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Applied and Environmental Microbiology, July 2000, p. 2988-2995, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The Common Nodulation Genes of Astragalus
sinicus Rhizobia Are Conserved despite Chromosomal
Diversity
Xue-Xian
Zhang,1,2,*
Sarah L.
Turner,3
Xian-Wu
Guo,1
Hui-Jun
Yang,1
Frédéric
Debellé,2
Guo-Ping
Yang,2
Jean
Dénarié,2
J. Peter
W.
Young,3 and
Fu-Di
Li1
Department of Microbiology, Huazhong
Agricultural University, Wuhan 430070, China1;
Laboratoire de Biologie Moléculaire des Relations
Plantes-Microorganismes, INRA-CNRS, 31326 Castanet-Tolosan Cedex,
France2; and Department of Biology,
University of York, York YO10 5YW, United
Kingdom3
Received 5 January 2000/Accepted 2 May 2000
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ABSTRACT |
The nodulation genes of Mesorhizobium sp.
(Astragalus sinicus) strain 7653R were cloned by functional
complementation of Sinorhizobium meliloti nod mutants. The
common nod genes, nodD, nodA, and
nodBC, were identified by heterologous hybridization and
sequence analysis. The nodA gene was found to be separated
from nodBC by approximately 22 kb and was divergently
transcribed. The 2.0-kb nodDBC region was amplified by PCR
from 24 rhizobial strains nodulating A. sinicus, which
represented different chromosomal genotypes and geographic origins. No
polymorphism was found in the size of PCR products, suggesting that the
separation of nodA from nodBC is a common feature of A. sinicus rhizobia. Sequence analysis of the
PCR-amplified nodA gene indicated that seven strains
representing different 16S and 23S ribosomal DNA genotypes had
identical nodA sequences. These data indicate that, whereas
microsymbionts of A. sinicus exhibit chromosomal diversity,
their nodulation genes are conserved, supporting the hypothesis of
horizontal transfer of nod genes among diverse recipient bacteria.
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INTRODUCTION |
Rhizobia are soil bacteria that can
form nodules, in which they fix nitrogen, on leguminous plants in a
host-specific manner. Nodulation (nod) genes have been
identified that control the specific infection and nodulation of the
plant hosts. The initial infection event is regulated by a NodD protein
or proteins which activate the transcription of other nod
genes in the presence of host-produced flavonoids (12, 25,
37). The nodABC genes are called common nod
genes because they are present in all rhizobia. Other nod genes, such as nodFE, nodH, nodSU, and
nodZ (12, 25, 37), are present in various
combinations in rhizobial species and are called host-specific
nod genes.
Expression of common and host-specific nod genes results in
the production of lipochitooligosaccharides (Nod factors) that act as
morphogenic signal molecules on specific legume hosts (12, 37). All Nod factors have a 
-1,4-linked N-acetyl
glucosamine oligosaccharide backbone ranging in length from 3 to 5 residues and substituted for by an N-acyl chain at the nonreducing end and other chemical groups on the glucosamine residues. The common nodABC gene products are involved in the synthesis of the
N-acylated oligosaccharide core, while the host-specific nod
gene products are involved in the decoration of this backbone with
substitutions that confer plant specificity. The nodABC
genes encode an acyltransferase, a chitin oligosaccharide deacetylase,
and a chitin oligosaccharide synthase, respectively (3, 33).
The common nod genes are also involved in determining host
range specificity to some extent. For example, different NodA proteins
recognize and transfer different fatty acid chains to the
chitooligosaccharide chain, the length of which is determined by NodC
(11, 27, 32). The common nodABC genes are
essential for nodule formation. Mutation in any of them abolishes the
ability to produce Nod factors and results in a nonnodulating
(Nod
) phenotype (12).
Astragalus sinicus L. (Chinese milk vetch) is an important
winter-growing green manure, traditionally grown in the rice fields of
southern China, Japan, and Korea. A. sinicus is a very
specific host and usually forms nodules only with rhizobia isolated
from itself (4), the only reported case of cross-inoculation
being with a rhizobial strain isolated from Astragalus
ciceri (24). Chen et al. (5) undertook a
taxonomic study of nine A. sinicus isolates and proposed a
new species, Rhizobium huakuii, for the rhizobia isolated
from this host. On the basis of 16S ribosomal DNA (rDNA) sequence data
and other taxonomic criteria, Jarvis et al. (21) proposed a
new genus, Mesorhizobium, to which R. huakuii was
transferred. Our previous work has shown that A. sinicus rhizobia are diverse (17, 42): of 204 strains analyzed, all are Mesorhizobium, but they belong to four different 16S
rDNA genotypes. These four genotypes can be subdivided into seven
genotypes when 16S and 23S rDNA data are combined.
Mesorhizobium sp. (A. sinicus) strain 7653R
belongs to the dominant 16S rDNA genotype, genotype 3, which is
different from that of the M. huakuii type strain,
CCBAU2609. Strain 7653R contains two large plasmids, and the
nod genes are carried by the larger plasmid (43).
The structures of the Nod factors produced by three
Mesorhizobium sp. (A. sinicus) strains, including
7653R, have been determined: the three strains produce identical
pentameric lipochitooligosaccharides that are O sulfated and partially
N glycolylated at the reducing end and N acylated at the nonreducing
end by a C18:4 fatty acid (40). The aims of this
study were to isolate and characterize the nod genes of
Mesorhizobium sp. (A. sinicus) strain 7653R, to
compare the common nod genes with those from other species
of rhizobia, and to look at nod gene variation among
A. sinicus nodule isolates representing the different
chromosome types (17, 42).
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
Rhizobia
were grown at 28°C on either tryptone-yeast (TY) medium (tryptone, 5 g/liter; yeast extract, 3 g/liter; CaCl2 · 2H2O, 0.87 g/liter) or yeast extract-mannitol (YEM) medium
(39). Escherichia coli strains were grown at
37°C on Luria-Bertani (LB) medium (28). Where appropriate,
antibiotics were added to the following concentrations (micrograms per
milliliter): streptomycin, 200; ampicillin and kanamycin, 100;
phleomycin, 20; and tetracycline, 10. The rhizobial strains from
A. sinicus are described in Table
1. The other rhizobial strains and
plasmids used in this study are shown in Table
2.
Construction of a cosmid gene library.
Total DNA of
Mesorhizobium sp. (A. sinicus) strain 7653R was
prepared according to the method of Ma et al. (23) and
partially digested with the restriction enzyme EcoRI.
Fragments of 20 to 30 kb were isolated by centrifugation (25,000 rpm on
a Beckman SW28 rotor; 20°C, 24 h) through a sucrose density
gradient (10 to 50% [wt/vol]), dialyzed to remove the sucrose, and
then concentrated to a suitable volume by precipitation with 2 volumes
of ethanol by resuspension in distilled water. DNA was then ligated
with a sevenfold excess of EcoRI-digested and
phosphatase-treated cosmid vector pLAFR3 (33). The ligated
DNA was packaged into lambda bacteriophage particles and introduced
into E. coli LE392 by transfection (28). Three
thousand single colonies were picked and stored separately in 20%
glycerol at 
70°C by using 96-well plates. The colonies were mixed
before use.
Conjugation and plant experiment.
The cosmid gene library
was introduced into Sinorhizobium meliloti mutant strains
through triparental mating with the help of plasmid pRK2013
(13); crosses were carried out on TY medium by the method
described by Christensen and Schubert (6). Medicago sativa seeds and nodules were surface sterilized by soaking in 95% ethanol for 5 min and in 0.2% acidified HgCl2 for 3 min and then being rinsed 10 times in sterile water. The
surface-sterilized seeds were germinated at 22°C in darkness
overnight, and the seedlings were aseptically grown in test tubes on
Jensen nitrogen-free agar slants (9). Two seedlings were put
into each tube.
DNA manipulations and sequence analyses.
Southern blots were
carried out on Hybond-N+ membranes (Amersham) according to
the manufacturer's instructions. The nod gene probes were
labeled with the Promega Multiprimer Labeling kit. The nodD
and nodC gene probes were retrieved from pRmSL42, which contains the nod genes of S. meliloti
(15). The nodA gene of Mesorhizobium
loti cloned in plasmid pPN25 (31) was used for nodA gene hybridization. A 0.6-kb SalI fragment
from pGMI174, internal to the S. meliloti 2011 nodE, was used as a nodE probe (8).
Sequencing of the common nod genes of
Mesorhizobium sp. strain 7653R was achieved by further
subcloning the 4.2-kb EcoRI and 10-kb HindIII
fragments as several smaller fragments (about 1 to 2 kb) into the
sequencing vector pBluescript KS+ (28). Sequencing reactions
were performed by Sanger's dideoxy chain termination method, and the
extension products were separated and detected on an Automated Laser
Fluorescent DNA Sequencer (Pharmacia). Primers were synthesized for
sequencing in order to get the whole sequences of both strands.
The DNA sequences were analyzed by using the GCG software version 7.1 (Genetics Computer Group, University of Wisconsin, Madison).
The
sequences used for phylogenetic studies were first aligned
by using
PILEUP, and phylogenetic trees were constructed by using
ClustalW
(
36), which uses the neighbor-joining algorithm with
the K2P
distance correction. The resultant trees were displayed
with TreeView
(
26). The
nodABC gene sequences of the following
species or strains used in phylogenetic analyses were obtained
from the
EMBL database (accession numbers in parentheses):
Azorhizobium caulinodans ORS571 (
L18897),
Rhizobium leguminosarum
bv. viciae
(
Y00548),
Rhizobium galegae (
X87578),
S. meliloti Rm1021
(
M11268),
S. meliloti Rm41 (
X01649),
Rhizobium etli CE-3
(
M58625 and
M58626),
Sinorhizobium sp. strain NGR234 (
X73362),
Sinorhizobium fredii USDA257 (
M73699),
M. loti
NZP2037 (
X52958),
Mesorhizobium sp. strain N33 (
U53327), and
Rhizobium tropici CFN299 (
X98514).
PCR amplifications.
The nodD-to-nodC
region of A. sinicus rhizobia was amplified by using primers
DC1 (5'-GTA CAG GAG GGC ATC GCG AA-3') and DC2 (5'-CTG CAG CTG CAG CGA
ATC TG-3'). Primer DC1 corresponds to positions 1244 to 1225 in the
Mesorhizobium sp. strain 7653R nodDBC sequence
(in nodD), and primer DC2 corresponds to positions 3189 to
3170 (in the nodC gene). The PCR was performed in a 60-µl
volume containing 1× reaction buffer (10 mM Tris-HCl, 50 mM KCl, 0.1% Triton X-100), 0.5 µM each primer, 1.25 mM MgCl2, 0.2 mM
deoxynucleoside triphosphates (dNTP), 30 ng of template DNA, and 2 U of
Taq polymerase (Sangon). The following temperature profile
was used: initial denaturation at 94°C for 3 min; 30 cycles of 94°C
for 1 min, 56°C for 1 min, and 72°C for 2 min; and final extension
at 72°C for 10 min.
Amplification of
nodA sequences with nodA-1 and nodA-2 used
the protocol of Haukka et al. (
18). Primers nodA-3 (5'-TCA
TAG
CTC YGR ACC GTT CCG-3') and nodA-4 (5'-ATC ATC KYN CCG GNN GGC
CA-3'), corresponding to positions 980 to 960 and 955 to 936 of
the
Mesorhizobium sp. strain 7653R
nodA sequence,
were designed
with this sequence and those present in the databases.
Where necessary,
these primers were used in a nested PCR, involving two
rounds
of amplification, with the product of the first amplification,
with primers nodA-1 and nodA-3, being used as a template for the
second
amplification, with primers nodA-1 and nodA-4. The same
amplification
conditions were used for each primer pair in 50-µl
volumes containing
1× reaction buffer, 50 pmol of each primer,
1.63 mM MgCl
2,
0.2 mM dNTP, and 1 U of
Taq polymerase (Promega).
The
following temperature profile was used: initial denaturation
at 97°C
for 2 min; 25 cycles of 92°C for 40 s, 55°C for 1 min,
and
72°C for 1.5 min; and final extension at 72°C for 5 min. Amplified
nodA sequences were cleaned by using the PCR Purification
kit
(Qiagen) and eluted in 30 µl of H
2O before sequencing
with the
Dye Terminator Ready Reaction kit (Perkin-Elmer) and an ABI
377
automated
sequencer.
Nucleotide sequence accession number.
The DNA sequences of
nodDBC and nodA are available in the EMBL
database under accession no. AJ249393 and AJ249353, respectively. The
nodA sequences are available in the EMBL database under
accession no. AJ25040 to AJ25042.
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RESULTS |
Cloning of common and specific nod genes from
Mesorhizobium sp. (A. sinicus) strain 7653R by
functional complementation.
Nod factors produced by M. huakuii strains have been shown to be similar to those from
S. meliloti (40): both are O sulfated at C-6 of
the reducing end and N acylated by 
,-polyunsaturated acyl chains
at the nonreducing end. We hypothesized that both species possess a
similar set of nodulation genes and that various S. meliloti
Nod mutants could be used to identify clones containing
their M. huakuii counterparts by heterologous functional
complementation by a genomic cosmid library of strain 7653R. The seven
S. meliloti 2011 nod mutant strains listed in
Table 2, which exhibit a clear Nod phenotype on alfalfa,
were used. The transconjugants of each cross were used to inoculate 200 seedlings of Medicago sativa, the macrosymbiont of S. meliloti. Efficient nodulation occurred with a high proportion (80 to 97%) of replicates for all crosses, except that of the
nodFL mutant (GMI6628), for which nodulation was found in
only 18% of tubes.
Cosmids carrying genes complementing the
S. meliloti nod
mutants were isolated from the surface-sterilized nodules and
characterized
by restriction analysis. A physical map of the nodulation
region
was constructed (Fig.
1). Cosmids
identical to pHN50 were isolated
from Nod
+ transconjugants
of the
S. meliloti nodA,
nodB,
nodC,
and
nodD1D2D3 mutants,
indicating that pHN50 contains all of these genes. Southern
hybridizations using the
nodD,
nodA, and
nodC probes located the
nodC and
nodD
genes to a 4.2-kb
EcoRI fragment and the
nodA
gene
to a 20-kb
EcoRI fragment and a 10-kb
HindIII fragment. These
results suggest that
nodA is separated from the
nodBC genes, in
contrast to the arrangement seen in most rhizobia. Cosmid pHN71,
isolated from a Nod
+ transconjugant of the
S. meliloti nodF nodL mutant (GMI6628),
and pHN50 share 5.0- and
0.8-kb
EcoRI fragments adjacent to the
20-kb
EcoRI fragment carrying
nodA. Southern
hybridization using
the
nodE probe indicated that the 5-kb
fragment contains a
nodE homolog. The pHN72 cosmid, isolated
from a Nod
+ transconjugant of the
nodH mutant
(GMI5619), overlaps pHN50 by
the 4.2-kb
EcoRI fragment
carrying the
nodDBC homologs. Partial
sequencing of the
adjacent 2.8-kb
EcoRI fragment of pHN72 indicated
that it
contains a
nodH homolog and a partial
nodI
homolog.
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FIG. 1.
Physical and genetic map of the nodulation
(nod) gene region of Mesorhizobium sp. strain
7653R. Black boxes indicate possible nod box sequences,
shaded arrows indicate sequenced genes, open arrows represent partially
sequenced genes, and the white box represents the truncated
nodB-like sequence downstream of nodA. The
positions of primers DC1 and DC2 are indicated by triangles. Only the
regions of cosmids pHN71 and pHN72 relevant to this study are shown
( ). E, EcoRI; S, SalI; B, BglII; P,
PstI; H, HindIII.
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Sequence analysis of the common nod genes of strain
7653R.
The 4.2-kb EcoRI fragment that hybridized with
the nodD and nodC probes was subcloned into
pBluescript KS+ (pHN51) and sequenced (accession no. AJ249393). This
revealed four open reading frames, which correspond to the full-length
nodD, nodB, and nodC genes and a
partial nodI gene (Fig. 1). There is a 47-bp nod
box (positions 1484 to 1530) in the nodDB intergenic region
(Fig. 2), but a full-length nodA gene is not present in this region. Alignment of
nodB gene sequences from 7653R and other rhizobia indicated
that the 7653R nodB gene has an extra 54 bp at its 5' end,
which shows 68.3% identity with the 5' end of the 7653R
nodA gene (Fig. 2).
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FIG. 2.
Organization of the 7653R nodDB intergenic
region. The 5' end of nodB is similar to the 5' end of 7653R
nodA (sequence written in italic letters). The nucleotide
sequence of the nodDB intergenic region is numbered as in
accession no. AJ249393. Arrows indicate start codons of the
nodD and nodB genes. Boldface letters indicate
the consensus nod box (29).
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The 10-kb
HindIII fragment carrying
nodA was
subcloned into pBluescript KS+ (pHN64). Southern hybridization with the
nodA probe and subcloning localized the gene to a 1.15-kb
PstI-
EcoRI
DNA fragment which was sequenced
(accession no.
AJ249353).
This region contains a full-length
nodA gene which has a recognizable
nod box
upstream (positions 242 to 288) (Fig.
3).
These results
confirm that
nodA is separated from
nodBC by a distance of approximately
22 kb. A 107-bp
truncated
nodB-like sequence (
nodB) was
identified
downstream of
nodA, which shares 50.5% sequence
identity with
the corresponding part of the full-length
nodB
gene (Fig.
3).
The
nodB start codon overlaps the
nodA stop codon (ATGA). This
kind of junction between
nodA and
nodB has been found in many
rhizobia.
The presence of
nodB immediately downstream of
nodA and the presence of a
nodA-like extension at
the 5' end of
nodB are molecular evidence that genome
rearrangements occurred at
the
nodA-nodB junction in an
ancestor of the nodulation region
now carried by strain 7653R.
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FIG. 3.
Organization of the 7653R DNA sequence upstream and
downstream of the nodA gene. DNA downstream nodA
shows similarity to a fragment of the nodB gene (sequence
written in italic letters). The nucleotide sequence is numbered as in
accession no. AJ249353. The start and stop codons of the
nodA gene are underlined. The consensus nod box
(29) is indicated by boldface letters.
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The organization of common nod genes varies among
Mesorhizobium species but is conserved in
Mesorhizobium isolated from A. sinicus.
To
determine whether the separation of nodA from
nodBC was unique to strain 7653R or was present in other
A. sinicus isolates, primers DC1 and DC2 were designed to
amplify the nodDC region, which corresponds to the 2.0-kb
PstI fragment of pHN51 (Fig. 1). Twenty-four representative
strains were selected from a collection of field isolates isolated from
six southern provinces of China. These strains had previously been
characterized by restriction fragment length polymorphism (RFLP)
analysis of PCR-amplified 16S rRNA genes and 16S-23S rDNA intergenic
spacers (IGS) (Table 1) (17, 42). PCR amplification of each
of the 24 strains, using primers DC1 and DC2, produced a single band of
2.0 kb. This is the same size as obtained from 7653R and indicates that
the separation of nodA from nodBC is a general
feature of A. sinicus rhizobia.
Additional evidence that
nodA is separated from
nodBC was provided by using primers nodA-1 and nodA-2. These
primers had been
used previously to amplify
nodA sequences
from tropical tree rhizobia
and correspond to residues 14 to 37 of
nodA and 65 to 43 of
nodB of the
S. meliloti 1021 sequence (M112684) (
18). Although a
nodB-like sequence follows the
nodA gene of
Mesorhizobium sp.
strain 7653R, the nodA-2 primer site in
this sequence is different,
and amplification would be unlikely. PCR
using the nodA-1 and
-2 primer pair did not produce visible products
with any of the
seven
A. sinicus isolates tested (Table
1).
This finding is further
evidence that
nodA is separated from
nodB in the
Mesorhizobium strains of
A. sinicus.
In contrast, the
M. ciceri,
M. mediterraneum,
M. plurifarium, and
M. tianshanense type strains
produced an amplification product
with the nodA-1-nodA-2 primer
combination, indicating that, in
these species, the
nodB
gene is located immediately downstream
of
nodA. The
nodA-1-nodA-2 primer pair did not amplify
nodA in
the
related species
M. loti. This is in agreement with the
previous
report showing that, in
M. loti, the
nodB gene is separated from
the
nodAC genes
(
31). The fact that the rearrangements in
M. huakuii and
M. loti are different suggests that these
rearrangements
occurred
independently.
Identity of nodA sequences in A. sinicus
rhizobia of various chromosomal types.
Having shown that the
general organization of the common nodulation genes was conserved among
A. sinicus rhizobia, we wanted to know whether the common
nod genes were also conserved at the DNA sequence level in
these rhizobia. For this work, we focused on the nodA gene
and used only seven strains that represent each of the seven genotypes
identified in an earlier study (42) (Table 1). Since the
nodA-2 primer could not be used to amplify nodA genes from
A. sinicus rhizobia, new reverse nodA PCR
primers, nodA-3 and nodA-4, were designed to match the 3' region of
aligned nodA sequences. PCR using the nodA-1-nodA-3 primer
pair produced weak amplification products with most of the A. sinicus isolates. However, a nested PCR using the nodA-1-nodA-4
primer pair and the nodA-1-nodA-3 PCR product as a template gave clean
amplification products for all seven A. sinicus isolates,
and each PCR product was sequenced on both strands. All seven
nodA sequences were identical to that of strain 7653R,
showing total conservation in spite of the diversity of geographical
origins and chromosome backgrounds.
Phylogenetic studies.
Phylogenetic relationships between the
nodA genes of A. sinicus rhizobia and the
nodA sequences available in the GenBank/EMBL database were
studied at both DNA and deduced protein levels. The results indicated
that the closest relatives of 7653R nodA genes are those of
two other Mesorhizobium strains, Mesorhizobium sp. (Oxytropis arctobia) strain N33 and M. loti.
In order to assess whether the nodA genes of all described
Mesorhizobium species clustered together, we sequenced the
nodA genes from type strains of M. tianshanense,
M. ciceri, M. mediterraneum, and M. plurifarium, after PCR amplification of nodA by using
the nodA-1 and nodA-2 primers. The dendrograms based on nodA
nucleotide sequence are shown in Fig. 4.
All Mesorhizobium nodA sequences, except the M. plurifarium sequence, lie in the same clade with 97% bootstrap support. The M. plurifarium type strain was isolated from
Acacia senegal, a tropical leguminous tree, which is
distantly related to the legume hosts of the other type strains
surveyed in this work (14). The nodA sequences of
M. ciceri and M. mediterraneum, two species
isolated from the same host plant (chickpea), differ at a single
position.
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FIG. 4.
Phylogenetic position of the Mesorhizobium
sp. (A. sinicus) strain 7653R nodA and
nodC sequences, calculated by using the neighbor-joining
algorithm and the Kimura two-parameter model. Percentage bootstrap
values obtained after 1,000 trials are shown, and the scale bars refer
to the number of substitutions per site. A., Azorhizobium;
S., Sinorhizobium; M., Mesorhizobium.
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Comparison of the 7653R
nodDBC gene sequences with the
corresponding sequences available in databases also indicated that
the
closest relatives of 7653R genes are those of
Mesorhizobium sp. (
Oxytropis arctobia) strain N33 and
M. loti.
The dendrogram
based on
nodC nucleotide sequences is shown
in Fig.
4.
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DISCUSSION |
The nod gene organization in A. sinicus
rhizobia is unusual.
The common nodulation genes in most rhizobial
species are organized in a single operon, nodABC, with a
copy of nodD upstream and divergently transcribed (15,
37). The exceptions described previously are R. etli
(38), M. loti (31), and
Mesorhizobium sp. strain N33 (7). M. loti strains NZP2037 and NZP2213 have nodB apart from
nodAC (Fig. 5). The
nodA gene is separated from nodBC in R. etli and Mesorhizobium sp. strain N33. However, in all
cases studied so far, the nodA, -B, and
-C genes are in the same orientation. Physical mapping and
sequence analysis of the common nodulation gene region of
Mesorhizobium sp. (A. sinicus) strain 7653R
revealed that nodA is separated from nodBC by
about 22 kb, and the two operons are divergently transcribed. Like
R. etli, M. loti, and Mesorhizobium
sp. strain N33, strain 7653R has a truncated nodB-like
sequence immediately downstream of the nodA gene (Fig. 5).
The complete 7653R nodB gene has extra sequence at the 5'
end that shows homology with the full-length nodA gene. These two observations support the hypothesis that nodABC
was the ancestral operon organization and that genome rearrangements have occurred.
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FIG. 5.
Schematic map of selected nodulation genes in rhizobial
species where rearrangements of the nodABC genes have been
detected (7, 31, 37). Black boxes indicate functional
nod genes. Shaded boxes indicate DNA fragments similar to
nod genes, while white boxes indicate nod boxes.
Arrows indicate the direction of transcription of the nod
genes. M., Mesorhizobium.
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Horizontal transfer of nodulation genes.
Genetically
dissimilar microsymbionts have frequently been isolated from a single
legume species (25). This is well documented in the case of
the symbionts of soybeans, which belong to two different genera,
Bradyrhizobium and Sinorhizobium (25),
and of beans which belong to five different species of
Rhizobium (R. leguminosarum, R. etli,
R. tropici, R. gallicum, and R. giardinii) (1). Our previous work indicated that
rhizobia belonging to four different 16S rDNA types nodulate A. sinicus (42). The high genetic diversity of A. sinicus rhizobia has been confirmed by the analysis of other
marker genes, such as the 23S rRNA gene (42),
recA, glnA, and glnII (our work
[unpublished data]). However, the results presented here show that
representatives of the different rhizobial rDNA types have the same
common nod gene organization and identical nodA
gene sequences. The chemical structures of Nod factors produced by
Mesorhizobium sp. (A. sinicus) strain 7653R and
two M. huakuii strains, Ra5 and Ra98, have been shown to be
the same (40). These findings suggest that the
nod genes of A. sinicus rhizobia are conserved in
contrast to their chromosomal diversity.
The phylogenetic positions of different 16S rDNA genotypes within
Mesorhizobium have been shown to be independent of host
plant origin (
22). Dendrograms based on both 16S and 23S
rDNA
indicate that
Lotus rhizobia, which are known to be
diverse, are
the closest relatives of the different rDNA types of
A. sinicus rhizobia studied in this paper (
42).
Sullivan et al. (
34)
observed transfer of the ability to
form effective nodules with
Lotus corniculatus between
different species of mesorhizobia within
soil communities. Although the
symbiotic genes of
Lotus rhizobia
are on the chromosome,
they can be transferred to nonsymbiotic
mesorhizobia in the field
environment because they are carried
on a symbiotic island
(
35). Some
Lotus rhizobia and
A. sinicus rhizobia have the same chromosomal background but
different host
ranges. For example,
Mesorhizobium sp.
(
A. sinicus) strain 7653R
and
M. loti NZP2037
belong to the same 16S rDNA genotype, which
is different from those of
both the
M. loti type strain, NZP2213,
and the
M. huakuii type strain, CCBAU2609. This situation is equivalent
to
that known for the three biovars of
R. leguminosarum, which
have the same chromosomal type, but have different symbiotic genes
and
host ranges. Like
R. leguminosarum, the nodulation genes of
A. sinicus rhizobia are carried on a large plasmid, which
could
spread within field populations by horizontal plasmid transfer
(
17). Rapid horizontal transfer would explain why identical
symbiotic genotypes are associated with different chromosomal
backgrounds. This work has also revealed that the
nodA
sequences
of the
M. mediterraneum and
M. ciceri
type strains are almost
identical. This finding parallels the results
for the
A. sinicus isolates: both the
M. ciceri
and
M. mediterraneum type strains
were isolated from the
same host plant,
Cicer arietinum (chickpea),
again
suggesting symbiotic gene transfer between chromosomal types
that are
sufficiently divergent to be considered separate
species.
These findings suggest that symbiotic transfers can occur between
different species of
Mesorhizobium, in agreement with the
findings of Sullivan et al. (
34) for
Lotus
rhizobia. Interspecies
transfer has also been documented for
Rhizobium species: the same
symbiotic genotypes have been
isolated from
R. leguminosarum bv.
phaseoli,
R. etli,
R. gallicum, and
R. giardini
(
1). If different
species of fast-growing rhizobia are
exchanging symbiotic genes
at detectable frequencies, other genes might
also be exchanged.
If this were the case, it would bring into question
the reliability
and usefulness of species designations. However, we
have recently
sequenced internal fragments of the
recA and
atpD genes of the
type strains of most rhizobium species,
and these data support
the 16S phylogeny (unpublished data). The three
genera
Rhizobium,
Sinorhizobium, and
Mesorhizobium are separated in all three phylogenies,
and
there is no evidence for intergenus gene exchange. Analysis
of the
glutamine synthetase genes (
glnA and
glnII) of
rhizobial
type strains may indicate an ancient exchange of genes
between
a
Rhizobium species and
M. huakuii but
does not suggest that intergenus
transfers of chromosomal genes are
common (
41).
We have shown that the functional
nodA sequences of
A. sinicus rhizobia are totally conserved between strains with
different
chromosomal backgrounds. While the amino acid sequence of
nodA is undoubtedly constrained by functional
considerations, the redundancy
of the genetic code means that many base
substitutions in the
DNA sequence will not alter the protein. Such
"silent" substitutions
usually have little selective effect and
accumulate relatively
rapidly over evolutionary time. Complete identity
at the DNA level
is therefore strong evidence for a recent common
ancestry of the
nodA genes. The nonfunctional
nodB sequence is presumably not
under selective pressure
and will accumulate mutations more rapidly
than a protein-coding gene.
The
nodB sequence could serve as
a molecular marker to
further investigate symbiotic gene transfers
among
A. sinicus rhizobia for population dynamic and evolutionary
studies.
If symbiotic gene transfers are rare, the phylogeny of
nodB would be more similar to the 16S rDNA (chromosomal)
phylogeny,
since both symbiotic and chromosomal genes will have had a
shared
history. If, on the other hand, there has been a recent rapid
spread of the
A. sinicus symbiotic genes, sequence variation
within
nodB will not reflect the chromosomal background.
The
nodB sequence
might therefore give an insight into
how common horizontal gene
transfer is in the genus
Mesorhizobium.
| |
ACKNOWLEDGMENTS |
We thank K. Lindström, Z. M. Zhang, and H. K. Chen for helpful discussion and advice.
This work was funded by IFS grant C/2305-2 and NSFC and EC programs
INCO-DC IC18-CT96-0103 and BIO4-CT.98-0483.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China. Phone and fax: 86 27 87396057. E-mail:
xxzhang{at}excite.com.
| |
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Abstract
Introduction
