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Appl Environ Microbiol, May 1998, p. 1845-1851, Vol. 64, No. 5
Institute of Genetic Ecology,
Received 21 July 1997/Accepted 20 February 1998
In a survey of DNA fingerprints of indigenous Bradyrhizobium
japonicum with the species-specific repeated sequences RS Insertion sequence (IS) elements are
discrete segments of DNA that are able to transpose to numerous sites
on bacterial plasmids and chromosomes, usually with an increase in
their copy number (7). IS elements can also promote
rearrangement of genomes and other replicons (7). Many IS
elements and uncharacterized repeated DNA sequences among
plant-associated gram-negative bacteria, including
Agrobacterium (5), Bradyrhizobium
(10, 11, 17), Rhizobium (3, 5, 35),
and Xanthomonas (2) spp., have been described.
These repeated elements often cause genomic instability affecting genes
responsible for plant associations (15, 23), and they have
been postulated to play a role in evolution and genomic instability
(1, 7, 17, 24, 29). Indeed, complete sequencing of a
symbiotic plasmid pNGR234a from a Rhizobium sp. demonstrated
that almost one-fifth of the total plasmid sequence is made up of IS
elements and a mosaic sequence structure including nodulation loci
(6).
Members of the genus Bradyrhizobium are slow-growing,
gram-negative, nitrogen-fixing heterotrophic bacteria which can form root nodules on several leguminous plants. In Bradyrhizobium
japonicum, several repeated DNA sequences (RS DNA fingerprints with RS In a previous paper (22) two B. japonicum
isolates, NC32a and NC3a, obtained from a Nakazawa field site showed
numerous bands of RS-specific hybridization. Of 213 isolates of soybean bradyrhizobia indigenous to six field sites in Japan (reference 22 and unpublished data), 19 isolates have been
found to exhibit numerous bands of RS-specific hybridization,
suggesting that the distribution of such isolates is ubiquitous. In
this study, we have genetically and phenotypically characterized field
isolates of soybean bradyrhizobia showing numerous bands of RS-specific hybridization as first steps toward gaining some understanding of their
ecological role.
Isolation of B. japonicum from fields.
Seeds of
soybean (Glycine max) cultivar Enrei were surface sterilized
by immersion in 0.5% sodium hypochlorite for 5 min followed by
washings with sterile water. Seeds were sown in sterile vermiculite and
then inoculated with a moist soil sample (1 g), which had been
collected from the plow layers of the Tokachi field at Tokachi Agricultural Station (Memuro, Tokachi, Hokkaido, Japan), the Nakazawa and Nagakura fields at Niigata Agricultural Experiment Station (Nagaoka, Niigata, Japan), the Ami field at the experimental farm of
Ibaraki University (Ami, Ibaraki, Japan), the Fukuyama field at the
Experimental Farm of Hiroshima University (Fukuyama, Hiroshima, Japan),
or the Ishigaki field at the experimental field of Ishigaki Island
Branch of Tropical Agriculture Research Center (Ishigaki, Okinawa,
Japan). The inoculation procedure was as described previously (22). The plants were cultivated in a greenhouse, and
nitrogen-free nutrient solution (20) was repeatedly
supplied.
Bacterial strains and media.
B. japonicum strains
USDA110, USDA122, and USDA123, obtained from H. H. Keyser of the
U.S. Department of Agriculture, Beltsville, Md., were used as standard
strains. A total of 213 soybean bradyrhizobia were isolated from the
six field sites described above, but the data shown in this work were
mainly obtained from three sites, i.e., Tokachi, Nakazawa, and
Nagakura, for which the prefixes T, NC, and NK were used for field
isolates, respectively. B. japonicum strains were grown
aerobically at 30°C in HM salt medium (4) supplemented
with 0.1% arabinose-0.025% yeast extract (Difco), referred to here
as HM medium, and in YM medium (20, 21). Escherichia
coli HB101 (recA DNA isolation and hybridization.
Total DNA isolation and
hybridization were carried out as described previously (21).
RS Estimation of copy numbers of RS Determination of mean generation time.
Precultures (0.5 ml)
of B. japonicum strains and the field isolates at a mid-log
stage were inoculated into 100 ml of HM medium. The turbidity
(A660) of cultures grown aerobically at 30°C
was measured every 6 h with a Shimadzu spectrophotometer (model
UV-1200). The mean generation time was calculated from the maximum
growth rate at an early exponential phase of duplicate cultures.
Determinations of serological and symbiotic phenotypes.
For
serotype determination, each isolate was tested for agglutination
reactions as previously described (22). Surface-sterilized soybean seeds (Glycine max cv. Enrei) were inoculated with a
culture of each isolate grown in YM medium. Plants were cultivated as described above. About 40 days after germination, the plants were subjected to analyses for nodulation, nitrogen fixation, and
hydrogenase activity. The number and fresh weight of nodules excised
from the roots were measured. To measure nitrogen-fixing activity, acetylene reduction was assayed by using the nodulated roots over a
period of 15 min as described by Hardy et al. (12).
Hydrogenase activity was determined amperometrically with homogenates
of soybean nodules as previously described (22).
16S rRNA gene sequencing.
Primers 27fR
(5'-CAGGAAACAGCTATGACCAGAGTTTGATCCTGGCTCAG-3') and 1069rU
(5'-TGTAAAACGACGGCCAGTCCAACATTCACACACGAG-3') were used for
16S rRNA gene sequencing. The sequences correspond to positions 8 to 27 and 1069 to 1086 in E. coli numbering, respectively
(18), and possess the sequences of M13 reverse and universal
sequencing primers at the 5' ends, respectively. Each 50-µl reaction
mixture contained 6.5 ng of total DNA of each isolate; 1.25 U of ExTaq polymerase (Takara Shuzo Co., Ltd.); 200 µM concentrations each of
dATP, dCTP, dGTP, and dTTP; and 800 nM concentrations of each primer in
a buffer recommended by the manufacturer. The temperature program was
30 s at 94°C and then 25 cycles of 40 s at 94°C, 40 s at 54°C, and 60 s at 72°C. After the PCR products were
purified with the QIA quick PCR purification kit (QIAGEN, Inc.), direct sequencing was performed by using the PCR products as templates with a
model 373A DNA sequencer and Taq dye primer cycle sequencing kits for 21M13 and M13Rev. (Applied Biosystems Co.). Sequences similar
to those determined for 16S rRNA genes were identified in DNA databases
(DDBJ/EMBL/GenBank). Based on these sequences, a phylogenetic tree was
constructed by the neighbor-joining method with Clustal W
(28).
Cloning and DNA sequencing of the PCR-amplified portion of the
RS Nucleotide sequence accession numbers.
The nucleotide
sequence data reported here will appear in the DDBJ, EMBL, and GenBank
nucleotide sequence databases with the following accession numbers:
AB004083 for RS B. japonicum isolates possess a high copy
number of RS
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
New Bradyrhizobium japonicum Strains
That Possess High Copy Numbers of the Repeated Sequence RS
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
and RS
, 21 isolates from three field sites showed numerous RS-specific hybridization bands. The isolates were designated highly reiterated sequence-possessing (HRS) isolates, and their DNA hybridization profiles were easily distinguished from the normal patterns. Some HRS
isolates from two field sites possessed extremely high numbers of RS
copies, ranging from 86 to 175 (average, 128), and showed shifts and
duplications of nif- and hup-specific
hybridization bands. The HRS isolates exhibited slower growth than
normal isolates, although no difference in symbiotic properties was
detected between the HRS and normal isolates. Nucleotide sequence
analysis of 16S rRNA genes showed that HRS isolates were strains of
B. japonicum. There was no difference in the spectra of
serological and hydrogenase groupings of normal and HRS isolates. Some
HRS isolates possessed a tandem repeat RS dimer that is similar to
the structure of (IS30)2, which was shown to
cause a burst of transpositional rearrangements in Escherichia
coli. The results suggest that HRS isolates are derived from
normal isolates in individual fields by genome rearrangements that may
be mediated by insertion sequences such as RS.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
, RS, RS
,
RS
, RS
, and RS
) have been identified (10, 11, 17).
At least one of these sequences, RS, has structural properties
similar to that of a prokaryotic IS element. Interestingly, the RS
copies are often clustered around the regions of nitrogen-fixation and
nodulation genes on the chromosome of B. japonicum USDA110
(17). An insertion sequence, HRS1, also was found to be
closely linked to common and genotype-specific nodulation genes in
B. japonicum serocluster USDA123 and USDA127 strains
(15, 27).
, RS, and HRS1 as probes revealed genetic
diversity within natural populations of B. japonicum that nodulated soybeans (13, 15, 22, 27), indicating that RS fingerprinting is useful for isolate or strain identification and is a
valuable tool for evaluating the genetic structure of indigenous
B. japonicum populations.
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
hsdR hsdM pro
leu Strr) was grown in Luria-Bertani medium
(19).
- and RS-specific probes and hup probe were prepared
from pRJ676 (14) and pHU52 (31), respectively, as
described previously (22).
and RS.
Each lane of
nylon membrane hybridized with 32P-labeled probes was cut
with a razor blade. The strip was placed into a scintillation vial
containing 10 ml of 0.4 M NaOH solution and then incubated at 45°C
for 30 min to dissolve the 32P-labeled probe in the
solution. The radioactivity of 32P was measured by Cerenkov
counting in a Beckman liquid scintillation counter (model LS6500). To
estimate the copy numbers of RS and RS, the radioactivity of each
isolate was compared to that of USDA110, which possesses 12 and 6 copies of RS and RS, respectively (17). Each result is
a mean of duplicate determinations.
tandem repeat.
Four oligonucleotide primers, i.e., primers
13 (5'-CGACAACCTCAACACCCATA-3'), 14 (5'-CTTCGTATAGATCGGCTGCT-3'), 17 (5'-ACGCATACAACGACAGAGCC-3'), and 18 (5'-TCAAATCGCGCTGCAACGTC-3'), were designed for PCR
amplification of the tandem repeat of RS on the basis of the
published nucleotide sequence of RS (17). Each 100-µl
reaction mixture contained 65 ng of total DNA or PCR-amplified DNA
fragment; 2.5 U of Ex Taq polymerase; 200 µM concentrations each of
dATP, dCTP, dGTP, and dTTP; and 400 nM concentrations of each primer in
a buffer recommended by the manufacturer. The temperature program was 1 cycle of 240 s at 95°C, 60 s at 55°C, and 60 s at
72°C; 30 cycles of 60 s at 94°C, 60 s at 55°C, and
60 s 72°C; and 1 cycle of 300 s at 72°C. PCR products
were separated by horizontal electrophoresis on 2% agarose gels, then
stained with ethidium bromide and photographed. Products were also
cloned into pCRTMII with a TA cloning kit (Invitrogen Co.)
according to the manufacturer's instructions. DNA sequence analysis
was performed with an A.L.F. DNA sequencer II (Pharmacia) on both
strands by using the M13 universal and M13 reverse primers.
tandem repeat of HRS isolate NC32a, AB004084 for
RS tandem repeat of HRS isolate NK5, AB004085 for RS tandem
repeat of HRS isolate T2, AB004086 for RS tandem repeat with 35-bp
spacer of RS tandem repeat in HRS isolate NK5, AB004807 for 16S rRNA
gene sequence of HRS isolate NK5, AB004808 for 16S rRNA gene sequence of HRS isolate NK6, and AB004806 for 16S rRNA gene sequence of normal
isolate NK2.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
and RS.
In addition to the two B. japonicum isolates, NC32a and NC3a, obtained from a Nakazawa field
site described in a previous paper (22), the DNA samples
from 19 additional isolates from Nagakura and Tokachi field sites were
found to show numerous bands of RS-specific hybridization, which were
easily distinguished from normal hybridization patterns exhibited by
USDA110 and USDA122. These isolates are designated highly reiterated
sequence-possessing (HRS) isolates.
and 6 copies of RS (17).
This analysis was based on the assumption that HRS and normal isolates
do not differ in their genome sizes and their intensities of
hybridization. The estimated copy numbers of RS and RS in HRS
isolates were much higher than those of the normal isolates (Table
1). According to the copy numbers of RS
elements, HRS isolates tested were categorized into two types, the
Niigata type and the Tokachi type (Table 1). Niigata-type HRS isolates
from Nakazawa and Nagakura field sites possessed a significantly higher
number of RS copies than those of Tokachi- type HRS and normal
isolates. The mean estimated copy numbers with standard deviations were
128 ± 25 (range, 86 to 175) for the RS copy and 33 ± 9 (range, 22 to 45) for the RS copy in the Niigata-type HRS isolates
and 21 ± 3 (range, 17 to 23) for the RS copy and 44 ± 6 (range, 35 to 51) for the RS copy in the Tokachi-type HRS isolates.
On the other hand, the copy number of normal isolates from the three
fields showed 7 ± 1 (range, 5 to 9) for the RS copy and 6 ± 3 (range, 2 to 9) for the RS copy, a finding that is similar to
the previously reported average number of RS-specific bands in 36 normal isolates of B. japonicum obtained from the Nakazawa
field site (RS, 9.4; RS, 7.6) (22). Strain USDA123, a
prevalent B. japonicum serotype strain indigenous to the
United States, was shown to have copy numbers of RS elements comparable
to the Tokachi-type HRS isolates (Table 1), although strains USDA110
and USDA122 fell into the category of normal isolates.
TABLE 1.
Estimated copy number of RS and RS and the mean
generation time in B. japonicum HRS and normal isolates
Shift and reiteration of nifDK- and hupLS-specific hybridization profiles. In general, DNA sequences in and around nifDK and hupLS genes of B. japonicum isolated from soybeans in fields are well conserved (21, 22). Hybridization specific for nifDK with HindIII-digested total DNAs from normal isolates from a field site at Ami consistently showed a 9.5-kb hybridization band (Fig. 1A). However, when B. japonicum field isolates from the Nagakura field site containing many HRS isolates were tested, we observed variation among the nifDK-specific hybridization bands in HRS isolates (Fig. 1A). Moreover, we observed reiteration of hupLS- specific bands when HindIII-digested total DNA of some isolates from the Nagakura field were hybridized (Fig. 1B). These results suggested that genomic rearrangements involving RS around the nif and hup genes of HRS isolates may have given rise to these band differences and reiterations.
|
Nucleotide sequences of 16S rRNA gene. To examine whether HRS isolates belong to a different genus or species from B. japonicum, we analyzed nucleotide sequences (ca. 1 kb) of the 16S rRNA genes of HRS isolates NK5 and NK6 and normal isolate NK2 and compared these with the corresponding published sequences of B. japonicum and its neighbors. Figure 2 shows a phylogenetic tree. Two HRS isolates, NK5 and NK6, and normal isolate NK2 could be grouped into a B. japonicum cluster that included USDA110 (L2330 and Z35330), indicating that these HRS isolates also belong to B. japonicum in terms of phylogeny based on 16S rRNA gene sequences.
|
Symbiotic phenotypes and growth rate.
Symbiotic phenotypes
showed no difference between the HRS and normal isolates with respect
to nodulation, nitrogen fixation, and hydrogen uptake (data not shown).
However, the growth rates of HRS isolates appeared to be slower than
those of normal isolates. Thus, we compared the mean generation time
between HRS and normal isolates (Table 1). The data indicated that the
generation time of HRS isolates was longer than that of normal
isolates. In particular, the Niigata-type HRS isolates, which possessed
extremely high copy numbers of RS (Table 1), showed significantly
longer generation times than the normal isolates and the normal strains
USDA110 and USDA122. The generation time of normal isolates was similar to the values (
6 h) reported previously (34).
Comparison of serogroup and hydrogenase phenotype.
To
investigate the origin of the B. japonicum HRS isolates, we
compared serogroups and hydrogenase types between normal and HRS
isolates from three field sites (Fig. 3).
The isolates tested did not react with antisera 76 and 94 of B. elkanii but did react with antisera of B. japonicum
strains USDA110, USDA122, USDA123, USDA129, and J5033. Although double
and triple cross-reactions were observed, the strains were classified
into six serogroups. Groupings based on a combination of serogroup and
hydrogenase phenotype of both HRS and normal isolates appeared to be
related to the field sites. Serogroups 110 and Hup+ were
dominant in both HRS and normal isolates from the Nakazawa and Nagakura
field sites, whereas serogroups 123-J5033 and Hup were
dominant in both HRS and normal isolates from the Tokachi field site.
These results suggested that HRS isolates are derived from indigenous
normal isolates or vice versa at the local site.
|
RS tandem repeat.
In recent years, it has been reported
that tandem repeats of the IS30 element can give rise to a
burst of DNA rearrangements, including site-specific deletions,
inversions, and intermolecular transposition in E. coli
(1, 24). The fact that Niigata-type HRS isolates possessed a
high copy number of RS prompted us to examine whether HRS isolates
have RS repeated sequences in tandem. Primers 13 and 14 were
designed to detect close or contiguous tandem repeats of RS
sequences. When the structure exists, the expected size of the
amplification product was 600 bp. PCR analysis with primers 13 and 14 showed that a 600-bp DNA fragment was amplified in Niigata-type HRS
isolates as expected, whereas there was no amplification product in
normal isolates from the Nakazawa and Nagakura field sites (Fig.
4). In Tokachi-type HRS isolates, which possessed a relatively low copy number of RS, the 600-bp DNA fragment was observed only in HRS isolate T2 (Fig. 4). To precisely determine the sequence at the junction of the RS tandem repeats, PCR
amplification was performed by using primers 17 and 18 and the 600-bp
products of HRS isolates NC32a, NK5, and T2 as templates. The
amplification products (ca. 240 bp) were then cloned and sequenced. The
resultant DNA sequences were aligned with RS7 (17) (Fig. 5). In these isolates, RS sequences
were formed which were tandemly repeated with four nucleotides
(5'-CTAG) shared between them (Fig. 5A). In isolate NK5, a second pair
of tandem RS repeats was found with a spacer region of 35 nucleotides between them (Fig. 5B). The fact that two RS units
shared the four nucleotides (5'-CTAG) in the tandem repeat structure
suggested that RS might target to the four nucleotides (5'-CTAG) of
the original RS, when the new RS is transposed in HRS isolates
(Fig. 5A). The 4-bp sequence is therefore likely to be a target site in
RS transposition.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Niigata-type HRS isolates are unique in that they have a tandem
repeat RS dimer and an extremely high RS copy number, abnormal patterns of hybridization with nif and hup genes,
a longer generation time, and a similar serotype grouping (serogroups
110 and 122). To our knowledge, this is the first report describing the
characteristics of Niigata-type HRS isolates of B. japonicum. On the other hand, Tokachi-type HRS isolates appear to
be found in the United States because strain USDA123 belonged to the
group of HRS isolates of this type in terms of the estimated copy
number of RS and RS. Rodriguez-Quinones et al. (27)
found hyper-reiterated DNA regions that are conserved among B. japonicum serocluster 123 strains, and they subsequently
determined the DNA region, HRS1, which has properties similar to those
of an IS element (15) and RS sequences.
The estimated copy numbers of RS in the 16 Niigata-type HRS isolates
ranged from 86 to 175, with a mean of 128 copies (Table 1). The RS
copy number in Niigata-type HRS isolates appeared to be significantly
higher than normally found for IS elements in bacteria (7).
Among natural isolates of E. coli, maximum and mean copy
numbers of IS1, IS2, IS3,
IS4, IS5, and IS30 were fewer than 27 and 8, respectively (7, 30). Comparably low numbers are
found in plant-associated bacteria such as Rhizobium (5, 35), Agrobacterium (5), and
Xanthomonas (2) spp.
However, it has been known that Shigella dysenteriae
(25), Acetobacter pasteurianus (16),
and Halobacterium spp. (9, 29) possess high copy
numbers of iso-insertion sequences of IS1,
IS1380, and ISHs, respectively, which are comparable to the number of RS copies found for the Niigata-type HRS isolates. The
high copy numbers of these IS elements are associated with DNA
rearrangements and genome instability in these bacteria. In Niigata-type HRS isolates possessing a high copy number of RS, the
variation in nifDK-specific hybridization bands and the
reiteration of hupLS-specific hybridization bands suggested
that DNA rearrangements might occur in the symbiotic regions.
It has been reported that tandem repeats of IS elements separated by a
few base pairs are active in the transposition of IS21 (26), IS3 (33), and IS30
(1, 24). Since the tandem repeat RS dimer
(RS)2 in HRS isolates is very similar to that of
(IS30)2, it is possible that this causes DNA
rearrangements in HRS isolates.
In general, the number of copies of IS elements in the genome increases without causing deletions because bacterial IS elements transpose conservatively. The corollary is that the IS copy number should generally increase in the evolution of strains of bacteria (7). It may be that HRS isolates have evolved from normal isolates by transposition and recombination events by amplification during DNA replication of arrays of tandemly repeated sequences which include the IS elements. The resultant HRS isolates are considered to exhibit extra-slow growth. Groupings based upon serogroup and hydrogenase phenotype suggested that such events have independently occurred in individual different field ecosystems.
Gross et al. (8) and Xu et al. (36) described extra-slow-growing (ESG) soybean bradyrhizobia that are indigenous to alkaline and Chinese soils, respectively. Xu et al. (36) proposed the name Bradyrhizobium liaoningens sp. nov. for the ESG strains from China based on many taxonomic features. The HRS isolates appear to resemble ESG strains. Both nodulated soybeans grew slowly in a free-living state (Table 1) and were sensitive to antibiotics such as tetracycline (data not shown). However, it is clear from the sequences of the 16S rRNA genes of the HRS isolates and the strain B. japonicum USDA110 that they are closely related. Moreover, the serotypes of HRS isolates were within the range of B. japonicum. Therefore, HRS isolates should be classified as B. japonicum.
The reason the Niigata-type HRS isolates were isolated only at two field sites of the Niigata Agriculture Experiment Station is not clear. The presence of B. japonicum HRS isolates in nature suggests that they might give rise to genetic diversification and adaptation of the bacteria to specific environments and so play an important role in the evolution of symbiotic bacteria including gene transfer. Further studies are necessary to understand the significance of B. japonicum HRS isolates in field populations.
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ACKNOWLEDGMENTS |
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This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan (no. 07660077) and the Joint Research Program of the Institute of Genetic Ecology, Tohoku University (942206).
We thank T. Mikami (Ibaraki University) for technical assistance in the serotype determinations and PCR analysis, H. Mitsui (Tohoku University) for technical assistance in the sequencing of the 16S rRNA genes, M. Sadowsky (University of Minnesota) for a critical reading of the manuscript, and T. Asami and M. Kubota (Ibaraki University) for their continuing interest.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Genetic Ecology, Tohoku University, Katahira, Aoba-ku, Sendai 980-77, Japan. Phone: 81-22-217-5684. Fax: 81-22-263-9845. E-mail: kiwamu{at}ige.tohoku.ac.jp.
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Abstract
Introduction
Materials & Methods
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Appl Environ Microbiol, May 1998, p. 1845-1851, Vol. 64, No. 5
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