 |
INTRODUCTION |
Denitrification is a facultative
trait whose manifestation depends on environmental factors.
Physiological studies have shown that the expression of denitrification
genes usually occurs in the absence of oxygen, or at least at a low
oxygen tension (pO2), and requires the simultaneous
presence of an N-oxide (reviewed in reference
33). The expression of denitrification in the strict sense is sequential in Pseudomonas stutzeri with respect to
nitrate respiration, since activation of the narGHJI operon
occurs at a higher partial oxygen pressure than that at which the other reductase genes are activated (24, 25). By monitoring the production of narH and nirS transcripts of
Paracoccus denitrificans, it has been shown that induction
of nitrate reductase precedes that of nitrite reductase (8).
In Escherichia coli, the transcription factor FNR mediates
the oxygen response while NarL, as part of a NarXL two-component sensor-response regulator system, mediates the nitrate signal. Both FNR
and NarL are required for the expression of the narGHJI operon. The numerous studies directed at these transcription factors have provided a detailed mechanistic picture of anaerobic and nitrate-dependent gene activation in a nitrate-respiring bacterium (reviewed in references 19 and
20). Moreover, a crystal structure for the nitrate
response regulator NarL has been reported recently (7).
It is unknown which N-oxide-sensitive regulatory system
controls the genes encoding the reductases of denitrifying bacteria that act on the substrates nitrite, nitric oxide, and nitrous oxide and
to what extent their activation depends on coordinately and/or
sequentially acting regulators. Advances in mRNA methodology prompted
us to study gene expression in P. stutzeri by monitoring the
kinetics of individual transcripts rather than by using reporter gene
fusions. In the present study, we investigated the signal and regulator
requirements for the transcriptional activation of nirS,
norCB, and nosZ (i.e., the structural genes for
the three reductases involved in nitrite denitrification) following a
shifting of the respiratory metabolism from oxygen to nitrate or
nitrite. By studying narX and narL deletion
mutants, we obtained evidence for the existence of a second nitrate-
and nitrite-responsive regulatory system in P. stutzeri that
is specific for denitrification. A preliminary account of this work has
appeared previously (21).
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
Wild-type traits of
P. stutzeri ATCC 14405 are represented in this study by the
spontaneously streptomycin-resistant strain MK21 (36).
P. stutzeri was grown on synthetic asparagine-citrate medium
at 30°C (36). For aerated cultures, 500 ml of medium in a
1-liter flask was inoculated with an overnight culture to an optical
density at 660 nm of about 0.2 and incubated in a gyratory shaker at
240 rpm. Denitrification was induced by adding sodium nitrate (0.1%)
or sodium nitrite (0.05%) and simultaneously shifting to strongly
O2-limited conditions by reducing the shaker speed to 120 rpm. When necessary, kanamycin, ampicillin, or streptomycin was added
at a final concentration of 50, 100, or 200 µg ml
1,
respectively. Nitrite in the culture medium was measured as an azo dye
(32).
Isolation of RNA and Northern blot analysis.
Total RNA was
extracted from batch cultures by the hot-phenol method, with 10- or
20-ml samples being taken at appropriate time intervals during the
transition from aerobic to denitrifying growth conditions
(1). Twenty micrograms of RNA from each sample was denatured
by glyoxal-dimethyl sulfoxide treatment and separated on a 1.2%
agarose gel (26). Equal loading of samples onto the gel was
verified by acridine orange staining of the rRNA. Transfer of RNA to a
positively charged nylon membrane (Boehringer Mannheim) was achieved by
upward capillary action. DNA probes were labeled with dUTP-digoxigenin
by random priming.
The nirS probe was derived by KpnI digestion of
plasmid pNIR44, a 1.9-kb PstI-BglII fragment of
plasmid pNOR161 served as the norCB probe, and the
nosZ probe was obtained as a 500-bp PstI fragment
from plasmid NS220. Details of these probes are described elsewhere
(31). DNA labeling and detection kits were from Boehringer Mannheim; they were used in accordance with the specifications of the
manufacturer. The dioxetane reagent CDP-Star [disodium 2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)-1-phenyl
phosphate] was used as the chemiluminescent substrate for
membrane-based detection of alkaline phosphatase conjugates. Biomax MR
(Kodak) exposures were quantitated by scanning laser densitometry with
an ImageMaster scanner and software (Pharmacia).
Primer extension analysis.
The 5' end of the nirS
transcript was mapped by primer extension (6). Reverse
transcription was initiated from the 
-32P 5'-labeled
primer 5'-ATCAGGCCAGCCAGGATAGG-3', which was complementary to the coding strand at positions 267 to 286 of the published nirS sequence (23). The nucleotide sequence was
obtained by the dideoxy chain termination method, using a
Thermo-Sequenase kit (United States Biochemical Corp.),
[35S]dATP (Amersham), and the same primer. The primer
extension and sequencing reaction products were analyzed on a 6%
denaturing polyacrylamide gel.
Construction of narL and narX deletion
strains.
The narXL gene region was obtained from cosmid
clones of Sau3A-digested genomic DNA of P. stutzeri by using the vectors pJA1 (12) and SuperCos1
(Stratagene). An 892-bp fragment of cosmid c164 with the complete
narL gene (22) was amplified with the primers
5'-GCAAAGCTTCGGCCTGAAGAACAGCG-3' and
5'-TCGAATTCTTGAGCGATTGCGCACAG-3'. The primers
added HindIII and EcoRI restriction sites
(nucleotides in boldface) to allow cloning into pUC18. The pUC
derivative with the narL gene was designated pUCnarL. The
kanamycin resistance (Kmr) cassette was excised from
plasmid pBSL15 (2) and used to replace a 358-bp
AvaI-HincII fragment in narL,
resulting in vector pUCnarL::Kmr. The
narX-carrying plasmid pBSXL was constructed by inserting a
2.4-kb AvaI fragment of the narX cosmid g279 into
pBluescriptII SK(
). The narX locus was mutated by
replacing a 520-bp internal Eco47III-HindIII
fragment with the Kmr cassette from pBSL15 to give plasmid
pBSXL::Kmr. Plasmids
pUCnarL::Kmr and pBSXL::Kmr
were transferred to MK21 by electroporation (Gene Pulser; Bio-Rad). The
narL mutant MRL118 and the narX mutant MRX119,
resulting from double-crossover events, were obtained by selection for
kanamycin resistance and ampicillin sensitivity. Mutational
inactivation of the genes was verified by sequencing and Southern
hybridization. MRL118 was negative with the
AvaI-HincII narL fragment as a probe and gave a single hybridizing 2.8-kb fragment with the Kmr
probe in a genomic PstI digest. The mutated 3.1-kb
narX fragment (wild-type size, 2.4 kb) was detected in a
genomic AvaI digest of DNA from MRX119 by hybridization,
using a 153-bp PCR fragment generated from the 3' end of the gene as
the probe (primer pair 5'-CAAGCATGCGGAAGCGAACC-3' and
5'-GGCGCGTTCTTGCAGG-3').
| |
RESULTS |
nirS is part of an operon.
We identified the
nirS transcript in the total-RNA fraction of cells grown
under denitrifying conditions to monitor the expression pattern of
nitrite reductase at the mRNA level. A 0.5-kb KpnI fragment
from the central region of nirS was used as hybridization probe (Fig. 1). In Northern blot
analyses, two transcripts, of 2 and 3.4 kb, were found which were
absent from aerobic cells. We interpret them to be the monocistronic
transcript of the nirS gene and the polycistronic message
from the nirSTB operon. Transcription upstream of
nirS, beginning with nirQ, proceeds in the
opposite direction (Fig. 1); thus, the large transcript cannot
originate from upstream genes. The monocistronic transcript may result
from early termination of transcription or from posttranscriptional processing of the polycistronic message. Several inverted repeats, possible stem-loop-forming mRNA structures, at the 3' ends of nirS and nirB (23) are candidates for
affecting transcription and/or processing. The resulting increase of
the nirS transcript relative to that of nirTB may
be necessary to control the amounts of NirS, NirT, and NirB proteins so
that they are in the appropriate balance for the denitrification
process. The genes nirT and nirB encode tetraheme
and diheme c-type cytochromes, respectively. Mutagenesis of
nirT demonstrated the requirement of the encoded heme
protein for an in vivo-functional nitrite-reducing system (23). NirT is similar to the NapC proteins encoding the
putative electron-transferring c-type cytochromes of
periplasmic nitrate reductases (33). Homologs of NirT have
also been found in nondenitrifying bacteria and ascribe a broader
significance to this protein (11, 13, 18, 27). Transcription
of nirB together with nirS indirectly provides
support for a role for the encoded diheme protein in nitrite reduction.
A proteolytically shortened NirB was shown to have peroxidase activity
(17).
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FIG. 1.
nirS is transcribed as an operon with two
downstream nir genes. Shown at the top are the physical map
and organization of the nir operon of P. stutzeri. The horizontal arrows indicate the extents of the two
nirS transcripts. The black bar shows the position of the
0.5-kb KpnI fragment used as the nirS probe in
Northern blotting. Vertical arrows point to the location of FNR boxes.
Restriction site abbreviation are as follows: B, BamHI; H,
HindIII; Hp, HpaI; and K, KpnI. At
the bottom of the figure is a Northern blot of 20-µg quantities of
total RNA from cells aerobically cultivated without nitrate addition
(lane 1) and from nitrate-denitrifying cells (lane 2). Hybridization
was done with the digoxigenin-labeled KpnI fragment. The
2.0-kb monocistronic nirS transcript and the 3.4-kb
transcript of the nirSTB operon are marked by arrows; the
16S and 23S rRNA species served as standards.
|
|
Mapping the nirS promoter.
The promoter of
nirS was mapped by primer extension analysis (Fig.
2). The sequence TAGCAT at position
10 shows similarity to sigma factor 
70-dependent
promoters. A potential FNR-binding site,
TTGAT-N4-GTCAA, that is almost identical to the
FNR-binding consensus sequence of E. coli is positioned at
43.5. FNR of E. coli binds to the partially palindromic
nucleotide sequence TTGAT-N4-ATCAA, known as the FNR box,
of FNR-activated promoters that is centered preferentially 41.5 bp
from the start site of transcription (20). Expression of
nirS depends on DnrD (formerly termed FnrD), which is a
member of the greater FNR-CRP family of regulators (30). We
presume that the FNR box of the nirS promoter is the target
of DnrD-dependent regulation. The second FNR box, located further
upstream, is considered to be required for the expression of
nirQ. In Pseudomonas aeruginosa, this gene was
shown to be expressed anaerobically and to possibly be regulated from
the single FNR box shared by the nirS and nirQ genes (4).
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FIG. 2.
The nirS promoter. (A) Primer extension analysis. Total
RNA was extracted from cells cultured under nitrate-denitrifying
conditions. The primer extension products from 10, 30, and 50 µg of
RNA (lanes 1 to 3, respectively) were separated on a sequencing gel
together with a sequence ladder generated with the primer shown in
panel B. The complementary sequence of the transcription initiation
site is represented; the 5' end of the nirS transcript is
indicated by the arrow. (B) The nirS promoter. The 5' end of
the transcript is labeled +1. Putative polymerase (10) and FNR
(43.5) binding sites are boxed. The oligonucleotide that was used for
primer extension is underlined. The first few N-terminal amino acids of
the NirS protein are shown in one-letter code. RBS, ribosome-binding
site.
|
|
Stability of transcripts.
The balance between mRNA synthesis
and decay has a profound effect on prokaryotic and eukaryotic gene
expression (10). A fast rate of turnover of mRNA is crucial
for a rapid response with a pattern of gene expression altered by
changing external growth conditions. The half-life of the
nirS transcript was determined with MK21 which had been
induced for denitrification over a 30-min period. Rifampin (200 µg
ml1 final concentration) was then added to prevent de
novo mRNA synthesis, and the amount of nirS transcript
remaining was determined by Northern blot analysis. We determined a
half-life of approximately 13 min for both the monocistronic and
polycistronic nirS messages (Fig.
3). Also, the transcripts of
norCB and nosZ were monitored under the same
conditions. The genes norCB, encoding the NO reductase complex, form an operon with a single 2-kb transcript (35). nosZ, the N2O reductase structural gene, is
transcribed from six promoters, but in each case as a monocistronic
unit (14). The norCB and nosZ mRNAs
each exhibited the same half-life as the nirS transcripts in
the denitrifying cell (data not shown). Hence, a differential stability
of transcripts is apparently not an element in the regulation of the
structural genes encoding the oxidoreductases of denitrification.
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FIG. 3.
Stability of nirS mRNA. A culture of P. stutzeri was induced for denitrification under
O2-limited conditions (shaker speed, 120 rpm) with sodium
nitrate (1 g liter1). At 2, 10, 15, and 20 min after
inhibition of transcription by addition of rifampin (200 µg
µl1 final concentration), total RNA was isolated and
separated electrophoretically, and transcripts were detected by
Northern blotting. Least-squares analysis gave a half-life of 12.6 min
for the nirS mRNA.
|
|
The stability of mRNA can be affected by the growth rate
(28). A half-life of 13 min was determined for mRNA in
denitrifying cells with a doubling time of about 2.5 h. Overall,
this qualifies the transcripts of denitrification genes as being of
intermediate to high stability. Reported half-life values for bacterial
mRNAs range from 0.5 to 50 min, typically being between 2 and 4 min (9). An extremely long half-life of 5 to 7 h has been
reported for hoxS mRNA (encoding a soluble hydrogenase) of
Ralstonia (Alcaligenes) eutropha cells
exhibiting a doubling time of 20 h (29).
ompA mRNA, encoding a porin, is another example of a rather
stable mRNA, with a half-life of 13 to 25 min in cells doubling every 40 min. However, this mRNA species becomes short-lived in slow-growing cells (28).
Temporal changes of nirS mRNA in the transition to
denitrification.
The time course of expression of nirS
in response to nitrate and nitrite was monitored by mRNA analysis
during the shift from aerobiosis to oxygen-limited and denitrifying
conditions. We have previously shown that complete anaerobiosis is not
required for expression of the denitrification system of P. stutzeri (25). The denitrification reductases appear
when the level of saturation of the medium with air falls below 17%
and an N-oxide is present. In a well-aerated culture (shaker
speed, 240 rpm), no transcripts from the nirS operon were
detected (Fig. 4). When the oxygen
tension was subsequently lowered by reducing the shaker speed to 120 rpm and excluding the N-oxide, both nirS
transcripts appeared within 15 min. At about 30 min after the onset of
oxygen limitation, the nirS transcripts had nearly
disappeared again, and they were not detected at subsequent sampling
points extending to 3 h (Fig. 4A). This shows that although oxygen
limitation alone causes a transient induction of nirS in
P. stutzeri, it is not sufficient for long-term induction in
the absence of an N-oxide. It is important to note that on
addition of nitrate or nitrite to well-aerated cells, no activation of
nirS transcription took place (data not shown).
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FIG. 4.
Kinetics of nirS induction by nitrate or
nitrite under low-oxygen-tension conditions. MK21 was grown aerobically
(shaker speed, 240 rpm) and checked for the absence of nirS
mRNA (left lanes, panels A and B). (A) The culture was shifted first to
low-oxygen-tension conditions (shaker speed, 120 rpm; lanes labeled low
pO2 in panels A and B) and then induced for denitrification
by addition of sodium nitrate (1 g liter1). (B) As in
panel A, but induction was with sodium nitrite (0.5 g
liter1). At the indicated time intervals after shifting
to low-pO2 conditions and adding the respiratory substrate,
the appearance of nirS transcripts was monitored by Northern
blot analysis. (C) Increases in cell mass, as determined by measurement
of optical densities at 600 nm (closed symbols), and changes in nitrite
concentrations (open symbols) were monitored in both experiments; data
were plotted on the absolute time scale. Circles correspond to panel A;
triangles correspond to panel B. Vertical arrows point to the effected
changes in growth conditions.
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|
Addition of nitrate to oxygen-limited cells rapidly induced
nirS transcription and cytochrome cd1
synthesis (Fig. 4A). A short period of accumulation of nitrite in the
culture medium accompanied the transition phase (Fig. 4C); this can be
rationalized from the sequential induction of nitrate reduction and
nitrite reduction (8, 24, 25). The level of nirS
mRNA reached a maximum within 15 min and remained constant during the
3-h period of observation (Fig. 4A). Since the half-life of
nirS mRNA is about 13 min, persistence of those transcripts
for hours can occur only if the nirS operon is continuously
activated. We repeated the experiment using nitrite instead of nitrate
and found the same temporal response of the nirS transcripts
(Fig. 4B). Toward the end of this experiment, the amount of
nirS mRNA was decreased in the 3-h measurement, which
coincided with the disappearance of nitrite (Fig. 4C). In parallel
experiments lasting up to 5 h, we correlated the disappearance of
the nirS transcripts with exhaustion of nitrite in the
medium, i.e., lack of the inducer. The amount of nir message
decreased within the time frame of the mRNA half-life.
We have performed the oxygen respiration-to-denitrification shift
experiment in an identical manner with the probes for norCB and nosZ. The induction patterns observed for
norCB and nosZ were the same as those seen for
the nirS operon (data not shown). In both cases there was a
short period of gene activation in response to lowering of the
pO2, whereas continuing transcription of norCB and nosZ again required the presence of nitrate or nitrite.
Within the limits of the temporal resolution obtained with 15-min
intervals of the initial sampling points, coordinate expression of
nirS, norCB, and nosZ was observed.
This is in agreement with the results from a previous immunochemical
study (24).
Evidence for a regulatory system other than NarXL that mediates
nitrate and nitrite induction of denitrification.
We have recently
found that P. stutzeri possesses a nitrate response
regulator, NarL. This regulator acts in concert with the sensor NarX as
part of a two-component system that mediates nitrate and nitrite
response in the expression of respiratory nitrate reductase
(22). The narL and narX genes were
mutated to generate the deletion strains MRL118 and MRX119,
respectively, as described in Materials and Methods. Under aerobic
growth conditions, no nirS transcripts were detected, but
under conditions of oxygen limitation, transcription of nirS
was induced by nitrate or nitrite in both mutants (Fig.
5A and B). The nitrate-challenged
narL strain MRL118 induced nirS mRNA within 15 min, and this mRNA then remained present at approximately the same
level during the observation period of 4 h. The same result was
obtained when the mutant was challenged with nitrite. The activation
kinetics of nirS transcription in the narL mutant
in response to nitrate or nitrite were indistinguishable from that of
the wild type. Transcription of the nirS operon in the
narX mutant MRX119 in response to nitrate and nitrite
exhibited again the same kinetic pattern as the narL mutant
or the wild type. Figure 5C shows exemplarily the activation of
nirS transcription by nitrite in an experiment involving a
shift from aerobic respiration to denitrification. Also, the expression
of norCB and nosZ was not altered in the mutants
MRL118 and MRX119. Since neither narX nor narL
inactivation affected expression of the reductases involved in
denitrification in the strict sense, we postulate the existence of one
or more signal transduction pathways triggered by nitrate or nitrite,
independent of the two-component sensor regulator system NarXL.
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FIG. 5.
Nitrate- or nitrite-induced nirS expression
is independent of NarL or NarX. The nitrate response regulator mutant
MRL118( narL) (A and B) and the nitrate sensor mutant
MRX119(narX) (C) were grown aerobically (shaker speed,
240 rpm), checked for the absence of nirS transcripts by
Northern blotting (data points labeled O2), and then
induced for denitrification by a shift to low pO2 (shaker
speed, 120 rpm) and the addition of nitrate (1 g of NaNO3
per liter) (A) or nitrite (0.5 g of NaNO2 per liter) (B and
C). The production of transcripts were monitored in each case for
4 h after the shift.
|
|
| |
DISCUSSION |
As shown in Fig. 4, oxygen withdrawal alone caused only a
transient effect on the nirS operon, whereas its continuing
expression and likewise that of the norCB and
nosZ genes, required activation by nitrate or nitrite.
Transcriptional activation of denitrification genes induced by
low-oxygen conditions cannot as yet be ascribed to a distinct regulator
in P. stutzeri. Although FnrA, an FNR-type regulator, of
this bacterium has been found earlier (15), it does not act
as a global regulator, which would induce the structural genes for
denitrification reductases in response to oxygen withdrawal, nor does
it act on DnrD, which is also necessary for the expression of
nirS and norCB (30). We presume that
the FNR box of the nirS promoter (Fig. 2) is the target of
DnrD. The dnrD gene itself is part of an operon that shows a
complex pattern of transcriptional response to oxygen and nitrate
(30). DnrD and its homologs from other bacteria belong into
a separate phylogenetic branch within the greater FNR family. This
group is unlikely to comprise oxygen-responsive elements, since all
lack the cysteine residues needed for complexing the 4Fe-4S clusters of
redox-active FNR proteins. In this respect, the situation in P. stutzeri is clearly different from that in P. aeruginosa, for which the FNR-type regulator ANR was suggested to
respond to oxygen and function in a hierarchical relationship with the
DNR regulator, targeting both nirS and norCB
(5).
The observations made in the studies with the narX and
narL mutants provide evidence for a pathway of nitrate and
nitrite regulation which we ascribe to a new regulatory circuit. Since inactivation of narX did not produce a phenotype with
respect to the induction of the denitrification genes proper, it is
clear that NarX functions independently from this system; i.e., the latter has a nitrate- and nitrite-sensory element distinct from NarX.
The genetic organization of narXL is suggestive of an operon structure (22), which means that the narX
mutation is polar on narL, but this will not affect the
conclusions noted above. Further work is needed to determine whether
the postulated new system belongs to the two-component paradigm, which
may comprise a system homologous to NarXL, such as NarQP of E. coli (16), but specific for denitrification, or whether
it comprises a novel type of nitrate- and nitrite-responsive system.
In an immunochemical study, we have previously found that the mutant
strain MK137, which lacks nitrate metabolism, requires nitrite for full
induction of cytochrome cd1. This observation suggests the presence of a nitrite-responsive element (34), which we can now attribute to the new regulatory system. The activator component of this system may directly recognize a promoter element of
nirS or act indirectly via a further transcriptional
regulator. Within this context, it is of interest that the
nirS promoter of P. aeruginosa was demonstrated
to be nitrite responsive by the use of lacZ fusions
(3). The regulator for this effect and the cognate promoter
element, however, were not identified. Experiments to isolate and
characterize the genes encoding the nitrate- and nitrite-responsive
components necessary for denitrification in P. stutzeri are
in progress.
We thank M. F. Alexeyev for kindly supplying plasmid pBSL15
and U. Schiek for providing a gene bank and the narL mutant.
The work was supported by the Deutsche Forschungsgemeinschaft and Fonds
der Chemischen Industrie.
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Abstract
Introduction
