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J Biol Chem, Vol. 273, Issue 47, 31463-31470, November 20, 1998
From the Instituto de Bioquímica Vegetal y
Fotosíntesis, Consejo Superior de Investigaciones
Científicas-Universidad de Sevilla, E-41092
Sevilla, Spain
Ammonium is an important nitrogen source for many
microorganisms and plants. Ammonium transporters whose activity can be
probed with [14C]methylammonium have been described
in several organisms including some cyanobacteria, and amt
genes encoding ammonium/methylammonium permeases have been recently
identified in yeast, Arabidopsis thaliana, and some
bacteria. The unicellular cyanobacterium Synechocystis sp.
PCC 6803 exhibited a [14C]methylammonium uptake activity
that was inhibited by externally added ammonium. Three putative
amt genes that are found in the recently published complete
sequence of the chromosome of strain PCC 6803 were inactivated by
insertion of antibiotic resistance-encoding gene-cassettes. The
corresponding mutant strains were impaired in uptake of
[14C]methylammonium. Open reading frame
sll0108 (amt1) was responsible for a high
affinity uptake activity (Ks for methylammonium, 2.7 µM), whereas open reading frames sll1017
(amt2) and sll0537 (amt3) made
minor contributions to uptake at low substrate concentrations. Expression of the three amt genes was higher in
nitrogen-starved cells than in cells incubated in the presence of a
source of nitrogen (either ammonium or nitrate), but amt1
was expressed at higher levels than the other two amt
genes. Transcription of amt1 was found to take place from a
promoter bearing the structure of the cyanobacterial promoters
activated by the nitrogen control transcription factor, NtcA.
Ammonium is a key compound in the assimilation of nitrogen in
numerous biological systems because it is the inorganic form of
nitrogen that is incorporated, usually via the glutamine
synthetase/glutamate synthase cycle, into carbon skeletons. Ammonium
present in the environment can be assimilated by many bacteria, yeast,
fungi, algae, and higher plants. Ammonium solutions always contain
ammonia (pKa [ammonium/ammonia], 9.25), which can
diffuse through biological membranes (1, 2). Diffusion of ammonia
followed by trapping of intracellular ammonium by glutamine synthetase can represent a significant process for nitrogen acquisition, especially in organisms like some bacteria, which can grow in alkaline
media. This process would be less important in fungi that grow in
acidic media. On the other hand, there is evidence for the presence of
ammonium transport systems in numerous organisms (2). The first report
of active ammonium uptake, by Hackette et al. (3), concerned
the fungus Penicillium chrysogenum. These authors introduced
the use of [14C]methylammonium as a probe for the
activity of the ammonium permease, a technique that has proven useful
to study ammonium transport in many other biological systems including
bacteria (4) (the pKa of methylammonium/methylamine
is 10.65). Bacterial ammonium/methylammonium permeases are commonly
repressed by high concentrations of ammonium in a process that, in the
enterobacteria, involves the nitrogen control Ntr system (Refs. 5 and
6; reviewed in Ref. 7).
A gene, amtA, putatively involved in ammonium transport has
been described in Escherichia coli (8), but it rather
appears to be involved in cysteine synthesis and has been renamed as
cysQ (9). On the other hand, genes encoding
ammonium/methylammonium permeases have been characterized in
Saccharomyces cerevisiae (MEP genes (10, 11)) and
Arabidopsis thaliana (AMT1 (12)). Genes that
would encode proteins homologous to the MEP and
AMT1 gene products are found in some bacteria like
Bacillus subtilis (nrgA (13)), Rhodobacter
capsulatus (14), E. coli (15), Corynebacterium
glutamicum (16), Azospirillum brasilense (17), and
Azotobacter vinelandii (18). Inactivation of the putative amt genes of C. glutamicum, A. brasilense, A. vinelandii, and E. coli has
confirmed that they encode permeases that can mediate methylammonium
transport (16-19).
Cyanobacteria are organisms that belong to the bacteria (or eubacteria)
group (20) and are characterized by their ability to perform oxygenic
photosynthesis. Sources of nitrogen used by many cyanobacteria include
nitrate, dinitrogen, urea, and ammonium (21). In cyanobacteria,
incorporation of ammonium into carbon skeletons takes place mainly
through the glutamine synthetase/glutamate synthase cycle (Ref. 22;
reviewed in Ref. 21). The pH values of cyanobacterial growth media are
usually above neutral (23). Diffusion of ammonia through cyanobacterial
cytoplasmic membranes has been demonstrated (24) and can provide,
pulled by glutamine synthetase, a mechanism for the net uptake of
ammonium (see e.g. Ref. 25). Expression of an
ammonium/methylammonium transport activity in some cyanobacterial
strains, including Synechococcus sp. PCC 7942, Anabaena azollae, and Anabaena variabilis, has
also been reported (26, 27). In Synechococcus sp. PCC 7942, the ammonium/methylammonium transport activity is repressed by growth in ammonium-containing medium (28) and requires an intact
ntcA gene to be expressed (29). NtcA is a cyanobacterial
transcriptional regulator, homologous to Crp from E. coli,
that activates the expression of ammonium-repressible genes in the
absence of ammonium (30, 31). The DNA target to which NtcA binds in the
promoter of the regulated genes has been characterized in
Synechococcus sp. PCC 7942 and contains the sequence
signature GTAN8TAC located about 22 bp1 upstream from a The whole sequence of the chromosome of the unicellular cyanobacterium
Synechocystis sp. PCC 6803 has been determined
(33)2 and shown to contain
three ORFs, namely sll0108, sll0537, and sll1017,
that would encode polypeptides homologous to the AMT1 and
MEP gene products cited above. Another ORF
(sll0895) described by Kaneko et al. (33) as
ammonium transport protein would actually encode a homologue of CysQ
(see above). In this work, we show that the three putative
amt genes of Synechocystis sp. PCC 6803 actually provide the cells with the capability to take up
[14C]methylammonium from the extracellular medium and are
subjected to repression by ammonium.
Strains and Growth Conditions--
Synechocystis sp.
strain PCC 6803 was grown axenically in BG11 (nitrate-containing)
medium (23) or in BG110 (nitrogen-free) medium supplemented
with 5 mM NH4Cl and 10 mM TES-NaOH
buffer (pH 7.5). For plates, the medium was solidified with 1%,
separately autoclaved agar (Difco). Cultures were grown at 30 °C in
the light with shaking (80-90 rpm) for liquid cultures.
Synechocystis sp. PCC 6803 mutants carrying gene-cassette
C.K3 (34) were routinely grown in medium supplemented with 30-50 µg
of Km·ml
E. coli strains DH5 Methylammonium Uptake Assays--
Wild-type or mutant
Synechocystis cells grown in nitrate- or ammonium-containing
medium or incubated in the absence of any nitrogen source for 6 h
were harvested by low speed centrifugation at room temperature, washed
with 20 mM KH2PO4, 10 mM NaHCO3-NaOH buffer (pH 7.1) and resuspended
in the same buffer. After a preincubation at 30 °C in the light (100 watt·m Intracellular Accumulation of Labeled
Methylammonium--
Filters containing cells that had been used in
uptake assays as described above were immediately immersed in 2 or 3 ml
of boiling water and incubated at 100 °C for 5 min. The filters were removed, and the resulting suspensions were centrifuged. Samples from
the supernatants were lyophilized and dissolved in a small volume of
water. Metabolites present in these samples were resolved by thin layer
chromatography using 0.1-mm cellulose plates (20 by 20 cm; Merck). The
solvent used was isopropanol/formic acid/water (40:2:10, v/v) (26). The
resulting radioactive areas were quantified in an InstantImager scanner
for DNA and RNA Isolation and Manipulation--
Isolation of genomic
DNA from cyanobacteria was carried out as described previously (38),
except for strain UTEX 1829 cells, which were frozen with liquid air
and broken by grinding with glass beads in a mortar. Isolation of total
RNA from Synechocystis sp. PCC 6803 was made as described
previously (39). Other molecular biology manipulations were carried out
by standard procedures (40).
Inactivation of ORFs sll0108, sll1017, and sll0537--
DNA
fragments corresponding to strain PCC 6803 ORFs sll0108,
sll1017, and sll0537 (33) were amplified by PCR
as described previously (41). The primers used (see Fig. 4 below) were:
Tr18, 5'-GCCCATTTCCAGAAGGATTGCCCG-3'; Tr19,
5'-GGAATCAGTTTCCTTAACAAAGCC-3'; Tr2a, 5'-GGGAGTTGATTTAGTATAGGCGATG-3';
Tr2b, 5'-TTACTAACGGTGTCCA ACCGGCG-3'; Tr3a,
5'-GTTTTCTGCCTACTTATTGGTGGCG-3'; and Tr3b,
5'-CTACGGTGGTGCCGGGCTTCAAAGG-3'.
PCR products were cloned in pGEM-T vector (Promega). Plasmids
containing PCR products generated with primers Tr18-Tr19, Tr2a-Tr2b, and Tr3a-Tr3b were named pCSX23, pCSX53, and pCSX52, respectively. Plasmid pCSX23 was digested with ApaI and PstI to
isolate the insert that was then cloned between the ApaI and
PstI sites of pBluescript SK(+), generating plasmid pCSX46.
The 1.1-kb Kmr gene-cassette C.K3 (34) excised with
SmaI was inserted into a unique HincII site that
is present in the strain PCC 6803-derived insert of pCSX46 to generate
plasmids pCSX47a and pCSX47b (both orientations). The 1.9-kb
Cmr gene-cassette C.C1 (34) digested with HincII
was inserted into the unique MscI site of pCSX53 and into
the unique MscI site of pCSX52 (these MscI sites
are present in the strain PCC 6803-derived inserts of the plasmids),
rendering plasmids pCSX57 and pCSX56, respectively. The orientation of
C.C1 in the inserts of pCSX56 and pCSX57 was not determined. Sequences
of the inserts of pCSX23, pCSX53, and pCSX52 were verified by using a
T7Sequencing kit (Amersham Pharmacia Biotech) and
[
Transformation of Synechocystis sp. PCC 6803 with pCSX47a,
pCSX47b, pCSX56, or pCSX57 as well as transformation of insertional mutant strain CSX47a with pCSX56 or pCSX57 was carried out as described
previously (41). Transformants were selected in BG11 solid medium
supplemented with 30 µg of Km·ml Southern and Northern Blotting and Hybridization--
Southern
analysis was carried out as described previously (41) using GeneScreen
Plus membranes (DuPont). For Northern blots, 10 to 15 µg of RNA were
loaded/lane and electrophoresed in 1% agarose denaturating
formaldehyde gels. Transfer to nylon membranes (Hybond N-plus, Amersham
Pharmacia Biotech; GeneScreen Plus, NEN Life Science Products),
prehybridization, and washes were carried out following the
manufacturer's recommendations; hybridization was performed at
42 °C in the presence of 50% formamide, 5× SSPE (1× SSPE is 0.18 M NaCl, 10 mM sodium phosphate, and 1 mM EDTA (pH 7.4)), 5× Denhardt's solution (40), 0.1%
SDS, and 50 µg of herring sperm DNA·ml
DNA probes used in the hybridizations were obtained by PCR using
pCSX23, pCSX53, and pCSX52 as templates and the corresponding oligonucleotides as primers. Probes were labeled with a DNA labeling kit (Ready to Go, Amersham Pharmacia Biotech) and
[ Comparative RNA Hybridization--
For hybridization of blots of
DNA from ORFs sll0108, sll1017, and
sll0537 (2 or 4 pmol of DNA fragments amplified by PCR as described above and resolved by electrophoresis in agarose gels) with
total cyanobacterial RNA (42), 15 µg of RNA from strain PCC 6803 was
partially hydrolyzed by incubation in 125 mM NaOH for 30 min at 0 to 4 °C and labeled with T4 polynucleotide kinase (Boehringer Mannheim) and [ Primer Extension Analysis--
A 525-bp fragment putatively
containing the promoter region of ORF sll0108 was amplified
using primers Am1 (5'-GGGAGCCACTAAAGTTCACAGG-3'; corresponds to
positions Band-shift Assays--
A 382-bp fragment putatively containing
the promoter region of ORF sll0108 was amplified by PCR
using pCSX49 as template and primers Am1 and Am3 (see above and Fig.
8A). This fragment was digested with BstXI, and
the resulting 138- and 244-bp fragments were isolated and used in
nonradioactive band-shift assays. Binding assays were carried out in a
final volume of 20 µl containing 6 mM HEPES-NaOH buffer
(pH 8.0), 7 mM Tris-HCl (pH 8.0), 42.5 mM KCl,
0.5 mM EDTA, 0.5 mM dithiothreitol, 0.375 mM MgCl2, 0.0025% gelatin, 0.001% Tween 20, 0.05 µg·µl Glutamine Synthetase Activity--
Glutamine synthetase activity
was measured by using the Transport of Labeled Methylammonium--
The cellular activity
level of uptake of [14C]methylammonium in
Synechocystis sp. PCC 6803 was higher in cells starved for
nitrogen than in nonstarved cells (grown with either nitrate or
ammonium). Additionally, growth in CO2-enriched air allowed
higher [14C]methylammonium uptake activities (Fig.
1). Determination of the rate of
[14C]methylammonium uptake in 1-min assays using a range
of substrate concentrations from 1 to 75 µM showed one
kinetic component with the following parameters: Ks,
2.7 µM; Vmax, 169 nmol·min Inactivation of Putative amt Genes--
Internal fragments from
strain PCC 6803 ORFs sll0108, sll0537, and
sll1017 were amplified by PCR and cloned. Gene-cassettes C.K3 (sll0108) or C.C1 (sll0537 and
sll1017) were inserted into the cloned DNA fragments, and
the resulting constructions (pCSX47a, pCSX47b, pCSX56, and pCSX57; see
Fig. 4) were transferred to strain PCC
6803 by transformation. Kmr and Cmr
transformants were selected and further grown in antibiotic-containing medium, and the presence of mutant and wild-type chromosomes in these
cells was studied by hybridization and PCR amplification. Clones that
were homozygous for the mutated chromosomes were chosen and named
CSX47a (sll0108::C.K3, that carries the
gene-cassette in the same orientation as sll0108), CSX47b
(sll0108::C.K3, that carries the gene-cassette in
the orientation oposite to sll0108), CSX56
(sll0537::C.C1), and CSX57
(sll1017::C.C1). Double mutants were then
generated by transformation of strain CSX47a with pCSX56 or pCSX57,
producing strains CSX200 (sll0108::C.K3,
sll0537::C.C1) and CSX201
(sll0108::C.K3,
sll1017::C.C1), respectively, each of them
containing only chromosomes carrying the two mutations.
Glutamine synthetase and the capability to take up 1 µM [14C]methylammonium were determined in
cells incubated for 6 h in BG110 medium for each of
the mutants described above and the wild-type strain PCC 6803. Similar
glutamine synthetase activity levels, 40 to 50 units·(mg of
Chl)
The effect of the concentration of [14C]methylammonium on
the rate of uptake was studied in mutant strains CSX47a, CSX200, and CSX201. The three mutants lacked the Ks 2.7 µM kinetic component, and no saturation kinetics was
observed in any of them for [14C]methylammonium
concentrations of up to 700 µM (at this concentration, uptake rate was ~175 nmol·min Analysis of Expression of the amt Genes--
The expression of
each amt gene was studied by Northern analysis using total
RNA isolated from cells of strain PCC 6803 grown with nitrate or
ammonium as the nitrogen source or grown with nitrate and incubated in
the absence of any source of nitrogen for 6 h. As a probe, a DNA
fragment generated by PCR as described above (see also Fig. 4) was used
for each amt gene.
A single mRNA of about 2 kb was detected for amt1(Fig.
6A). The relative levels of
this transcript in the tested nitrogen regimes were 1:1.76:30
(ammonium:nitrate:minus nitrogen). An mRNA of about 1.5 kb was
observed for amt2 only in nitrogen-starved cells (Fig.
6B). (A band of about 2.6 kb was also detected with the
amt2 probe and RNA from the three different nitrogen
regimes. It is possible, however, that this band represents nonspecific hybridization with some rRNA.) Although hardly visible, a 2.6-kb mRNA was detected only in nitrogen-starved cells for
amt3 (Fig. 6C). These results showed that the
three amt genes of strain PCC 6803 are expressed and that,
in the three cases, expression is activated under nitrogen stress.
Indeed, activation of expression took place quickly in response to
nitrogen starvation, as shown in Fig. 7
for amt1 (similar results were obtained with an
amt2 probe, whereas the amt3 transcript was
hardly visible at any incubation time (not shown)).
Because the amt1 transcript was more readily detectable than
the amt2 or amt3 transcripts, amt1
appears to be expressed at higher levels than the other two genes. To
corroborate this observation, we performed experiments where
filter-bound, PCR-generated DNA fragments from the three amt
genes were hybridized to 32P-labeled total RNA. The RNA was
isolated from cells of strain PCC 6803 grown with ammonium or nitrate
as the nitrogen source or grown with nitrate and starved for nitrogen
for 6 h. With RNA isolated from nitrogen-starved cells,
hybridization was detected for the three genes, and the relative level
of labeling was 1:6:54 (amt3:amt2:amt1) (Table
II). With RNA isolated from ammonium- or
nitrate-grown cells, only hybridization to DNA corresponding to the
amt1 gene was detected.
Possible NtcA-dependent Transcription Start Point for
the amt1 Gene--
The possible tsp of amt1 was
investigated by primer extension analysis. Two oligonucleotides, Am2
and Am3, complementary to sequences located close to the 5' end of the
amt1 gene (Fig. 8A) were used as primers. An extension product whose 3' end corresponded to
a T doublet located 142 nucleotides upstream from the amt1 start codon was detected with the Am3 primer. This extension product was much more efficiently obtained with RNA isolated from
nitrogen-starved cells than from cells grown with nitrate and more with
RNA from nitrate- than from ammonium-grown cells (Fig. 8B).
The possible tsp of the amt1 gene can thus be
localized to the A doublet indicated in Fig. 8A that is
preceded by a putative
Binding of NtcA to that putative NtcA-binding site was tested by using
an extract of an E. coli strain carrying a plasmid bearing
the Anabaena sp. PCC 7120 ntcA gene as a source
of NtcA and a 138-bp DNA fragment corresponding to the first 138 nucleotides shown in Fig. 8A. Binding to a 244-bp fragment
containing the putative NtcA-binding site that is located immediately
upstream of the amt1 gene was also tested (see Fig.
8A). In both cases, a nonrelated, control DNA was included
in the assay. As shown in Fig. 8C, retardation was only
observed with the 138-bp DNA fragment containing the putative
amt1 promoter. On the other hand, no retardation was
observed in binding assays carried out with extracts of an E. coli strain bearing expression vector pQE9 (not shown). These
results suggest that the 138-bp DNA fragment bears a real NtcA-binding
site, whereas the significance, if any, of the putative NtcA-binding
site present in the 244-bp DNA fragment remains to be investigated.
Occurrence of amt Homologous Sequences in Other
Cyanobacteria--
The same DNA fragments corresponding to the three
amt genes used to probe Northern blots were used in Southern
blot analyses to investigate the presence of putative amt
homologues in some other cyanobacteria (not shown). Hybridization with
the amt1 probe was observed for every cyanobacterium tested,
including strains of taxonomic sections I (Synechococcus sp.
PCC 7942), III (Pseudanabaena sp. PCC 6903), IV
(Anabaena sp. PCC 7120, Calothrix sp. PCC 7601, and Nostoc sp. strains PCC 7107 and PCC 7413), and V
(F. muscicola UTEX 1829). Appreciable hybridization with the
amt2 probe was observed with DNA from all the strains but
UTEX 1829 and PCC 7120. A clear, though weak, hybridization signal with
the amt3 probe was only observed with DNA from UTEX 1829.
Synechocystis sp. PCC 6803 shows an activity of
[14C]methylammonium uptake that exhibits characteristics
similar to those of methylammonium uptake in other cyanobacteria
including Synechococcus sp. PCC 7942 and A. variabilis ATCC 29413 (26, 27). Thus, [14C]methylammonium uptake would result from an initial
transport of methylammonium followed by further transport and
metabolism via glutamine synthetase. Under our experimental conditions,
an accumulation of [14C]methylammonium within the cells
representing an intracellular to extracellular ratio of methylammonium
of up to about 60 was observed. Ratio values of up to 200 or 40 have
been reported for strains PCC 7942 and ATCC 29413, respectively (26,
27). An intracellular to extracellular ratio of 60 would correspond,
under the incubation conditions used in this work, to a free energy change for [14C]methylammonium transport equivalent to
+107 mV. Because the membrane potential of photosynthetically active
cyanobacterial cells is known to be in the range of Inactivation of each of the three putative amt genes of
Synechocystis sp. PCC 6803, namely sll0108
(amt1), sll0537 (amt3), and
sll1017 (amt2), impairs
[14C]methylammonium uptake (Table I, Fig. 5). Tight
inhibition by ammonium of [14C]methylammonium uptake (see
Fig. 3 and Refs. 3, 12, 16, 26, 53, 54) suggests that ammonium is the
natural substrate for the permease(s) taking up methylammonium,
especially in organisms for which methylammonium is not a nutrient. The
affinity of those permeases for ammonium would be higher than for
methylammonium (Ks can be 40 to 100 times lower for
ammonium than for methylammonium (Refs. 3, 11, 53)). The Amt1 permease,
which appears to be responsible for the Ks 2.7 µM methylammonium uptake kinetic component exhibited by
strain PCC 6803, would thus represent a transport system with a very
high affinity for ammonium and would therefore be able to mediate the
uptake of ammonium that may be found at very low concentrations in some
natural habitats. Amt2 and Amt3, on the other hand, make only a limited
contribution to uptake of methylammonium in the µM range
and could therefore represent permeases with a lower affinity for
ammonium/methylammonium than Amt1. The Synechocystis amt
mutants isolated in this work, including strains that bear only one
functional amt gene (strain CSX200:
amt1 The regulatory pattern of expression of the amt genes
suggests a major role for the Amt permeases in uptake of ammonium
present at low concentrations in the extracellular medium.
[14C]Methylammonium uptake activity, representing
transport plus metabolism via glutamine synthetase, is repressed by
ammonium and is maximal in cells starved for nitrogen in
CO2-enriched air (Fig. 1). Consistently, the three
amt genes are preferentially expressed in nitrogen-starved
cells (Fig. 6). Expression of amt1, however, is much higher
than expression of amt2 or amt3 under any of the
tested nitrogen regimes. This resembles the situation in S. cerevisiae where the gene encoding the permease with highest affinity for ammonium, MEP2, is expressed at a much higher
level than the MEP1 and MEP3 genes encoding lower
affinity permeases (11).
We have further studied the expression of amt1 and have
found that transcription of this gene takes place from a promoter that
shows the structure of the cyanobacterial NtcA-activated promoters
(31). Additionally, a DNA fragment carrying this promoter binds NtcA
in vitro (Fig. 8). Like some other genes characterized in
Synechococcus sp. PCC 7942 and Anabaena sp. PCC
7120 that are subjected to repression by ammonium (29, 42, 55-58),
amt1 appears to belong to the NtcA regulon. A
Synechocystis sp. PCC 6803 ntcA mutant is not yet
available to confirm NtcA-dependent gene expression in this
cyanobacterium. Nonetheless, NtcA-mediated nitrogen control can be
important also in Synechocystis sp. PCC 6803, because a number of genes of this strain have been shown to be transcribed from
NtcA-type promoters that bind NtcA in vitro. These include, in addition to amtl, icd coding for isocitrate
dehydrogenase (59), glnA for glutamine synthetase (60), and
glnB for the cellular nitrogen status signaling protein
PII (39).
The sizes of the transcripts for the three Synechocystis amt
genes (Fig. 6) were analyzed in relation to the DNA sequences surrounding these genes (Ref. 33; see also Fig. 4). The amt1 gene, which is composed of 1521 bp, would be transcribed as a monocistronic mRNA (observed transcript size, 2 kb). For
amt2, a gene consisting of 1326 bp, the observed 1.5-kb
hybridization band would also correspond to a monocistronic transcript.
The amt3 gene, which is composed of 1623 bp, is found
downstream of an ORF (sll0536) with which it shows a
4-nucleotide overlap. A transcript containing both ORFs
(amt3 and sll0536) would have a size of, at
least, 2693 nucleotides, which roughly corresponds to the size of the
mRNA detected with the amt3 probe (2.6 kb). Interestingly, sll0536 would encode a polypeptide that
shares homology with a putative potassium channel protein of E. coli (accession number P31069). Whether this implies a coordinated function of Amt3 and a potassium channel remains to be investigated.
The Synechocystis Amt proteins as well as their homologues
from other biological sources are highly hydrophobic polypeptides that
show 10 to 12 putative membrane-spanning regions. They appear to
constitute monocomponent permeases whose activity would not depend on
ATPase subunits or periplasmic-binding proteins. Consistently, the
methylammonium uptake activity of Synechococcus sp. PCC 7942 is preserved in spheroplasts (26). As deduced from data found in
currently available data banks, the Amt family would comprise proteins
from very diverse biological groups. In addition to the three Amt
permeases from Synechocystis sp. PCC 6803, some other members of this protein family are the three MEP proteins from yeast
(accession numbers P40260, P41948, and P53390, respectively), AMT1 from
A. thaliana (P54144), Amt from C. glutamicum
(P54146), A. brasilense (AF005275), A. vinelandii (U91902), and E. coli (P37905), and putative ammonium
permeases of B. subtilis (NrgA; Q07429), Mycobacterium
tuberculosis (Q10968), Methanococcus jannaschii (Q58739
and Q60366), and Caenorhabditis elegans (P54145).
Phylogenetic analyses of these proteins can be found elsewhere (11,
17). Interestingly, the three Synechocystis Amt proteins
show a higher identity degree to each other (40 to 43% identity) than
to any of their homologues from other organisms listed above (27 to
37% identity).
We thank Mario
García-Domínguez for help with some RNA preparations
and Georg Schmetterer for a critical reading of the manuscript.
*
This work was supported by Dirección General de
Enseñanza Superior, Spain Grants PB94-0074 and PB95-1267.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
bp, base pair(s); Ap, ampicillin; Chl, chlorophyll a; Cm, chloramphenicol; Km, kanamycin; ORF, open reading frame; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; tsp, transcription start point; PCR, polymerase chain
reaction; kb, kilobase pair(s).
3
A. M. Muro-Pastor, A. Herrero, and E. Flores, unpublished data.
2
The www address of the sequence reported in Ref.
33 is: http://www.kazusa.or.jp/cyano/cyano.html.
Ammonium/Methylammonium Permeases of a Cyanobacterium
IDENTIFICATION AND ANALYSIS OF THREE NITROGEN-REGULATED
amt GENES IN SYNECHOCYSTIS sp. PCC 6803*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
10,
Pribnow box in the form TAN3T (31). This promoter structure
for NtcA-regulated genes is predicted to be valid for other
cyanobacteria as well, because the putative helix-turn-helix motif for
binding to DNA is identical in the NtcA polypeptides from different
sources, including Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 (32).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1, and mutants carrying gene-cassette C.C1 (34)
were grown in medium supplemented with 10-80 µg of
Cm·ml1. Other cyanobacterial strains used in this work
(Anabaena sp. PCC 7120, Nostoc sp. PCC 7107, Nostoc sp. PCC 7413, Calothrix sp. PCC 7601, Fischerella muscicola UTEX 1829, Pseudanabaena
sp. PCC 6903, and Synechococcus sp. PCC 7942) were grown in
BG11 medium. Cultures used for RNA isolation and for some of the uptake
assays shown in Fig. 1, as indicated, were grown in BG11C (BG11
supplemented with 0.84 g·l1 NaHCO3) or
BG110C medium supplemented with 15 mM
NH4Cl and 30 mM TES-NaOH buffer (pH 7.5)
bubbled with a mixture of CO2 (1.5% v/v) and air. To
achieve nitrogen starvation of cultures, nitrate-grown cells
(ammonium-grown cells in the experiment shown in Fig. 7) were harvested
at room temperature, washed with and resuspended in BG110
medium (or BG110C medium, in the case of cultures used for
RNA isolation), and incubated for 6 h under culture conditions. Cyanobacterial cell mass was estimated by measuring the concentration of Chl of the cultures. Chl was determined in methanolic extracts (35).
, GM48, and BL21 were grown in LB
medium with, when necessary, 50 µg of Ap·ml1, 50 µg
of Km·ml1, or 25 µg of Cm·ml1.
2, white light) for 5 to 30 min, the assays were
started by mixing the suspension of cells (4 to 15 µg of
Chl·ml1) with a solution of
[14C]CH3NH2·HCl (50 µCi·µmol1; ICN Pharmaceuticals, Inc.) in
phosphate-bicarbonate buffer. After incubation for the time periods
indicated in each experiment, 0.1- to 1-ml samples were filtered
(0.45-µm pore size Millipore HA filters were used) and washed with 2 to 5 ml of phosphate-bicarbonate buffer. The filters carrying the cells
were then immersed in scintillation mixture, and their radioactivity
was measured. Retention of radioactivity by boiled cells was used as a
blank. In some experiments, as indicated, 1 mM
L-methionine-D,L-sulfoximine was
added to the cell suspension 25 min before the assay was started.
particles (Packard). To calculate the intracellular
concentration of [14C]methylammonium, an intracellular
volume of 125 µl·mg1 Chl was assumed (36, 37).
-35S]thio-dATP.
1 or 10 µg of
Cm·ml1. Kmr and Cmr
transformants were then grown in medium supplemented with 50 µg of
Km·ml1 or 20-80 µg of Cm·ml1. To
test whether the resulting mutant strains were homozygous for the
mutant chromosomes, PCR amplification with genomic DNA from the mutants
as templates and the corresponding primers was carried out; additional
testing was made by hybridization.
1. Filters were
washed twice at room temperature with 2× SSPE and 0.1% SDS for 10 min
and once with 1× SSPE and 0.1% SDS at 65 °C for 15 min. Total cpm
of radioactive areas in Northern blot hybridizations were determined
with an InstantImager scanner.
-32P]dCTP.
-32P]dATP (43).
Hybridization was performed at 65 °C in a solution containing: 50 mM Tris-HCl, pH 8.0, 0.2% bovine serum albumin, 0.2%
Ficoll, 0.1% sodium pyrophosphate, 1% SDS, 1 M NaCl, and 100 µg·ml1 yeast tRNA (Boehringer) (44). Filters were
washed twice at 65 °C for 30 min with 1× SSC and 1% SDS and once
at room temperature for 15 min with SSC 0.2× (1× SSC is 0.15 M NaCl and 15 mM sodium citrate (pH 7.0)).
Radioactivity of bands in filters was quantified with an InstantImager scanner.
236 to 215 with respect to the translational start of ORF
sll0108) and Am2 (5'-CGGCTATCAAAATCCAGATGGC-3';
complementary to nucleotides +289 to +268 with respect to the
translational start of ORF sll0108). The PCR product was
cloned in pGEM-T vector, rendering plasmid pCSX49. This plasmid was
used to generate dideoxy-sequencing ladders for primer extension
analysis. Oligonucleotides used for primer extension were Am2 (see
above) and Am3 (5'-GGAACACAGGCCAACCAGGGAG-3'; complementary to
nucleotides +146 to +125 with respect to the translational start of ORF
sll0108). Oligonucleotides were end-labeled with T4
polynucleotide kinase (Boehringer Mannheim) and
[-32P]dATP as described (45) and annealed with 50 µg
of total RNA from Synechocystis sp. PCC 6803 in the presence
of 10 mM Tris-HCl (pH 8.0) containing 150 mM
KCl and 1 mM EDTA. Annealing mixtures were incubated first
at 85 °C for 10 min for denaturation of RNA and then at 50 °C for
3 h for annealing. The extension reactions were carried out at
47 °C for 1 h in a final volume of 45 µl containing the whole
annealing reaction, 0.15 µg·µl1 actinomycin D, 0.75 mM each deoxynucleoside triphosphate, 0.15 units·µl1 avian myeloblastosis virus reverse
transcriptase (Boehringer Mannheim), and buffer (Boehringer Mannheim).
Reaction mixtures were then treated with RNase A (DNase-free,
Boehringer Mannheim) and extracted with phenol, and the DNA was
precipitated with sodium acetate and ethanol and resuspended in
formamide loading dye. These preparations were loaded onto 6%
polyacrylamide sequencing gels next to the corresponding
dideoxy-sequencing ladders.
1 bovine serum albumin, 8% glycerol,
75-100 ng of the DNA fragment to be tested, 75 ng of a control DNA
fragment, and 5 µl of a diluted E. coli BL21 extract in
which the NtcA protein from Anabaena sp. PCC 7120 had been
overproduced from the
isopropyl--D-thiogalactopyranoside-inducible promoter of
vector pQE9 (Quiagen, Inc).3
Extract of E. coli BL21 was prepared according to the
manufacturer's recommendations, and 1:100 and 1:200 dilutions of an
extract containing 56.8 µg·µl1 total protein were
used in the experiments. The DNA fragment used as nonrelated, control
DNA was a 223-bp fragment of pBluescript SK(+) obtained by PCR
amplification using the Universal M13 -20 and Reverse primers. Assay
mixtures were incubated at room temperature for 30 min, loaded in a
nondenaturing 6% polyacrylamide gel, and electrophoresed at 200 V. Gels were stained with ethidium bromide for visualization of DNA bands.
-glutamyltransferase assay as described
previously (46). One activity unit corresponds to one µmol of
-glutamylhydroxamate produced/min.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1.(mg of Chl)1. Methylammonium
does not serve as a nitrogen source in strain PCC 6803 (not shown), and
as is the case in some other cyanobacteria (26, 27), intracellular
radioactivity derived from [14C]methylammonium was
distributed into two metabolites (Fig.
2). One of these metabolites was
identified as methylammonium itself by co-chromatography with authentic
[14C]methylammonium. Production of the second metabolite
was hampered (96.4 and 97.5% inhibition in two independent
experiments, respectively) by treatment of the cells with
L-methionine-D,L-sulfoximine, an inhibitor of glutamine synthetase, putatively identifying that metabolite as -glutamylmethylamide (47, 48). In experiments where
[14C]methylammonium was supplied at 1 µM,
intracellular methylammonium concentrations of about 50 µM, which represented, at the time of sampling, an
intracellular to extracellular methylammonium ratio of close to 60, were detected. The process of [14C]methylammonium uptake
was completely inhibited by the addition of ammonium (Fig.
3). Inhibition by ammonium was
reversible, and the lag period before [14C]methylammonium
uptake commenced was almost proportional to the added concentration of
ammonium. Assuming that [14C]methylammonium uptake
started when ammonium was exhausted from the incubation medium, an
ammonium uptake rate of 780 nmol·min1.(mg of
Chl)1 can be calculated that is similar to the figures
obtained in direct determinations of ammonium uptake (not shown).
View larger version (25K):
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Fig. 1.
Time course of
[14C]methylammonium uptake in Synechocystis
sp. PCC 6803. Cells grown in shaken cultures (open
symbols) or in cultures bubbled with CO2-enriched air
(closed symbols) with ammonium (circles) or
nitrate (triangles) or starved for nitrogen for 6 h
(squares) were used in uptake assays with 6 µM
[14C]methylammonium as a substrate.
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Fig. 2.
Fate of [14C]methylammonium in
Synechocystis sp. PCC 6803. Nitrogen-starved cells were used in a 1-min uptake assay with 8 µM [14C]methylammonium. Metabolites were
extracted and analyzed by thin layer chromatography as described
under "Experimental Procedures." O, origin;
GMA, -glutamylmethylamide; MA,
methylammonium.
View larger version (22K):
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Fig. 3.
Inhibition by ammonium of
[14C]methylammonium uptake. Nitrogen-starved cells
(6.4 µg of Chl·ml 1) were used in uptake assays with 6 µM [14C]methylammonium and the indicated
NH4Cl concentration, which was added simultaneously with
the methylammonium. 
, no NH4Cl added; 
, 20 µM NH4Cl; 
, 40 µM
NH4Cl; 
, 60 µM NH4Cl.
View larger version (13K):
[in a new window]
Fig. 4.
Schematic representation of the
Synechocystis sp. PCC 6803 genomic regions where the
putative amt genes (sll0108,
sll1017, and sll0537) are located (derived from
Kaneko et al. (33)). Restriction sites where the C.K3
or C.C1 gene-cassette was inserted are shown along with the names of
the plasmids carrying those constructions. The approximate locations of
the primers used to amplify by PCR fragments of the putative
amt genes are also indicated.
1, were found in all the strains. On the other hand,
inactivation of ORF sll0108 abolished
[14C]methylammonium uptake by about 97%, whereas
inactivation of sll0537 or sll1017 had only
minor effects (Table I). Nonetheless, time course assays carried out with low substrate concentrations (5 to
270 nM) confirmed that inactivation of sll0537
or sll1017 further impaired
[14C]methylammonium uptake in the
sll0108-inactivated mutant (see data for 50 nM
[14C]methylammonium in Fig.
5). The three investigated ORFs appear therefore to encode permeases that mediate methylammonium uptake. We
have named them as amt1 (sll0108),
amt2 (sll1017), and amt3 (sll0537), respectively.
Methylammonium uptake activities of wild-type Synechocystis sp. PCC
6803 and some derivative insertional mutants
View larger version (25K):
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Fig. 5.
Uptake of [14C]methylammonium
by mutant strains CSX47a (sll0108::C.K3) ( ),
CSX200 (sll0108::C.K3,
sll0537::C.C1) (), and CSX201
(sll0108::C.K3,
sll1017::C.C1) (). Nitrogen-starved cells
were supplemented with 50 nM
[14C]methylammonium and assayed for methylammonium
uptake.
1.(mg of
Chl)1 in the three mutants). No difference in uptake rate
between strains CSX200 or CSX201 and CSX47a was found above 18 µM [14C]methylammonium. This suggests that,
at these substrate concentrations, inward diffusion of
[14C]methylamine may exceed
[14C]methylammonium transport mediated by Amt2 or Amt3,
thus impeding the determination of kinetic parameters of uptake in
those mutants.
View larger version (43K):
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Fig. 6.
Northern blot analysis of the expression of
the amt genes in Synechocystis sp.
PCC 6803. Total RNA, isolated from cells grown with ammonium
(NH4+) or nitrate
(NO3 ) or grown with nitrate and incubated for
6 h in the absence of any nitrogen source (N), was hybridized
with a PCR-generated probe of amt1 (A), amt2 (B),
or amt3 (C). See "Experimental Procedures" for
conditions of hybridization and for generation of DNA probes. Sizes in
kilobases are indicated on the left.
View larger version (111K):
[in a new window]
Fig. 7.
Time course of expression of the amt1
gene in response to nitrogen starvation in Synechocystis
sp. PCC 6803. Total RNA, isolated from cells grown with
ammonium (NH4+) or grown with ammonium and
incubated for the indicated times in the absence of any nitrogen
source, was hybridized with a PCR-generated probe of amt1.
Sizes in kilobases of some size markers are indicated on the
left.
Hybridization of 32P-labeled RNA to DNA fragments of the strain
PCC 6803 amt genes
10 box in the form TTGAAT. Seventeen
nucleotides upstream from this box a TACAGA hexamer is found that might
be considered a poor 35 promoter box. However, 20 nucleotides
upstream from the 10 box a nucleotide sequence,
AAAAGTAN8TAC, is found that would represent a perfect NtcA-binding site (Fig. 8A).
View larger version (36K):
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Fig. 8.
Analysis of the amt1
promoter. A, nucleotide sequence of the 5' end
and sequences upstream of amt1 (taken from Kaneko et
al. (33)). The location of primers Am1, Am2, and Am3 as well as of
the possible tsp (+1) and promoter elements ( 10 box and
NtcA-binding site) of the amt1 gene is indicated. Note a
second putative NtcA-binding site (GTAN8TAC) just in front
of the ORF start. B, primer extension using the Am3 primer
and total RNA isolated from cells grown with ammonium (1) or
nitrate (2) or grown with nitrate and incubated for 6 h
in the absence of any nitrogen source (3). The
arrow points to the extension product identifying the
putative tsp. C, band-shift assay of a DNA
fragment containing the putative promoter of the amt1 gene
(138-bp fragment, from the start of the sequence shown in A
to the BstXI site) (lanes 1 to 3) or a
DNA fragment carrying the 5' end and some sequences upstream of the
amt1 gene (244-bp fragment, corresponding to nucleotides 139 through 382 of the sequence shown in A, i.e. from
the BstXI site to the Am3 primer) (lanes 4 to
6). In addition, as a control, all assays contained a
nonrelated DNA fragment from pBluescript SK(+) (223-bp fragment). As a
source of NtcA protein, a cell-free extract from an E. coli
strain carrying an Anabaena sp. PCC 7120 ntcA
clone was used (see "Experimental Procedures"). Cell-free extract
added: 0 µg of protein (lanes 1 and 4), 1.4 µg of protein (lanes 2 and 5), and 2.8 µg of
protein (lanes 3 and 6).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
110 to 130 mV
(49, 50), the observed accumulation of methylammonium is close to that
which would be permitted by the membrane potential. The effects of some metabolic inhibitors on methylammonium uptake have also been
interpreted in terms of methylammonium transport being dependent on the
membrane potential of the cells (1, 16, 26, 27). Interestingly, methylammonium influx has been shown to decrease the membrane potential
in some algae (51, 52). It should be noted, however, that the observed
accumulation of [14C]methylammonium may represent an
underestimation of the methylammonium transport activity of the cells,
because some of the [14C]methylammonium taken up may be
leaking out from the cells by diffusion as
[14C]methylamine (24).
, amt2+,
amt3; strain CSX201: amt1,
amt2, amt3+), are
however still able to grow using ammonium as a nitrogen source (not
shown). It is currently unknown whether diffusion of ammonia or
transport of ammonium via Amt2 or Amt3 is responsible for
ammonium-dependent growth of those mutants. An impairment in ammonium-dependent growth has only been reported in a strain of S. cerevisiae lacking the three MEP genes
present in this organism (11) and in an E. coli amtB mutant
when the cells were incubated at pH values below 7 (19).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Instituto de
Bioquímica Vegetal y Fotosíntesis, Centro de
Investigaciones Científicas Isla de la Cartuja, Avda.
Américo Vespucio s/n, E-41092 Sevilla, Spain. Tel.:
+34-95-448.9523; Fax: +34-95-4460065; E-mail: flores{at}cica.es.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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