Applied and Environmental Microbiology, May 1999, p. 2015-2019, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Enhanced Nitrogen Fixation in a Rhizobium
etli ntrC Mutant That Overproduces the Bradyrhizobium
japonicum Symbiotic Terminal Oxidase
cbb3
Mario
Soberón,1,*
Oswaldo
López,1
Claudia
Morera,1
Maria de Lourdes
Girard,2
Maria Luisa
Tabche,1 and
Juan
Miranda1
Departamento de Biología Molecular de
Plantas, Instituto de Biotecnología, Universidad Nacional
Autónoma de México, Cuernavaca, Morelos
62271,1 and Centro de
Investigación sobre Fijación de Nitrógeno,
Universidad Nacional Autónoma de México, Cuernavaca,
Morelos,2 México
Received 3 December 1998/Accepted 11 March 1999
 |
ABSTRACT |
The ntrC gene codes for a transcriptional activator
protein that modulates gene expression in response to nitrogen. The
cytochrome production pattern of a Rhizobium etli ntrC
mutant (CFN2012) was studied. CO difference spectral analysis of
membranes showed that CFN2012 produced a terminal oxidase similar to
the symbiotic terminal oxidase of bacteroids in free-living cells under
aerobic conditions, with a characteristic trough at 553 nm. CFN2012
produced two c-type cytochromes with molecular masses of 27 and 32 kDa, in contrast with the wild-type strain, which produced only
a 32-kDa c-type cytochrome. The expression levels of the
R. etli fixNOQP operon, which codes for terminal oxidase
cbb3, were not affected by the ntrC
mutation. However, the production levels of the two c-type cytochromes (27 and 32 kDa) were enhanced at least eightfold when the
Bradyrhizobium japonicum fixNOQP operon was expressed in
CFN2012 from the nptII promoter (pMSfixc),
suggesting that these proteins are subunits FixO (27 kDa) and FixP (32 kDa) of cbb3 and that
CFN2012/pMSfixc overproduced this terminal oxidase.
CFN2012/pMSfixc showed a significant increase in its
symbiotic performance as judged by the determination of nitrogenase
activities of plants inoculated with this strain, suggesting that the
overproduction of cbb3 terminal oxidase
correlates with an enhancement in symbiotic nitrogen fixation.
| |
INTRODUCTION |
In free-living diazotrophic bacteria
combined nitrogen regulates the expression of the nitrogenase
structural genes, inhibiting nitrogen fixation (10). The
general nitrogen regulatory system (ntr system) activates
the transcription of nitrogenase structural genes when combined
nitrogen is not available (3). In contrast, in symbiotic
nitrogen-fixing bacteria (genera Bradyrhizobium and Rhizobium) combined nitrogen has no effect on the expression
of nitrogenase structural genes (8). Bacteria of these
genera may establish a specific symbiotic relationship with their
legume host plant. These bacteria elicit the formation of new organs, i.e., root nodules, in which bacteroids reduce atmospheric nitrogen to
ammonia and supply the host plant with combined nitrogen. For the
induction of root-nodules, members of Rhizobium express
nod genes which code for enzymes involved in the formation
of lipooligosaccharide factors (nod factors). However, in the presence
of combined nitrogen, the formation of the root nodules is inhibited.
It has been demonstrated that nitrogen negatively regulates bacterial
nod gene expression (6, 7, 14, 27). In
Rhizobium etli nitrogen repression of nod gene
expression is mediated by the ntr system (14).
Symbiosis requires a respiratory chain that has a high affinity for
O2 and is efficiently coupled to ATP production since nitrogen fixation is an energy-consuming process, requiring up to 20 ATP molecules to reduce just one molecule of N2. The genes of Bradyrhizobium japonicum which code for the bacteroid
terminal oxidase have been identified as the fixNOQP operon
(20). The sequence analysis of these genes, and biochemical
characterization of the purified enzyme, showed that they code for a
three-subunit terminal oxidase (cbb3) (11,
20, 21). FixN is a b-type heme- and copper-containing
subunit, FixO is a single-heme-containing c-type cytochrome,
and FixP is a diheme-containing c-type cytochrome (11,
20, 21). In R. etli, mutants that produce the
cbb3 terminal oxidase under free-living
conditions showed enhanced symbiotic nitrogen fixation (15).
In an attempt to study if nitrogen regulation affects respiration in
R. etli, we analyzed the cytochrome production pattern of an
R. etli mutant with change affecting the ntr
system. A mutation in ntrC, which codes for a
transcriptional activator protein that modulates gene expression in
response to nitrogen (16), was analyzed. This analysis
showed that the ntrC mutant produces a terminal oxidase of
the cbb3 type in free-living cultures. Analysis of the expression of the R. etli fixNOQP genes showed that
the expression of these genes was not affected by the ntrC
mutation. However, expression of the B. japonicum fixNOQP
operon from a constitutive promoter in the R. etli ntrC
mutant greatly enhanced the production level of the
cbb3 terminal oxidase and also the symbiotic
performance of this strain. These data indicate that symbiotic nitrogen
fixation can be improved by the overproduction of the symbiotic
terminal oxidase cbb3.
| |
MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
Bacterial strains and plasmids used are listed in Table
1. R. etli cells were cultured
in minimal medium (MM), peptone yeast extract (PY) medium
(17), or yeast extract succinate (YS) (15). To
achieve microaerobic cultures (O2 pressure, 2 kPa) 2 ml of active culture was used to inoculate 40 ml of medium. These cultures had previously been evacuated and flushed with a 1,200-ml · min
1 sterile N2 stream for 10 min. Calculated
volumes (9.8 ml) of sterilized, high-purity commercial air were
injected by making use of disposable syringes, following extraction of
the same volume of N2. Escherichia coli was
grown in Luria broth medium. Antibiotics were used at the following
concentrations: rifampin, 50 mg/liter; tetracycline, 5 mg/liter;
kanamycin, 30 mg/liter; and streptomycin, 100 mg/liter.
DNA manipulations.
Cloning, restriction mapping,
transformation, plasmid isolation, and 
-galactosidase measurements
were done as described (12). SalI and
PstI clones from cGD101 were subcloned into pBluescript SK(+) vector and sequenced (4,832 bp; GenBank accession no. U76906) at
the automated DNA sequencing facility at the Molecular Genetics Core
Facility in the Department of Microbiology and Molecular Genetics,
University of Texas
Houston Medical School. Computer-assisted sequence
analysis and comparisons with the GenBank sequence were done using the
Gene Works 2.6 program from Intelligenetics, Inc.
Spectral and electrophoretic analysis of cytochromes.
Cells
were grown overnight on PY medium. Cells were washed and diluted
50-fold on fresh YS medium and grown on a rotary shaker (200 rpm) at
30°C for 36 h. Cells were harvested by centrifugation, washed,
and suspended to 30% (wt/vol) in 50 mM Tris hydrochloride (pH 7.4)-5
mM CaCl2-5 mM MgCl2. Cytochrome spectra of
whole cells or membrane preparations in an SLM Aminco Midan II
spectrophotometer were recorded. Samples were reduced with dithionite
(a few grains) or oxidized with ammonium persulfate. Carbon monoxide
difference spectra were obtained by bubbling with CO (2 min) to reduce
a cell sample, and spectra were recorded against a reduced sample. Spectra were obtained at room temperature with 1.0-cm light path cuvettes. c-type proteins were analyzed after sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of membrane
samples, which were prepared mechanically as previously described
(23). Protein blotting and heme peroxidase detection were
done as reported (26), except that in Western blotting the
detection reagents used for peroxidase detection were obtained from
Pierce (SuperSignal Substrate; Rockford, Ill.); the use of these
chemicals greatly enhanced the sensitivity of this technique. Protein
was determined as described (13).
Nitrogen fixation determination.
For acetylene reduction
measurements, Phaseolus vulgaris cv. negro jamapa was
surface sterilized in hypochlorite and germinated on moist sterile
filter paper. Three-day-old seedlings were transferred to plastic
growth pots, inoculated with a bacterial suspension in PY medium, and
grown with nitrogen-free salts in a greenhouse (23).
Nitrogenase was determined by measuring the acetylene reduction of
nodulated plant roots transferred to tubes with rubber seal stoppers by
injecting acetylene to a final concentration of 10% of the gas phase.
Samples were incubated 40 min at room temperature, and ethylene
production was determined by gas chromatography in a Packard model 430 chromatograph (23). For each strain, the total nitrogen of
60-day-old nodulated plants was determined for three plants in four
pots each (total, 12 plants) using an Antek 720 nitrogen detector as
previously described (4).
Bacteroid preparation.
Nodules were harvested 30 days after
inoculation. Bacteroids were isolated by layering a nodule extract on a
sucrose gradient as previously reported (23).
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RESULTS |
Cytochrome production in an ntrC mutant of R. etli.
Cytochrome production was analyzed in cells cultured
aerobically to the stationary phase of growth and in bacteroids of the wild-type (CE3) and the ntrC mutant (CFN2012) strains.
Figure 1A shows difference spectra
(spectra for reduced cells minus spectra for oxidized cells) of
free-living cells, showing that CE3 produced c-type
cytochromes (peak at 553 nm) and b-type cytochromes
(shoulder at 562 nm) but no aa3 cytochromes
(peak at 603 nm) under this culture condition. CFN2012 produced
c-type, b-type, and aa3
cytochromes (Fig. 1A). Carbon monoxide (CO) difference spectra were
obtained since CO reacts specifically with cytochrome terminal
oxidases. CO difference spectra showed that the CE3 cells produced only cytochrome terminal oxidase o (peaks at 544 and 572 nm and
trough at 562 nm) and no cytochrome aa3 (trough
at 610 nm). In contrast, CFN2012 produced a different CO-reactive
cytochrome with a spectrum signal showing a characteristic trough at
553 nm and no cytochrome aa3 (Fig. 1A). The lack
of evidence for cytochrome aa3 in CO difference spectra of CFN2012 membranes suggests that the absorption peak at 603 nm found in difference spectra (spectra for reduced cells minus those
for oxidized cells) of CFN2012 could be due to a different heme-containing protein that absorbs near 600 nm (e.g., catalase).
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FIG. 1.
Difference spectra (spectra for reduced cells minus
those for oxidized cells) (spectra 1 and 2 in panels A and B) and CO
difference spectra (spectra 3 and 4 in panels A and B) of R. etli whole cells. (A) Spectra 1 and 3 are for CE3 cells cultured
for 36 h (18.7 mg of protein ml1), and spectra 2 and
4 are for CFN2012 cells cultured for 36 h (23.5 mg of protein
ml1). (B) Spectra of bacteroids of CE3 (17.7 mg of
protein ml1) (spectra 1 and 3) and CFN2012 (25.5 mg of
protein ml1) (spectra 2 and 4). Free-living cells were
cultured aerobically in YS medium.
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Bacteroids of the CE3 and CFN2012 strains had similar cytochrome
production patterns, producing c-type (peak at 551 nm),
b-type (shoulder at 562 nm), and aa3
(peak at 603 nm) cytochromes (Fig. 1B). Figure 1B also shows that
bacteroids of these strains produced a CO-reactive cytochrome with a
spectrum with a trough at 553 nm, very similar to the CO-reactive
cytochrome produced by CFN2012 in free-living cultures, and also
cytochrome aa3 (trough at 610 nm).
c-type cytochrome production by CFN2012.
The
c-type cytochromes produced by CE3 and CFN2012 strains
cultured aerobically in YS medium to the stationary growth phase were
analyzed. CE3 and CFN2012 cells harboring plasmid pMSfixc,
which contains the B. japonicum fixNOQP operon fused to the nptII promoter (24), were included as controls
for the production of FixO (27 kDa) and FixP (32 kDa) c-type
cytochrome subunits of terminal oxidase cbb3.
Figure 2 shows that CE3 cell membranes contained detectable levels of a 32-kDa c-type cytochrome,
as was previously reported (23); in contrast, CFN2012
produced higher levels of the 32-kDa protein and produced an additional c-type cytochrome of 27 kDa (Fig. 2).
CE3/pMSfixc produced both the 32- and 27-kDa proteins,
while CFN2012/pMSfixc showed very high production levels of
both c-type cytochromes and also detectable levels of a
20-kDa c-type cytochrome (Fig. 2). The production of the two
c-type cytochromes by the ntrC mutant, as well as
the results of the CO difference spectral analysis (Fig. 1A), suggests
that in this strain a terminal oxidase similar to a
cbb3 terminal oxidase was produced in aerobic
cultures of free-living stationary-phase cells. We quantified the
production levels of cbb3 terminal oxidase by
measuring the 32- and 27-kDa c-type proteins produced when
fixNOQP was transcribed from the nptII promoter.
This analysis showed that CFN2012 produced eightfold-higher levels of
this oxidase than CE3.
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FIG. 2.
Proteins containing c-type heme from membrane
particles. Each lane contains 50 µg of protein from membrane
particles from CE3 cells (lane 1), CFN2012 cells (lane 2),
CE3/pMSfixc cells (lane 3), or CFN2012/pMSfixc
cells (lane 4) grown aerobically in YS medium for 36 h.
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Expression of R. etli fixNOQP.
In order to further
analyze the free-living cell production of the
cbb3 terminal oxidase in CFN2012, we decided to
study the expression of the R. etli fixNOQP operon. A cosmid
clone (cGD101) of the R. etli symbiotic plasmid pd
(9), which hybridized against heterologous gene probes from
fixK (from Sinorhizobium meliloti) (1)
and fixN (from B. japonicum) (20), was
identified (data not shown). In order to localize the
fixNOQP operon, 4,832 bp were sequenced (GenBank accession
no. U76906).
Five open reading frames were identified as the R. etli fixK
and fixNOQP operon (Fig. 3). The predicted FixK protein is
comprised of 239 residues. FixK is a transcriptional activator involved in fixNOQP induction in S. meliloti
(1). This protein has 63% identity with FixK of
Rhizobium leguminosarum bv. viciae (19) (GenBank
accession no. Z70305) and lacks the cysteine-amino-terminal domain
present in Fnr. The predicted FixN protein is comprised of 540 residues. R. etli FixN has significant homology with FixN from different organisms, i.e., 78% identity with FixN of S. meliloti (GenBank accession no. Z21854) and 92% identity with
both copies of fixN gene products in R. leguminosarum (GenBank accession no. Z80339 and Z80340). FixN
contains the six conserved histidine residues proposed to bind the
low-spin b-type heme (H117 and H406), CuB (H266, H136, and
H317), and the high-spin b-type heme (H404). FixO, comprised
of 244 residues, has 86% identity with FixO of R. leguminosarum and 75% identity with FixO of S. meliloti. FixO contains two cysteine residues, at positions 69 and
72, which are likely to be involved in the attachment of
c-type heme. FixQ, comprised of 50 residues, has 88%
identity with FixQ from R. leguminosarum and 66% identity
with FixQ from S. meliloti. Finally, FixP, comprised of 287 residues, has identities of 81 and 61% with FixP of R. leguminosarum and of S. meliloti, respectively, and
contains the four conserved cysteine residues involved in the
attachment of two c-type hemes (C121, C124, C216, and C219).
Similar to R. leguminosarum bv. viciae (19), in
R. etli fixK and fixNOQP are transcribed in
opposite directions with a spanning DNA region of 230 bp (Fig.
3). These data suggest that the promoters of both genes are contained in this DNA region. The "anaerobox" sequence (TTGATGTAGATCAA) is located 88 bp in front of the
beginning of fixN (Fig. 3).
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FIG. 3.
Physical-genetic map of the fixK-fixNOQP
sequenced region. (A) Physical-genetic map of the R. etli
fixK-fixNOQP genetic region. The restriction sites used in
subcloning for sequencing are shown. Sc, SacI; P,
PstI; S, SalI. The position of the anaerobox
sequence is shown. (B) Diagram of fixN-lacZ transcriptional
gene fusion construction. S.D., Shine-Dalgarno sequence. Arrows
indicate the directions of transcription.
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In order to generate transcriptional lacZ gene fusions of
fixN (plasmid pOLfix10) promoter, a 1.1-kb PstI
fragment containing the amino-terminal part of FixN and FixK was cloned
in plasmid pMP220, which carries the E. coli lacZ gene
(25) (Fig. 3B).
The expression of fixNOQP in CE3 and CFN2012 strains
cultured aerobically or microaerobically was studied. The expression of
fixN was induced 15-fold in cells cultured microaerobically, as has been shown in several other Rhizobium species,
showing that in R. etli as well, oxygen is the most
important metabolic signal triggering fixNOQP expression.
The level of expression of fixN was slightly lower in the
wild-type than in the CFN2012 strain under stationary-phase aerobic
(60%) and microaerobic (70%) conditions. This analysis revealed that
the production of cbb3 terminal oxidase by
CFN2012 was not due to the enhanced expression of fixNOQP,
suggesting that NtrC modulates cbb3 production
at a level different from fixNOQP transcription. When cells
were grown on YS medium under atmospheric O2 tension for
8 h, under atmospheric O2 tension for 36 h, and
under 2 kPa O2 tension for 8 h, the CE3 strain
carrying the fixN-lacZ(pOLfix10) reporter gene fusion showed
-galactosidase activities of 474, 342, and 6,199 U/mg of protein,
respectively, and the CFN2012 strain carrying the same fusion showed
activities of 526, 557, and 8,719 U/mg of protein, respectively (values
are expressed after subtraction of activities of strain without any
plasmid [range, 50 to 90 U/mg of protein]).
Symbiotic nitrogen fixation of plants inoculated with different
R. etli strains.
P. vulgaris cv. negro jamapa
plants were inoculated with CE3 and CFN2012 strains and the same
strains harboring pMSfixc, and nitrogenase activity was
determined at different days after inoculation. Figure
4 shows that for plants inoculated with
CE3 the highest nitrogenase activity (23 nmol of ethylene produced h1 per plant) was reached at 52 days after inoculation
and decreased gradually afterwards. No effect of plasmid
pMSfixc was found in this strain. Plants inoculated with
CFN2012 demonstrated nitrogenase activity more rapidly than those
inoculated with CE3, but similar nitrogenase activities were observed.
CFN2012 strain harboring pMSfixc induced nitrogenase
activity more rapidly and achieved a twofold-higher nitrogenase
activity than the CE3 strain (Fig. 4). In addition, nitrogen fixation
was estimated by the total nitrogen content of plants 60 days after
inoculation with the different strains. CE3/pMSfixc-inoculated plants contained 90% (3.51 ± 0.092 mg of nitrogen [100 mg dry weight]1; n = 4) of the nitrogen found in CE3-inoculated plants (3.90 ± 0.031 mg of nitrogen [100 mg dry weight]1; n = 4), whereas CFN2012- and CFN2012/pMSfixc-inoculated
plants contained 8% (4.25 ± 0.102 mg of nitrogen [100 mg dry
weight]1; n = 4) and 18% (4.57 ± 0.065 mg of nitrogen [100 mg dry weight]1; n = 4) more nitrogen, respectively, than CE3-inoculated plants.
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FIG. 4.
Acetylene reduction activity in plants inoculated with
strains CE3 ( ), CE3/pMSfixc ( ), CFN2012 ( ), and
CFN2012/pMSfixc ( ). Acetylene reduction activity for
four plants in each of two pots was determined on the specified days.
Data are means for two pots (eight plants); variations were 30% or
less.
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DISCUSSION |
Symbiotic nitrogen fixation is an energy-consuming process
requiring up to 20 ATP molecules to reduce one molecule of
N2. Also, symbiotic nitrogen fixation occurs at very low
oxygen tensions (2). Therefore, a high-affinity oxidase, of
the cbb3 type, efficiently coupled to ATP
production is produced during symbiosis. During free life,
Rhizobium species do not produce the
cbb3 terminal oxidase, mainly due to a low level
of expression of the fixNOQP operon in the presence of
O2 (2).
In this work we present data showing that, in R. etli, in
addition to oxygen regulation, NtrC represses the free-living cell production of the cbb3 symbiotic terminal
oxidase. The analysis of cytochrome production revealed that in aerobic
cultures an ntrC mutant produced a terminal oxidase very
similar to that produced in bacteroids, in contrast with the wild-type
strain. This suggests that nitrogen availability could modulate
symbiotic cytochrome production in R. etli. The molecular
mechanism by which NtrC represses cbb3
production is still unclear. However, two results indicate that this
regulation is exerted after transcription of the structural fixNOQP genes: (i) no difference was found in
fixNOQP expression between CFN2012 and CE3; and (ii) under
conditions where fixNOQP was expressed under a strong
promoter, CFN2012 produced at least eightfold-higher levels of
cbb3 than CE3, suggesting that a
posttranscriptional step involved in cbb3
biogenesis was negatively regulated by NtrC. In several
Rhizobium species, including R. etli, several
genes that participate in the correct assembly of the
cbb3 terminal oxidase have been identified.
These include genes necessary for the covalent attachment of heme to
c-type apoproteins (5) and genes
(fixGHIS) that are probably involved in the transport of copper ions, which are essential for oxygen reduction by
cbb3 (5, 22).
The ntrC mutation in CFN2012 had a positive effect on
nitrogenase activity only when the fixNOQP genes were
expressed from a strong promoter; also, CFN2012/pMSfixc
produced an eightfold-higher level of cbb3
terminal oxidase than CE3/pMSfixc in culture. These data
suggest that increased cbb3 terminal production correlates with enhanced nitrogen fixation. However, because
ntrC is known to be global in its regulatory role, it cannot
be ruled out that other effects of ntrC mutation, in
addition to the elevated levels of the terminal oxidase, could also
participate in the enhanced nitrogen fixation capacity of this strain.
In fact plants inoculated with CFN2012 accumulated more nitrogen than
CE3-inoculated plants. It is important to study the respiratory
physiology of ntrC mutant-derived bacteroids.
Total nitrogen determinations of plants inoculated with the different
strains correlated roughly with nitrogenase activities. However,
plasmid pMSfixc had a negative effect on nitrogen
accumulation for plants inoculated with the CE3 strain even though
plants inoculated with these strains showed very similar nitrogenase
activities. Therefore, we cannot rule out the possibility that in
CFN2012/pMSfixc-inoculated plants nitrogen accumulation was
also affected by the presence of this plasmid. At this moment we have
no explanation for the negative effect of this construct, although the
process of replication of the plasmid or enhanced transcription could compete for ATP with nitrogen fixation. It is important to introduce the nptII-fixNOQP construct into the genome to determine the
reason for the negative effect of plasmid pMSfixc on plant
nitrogen accumulation.
Two possible reasons for the enhanced symbiotic nitrogen fixation
observed for CFN2012/pMSfixc can be proposed: one is a
higher nitrogenase activity due to enhanced supply of ATP to
nitrogenase, and the other is a more-efficient performance during
infection and bacteroid development. R. etli bacteroids
repress ntrC expression, and NtrC protein could not be
detected (18). This explains the similar cytochrome
production patterns found in bacteroids of CE3 and CFN2012 strains.
However, it was shown that bacteria inside the infection thread and
young bacteroids could express ntrC (18). Since
NtrC is not produced in well-developed bacteroids, it seems possible
that the difference in nitrogenase activity between
CE3/pMSfixc and CFN2012/pMSfixc could be due to
a better performance of CFN2012 during infection. This could suggest
that the repression of cbb3 terminal oxidase production by NtrC could be relevant during the infection process whereas another terminal oxidase could support growth. Alternatively, the negative regulation of the production of the symbiotic terminal oxidase by NtrC could be an additional control point for the repression of symbiosis when combined nitrogen is available.
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ACKNOWLEDGMENTS |
We thank Alejandra Bravo for revising the manuscript, Guadalupe
Espín for providing us the CFN2012 strain, Yolanda Mora for total nitrogen determinations, and Jose Luis Zitlalpopoca for technical assistance.
This work was partially supported by the European Communities through
the International Scientific Co-operation Program, contract CI1*-CT94-0042, by CONACyT contract no. 3372-N9309, and by DGAPA contract no. IN204697.
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FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Biología Molecular de Plantas, Instituto de
Biotecnología, U.N.A.M., Apdo Postal 510-3, Cuernavaca, Morelos
62271, México. Phone: 52 5 6227618. Fax: 52 73 172388. E-mail:
mario{at}ibt.unam.mx.
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Applied and Environmental Microbiology, May 1999, p. 2015-2019, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
