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Journal of Bacteriology, June 1999, p. 3658-3665, Vol. 181, No. 12
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Nitrate and Nitrite Control of Respiratory Nitrate
Reduction in Denitrifying Pseudomonas stutzeri by a
Two-Component Regulatory System Homologous to NarXL of
Escherichia coli
Elisabeth
Härtig,
Ulrike
Schiek,
Kai-Uwe
Vollack, and
Walter G.
Zumft*
Lehrstuhl für Mikrobiologie der
Universität zu Karlsruhe, Karlsruhe, Germany
Received 10 February 1999/Accepted 6 April 1999
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ABSTRACT |
Bacterial denitrification is expressed in response to the
concurrent exogenous signals of low-oxygen tension and nitrate or one
of its reduction products. The mechanism by which nitrate-dependent gene activation is effected was investigated in the denitrifying bacterium Pseudomonas stutzeri ATCC 14405. We have
identified and isolated from this organism the chromosomal region
encoding the two-component sensor-regulator pair NarXL and found that
it is linked with the narG operon for respiratory nitrate
reductase. The same region encodes two putative nitrate or nitrite
translocases, NarK and NarC (the latter shows the highest similarity to
yeast [Pichia] and plant [Nicotiana]
nitrate transporters), and the nitrate-regulated transcription factor,
DnrE, of the FNR family. The roles of NarX and NarL in nitrate
respiration were studied with deletion mutants. NarL activated the
transcription of narG, narK, and
dnrE but did not affect the denitrification regulons for
the respiratory substrates nitrite, nitric oxide, and nitrous oxide.
The promoters of narG, narK, and
dnrE carry sequence motifs, TACYYMT, which correspond to
the NarL recognition sequence established for Escherichia
coli. The cellular response toward nitrate and nitrite was
mediated by the sensor protein NarX, which discriminated weakly between
these oxyanions. Our data show that the NarXL two-component regulatory
system has been incorporated into the bacterial denitrification process
of P. stutzeri for selective regulation of nitrate respiration.
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INTRODUCTION |
Denitrification by prokaryotes is
part of the global nitrogen cycle, where it is responsible for the
balance of the nitrogen budget of the biosphere. In a pathway of four
reaction steps, nitrate is successively reduced via nitrite, nitric
oxide (NO), and nitrous oxide (N2O) to dinitrogen.
Denitrification genes are usually expressed in response to nitrate or
nitrite and a low oxygen level (although aerobic denitrification exists
in specialized cases) (for a review, see reference
45). This requires the activation of sensory devices
and signal transduction pathways by these respiratory substrates or
their reduction products. We were interested in the mechanisms by which
a denitrifying bacterium that is deprived of oxygen and shifted to N
oxide utilization senses nitrate or nitrite and activates genes for
anaerobic respiration.
In Escherichia coli the transcription factor NarL is an
important regulator in cellular bioenergetics. NarL activates the operon for respiratory nitrate reductase, narGHJI, and other
operons of ancillary systems required for nitrate respiration. At the same time, the factor acts as a repressor of operons for alternative modes of respiration. NarL is part of a two-component regulatory system, NarXL (reviewed in reference 13). The
sensor-regulator pair is duplicated in NarQP, which exhibits a
specificity toward target genes somewhat different from that of NarXL.
Putative NarX and NarL homologs, requiring functional analysis, have
surfaced as the result of projects to sequence the genomes of
Haemophilus influenzae, Neisseria gonorrhoeae,
Yersinia pestis, Bacillus subtilis, and
Pseudomonas aeruginosa. Here we identify by a targeted
approach the narXL genes of the denitrifying bacterium
Pseudomonas stutzeri and study their phenotypic
manifestations in deletion strains. The function of NarXL is to
activate the operon encoding the initiator reaction for
denitrification, i.e., respiratory nitrate reduction, but not to act as
a global regulatory system for the overall denitrification process.
(Preliminary accounts of this work have been presented previously
[21, 45].)
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
Wild-type P. stutzeri (ATCC 14405), the mutant strain MK21, which is a
spontaneously Smr mutant but otherwise represents wild-type
traits, and the MK21 derivatives MRL118 (
narL
Kmr Smr) and MRX119 (narX
Kmr Smr) were cultured at 30°C in synthetic
medium with asparagine and citrate as major ingredients
(10). The construction of the mutant strains MRL118 and
MRX119 has been described previously (22). E. coli DH10B and XL1-Blue MR were grown at 37°C in Luria-Bertani medium. Where necessary, kanamycin, ampicillin, or streptomycin was
added at a final concentration of 50, 100, or 200 µg
ml
1, respectively. Cultures from which total RNA was
prepared were grown in a 1-liter flask equipped with baffles and filled
with 500 ml of medium. The optical density at 660 nm upon inoculation was about 0.3. The shaker speed of the gyratory incubator used was set
at 240 rpm. Initial air saturation was estimated to be about 95% with
a Clark-type electrode. Samples of oxygen-respiring cells were drawn
after 3 h. For a shift to denitrifying conditions, cells were
induced by nitrate (1 g/liter) for 1 h under O2
limitation by decreasing the shaker speed to 120 rpm, which lowered air
saturation to about 0.5%. Full anaerobiosis is not required for the
expression of the denitrification system of P. stutzeri
provided that nitrate or nitrite is present (26). Samples
for RNA extraction were drawn from cell suspensions that had reached an
optical density of about 0.6 at 660 nm.
Purification of nitrate reductase and immunoblotting.
Nitrate reductase from P. stutzeri was solubilized by heat
and purified as described previously (6). The subunits were
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), and the large subunit, NarG, was blotted onto a polyvinyl
difluoride membrane. An N-terminal sequence, NRKQGEFADGHGETR, was
obtained at the Protein Sequencing Facility, University of Konstanz.
For immunoblot analysis of enzymes, aerobically grown cells were
transferred to fresh medium and induced for 8 h under
O
2 limitation with sodium nitrate (1 g/liter) or sodium
nitrite (0.5
g/liter). Cells were harvested by centrifugation and
washed twice
with 25 mM Tris-HCl (pH 7.5)-10 mM MgCl
2.
They were suspended
in the same buffer without MgCl
2 and
broken in the cold by sonication
(Branson). The supernatant from
centrifugation for 20 min at 39,000
×
g was used as a
cell extract for SDS-PAGE (
27). Proteins were
transferred to
a nitrocellulose membrane by semidry electrotransfer.
For Western
blotting (
38), polyclonal antisera were raised against
the
purified oxidoreductases. Quantitation was done by scanning
laser
densitometry with an ImageMaster scanner and software (Amersham
Pharmacia Biotech). Protein concentration was determined by the
Lowry
procedure with bovine serum albumin as the
standard.
Cloning of the narXL region.
A narL
fragment was amplified from genomic DNA of strain MK21 with the primer
pair 5'-CAAAGCTTSGACGACCACCCSMT-3' and 5'-TCGAATTCARGTARCCGTCSGCRC-3', designed from
conserved NarL and NarP sequences and observing the codon preference of P. stutzeri genes. The restriction sites
HindIII and EcoRI (boldfaced nucleotides in
primer sequences) were added to allow the subsequent cloning of the PCR
product. The PCR was carried out at an annealing temperature of 45°C.
Amplification products were separated by electrophoresis, blotted onto
a nylon membrane, and hybridized (16) with the
narP probe of E. coli. A 285-bp fragment was
isolated, cloned into pBluescript II SK(+) to give plasmid pBSnarL, and verified by sequencing as being homologous to narL from
E. coli.
A genomic cosmid library of wild-type
P. stutzeri was
constructed with the SuperCos1 vector and
E. coli XL1-Blue
MR as the
host (Stratagene). DNA was purified by a CsCl gradient and
partially
digested with
Sau3A under conditions that yielded
fragments of
30 to 50 kb. These fragments were cloned into the
BamHI site of
the vector by following the protocol of the
supplier. Packaging
was performed by using the GigapackIII XL packaging
extract (Stratagene).
For screening of the library by colony
hybridization, we used
an internal
narL probe of 219 nucleotides, which was amplified
from plasmid pBSnarL with the primers
5'-GCGTGACCTGCTGGATCTG-3'
and
5'-GCACATGGCTCTGCTCGTC-3'. For digoxigenin (DIG) labeling
of
probes, the PCR mixture was made up to contain 7 µM DIG-11-dUTP.
Cloning of a narG fragment, gene probes, and nucleic
acid manipulations.
We translated the amino acid sequence
GEFADGH, obtained from N-terminal sequencing of the NarG
subunit, into the degenerate forward primer
5'-GGYGARTTCGCSGACGGYCAC-3'. The reverse primer 5'-TSGCCGGGATCGGSGAGAAGCC-3' was designed from the conserved
sequence GFSPIPAM, encoded at the 5' ends of the narG genes
of E. coli (positions 188 to 195) (5) and
B. subtilis (positions 192 to 199) (24). A PCR
fragment of 530 bp was amplified from genomic DNA of P. stutzeri MK21 at an annealing temperature of 55°C. The putative
narG fragment was identified by Southern hybridization (16) with the E. coli narG gene. The ends were
filled in with Klenow polymerase, and the fragment was cloned by
blunt-end ligation into the EcoRV-cleaved plasmid
pBluescript II SK(+). The insert in the resulting plasmid, pBSnarG, was
sequenced with M13 universal and reverse primers to ensure its
identity. An internal 356-bp PCR probe for Southern and Northern
hybridizations was prepared from pBSnarG with the primer pair
5'-ATCCGCTCGCGCTGGCAGTA-3' and 5'-CCATGCCGCGCTTGCTCTTG-3' and was labeled with digoxigenin.
The 881-bp
narG probe of
E. coli was excised with
PstI from plasmid pSL962 (
33). A 410-bp fragment
of
narP was prepared
by PCR with plasmid pVJS334 as the
template (
31) and the primers
5'-TCCTGGCTCTGAAGTGGTCG-3' and 5'-CAAGCTGCAGAACATCC-3'.
The digoxigenin-labeled
probes for
nirS and
norB were amplified from cosmid c146 (
41)
with
primer pairs located at positions 456 to 473 (5'-ACCGAAGCGGATGCAAG-3')
and 1127 to 1144 (5'-TGCGAAGCGACGATGGAC-3') of the published sequence
of
nirS (
25) and at positions 1192 to 1209 (5'-TCTGATCGGCCTGGCAGT-3')
and 1868 to 1885 (5'-ACCAGCAGCGGTGAGGAA-3') of the published sequence
of
norB (
46). The probe for the
nosZ gene
was a
PstI digest
from plasmid pNS220 (
42).
DNA sequencing of cosmid g279 and PCR fragments was carried out by the
dideoxy chain termination method with the Thermo Sequenase
cycle-sequencing kit (Amersham Pharmacia Biotech) and
35S-labeled dATP (ICN). Unless specified otherwise,
standard procedures
were followed for DNA manipulations
(
32).
RNA isolation, Northern blotting, and primer extension
analysis.
Total RNA was extracted from 12 ml of cell suspensions
of MK21, MRL118, and MRX119 by the hot phenol method (1).
Samples (10 µg) were separated electrophoretically in 1.2% agarose
gels containing 0.4 M formaldehyde (2). RNA was transferred
to a positively charged nylon membrane by downward capillary transfer (9). The dioxetane derivative CDP-Star was used as a
chemiluminescent substrate for membrane-based detection of alkaline
phosphatase conjugates.
In the primer extension analysis (
2), 50 µg of RNA was
used to map the 5' end of the
narG transcript. Reverse
transcription
was initiated from the
-
32P-end-labeled
primer, 5'-TCCTGTTGAAGAAGCGCAGTTGATCGAG-3', complementary
to
the 5' end of the
narG coding region. The sequencing
reaction
was performed with the same primer. The primer extension
products
and the sequencing reactions were analyzed on a 6% denaturing
polyacrylamide gel. For mapping the
narK transcript
initiation
sites, we used the primer
5'-AATCCAGAGGTTGCGATTGGCGATCC-3' and
the same conditions as
for
narG.
Nucleotide sequence accession number.
The narXL
sequence data have been deposited with the DDBJ/EMBL/GenBank databases
under accession no. AJ131854.
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RESULTS |
Isolation of the narXL region and linkage with the
narG operon.
We based our strategy for the isolation
of narXL from P. stutzeri on designing primers on
the basis of a comparison of the amino acid sequences of NarL and NarP
from E. coli (20, 31, 34) with those of the
hypothetical NarP protein from H. influenzae, encoded by the
open reading frame (ORF) HI0726 (19). In addition to the
domains for DNA binding and phosphorylation, NarL and NarP proteins
have the conserved sequences I(V)DDHPL(M) and GADGYL. These regions
were translated into a degenerate primer pair to be used to amplify a
narL fragment from genomic DNA of P. stutzeri (see Materials and Methods). A 285-bp fragment was detected with the
narP probe of E. coli among the amplification
products. This PCR fragment was isolated and served as a template for
the preparation of a genuine narL probe of 219 nucleotides.
The probe was used to locate the narL gene in a genomic
library on cosmid g279. Genes encoding NarXL of E. coli are
clustered with the narG operon and narK, encoding
a putative nitrite transporter. To explore a possible linkage of
narL with narG in P. stutzeri, we
hybridized cosmid g279 with the narG probe; narG
was found on this cosmid also.
Having established a linkage of the two
nar functions, we
determined a double-stranded sequence of about 8 kb by using
sequence-derived
primers. The physical map of the
narXL
region is shown in Fig.
1. The
narL-encoding ORF spans 657 bp. It is followed by ORF235,
which has no noteworthy similarity with current sequence entries
in
data banks. The next ORF encodes the transcription factor DnrE,
which
belongs to the FNR family (
41). In the 5' direction
narL overlaps 71 bp with an ORF that encodes a homolog of
the nitrate
sensor protein NarX. Upstream of
narX two ORFs
encode the hypothetical
transporters, NarK and NarC, which show
similarity to NarK of
E. coli and fungal or plant nitrate
transporters, respectively.
Both belong to the family of major
facilitator permeases (
39).
The highest similarity of NarC
was found with putative nitrate
transporters of the yeast
Pichia
angusta (26% identity in a 454-amino-acid
overlap; accession no.
Q92240) and the higher plant
Nicotiana plumbaginifolia (29%
identity in a 525-amino-acid overlap; accession
no.
O04431). A
comparison with the products of two
narK genes
of
P. aeruginosa, NarK
1 and NarK
2 (accession no.
Y15252), shows
that NarK
2 is homologous to NarK of
P. stutzeri, whereas NarK
1 shows more similarity to the
deduced nitrate or nitrite transporters
NarK, NasA, and NarT of the
gram-positive bacteria
B. subtilis (
11,
29) and
Staphylococcus carnosus (
17). In principle,
the
presence of two transporters would satisfy the requirement
of movement
of nitrate from the periplasm to the cytoplasm and
the opposite
translocation of nitrite. This makes the existence
of NarC and NarK and
their roles in denitrification and perhaps
also nitrate assimilation an
intriguing prospect. The third product
of the
narK region,
encoded by ORF134, is weakly similar to the
so-called conserved protein
MTH153 of
Methanobacterium thermoautotrophicum (accession
no.
AE000803) and the hypothetical protein ORF138
of
Wolinella
succinogenes (accession no.
AJ000662), both of
unknown function.
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FIG. 1.
Organization of the narXL region of P. stutzeri and physical map of mutants. The map covers approximately
9.2 kb. narX overlaps narL by 71 bp. The maps for
the narX and narL mutants are shown with the
extent of deletions and orientation (arrowheads) of the kanamycin
cassette. PL, NarL-regulated promoters. ORFs 235 and 134 are labeled according to the number of amino acids of their
hypothetical gene products.
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Properties of the derived NarL and NarX proteins.
NarL of
P. stutzeri (NarLPs) consists of 218 amino
acids, Mr 24,378; the protein has 51 and 47%
positional identity with the E. coli proteins NarL and NarP,
respectively. Because of the slightly higher amino acid identity with
NarL of E. coli (NarLEc) and its function as
regulator of the narG operon, we termed the newly isolated
P. stutzeri gene narL. Figure
2 shows an alignment of NarLPs with homologous proteins. The crystal structure of
NarLEc became known recently (3, 4). The high
similarity of NarLPs with the E. coli protein
allows predictions of secondary structure as shown in Fig. 2.
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FIG. 2.
Structural features of NarL and NarX. Sequences
were aligned with the CLUSTAL W program (37). Identical
amino acids are marked by asterisks; similar amino acids are marked by
colons. (A) Alignment of NarL and NarP proteins, identified in the
bottom row. The structural predictions for NarLPs as
deduced from the E. coli protein (4) are shown
for the 10  -helices and 5  -strands. Helices 8 and 9 form the
DNA-binding region. Boldfaced letters, E. coli residues
important for phosphoryl transfer and the equivalent positions of
NarLPs (aspartic acid residues 13, 14, and 59 and lysine
109) and homologous proteins. (B) Alignment of NarX and NarQ proteins,
identified in the bottom row. The regions forming distinct structural
elements are boxed and are discussed in the text. The predicted
transmembrane helices for P. stutzeri and E. coli
are boldfaced and are labeled TM1 and TM2.
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The NarL
Ps residues Asp13, Asp14, Asp59, and Lys109
correspond to a set of conserved amino acids found in response
regulator
proteins. The aspartic acid residues form an acidic pocket
which
is part of the phosphoryl acceptor chemistry (
30,
35).
Mutation
of Asp59 of NarL
Ec, the site of protein
phosphorylation, has shown
that this residue is necessary for the
expression of formate dehydrogenase
(the
fdnG gene) or the
repression of fumarate reductase (the
frdA gene) in response
to nitrate (
15).
The derived NarX polypeptide of
P. stutzeri
(NarX
Ps) consists of 648 amino acids,
Mr 71,791. NarX
Ps has 31%
positional identity
each with NarX and NarQ of
E. coli.
Hydropathy analysis and transmembrane
prediction suggest two
membrane-spanning regions (TM1 and TM2
[Fig.
2]) that delimit an
internal periplasmic domain and a carboxy-terminal
cytoplasmic domain.
The latter exhibits the conserved regions,
termed H, N, and D from the
presence of key amino acids in these
regions, which are characteristic
for the histidine protein kinase
family (
36). The asparagine
and histidine residues, which were
identified by site-directed
mutagenesis to be essential for kinase
activity of NarX
Ec
(
8), are present in NarX
Ps. The conserved
histidyl residue, which is subject to autophosphorylation, resides
within the H region. In addition to the common characteristics
of the
members of the kinase family, the periplasmic P region,
also known as
the P-box, and the cytoplasmic C region, a stretch
of conserved
residues intercalated between the H and N regions,
are conserved in
nitrate- and nitrite-responsive sensory kinases.
The P region is
involved in binding of and distinguishing between
nitrate and nitrite
(
7,
43), whereas the C region is a common
feature of sensor
proteins and is thought to be important in conferring
specificity on
sensor-response regulator interaction (
30).
NarX
Ps is C-terminally extended vis-à-vis NarX and
NarQ from other sources.
Certain sensor proteins with similarity to
NarX
Ps, such as FixL,
PhoR, EnvZ, and CpxA, also have
extended C termini that show no
sequence
conservation.
NarX senses nitrate and nitrite, with some preference for
nitrate.
Nitrate and nitrite are the principal substrates for
denitrification, and at a low oxygen tension, both induce the complete pathway of four consecutive enzymatic reactions. We were interested, therefore, in the specificity of NarXPs toward both
substrates. We used the previously constructed narX deletion
mutant MRX119, which has a 520-bp internal
Eco47III-HindIII fragment replaced by a
kanamycin resistance cassette (Fig. 1), to study by immunochemical means the expression of the structural genes for nitrate reductase, cytochrome cd1 nitrite reductase, NO reductase,
and N2O reductase. MRX119 and the control strain MK21 were
induced to denitrify nitrate or nitrite as described in Materials and
Methods. The protein pattern of cell extracts was analyzed by SDS-PAGE,
and the four denitrifying reductases were detected with polyclonal
antisera (Fig. 3). Nitrate and nitrite
both induced the synthesis of nitrate reductase in the wild type.
Nitrite was about half as active an inducer as nitrate, as judged from
the amount of NarG detected by Western blot analysis. The level of
nitrate reductase was strongly reduced in MRX119 irrespective of which
growth-supporting N oxide was present, with NarH falling below the
detection limit. Lack of expression of nitrate reductase at wild-type
levels in the narX mutant with nitrite as a substrate
indicated that the nitrite signal is also processed by
NarXPs. Induction of the other three denitrification
enzymes elicited by either nitrate or nitrite was not affected by the
disruption of narX, and they were present at wild-type
levels (Fig. 3). The weak expression of nitrate reductase in MRX119 may
depend on an alternative N oxide-responsive regulatory system, for
which indirect evidence exists (22).
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FIG. 3.
NarX functions as a sensory component with a preference
for nitrate. Immunolabeling was done with polyclonal antisera raised
against the purified nitrate reductase (NarGH; upper bands represent
the NarG subunit, and lower bands represent the NarH subunit),
cytochrome cd1 nitrite reductase (NirS), the
cytochrome b subunit of NO reductase (NorB), and
N2O reductase (NosZ). Lanes 1 and 2, cells cultured for
8 h with nitrate and nitrite, respectively (see Materials and
Methods). Each panel shows the results obtained with strain MK21 (WT)
and strain MRX119 (NarX). Detection was carried out with
a protein A-peroxidase conjugate and chloronaphthol (28).
The NarG levels obtained by nitrate or nitrite induction differ in this
experiment by a factor of 2.3. Amounts of cell extracts used were 48 µg each for NarGH and NorB and 6 µg each for NirS and NosZ. Mass
references (in kilodaltons) were derived from the SeeBlue standard
(Novex).
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NarL acts selectively in denitrification as a transcriptional
activator of the narG operon.
To study the role of
NarL, we used the mutant MRL118 and monitored the expression of
the four structural reductase genes for denitrification at
the mRNA level. narL of MRL118 lacks a 358-bp HincII-AvaI fragment and carries a
Kmr marker instead (Fig. 1). For comparative studies of
gene expression, mutant MRX119 was included. If narXL is
organized as an operon and NarL cannot be replaced by a homologous
component, the phenotypes of narL and narX
mutants should be indistinguishable.
We used an internal fragment from
narG as a probe to
detect transcription from the
narG operon of
P. stutzeri in Northern
blot analysis. The operon from this
bacterium has not yet been
sequenced. However,
nar sequences
from
P. aeruginosa (accession
no.
Y15252),
Paracoccus denitrificans GB17 (subjective synonym,
Thiosphaera pantotropha) (
Q56356),
Mycobacterium
tuberculosis (
O06559),
Thermus thermophilus
(
Y10124),
B. subtilis (
X91819),
S. carnosus
(
AF029225), and
Streptomyces coelicolor (
AL031515)
show that without exception, nitrate-respiring and denitrifying
bacteria, both gram negative and gram positive, have the nitrate
reductase structural genes
narG,
narH, and
narI, as well as a
chaperone-like protein, encoded by
narJ, in an invariant
narGHJI gene cluster,
probably in each case, as in
E. coli (
X16181),
as an operon
of four
cistrons.
Specimens of total RNA from cells grown aerobically and from those
shifted to denitrifying conditions were analyzed by RNA-DNA
hybridization. The
narG transcript was found only in
denitrifying
cells but not in O
2-respiring wild-type
P. stutzeri (Fig.
4).
We noted
mRNA instability on Northern blots in the form of considerable
streaking, which was not observed with the transcripts of the
other
oxidoreductase genes. The size of the largest
narG signal,
6.9 kb, corresponded to that expected from a
narGHJI operon
but
would not be large enough to also include genes encoding the
nitrate
or nitrite transporters upstream, or other genes downstream of
this operon. No
narG transcripts were detected under
denitrifying
conditions in the
narL and
narX
mutants MRL118 and MRX119, respectively.
On the other hand, transcripts
of the
nirSTB operon (encoding
cytochrome
cd1 and two low-molecular-mass
c-type
cytochromes),
the
norCB operon (encoding the NO reductase
complex), and the
nosZ gene (encoding N
2O
reductase) were all present in both mutants
(Fig.
4). No transcripts
were detected in RNA isolated from aerobically
grown cells, in
agreement with the notion that induction of the
denitrification
apparatus occurs at a low oxygen tension. Our
results show that NarL
acts at the transcriptional level and activates
the
narG
operon but not the other structural genes of denitrification
oxidoreductases. We were unable to detect
narXL transcripts,
but
the overlapping gene organization (Fig.
1) and the mutational
results are indicative of a
narXL operon.
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FIG. 4.
NarL acts as a transcriptional activator for the
narG operon. The DNA probe used for Northern blot analysis
is given at the top of each panel. Total RNA was extracted from MK21
(lanes 1), MRL118 (narL) (lanes 2), and MRX119
(narX) (lanes 3). Cells were grown for 3 h with
oxygen in the absence of nitrate (+O2) and then shifted to
nitrate-denitrifying conditions and extracted 1 h after the shift
(denitrifying). Transcripts from the nir operon are found as
monocistronic nirS and polycistronic nirSTB
messages (22). Size standards are the RNA molecular weight
marker no. I (Boehringer GmbH, Mannheim, Germany) and the 16S and 23S
rRNA species.
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NarL-regulated promoters.
With the aim of identifying
potential binding sites for the NarL regulator, we located the
promoters of narG and narK by primer extension
analysis (Fig. 5). The coding region of
narG was independently identified from the N-terminal amino
acid sequence obtained from the purified NarG subunit. The comparison
of the nucleotide sequence-derived protein and the N terminus obtained
from sequencing the isolated NarG subunit revealed that the first 11 amino acids were missing in the protein (Fig.
6A). The reason for this modification is unclear. We assume that the N terminus is processed by a protease, possibly activated during the heat treatment used for the isolation of
nitrate reductase. The proteolytic activity, cleaving at the carbonyl
site of phenylalanine, exhibits chymotrypsin specificity. The
heterogeneity of the NarH subunit is a known phenomenon for heat-solubilized nitrate reductase (reference 6 and
citations therein) but has not been reported so far for the NarG
subunit. The 5' end of narG was determined by primer
extension (Fig. 5). Divergently oriented NarL sites centered around
100 nucleotides from the start site of transcription, as well as a
degenerate FNR site, at a distance of 45.5 nucleotides, and an FNR
half-site, TTGAT, downstream of position +1, form part of the
promoter region (Fig. 6A). The FNR site of the P. stutzeri
narG promoter may be involved in the anaerobic expression of this
operon. Although an unprecedented multiplicity of four FNR factors has
been identified in P. stutzeri, a candidate regulator to
activate the narG operon in response to oxygen withdrawal is
still missing (41).
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FIG. 5.
Determination of the 5' ends of the narG and
narK transcripts by primer extension analysis. Total RNA was
obtained from wild-type cells (MK21) grown aerobically (lane 1), under
O2 limitation (lane 2), or under nitrate-denitrifying
conditions (O2 limitation in the presence of nitrate)
(lanes 3). The right panel for narK shows the lack of
extension products of RNA from MRL118 (narL) (lane 4),
which had been induced for denitrification identical to that of the
wild type. Primer extension was performed with oligonucleotides
complementary to the 5' ends of the coding regions of narG
and narK shown in Fig. 6. Lanes A, C, G, and T show the
results of dideoxy sequencing reactions carried out with the same
primers. For MRL118 only the dideoxyadenine reaction is shown.
|
|
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|
FIG. 6.
NarL-dependent promoters of narG (A) and
narK (B). The transcription start sites obtained from primer
extension analysis are marked +1. Potential NarL sites are marked by
half-arrows; nucleotides that correspond to the E. coli
consensus are boldfaced. Putative FNR sites are boxed, and nucleotides
within those sites that correspond to the E. coli consensus
are highlighted. The oligonucleotides used for primer extension are
underlined. RBS, ribosome binding site. The amino acid sequence
obtained from the purified NarG subunit is shown in boldface in panel
A.
|
|
Figure
5 shows that the determination of the transcription start site
of
narK revealed two putative promoters. Under aerobic
conditions no transcripts were detected, but both promoters were
weakly
activated under O
2-limited conditions. Transcription was
enhanced by the addition of nitrate, and promoter P1 showed the
stronger response. Since the pattern of appearance of P2 followed
that
of P1, we cannot rule out the possibility that the RNA species
generating P2 is a processing or degradation product from the
RNA
giving rise to P1. In the
narL mutant MRL118, no
narK transcripts
were detected (Fig.
5). The promoter P1 of
narK shows sequence
motifs corresponding to recognition
sites for NarL and FNR at
typical distances of these binding elements
from the transcription
initiation site (Fig.
6B). Again, this is in
conformity with the
observed induction pattern for
denitrification.
| |
DISCUSSION |
We have argued that the denitrification process consists of three
to four modules, i.e., partly independent respiratory systems utilizing
nitrate, nitrite, nitric oxide, or N2O (44, 45). Only nitrite reduction is tightly coupled with the subsequent reaction,
the reduction of NO to N2O, presumably to maintain NO at a
low steady-state level and to limit the toxic effects of this radical.
Our data show an autonomous element for regulating nitrate respiration
in the form of the NarXL two-component system, distinct from regulators
affecting nitrite denitrification, i.e., the reduction of nitrite to a
gaseous product. The regulatory independence of denitrification in the
strict sense from the initiator reaction supports our concept of a
modular design.
A phylogenetic analysis by the CLUSTAL W program of the known NarL and
NarP proteins, and putative homologs deduced from genomic sequencing
projects, shows that the NarL proteins of the pseudomonads are most
closely related to NarLEc (data not shown). NarL of
E. coli binds to cognate promoters via heptameric sequences
whose consensus is TACYYMT (12, 14, 40). The location of
the NarL site is typically variable with respect to distance from and
orientation toward the start of transcription (13). In
anaerobically, nitrate-regulated promoters, the NarL site is usually
found at greater distances from the transcription start site than the
FNR site for binding the anaerobic regulator. We have shown so far that
in P. stutzeri, transcription of narG,
narK, and dnrE is activated by nitrate. DnrE is a
transcription factor under the putative control of NarL. It belongs to
the new DNR branch of regulators of the greater FNR family, which lack
the cysteine motif for binding a [4Fe-4S] center as their major
distinction (41). Those three genes show potential NarL
sites which match or are highly similar to the consensus derived for
E. coli (Table 1). DNA
footprinting or mutational analysis is still required to attribute
functionality to the heptameric motifs.
The P and C regions are specific for NarX-type sensor proteins. They
show a remarkable degree of conservation among the five proteins
compared in Fig. 2. The P region was shown in an elegant mutational
study to be responsible for the binding of nitrate and nitrite and to
harbor elements essential for the discrimination of these ions. Whereas
NarXEc is strongly biased towards nitrate (narG
expression in a narQ null mutant is induced 100-fold by nitrate but only 4-fold by nitrite), no such bias is incorporated in
NarQEc (43). The preference of
NarXPs for nitrate is only about twofold. Extending a
comparison of the P-box sequences of NarX and NarQ to include the
sensor proteins of the pseudomonads lowers the identity score to 10 (from 15) of 18 consecutive amino acids. The amino acids Ser43, His45,
and Lys49 of NarX (E. coli sequence numbering), supposedly
of discriminatory quality compared with the positionally equivalent
residues of NarQ proteins, Asp, Glu, and Ile, lose their
differentiating value, but it is still to be noted that the P-box of
NarXPs resembles that of NarQEc more closely
than that of NarXEc. As suggested previously, elements outside of the P-box are also likely to participate in discriminating between nitrate and nitrite (43).
An important element of the P-box is the conserved Arg54, whose
mutagenesis results in a ligand-unresponsive protein (7, 43). When nitrate or nitrite interact with a protein usually they
require a transition metal for binding and their catalytic transformation. We have previously drawn attention to structures of
protein nitrate complexes, even though they are unrelated to nitrate
sensing, suggesting a mode of nitrate binding for the sensory domain of
NarX (45). In Limulus polyphemus, hemocyanin nitrate occupies the site of the allosteric effector chloride (23), whereas in the tyrosine phosphatase of Yersinia
enterocolitica, which takes part in the cellular regulation of
pathogenicity, nitrate is bound by the phosphate-binding peptide loop
(18). In each of these crystal structures, nitrate is
hydrogen-bonded with two oxygens to the N
and
N
atoms of an arginine residue. Further hydrogen bonds
extend from the oxygen atoms of nitrate to the hydroxyl group of serine
in hemocyanin or to amide nitrogens of the phosphate loop in tyrosine phosphatase. Both in hemocyanin and in the phosphatase, conformational changes distant from the binding site of nitrate are induced by anion
binding. Thus, these models indicate the possibility that the binding
of nitrate to the conserved arginine residue in the P region of NarX
and transmembrane signaling may take place without a requirement for a
transition metal in the nitrate sensor.
| |
ACKNOWLEDGMENTS |
We are indebted to John A. DeMoss and Valley Stewart for kindly
providing plasmids and to the late I Rasched for protein sequencing. We
thank H. Körner for a gift of NarG protein, B. Schreckenberger for technical assistance, and D. Jahn for sharing sequence information prior to publication.
The generous financial support of the Deutsche Forschungsgemeinschaft
and the Fonds der Chemischen Industrie is gratefully acknowledged.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Lehrstuhl
für Mikrobiologie, Universität Karlsruhe, PF 6980, D-76128
Karlsruhe, Germany. Phone: 49 (721) 6080. Fax: 49 (721) 608 4290. E-mail: dj03{at}rz.uni-karlsruhe.de.
| |
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Abstract
Introduction
