Previous Article | Next Article 
Appl Environ Microbiol, March 1998, p. 902-906, Vol. 64, No. 3
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Lipopolysaccharide Profiles from Nodules as Markers
of Bradyrhizobium Strains Nodulating Wild Legumes
Mónica
Santamaría,1
Ángel
M.
Gutiérrez-Navarro,2 and
Javier
Corzo1,*
Departamento de Bioquímica y
Biología Molecular,1 and
Departamento de Microbiología y Biología
Celular,2 Universidad de La Laguna, 38071 Tenerife, Spain
Received 10 March 1997/Accepted 7 December 1997
 |
ABSTRACT |
To develop the use of electrophoretic lipopolysaccharide profiles
for Bradyrhizobium strain identification, we studied the feasibility of using electrophoresis of whole legume nodule homogenates to obtain distinctive lipopolysaccharide profiles. The electrophoretic patterns were the same whether we used nodule extracts, bacteroids, or
cultured bacteria as samples, and there was no evidence of changes in
the ladder-like pattern during the nodulation process. To assess the
reliability of using lipopolysaccharide profiling performed with
individual nodules for studying the diversity and microdistribution of
the rhizobia nodulating wild shrub legumes, we used a population of
Adenocarpus foliolosus seedlings. We obtained 75 different
profiles from the 147 nodules studied. There was no dominant profile in
the sample, and a plant with different nodules generally produced
different profiles. Electrophoresis of legume root nodules proved to be
a fast and discriminating technique for determining the diversity of a
bradyrhizobial population, although it did not allow the genetic
relationships among the nodulating strains to be studied.
| |
INTRODUCTION |
The usual way to study natural
populations of rhizobia to collect soil samples and then catch the
rhizobia by using the appropriate legume host as a trap. After this,
the bacteria are isolated from the nodules, and their diversity is
evaluated by different methods. This approach is useful for isolating
nodulating rhizobial strains present in the soil and for evaluating
their competition under controlled conditions. However, all of the
information concerning the original short-range distribution of the
bacteria in the undisturbed soil is lost. In fact, the distribution and
diversity of rhizobia in soil at the microscale level have received
little attention (2), and little is known about the
distribution of bacteria nodulating perennial legumes in the wild,
including the microdistribution of different nodule strains in the
soil, the presence of different strains in the same plant, and the
frequency of nodule occupation by individual strains.
A strain identification procedure performed directly with individual
nodules is of great interest for studies involving large numbers of
samples, as is analysis of natural populations. Serology (1)
and different types of PCR-based methods, such as enterobacterial repetitive intervening consensus sequence (ERIC)-PCR (15),
have been used to identify strains directly from nodule homogenates. Here we describe the use of lipopolysaccharide (LPS) profiling of
nodule squashes as a reliable and discriminating technique for
identifying the strains in nodules from a natural population. The
rationale for using LPS profiles is as follows: (i) it is feasible to
use LPS profiling to study the diversity of rhizobia and other
gram-negative bacteria (8-10, 18, 19, 25, 27); (ii) LPS
molecules are present exclusively in gram-negative bacteria, so
contamination by plant components is not possible; and (iii) the number
of rhizobia inside the nodules and the sensitivity of the technique
allowed us to obtain LPS profiles directly from the nodules without
amplification of the bacteria or the molecules themselves. Thus, nodule
LPS profiling could be fast, cheap, and not subject to artifacts due to
contamination or to a lack of specificity of the amplification
procedure.
To determine the reliability of using LPS profiling performed with
individual nodules to study the diversity and microdistribution of the
rhizobia nodulating wild shrub legumes, we used an undisturbed plot in
the pine forest of Tenerife Island in which a perennial legume shrub,
Adenocarpus foliolosus, is the dominant plant in the
undergrowth.
| |
MATERIALS AND METHODS |
Reference strains and culture conditions.
The strains used
in this study were Bradyrhizobium japonicum USDA 110 and
Bradyrhizobium strains isolated from various Canary Island
legume shrubs. Bradyrhizobium (Chamaecytisus)
strains BGA-1 and BTA-1 (17) and Bradyrhizobium
strains BCO-1, BES-2, BES-3, BES-6, BGA-2, BRT-3, BRT-5, and BTA-2
(25) have been described previously. Cells were maintained
at 
80°C in yeast extract-mannitol (YM) medium (30)
containing 20% glycerol and were grown in YM medium. The samples used
for electrophoresis were obtained from cultures grown in YM broth for 5 days at 28°C. The bacteria were harvested by centrifugation, and the
sediments were washed with 0.85% NaCl in water. To obtain nodules,
plants were axenically grown from germinating seeds (soybean cultivar
Williams for B. japonicum and Chamaecytisus
proliferus for the Canary Islands strains) in plastic boxes by
using Hewitt medium (13) and vermiculite-sand (1:1) as the
substrate. The plants were inoculated twice, once at the moment of
sowing and then 12 days later. After 3 months (1 month for soybeans)
cultured plants were removed, and the nodules were used for
electrophoresis.
Preparation of samples, electrophoresis, and silver
staining.
The LPS were purified as described by Westphal and Jann
(31). B. japonicum USDA 110 cells from 7-day-old
YM medium cultures and purified B. japonicum USDA 110 bacteroids were used as LPS sources. The bacteroids were obtained from
soybean nodules by the method of Tao et al. (28). The
proteinase K-treated bacterial samples used for electrophoresis were
prepared as described by Siverio et al. (27).
Electrophoresis was carried out by the method of Laemmli
(16) in the presence of 1% sodium dodecyl sulfate (SDS) at
25°C in 0.5-mm-thick 12.5% polyacrylamide slab gels. Silver staining
of the LPS was performed by the method of Tsai and Frasch
(29). The stained gels were examined with a Pharmacia-LKB
Ultroscan XL densitometer, and the software used for gel analysis and
comparison was Pharmacia-LKB Analysis GelScan XL software. The
comparison of the profiles involved the following three steps: (i)
normalization of the profiles to the same length; (ii) grouping of the
profiles on the basis of their main characteristics, including
ladder-like patterns (see Fig. 3, lanes 1, 3, 4, and 5), groups of
stacked bands (see Fig. 3, lane 2), and profiles without any
discernible periodicity (see Fig. 3, lane 12); and (iii) direct
comparison of profiles of each group by superimposing the normalized
densitometric traces. Two profiles were considered identical when 95%
of the bands in the profiles coincided.
Location studied and naturally growing nodule collection.
Nodules were taken from A. foliolosus seedlings growing in a
plot (75 by 75 m) in Chipeque (Tenerife, Canary Island; UTM
coordinates 28RCS5639; altitude, 1,920 m). This plot is in a pine
forest that was planted between 1949 and 1952 and has not been
cultivated since. Seedlings were harvested twice; 55 plants were
harvested at the end of the growth season in April, and 51 plants were
harvested in July during the summer resting period. The shoot length
ranged from 3 to 12 cm, and the main root length ranged from 3 to 14 cm. The samples taken in April had swollen pink nodules; the roots were
cleaned, and the nodules were processed immediately after harvesting.
In contrast, the nodules obtained in July were obtained under drought
conditions, and homogenization of these nodules was difficult, so the
plants were placed in water for 12 h to allow the nodules to swell
before processing.
Nodule LPS electrophoresis.
Nodules were washed, crushed
individually in water (15 µl/mm of nodule diameter) by using a 1-ml
glass potter (Tissue Grind Micro; Kontes Scientific Glassware,
Vineland, N.J.), and suspended (1:1, vol/vol) in 10%
dithiothreitol-4% SDS-20% glycerol-0.035% bromphenol blue in 1 M
Tris (pH 8.0). The procedure used was the procedure used for the
cultured cells. Nodules that had a diameter of about 1 mm produced
enough sample for five analyses.
| |
RESULTS AND DISCUSSION |
Usefulness of nodule LPS profile analysis for bacterial
identification.
Analysis of the LPS profiles of whole-cell
homogenates of cultured rhizobia has been used previously as a highly
discriminating technique for strain identification (8, 9, 19,
25). However, there are no previously published data on the use
of nodule squashes as samples for electrophoresis. Figures
1 to
3 show LPS profiles obtained by
performing polyacrylamide gel electrophoresis (PAGE) with nodules, and
these profiles prove that it is possible to obtain clear and easily
distinguishable LPS profiles directly from nodules.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 1.
Electrophoretic profiles of the LPS from B. japonicum USDA 110. (a) Densitometric traces of the patterns. (b)
Relationship between the number of the band and its
Rf value. The samples used were a nodule squash
lysate (A), a bacteroid lysate (B), a free-living cell lysate (C),
purified LPS from free-living bacteria (D), and purified bacteroid LPS
(E). The characteristic triplets of many Bradyrhizobium
strains are clearly present in the zone between
Rf 0.4 and Rf 0.7.
|
|
View larger version (127K):
[in this window]
[in a new window]
|
FIG. 2.
Electrophoretic profiles of LPS from free-living and
nodule squashes from various Bradyrhizobium
(Chamaecytisus) strains isolated from Canary Island legumes
and B. japonicum USDA 110. Lanes C, Whole-cell lysate; lanes
N, nodule squash lysate.
|
|
View larger version (121K):
[in this window]
[in a new window]
|
FIG. 3.
LPS profiles of some A. foliolosus nodules.
Lanes 15 to 20 contained samples from six nodules of one seedling. The
profiles in lanes 15 to 18 and 22 were ascribed to the same strain; the
profiles in all other lanes were different, so they were adscribed to
different strains.
|
|
A second question concerns the similarity between the LPS profiles of
free-living bacteria and the LPS profiles of nodules.
As Fig.
1 shows,
the profiles of purified LPS from bacteria and
from bacteroids are
identical; the same is true for the LPS profiles
of whole-cell lysates,
bacteroids, or nodule squashes. This suggests
that no changes occur
during nodulation, although the LPS of bacteria,
bacteroids, or nodules
produced a band in the front of the lane
that is not present in the
profiles of purified LPS. Despite this
difference, all of the patterns
were identical in the zone examined,
with bands having
Rf values between 0.15 and 0.8 (Fig.
1b). Figure
2 shows the LPS profiles of free-living bacteria and nodules induced
in
their host plants; samples of the roots, treated in the same
way as the
nodules, produced a faint streak in the gel without
any noticeable band
(data not shown). Like the
B. japonicum USDA
110 trace, the
densitometric traces (data not shown) revealed
that there were no
differences in the profiles except in the high-mobility
zone, where
some of the profiles contained a wide streak.
Our results could disagree with the accepted claim that rhizobial LPS
change in the nodules, but this discrepancy is only
fictitious. There
are previously published data which show that
there are differences in
the LPS profiles of bacteria and bacteroids;
however, in many instances
these differences are detectable only
when monoclonal antibodies are
used, and there is no alteration
of the electrophoretic pattern
(
22,
28). In other cases the
differences in the LPS profiles
are related to differences in
host specificity or to the loss of a
symbiotic plasmid (
7,
26). Thus, the silver-stained LPS
profiles from nodules have
the same value as the isolated cell LPS
profiles for strain identification
purposes.
What is the meaning of an electrophoretic LPS profile?
Both
multilocus enzyme electrophoresis and nucleic acid comparison
procedures allow simultaneous differentiation between bacterial strains
and deduction of genetic relationships between strains. This is not the
case for LPS profile comparison, and is the main limitation of the
technique. The LPS structure is due to the specificity of the enzymes
involved in the synthesis of LPS (11, 32), so different LPS
profiles reflect differences in such enzymes. In this sense LPS profile
comparison is similar to multilocus enzyme electrophoresis analysis,
but the lack of a direct relationship between enzyme specificity and
LPS profile precludes any genetic analysis. In addition, the positions
of the bands in an LPS profile are not mutually independent, and, as a
consequence, the software used to quantify differences between
restriction fragment length polymorphism or similar electrophoretic
profiles, which assumes that each band is independent of the other
bands in the same lane, is not useful for quantitative comparison of
LPS profiles. This is due to the mechanism of LPS biosynthesis, which
involves the sequential addition of identical subunits to a growing
O-antigen chain, and to the fact that LPS are separated by SDS-PAGE on
the basis of their sizes (23). This means that the position
of band n is related by a monotonous function to the
positions of n-1, n-2, and other bands (Fig. 1)
(11, 23). However, this does not apply to the LPS profiles
without a regular ladder-like pattern, such as the LPS profiles of many
Rhizobium strains (25).
Study of a natural population: a real application of the
method.
To assess the reliability of the LPS profile technique for
studying the nodule occupants in a legume population, we used naturally nodulated A. foliolosus seedlings from a small forest plot.
There were no differences in plant size or in the number of nodules per
plant between the samples taken in April and the samples taken in July.
The seedlings were poorly nodulated; of the 106 seedlings studied, 25 lacked any nodules, 46 had only one nodule, 13 had two nodules, 11 had
three nodules, 9 had four nodules, and 2 had six nodules. This could
have been due to the fact that these symbiotic couples have an
intrinsically low nodulation rate. Three strains isolated from root
nodules obtained in Chipeque were tested for nodulating ability under
presumably optimal conditions, and the numbers of nodules recovered
from 3-month-old plants were as follows: 0 to 9 nodules (mean, 3.6 nodules) for isolate BES-2, 1 to 20 nodules (mean, 9.1 nodules) for
isolate BES-3, and 0 to 4 nodules (mean, 1.4 nodules) for isolate
BES-6. These figures are in contrast to those obtained for cultured
legumes, for which the numbers of nodules both in soil and in synthetic
media are much greater. A possible explanation for this is the
different life cycles of the legumes studied. Cultured annual legumes
are fast growers with short life cycles, whereas in one growing season
Adenocarpus or Chamaecytisus seedlings reach only
a small size, so a few nodules could be enough to supply their low
nitrogen requirements.
We obtained 155 nodules from the population studied; a sample of the
LPS profiles obtained from these nodules is shown in
Fig.
3. The
nodules obtained in the summer under drought conditions
generally
produced LPS profiles that were less well-resolved than
the LPS
profiles obtained in the spring, but all but seven nodules
could be
analyzed. These seven nodules produced only faint streaks
with no
evidence of LPS; one nodule from a spring sample also
failed to produce
a clear LPS pattern. These samples were not
used in the analysis
described below. The remaining 147 nodules
produced 75 different LPS
profiles. All of the profiles were typical
Bradyrhizobium
profiles, with a ladder-like pattern and no evidence
of the
characteristic
Rhizobium LPS-I (
25). There was
not a
clearly dominant profile in the population; 43 profiles appeared
only once, 16 profiles appeared twice, 7 profiles appeared three
times,
2 profiles appeared four times; 1 profile appeared five
times, 5 profiles appeared six times, and 1 profile appeared seven
times. A
plant with multiple nodules was generally nodulated by
more than one
strain (Table
1), and neighboring nodules
on the
same plant were generally formed by different strains. When a
strain appeared more than once, it was found in different plants.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Number of different LPS profiles obtained
with A. foliolosus seedlings in which more than one
nodule was found
|
|
However, a word of caution must be given: the number of LPS profiles
can overestimate the actual number of strains. Diversity
studies based
on whole-nodule lysates performed by using LPS profiling,
serology, or
ERIC-PCR have the problem that multiple nodule occupation
could result
in complex patterns that can be wrongly ascribed
to new strains. For
instance, Labes et al. (
15) studied pea
nodules by ERIC-PCR
and concluded that mixed nodule infections
caused by two strains could
result in classification of each resulting
nodule as a nodule formed by
a new strain. In this regard the
results of Noel and Brill are
noteworthy; in a sample of nine
soybean nodules taken at random, seven
contained two or more different
B. japonicum strains, as
defined by protein profiles on PAGE gels
(
21). Clearly, if
this situation is common, any technique that
depends on a comparison of
band sets is prone to produce an elevated
number of false strains. To
evaluate the sensitivity of LPS profiling
to this problem, we
electrophoresed mixtures of two strains in
different proportions; the
results depended on the differences
between the profiles, but in
general if the second strain accounted
for less than 20% of the
population, the profile of the main strain
did not change. With 50:50
mixtures, the resulting profiles were
clearly different and more
complex than the profiles obtained
for isolated samples (Fig.
4). If we take this information into
account, of the 75 different profiles that we found in our sample,
10 could be produced by the presence in the same nodule of two
or more
strains; all 10 of these profiles appeared only once in
the sample.
Thus, we concluded that the number of actually different
strains in the
147 nodules studied was between 65 (if we assumed
that all complex
patterns were due to multiple nodule occupation)
to 75 (if we assumed
that there was no multiple occupation). LPS
profiling of nodules
increases our knowledge of the spatial distribution
of the nodulating
bradyrhizobia in an undisturbed soil. Our sample
was composed of a
large number of different strains, each one
recovered from only one or
a few nodules, and in many instances
neighboring nodules were formed by
different strains. This could
be explained by assuming that the
population is a mosaic of different
strains, each one occupying small
areas in the soil; an alternative
explanation is that the rhizobial
population is distributed more
or less evenly in the soil, but each
nodule is formed by a random
strain from the whole population. Our data
did not support either
of these alternatives. The diversity that we
have found in the
population studied was higher than the diversity
found in other
studies (
3,
4,
6,
12,
14,
15,
20). As any
individual
method detects only a fraction of the total strains in a
population
(
24) and the strains recovered from nodules may
be only a fraction
of soil population (
5), the number of
different
Adenocarpus-nodulating
strains in the Tenerife
forest could be enormous.
View larger version (100K):
[in this window]
[in a new window]
|
FIG. 4.
Effect of mixing whole-cell extracts of isolated
Bradyrhizobium (Chamaecytisus) strains on LPS
profiles. The centers of the electrophoretic gels, in which the
differences are clearer, are shown. Note that the isolated strains
produced a pattern in which triplets of bands are clearly evident,
whereas the mixtures produced more complex patterns containing groups
of four to six bands. The profiles in the lanes marked with the same
letter were ascribed to the same strain in a blind assay.
|
|
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the Canary Islands
Government.
We thank E. Meléndez-Hevia for the use of his facilities for the
densitometric analysis and for helpful discussions and Carlos Corzo for
help with nodule collection.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Bioquímica y Biología Molecular, Universidad de La
Laguna, 38071 Tenerife, Spain. Phone: 34-22-603726. Fax:
34-22-603724. E-mail: FCORZO{at}ULL.es.
| |
REFERENCES |
| 1.
|
Asanuma, S.,
G. Thottappilly,
A. Ayanaba, and V. R. Rao.
1985.
Use of the enzyme-linked immunosorbent assay (ELISA) in the detection of Rhizobium both in culture and from root nodules of soybeans and cowpeas.
Can. J. Microbiol.
31:524-528.
|
| 2.
|
Barnet, Y. M.
1991.
Ecology of legume root-nodule bacteria, p. 199-228. In
M. J. Dilworth, and A. R. Glenn (ed.), Biology and biochemistry of nitrogen fixation.
Elsevier Science Publishers B.V., Amsterdam, The Netherlands.
|
| 3.
|
Bottomley, P. J.,
H. Cheng, and S. R. Strain.
1994.
Genetic structure and symbiotic characteristics of a Bradyrhizobium population recovered from a pasture soil.
Appl. Environ. Microbiol.
60:1754-1761[Abstract/Free Full Text].
|
| 4.
|
Bromfield, E. S. P.,
I. B. Sinha, and M. S. Wolynetz.
1986.
Influence of location, host cultivar, and inoculation on the composition of naturalized populations of Rhizobium meliloti in Medicago sativa nodules.
Appl. Environ. Microbiol.
51:1077-1084[Abstract/Free Full Text].
|
| 5.
|
Bromfield, E. S. P.,
L. R. Barran, and R. Wheatcroft.
1995.
Relative genetic structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of alfalfa (Medicago sativa) and sweet clover (Melilotus alba).
Mol. Ecol.
4:183-188.
|
| 6.
|
Brunel, B.,
S. Rome,
R. Ziani, and J. C. Cleyet-Marel.
1996.
Comparison of nucleotide diversity and symbiotic properties of Rhizobium meliloti populations from annual Medicago species.
FEMS Microbiol. Ecol.
19:71-82.
|
| 7.
|
Carlson, R. W., and M. Yadav.
1985.
Isolation and partial characterization of the extracellular polysaccharides and lipopolysaccharides from fast-growing Rhizobium japonicum USDA 205 and its Nod mutant, HC205, which lacks the symbiotic plasmid.
Appl. Environ. Microbiol.
50:1219-1224[Abstract/Free Full Text].
|
| 8.
|
Casella, S.,
N. Rossi, and A. Toffanin.
1992.
Cell surface lipopolysaccharides of different rhizobia.
FEMS Microbiol. Lett.
93:213-220.
|
| 9.
|
De Maagd, R. A.,
C. van Rossum, and B. J. J. Lugtenberg.
1988.
Recognition of individual strains of fast-growing rhizobia by using profiles of membrane proteins and lipopolysaccharides.
J. Bacteriol.
170:3782-3785[Abstract/Free Full Text].
|
| 10.
|
De Weger, L. A.,
B. Jann,
K. Jann, and B. Lugtenberg.
1987.
Lipopolysaccharides of Pseudomonas spp. that stimulate plant growth: composition and use for strain identification.
J. Bacteriol.
169:1441-1446[Abstract/Free Full Text].
|
| 11.
|
Goldman, R. C., and F. Hunt.
1990.
Mechanism of O-antigen distribution in lipopolysaccharide.
J. Bacteriol.
172:5352-5359[Abstract/Free Full Text].
|
| 12.
|
Hartmann, A., and N. Amarger.
1991.
Genotypic diversity of an indigenous Rhizobium meliloti field population assessed by plasmid profiles, DNA fingerprinting, and insertion sequence typing.
Can. J. Microbiol.
37:600-608.
|
| 13.
|
Hewitt, E. J.
1952.
.
Sand and water culture methods used in the study of plant nutrition. Technical Communication 22.
Farnhan Royal Brahs Commonwealth Agricultural Bureau, United Kingdom.
|
| 14.
|
Kamicker, B. J., and W. J. Brill.
1986.
Identification of Bradyrhizobium japonicum nodule isolates from Wisconsin soybean farms.
Appl. Environ. Microbiol.
51:487-492[Abstract/Free Full Text].
|
| 15.
|
Labes, G.,
A. Ulrich, and P. Lentzsch.
1996.
Influence of bovine slurry deposition on the structure of nodulating Rhizobium leguminosarum bv. viciae soil populations in natural habitat.
Appl. Environ. Microbiol.
62:1717-1722[Abstract].
|
| 16.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[Medline].
|
| 17.
|
León-Barrios, M.,
A. M. Gutiérrez-Navarro,
R. Pérez-Galdona, and J. Corzo.
1991.
Characterization of Canary Island isolates of Bradyrhizobium sp. (Chamaecytisus proliferus).
Soil Biol. Biochem.
23:487-489.
|
| 18.
|
Lindström, K.,
P. Lipsanen, and S. Kaijalainen.
1990.
Stability of markers used for identification of two Rhizobium galegae inoculant strains after five years in the field.
Appl. Environ. Microbiol.
56:444-450[Abstract/Free Full Text].
|
| 19.
|
Lindström, K., and H. H. Zahran.
1993.
Lipopolysaccharide patterns in SDS-PAGE of rhizobia that nodulate leguminous trees.
FEMS Microbiol. Lett.
107:327-330.
|
| 20.
|
Madrzak, C. J.,
B. Golinska,
J. Króliczak,
K. Pudelko,
D. Lazewska,
B. Lampka, and M. J. Sadowsky.
1995.
Diversity among field populations of Bradyrhizobium japonicum in Poland.
Appl. Environ. Microbiol.
61:1194-1200[Abstract].
|
| 21.
|
Noel, K. D., and W. J. Brill.
1980.
Diversity and dynamics of indigenous Rhizobium japonicum populations.
Appl. Environ. Microbiol.
40:931-938[Abstract/Free Full Text].
|
| 22.
|
Noel, K. D.,
D. M. Duelli,
H. Tao, and N. J. Brewin.
1996.
Antigenic change in the lipopolysaccharide of Rhizobium etli CFN42 induced by exudates of Phaseolus vulgaris.
Mol. Plant Microbe Interact.
9:180-186.
|
| 23.
|
Palva, E. T., and P. H. Mäkelä.
1980.
Lipopolysaccharide heterogeneity in Salmonella typhimurium analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis.
Eur. J. Biochem.
107:137-143[Medline].
|
| 24.
|
Raffetti, D.,
C. Scotti,
S. Gnocchi,
S. Fancelli, and M. Bazzicalupo.
1996.
Genetic diversity of an Italian Rhizobium meliloti population from different Medicago sativa varieties.
Appl. Environ. Microbiol.
62:2279-2285[Abstract].
|
| 25.
|
Santamaría, M.,
J. Corzo,
M. León-Barrios, and A. M. Gutiérrez-Navarro.
1997.
Characterisation and differentiation of indigenous rhizobia isolated from Canarian shrub legumes of agricultural and ecological interest.
Plant Soil
190:143-152.
|
| 26.
|
Shindu, S. S.,
N. J. Brewin, and E. L. Kannenberg.
1990.
Immunochemical analysis of lipopolysaccharides from free-living and endosymbiotic forms of Rhizobium leguminosarum.
J. Bacteriol.
172:1804-1813[Abstract/Free Full Text].
|
| 27.
|
Siverio, F.,
M. Cambra,
M. T. Gorris,
J. Corzo, and M. M. López.
1993.
Lipopolysaccharides as determinants of serological variability in Pseudomonas corrugata.
Appl. Environ. Microbiol.
59:1805-1812[Abstract/Free Full Text].
|
| 28.
|
Tao, H.,
N. J. Brewin, and K. D. Noel.
1992.
Rhizobium leguminosarum CFN42 lipopolysaccharide antigenic changes induced by environmental conditions.
J. Bacteriol.
174:2222-2229[Abstract/Free Full Text].
|
| 29.
|
Tsai, C. M., and C. E. Frasch.
1982.
A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels.
Anal. Biochem.
119:115-119[Medline].
|
| 30.
|
Vincent, J. M.
1970.
.
A manual for the practical study of the root nodule bacteria. IBP handbook.
Blackwell Scientific Publications, Ltd., Oxford, United Kingdom.
|
| 31.
|
Westphal, O., and K. Jann.
1965.
Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure.
Methods Carbohydr. Chem.
5:83-91.
|
| 32.
|
Whitfield, C.
1995.
Biosynthesis of lipopolysaccharide O antigens.
Trends Microbiol.
3:178-185[Medline].
|
Appl Environ Microbiol, March 1998, p. 902-906, Vol. 64, No. 3
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Top
Abstract
Introduction
