Previous Article | Next Article 
Journal of Bacteriology, August 1999, p. 5099-5102, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Disruption of narG, the Gene Encoding the Catalytic
Subunit of Respiratory Nitrate Reductase, Also Affects Nitrite
Respiration in Pseudomonas fluorescens YT101
Jean-François
Ghiglione,1
Laurent
Philippot,2
Philippe
Normand,1
Robert
Lensi,1 and
Patrick
Potier1,*
Laboratoire d'Ecologie Microbienne du Sol,
UMR C.N.R.S. 5557, Université Claude Bernard, Lyon 1, 69622 Villeurbanne Cedex,1 and Laboratoire de
Microbiologie des Sols, INRA-CMSE, B.V. 1540, 21034 Dijon
Cedex,2 France
 |
ABSTRACT |
The Pseudomonas fluorescens YT101 gene
narG, which encodes the catalytic 
subunit of the
respiratory nitrate reductase, was disrupted by insertion of a
gentamicin resistance cassette. In the Nar
mutants,
nitrate reductase activity was not detectable under all the conditions
tested, suggesting that P. fluorescens YT101 contains only
one membrane-bound nitrate reductase and no periplasmic nitrate
reductase. Whereas N2O respiration was not affected,
anaerobic growth with NO2 as the sole electron acceptor was
delayed for all of the Nar mutants following a transfer
from oxic to anoxic conditions. These results provide the first
demonstration of a regulatory link between nitrate and nitrite
respiration in the denitrifying pathway.
| |
TEXT |
Dissimilatory denitrification refers
to the respiratory reduction of nitrate and/or nitrite to dinitrogen
via nitric oxide and nitrous oxide. This alternative anaerobic process
of energy conservation is phylogenetically widespread in bacteria. The
first step, reduction of nitrate to nitrite, can be performed not
only by most denitrifiers but also by nitrate reducers, such
as Escherichia coli, that are unable to reduce
the nitrite produced into gas. It has been shown that the reaction
sequence
NO2
NON2ON2
forms a functional unit since several steps of the reduction chain are
interdependent in various bacteria (1, 6, 9). For example,
mutation in the nir genes of Pseudomonas stutzeri
resulted in the simultaneous loss of nitrite and nitric oxide reduction
(10). Similarly, a significant decrease in nitric oxide
reductase activity was observed after Tn5 insertion in the nirS gene of Pseudomonas fluorescens YT101
(21). Finally, inactivation of the norQ gene of
Paracoccus denitrificans encoding the nitric oxide reductase
eliminated both nitrite reduction and nitric oxide reduction
(20). To date, regulatory links between nitrate reductases and the other reductases of the denitrifying pathway have never been
demonstrated, and it should therefore be interesting to determine if
nitrate respiration is a part of this complex regulatory network. To
address this possibility, we constructed and characterized isogenic
mutants of P. fluorescens unable to synthesize the
membrane-bound nitrate reductase A (NRA). Of the different structural
genes encoding this enzyme, narG was chosen as the target
for mutagenesis since it encodes the catalytic subunit, and the
chromosomal copy of narG was replaced with an in
vitro-inactivated copy of this gene. To our knowledge, this is the
first time that mutants deficient in the synthesis of the NRA have been
obtained by allelic exchange of the narG gene in a
denitrifying bacterium.
Construction of Nar mutants of P. fluorescens.
P. fluorescens YT101 is a Rifr derivative of
the strain AK15 (21). To obtain the Nar
mutants, the wild-type chromosomal copy of narG of P. fluorescens YT101 was replaced after homologous recombination by a
copy of the deleted gene with an insertion of the apra3
gentamicin resistance gene. DNA restriction, agarose gel
electrophoresis, ligation, and transformation were carried out by
standard methods (16). Plasmid pNR25 (11)
carrying 3.3 kb of narG was digested with NarI,
and ends were made blunt by the action of Klenow and T4 DNA polymerase.
The SmaI fragment containing the 1.8-kb apra3 gene from plasmid pHP45
(12) was blunt ended and ligated
into the NarI-digested pNR25. The presence of the
apra3 gene was confirmed by restriction enzyme analysis. The
narG::apra3 construct was then
excised from pSDNG3 by using the ClaI and SpeI
enzymes and cloned into the EcoRI site of pLAFR3
plasmid (19), yielding pLDNR5. The pLDNR5 plasmid was
then mobilized from E. coli to P. fluorescens YT101 by triparental mating by using the
conjugative plasmid helper pRK2013 (11).
Transconjugants were then selected on Luria-Bertani (LB) medium
containing rifampin (50 µg/ml), gentamicin (10 µg/ml), and
tetracycline (10 µg/ml). Recombinants showing double crossover were
identified after several rounds of growth in LB medium containing
gentamicin at 4°C to cure the pLDNR5 and scoring for Tcs
Gmr Rifr colonies. Three mutants, namely
LP59JG, LP37JG, and LP15JG, were obtained from independent experiments.
Replacement of the wild-type chromosomal copy of the narG
gene by the deleted copy containing the apra3 gentamicin
resistance gene was verified by Southern blot analysis of chromosomal
DNA of the wild-type strain YT101 and of the Nar mutants
digested with BglII. The 3.3-kb fragment of the
narG gene, the deleted fragment of the narG gene,
and the 1.8-kb apra3 gene were used as probes and labeled
with digoxigenin-11-dUTP by using the PCR DIG Probe Synthesis kit
(Boehringer, Mannheim, Germany) as described by the manufacturer, and
the results were in complete agreement with the predicted patterns.
Southern blot analysis of DNA of Nar mutants using
labeled pLAFR3 as a probe allowed us to verify that no additional
insertion of the vector occurred (data not shown).
Expression of NRA was compared in wild-type and mutant strains by
immunoblotting techniques. For this purpose, the NRA catalytic subunit of P. fluorescens YT101 was purified to homogeneity
as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining according to the procedure described previously (11). The identification of the catalytic
subunit was confirmed by determining the amino-terminal sequence by
automated gas phase Edman degradation (Centre d'Analyse du CNRS,
Solaize, France). An antiserum was raised in rabbits (Valbex,
Villeurbanne, France) and was used to detect NRA expression by
conventional immunoblotting techniques. As shown in Fig.
1, the antiserum recognized a 118-kDa
protein in total protein extracts from the wild-type strain incubated
for 4 h under anaerobiosis in the presence of 20 mM nitrate (lane
1). As expected, this peptide was not detected in Nar
mutants incubated for 4 h or more under inducing conditions (lanes 2, 3, and 4).
View larger version (27K):
[in this window]
[in a new window]
|
FIG. 1.
Western blot of total proteins from the wild-type strain
YT101 (lane 1) and LP59JG (lane 2), LP37JG (lane 3), and LP15JG mutants
(lane 4), and LPCJG21 (lane 5) complemented strain. The blot was
developed with polyclonal antibodies raised against the subunit of
nitrate reductase purified from P. fluorescens YT101.
Molecular sizes are indicated on the left in kilodaltons.
|
|
Furthermore, nitrate reductase activities were compared in dense
cultures and in cell extracts of the wild-type strain and Nar mutants by using methyl viologen or benzyl viologen
as an electron donor (8). In the case of the wild-type
strain, no nitrate reductase activity could be detected with either
electron donor when cells were grown aerobically with or without
nitrate. A nitrate reductase activity was observed with benzyl viologen
as the electron donor for wild-type cells incubated anaerobically with
nitrate (10 mM), but no activity could be detected under the same
conditions when methyl viologen was the electron donor. For the mutants
strains, no activity was detected under all the conditions tested with either electron donor (results not shown). The existence of an additional periplasmic nitrate reductase has been reported in a
wide variety of phylogenetically unrelated bacteria (2-5, 7, 13-15, 17, 18). Since all of our Nar mutants
presented a total lack of nitrate reductase activity whatever the
conditions tested, this strongly suggests that P. fluorescens YT101 contains only one nitrate reductase. Therefore, the existence of more than one nitrate reductase in nitrate-reducing or
denitrifying bacteria cannot be generalized.
Effects of narG disruption on growth characteristics of
Nar mutants.
The ability of Nar
mutants to use nitrate, nitrite, or nitrous oxide as the sole electron
acceptor to sustain growth was compared to that of the wild-type strain
YT101. For this, 150-ml plasma flasks containing 38 ml of LB medium
supplemented with either 20 mM KNO3 or 10 mM
KNO2 were made anaerobic by evacuation and flushing three
times with helium. Flasks containing 20 mM N2O were
prepared by replacing (after flushing) 18 ml of the atmosphere of
150-ml plasma flasks containing 38 ml of LB medium with 18 ml of
gaseous N2O. After inoculation with either the wild-type strain YT101 or the Nar mutant strains, flasks were
incubated at 28°C on an orbital shaker. Each treatment was tested in
triplicate, and bacterial growth was monitored at 580 nm. Nitrate
and nitrite concentrations were determined by ionic
chromatography (Centre d'Analyse du CNRS). Nitrous oxide
concentration was determined by using a GIRDEL 30 gas chromatograph
equipped with a thermal conductivity detector.
Under aerobic conditions, the wild-type strain and the
Nar
mutants had similar growth rates (result not
shown). The growth
characteristics of the wild-type strain and
the Nar
mutants under anaerobic conditions with various
nitrogen oxides
as electron acceptors are given in Fig.
2. When nitrate was the
sole electron
acceptor, Nar
mutants were unable to grow while the
wild-type strain reached
optical density at 580 nm of 0.75 within
30 h (Fig.
2A). Moreover,
no significant reduction of nitrate was
observed for the Nar
mutants after 50 h while
nitrate decreased below the detection
threshold within 25 h for
the wild-type strain. When nitrous oxide
was the sole electron
acceptor, the wild-type strain and Nar
mutants had
similar growth curves and nitrous oxide was similarly
reduced within
40 h in all cultures (Fig.
2C). When nitrite was
the sole electron
acceptor, the wild-type strain reached an optical
density at 580 nm of
0.18 within 30 h with a concomitant decrease
of nitrite
concentration in the culture medium (Fig.
2B). A 14-h
lag was observed
for both growth resumption and nitrite consumption
for all
Nar
mutants. The generation times of the wild type and
mutants were
similar, i.e., 16 h 30 min and 17 h,
respectively. At the end
of this experiment, mutant cells were
reinoculated into fresh
medium containing nitrite and anaerobic growth
was monitored.
In this case, the lag period was no longer observed, and
both
the wild-type strain and mutant strains reinitiated growth
immediately
with the same generation time, 7 h 50 min (Fig.
3A). This could
be due to the selection
of variants or spontaneous mutants in
which the characteristics of
anaerobic growth with nitrite have
been restored. This possibility was
ruled out by showing that
the delay reappeared when cells grown twice
under anaerobiosis
with nitrite were then grown aerobically before
being recultivated
under anoxic conditions with nitrite (Fig.
3B). One
of the Nar
mutants (strain LP59JG) containing the
narG::
apra3 gene was
restored to the
wild-type phenotype by complementing the
narG gene in
cis by using plasmid pLNR4. After introduction of this
plasmid into the LP59JG Nar
mutant, transconjugants were
then screened for their abilities
to grow anaerobically with nitrate as
the sole electron acceptor.
NarG synthesis by the complemented strain
LPCJG21 was confirmed
by Western blotting (Fig.
1, lane 5) and by
measuring nitrate
reductase activity with benzyl viologen as an
electron donor.
When oxygen, nitrate, nitrite, and nitrous oxide were
used as
electron acceptors the complemented strain exhibited growth
patterns
that were identical to those of the wild-type strain (data not
shown). In particular, the delay before growth resumption observed
under anaerobiosis with nitrite as the sole electron acceptor
was no
longer observed. Therefore, the phenotypic changes observed
for
Nar
mutants can be clearly attributed to the insertional
inactivation
of
narG.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 2.
Anaerobic growth (top) and electron acceptor consumption
(bottom) of the wild-type strain YT101 ( ) and the LP59JG ( ),
LP37JG ( ), and LP15JG ( ) mutants in LB medium supplemented with
KNO3 (20 mM) (A), KNO2 (10 mM) (B), and
N2O (20 mM) (C). When not visible, the standard deviation
(vertical bar) is within symbol dimensions. O. D., optical
density.
|
|
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 3.
Anaerobic growth in LB medium with 10 mM
KNO2 as the sole electron acceptor of wild-type YT101
strain () and the LP57JG (), LP37JG (), and LP15JG ()
mutants. Cells were precultured anaerobically with 10 mM
KNO2 (A), or cells were precultured anaerobically with 10 mM KNO2 to late exponential phase and then grown
aerobically before being recultivated under anaerobiosis with 10 mM
KNO2 (B). When not visible, the standard deviation
(vertical bar) is within symbol dimensions. O. D., optical
density.
|
|
The effects of
narG mutation on the denitrifying pathway
were limited to nitrite respiration. Since the DNA fragment that
was
used to disrupt
narG contains a transcription termination
sequence (hairpin) which is oriented so as to block transcription
coming out from the
apra3 fragment (
12), it can
be expected
that transcription of downstream
narG sequences
was inhibited
or dramatically reduced. Therefore, the phenotype of the
Nar
mutants can be attributed either to the lack of the
product of
narG itself or to the lack of the product(s) of
the gene(s) located
downstream of
narG in the
narGHJI operon.
The growth resumption delay observed in all the Nar
mutants of
P. fluorescens after the transfer from aerobic to
anaerobic
conditions with nitrite as the sole electron acceptor
suggests
that there is a genetic and/or functional relationship between
the dissimilatory reduction of nitrate and that of nitrite. Since
nitrite respiration was not inhibited but only delayed in the
Nar
mutants, the reasons for this phenotypic change are
likely to
be complex and may include the following. (i) Some
specific gene(s)
may exist in the
nar operon of
P. fluorescens, whose product(s)
is involved in
the regulation of
nir gene expression. Therefore,
either the
synthesis of nitrite reductase and/or a specific nitrite
transport
system could be delayed or could proceed at a slower
rate in
Nar
mutants. In the latter case, growth on nitrite should
occur only
when a sufficient cellular concentration of nitrite
reductase
and/or nitrite carrier is reached within the cells. (ii) The
genes
coding for nitrate and nitrite reduction in
P. fluorescens YT101
may be localized on the same cluster, and
disruption of a single
transcriptional unit could affect all the
regulatory circuits
of this denitrification gene cluster. In support of
this hypothesis,
Ye et al. (
22) have recently identified a
DNA region involved
in reduction of nitrate, nitrite, and nitric oxide
by the denitrifying
bacterium
Pseudomonas sp. strain G-179.
(iii) Besides this putative
transcriptional control of
nir
genes by the products of the
nar operon, some enzyme
regulation at the activity level may also
exist. Given that the
membrane-bound nitrate reductase faces the
cytoplasm whereas nitrite
reductase is periplasmic, a protein-protein
interaction seems unlikely.
(iv) Finally, tolerance to nitrite
may have been decreased in
Nar
mutants. It is therefore possible that following a
shift from
oxic to anoxic conditions, anaerobic growth with nitrite may
require
a physiological acclimation, a process in which a functional
nitrate
reductase could be involved. In mutants lacking nitrate
reductase,
this acclimation process would be hindered, resulting in a
longer
lag phase before growth resumption. This hypothesis is supported
by the fact that when wild-type and mutant cells were cultivated
twice
under anaerobiosis with nitrite, growth occurred at a rate
which was
half that of cells precultured under oxic conditions.
The data,
however, do not allow us to distinguish among the various
explanations.
Our results have highlighted intriguing questions, not the least being
the nature of the linkage between the respiratory nitrate
reductase and nitrite reduction. The Nar
mutants
constructed in this study may facilitate further investigation.
The
relationship between nitrate and nitrite reduction observed
in
P. fluorescens YT101 raises the question of whether it is similar
in
other denitrifying
bacteria.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire
d'Ecologie Microbienne du Sol, UMR C.N.R.S. 5557, Université
Claude Bernard, Lyon 1, 43 bd. du 11 Novembre 1918, 69622 Villeurbanne
Cedex, France. Phone: 33 4 72 43 13 78. Fax: 33 4 72 43 12 23. E-mail: potier{at}biomserv.univ-lyon1.fr.
| |
REFERENCES |
| 1.
|
Arai, H.,
Y. Igarashi, and T. Kodama.
1994.
Structure and ANR-dependent transcription of the nir genes for denitrification from Pseudomonas aeruginosa.
Biosci. Biotechnol. Biochem.
58:1286-1291[Medline].
|
| 2.
|
Blattner, F. R.,
G. Plunkett III,
C. A. Bloch,
N. T. Perna,
V. Burland,
M. Riley,
J. Collado-Vides,
J. D. Glasner,
C. K. Rode,
G. F. Mayhew,
J. Gregor,
N. W. Davis,
H. A. Kirkpatrick,
M. A. Goeden,
D. J. Rose,
B. Mau, and Y. Shao.
1997.
The complete genome sequence of Escherichia coli K-12.
Science
277:1453-1474[Abstract/Free Full Text].
|
| 3.
|
Carter, J. P.,
Y. H. Hsiao,
S. Spiro, and D. J. Richardson.
1995.
Soil and sediment bacteria capable of aerobic nitrate respiration.
Appl. Environ. Microbiol.
61:2852-2858[Abstract].
|
| 4.
|
Choe, M., and W. S. Reznikoff.
1991.
Anaerobically expressed Escherichia coli genes identified by operon fusion techniques.
J. Bacteriol.
173:6139-6146[Abstract/Free Full Text].
|
| 5.
|
Choe, M., and W. S. Reznikoff.
1993.
Identification of the regulatory sequence of anaerobically expressed locus aeg-46,5.
J. Bacteriol.
175:1165-1172[Abstract/Free Full Text].
|
| 6.
|
deBoer, A. P. N.,
W. N. M. Reijnders,
J. G. Kuenen,
A. H. Stouthamer, and R. J. M. van Spanning.
1994.
Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans.
Antonie Leeuwenhoek
66:111-127[Medline].
|
| 7.
|
Grove, J.,
S. Tanapongpipat,
G. Thomas,
L. Griffiths,
H. Crooke, and J. Cole.
1996.
Escherichia coli K-12 genes essential for the synthesis of c-type cytochromes and a third nitrate reductase located in the periplasm.
Mol. Microbiol.
19:467-481[Medline].
|
| 8.
|
Jones, R. W., and P. B. Garland.
1997.
Sites and specificity for the reduction of bipyridilium compounds with anaerobic respiratory systems of Escherichia coli. Effects of permeability barriers imposed by the cytoplasmic membrane.
Biochem. J.
190:79-94.
|
| 9.
|
Jüngst, A.,
S. Wakabayashi,
H. Matsubara, and W. G. Zumft.
1991.
The nirSTBM region coding for cytochrome cdl-dependent nitrite respiration of Pseudomonas stutzeri consist of a cluster of mono-, di- and tetraheme proteins.
FEBS Lett.
279:205-209[Medline].
|
| 10.
|
Jüngst, A., and W. G. Zumft.
1992.
Interdependence of respiratory NO reduction and nitrite reduction revealed by mutagenesis of nirQ, a novel gene in the denitrification gene cluster of Pseudomonas stutzeri.
FEBS Lett.
314:308-314[Medline].
|
| 11.
|
Philippot, L.,
A. Clays-Josserand,
R. Lensi,
I. Trinsoutrot,
P. Normand, and P. Potier.
1997.
Purification of the dissimilative nitrate reductase of Pseudomonas fluorescens and cloning and sequencing of its corresponding genes.
Biochim. Biophys. Acta
1350:272-276[Medline].
|
| 12.
|
Prentki, P., and H. M. Krish.
1984.
In vitro insertional mutagenesis with a selectable DNA fragment.
Gene
29:303-313[Medline].
|
| 13.
|
Reyes, F.,
M. D. Rodan,
W. Klipp,
F. Castillo, and C. Moreno-Vivian.
1996.
Isolation of periplasmic nitrate reductase genes from Rhodobacter sphaeroides DSM 158: structural and functional differences among prokaryotic nitrate reductases.
Mol. Microbiol.
19:1307-1318[Medline].
|
| 14.
|
Reyes, F.,
M. Gavira,
F. Castillo, and C. Moreno-Vivian.
1998.
Periplasmic nitrate-reducing system of the phototrophic bacterium Rhodobacter sphaeroides DSM 158: transcriptional and mutational analysis of the napKEFDABC gene cluster.
Biochem. J.
331:897-904.
|
| 15.
| Richterich, P., N. Lakey, G. Gryan, L. Jaehn, L. Mintz,
K. Robinson, and G. M. Church. 1994. Automated multiplex
sequencing of the E. coli genome. Unpublished DNA sequence.
GenBank accession no. U00008.
|
| 16.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 17.
|
Sears, H. J.,
S. J. Ferguson,
D. J. Richardson, and S. Spiro.
1993.
The identification of a periplasmic nitrate reductase in Paracoccus denitrificans.
FEMS Microbiol. Lett.
113:107-112.
|
| 18.
|
Siddiqui, R. A.,
U. Warnecke-Eberz,
A. Hengsberger,
B. Schneider,
S. Kostka, and B. Friedrich.
1993.
Structure and function of a periplasmic nitrate reductase in Alcaligenes eutrophus H16.
J. Bacteriol.
175:5867-5876[Abstract/Free Full Text].
|
| 19.
|
Staskawicz, B.,
D. Dahlbeck,
N. Keen, and C. Napoli.
1987.
Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea.
J. Bacteriol.
169:5789-5794[Abstract/Free Full Text].
|
| 20.
|
Stouthamer, A. H.,
A. P. N. de Boer,
J. van der Oost, and R. J. M. van Spanning.
1997.
Emerging principles of inorganic nitrogen metabolism in Paracoccus denitrificans and related bacteria.
Antonie Leeuwenhoek
71:33-41.
|
| 21.
|
Ye, R. W.,
A. Arunakumari,
B. A. Averill, and J. M. Tiedje.
1992.
Mutants of Pseudomonas fluorescens deficient in dissimilatory nitrite reductase are also altered in nitric oxide reduction.
J. Bacteriol.
174:2560-2564[Abstract/Free Full Text].
|
| 22.
| Ye, R. W., L. Bedzyk, and T. Wang. 1998. Identification and characterization of a DNA region involved in
reduction of nitrate, nitrite, and nitric oxide by the denitrifying
bacteria Pseudomonas sp. G-179. Unpublished DNA sequence.
GenBank accession no. AF083948.
|
Journal of Bacteriology, August 1999, p. 5099-5102, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Palmer, K. L., Brown, S. A., Whiteley, M.
(2007). Membrane-Bound Nitrate Reductase Is Required for Anaerobic Growth in Cystic Fibrosis Sputum. J. Bacteriol.
189: 4449-4455
[Abstract]
[Full Text]
-
Baek, S.-H., Shapleigh, J. P.
(2005). Expression of Nitrite and Nitric Oxide Reductases in Free-Living and Plant-Associated Agrobacterium tumefaciens C58 Cells. Appl. Environ. Microbiol.
71: 4427-4436
[Abstract]
[Full Text]
-
Delorme, S., Philippot, L., Edel-Hermann, V., Deulvot, C., Mougel, C., Lemanceau, P.
(2003). Comparative Genetic Diversity of the narG, nosZ, and 16S rRNA Genes in Fluorescent Pseudomonads. Appl. Environ. Microbiol.
69: 1004-1012
[Abstract]
[Full Text]
-
Demanèche, S., Kay, E., Gourbière, F., Simonet, P.
(2001). Natural Transformation of Pseudomonas fluorescens and Agrobacterium tumefaciens in Soil. Appl. Environ. Microbiol.
67: 2617-2621
[Abstract]
[Full Text]
-
Ghiglione, J.-F., Gourbiere, F., Potier, P., Philippot, L., Lensi, R.
(2000). Role of Respiratory Nitrate Reductase in Ability of Pseudomonas fluorescens YT101 To Colonize the Rhizosphere of Maize. Appl. Environ. Microbiol.
66: 4012-4016
[Abstract]
[Full Text]
Top
Abstract
Text
