Organismes Photosynthétiques et Environnement, CNRS FRE 2433, Département de Biologie, École Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France
Correspondence
Jean Houmard
jhoumard{at}biologie.ens.fr
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ABSTRACT |
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INTRODUCTION |
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NblA (nbl stands for non-bleaching) is a polypeptide of Mr 7000 required for phycobilisome degradation in non-diazotrophic cyanobacteria (Baier et al., 2001
; Collier & Grossman, 1994
). In such cyanobacteria, phycobilisome degradation under nitrogen-limited conditions can provide an intracellular source of nitrogen, and reduce both photodamage and oxygen evolution (Collier et al., 1994
; Ochoa de Alda et al., 1996
; Sauer et al., 2001
). In Synechococcus sp. strain PCC 7942 and Synechocystis sp. strain PCC 6803, high levels of nblA transcripts (Grossman et al., 2001
; Luque et al., 2001
; Richaud et al., 2001
), as well as proteins (Baier et al., 2001
) accumulate under macronutrient limitation. For filamentous diazotrophic cyanobacteria belonging to sections IV and V (Rippka & Herdman, 1992
), the lack of a combined nitrogen source leads to differentiation of heterocysts (cells specialized for aerobic N2 fixation). Phycobiliprotein content within the heterocysts seems to vary with strain ecophysiology (Meeks & Ehlai, 2002
). In the heterocysts prepared from Azolla symbioses with either Anabaena pinnata or Anabaena caroliniana, total phycobiliprotein complement does not seem to differ from that of the vegetative cells (Kaplan et al., 1986
). In contrast, for free-living heterocyst-forming species phycobiliprotein degradation has been reported (Wood & Haselkorn, 1980
). The rationale for such a degradation was that it participates in the turning down of photosystem II activity and O2 production within the heterocysts (Wolk, 1996
). The much lower photosynthetic activity observed in situ for the cyanobacterial partner within the symbioses could be considered as an alternative strategy that has been selected by symbiotic species.
The filamentous cyanobacterium Tolypothrix sp. PCC 7601 has been widely used as a model strain, in particular because it can modulate its phycobiliprotein content according to the available light wavelengths, a phenomenon known as complementary chromatic adaptation (Grossman et al., 2001; Tandeau de Marsac & Houmard, 1993
). Although this free-living strain is unable to form functional heterocysts, a spontaneous revertant of it, strain PCC 7601/1, can form fully differentiated heterocysts and fix N2 aerobically (Rippka & Herdman, 1992
). We thus chose to use these strains to study the function of NblA in free-living filamentous heterocystous cyanobacteria. Complete genomic sequences revealed that in some strains there may exist up to four nblA gene copies. Based on sequence similarities, the Tolypothrix PCC 7601 nblA gene that we have studied was designated nblAI (Luque et al., 2003
), and this name will be used throughout. Anti-NblAI antibodies were raised and used to probe extracts from cells grown under nitrogen-depleted and nitrogen-replete conditions. From the data obtained it is concluded that NblAI has a specific role in heterocyst physiology.
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METHODS |
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To achieve nitrogen starvation, exponentially growing cells (OD750 0·75) were harvested by centrifugation (5000 g, 10 min at room temperature), washed in an equal volume of BG-110 medium, recentrifuged and resuspended in BG-110 at the original cell concentration. Non-N2-fixing cultures bleached within 1 day, as a result of a 50 % loss of phycoerythin, phycocyanin and allophycocyanin. In contrast, chlorophyll a and protein contents of the cultures remained constant during this time period (Guétat & Stanier, 1979).
Cyanobacterial cell mass was estimated by measuring the chlorophyll a concentration of the cultures. Chlorophyll a was determined in 90 % acetonic extracts (Porra, 1991). Whole-cell spectra were recorded from 350 to 800 nm with an Aminco DW-2 spectrophotometer.
Production and purification of His6-tagged NblAI.
The nblAI gene was PCR amplified as a 295 bp fragment using the following primers: forward [5'-C AGG GCG CCA TGG ACA TCC CAA TGG-3'], reverse [5'-CCA TTA GAG AAA GGA TCC GGC TTG CAG TCT-3'] (NcoI and BamHI sites underlined). After restriction by NcoI and BamHI, it was cloned into NcoI/BamHI-restricted pProEX-Htb plasmid (Gibco-BRL). The ligation mixture was used to transform Escherichia coli DH5 cells, and selection was achieved on ampicillin plates. One transformant E. coli DH5
(pProNblAI) was selected for further use, and DNA sequencing confirmed the identity of the insert to the genomic sequence.
E. coli DH5(pProNblAI) cells were grown at 37 °C to OD590
0·5 and induced for 5 h to produce His6-NblAI, by adding 0·6 mM IPTG. After centrifugation, cell pellets were resuspended (1/10 initial volume) in 50 mM Tris/HCl pH 7·8, 1 mM DTT and 1 mM Pefabloc. The cell suspension was sonicated four times for 25 s, and cell debris removed by centrifugation at 17 000 g for 20 min. The supernatant was loaded on Ni-columns according to the manufacturer's instructions, and the recombinant protein eluted with 250 mM imidazole in 50 mM sodium phosphate buffer pH 8 containing 300 mM NaCl. His6-tagged NblAI was treated with the TEV protease to remove the His-tag. Proteins were analysed by LiDS-PAGE.
Cell fractionation and PAGE.
Samples (1 ml) of cells from the late exponential phase (OD750 0·75), and from 1 day-bleached batch cultures exhibiting the same chlorophyll a concentration, were centrifuged at 2500 g, for 15 min. Pellets were washed twice with buffer (50 mM Tris/HCl pH 8·3, 0·1 mM Pefabloc, 1 mM EDTA), and stored at 20 °C until use. For electrophoresis, pellets were resuspended in 150 µl loading buffer (30 mM Tris/HCl, pH 6·8, 8 M urea, 10 %, v/v, glycerol, 5 % SDS, 5 % -mercaptoethanol and 0·005 % bromophenol blue). Samples were boiled for 5 min and centrifuged at 15 000 g for 5 min prior to loading. Proteins (10 µl samples containing the same amount of chlorophyll) were separated using Tris-Tricine gels (Schagger & von Jagow, 1987
) with a resolving gel (16·5 % T, 2·6 % C), a spacer gel (10 % T, 2·6 % C) and a stacking gel (8 % T, 2·6 % C). Electrophoresis was run at 30 V for 1 h, and then at 100 V. LiDS-PAGE was performed using the same Tris-Tricine buffer system at constant voltage (70 V) at 4 °C for 16 h.
Polyclonal antibodies.
A 14 amino acid synthetic peptide corresponding to a less-conserved region of the Tolypothrix PCC 7601 NblAI, NH2-EEQIKNLNQEQSQKC-COOH (a Cys residue was added at the carboxy-terminal end; Fig. 1), was produced by Synt:em (Nîmes, France). The peptide was purified by HPLC and conjugated to maleimide-activated key-hole limpet haemocyanin via covalent linkage to the carboxy-terminal cysteine. Two rabbits were immunized with this conjugate, using a combination of Freund's complete and incomplete adjuvants. One of these antisera (P4236) was used for the present studies, and IgGs from this serum were purified using the Avichrom kit (Sigma).
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Electron microscopy and immunogold labelling.
Tolypothrix sp. cells were centrifuged at room temperature and fixed at 4 °C for 60 min in glutaraldehyde (2 %, v/v) in 0·1 M sodium phosphate buffer pH 7·4 containing 0·4 M sucrose, and embedded in LR White medium-grade resin after dehydration. Immunolabelling reactions were performed as previously described (Lichtlé et al., 1992), using anti-NblAI antibodies at a 1/1000 dilution for 2 h, and gold-labelled goat anti-rabbit antibodies (10 and 15 nm gold particles, Bio Cell Gold Conjugates) at a 1/30 dilution for 60 min. Thin sections were examined with a JEOL CX2 electron microscope. Quantifications were performed on thin sections labelled with the 10 nm gold particles. For each strain and each culture conditions, two grids were independently labelled with 10 nm gold particles. At least 14 sections were analysed for each labelling condition and each sample. Vegetative cells, hormogonia and heterocysts, as well as a similar surface devoid of cells that represented background, were cut out from the pictures, and the corresponding surfaces weighed. For each sample, the number of gold particles was counted, and arbitrary units defined as the number of gold particles per g paper. Data were analysed using the SPSS comprehensive statistical software.
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RESULTS AND DISCUSSION |
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The gene product is predicted to have an Mr of 6654 and a theoretical pI of 5·7. Fig. 1(C) shows an alignment of some of the presently available sequences for NblA, a protein only found to date in cyanobacteria and red algae. A central part of 50 residues can be aligned, with only few amino acids conserved in all of the sequences. The closest homologue of the Tolypothrix PCC 7601 polypeptide [60 % identity (89 % similarity)] is a member of the Nostoc punctiforme (PCC 73102) nblA multigene family (genome sequence available at http://www.jgi.doe.gov). Because of its high similarity with the Nostoc punctiforme NblAI, the Tolypothrix PCC 7601 gene product will hereafter be designated NblAI. It is only 35 % identical to the NblA of the unicellular Synechococcus PCC 7942 (Fig. 1C
). The rather poor strict sequence conservation of the NblA polypeptides well agrees with the result of the hybridization experiment.
Expression of NblAI
To raise antibodies against NblAI, we chose a poorly conserved region of the protein so as to avoid, assuming that a nblA gene family may exist, possible cross-reactions with other NblA-like polypeptides. A synthetic peptide corresponding to the least-conserved region in the NblA protein family (Fig. 1B) was used to raise antibodies. BLAST searches for short nearly exact matches showed no significant overlap of the immunizing peptide with any known protein of the non-redundant database (release April 2003). The antibodies recognized the synthetic peptide, either free or conjugated to the key-hole limpet haemocyanin carrier used during the immunization process.
In parallel, the E. coli DH5(pProNblAI) strain was engineered so as to overexpress a recombinant His6-NblAI protein. After induction with IPTG, the His6-tagged NblAI produced in E. coli can easily be visualized after Coomassie blue staining, both in crude extract (Fig. 2
A, lane 1) and after purification by affinity chromatography on Ni-columns (Fig. 2A
, lane 3). After proteolysis with the rTEV protease and passage through a Ni-column, untagged NblAI was recovered in the filtrate (Fig. 2A
, lane 4). Immunodetection experiments were performed using anti-NblAI IgGs previously immunopurified by incubation with E. coli total proteins. The recombinant NblAI polypeptide, as well as its His-tag-less derivative (Fig. 2B
, lanes 3 and 4) were the only polypeptides to be recognized by the antibodies.
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Immunocytological detection of NblAI
Tolypothrix sp. PCC 7601/1 grows as long filaments of vegetative cells in a nitrate-containing medium, but under N2-fixing conditions it develops terminal heterocysts and terminal hormogonia spaced by about 200 vegetative cells. For each culture condition, two grids were independently labelled with 10 nm gold particles, and analysed. The results of the statistical analysis are shown in Table 1. The number of gold particles present in the Tolypothrix sp. PCC 7601/1 heterocysts is about six times that in vegetative cells, and this enhancement is specific to the heterocyst since we did not detect any significant NblAI accumulation within hormogonial cells (Table 1
).
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Conclusion
The stimuli that lead to phycobilisome degradation may vary depending on the organism (Richaud et al., 2001). At present NblA proteins have been studied almost exclusively in unicellular non-N2-fixing cyanobacteria, for which phycobilisome degradation appears to be a very important adaptative mechanism that improves survival of the cyanobacteria under high light conditions and during nutrient limitation (Görl et al., 1998
; Schwarz & Grossman, 1998
; Sauer et al., 2001
; van Waasbergen et al., 2002
). For the heterocyst-forming strains, nitrogen starvation leads to the switching on of the N2-fixation process and the shut-off of O2 evolution which is necessary for the functioning of the nitrogenase. Our results show that, although nblAI expression is indeed enhanced by nitrogen starvation, NblAI is also present in cells grown under nitrogen-replete conditions. These data are in agreement with the hypothesis that NblAI is a necessary cofactor recruited for phycobilisome degradation but not the triggering factor, unless a threshold concentration would be necessary (Luque et al., 2003
). In filamentous heterocystous cyanobacteria, differential expression of nblAI could thus be part of the developmental cascade of events that accompanies heterocyst differentiation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 16 December 2003;
revised 12 January 2004;
accepted 14 January 2004.
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