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Applied and Environmental Microbiology, June 1999, p. 2716-2722, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Increase in Alfalfa Nodulation, Nitrogen Fixation, and Plant
Growth by Specific DNA Amplification in Sinorhizobium
meliloti
Marcela
Castillo,
Margarita
Flores,
Patrick
Mavingui,
Esperanza
Martínez-Romero,
Rafael
Palacios, and
Georgina
Hernández*
Centro de Investigación sobre
Fijación de Nitrógeno, UNAM, Cuernavaca, Morelos,
México
Received 14 December 1998/Accepted 11 March 1999
 |
ABSTRACT |
To improve symbiotic nitrogen fixation on alfalfa plants,
Sinorhizobium meliloti strains containing different average
copy numbers of a symbiotic DNA region were constructed by specific DNA
amplification (SDA). A DNA fragment containing a regulatory gene
(nodD1), the common nodulation genes (nodABC),
and an operon essential for nitrogen fixation (nifN) from
the nod regulon region of the symbiotic plasmid pSyma of
S. meliloti was cloned into a plasmid unable to replicate
in this organism. The plasmid then was integrated into the homologous
DNA region of S. meliloti strains 41 and 1021, which
resulted in a duplication of the symbiotic region.
Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an
antibiotic marker present in the integrated vector. Derivatives of
strain 41 containing averages of 3 and 6 copies and a derivative of
strain 1021 containing an average of 2.5 copies of the symbiotic region
were obtained. In addition, the same region was introduced into both
strains as a multicopy plasmid, yielding derivatives with an average of
seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen
content, and plant growth were analyzed in alfalfa plants inoculated
with the different strains. The copy number of the symbiotic region was
critical in determining the plant phenotype. In the case of the strains
with a moderate increase in copy number, symbiotic properties were
improved significantly. The inoculation of alfalfa with these strains
resulted in an enhancement of plant growth.
| |
INTRODUCTION |
The construction of bacterial
strains with an increased copy number of specific genes has been widely
used for biotechnological applications. The most common procedure is to
introduce the DNA of interest as a multicopy plasmid. In some cases
gene amplification has been obtained through the natural mechanisms of
homologous recombination, which usually occurs between reiterated
sequences present in direct orientation (3).
DNA amplification is a common feature of the genome of prokaryotic
organisms (3, 26). In some cases natural gene amplifications are associated with adaptive responses in different bacteria
(26). Amplification may be induced in any part of the genome
by cointegrating a plasmid carrying the region of interest into the
homologous region of the genome. This results in a direct repeat of the
region interposed by the vector used and constitutes an amplifiable or amplicon structure. Homologous recombination between the repeats leads
to duplication and further amplification of the whole amplicon structure. This methology has been used to enhance the production of
specific compounds (2, 13).
We have studied gene amplification phenomena in bacteria of the genus
Rhizobium (27). These bacteria establish
symbiosis with leguminous plants and elicit the formation of
nitrogen-fixing nodules. By using random DNA amplification
(RDA) in the symbiotic plasmid of Rhizobium tropici we have
recently shown that DNA amplification may be used to obtain strains
with enhanced competitiveness for nodulation (22).
Bacteria belonging to Rhizobium and related genera establish
nitrogen-fixing symbiosis with legumes. The symbiosis is the result of
the differentiation of both bacteria and plant cells mediated by an
exchange of chemical signals between the partners. Initially, flavonoid
compounds from the plant activate a regulatory circuit in the
microsymbiont which results in the synthesis of signals known as
nodulation (Nod) factors. These compounds are lipooligosaccharides that
induce the initial steps of the differentiation of the plant cells
(19).
The genome of Sinorhizobium meliloti, the microsymbiont of
alfalfa, is comprised of a chromosome of 3.7 Mb and two megaplasmids of
1.4 and 1.7 Mb (10). Most of the nodulation (nod)
and nitrogen fixation (nif and fix) genes are
located in the 1.4-Mb replicon, which is known as the symbiotic plasmid
(pSyma). An approximately 90-kb region of this replicon contains the
different structural and regulatory genes that constitute the
nod regulon. The nod regulon contains several
operons that code for the enzymes that synthesize the Nod factors.
Regulation is exerted by a complex circuit that includes four genes of
the nod regulon: nodD1, nodD2, nodD3, and SyrM (17, 21, 24). Several
nif and fix genes are located in the same region,
interspersed with the elements that constitute the nod regulon.
With the aim of improving nodulation and nitrogen fixation in alfalfa
plants, we have now induced amplification in a specific region of the
symbiotic plasmid of S. meliloti. In contrast to the
RDA strategy, we used specific DNA amplification (SDA) for this
experimental approach.
We chose a particularly interesting DNA fragment from the
nod regulon region and amplified it to different copy
numbers. This fragment contains the regulatory gene nodD1;
the nodulation genes nodA, nodB, and
nodC, which encode the enzymes responsible for the synthesis
of the core structure of Nod factors; and an operon which is essential
for nitrogen fixation. To manipulate the copy number of this DNA
fragment two strategies were used: (i) its cointegration into the
homologous region of pSyma and amplification by the SDA procedure and
(ii) its introduction on a multicopy plasmid. Derivatives of S. meliloti containing this fragment with an average copy number of
2.5 to 3 showed a significant increase in nodulation and nitrogen
fixation and promoted alfalfa growth.
| |
MATERIALS AND METHODS |
DNA manipulations.
Total DNA was isolated from
Sinorhizobium strains by using a DNA/RNA isolation kit
(USB/Amersham, Cleveland, Ohio). Standard DNA manipulations such as
restriction, agarose gel electrophoresis, and filter blot hybridization
were performed as described previously (7). Probes were
radiolabelled with 32P by using a random priming DNA
labelling kit (rediprime; Amersham).
Construction of S. meliloti strains carrying DNA
amplifications in pSyma.
The 8.5-kb EcoRI fragment from
pKSK5 (16), kindly provided by E. Kondorosi, was purified
and ligated into the EcoRI site of pSUP202 (30),
generating plasmid pCH1 of 17 kb. This plasmid was introduced into
Escherichia coli DH5
by transformation. From E. coli, pCH1 was mobilized to S. meliloti strains 41 (15) and 1021 (23) by triparental mating by using
E. coli HB101(pRK2013) (6) as a helper.
Sinorhizobium transconjugants were selected in minimal
medium containing 5 µg of tetracycline (Tc) and 50 µg of
spectinomycin per ml (for strain 41 derivatives) or 100 µg of
spectinomycin per ml (for strain 1021 derivatives). These transconjugants contained a cointegration of pCH1 into the homologous region in pSyma. Sinorhizobium derivatives containing a
higher level of amplification were selected by plating in different
concentrations of Tc. Strains CFNM101 and CFNM103 were obtained from
plates with 7.5 µg of Tc per ml, and strain CFNM102 was obtained from
plates with 30 µg of Tc per ml. Strains CFNM104 and CFNM105 were
obtained by mobilizing pKSK5 from E. coli DH5 into
S. meliloti 41 or 1021, respectively, by triparental mating
with E. coli HB101(pRK2013) (6) as a helper.
Determination of copy number.
Southern blots of
KpnI-digested total DNA from Sinorhizobium
strains were hybridized against an intragenic PCR product of the nodA gene. The copy number of the symbiotic fragment
used in this study was quantified by densitometric integration of
the corresponding hybridization signals on the autoradiograms. The intensity of the 10-kb KpnI wild-type fragment was set
at unity. The intensity of the recombinant KpnI fragment
from strains CFNM101, CFNM102, and CFNM103 (17 kb) and from strains
CFNM104 and CFNM105 (25 kb) (see below) was divided by that of
the 10-kb fragment to obtain the copy number of the corresponding
recombinant fragment. The total copy number in each strain was the copy
number of the recombinant fragment plus one.
Nod factor production.
The lipooligosaccharides produced by
S. meliloti wild-type and amplified strains were examined by
reversed phase thin-layer chromatography (32) by using
bacterial supernatants according to the procedures described previously
(18). Cultures were grown in the absence or in the presence
of the inducer luteolin (1.2 µM). Cultures for Nod factor analysis
were labelled with [14C]glucosamine (0.5 µCi/ml, 52.6 µCi/mmol; Amersham), adjusting the same amounts of bacteria in each culture.
Plant growth conditions.
Seeds from alfalfa (Medicago
sativa) var. Iroquois were surfaced sterilized, germinated, and
grown either in 12.5-cm-wide by 15.5-cm-high plastic growth pouches
(Seed-Pack Growth Pouch; Vaughan's Seed Company, Downers Grove, Ill.)
or in 20-cm-diameter by 16-cm-high plastic pots containing sterile
vermiculite. Ten plants were grown in each growth pouch, and twenty
plants were grown in each pot. Four days after germination, plants were
inoculated with the S. meliloti strains. The bacterial
inoculum was prepared from an overnight culture grown on rich medium
plus the appropriate antibiotic, then washed, and adjusted to give an
inoculum of ca. 109 cells per plant. Plants were incubated
at 21°C and 80% humidity, with a 12-h photoperiod provided by cool
white fluorescent lights at 110.4 µmol m
2
s1, and were watered with Jensen liquid medium
(30) as necessary.
Nitrogenase activity and total nitrogen determinations.
Alfalfa plants were harvested 60 days after inoculation and combined at
random into samples of five plants each. Nitrogenase activities were
determined in seven samples (35 plants) from each condition by the
acetylene reduction method. The total nitrogen content of dry foliar
samples was determined with an ANTEK 7000 (Antek Instruments, Inc.,
Houston, Tex.) nitrogen analyzer, as reported previously
(5).
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RESULTS |
Construction of S. meliloti derivatives with different
copy numbers of a symbiotic DNA region.
An EcoRI
fragment of 8.5 kb from the pSyma of S. meliloti 41 (Fig.
1A) contains several genes that
participate in the establishment of symbiosis and in nitrogen fixation.
These include the regulatory gene nodD1; the structural
nodulation genes nodA, nodB, and nodC and their regulatory element nod box n1; and an operon
essential for nitrogen fixation composed of gene nifN
(previously called fixF) and an open reading frame (ORF)
that codes for a protein of yet unassigned function (ORF110). In
addition, the fragment contains genes nolI and
nodN, which are part of an operon whose transcription is
regulated by nod box n4, located 4 kb upstream of the
fragment.
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FIG. 1.
Construction and characterization of S. meliloti derivatives with different copy numbers of a symbiotic
DNA region. (A to E) Diagrams of relevant structures. Hatched areas
indicate EcoRI fragment containing nodulation and nitrogen
fixation gene sequences; the heavy solid line indicates the vector DNA
sequences; the dashed line shows pSyma DNA sequences bordering the
EcoRI fragment. Abbreviations: R, EcoRI sites; K,
KpnI sites; K1, wild-type KpnI fragment; K2,
recombinant KpnI fragment; O, origin of replication; Tc,
tetracycline resistance marker. Arrows indicate the positions and
directions of transcription of the genes present in the
EcoRI fragment: 1, nolI; 2, nodN; 3, nifN; 4, ORF110; 5, nodD1; 6, nodA; 7, nodB; 8, nodC. Panels: A, EcoRI
fragment; B, pCH1; C, S. meliloti wild-type strain; D,
S. meliloti containing a cointegration of pCH1 into the
homologous region of pSyma; E, S. meliloti containing an
amplification of the EcoRI fragment with a copy number of
four. (F) Southern blots of KpnI-digested total DNA
hybridized with a nodA probe from (lanes): a and b, S. meliloti 41; c and d, CFNM101; e and f, CFNM102; g, CFNM104; h and
i, S. meliloti 1021; j and k, CFNM103; l, CFNM105. In lanes
a, c, e, g, h, j, and l the DNA was extracted from in vitro bacterial
cultures. In lanes b, d, f, i, and k the DNA was extracted from
nodule-recovered bacteria. The KpnI fragments K1 and K2 and
the pKSK5 plasmid are indicated.
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|
The fragment was purified from plasmid pKSK5 (
16) and cloned
into the
EcoRI site of pSUP202 (
30) to obtain
plasmid pCH1
(Fig.
1B). This plasmid contains an origin of replication
that
is active in
E. coli but not in
S. meliloti,
a mob site, and Tc
and ampicillin resistance markers. pCH1 was
mobilized from
E. coli into
Sinorhizobium by
conjugation, and Tc-resistant transconjugants
were selected. The
transconjugants contained a cointegration of
pCH1 into the homologous
region of pSyma of
S. meliloti. The resulting
structure is a
direct repeat of the 8.5-kb region separated by
the cloning vector
(Fig.
1D). Homologous recombination between
the direct repeats may lead
to further amplification. Amplified
strains were selected by increasing
the Tc concentration in the
cell culture. These strains contain tandem
repeats of the whole
structure (Fig.
1E). Derivatives of
S. meliloti 41 containing
the
EcoRI fragment in average
copy numbers of 3 (CFNM101) and
6 (CFNM102) and a derivative of strain
1021 with an average copy
number of 2.5 (CFNM103) were used for further
experiments. In
addition, pKSK5, which contains the same
EcoRI fragment in a vector
that replicates in
S. meliloti, was introduced into both strains
by conjugation from
E. coli. This generated strains CFNM104 (from
41) and
CFNM105 (from 1021) containing the fragment in an average
copy number
of
7.
To characterize the relevant structures of the amplified strains, total
DNA was digested with restriction endonuclease
KpnI
and
subjected to gel electrophoresis. The
EcoRI fragment
contains
one
KpnI site upstream of gene
nodD1,
while the vector of pCH1
does not contain
KpnI sites.
Southern blots were hybridized by
using an intragenic PCR product from
S. meliloti nodA as a probe
(Fig.
1F). The wild-type strains
show one
KpnI fragment (K1) of
10 kb (Fig.
1F). As expected,
the strains containing amplifications
of the 8.5-kb
EcoRI
fragment show the same
KpnI fragment as the
wild type and a
new recombinant fragment of 17 kb (K2), whose
relative intensity
increases with the level of amplification (Fig.
1F). The strains that
harbor the symbiotic region in a multicopy
plasmid show the wild-type
fragment and a fragment of 28.5 kb
that corresponds to the linearized
pKSK5. The copy number of the
region was calculated from the relative
intensities of both signals
in each strain. Figure
1F shows the
autoradiograms of the Southern
blots from the wild-type strains and
their
derivatives.
Nod factor production.
The patterns of Nod factor production
were analyzed in the wild type and amplified derivatives of S. meliloti. The results obtained were in general agreement with what
has been reported for S. meliloti (28). In the
wild-type strains Nod factor production was faintly observed. With a
moderate increase in copy number of the symbiotic region (strains
CFNM101 and CFNM103), Nod factor production was clearly observed. The
highest production of Nod factors was obtained with strain CFNM102,
bearing the greatest number of copies of the amplified region, and
strains CFNM103 and CFNM105, which contain the region in a multicopy
plasmid. Data from S. meliloti 41 and its derivatives are
shown in Fig. 2.
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FIG. 2.
Autoradiogram of a reversed-phase thin-layer
chromatography of Nod factors labelled with
[14C]glucosamine. Lanes 1, 3, 5, and 7, luteolin-induced
S. meliloti; lanes 2, 4, 6, and 8, noninduced
S. meliloti wild-type strain and derivatives. Lanes: 1 and
2, S. meliloti 41; 3 and 4, CFNM101; 5 and 6, CFNM102; 7 and
8, CFNM104.
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|
Symbiotic phenotype of alfalfa plants inoculated with the
S. meliloti strains constructed.
The
ability of the different S. meliloti strains to
nodulate alfalfa plants was determined. The nodulation kinetics of
alfalfa plants inoculated in growth pouches are shown in Fig.
3. Strain CFNM101 shows a
significant (P 
0.03) increase in nodulation, while
strains CFNM102, CFNM104, and CFNM105 show a similar and significant (P 0.02) decrease in nodulation compared
to the respective wild-type strains (Fig. 3). At late postinoculation time points, strain CFNM103 shows a significant (P 0.01) increase in nodulation with respect to the 1021 wild-type
strain (Fig. 3B).
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FIG. 3.
Nodulation kinetics of alfalfa plants inoculated with
different S. meliloti strains. Points are mean values ± the standard error from 100 plants inoculated with each strain. (A)
 , S. meliloti 41;  , CFNM101;  , CFNM102;  ,
CFNM104. (B) , S. meliloti 1021; , CFNM103; ,
CFNM105. In panel A, the open squares and triangles correspond to the
two lowest lines; their values for each data point are almost
identical, and therefore the symbols overlap, obscuring the fact that
they should appear to be open, not solid.
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The symbiotic phenotype of alfalfa plants inoculated in pots with
vermiculite in environmentally controlled growth chambers
was
determined. Plants were harvested after 60 days. Three independent
experiments were performed, and similar results were obtained
among
them. Data from one such experiment are presented in Fig.
4. In agreement with the data from Fig.
3, the plants inoculated
with CFNM101 or with CFNM103 show an increase
in nodulation of
2.2- to 2.5-fold. These plants show a 2.6- to 2.8-fold
significant
increase (
P 0.01) in acetylene reduction
activity per plant
and a 2-fold significant increase (
P 0.01) in root and in foliar
dry weight compared to the wild-type
strain 41 or 1021, respectively.
In addition, these plants have a
significantly (
P 0.01) higher
foliar nitrogen
content per plant (2- to 2.2-fold). When alfalfa
plants were inoculated
with CFNM102, CFNM104, or CFNM105, they
had significantly
(
P 0.01) fewer nodules (33%), lower acetylene
reduction activity (44%), decreased root and foliar dry weight
(38 to
50%), and lower foliar nitrogen content (25 to 38%) compared
to the
respective wild-type strain. In fact, the values of growth
(per dry
weight basis) and of nitrogen content obtained for alfalfa
plants
inoculated with the latter strains containing higher copy
numbers of
the symbiotic region were similar to those obtained
for control
uninoculated plants watered without nitrogen (Fig.
4C).
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FIG. 4.
Phenotype of alfalfa plants inoculated with different
S. meliloti strains. The values of all the parameters are
mean values ± the standard deviation from 35 plants; results were
determined at 60 days postinoculation. Columns (A): , S. meliloti 41;  , CFNM101;  , CFNM102; ,
CFNM104. Columns (B): , S. meliloti 1021; , CFNM103;
,
CFNM105. The open column in part C shows results for uninoculated
control plants watered without nitrogen.
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Figure
5 shows representative alfalfa
plants from the experiment described above. The improved growth and
increased nodulation
of alfalfa plants inoculated with strain CFNM101
is evident. In
contrast, plants inoculated with strain CFNM102 or
CFNM104 show
poor growth, which is very similar to the growth of
uninoculated
plants watered without nitrogen, and they show decreased
nodulation.
The phenotypes of the alfalfa plants inoculated with the
1021
derivatives, CFNM103 or CFNM105 (not shown), were very similar
to
those inoculated with CFNM101 or CFNM104, respectively.
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FIG. 5.
Alfalfa plants inoculated with different S. meliloti strains. Representative plants from the experiment
analyzed in Fig. 3 are shown. (A) An uninoculated control plant watered
without nitrogen and plants inoculated with strains 41, CFNM101,
CFNM102, and CFNM104 are shown from left to right. (B to E) Nodulated
roots from plants inoculated with 41 (B), CFNM101 (C), CFNM102 (D), or
CFNM104 (E). Bar, 1 cm.
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Southern blot analysis and densitometric quantification were performed
with bacterial colonies recovered from surfaced sterilized
alfalfa
nodules in order to determine the stability of the amplified
symbiotic region during symbiosis. The results shown in Fig.
1F
indicate that each strain recovered from alfalfa nodules showed
copy
numbers similar to those of the strains used as an
inoculum.
| |
DISCUSSION |
Together with previously reported results (22), our
studies here show the potential of enhancing symbiotic properties by DNA amplification. At least under laboratory conditions, the
manipulation of the S. meliloti genome by SDA may be a
powerful tool for increasing nodulation, nitrogen fixation, and growth
in alfalfa plants. When the DNA sequence and gene organization are
unknown, RDA may be used. When the molecular basis and DNA organization
of a particular function are known, as is the case for symbiotic
nitrogen fixation in S. meliloti, a more direct approach
(SDA) may be a better choice.
The fact that most nodulation and nitrogen fixation genes are
interspersed, yet clustered in a region of pSyma of S. meliloti, provides a good opportunity for increasing symbiotic
nitrogen fixation by SDA. We chose a region containing three elements
with key roles in symbiotic nitrogen fixation. The nodD1
gene, activated by flavonoids, recognizes nod boxes upstream
of the nod genes and positively regulates their expression
(9, 11). The structural nodulation genes nodABC
participate in the synthesis of the basic structure of the Nod factors.
NodC catalyzes the polymerization of UDP-glucosamine
(8), NodB is a de-N-acetylase (12) and NodA is a
N-acyltransferase (25) which adds the acyl chain to the
oligosaccharide core. nodABC mutants do not form nodules
(20). The nitrogen fixation gene nifN is involved
in nitrogenase FeMo cofactor biosynthesis, and nifN mutants
do not fix nitrogen (1). Other components of this region are
nodulation genes nolI and nodN. The transcription
of these genes is regulated and starts in a region located 4 kb
upstream of the EcoRI fragment. For this reason we assume
that in the amplified state only one copy of these genes is transcribed.
The actual molecular mechanism of the observed symbiotic phenotypes is
beyond the scope of this study. The increase in copy number of the
regulatory gene nodD1 and/or of the structural genes nodABC might result in an increase in nodulation. The
increase in nodulation may be in relation to a slight increase in Nod
factor production observed in strains CFNM101 and CFNM103 in
relation to the respective wild-type strains. Strains CFNM102, CFNM104, and CFNM105, with higher copy numbers, showed a greater amount of
Nod factor production, but they showed reduced nodulation. The latter
observation is consistent with the previous observation that strains
bearing multicopy plasmids with nod genes have reduced nodulation (14). An increase in nodulation could lead to an increase in nitrogen fixation. In addition, the higher dosage of the
nifN operon might have a direct effect on nitrogen fixation.
There are examples that show that the manipulation of structural or
regulatory nod genes in rhizobia affects their symbiotic capacities. A Rhizobium leguminosarum strain carrying extra
copies of structural nodulation genes (nodABC) in a
multicopy plasmid was reported to have a reduced nodulation capacity on
Vicia plants (14). The constitutive expression in
different rhizobia of a chimeric nodD1 gene, consisting of
75% of the nodD1 gene of S. meliloti at the 5'
end and 27% of the nodD gene of Rhizobium
trifoli, affected nodulation and nitrogen fixation when introduced
into different rhizobia (31). Its expression in R. trifoli and S. meliloti resulted in an extension of the
host range for nodulation to the tropical legumes Macroptilium
atropurpureum, Lablab purpureus, and Leucaena
leucocephala (31). Its expression in R. trifoli and R. leguminosarum resulted in a significant
increase in nitrogen fixation during symbiosis with Vicia
sativa and Trifolium repens (31).
When gene amplification is used for enhancing relatively simple
functions such as the production of specific compounds, the increase in
copy number is usually achieved by the introduction of multicopy
plasmids. This approach has the disadvantage that the copy number
depends on the replication characteristics of the vector used and is
not easy to manipulate. When complex and tightly regulated functions
are involved, the copy number may be critical. In these cases the
approach based on amplification by recombination with the homologous
DNA region is more convenient. This situation is clearly exemplified by
the present study. When the symbiotic DNA region was introduced in a
multicopy plasmid, the copy number was set to about seven; this
actually resulted in a decrease in nodulation and nitrogen fixation. In
contrast, induced DNA amplification in the homologous genomic region
produced strains with different copy numbers. As shown here, a moderate increase in copy number (ca. three) produced an increase in nodulation, nitrogen fixation, and plant growth, while a higher copy number (ca.
six) resulted in a decrease in these functions.
Efforts have been made to improve the symbiotic capability of S. meliloti in alfalfa. Genetically modified strains containing extra
copies of dct (dicarboxylic transport) genes and a stronger nifA gene promoter show moderately increased nitrogen
fixation abilities under some field conditions (4). In this
case, the engineered strains caused a limited alfalfa yield increase
over plants inoculated with the wild type, but the effects could
also be attributed to the sites of insertion of the
dct and nif genes (29). In this work
we describe S. meliloti strains CFNM101 and CFNM103
which contain an average of 2.5 to 3 copies of the symbiotic region studied and show improved symbiotic abilities that result in an
increase in nodulation, nitrogen fixation, and growth of alfalfa plants
under environmentally controlled conditions. It will be interesting to
determine whether the experimental approaches of RDA or SDA could be
used for agricultural applications. In this regard, it is important to
consider the two principal requirements for the development of improved
strains for use as effective inoculants: a high symbiotic nitrogen
fixation capacity and high competitive ability. Therefore, a good
strategy may be to apply RDA and/or SDA to native S. meliloti strains in order to obtain improved engineered strains
which are competitive and persistent in certain areas where alfalfa is cultivated.
| |
ACKNOWLEDGMENTS |
This work was supported in part by CONACyT (México) grant
number L0013N.
We are grateful to Yolanda Pérez-Tejada, César
Rodríguez, and Angeles Moreno for technical assistance; to Eva
Kondorosi for providing a plasmid used; and to Michael Dunn for
critically reading the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centro de
Investigación sobre Fijación de Nitrógeno, UNAM, Ap.
Postal 565-A, Cuernavaca, Morelos, México. Phone: (52) (73)
13-98-77. Fax: (52) (73) 11-67-10. E-mail:
gina{at}cifn.unam.mx.
| |
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Applied and Environmental Microbiology, June 1999, p. 2716-2722, Vol. 65, No. 6
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
