Unité; des Cyanobacté;ries, CNRS URA 2172, Département de Microbiologie Fondamentale et Mé;dicale, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris Cedex 15, France1
Laboratoire de Chimie Bacté;rienne, CNRS, 31 chemin Joseph Aiguier, BP71 13277, Marseille Cedex 9, France2
Station Biologique de Roscoff, CNRS UMR 1931, 29682 Roscoff Cedex, France3
Author for correspondence: Nicole Tandeau de Marsac. Tel: +33 1 45 68 8415. Fax: +33 1 40 61 3042. e-mail: ntmarsac{at}pasteur.fr
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ABSTRACT |
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Keywords: Prochlorales, glnB and glnK genes, lack of PII phosphorylation, inorganic carbon uptake, Prochlorococcus marinus MED4
Abbreviations: Ci, inorganic carbon; MSX, L-methionine-D,L-sulfoximine
a The GenBank accession number for the glnB gene sequence reported in this paper is AJ271089.
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INTRODUCTION |
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The PII protein (glnB gene product) is one of the most widespread signal transducers in the control of nitrogen metabolism (Arcondéguy et al., 2001 ; Ninfa & Atkinson, 2000
; Merrick & Edwards, 1995
). It has been found in archaea and bacteria, as well as in algae and plants. In Escherichia coli, the paradigm for PII structure and function in bacteria, PII is modified by uridylylation at a conserved Y51 residue, located at the tip of a solvent-exposed loop (Cheah et al., 1994
; Jaggi et al., 1996
). In contrast, the PII protein of the cyanobacterium Synechococcus sp. PCC 7942 is phosphorylated at residue S49 (Forchhammer & Tandeau de Marsac, 1994
). In both organisms, the trimeric PII protein binds the small effectors ATP and 2-oxoglutarate in a synergistic manner (Forchhammer & Hedler, 1997
; Jiang & Ninfa, 1999
; Kamberov et al., 1995
). However, the PII signal transduction pathway has diverged during evolution. In E. coli, the main signal for cellular nitrogen status is glutamine, the level of which is sensed by the uridylyl-transferase/uridylyl-removing (UTase/UR) enzyme complex (Jiang et al., 1998a
, b
; Reitzer, 1996
). PII transduces the perceived signal to proteins that modulate glutamine synthetase activity and control the expression of genes regulated by nitrogen availability (Jiang et al., 1998b
, c
; Jiang & Ninfa, 1999
). In cyanobacteria, as exemplified by Synechococcus sp. PCC 7942, a protein serine kinase and a phosphatase regulate the phosphorylation state of PII (Forchhammer, 1999
; Irmler et al., 1997
). Furthermore, 2-oxoglutarate rather than glutamine may serve as the main signal for the cellular status of nitrogen. High levels of 2-oxoglutarate favour the phosphorylation of PII, and low levels lead to its dephosphorylation (Forchhammer, 1999
; Forchhammer & Tandeau de Marsac, 1995a
; Irmler et al., 1997
; Muro-Pastor et al., 2001
). In the presence of the unphosphorylated form of PII, under an ammonium regime, an inhibition of nitrate and nitrite uptake is observed, whereas in the absence of ammonium the phospho-PII, liganded to an effector (probably 2-oxoglutarate), alleviates this inhibition (Lee et al., 1998
, 1999
). In Synechocystis sp. PCC 6803, the redox state of the cells may act as a trigger for PII phosphorylation (Hisbergues et al., 1999
). Moreover, under a high inorganic carbon (Ci) regime, the PII system relieves inhibition of nitrate uptake and inhibits the high affinity transport system for bicarbonate. While it is now established that in two unicellular freshwater cyanobacteria, Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803, the role of PII is to co-ordinate nitrogen and carbon metabolism, in filamentous strains the role of this protein remains unclear (Hanson et al., 1998
; Liotenberg et al., 1996
).
A second PII protein, termed GlnK, has been identified in E. coli (van Heeswijk et al., 1996 ). PII and GlnK are structurally similar, but in part functionally distinct proteins (Atkinson & Ninfa, 1998
). A PII-like protein has also been identified in the chloroplast genome of the red alga Porphyra purpurea (Reith & Munholland, 1993
) and of higher plants such as Arabidopsis thaliana and Ricinus communis (Hsieh et al., 1998
). In eukaryotes, PII may serve as part of a complex signal transduction network involved in the recognition of the status of carbon and nitrogen, as has been proposed for PII in cyanobacteria (Forchhammer & Tandeau de Marsac, 1995b
; Hisbergues et al., 1999
).
We report here the characterization of the PII protein (glnB gene product) and some aspects of carbon and nitrogen assimilation of the marine cyanobacterium Prochlorococcus marinus PCC 9511, which grows in the presence of ammonium or urea, but not with nitrate or nitrite. During the course of the present work, the genome sequence of P. marinus MED4, a strain identical at the subspecies level to P. marinus PCC 9511 (Laloui et al., 2002 ; Rippka et al., 2000
), was completed. This provided a means to confirm the biochemical results obtained with P. marinus PCC 9511 and helped us to obtain deeper insights into the putative role of PII in a strain not yet amenable to genetic studies.
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METHODS |
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Plasmids were maintained in E. coli strain DH5 Mcr-. Recombinant E. coli strains were grown at 37 °C in LuriaBertani medium supplemented with 100 µg ampicillin ml-1.
Nucleic acid methods.
DNA was extracted from pooled pellets corresponding to a total culture volume of 1 l and an OD750 of about 0·150·17. After two washes in 10 ml NET solution (6 mM Tris/HCl, pH 8, 1 M NaCl and 100 mM Na2-EDTA, pH 8), the cells lysed following resuspension in 10 ml 10 mM Tris/HCl, 20 mM EDTA, pH 8. Two extractions were performed with phenol/chloroform (1:1, v/v) followed by two with chloroform/isoamylalcohol (24:1, v/v). DNA was ethanol-precipitated, washed once with 70% (v/v) ethanol, air-dried and resuspended in 10 mM Tris/HCl, 0·1 mM EDTA, pH 8.
DNA gel electrophoresis, blotting and hybridizations were carried out as described by Damerval et al. (1989) . Prehybridization (4 h) and hybridization (16 h) experiments were performed at 65 and 55 °C, respectively. An internal HindIIIBsaB1 fragment (236 bp) of the glnB gene from Calothrix sp. PCC 7601 (accession no. X97327) was used as a probe. The probe was labelled with [
-32P]dATP (110 Tbq mmol-1) by using a Megaprime random labelling kit (Amersham).
A partial library was constructed by ligating XbaI DNA fragments of approximately 4 kb into the dephosphorylated pBluescript SK- vector as described by Sambrook et al. (1989) . Ligated DNA was transformed by electroporation (Bio-Rad) into E. coli strain DH5
Mcr_ (Dower et al., 1988
). The recombinant plasmid DNA from the clone carrying the proper insert was purified with the QIA filters Qiagen kit (ref. 12262) and sequenced on both strands (Genome Express).
PII analysis.
Cultures of P. marinus PCC 9511 were grown to an OD750 of about 0·150·17. Cells were harvested by centrifugation at 12000 g for 15 min at 18 °C and resuspended to an OD750 of approximately 1·5 in the medium used for the experimental tests, containing 400 µM (NH4)2SO4, no nitrogen source, 100 µM NaNO3 without CO2 enrichment or 100 µM NaNO3 with 3% (v/v) CO2. After 18 h incubation under these experimental conditions, cell suspensions were concentrated approximately 10 times by centrifugation and incubated for an additional period of 6 h before proceeding to the preparation of the cell-free extracts as described by Forchhammer & Tandeau de Marsac (1994) . For PII analyses of Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803, cell extracts were prepared from experimental cultures incubated as described by Lee et al. (1998)
. Protein content in cell-free extracts was estimated by using the Bio-Rad protein assay with BSA (Sigma A-9647) as standard. Native PAGE, immunoblotting and detection with Synechococcus sp. PCC 7942 PII antisera were carried out as described by Forchhammer & Tandeau de Marsac (1994)
. PII was visualized using the ECL detection system of Amersham in which skim dried milk, used in the blocking reagent, was replaced by BSA and the solutions for primary and secondary antibodies contained 0·25% (v/v) Tween (Sigma P-4675) and BSA at final concentrations of 3 and 1% (w/v), respectively, in 150 mM NaCl, 15 mM Tris/HCl, 1mM EDTA, pH 7·5.
Chlorophyll determination.
The concentration of chlorophylls a and a2 were determined spectroscopically from methanolic extracts using the extinction coefficient of chlorophyll a at 665 nm (74·5; Mackinney, 1941 ).
Enzyme assays.
Nitrate and nitrite reductase activities were determined as described by Lee et al. (2000) .
The nitrate uptake assay was performed as described by Lee et al. (1998) , except that cells were resuspended in standard N-free growth medium and incubated at 20 °C under a photosynthetic photon flux density of 50 µmol quanta m-2 s-1.
For the bicarbonate uptake assay, cells were resuspended in artificial seawater (Turks Island Salt Solution; Merck Index no. 9954; Rippka et al., 2000 ) buffered with 50 mM HEPES, pH 8, to a concentration of 1012 µg chlorophyll a2 ml-1 (equivalent to an OD750
1). The concentrated cell suspension was preincubated in a Clark electrode, under a photosynthetic photon flux density of 400 µmol quanta m-2 s-1, until carbon storage was exhausted as shown by the levelling off of the O2 emission rate. Bicarbonate uptake activity was then measured as described by Bédu et al. (1995)
on cells incubated under 70 µmol quanta m-2 s-1.
Phylogenetic analysis.
Distance analysis (neighbour-joining) and maximum-parsimony methods were performed using the PHYLIP package (version 3.57c) (Felsenstein, 1988 ). The Dayhoff option was employed to compute evolutionary distances. Bootstrap analyses (100 replicates; Felsenstein, 1985
) were performed for both analyses.
In silico analysis of the P. marinus MED4 genome.
The search for orthologues in the genome of strain MED4 (http://genome.ornl.gov) was performed by using the Synechocystis sp. PCC 6803 genome database (CyanoBase; http://www.kazusa.or.jp/cyano/) and TBLASTN version 2.2.1. Sequences from other micro-organisms were extracted from GenBank or SWISS-PROT.
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RESULTS AND DISCUSSION |
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In contrast to proteobacteria, the presence of additional glnB-like genes has not been reported in cyanobacteria so far. In agreement, only one band was detected in Southern hybridizations using the glnB gene of P. marinus PCC 9511 as probe, irrespective of the restriction enzymes employed (data not shown) and, like in other cyanobacterial genomes, no equivalent of glnK was found in strain MED4.
Analysis of PII modification
The isoforms of the PII protein can be separated by electrophoresis on native PAGE. The molecular masses of the PII proteins from P. marinus PCC 9511, Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942 are nearly identical (12314·18, 12397·38 and 12391·36 Da, respectively), but their isoelectric points differ (5·28, 6·33 and 7·95, respectively) leading to the difference in electrophoretic mobility observed in Fig. 2 (http://kr.expasy.org/tools/).
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The lack of in vivo modification of PII in P. marinus PCC 9511 is in contrast to the results obtained with the unicellular freshwater strains Synechococcus sp. PCC 7942 (Forchhammer & Tandeau de Marsac, 1994 , 1995a
; Lee et al., 1999
) and Synechocystis sp. PCC 6803 (Hisbergues et al., 1999
). In these cyanobacteria, the degree of PII phosphorylation depends on the nitrogen source (Forchhammer & Tandeau de Marsac, 1994
). In the presence of ammonium, PII is rapidly and completely dephosphorylated, whereas in the presence of nitrate different degrees of PII phosphorylation can be observed, depending on the supply of CO2 to the cells (Lee et al., 1999
). Modification of the PII protein was not observed in the filamentous heterocystous strains, Nostoc sp. PCC 73102 (Hanson et al., 1998
) and Calothrix sp. PCC 7601 (Liotenberg et al., 1996
). In the latter strain, however, and in contrast to P. marinus PCC 9511, a very low level of modification of PII was achieved after incubation of the cells in the simultaneous presence of ammonia and MSX.
A number of ORFs from Synechocystis sp. PCC 6803 have no orthologues in P. marinus MED4 which possesses a much smaller genome (3·6 and 1·7 Mb, respectively). In particular, no orthologues of the 11 genes encoding potential serine/threonine eukaryotic kinases in Synechocystis sp. PCC 6803 were found. However, three orthologues of the five genes encoding ABC1-like proteins could be identified (Tables 1 and 2
). According to Shi et al. (1998)
, the latter family of proteins contains the minimum complement of the amino acid sequence features essential for phosphotransferase activity, though their target and kinase activity remain to be demonstrated. Of the 13 putative phosphatases present in Synechocystis sp. PCC 6803, only two shared significant similarities with ORFs identified in the P. marinus MED4 genome (Tables 1
and 2
). None of the latter corresponds, however, to the phosphatase PP2C-type protein sll1771 shown by Irmler & Forchhammer (2001)
to specifically dephosphorylate the PII protein in Synechocystis sp. PCC 6803. These analyses are in favour of a limited PII-specific kinase/phosphatase system in members of Prochlorococcus and in full agreement with the lack of PII modification observed by biochemical means.
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In strain MED4, the genes for the reductases and the uptake system for nitrate and nitrite are lacking (Table 2). The same holds true for nrtP, a gene encoding a novel type of nitrate/nitrite permease first described for the coastal cyanobacterium Synechococcus sp. PCC 7002 (Sakamoto et al., 1999
). P. marinus strains MED4 and PCC 9511 are therefore naturally deficient for assimilation of these nitrogen sources. In contrast, the ure genes encoding the urease complex that have been characterized in strain PCC 9511 (Palinska et al., 2000
) and amt1, one of the three ORFs encoding ammonium permeases in Synechocystis sp. PCC 6803, are conserved in strain MED4 (Tables 1
and 2
).
Kinetics of bicarbonate uptake
Cells of P. marinus PCC 9511 incubated under experimental conditions as described in Methods, display a low (Km,app 240 µM) and a high (Km,app 4 µM) affinity bicarbonate uptake system (Fig. 3). In the cyanobacterium Synechocystis sp. PCC 6803, the activity of a high affinity Ci transport system, expressed under a low Ci concentration, is under the control of PII. In this strain, a function of PII as 2-oxoglutarate sensor was proposed, with the 2-oxoglutarate-PII complex inhibiting, directly or indirectly, the high affinity Ci transport system independently of the phosphorylation state of PII. Such a control of Ci acquisition may also be exerted in P. marinus PCC 9511, incapable of PII phosphorylation.
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Concluding remarks
In both Synechococcus PCC 7942 and Synechocystis sp. PCC 6803, the signal transducer PII coordinates both nitrogen and carbon metabolism (Hisbergues et al., 1999 ; Lee et al., 2000
). The nitrate and nitrite assimilation pathway is absent in P. marinus MED4, a subspecies identical to PCC 9511 (Rippka et al., 2000
). Moreover, the number of putative serine/threonine kinases and phosphatases is limited. Finally, if PII exerts a regulatory function in members of Prochlorococcus, its interplay will need to proceed without post-translational modification of the protein.
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ACKNOWLEDGEMENTS |
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Received 20 December 2001;
revised 11 February 2002;
accepted 16 April 2002.