared to undergo degradation and to accumulate numerous membraneous vesicles. The degeneration of the bacterial and
host cell cytosol and the lack of intact peribacteroid
membranes indicated an impaired root-nodule symbiosis.
Discussion
The present work is the ®rst study of the exoB gene of R.
leguminosarum bv trifolii, involved in polysaccharide
synthesis. This gene is chromosomally located, and the
original cosmid that contained the gene complemented
none of the R. meliloti exoA, exoF or exoM mutants. In
R. meliloti 1021, exoB maps in a cluster of exo genes that
includes exoA, exoF, and exoM, on the second symbiotic
megaplasmid (Leigh and Walker 1994). Therefore, the
genomic distribution of the exo loci di€ers in these two
Rhizobium species. As in R. leguminosarum bv trifolii,
exoB maps on the chromosome of other Rhizobiaceae
species including one that nodulates H. coronarium
(Ollero et al. 1994), and one that nodulates Acacia
(Lopez-Lara et al. 1995) and Agrobacterium tumefaciens
(Cangelosi et al. 1987). Despite the high divergence in
genomic organization of the exo loci between R. meliloti
and R. leguminosarum bv trifolii, DNA sequence similarity and functional interchangeability indicated parallel evolution of the exoB genes in both species.
The EPS produced by R. leguminosarum bv trifolii
wild-type strain corresponded to the Type 2 EPS synthesized by one of the major groups of R. leguminosarum
bv trifolii strains. This EPS bears a single acetyl group
on O-3 of one of the glucuronic acid residues in the
backbone and contains only half as much 3-hydroxybutyryl groups as R. leguminosarum bv viciae (PhilipHollingsworth et al. 1989). The 1H-NMR and GLC/MS
analyses of the acidic EPS produced by RS800 and RS12
exoB mutant strains were fully consistent with the
assumed function of ExoB. Since the exoB mutant
c
Fig. 5 Structure of R. leguminosarum bv trifolii RS800 exopolysaccharide (EPS). Diagram showing the octasaccharide repeated unit of
RS800 EPS. (Glc Glucose, Gal galactose, GlcA glucuronic acid, Pyr
pyruvyl group, OAc acyl group, 3HB 3-hydroxybutyryl group)
Fig. 6 Light micrographs of longitudinal nodule sections of Trifolium
subterraneum inoculated with R. leguminosarum bv trifolii wild type,
RS800 (A, B) or the exoB mutant, RS12 (C±F). B, D, and F are higher
magni®cations of the center of nodule sections in A, C, and E,
respectively. (Bar scales 20 lm (B, D, F) and 100 lm (A, C, E)
137
138
Fig. 7 Transmission electron micrographs of nodule sections of T. subterraneum inoculated with R. leguminosarum bv trifolii wild type, RS800
(A), or the exoB mutant, RS12 (B±D). Bar scales are 1 lm in A, C, and D; 2 lm in B
cannot make the precursor for galactose incorporation,
the EPS repeat unit that it makes lacks the terminal
galactose in the branch, thus eliminating the site for
attachment of the 3-hydroxybutyrate substitution and
one of the pyruvate substitutions. This explains how
mutation of a gene responsible for interconversion of a
glycosyl component of EPS a€ects not only the incorporation of that carbohydrate residue but also of noncarbohydrate substitutions attached to it in the synthesis
of the EPS repeat unit structure.
Although a chemical analysis of LPS in the exoB
mutant was not performed, a possible model to account
for its altered electrophoretic pro®le is that the exoB
mutation blocked the synthesis of the galactose-containing tetrasaccharide core (Hollingsworth et al. 1989)
to which the O-antigen is normally attached. Because
ExoB is required for the biosynthesis of precursors of
EPS and LPS, a pleiotropic e€ect on both polymers has
also been described for exoB mutants of R. leguminosarum bv viciae (Canter Cremers et al. 1990), a
Rhizobium species that nodulates H. coronarium (Ollero
et al. 1994), and one that nodulates Acacia (Lopez-Lara
et al. 1995). Since EPS and LPS of RS12 were a€ected by
the exoB mutation, the symbiotic defect of this mutant
could not be attributed to alterations in any one of these
two surface glycoconjugates.
R. leguminosarum bv viciae exoB mutants fail to induce nodules or nodule-like structures on host plants
(Canter Cremers et al. 1990). In contrast, the R. leguminosarum bv trifolii exoB mutant induced nodule
139
formation on clovers, but their di€erentiation and distribution on the root di€ered considerably from nodules
induced by the wild-type parent. Thus, the symbiotic
phenotypes of exoB mutants of these closely related
rhizobia are not identical. This implies that the requirement(s) for galactosyl-containing glycoconjugates
of rhizobia may be more stringent in certain symbioses
than others. Our results are consistent with the data of
Derylo et al. (1986) which showed that exopolysaccharide de®cient mutants derived from R. leguminosarum bv
trifolii 24AR5 were able to induce early steps of nodulation, although only a small number of bacteria were
released from infection threads and some of them appeared to be lysed. Moreover, our results on the majority of RS12-induced nodules are in accordance with
the analysis of R. meliloti 1021 exo mutant-induced
nodules (Yang et al. 1992). These nodules also develop
more frequently on secondary roots than on the primary
root. The exo mutant-induced infection threads abort
within the peripheral cells, and the nodules lack a wellde®ned apical meristem.
In summary, our studies show that mutation of the
exoB gene in R. leguminosarum bv trifolii leads to important alterations in EPS and LPS structures, and to
symbiotic defects in nodule development, invasion of
nodule host cells, and sustained maintenance of an intracellular endosymbiotic state.
Acknowledgements G. C. Walker is gratefully acknowledged for
providing strains and plasmids, and for receiving B.S.-A. for a
short stay in his laboratory. We thank Ana BuendõÂ a for her collaboration in LPS analysis and AsuncioÂn Fernandez from the
Servicio de MicroscopõÂ a ElectroÂnica-Universidad de Sevilla for
preparation of samples for microscopical study. B.S.-A. and C.C.
were supported by the Ministerio de EducacioÂn y Ciencia. This
work was funded by grants BIO90-0521 and BIO93-0427 from the
ComisioÂn EspanÄola Interministerial de Ciencia y TecnologõÂ a.
Portions of this work were supported by the Michigan Agricultural
Experiment Station, USDA-NRI grant 94±03537, and NSF grant
BIR 9120006 to the MSU Center for Microbial Ecology.
References
Ausubel FM, Brent R, Kingston RE, Moore DD, Seichnan JG,
Smith JA, Struhl F (1989) Current protocols in molecular biology. Greene and Wiley Interscience, New York
Beringer JE (1974) R-factor transfer in Rhizobium leguminosarum. J
Gen Microbiol 84:188±198
Bhagwat AA, Tully RE, Kesister DL (1991) Isolation and characterization of a competition-defective Bradyrhizobium japonicum mutant. Appl Environ Microbiol 57:3496±3501
Boyer HW, Roulland-Dussoix D (1969) A complementation
analysis of restriction and modi®cation in Escherichia coli. J
Mol Biol 41:459±472
Breedveld MW, Canter Cremers HCJ, Batley M, Posthumus MA,
Zevenhuizen LPTM, Wij€elman CA, Zehnder AJB (1993)
Polysaccharide synthesis in relation to nodulation behavior of
Rhizobium leguminosarum. J Bacteriol 175:750±757
BuendõÂ a AM, Enenkel B, KoÈplin R, Niehaus K, Arnold W, PuÈhler
A (1991) The Rhizobium meliloti exoZ/exoB fragment of
megaplasmid 2: ExoB functions as a UDP-glucose 4-epimerase
and ExoZ shows homology to NodX of Rhizobium leguminosarum biovar viciae strain TOM. Mol Microbiol 5:1519±
1530
Cangelosi GA, Hung L, Puvanesarajah V, Stacey G, Ozga DA,
Leigh JA, Nester EW (1987) Common loci for Agrobacterium
tumefaciens and Rhizobium meliloti exopolysaccharide synthesis
and their roles in plant interactions. J Bacteriol 169:2086±2091
Canter Cremers HCJ, Batley M, Redmond JW, Eydems L,
Breedveld MW, Zevenhuizen LPTM, Pees E, Wij€elman CA,
Lugtenberg BJJ (1990) Rhizobium leguminosarum exoB mutants
are de®cient in the synthesis of UDP-glucose 4¢ epimerase. J
Biol Chem 265:21122±21127
Carlson RW (1984) The heterogeneity of Rhizobium lipopolysaccharides. J Bacteriol 158:1012±1017
Chakravorty A, Zurkowski W, Shine J, Rolfe BG (1982) Symbiotic
nitrogen ®xation: molecular cloning of Rhizobium genes involved in exopolysaccharide synthesis and e€ective nodulation.
J Mol Appl Genet 1:585±596
Corbin D, Ditta GS, Helinski DR (1982) Clustering of nitrogen
®xation (nif) genes in Rhizobium meliloti. J Bacteriol 149:221±228
Coronado C, Sanchez-Andujar B, Palomares AJ (1996) Rhizobium
extracellular structures in symbiosis. World Microbiol Biotechnol 12:127±136
Dazzo FB (1982) Leguminous root nodules. In: Burns RG, Slater
JH (eds) Experimental microbial ecology. Blackwell Scienti®c
Publications, Oxford, pp 431±446
De Lorenzo V, Herrero M, Jakubzik U, Timmis UK (1990) MiniTn5 transposon derivatives for insertion mutagenesis, promoter
probing, and chromosomal insertion of cloned DNA in gramnegative eubacteria. J Bacteriol 172:6568±6572
Derylo M, Skorupska A, Bednara J, Lorkiewicz Z (1986) Rhizobium trifolii mutants de®cient in exopolysaccharide production.
Physiol Plant 66:699±704
De Troch P, Keijers V, Vanderleyden J (1994) Sequence analysis of
the Azospirillum brasilense exoB gene, encoding UDP-glucose
4¢-epimerase. Gene 144:143±144
Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of
sequence analysis programs for the VAX. Nucleic Acids Res 12:
387±395
Diebold R, Noel KD (1989) Rhizobium leguminosarum exopolysaccharide mutants: biochemical and genetic analyses and
symbiotic behaviour on three hosts. J Bacteriol 171:4821±4830
Ditta G (1986). Tn5 mapping of Rhizobium nitrogen ®xation genes.
Methods Enzymol 118:519±528
Ditta G, Stan®eld S, Corbin D, Helinski DR (1980). Broad hostrange DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad
Sci USA 77:7347±7351
Ditta G, Schmidhauser T, Yakobson E, Lu P, Liang XW, Finlay
DR, Guiney D, Helinski DR (1985) Plasmid related to the
broad host-range vector, pRK290, useful for gene cloning and
for monitoring gene expression. Plasmid 13:149±153
Eckhardt T (1978) A rapid method for the identi®cation of plasmid
deoxyribonucleic acid in bacteria. Plasmid 1:584±588
Heniko€ S (1984) Unidirectional digestions with exonuclease III
creates targeted breakpoints for DNA sequencing. Gene
28:351±359
Hollingsworth R, Abe M, Sherwood J, Dazzo FB (1984) Bacteriophage-induced acidic heteropolysaccharide lyases that convert the acidic heteropolysaccharides of Rhizobium trifolii into
oligosaccharide units. J Bacteriol 160:510±516
Hollingsworth R, Carlson RW, Garcia F, Gage DA (1989) A new
core tetrasaccharide component from the lipopolysaccharide
from Rhizobium trifolii ANU843. J Biol Chem 264:9300±9304
Ish-Horowicz D, Burke JF (1981) Rapid and ecient cosmid
cloning. Nucleic Acids Res 9:2989±2998
Kittelberger R, Hilbink F (1993) Sensitive silver-staining detection
of bacterial lipopolysaccharides in polyacrylamide gels. J Biochem Biophys Methods 26:81±86
Kondorosi A, Kondorosi E, Pankhurst CE, Broughton W, Banfalvi S (1982) Mobilization of a Rhizobium meliloti megaplasmid carrying nodulation and nitrogen ®xation genes into other
rhizobia and Agrobacterium. Mol Gen Genet 188:433±439
KoÈplin R, Wang G, HoÈtte B, Priefer UB, PuÈhler A (1993) A 3.9 kb
DNA region of Xanthomonas campestris pv campestris that is
140
necessary for lipopolysaccharide production encodes a set of
enzymes involved in the synthesis of dTDP-rhamnose. J Bacteriol 175:7786±7792
Leigh JA, Lee CC (1988) Characterization of polysaccharides of
Rhizobium meliloti exo mutants that form ine€ective nodules. J
Bacteriol 170:3327±3332
Leigh JA, Walker GC (1994) Exopolysaccharides of Rhizobium:
synthesis, regulation and symbiotic function. Trends Genet 10:
63±67
Leigh JA, Signer ER, Walker GC (1985) Exopolysaccharide-de®cient mutants of Rhizobium meliloti that form ine€ective nodules. Proc Natl Acad Sci USA 82:6231±6235
Lemaire HG, MuÈller-Hill B (1986) Nucleotide sequences of the
galE gene and the galT gene of E. coli. Nucleic Acids Res
14:7705±7711
Long S, Reed JW, Himawan J, Walker GC (1988) Genetic analysis
of a cluster of genes required for synthesis of the Calco¯uorbinding exopolysaccharides of Rhizobium meliloti. J Bacteriol
170:4239±4248
Lopez-Lara IM, Orgambide G, Dazzo FB, Olivares J, Toro N
(1995) Surface polysaccharide mutants of Rhizobium sp. (Acacia) strain GRH2: major requirement of lipopolysaccharide for
successful invasion of Acacia nodules and host range determination. Microbiology 141:573±581
Meade HM, Long SR, Ruvkun GB, Brown SE, Ausubel FM
(1982) Physical and genetic characterization of symbiotic and
auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149:114±122
Megias M, Caviedes MA, Andres M, Sousa C, Ruiz-Berraquero F,
Palomares AJ (1988) Localization of his genes on the Rhizobium
trifolii RS800 linkage map. Mol Gen Genet 211:369±372
Ollero FJ, Valverde MA, SaÂnchez-PalazoÂn L, Villalobo E, Espuny
MR, BellogõÂ n R (1994) An exoB mutant of Rhizobium sp. is
e€ective in indeterminate nodules of Hedysarum coronarium.
Microbiology 140:1389±1394
Orosz L, Svab Z, Kondorosi A, Silk T (1973) Genetic studies on
Rhizobiophage 16-3. I. Genes and functions on the chromosome. Mol Gen Genet 125:341±350
Philip-Hollingsworth S, Hollingsworth R, Dazzo FB (1989) Hostrange related structural features of the acidic extracellular
polysaccharides of Rhizobium trifolii and Rhizobium leguminosarum. J Biol Chem 264:5710±5714
Quandt J, Hynes MF (1993) Versatile suicide vectors which allow
direct selection for gene replacement in gram-negative bacteria.
Gene 127:15±21
Rodriguez-QuinÄones F, Fernandez-Burriel M, Banfalvi Z, Megias
M, Kondorosi A (1989) Identi®cation of a conserved, reiterated
DNA region that in¯uences the eciency of nodulation in
strain RS1051 of Rhizobium leguminosarum bv trifolii. Mol.
Plant-Microbe Interact 2:75±83
Rolfe BG, Gressho€ PM, Shine J, Vincent JW (1980) Interaction
between a non-nodulating and an ine€ective mutant of Rhizobium trifolii resulting in e€ective (nitrogen ®xing) nodulation.
Appl Environ Microbiol 39:449±452
Rosenberg C, Casse-Delbart F, Dusha I, David M, Boucher C
(1982) Megaplasmid in the plant-associated bacteria Rhizobium
meliloti and Pseudomonas solanacearum. J Bacteriol 150:402±406
Simon R, Quandt J, Klipp W (1989) New derivatives of transposon
Tn5 suitable for mobilization of replicons, generation of operon
fusions and induction of genes in gram-negative bacteria. Gene
80:161±169
Yang C, Signer ER, Hirsch AM (1992) Nodules initiated by Rhizobium meliloti exopolysaccharide mutants lack a discrete,
persistent nodule meristem. Plant Physiol 98:143±151
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage
cloning vectors and host strains: nucleotide sequence of the
M13mp18 and pUC19 vectors. Gene 33:103±119
Zevenhuizen LPTM, Bertocchi C, Van Neerven ARW (1986)
Congo red absorption and cellulose synthesis by Rhizobiaceae.
Antonie Van Leeuwenhoek 52:381±386