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Applied and Environmental Microbiology, August 1998, p. 3092-3095, Vol. 64, No. 8
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Requirement of RpoN (Sigma Factor 
54) in
Denitrification by Pseudomonas stutzeri Is Indirect and
Restricted to the Reduction of Nitrite and Nitric Oxide
Elisabeth
Härtig and
Walter G.
Zumft*
Lehrstuhl für Mikrobiologie der
Universität Karlsruhe, D-76128 Karlsruhe, Germany
Received 15 January 1998/Accepted 24 May 1998
 |
ABSTRACT |
The rpoN region of Pseudomonas stutzeri was
cloned, and an rpoN null mutant was constructed. RpoN was
not essential for denitrification in this bacterium but affected the
expression levels and enzymatic activities of cytochrome
cd1 nitrite reductase and nitric oxide reductase, whereas those of respiratory nitrate reductase and nitrous
oxide reductase were comparable to wild-type levels. Since the
transcription of the structural genes nirS and
norCB, coding for nitrite reductase and the nitric oxide
reductase complex, respectively, proceeded unabated,
our data indicate a posttranslational process for the two key enzymes
of denitrification depending on RpoN.
| |
TEXT |
Denitrification is an alternative
way of energy conservation for many facultative anaerobic bacteria. The
process is thought to be organized in a tripartite modular way, in
which the respiratory systems utilizing nitrate, nitrite and
nitric oxide (NO), and nitrous oxide (N2O) have to be
induced by environmental signals to form a functional unit for the
complete denitrification pathway (for a review, see reference
24). To achieve this, the expression of the genes
encoding the four reductases and accessory proteins have to be
regulated in a concerted way, which may be controlled by a distinct
sigma factor. Sigma factors have been found as coordinating elements for the transcription of specific sets of genes in response to
environmental stimuli.
Sigma factor 
54 (encoded by the rpoN gene)
was originally described as a factor involved in the expression of
nitrogen-regulated genes; since then multiple and diverse physiological
functions have been found to depend on this factor (12). The
role of 54 in denitrification is still insufficiently
clarified. The factor is required in Ralstonia eutropha
(formerly Alcaligenes eutrophus), a denitrifying hydrogen
bacterium, for anaerobic growth on nitrate (18). However, it
is not clear whether critical genes for denitrification depend on RpoN
or whether the requirement is indirect in nature. In Pseudomonas
aeruginosa RpoN controls diverse sets of genes, such as those for
glutamine synthetase, urease, and flagellin, but an rpoN
mutant grows anaerobically on nitrate (20). In this case it
is not known whether the expression of genes for the entire denitrification pathway is independent of RpoN. A comparison of promoters of several denitrification genes of Pseudomonas
stutzeri did not provide sequence-specific clues with respect to a
dependence on RpoN (4). In Bradyrhizobium
japonicum the expression of genes for nitrate respiration again is
independent of rpoN (for a review, see reference
6), but as for P. aeruginosa the role of
54 in the proper denitrification system remains to be
investigated. An rpoN mutant of the diazotrophic denitrifier
Azospirillum brasilense is defective in nitrate
assimilation, yet a possible effect on denitrification has not been
explored (17).
Here we describe the isolation of the rpoN gene region from
P. stutzeri. To show which step of denitrification is
regulated by RpoN we constructed an rpoN mutation by gene
replacement and analyzed the mutant for the expression of the four
terminal reductases of denitrification at the transcriptional and
translational levels.
Isolation and cloning of rpoN.
The experimental strain
was P. stutzeri MK21, a spontaneously streptomycin-resistant
derivative of strain ATCC 14405. The presence of an rpoN
gene was demonstrated by Southern hybridization of genomic DNA
with a probe from rpoN of Pseudomonas putida. A genomic cosmid library of MK21 was screened with a 1.4-kb
SacI-HindIII fragment derived from the
rpoN-carrying plasmid pNTR1 (10). Cosmid DNA of
264 clones was isolated (5) and digested with SmaI. The DNA was separated on 0.75% agarose gels and
blotted onto nitrocellulose membranes. Hybridization was performed at 65°C as described elsewhere (19). The rpoN gene
of P. stutzeri was found on a 3.4-kb HindIII
fragment on cosmid c167. It was cloned as two
EcoRI-HindIII fragments of 1.6 and 1.8 kb
into the vector pUC18. The 1.8-kb clone, pRpoN1.8, carried the complete rpoN gene. Occasionally a bacterium harbors two gene copies
of rpoN (11). Since hybridization of
genomic DNA with the homologous 1-kb XhoI probe (see
below) at low stringency (45°C) gave a single signal, we assume that
P. stutzeri possesses only one copy of rpoN.
Sequence analysis of rpoN and its flanking
regions.
The nucleotide sequence of rpoN was determined
by sequencing plasmid pRpoN1.8, and the rpoN-flanking
regions were obtained by direct sequencing of cosmid c167. For this
purpose the dideoxy chain termination method with universal and
sequence-specific primers was used together with a Thermo-Sequenase kit
(United States Biochemical Corp.) and [35S]dATP
(Amersham). The sequence revealed four open reading frames (ORFs),
whose derived products were similar to those derived from the
rpoN region of other denitrifiers (Fig.
1). The rpoN sequence extends
over 1,503 bp and encodes a protein of 502 amino acids with a
Mr of 56,843. The derived amino acid sequence
exhibits the three regions which have been determined to be typical of 54 factors (for a review, see reference
15). The N-terminal region has the domain of 50 amino acids, rich in glutamine and leucine, followed by 110 residues
with a prevalence of acidic amino acids. The carboxy-terminal region
exhibits the helix-turn-helix structure (amino acid positions 387 to
412, P. stutzeri count) and the invariant sequence
ARRTVAKYR (positions 479 to 487), known as the RpoN box (21).
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FIG. 1.
Organizational conservation of rpoN regions
in denitrifying bacteria. ORFs are shown as arrow boxes to indicate the
direction of transcription. Numbers of amino acids of the derived gene
products are shown. Homologous components are indicated by identical
patterns: ATP-binding proteins of ABC transporters ( ), modulators of
54 function ( ), and PTS proteins, EIIA
( ). Information about the following organisms was taken from the
following references: P. aeruginosa (9), B. japonicum (11), R. eutropha (22),
A. brasilense (17). Incompletely sequenced ORFs
are shown as open boxes.
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|
ORF241, upstream of
rpoN, is transcribed in the same
direction and potentially encodes a 241-amino-acid protein with a
Mr of 26,420. Downstream of
rpoN,
ORF102 may encode a 102-amino-acid
polypeptide with a
Mr of 11,752. The next ORF was sequenced only
partially to cover 120 amino acids. The product of this ORF is
a
homolog of the protein E
IIA of the phosphotransferase
system.
Mutations in the corresponding ORFs 95 and 154 downstream of
rpoN of
Klebsiella pneumoniae increase
transcription from RpoN-dependent
promoters, suggesting that the gene
products may act as modulators
of
54 activity
(
16).
Primer extension analysis was used to locate the promoter of
rpoN. Total RNA was prepared from MK21 cells grown in
asparagine-citrate
(AC) medium under oxygen-limited conditions and
supplemented with
1 g of NaNO
3 per liter
(
3). The RNA was extracted by a method
described elsewhere
(
1), and primer extension was done by a
standard protocol
(
2). We found two transcript initiation sites
spaced by only
two nucleotides. Upstream of those sites the sequences
TATAAT
and TAGGCA are thought to be
10 and
35 binding
motifs,
respectively. The
10 sequence is identical to the
70 consensus sequence of
Escherichia coli
(
8), whereas the
35
sequence varies somewhat from the
TTGACA consensus sequence. The
presence of these motifs
suggests that
rpoN of
P. stutzeri is
under the
control of the principal sigma factor
70, encoded by
rpoD. Interestingly, the
rpoN promoter of
P. stutzeri is identical from positions +2 to
21 to the
promoter sequence
of
rpoN of
P. aeruginosa and
extends further to an identical
35
motif (Fig.
2). Overall the derived
P. stutzeri and
P. aeruginosa RpoN proteins are 84.5%
identical.
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FIG. 2.
Nucleotide sequence of the rpoN promoter. The
transcriptional start of rpoN was determined by primer
extension analysis; the oligonucleotide used for primer extension is
underlined. The 5' ends of transcripts are marked by arrows and +1.
Putative 35 and 10 motifs are boxed. RBS, ribosome binding site.
The promoter regions identical to that of P. aeruginosa are
printed in lowercase letters. The C-terminal and N-terminal
amino acid sequences of ORF241 and RpoN, respectively, are given below
the nucleotide sequence in one-letter code.
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Construction of an rpoN mutation by gene
replacement.
We constructed a null mutant to analyze the role of
RpoN in denitrification of P. stutzeri. An internal
1-kb XhoI fragment which covers 66% of the rpoN
gene was deleted from pRpoN1.8. The kanamycin resistance
(Kmr) cassette from plasmid pUC4-Kiss (Pharmacia) with the
aminoglycoside 3'-phosphotransferase gene of transposon
Tn903 was inserted in the opposite orientation into
rpoN, yielding plasmid pRpoN::Kmr. The
replacement construct was linearized at the EcoRI site and electroporated into competent P. stutzeri cells. For
electroporation the bacterium was grown in 3.2% Bacto Tryptone-2%
yeast extract-0.5% NaCl, pH 7.5, for 16 h at 30°C. The cells
were washed twice with 15% glycerol in 1 mM MOPS
(morpholinepropanesulfonic acid), pH 7.5. Conditions for
electroporation in a 0.2-cm cuvette were 2.5 kV, 25 µF, 200 
(Bio-Rad Gene Pulser). Disruption of the wild-type gene by homologous
recombination in mutant MK516 was selected for on the basis of
kanamycin resistance (final concentration, 200 µg ml
1)
and ampicillin sensitivity (100 µg ml1).
Genomic DNA from MK516 was cleaved with HindIII. The
digest was separated on a 0.8% agarose gel, transferred to a nylon
membrane, and hybridized with the 1-kb XhoI fragment of
rpoN and the 1.3-kb Kmr cassette to verify gene
inactivation. Because of the effected deletion the XhoI
probe gave no signal, whereas the Kmr probe yielded two
signals of 1.5 and 2.6 kb, due to the internal HindIII
site of the inserted cassette (data not shown).
Effect of the rpoN mutation on denitrification
enzymes.
A striking phenotype of the rpoN mutant MK516
was its loss of growth on AC minimal medium. Since the structural gene
for glutamine synthetase is under RpoN control in P. aeruginosa (20), we supplemented AC medium with
glutamine but were unsuccessful in restoring growth with or without
nitrate either under aerobic or anaerobic conditions. The mutant was
also unable to use histidine or proline as the sole nitrogen source.
The lack of growth of the rpoN mutant on minimal medium
indicates that the absence of 54 must affect amino acid
metabolism and other routes of intermediary metabolism of
P. stutzeri. MK516 grew in Luria-Bertani medium with nitrate, aerobically as well as anaerobically, but under the
latter conditions grew more slowly than the wild type, demonstrating that rpoN exerts a general effect on denitrification. MK516
also lost its motility on 0.3% swarm agar, which indicates that RpoN may be required for flagellin synthesis, as had been demonstrated for
P. aeruginosa (20).
To find out which step in the denitrification pathway was affected by
rpoN, we measured the in vitro activities of the reductases
for nitrate, nitrite, and NO of MK516 in comparison to those of
MK21,
representing wild-type traits (Table
1).
Denitrification
of MK516 was induced by a shift from aerobic to
O
2-limited growth
conditions in the presence of nitrate.
Luria-Bertani medium (500
ml in a 1-liter flask) was inoculated with an
aerobic overnight
culture to result in an optical density at 660 nm
(OD
660) of about
0.2. The culture was incubated for 5 h on a gyratory shaker at
240 rpm, 30°C, until the OD
660
was
0.4. Denitrification was induced
by adding NaNO
3
(final concentration, 1 mg ml
1) and reducing the shaking
speed to 120 rpm. Cells were harvested
after 16 h by
centrifugation and broken in a French press, and
cell extract was
obtained as the supernatant from centrifugation
(10 min, 20,000 ×
g). MK516 showed reduced activities of cytochrome
cd1 nitrite reductase and NO reductase, whereas
the activity of
nitrate reductase corresponded to that of the wild type
(Table
1). Also, N
2O reductase was not affected in MK516,
measured as
in vivo activity of whole cells by gas chromatography (data
not
shown). Next, we complemented the mutation in MK516 with the
rpoN gene to verify that the lower activity found with
nitrite and
NO reductases was a direct effect of
rpoN
deletion and not a polar
effect of genes located downstream of
rpoN. The 1.8-kb
EcoRI-
HindIII
fragment, containing the complete copy of
rpoN, was cloned
into
vector pUCP24, a broad-host-range vector, that is able to
replicate
in the genus
Pseudomonas (
23). The
construct was transferred
to MK516 by electroporation. The complemented
cell line MK516c
exhibited increased enzyme activities, coming close to
the wild-type
level and sometimes even surpassing it, indicating that
the diminished
reductase activities of MK516 were a direct result of
the
rpoN mutation (Table
1).
Whether the effect on nitrite and NO reductases was caused by a
decreased expression of the respective structural genes was
investigated by comparing the amount of transcripts of
nirS
(nitrite
reductase) and of
norCB (NO reductase complex) of
MK516 with that
of the wild type (Fig.
3). Both structural gene sets are
organized
in independent transcriptional units (
24).
nirS is transcribed
both from the
nirSTB operon
and as a monocistronic message and
thus yields two signals on Northern
blot analysis. Aerobically
cultivated cells exhibited no transcripts.
In cells grown under
denitrifying conditions the
nirS and
norCB transcripts were readily
detectable. The levels found
in denitrification-induced MK516
were not decreased versus that in
MK21. This rules out a direct
dependence of
nirS and
norCB transcription on
54. Finally, we
determined the level of denitrification enzymes,
since the lower
activities of nitrite and NO reductases could
be due to a
posttranscriptional mechanism that depends on
54 and
affects the concentration of enzymes. Cells were shifted
to
denitrification, and a cell extract was prepared as described
above.
The protein pattern was analyzed by sodium dodecyl
sulfate-polyacrylamide
gel electrophoresis, and the reductases were
detected with the
respective polyclonal antisera by immunoblotting. The
rpoN mutant
showed substantially lower levels of both
nitrite reductase and
NO reductase, whereas those of nitrate reductase
and N
2O reductase
corresponded to that of MK21 (Fig.
4). On complementation of MK516
by
rpoN, wild-type levels of all four reductases were detected.
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FIG. 3.
The rpoN null mutant is not affected in the
transcription of nirS and norCB. Total RNA from
MK516 and MK21 was prepared according to the method of Aiba et al.
(1) from cells grown aerobically (lane 1 of each panel) or
under denitrifying conditions (lane 2 of each panel). Induction of
denitrification was as described in the text for the activity
measurements. Samples (20 µg of RNA) were denatured by
glyoxal-dimethyl sulfoxide treatment and separated on a 1.2% agarose
gel (14). After transfer to a nylon membrane, the
nirS and the norCB transcripts were detected by
hybridization with digoxigenin-labeled probes (labeling kit from
Boehringer Mannheim, following the instructions of the manufacturer). A
500-bp KpnI fragment of the nirS gene and a 2-kb
PstI-BglII fragment of the norCB
operon were used as probes. nirS exhibited mono- and
polycistronic transcripts of 2 and 3.4 kb, respectively; the
norCB transcript was 2 kb. Equal gel loading was verified by
staining with acridine orange. The 16S and 23S rRNA species served as
standards.
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FIG. 4.
An rpoN mutation reduces the cellular level
of nitrite reductase (NirS) and NO reductase (assayed as the NorB
subunit). Cell extracts of strains MK21 (lanes 1), MK516 (lanes 2), and
the complemented mutant MK516c (lanes 3), all induced for
denitrification, were separated electrophoretically on a sodium dodecyl
sulfate-12.5% polyacrylamide gel and blotted onto nitrocellulose, and
the reductases were detected immunochemically. NarGH, nitrate
reductase; NosZ, N2O reductase. Size markers (in
kilodaltons) are indicated to the left of each panel.
|
|
In conclusion,
54 is not involved in the expression of
the denitrification system of
P. stutzeri by acting as a
transcription
factor for one or several reductase genes, yet it
affected the
cellular concentrations of nitrite reductase and NO
reductase.
Since the transcription of both
nirS and
norCB proceeded unabated
in the
rpoN strain, the
enzyme levels seem to be affected by a
posttranslational mechanism
involving one or several products
of
54-dependent gene
expression, hence leading to diminished enzyme
concentrations and
concomitantly decreased denitrification rates.
The effect is restricted
to the two key enzymes of denitrification,
nitrite reductase and NO
reductase, and does not affect nitrate
and nitrous oxide respiration.
Our findings underline once more
that the respiratory systems utilizing
nitrite and NO are interlaced
at several levels of regulation and
further support the modular
view of the denitrification process that we
have detailed elsewhere
(
24).
Nucleotide sequence accession number.
The rpoN
sequence reported here is available under the accession number
AJ223088 in the EMBL/GenBank/DDBJ data banks.
| |
ACKNOWLEDGMENTS |
We thank S. Harayama for a gift of rpoN from P. putida, H. Cuypers for his participation in the early stage of
this project, and B. Schreckenberger for excellent technical
assistance.
The work was supported by the Deutsche Forschungsgemeinschaft and Fonds
der Chemischen Industrie.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Lehrstuhl
für Mikrobiologie, Universität Karlsruhe, P.O. Box 6980, D-76128 Karlsruhe, Germany. Phone: 49 (721) 608 3474. Fax: 49 (721) 608 4920. E-mail: dj03{at}rz.uni-karlsruhe.de.
| |
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Applied and Environmental Microbiology, August 1998, p. 3092-3095, Vol. 64, No. 8
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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