Department of Applied Biological Sciences,
School of Agricultural Sciences, Nagoya University, Nagoya 464-01, Japan
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INTRODUCTION |
Nitrate, a major source of nitrogen
for cyanobacteria in their natural environment, is transported into the
cells by an active transport system (NRT) and reduced to ammonium by
the sequential action of nitrate reductase (NR) and nitrite reductase
(NiR). The resulting ammonium is fixed into amide group of glutamine by
glutamine synthetase, and the amide nitrogen is subsequently transferred to other compounds by various glutamine
amidotransferases to form various nitrogenous compounds. In the
unicellular cyanobacterium Synechococcus sp. strain PCC
7942, the genes encoding NRT (nrtABCD) (26, 27,
30), NR (narB) (4, 18), and NiR
(nirA) (21, 38) form an operon,
nirA-nrtABCD-narB (nirA operon)
(38), the transcription of which is repressed by ammonium.
Transcription of the nirA operon is induced by
removal of ammonium from the medium or by inhibition of ammonium
assimilation with L-methionine-DL-sulfoximine (MSX; an inhibitor of glutamine synthetase) or
6-diazo-5-oxo-L-norleucine (DON; an inhibitor of glutamine
amidotransferases) (38). Upstream of the nirA
operon are the two genes required for maximum nitrate assimilation (nirB and ntcB), which form
another ammonium-repressible operon (nirB
operon) transcribed divergently from the nirA
operon (36). Under inducing conditions,
nitrite, either added exogenously or generated internally from nitrate,
further activates transcription of the nirA operon
but not that of the nirB operon (2, 17). Since nitrite is reduced by NiR and the resulting ammonium negatively regulates nirA operon transcription, the
nitrite-promoted positive regulation of the nirA
operon is clearly discernible only when ammonium fixation is
inhibited (17) or when cells are exposed to prolonged stress
of nitrogen deficiency (2).
The nirA and nirB operons and other
ammonium-repressible transcription units require the Crp-type
DNA-binding protein NtcA (40) for their expression. In
the promoter region, the NtcA-dependent transcription units have
the consensus sequence
GTAN8TACN22TAN3T, in which
GTAN8TAC is the NtcA-binding site and TAN3T is
presumably the 
10 promoter element (20). There are
three NtcA-binding sites, designated nirI,
nirII, and nirIII (20), in the 286-bp region between nirA and nirB (36). Two
of these, nirI and nirIII, are thought to
constitute the promoter of the nirA (20) and nirB (36) operons, respectively, but the
role of the other NtcA-binding site (nirII), which is
located in the middle of the nirA-nirB intergenic region,
remains unclear. The nitrite-promoted positive regulation of
nirA operon transcription, on the other hand,
requires a LysR-type DNA-binding protein encoded by the ntcB
gene (2), but the cis-acting sequence involved is
yet to be identified. The binding sites of the transcriptional
regulators of LysR family generally have an inverted repeat structure
built around the motif TN11A (LysR motif
[11]). There are two such inverted repeats in the
nirA-nirB intergenic region, both of which are located between nirI and nirII (designated L1 and L2
[Fig. 1]). In this study, we used
luxAB transcriptional fusions to monitor the activities of fragments of the nirA and nirB regulatory
regions under various nitrogen conditions. One of the potential binding
sites for a LysR-type protein (L1), having the sequence
TGCAN5TGCA, and the NtcA-binding site nirII are
shown to be involved in the nitrite-responsive regulation of the
nirA operon. We show that NtcB and nitrite do not promote transcription of the nirA operon but
enhance transcription once it is induced by the action of
NtcA. The physiological significance of the nitrite-responsive
enhancement of nirA operon transcription during
nitrate-limited growth is discussed.
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FIG. 1.
(A) Schematic representation of the insertion of
transcriptional luxAB fusions into the cmp locus
of the Synechococcus chromosome. The recombinational plasmid
pYK5, which cannot replicate in the cyanobacterium, contains a
promoterless luxAB gene cluster, a spectinomycin resistance
cassette (sper), and a
PlacIq::ntcA
transcriptional fusion, which are flanked by Synechococcus
cmpB and cmpC genes. Various fragments of the
nirA-nirB intergenic region were cloned between the
SpeI (Sp) and BglII (Bg) sites preceding the
luxAB gene cluster. Transformation of
Synechococcus with the plasmid resulted in
spectinomycin-resistant strains in which homologous recombination had
transferred the reporter construct to the cyanobacterial chromosome.
(B) The nirA-nirB intergenic region of
Synechococcus sp. strain PCC 7942, showing the nucleotide
sequences of both DNA strands. Numbers above the sequences indicate
positions of the nucleotides with respect to the translation start site
of nirA. The transcription start sites of the
nirA and nirB operons are indicated by
filled triangles. The putative 10 elements of the promoters of the
nirA and nirB operons are underlined. The
three NtcA-binding sites (nirI, nirII, and
nirIII) and the two potential binding sites for LysR-type
DNA-binding proteins (L1 and L2) are boxed. The letters above and below
the sequences represent the base replacements created in strains YKA4,
YKA5, YKA6, YKA7, YKA9, and YKB4. Sites of the luxAB fusion
are also indicated.
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MATERIALS AND METHODS |
Strains and growth conditions.
A derivative of
Synechococcus sp. strain PCC 7942, which is cured of the
resident small plasmid pUH24 (strain R2-SPc [19]; hereafter designated simply strain PCC 7942), is the parental strain of
all of the cyanobacterial strains used in this study. A deletion mutant
of the nrtABCD genes (NA3 [23]), lacking
specifically the high-affinity nitrate-nitrite transporter, was the
genetic background into which reporter gene fusions were introduced by transformation. An ntcA deletion mutant (NIC2) was
constructed from strain PCC 7942 by deleting the ntcA coding
region and 108 and 18 bp, respectively, of its 5' and 3' flanking
sequences from the genome by the marker exchange-eviction mutagenesis
method (32), using a 3.8-kbp nptI-sacB cartridge
excised from pRL250 (6) as the selection marker. All
Synechococcus strains were grown photoautotrophically under
continuous illumination provided by fluorescent lamps (70 microeinsteins m
2 s1) at 30°C. The basal
medium used was a nitrogen-free medium obtained by modification of
BG11 medium (35) as previously described (39).
Ammonium-containing medium and nitrate-containing medium were prepared
by addition of 3.75 mM (NH4)2SO4
and 60 mM KNO3, respectively, to the basal medium.
Both media were buffered with 20 mM HEPES-KOH (pH 8.2). When
appropriate, spectinomycin was added to the media at 1 µg/ml. The
cultures were aerated with 2% (vol/vol) CO2 in air.
Escherichia coli DH5
and NM522, used as hosts for plasmid
constructions and protein expression, respectively, were grown on
Luria-Bertani medium supplemented with ampicillin (50 µg/ml) and/or
spectinomycin (15 µg/ml) when appropriate.
Transcription of the nirA operon and the reporter
gene fusions was induced by treatment of ammonium-grown cyanobacterial
cells with MSX or DON or by transfer of the ammonium-grown cells to ammonium-free media. MSX and DON were added to cyanobacterial cultures
in the mid-logarithmic phase of growth with or without simultaneous
addition of NaNO2. The final concentrations of MSX, DON,
and NaNO2 were 0.1, 0.15, and 5 mM, respectively. For
transfer of the cells to ammonium-free media, the ammonium-grown cells were collected by centrifugation at 5,000 × g for 5 min at 25°C, washed twice with the basal medium by resuspension and
recentrifugation, and inoculated into the basal medium and the
nitrate-containing medium.
Plasmids used for integration of luxAB
transcriptional fusions into the Synechococcus genome.
Fusions of the nirA and nirB regulatory sequences
and a promoterless luxAB gene cluster were constructed on
plasmids and transferred into the cmp locus of the
Synechococcus genome (25) through homologous
recombination between the transforming plasmid DNA and the recipient
cyanobacterial chromosome. The plasmids used as the donors of the
luxAB transcriptional fusions were derivatives of pYK5 (Fig.
1A), which is based on pUC119 and does not replicate in
Synechococcus. For construction of pYK5, a 1.8-kbp
BglII-SphI fragment of Synechococcus
DNA carrying the cmpB-cmpC region (25) was cloned
into pUC119 to form pG23. A 2.1-kbp DNA fragment carrying a
promoterless luxAB gene cluster of Vibrio harveyi
(from nucleotide 12 with respect to the luxA initiation
codon to nucleotide +66 with respect to the luxB termination
codon [8] and [15]) was amplified by
PCR as a BglII-NheI fragment. A
PlacIq::ntcA
transcriptional fusion, in which ntcA coding region is fused
to the lacIq promoter (nucleotides 247 to 1
with respect to the lacIq initiation codon), was
prepared by overlap extension PCR (14), using plasmid
pTrc99A (3) and Synechococcus chromosomal DNA as
templates. A spectinomycin resistance cassette flanked by strong transcriptional terminators (31) was excised from plasmid
pRL463 (9) by digestion with SalI. The
PlacIq::ntcA
transcriptional fusion, the promoterless luxAB gene cluster, and the spectinomycin resistance cassette were sequentially cloned into
pT7Blue T-Vector (Novagen) to form a 5.0-kbp insert flanked by
NheI recognition sites, which was subsequently excised from the plasmid and ligated into the XbaI site in the
cmpC fragment in pG23 to form pYK5. The interrupted
cmpBC sequence in pYK5 allows homologous recombination
between the plasmid and the cyanobacterial chromosome, and the
spectinomycin resistance gene confers a selection marker for
transformants. The
PlaqIq::ntcA
transcriptional fusion was introduced to allow constitutive expression
of NtcA. When transferred from pYK5 to the genome of the
ntcA deletion mutant NIC2, the
PlaqIq::ntcA fusion
complemented the defect of the mutant in nitrate assimilation,
verifying functional expression of NtcA. The nirA and
nirB regulatory sequences were cloned between the unique
SpeI and BglII recognition sites of pYK5, which
are located 48 and 12 bases, respectively, upstream of the
luxA start codon (Fig. 1A).
For preparation of the nirA and nirB regulatory
sequences to be inserted into pYK5, a 261-bp segment of the 286-bp
nirA-nirB intergenic region, carrying nucleotides 275 to
15 with respect to the translation start site of nirA
(i.e., nucleotides 272 to 12 with respect to the translation start
site of nirB [Fig. 1B]), was amplified by PCR and cloned
into pT7Blue T-Vector in two orientations. Deletions and subcloning
from the cloned DNA segments, using the internally located
EcoRI and EcoT22I restriction sites and the other
restriction sites in the polycloning site of the vector, yielded
various fragments of the nirA-nirB intergenic region
carrying one or two of the three NtcA-binding sites (see Fig. 3), which
were flanked by SpeI and BamHI recognition
sequences originating from the pT7Blue vector. The nirA and
nirB regulatory sequences with base substitutions were
generated by overlap extension PCR using oligonucleotide primers
carrying mismatches with the wild-type sequence (13) and
cloned into pT7Blue T-Vector. After confirmation of the nucleotide
sequence, the cloned regulatory regions were excised from the plasmids
with SpeI and BamHI and cloned between the
SpeI and BglII sites of pYK5 (Fig. 1A). The resulting plasmids were used to transfer the luxAB
transcriptional fusions into the chromosome of the NA3 mutant to yield
strains YKA1-YKA9 and YKB1-YKB4. pYK5 was used to transform NA3 to
yield strain YKC, which was used as a promoterless control for the
luxAB reporter.
Expression of plasmid-borne ntcA and ntcB
in Synechococcus.
A PCR-amplified ntcA gene, in
which an NcoI recognition site had been created at the
translation start site, was cloned into pT7Blue T-Vector. After
verification of nucleotide sequence, the ntcA gene was
excised from the plasmid with NcoI and XbaI and cloned between the NcoI and XbaI sites of the
shuttle expression vector pSE2 (2). The resulting plasmid
(pNTCA), encoding a modified NtcA protein in which the second amino
acid residue had been changed from Leu to Val, was used for expression
of ntcA in the ntcA deletion mutant NIC2. Another
pSE2 derivative carrying ntcB, pNTCB (2), was
used for NtcA-independent expression of ntcB in NIC2.
Complementation of the defects of the mutants of ntcA and
ntcB with pNTCA (see Results) and pNTCB (2),
respectively, in the absence of
isopropyl-
-D-thiogalactopyranoside (IPTG) indicated IPTG-independent expression of the genes from the plasmids.
Preparation of NtcA and NtcB proteins and gel shift assay.
The PCR-amplified ntcA (see above) and ntcB
(2) genes cloned into pT7Blue T-Vector were excised from the
plasmids with NcoI and XbaI and, after blunting
of the termini, cloned individually into the SalI site in
the polylinker of the expression vector pMAL-c2 (33). The
resulting plasmids, designated pMNTCA and pMNTCB, carried chimeric
genes encoding translational fusions of the maltose-binding protein
with NtcA and NtcB, respectively. Cells of E. coli NM522
transformants carrying pMAL-c2, pMNTCA, and pMNTCB were grown in
Luria-Bertani medium. Expression of the chimeric genes was induced by 1 mM IPTG, and the recombinant proteins were purified on amylose resin
(33). The purified fusion proteins were cleaved with factor
Xa and used for gel retardation assays.
The DNA fragment used as the probe, carrying nucleotides 173 to 15
with respect to the nirA start codon, was labeled at both
termini with 32P, using the Klenow fragment of DNA
polymerase I and [-32P]dCTP. Gel retardation assays
were performed essentially as described by Buratowski and Chodosh
(5), with supplementation of 5 mM nitrite to the buffers
used for the binding reactions, preparation of the gels, and
electrophoresis. Gels containing 4% polyacrylamide were used for
separation of the free probe and the protein-DNA complexes. Gels were
dried, and the signals were detected with a Bio-image analyzer (Fuji
Photo Film).
Isolation and analysis of DNA and RNA.
Chromosomal DNA was
extracted and purified from the cells of the wild-type and the reporter
strains of Synechococcus sp. strain PCC 7942 as described by
Williams (41). Manipulations and analyses of DNA were
performed according to standard protocols (34). Total RNA
was extracted and purified from Synechococcus cells by the
method of Aiba et al. (1). For Northern hybridization analysis, RNA samples (10 µg per lane) were denatured by treatment with formaldehyde, fractionated by electrophoresis in 1.2% agarose gels that contained formaldehyde, transferred to positively charged nylon membranes (Hybond N+; Amersham), and hybridized with the following 32P-labeled double-stranded DNA probes: a 640-bp
NcoI-AvaI fragment of nirA
(38) and a 1.3-kbp PCR-amplified fragment of ndhB
(24) from Synechococcus sp. strain PCC 7942.
Measurement of in vivo bioluminescence.
For measurement of
in vivo luminescence from Synechococcus cells carrying
luxAB transcriptional fusions, 1 ml of cell culture containing 0.001 to 0.5 µg of chlorophyll (Chl) was transferred to a
test tube and mixed with 20 µl of 0.1% n-decanal
emulsion. Bioluminescence of the cell suspension was measured with a
luminometer (ARGUS-50; Hamamatsu Photonics) immediately after the
addition of n-decanal. Intensity of bioluminescence was
expressed in counts of photons per minute per microgram of Chl. Chl was
determined as described by Mackinney (22).
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RESULTS |
Use of luxAB as a reporter of the activity of the
promoter of the nitrate assimilation operon.
To monitor
transcriptional activities and regulation of the nirA and
nirB operon promoters, fusions of various fragments
of the nirA-nirB intergenic region to promoterless
luxAB were constructed on plasmid pYK5 and integrated into
the cmpC gene of the Synechococcus chromosome
(Fig. 1A). The cmp locus was chosen as the target of integration of the promoter-reporter fusions because the
cmpABCD operon is expressed only under
carbon-limited conditions and is not required for normal growth of the
cyanobacterium under the high-CO2 conditions (2%, vol/vol)
used in this study (28, 29). Since the steady-state level of
nirA and nirB operon transcription in
nitrate-utilizing cells of Synechococcus sp. strain PCC 7942 is low due to negative feedback by the ammonium generated internally from nitrate (36, 38, 39), a mutant (NA3) of
Synechococcus lacking the active nitrate transporter
(23) was used as the standard host for luxAB
fusions, so that a high level of nirA operon
transcription is maintained in nitrate-containing medium because of the
constant stress of nitrogen deficiency. All of the
Synechococcus reporter strains constructed from NA3 grew
normally in ammonium-containing medium; because of nitrogen limitation, growth was slower in nitrate (60 mM)-containing medium, with reduced pigmentation.
Figure 2 shows the time course of
bioluminescence from cells of
PnirA::luxAB reporter strain YKA1,
carrying nearly the entire nirA-nirB intergenic region fused
to luxAB (Fig. 3), after
derepression of nirA operon transcription.
Luciferase activity was low in ammonium-grown cells and was not
affected by transfer of the cells to fresh ammonium-containing medium. Transfer of the cells to nitrogen-free medium induced expression of luciferase, resulting in a 25-fold increase in in vivo
luminescence in 24 h. When transferred to nitrate (60 mM)-containing medium, the cells expressed a much higher level of
luciferase activity (2 × 107 counts per µg of Chl
per min) in 24 h and maintained it during growth with nitrate
(Fig. 3). The YKA1a strain, carrying the same promoter-reporter fusion
as YKA1 in a wild-type background, expressed only low luciferase
activity (ca. 105 counts per µg of Chl per min) when
grown in the nitrate-containing medium (not shown), indicating that the
constant nitrogen stress in nitrate-grown YKA1 led to the
high-level expression of the luxAB reporter. Luciferase
activity of YKA1 was induced also by addition of DON, an inhibitor of
glutamine amidotransferases, to the ammonium-grown cultures (Fig. 2).
Nitrite, when added with DON to the ammonium-grown culture, caused a
20-fold increase of luminescence compared to the level obtained by
treatment with DON alone. The regulation of luciferase expression,
i.e., induction by removal of ammonium or by inhibition of nitrogen
assimilation, and activation by nitrite, is essentially the same as
that of nirA operon transcription, as demonstrated
by direct determination of nirA mRNA (17, 38).
These results verified that the luciferase encoded by luxAB
can be used as the reporter of the activity of the nirA
operon promoter. The results also confirmed that the nirA-nirB intergenic region contains all of the
cis-acting elements required for the negative and positive
regulation of nirA operon expression.
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FIG. 2.
Effects of various nitrogen conditions on expression of
luciferase in PnirA::luxAB reporter
strain YKA1, which carries nearly the entire nirA-nirB
intergenic region (nucleotides 275 to 15 with respect to the
nirA translation start site [Fig. 1B and 3]) fused to
promoterless luxAB. Cells were grown with ammonium, the
culture was separated into five portions, and changes in luciferase
activity after addition of DON alone ( ) and DON plus nitrite ( )
and after transfer of the cells to nitrogen-free medium ( ),
nitrate-containing medium ( ), and ammonium-containing medium ( )
were monitored by measuring in vivo bioluminescence in the presence of
n-decanal.
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FIG. 3.
Effects of upstream regions of nirA and
nirB on luxAB expression in ammonium- and
nitrate-containing media. Fragments of the nirA-nirB
regulatory region having the indicated endpoints relative to the
translational start site of nirA were fused individually to
the luxAB gene in pYK5 and transferred to the chromosome of
Synechococcus mutant NA3 to generate
PnirA::luxAB and
PnirB::luxAB reporter strains. The
three NtcA-binding sites, nirI, nirII, and
nirIII, are indicated by open, dotted, and closed boxes,
respectively, and the two potential binding sites for LysR-type
DNA-binding proteins, L1 and L2, are indicated by filled circles. The
bioluminescence data are the means of five measurements, with standard
deviations indicated.
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Promoter and other cis-acting sequences of
nirA and nirB.
Figure 3 summarizes the
bioluminescence data from various
PnirA::luxAB and
PnirB::luxAB reporter strains grown
continuously in ammonium-containing and nitrate-containing media. When
grown with ammonium, luciferase activity in all of the reporter strains was low, being similar to that in ammonium-grown cells of YKC used as
the promoterless control for luxAB expression. The
luciferase activity of nitrate-grown YKC cells was 4.3 times higher
than that of the ammonium-grown cells (104 counts per µg
of Chl per min), presumably because of the lower growth rate and
decreased pigmentation of the nitrate-grown cells than of the
ammonium-grown cells. Nitrate-grown cells of the
PnirA::luxAB reporter strain YKA3,
which carries nucleotides 95 to 15 fused to luxAB,
showed luciferase activity 110 times higher than that of the
ammonium-grown cells, indicating that the 81-bp DNA segment carries the
promoter that directs nitrogen-responsive gene expression. YKA2 and
YKA1 showed stronger luminescence than YKA3 when grown with nitrate
(140 and 200 times that of YKA3, respectively) but not when grown with
ammonium (1.1 and 1.2 times that of YKA3, respectively). These results
suggested that the region from nucleotides 173 to 96 enhances
transcription from the nirA operon promoter.
When grown with nitrate, the
PnirB::luxAB reporter strain YKB3
exhibited luminescence 38 times stronger than that of the
ammonium-grown cells (Fig. 3), showing that the DNA segment from
nucleotides 275 to 170 with respect to the nirA
translation start site (nucleotides 117 to 12 with respect to the
nirB translation start site), carrying nirIII and
the putative 10 element, can promote nitrogen-responsive gene
expression. The DNA region farther upstream, from nucleotides 170 to
15 with respect to the nirA start codon, did not affect transcription from the nirB operon promoter (Fig. 3,
YKB1 and YKB2).
cis-acting elements involved in nitrite-responsive
regulation of the nirA operon.
To further
characterize the cis-acting element(s) regulating
transcription of the nirA operon, effects of DON and
nitrite on expression of luciferase activity were examined in various PnirA::luxAB reporter strains (Fig.
4). When nitrogen assimilation was
inhibited by DON treatment, YKA1, YKA2, and YKA3 expressed similar
levels of luciferase activity, confirming the presence of the
nitrogen-responsive promoter in the region from nucleotides 95 to
15 with respect to the nirA start codon. Site-specific modification of nirI in this region abolished luciferase
expression in response to DON (YKA4), confirming that the NtcA-binding
site is an essential element of the nirA operon
promoter.
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FIG. 4.
Effects of DON and nitrite on expression of
luxAB in various
PnirA::luxAB and
PnirB::luxAB reporter strains.
Bioluminescence was measured before (control) and 12 h after
addition of DON alone and of DON plus nitrite to ammonium-grown
cultures of the Synechococcus reporter strains. The three
NtcA-binding sites and the two putative binding sites for LysR-type
proteins are indicated as in Fig. 3. X indicates the NtcA-binding
motifs and the LysR motifs where site-specific nucleotide changes were
created. The bioluminescence data are the means of five measurements,
with standard deviations indicated.
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The presence of nitrite during DON treatment increased the
luciferase activity of YKA2 and YKA1 15- and 23-fold, respectively but
decreased that of YKA3 by 50% (Fig. 4). In YKA1b and YKA2b, which
carry the same promoter-reporter fusions as YKA1 and YKA2, respectively, in an ntcB background
(
ntcB::kan nrtABCD), DON induced luciferase expression but nitrite decreased luciferase activity
as in YKA3 (not shown). These results confirmed that the
nitrite-promoted increase of luciferase activity in YKA1 and YKA2
represents positive regulation by NtcB and nitrite of the nirA operon promoter, as shown previously by the
determination of nirA mRNA (2).
The nitrite activation of luciferase expression in YKA2 but not in YKA3
indicated that the regulatory sequence essential for the
nitrite-promoted activation of nirA operon
transcription is located in the region from bases 173 to 96, which
carries the NtcA-binding site nirII and the two putative
binding sites for LysR-type DNA-binding proteins, L1 and L2 (Fig. 1B).
Site-specific modification of either the left half or the right half of
the L1 site in YKA2 (Fig. 1B) abolished the nitrite-promoted increase of luciferase expression without affecting the luciferase induction by
DON treatment (Fig. 4, YKA5 and YKA6), whereas site-specific modification of L2 in YKA2 (Fig. 1B) had no effect on the regulation of
luciferase expression (YKA7). Removal of the NtcA-binding site nirII (YKA8) or its site-specific modification (YKA9) also
resulted in loss of the response to nitrite. These findings suggested
that the NtcA-binding site nirII and one of the potential
binding sites for LysR-type proteins, L1, are required for the
nitrite-responsive regulation of nirA operon
transcription.
When treated with DON, the PnirB::luxAB
reporter strains YKB1, YKB2, and YKB3 expressed essentially the same
level of luciferase activity (Fig. 4). None of the
PnirB::luxAB reporter strains
showed any nitrite-responsive activation of luciferase expression,
consistent with our previous conclusion that nitrite does not affect
nirB operon transcription (2). These
findings showed that transcription from the nirB
operon promoter is regulated simply by repression/derepression and that the region from bases 112 to 17 with respect to the nirB start codon carries all of the elements required for
regulation. Site-specific modification of nirIII abolished
the luciferase expression in response to DON treatment (YKB4), showing
that the NtcA-binding site is essential for the activity of the
nirB operon promoter.
Dependence on NtcA of the nitrite-responsive, NtcB-dependent
activation of nirA operon transcription.
Although it has a functional nirB-ntcB operon,
strain YKA4, carrying the
PnirA::luxAB fusion with
modified nirI, did not express luciferase when treated
with DON and nitrite (Fig. 4). This indicated that NtcB and nitrite
cannot induce transcription of the reporter genes by themselves even
under nitrogen starvation and that induction by NtcA is a prerequisite
for enhancement by NtcB of the transcription.
Dependence on NtcA of the action of NtcB was confirmed by determination
of the nirA mRNA in the ntcA deletion mutant NIC2 (Fig. 5). Since induction of the
nirB-ntcB operon is dependent on NtcA, a
plasmid-borne ntcB was used for constitutive expression of
NtcB in the mutant. In the wild-type strain PCC 7942, MSX treatment of
ammonium-grown cells induced nirA operon
transcription (Fig. 5A, lane 2) and nitrite further enhanced the
transcription as previously shown (Fig. 5A, lane 3). The hybridization
signal was smeary as previously shown, representing rapid degradation
of the primary transcript (38). The level of the transcript
of ndhB, encoding an NADH dehydrogenase subunit, was not
affected by the nitrogen conditions tested (Fig. 5B). In the
ntcA deletion mutant NIC2, there was no induction of
nirA operon transcription by treatment of
ammonium-grown cells with MSX alone or with MSX plus nitrite (Fig. 5A,
lanes 4 to 6). Introduction into NIC2 of a plasmid carrying
ntcA restored the abilities of the cells to express
nirA operon upon MSX treatment and to respond to
nitrite (Fig. 5A, lanes 7 to 9), whereas introduction of pNTCB, which allows NtcA-independent expression of NtcB (2), restored
neither of these abilities (Fig. 5A, lanes 10 to 12). Thus, NtcB and
nitrite cannot activate nirA operon transcription in
the absence of NtcA.
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FIG. 5.
Northern blot analysis of RNA from wild-type PCC 7942 strain (WT; lanes 1 to 3), the ntcA deletion mutant (NIC2;
lanes 4 to 6), the NIC2 mutant transformed with a plasmid carrying
Ptrc::ntcA transcriptional fusion
(NIC2[pNTCA]; lanes 7 to 9), and the NIC2 mutant transformed with
a plasmid carrying Ptrc::ntcB transcriptional
fusion (NIC2[pNTCB]; lanes 10 to 12), showing the requirement for
ntcA of the activation of nirA operon
transcription by NtcB and nitrite. Cells were grown with ammonium, the
culture was separated into three portions, and total RNA was extracted
from the cells 60 min after addition of nothing (control; lanes 1, 4, 7, and 10), MSX (lanes 2, 5, 8, and 11), and MSX plus nitrite (lanes 3, 6, 9, and 12). The RNA samples (10 µg per lane) were denatured,
fractionated by electrophoresis, transferred to positively charged
nylon membranes, and hybridized with the probes specific to
nirA (A) and ndhB (B). The arrow in panel A
indicates the calculated size of the full-length mRNA of the
nirA operon, whereas that in panel B shows the
position of the ndhB transcript.
|
|
Gel shift assay.
To examine whether NtcB binds to the
nirA operon regulatory region, gel shift assays were
performed with NtcB and/or NtcA, using the DNA segment carrying
nucleotides 173 to 15 with respect to the nirA
start codon as the probe. Since translational fusions of NtcB to
glutathione S-transferase and to a
His6-containing amino acid segment yielded insoluble
materials which could not be solubilized, a MalE-NtcB fusion, expressed
in E. coli as a soluble protein, was purified to near
homogeneity (Fig. 6A), cleaved with
factor Xa (Fig. 6B), and used for the experiments (Fig.
7). Samples of NtcA were also obtained by
cleavage of a MalE-NtcA fusion with factor Xa (Fig. 6). While addition
of NtcA to a concentration of 15 nM yielded two clearly retarded bands
representing DNA-protein complexes as previously reported by Luque et
al. (20) (Fig. 7, lanes 7, 13, and 18), addition of NtcB up
to 500 nM did not affect the electrophoretic mobility of the DNA probe
(lanes 3 to 6) or of the probe-NtcA complexes (lanes 8 to 11, 14 to 17, and 19 to 22). These results showed that the NtcB protein does not bind
to the nirA operon regulatory region under the given conditions.
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FIG. 6.
Preparation of recombinant NtcA and NtcB proteins. (A)
Expression in E. coli and purification of MalE (lanes 1 to
4) and the MalE-NtcA (lanes 5 to 8) and MalE-NtcB (lanes 9 to 12)
fusions. Proteins were separated on a sodium dodecyl sulfate-10%
polyacrylamide gel and stained with Coomassie brilliant blue. Lanes 1, 5, and 9, total protein from the E. coli expression strains
before IPTG treatment; lanes 2, 6, and 10, total protein from the
expression strains after a 1-h treatment with IPTG; lanes 3, 7, and 11, soluble fraction from the IPTG-induced expression strains; lanes 4, 8, and 12, proteins purified on amylose resin. (B) Cleavage of the
MalE-NtcA (lanes 1 and 2) and MalE-NtcB (lanes 3 and 4) fusions with
factor Xa. Protein profiles before (lanes 1 and 3) and after (lanes 2 and 4) treatment with factor Xa were compared. Proteins were separated
on sodium dodecyl sulfate-12.5% (lanes 1 and 2) and 10% (lanes 3 and
4) polyacrylamide gels and stained with Coomassie brilliant blue. Lanes
M, molecular mass markers (masses are indicated at the left).
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FIG. 7.
Mobility shift assays showing retardation in 4%
polyacrylamide gel of the 32P-labeled nirA
promoter segment (nucleotides 173 to 15 with respect to the
nirA translation start site) by NtcA but not by NtcB.
Samples of MalE (Fig. 6A, lane 4) and the MalE-NtcA and MalE-NtcB
fusions cleaved with factor Xa (Fig. 6B, lanes 2 and 4) were added to
the reaction mixtures to give the indicated concentrations of NtcA,
NtcB, and MalE. C1 and C2, DNA-protein complexes; F, free probe.
|
|
Structures of the nirA operon promoters of
other cyanobacteria.
Figure 8
compares the nucleotide sequences of the nirA regulatory
regions of four species of cyanobacteria, including
Synechococcus sp. strain PCC 7942. As previously
reported, the nirA operons of Plectonema
boryanum (37) and Anabaena sp.
strain PCC 7120 (10) have the consensus sequence of the
NtcA-dependent, ammonium-repressible promoter, i.e.,
GTAN8TACN22TAN3T. The
nirA gene of Synechocystis sp. strain PCC
6803, which was identified by the genome sequencing project
(16), also has an NtcA-binding motif and the putative 10
element in its promoter region (Fig. 8). The L1 sequence of Synechococcus sp. strain PCC 7942 is centered at nucleotide
23 with respect to the conserved NtcA-binding site (Fig. 8).
The other species of cyanobacteria were found to have an L1-like
inverted repeat containing a LysR motif (TN11A) at
the same location as that of L1 with respect to the conserved
NtcA-binding site (Fig. 8). On the other hand, the NtcA-binding motif
corresponding to nirII of Synechococcus sp.
strain PCC 7942 was not present in the other species of cyanobacteria
(not shown).
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FIG. 8.
Alignment of the promoter regions of the nirA
operons from various species of cyanobacteria. The regions of
the putative binding site for a LysR-type DNA-binding protein, the
NtcA-binding site, and the 10 sequence are boxed. The nucleotides
forming an inverted repeat with a LysR motif (TN11A) are
shaded. Asterisks indicate the nucleotides conserved in the four
promoter sequences. Gaps have been introduced into the sequences to
maintain optimal alignment. Numbers at the right indicate positions of
the rightward-most bases with respect to the translation start site of
nirA. Strains: 7942, Synechococcus sp. strain PCC
7942; 6803, Synechocystis sp. strain PCC 6803; 7120, Anabaena sp. strain PCC 7120; P.b., Plectonema
boryanum.
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|
| |
DISCUSSION |
Transcriptional luxAB fusions integrated into the
chromosome of Synechococcus at the cmp locus
allowed us to characterize promoters and cis-controlling
elements regulating expression of nirA and
nirB operons. In accordance with the essential
role of NtcA in activation of the cyanobacterial ammonium-repressible transcription units (20), the
PnirA::luxAB and
PnirB::luxAB reporters with modified
nirI and nirIII, respectively, did not express
luciferase activity when treated with DON (Fig. 4, YKA4 and YKB4),
verifying that the conserved NtcA-binding site of
ammonium-repressible transcriptional units (20) is an
essential promoter element. YKA4 cells treated with DON plus nitrite
did not express luciferase activity despite the presence of a
functional ntcB gene (Fig. 4), indicating that the positive
regulation by NtcB and nitrite requires prior induction by NtcA of
nirA operon transcription. The action of NtcB and
nitrite on the nirA operon is therefore deduced to
be enhancement of the transcription. Site-specific mutagenesis showed
that the NtcA-binding site nirII, which is located in the
middle of the nirA-nirB intergenic region, and the putative
binding site (L1) for a LysR-type protein, which is located between
nirI and nirII, are required for the
nitrite-responsive, NtcB-dependent enhancement of nirA
operon transcription (Fig. 4). Among the known
NtcA-dependent transcription units of Synechococcus sp. strain PCC 7942, none but the nirA operon has
the L1 motif in the regulatory region (7, 12, 36, 40). In
accordance with this finding, nitrite has no effect on the
transcription of ntcA, glnA, and the
nirB-ntcB operon (2). NtcB and nitrite thus seem to specifically activate the nirA operon
among the transcription units under the global nitrogen control.
Although two cis-acting sequences involved in the
nitrite-responsive, NtcB-dependent enhancement of nirA
operon transcription have been identified, the
molecular mechanism of the regulation remains unclear. Dependence on
the LysR-type protein NtcB and the requirement for a sequence motif
conforming to the structure of the binding sites for LysR-type proteins
seemed to suggest that NtcB binds to the L1 site in response to nitrite
and thereby enhances transcription. However, NtcB samples obtained by
cleavage of a MalE-NtcB fusion did not bind to the L1 site in vitro
irrespective of the presence of nitrite and NtcA (Fig. 7), which
suggests that one or more additional factors are involved in the
regulation. For example, nitrite may modify the DNA-binding activity of
NtcB through the action of another protein. It is also possible that NtcB activates transcription of another LysR-type protein, which in
turn binds to the L1 motif and regulates nirA operon
transcription. Further work, including study of the interaction of NtcB
and nitrite, is required for elucidation of the molecular mechanism of
the regulation.
The use of an NRT-deficient cyanobacterial strain (NA3) as the
recipient of the luxAB reporter fusions led to expression of high luciferase activity during growth of the reporter strains with
nitrate, when the regulatory sequence included the region involved in
the nitrite-responsive positive regulation (YKA1 and YKA2 [Fig. 2 and
3]). However, the level of luciferase expression in nitrate-grown
YKA3, which lacks the cis-acting elements essential for the
nitrite enhancement of transcription, was much lower than that in YKA1
and YKA2 (Fig. 3) and was similar to the level obtained simply by
derepression of the transcription by DON treatment (Fig. 4). Thus, the
high luciferase activity seen in nitrate-grown YKA1 and YKA2 is
ascribed to the operation of the nitrite-responsive positive regulation
mechanism of nirA operon transcription. Due to the
limitation of nitrate uptake, the intracellular concentration of
nitrite in the reporter strains is believed to be too low to form the
level of ammonium that causes repression of the nirA operon promoter. The operation of the nitrite-responsive
mechanism under these conditions indicates that the nitrite-responding
mechanism has a high sensitivity to nitrite. In our previous studies,
the positive effect of nitrite on nirA operon
transcription was clearly discernible after inhibition of ammonium
fixation (17) or after exposure of the cells to prolonged
nitrogen deficiency (2). The present results, obtained
with NRT-deficient strains, predict that the positive
regulation mechanism will greatly enhance nirA operon transcription even during growth of the wild-type
strain, provided that the nitrate levels in the medium are not
saturating for uptake and reduction. Since cyanobacterial NRT has an
apparent Km(NO3) of
about 1 µM (30), endogenously formed nitrite would enhance expression of the nitrate assimilation operon in the wild-type cells at external nitrate concentrations of 1 µM or lower. Such low
concentrations of nitrate are barely maintained during growth of
cyanobacterial cells in laboratory cultures but would be common in the
natural environment.
We previously showed that nitrite positively regulates nirA
operon transcription in P. boryanum as well as in
Synechococcus sp. strain PCC 7942 (17). The
L1-like inverted repeat of P. boryanum, which is
present at the same location in the nirA regulatory region
as in strain PCC 7942 (Fig. 8), is hence likely to be involved in the
nitrite stimulation of nirA operon transcription in
this cyanobacterium. The absence in P. boryanum of the
NtcA-binding site corresponding to nirII indicates that
nitrite regulation of the nirA operon does not
require the additional NtcA-binding site in P. boryanum. The nirA regulatory regions of
Anabaena sp. strain PCC 7120 and Synechocystis
sp. strain PCC 6803 also have no nirII NtcA-binding site.
However, the occurrence of L1-like motifs in these two strains (Fig. 8)
and the identification of a ntcB homolog in strain PCC 6803 (16) suggest the common occurrence of
nitrite-promoted, NtcB-dependent enhancement of
nirA operon transcription in cyanobacteria.
This work was supported by Grants-in-Aid for Scientific Research
in Priority Areas (09274101 and 09274103) from the Ministry of
Education, Science and Culture, Japan.
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Abstract
Introduction
