Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Avda. Américo Vespucio s/n, E-41092 Sevilla, Spain1
Author for correspondence: Enrique Flores. Tel: +34 954 489523. Fax: +34 954 460065. e-mail: flores{at}cica.es
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ABSTRACT |
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Keywords: ammonium transport, Amt permease, cyanobacteria, nitrogen control
Abbreviations: Chl, chlorophyll a; Cm, chloramphenicol; Km, kanamycin; Nm, neomycin
The GenBank accession number for the nucleotide sequence of the amt1 gene described in this paper is AJ311900.
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INTRODUCTION |
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Cyanobacteria are prokaryotes that carry out oxygenic photosynthesis, fixing CO2 through the reductive pentose phosphate cycle, and widely use inorganic ions as nitrogen sources. Ammonium transport in cyanobacteria, including Synechococcus sp. PCC 7942, Anabaena azollae, Anabaena variabilis and Synechocystis sp. PCC 6803, has been characterized with the use of [14C]methylammonium as a probe (Boussiba et al., 1984 ; Rai et al., 1984
; Montesinos et al., 1998
). It has been shown that [14C]methylammonium transport in these organisms is a concentrative process that is affected by metabolic inhibitors and relies upon the cell membrane potential. Uptake of [14C]methylammonium in cyanobacteria is quickly followed by its incorporation into glutamate, catalysed by glutamine synthetase, to generate
-glutamylmethylamide (Boussiba et al., 1984
; Rai et al., 1984
; Montesinos et al., 1998
). Thus, standard [14C]methylammonium uptake assays involve both transport and metabolism of methylammonium.
In Synechococcus sp. PCC 7942, the Ks for methylammonium of the uptake system is 7 µM, while the Ks for ammonium would be lower (Boussiba et al., 1984 ). In this cyanobacterium, the [14C]methylammonium uptake activity is not expressed in an ntcA mutant (Vega-Palas et al., 1990
). NtcA, which belongs to the CAP family of transcriptional regulators, is a nitrogen-control transcription factor in cyanobacteria (Herrero et al., 2001
). The structure of the NtcA-activated promoters is constituted by a -10 box in the form TAN3T and an NtcA-binding site which carries the palindromic sequence GTAN8TAC centred at about -41·5 with respect to the transcription start point (Luque et al., 1994
; Herrero et al., 2001
). Three nitrogen-regulated amt genes have been identified in Synechocystis sp. PCC 6803 and one of them, amt1, has been characterized and found to be preceded by an NtcA-type promoter sequence to which NtcA can bind in vitro (Montesinos et al., 1998
). However, because an ntcA mutant is not available for Synechocystis sp. PCC 6803, the NtcA-dependence of amt1 expression in this cyanobacterium could not be established.
In this paper we report the cloning and characterization of the amt1 gene from Synechococcus sp. PCC 7942. Our characterization of this gene has included aspects of the physiology of amt1 expression and of Amt1 function that have not been previously addressed for any other cyanobacterial ammonium transporter.
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METHODS |
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Cyanobacterial cell mass was estimated by measuring the concentration of Chl of the cultures, determined in methanolic extracts of the cells (Mackinney, 1941 ). Concentration of protein was determined by the Bradford assay (Bradford, 1976
), using ovalbumin as a standard.
E. coli strains DH5 and MC1061 were grown in LB medium with, when necessary, 50 µg ampicillin ml-1 and 25 µg Cm ml-1.
DNA and RNA isolation, manipulation and analysis.
Isolation of genomic DNA from cyanobacteria was carried out as described by Cai & Wolk (1990) . Isolation of total RNA from Synechococcus sp. PCC 7942 and from the ntcA mutant CS37 was done as described previously (Luque et al., 1994
). Other molecular biology manipulations were performed by standard procedures (Sambrook et al., 1989
).
Southern analysis was carried out as described previously (Frías et al., 1993 ) using Hybond-N+ membranes (Amersham Pharmacia Biotech). Heterologous hybridization and in situ hybridization of colonies were performed under low-stringency conditions (Ausubel et al., 2001
), using as a probe a 1 kb internal DNA fragment from the Synechocystis sp. PCC 6803 amt1 gene, obtained by PCR with the primers Tr18 (5'-GCC CAT TTC CAG AAG GAT TGC CC-3') and Tr19 (5'-GGA ATC AGT TTC CTT AAC AAA GCC-3') and plasmid pCSX23 (Montesinos et al., 1998
) as template. Transfer to nylon membranes and washing of the membranes were carried out following the manufacturers recommendations.
For Northern blots, 30 µg RNA was loaded per lane and electrophoresed in 1% agarose denaturing formaldehyde gels. As a probe, an internal DNA fragment of 334 bp from the Synechococcus sp. PCC 7942 amt1 gene, obtained by PCR amplification with primers amt2 (5'-CCG CAG ACC AAA GTG AAC C-3') and amt4 (5'-CGG CTG GAC GGC TTC AAC-3') and plasmid pCSF50 as template, was used. Hybridization was done at 65 °C in 5xSSPE (1xSSPE is 0·18 M NaCl, 10 mM sodium phosphate and 1 mM EDTA, pH 7·4), 5xDenhardts solution, 1% SDS, and 100 µg herring sperm DNA ml-1. Filters were washed twice at room temperature with 2xSSPE for 15 min and, if necessary, once at 65 °C with 2x SSPE and 2% SDS. As a control for RNA loading and transfer efficiency, the filters were reprobed with a 0·57 kb XhoIPstI fragment that contains the RNase P RNA gene (rnpB) from Synechococcus sp. PCC 7942 (Banta et al., 1992 ). For slot blots, 20 µg of each RNA sample was blotted on a filter. Probes were labelled with a DNA labelling kit (Ready to Go, Amersham Pharmacia Biotech) and [
-32P]dCTP. Radioactive areas in Northern or slot-blot hybridizations were visualized and quantified with a Cyclone storage phosphor system (Packard).
To generate plasmid pCSF50, plasmid pCSF48 (see Results) carrying a 4 kb DNA fragment from Synechococcus sp. PCC 7942 was digested with PstIEcoRI and a resulting 2·8 kb DNA fragment was cloned between the PstI and EcoRI sites of pBluescript SK(+). The insert of plasmid pCSF50 was sequenced using a T7 Sequencing kit (Amersham Pharmacia Biotech) and [35S]dATPS.
Inactivation of amt1 and construction of an amt1::lacZ fusion.
The 1·3 kb CmR gene cassette C.C2, from pRL479 (Elhai & Wolk, 1988 ), excised with HindIII and filled with the Klenow enzyme, was inserted into a unique NcoI site that is present in the pCSF50 insert, after filling it with the Klenow enzyme, to generate plasmids pCSF72 and pCSF73 (both orientations). The 5·1 kb lacZ neomycin/kanamycin-resistant (Nm/Km)R gene cassette (Thiel et al., 1995
), excised with SmaI from pPE20, was also inserted into the Klenow-enzyme-filled NcoI site of pCSF50, and a construct, pCSP3a, in which lacZ was in the same orientation as amt1, was chosen for further work.
Transformation of Synechococcus sp. PCC 7942 with plasmids pCSF72, pCSF73 and pCSP3a was performed as described by Golden & Sherman (1984) . Transformants were selected in BG110 solid medium supplemented with 2·5 mM NH4Cl + 5 mM TES/NaOH (pH 7·0) and 7 µg Cm ml-1 for pCSF72 and pCSF73, and in BG11 medium supplemented with 10 µg Km ml-1 for pCSP3a. To test whether the resulting mutant strains were homozygous for the mutant chromosomes, Southern analysis was done for pCSF72 and pCSF73 transformants using as a probe a 334 bp DNA fragment generated by PCR with primers amt2 and amt4 (see above) and plasmid pCSF50 as template, and PCR analysis was performed for pCSP3a transformants with primers amt2 (see above) and amt12 (5'-GGG GCG AAC TGT AAC ATC GAT TAC AAA AC-3'). Double recombinant, homozygous clones were obtained for the three constructs and named strain CSF72 (pCSF72 transformant), CSF73 (pCSF73 transformant), and CSP2 (pCSP3a transformant), respectively.
Methylammonium-uptake assays, glutamine synthetase activity analysis, and growth tests.
Methylammonium-uptake assays were performed essentially as described by Montesinos et al. (1998) . High-carbon wild-type or mutant Synechococcus cells grown in ammonium-containing medium and incubated with the indicated nitrogen source for 1·5 h were harvested at room temperature, washed with 20 mM KH2PO4, 10 mM NaHCO3NaOH buffer (pH 7·1) and resuspended in the same buffer to give a cell density corresponding to 512 µg Chl ml-1. After preincubation at 30 °C in the light (180 µE m-2 s-1) for 1015 min, the assays were started by mixing the suspension of cells with a solution of 14CH3NH2.HCl (1·9x106 Bq µmol-1; NEN) in phosphate/bicarbonate buffer. After incubation for the time periods indicated in each experiment, 0·11 ml samples were filtered (0·45 µm pore size Millipore HA filter was used) and washed with 25 ml phosphate/bicarbonate buffer. The filters carrying the cells were then immersed in a scintillation cocktail and their radioactivity was measured. Retention of radioactivity by boiled cells was used as a blank.
Glutamine synthetase activity was measured by using the -glutamyltransferase assay as described by Shapiro & Stadtman (1970)
. One activity unit corresponds to 1 µmol
-glutamylhydroxamate produced min-1.
To test growth of the mutants on solid medium, plates of BG110 medium unsupplemented or supplemented with 2·5mM NH4Cl, and buffered with 10 mM TES at pH 5, 6 and 7, were prepared. Because it includes ferric ammonium citrate, BG110C contains an unspecified concentration of ammonium that may reach a value of 20 µM. Drops of 10 µl cell suspensions of strains PCC 7942, CSF72 and CSF73, at 10 µg Chl ml-1, were spotted on the medium and the plates were incubated under culture conditions.
ß-Galactosidase activity.
This activity was determined in chloroform-treated cells in 10 mM Tricine/NaOH buffer (pH 7·5) with 0·72 mg ONPG ml-1 as substrate. The reaction was developed at 30 °C for 10 min and stopped by addition of Na2CO3 at 0·74 M (final concentration). One activity unit corresponds to the production of 1 µmol o-nitrophenol min-1.
Purification of NtcA from induced extracts of E. coli DH5(pCSI26).
The NtcA recombinant protein from Synechococcus sp. PCC 7942 (Luque et al., 1994 ) was purified from extracts of induced cells of E. coli DH5
(pCSI26), which overexpressed NtcA from the trc promoter, a synthetic IPTG-inducible promoter. E. coli DH5
cells carrying plasmid pCSI26 were grown overnight in LB medium supplemented with 100 µg ampicillin ml-1. Thirty millilitres of this saturated culture was used to inoculate 500 ml fresh medium that was incubated at 37 °C with shaking (200 r.p.m.) for 90 min. IPTG was then added to a final concentration of 1 mM and the cells were further incubated for 2 h under the same conditions. Cells were harvested at 10000 g for 5 min at 4 °C and resuspended in cold buffer A, containing 20 mM sodium phosphate (pH 7·0), 0·2 M NaCl and 10% (v/v) glycerol, at a ratio of 5 ml buffer A per g cells. The cells were then disrupted by four cycles of 30 s sonication, with a Branson 250 sonifier at 60 W, followed by 30 s incubation on ice. Soluble fractions were obtained after two successive centrifugations at 31000 g for 30 min and 142000 g for 90 min, both at 4 °C.
Cell-free extracts from the cells of 12 l induced culture were applied to a 4 ml heparin-Sepharose column equilibrated with buffer A, at a flow rate of 0·2 ml min-1, washed with 10 column volumes of the same buffer and, finally, eluted with a 0·21 M NaCl gradient, followed by 10 ml buffer A supplemented with 1 M NaCl, at 0·06 ml min-1. NtcA was eluted at a salt concentration of 0·85 M. Fractions containing NtcA were mixed, diluted with 20 mM sodium phosphate and 10% glycerol to decrease salt concentration, and subjected again to the same chromatography process.
Band-shift assays.
A 159 bp DNA fragment containing the putative promoter from the Synechococcus sp. PCC 7942 amt1 gene, obtained by PCR using primers amt9 (5'-CCC CTC GAC TTA TCT ATG-3') and amt10 (5'-CTA GGT ACC GCT GAG CAG-3') and plasmid pCSF50 as template, was end-labelled using [-32P]ATP and T4 polynucleotide kinase (Boehringer Mannheim). The reaction was carried out in the buffer supplied by the manufacturer, with 0·51 U enzyme µl-1 and 9 x 105 Bq [
-32P]ATP. After incubation for 1 h at 37 °C, labelled DNA was precipitated with ethanol and non-incorporated nucleotides were removed by consecutive washes with 70% (v/v) ethanol. Finally, the DNA was resuspended in 10 mM Tris/HCl and 0·1 mM EDTA (pH 7·0) buffer. Binding assays were performed essentially as described previously (Luque et al., 1994
) using purified NtcA protein isolated from E. coli DH5
(pCSI26) (see above). For competition experiments, a 160 bp fragment of the polylinker of plasmid pGEM-T, obtained by PCR amplification with reverse and universal primers, was used as unrelated DNA.
Primer extension analysis.
Plasmid pCSF50 was used to generate dideoxy-sequencing ladders for extension analysis. Oligonucleotides used for primer extension were amt7 (5'-CGC AGG CGC TAC ACC TCG AGG C-3'; complementary to nucleotides +24 to +2 with respect to the translation start of the Synechococcus amt1 gene) and amt8 (5'-GAA AGT CAG GAA CAG CAG TGG-3'; complementary to nucleotides +81 to +60). Oligonucleotides were end-labelled with T4 polynucleotide kinase (Boehringer Mannheim) and [-32P]ATP as described by Ausubel et al. (2001)
and annealed with 1025 µg total RNA from Synechococcus sp. PCC 7942 and mutant strain CS37, in the presence of 10 mM Tris/HCl (pH 8·0), 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 1 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, 0·25 mM each deoxynucleoside triphosphate, 200 U reverse transcriptase (Superscript II, Gibco-BRL) and buffer (Gibco-BRL). Reaction mixtures were then treated with RNase A (DNase-free, Boehringer) and extracted with phenol. The extended fragments were precipitated with sodium acetate and ethanol, resuspended in loading dye, and loaded onto 6% polyacrylamide-urea sequencing gels next to the corresponding sequencing ladder. Images of radioactive gels were obtained using a Cyclone storage phosphor system (Packard).
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RESULTS |
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Inactivation of the putative amt1 gene
The 1·3 kb gene cassette C.C2 was inserted into an internal NcoI site of the putative Synechococcus amt1 gene, in both orientations, rendering plasmids pCSF72 (gene cassette in opposite orientation to amt1) and pCSF73 (amt1 and gene cassette in the same orientation), respectively. These constructs were transferred to strain PCC 7942 by transformation. CmR transformants were selected and their genomic structure was studied by hybridization. Clones homozygous for the mutated chromosomes were named CSF72 and CSF73, respectively. Glutamine synthetase activity and the activity of uptake of 5 µM [14C]methylammonium were determined in cells from high-carbon cultures grown with ammonium and incubated for 1·5 h in the presence of ammonium or nitrate or without any nitrogen source under the same carbon conditions. While similar glutamine synthetase activity levels [43·260·7 U (mg Chl)-1 in the presence of ammonium, 69·881·3 U (mg Chl)-1 in the presence of nitrate, and 74·483·7 U (mg Chl)-1 in the absence of nitrogen] were found for the wild-type strain and the amt1 mutants, the mutants showed only 12·4% [14C]methylammonium uptake activity as compared to the wild-type (Table 1). The time-course of [14C]methylammonium uptake was studied in cells of strains PCC 7942, CSF72 and CSF73 grown with ammonium and incubated for 1·5 h in medium lacking combined nitrogen, showing again that [14C]methylammonium uptake was drastically hampered in the mutants (Fig. 1
). These results indicated that the product of the cloned ORF is indeed required for methylammonium transport in Synechococcus sp. PCC 7942 and identified that ORF as the amt1 gene.
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Expression of amt1
The expression of the amt1 gene was studied by Northern analysis using RNA of Synechococcus sp. PCC 7942 and the ntcA mutant strain CS37. Total RNA was isolated from cells grown on ammonium under high-carbon conditions and incubated under the same carbon conditions for 1·5 h in the presence of ammonium or nitrate or in the absence of any nitrogen source. As a probe, an internal DNA fragment of Synechococcus amt1 was used. A single transcript of 2 kb was detected with RNA from the wild-type whose abundance strongly increased in the absence of ammonium, but it was not detected with RNA from strain CS37 (Fig. 3a). These results indicated that amt1 expression is subjected to nitrogen control and is dependent on the global nitrogen regulator NtcA.
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To investigate further the influence of the carbon/nitrogen balance on amt1 expression, a gene cassette consisting of a promoterless lacZ gene followed by a (Nm/Km)R encoding gene (Thiel et al., 1995 ) was inserted into the NcoI site of Synechococcus amt1. A Synechococcus clone homozygous for mutant chromosomes in which the lacZ-(Nm/Km)R insert was located such that the amt1 promoter drives lacZ was isolated and named CSP2. After transfer of ammonium-grown CSP2 cells to fresh medium, cellular levels of ß-galactosidase activity increased linearly for at least 56 h. Rates of ß-galactosidase expression in this strain could therefore be investigated under different carbon- and nitrogen-regimes. Cells grown and incubated under high-carbon supply showed a normal pattern of nitrogen regulation, with higher rates of ß-galactosidase expression being observed in the absence than in the presence of ammonium (Table 2
). However, when high-carbon-grown cells were transferred to air levels of CO2, no ß-galactosidase induction was observed even in the absence of ammonium. On the other hand, when low-carbon-grown CSP2 cells were transferred to the same low-carbon conditions, ß-galactosidase induction was observed, although the highest rate of ß-galactosidase expression was observed when the low-carbon-grown cells were transferred to a medium exposed to high-carbon conditions and lacking any source of nitrogen (Table 2
). These results stress the importance of the carbon supply for amt1 expression.
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DISCUSSION |
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The Amt1 permease is dispensable for growth in nitrogen-replete media, and the amt1 mutants were indeed isolated in media containing 2·5 mM ammonium. We have obtained evidence, however, for a role of Amt1 in scavenging of ammonium from the medium when it is found at low concentrations such as those present in BG110 medium (20 µM). Amt1 may also have a role in recapture of ammonium leaked out from the cells when a nitrogen source such as nitrate is being used. Because ammonium solutions always contain ammonia, and because of the high permeability of lipid bilayers for ammonia (Howitt & Udvardi, 2000
), this compound may leak out from the cells when they are incubated in media containing concentrations of ammonium lower than those present within the cells (i.e. those generated during the assimilation of other nitrogen sources). Amt1 appears therefore to be involved in nitrogen nutrition when the cells are exposed to a limiting ammonium concentration or to alternative nitrogen sources. This proposed role for the Amt1 protein is consistent with the observed regulation of the amt1 gene, whose expression is increased in the absence of added ammonium. The concentration of ammonia present in solutions of high ammonium concentration appears to be sufficient to permit entrance into the cell, by diffusion, of an amount of ammonia high enough to support cell growth (Soupene et al., 1998
).
A Synechococcus ntcA mutant, affected in global nitrogen regulation, was originally described to lack expression of methylammonium transport activity (Vega-Palas et al., 1990 ). We have now shown that amt1 expression is NtcA-dependent and that amt1 is transcribed from an NtcA-activated promoter. As is the case for other NtcA-activated genes, NtcA activates transcription of amt1 in response to ammonium withdrawal. However, in cells using nitrate as the nitrogen source, detection of amt1 mRNA also depends on the supply of inorganic carbon. A similar behaviour has been described for an NtcA-dependent promoter of the Synechococcus glnB gene encoding the signal transduction protein PII (Lee et al., 1999
). Although the degree of induction of amt1 detected with an amt1::lacZ transcriptional fusion was lower than that estimated by DNARNA hybridization, studies on the rate of ß-galactosidase expression in the fusion strain CSP2 also indicated the importance of the carbon supply for determining the level of amt1 expression. Thus, low-carbon-grown CSP2 cells, which would be adapted to assimilate air levels of CO2, show nitrogen-regulated ß-galactosidase expression when incubated under low- as well as under high-carbon conditions. In contrast, high-carbon-grown CSP2 cells do not induce ß-galactosidase in response to nitrogen step-down when they are incubated under low-carbon conditions, likely reflecting a transient situation of carbon stress due to the absence of a carbon-concentrating system(s) in these cells. Our results suggest that, rather than just to ammonium, amt1 expression responds to the carbon/nitrogen balance of the cell. Because expression of amt1 is under the control of NtcA, it may be suggested that in the Synechococcus cells NtcA responds to signals of carbon as well as of nitrogen availability, thus integrating nitrogen assimilation with the photosynthetic assimilation of CO2.
Although lower than in cells incubated in the absence of ammonium, a substantial activity of [14C]methylammonium uptake was observed in ammonium-grown cells (Table 1). Inactivation of amt1 resulted in a drastic reduction of [14C]methylammonium uptake in ammonium-grown cells, indicating that the uptake activity detected in ammonium-grown wild-type cells is mediated mainly by Amt1. Wild-type cells incubated in the absence of ammonium showed uptake activities 2·22·4-fold higher than ammonium-grown cells, whereas amt1 mRNA levels under high-carbon conditions were about fivefold higher in the absence than in the presence of ammonium. These differences between activity and mRNA levels could result from differences in stability of the protein, as compared to that of mRNA, under the different nitrogen conditions. Lack of correlation between yeast MEP2 gene expression (Marini et al., 1997
) and accumulation of the MEP2 protein (Lorenz & Heitman, 1998
) has also been observed, with the MEP2 protein being present at levels higher than expected in cells grown with high concentrations of ammonium. An alternative possibility to explain lack of correlation between amt1 mRNA and methylammonium transport activity levels could be that, for induced cells, a factor other than Amt1 poses an upper limit to total methylammonium uptake. Since uptake involves the transport and metabolism of methylammonium, such a factor could obviously be glutamine synthetase activity.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 31 August 2001;
revised 12 November 2001;
accepted 19 November 2001.
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