Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M503153200 on August 11, 2005

J. Biol. Chem., Vol. 280, Issue 40, 33935-33944, October 7, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/40/33935    most recent
M503153200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cadoret, J.-C.
Right arrow Articles by Houmard, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cadoret, J.-C.
Right arrow Articles by Houmard, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Cyclic Nucleotides, the Photosynthetic Apparatus and Response to a UV-B Stress in the Cyanobacterium Synechocystis sp. PCC 6803*{boxs}

Jean-Charles Cadoret{ddagger}1, Bernard Rousseau{ddagger}, Irène Perewoska{ddagger}, Cosmin Sicora§, Otilia Cheregi§, Imre Vass§, and Jean Houmard{ddagger}2

From the {ddagger}Organismes Photosynthétiques et Environnement, CNRS FRE 2433, Département de Biologie, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France and §Institute of Plant Biology, Biological Research Centre, P. O. Box 521, 6701 Szeged, Hungary

Received for publication, March 22, 2005 , and in revised form, July 22, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclic nucleotides cAMP and cGMP are ubiquitous signaling molecules that mediate many adaptative responses in eukaryotic cells. Cyanobacteria present the peculiarity among the prokaryotes of having the two types of cyclic nucleotide. Cellular homeostasis requires both cyclases (adenylyl/guanylyl, for their synthesis) and phosphodiesterases (for their degradation). Fully segregated null mutants have been obtained for the two genes, sll1624 and slr2100, which encode putative cNMP phosphodiesterases. We present physiological evidence that the Synechocystis PCC 6803 open reading frame slr2100 could be a cGMP phosphodiesterase. In addition, we show that Slr2100, but not Sll1624, is required for the adaptation of the cells to a UV-B stress. UV-B radiation has deleterious effects for photosynthetic organisms, in particular on the photosystem II, through damaging the protein structure of the reaction center. Using biophysical and biochemical approaches, it was found that Slr2100 is involved in the signal transduction events which permit the repair of the UV-B-damaged photosystem II. This was confirmed by quantitative reverse transcriptase-PCR analyses. Altogether, the data point to an important role for cGMP in signal transduction and photoacclimation processes during a UV-B stress.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Light provides to photosynthetic organisms the energy for life and, thus, is a key environmental factor. However, it may also produce important damages when the incoming flux of excitation energy over-whelms the metabolic capacities of the cells. A variety of cellular and molecular responses exist that allow the organisms, among which are cyanobacteria, to cope with and adapt to changes in the intensity and/or spectral quality of light (1, 2). Extensive research efforts have focused on the elucidation of the molecular mechanisms that regulate acclimation and adaptation of photosynthetic organisms to high light and UV radiations. The latter, through UV-B-generated radicals in particular, have a number of negative impacts on cell physiology, damaging nucleic acids, proteins, and/or lipids (3–5). Among their effects, UV-B photons are known to cause important damages to the photosynthetic apparatus, leading to decreased oxygen evolution and CO2 fixation (6–8) and decreases in biomass production, secondary sugars, and chlorophyll content as well as to inactivation of ATPase (9).

In cyanobacteria more than 99% of the UV-B is absorbed by chlorophyll-binding proteins and phycobilisomes, the light-harvesting complexes (10). One of the main targets of the UV-B radiation is Photosystem II (PSII),3 whose electron transport is inhibited and D1 and D2 reaction center proteins are degraded (11–18). The ensuing degradation of the D1 and D2 proteins, which compose the heart of the PSII reaction center, leads to an inhibition of the PSII activity (19). Among the strategies developed to cope with UV-B, cyanobacteria increase their de novo synthesis of the D1 and D2 subunits to repair PSII (20, 21).

To adapt to new conditions, cells must first perceive the environmental signals and then transduce them to the response apparatus so as to modify their metabolism accordingly. Second messengers such as cyclic nucleotides (cNMPs) play key roles in the transduction steps (22). Cyanobacteria are the only prokaryotes that, like eukaryotes, possess both cAMP and cGMP, their precise role being, however, poorly documented at present (23, 24). Information has been obtained from different strains, and currently only an incomplete picture of the role of cNMPs is available. For Spirulina cells, which excrete cAMP under standard growth conditions, cAMP stimulates respiration and gliding motility and triggers mat formation (24). In Anabaena flos-aquae, the extracellular cAMP concentration was shown to be 10-fold higher in stationary than in exponential phase (25). In Anabaena cylindrica, light-off and light-on signals modulate the intracellular cAMP concentration (26). cAMP-mediated photosignaling through phytochrome-like proteins has now been demonstrated in Anabaena PCC 7120 (27). In contrast to what happens in A. cylindrica, the cellular cAMP concentration increases upon a shift from dark to light, especially blue light, in Synechocystis PCC 6803 (24, 28). We also know that in that strain, both cAMP and its receptor protein (Sycrp1) are required for motility. Mutants in either the adenylyl cyclase or Sycrp1 are indeed sessile, motility for cya1 mutants being recovered by the addition of exogenous cAMP (29, 30). Finally, in that same strain the cGMP concentration increases when cells are grown photoheterotrophically and starved for nitrogen (31).

Cyclic nucleotide homeostasis requires both cyclases to achieve their synthesis from ATP or GTP and phosphodiesterases for their degradation to AMP or GMP. By complementation of Escherichia coli cya mutants, a few cyanobacterial genes coding for adenylyl cyclases have been cloned (32). The recent availability of complete genome sequences for 13 cyanobacterial strains has revealed that many putative adenylyl cyclases may exist within a single species, with at least 6 present in Anabaena/Nostoc PCC 7120 (33) and up to 13 in Trichodesmium erythraeum.4 Both an adenylyl and a putative guanylyl cyclase have been described for Synechocystis PCC 6803 (29, 34). Although phosphodiesterase enzymatic activities have been measured for cAMP degradation in a few species (35), no orthologue of bacterial-type phosphodiesterases could, however, be recognized by in silico approaches in Synechocystis PCC 6803 (36).

Two Synechocystis PCC 6803 open reading frames (slr2100 and sll1624) carry a so-called HD (or phosphohydrolase) domain. Belonging to the HD protein family, they were proposed to be putative cNMP phosphodiesterases (36). The questions we addressed were: do these molecules regulate the intracellular level of cAMP and/or cGMP, and what is their in vivo function? To get an answer, the two genes were inactivated, and the phenotype of the corresponding mutants was studied. Under steady-state standard conditions, both grow at a rate similar to that of the wild type strain. However, the slr2100 mutant contains slightly lesser amounts of the photosynthetic apparatus components and higher amounts of cGMP and is more sensitive to a UV-B stress than the wild type. We found that it is impaired in its ability to repair the damaged PSIIs under UV-B radiation. Altogether, the data show that cGMP could play an important role in the adaptation, regulation, and functioning of the photosynthetic apparatus in Synechocystis PCC 6803.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Growth Conditions—Synechocystis sp. PCC 6803 was obtained from the Pasteur Culture Collection. Liquid cultures were routinely grown photoautotrophically at 30 °C under white light (30 µmol of photon·m-2·s-1, cool-white Philips fluorescent tubes) in BG11 medium (37) supplemented with 10 mM NaHCO3 and buffered with 10 mM Hepes-NaOH, pH 8.0, under a 1% (v/v) CO2-enriched atmosphere in an illuminated rotatory shaker (120 rpm). For growth on plates, 1% of separately autoclaved agar was added. Whenever appropriate, antibiotics (spectinomycin (30 µg·ml-1), kanamycin (75 µg·ml-1), or chloramphenicol (7 µg·ml-1)) were added to the plates.

Cyanobacterial growth was followed by recording optical densities at 750 nm. Cyanobacterial cell mass was estimated by measuring the Chla concentration of the cultures. Chla was determined in 90% acetone extracts (38) or using the Chlorophyll 1.03 program (bilbo.bio.purdue.edu/www-cyanosite/protocols/chl103.html). Whole cell spectra were recorded from 400 to 750 nm with an Aminco DW-2 spectrophotometer. The E. coli strain routinely used as host, DH5{alpha}, was grown at 37 °C in Luria broth medium with appropriate antibiotics whenever necessary (ampicillin and/or chloramphenicol at 50 or 30 µg·ml-1, respectively).

UV-B Treatment—UV-B irradiation was performed at 30 °C in open, rectangular glass containers in which 11-mm-height layers of cells were maintained in suspension by magnetic agitation. UV-B light was provided by a Vilbert-Lourmat VL-215M lamp in combination with a 0.1-mm cellulose acetate filter (Clarfoil, Courtaluds Chemicals, UK), yielding an intensity of 6 µmol of photons·m-2·s-1 at the surface of the samples. Incident wavelengths range from 290 to 340 nm, with a maximum at 312 nm. Cells were used at a concentration of 6.5 µg Chla·ml-1.

Oxygen Evolution Measurements—Steady-state rates of oxygen evolution were measured using a Hansatech DW2 O2 electrode at a light intensity of 1000 µmol of photons·m-2·s-1 of photosynthetically active radiation (400–700 nm) in the presence of 0.5 mM 2,5-dimethyl-p-benzoquinone as electron acceptor. Light was provided by a Halogen-Bellaphot (12 V, 100 watts, Osram, Germany) lamp. Typically, 2 ml of cells at 6.5 µg of Chla·ml-1 were used in each measurement. Results are expressed in relative value, 100% as the steady-state oxygen evolution at time 0.

Fluorescence Relaxation Decay—Flash-induced increase and subsequent decay of chlorophyll fluorescence yield was measured with the P.S.I. double-modulated fluorometer FL-100 (P.S.I., Brno, Czech Republic) and was performed in the 150 µs to 100 s range as described (19).

Plasmid Constructions—Standard cloning procedures were used according to Sambrook et al. (39). A 2680-bp DNA fragment containing the entire slr2100 (rre20) open reading frame and two flanking regions (704 bp upstream and 870 bp downstream, i.e. from nucleotides 1567613 to 1570293 in Cyanobase; www.kazusa.or.jp/cyano) was amplified by PCR from genomic DNA using two specific primers that were designed from the sequences available in Cyanobase (TABLE ONE). For oriented cloning into pBluescriptSK+, BamHI and PstI restriction sites were added at the 5'- and 3'-ends of the sequence, respectively. The PCR product was obtained using 0.1 µg of purified Synechocystis genomic DNA after 35 cycles of amplification with the Expand High Fidelity system (Roche Applied Science), with cycles set up as 94 °C (30 s), 63 °C (30 s), and 68 °C (3 min). The PCR fragment was digested by BamHI and PstI and cloned into a pBluescript SK+vector (Stratagene) digested by the same enzymes, producing pSlr2100. A similar protocol was used to construct pSll1624, the BamHI-PstI insert (2427 bp) containing the entire sll1624 (rre18) open reading frame and two flanking regions (782 bp upstream and 640 bp downstream, i.e. from nucleotides 1320195 to 1322621 in Cyanobase).


View this table:
[in this window]
[in a new window]
  		TABLE ONE
Oligonucleotides used for mutant constructions Restriction sites used for cloning are in bold, and non-cyanobacterial sequences are underlined.

 
The p{Delta}Slr2100 derivative was constructed by substituting the entire slr2100-coding sequence with the {Omega} cassette (aadA gene), which confers resistance to both spectinomycin and streptomycin. The slr2100-coding region was removed by performing a reverse PCR on pSlr2100 using primers specific for the slr2100-flanking regions Del2100-Fw and Del2100-Rev and the above-described protocol. Both primers were designed to generate a XmaI site at the extremity of the PCR product site used to insert the {Omega} cassette (2-kilobase-long XmaI fragment from pHP45) after dephosphorylation with shrimp alkaline phosphatase.

The p{Delta}Sll1624 derivative was constructed by substituting the entire sll1624-coding sequence with the cassette from pUC4K, which confers resistance to kanamycin. The two flanking sequences were prepared by PCR and ligated after restriction by SmaI to pBluescript SK+. Then the kanamycin cassette was inserted into the SmaI site.

Construction of the Synechocystis Mutants—Wild type Synechocystis PCC 6803 cells were transformed separately with plasmids p{Delta}Slr2100 and p{Delta}Sll1624, which do not replicate in the cyanobacterium, according to Golden et al. (40). Cells were incubated for 48 h on nitrocellulose filters (Nuclepore REC-85) without any selection, and the transformants were selected after transfer to antibiotic-containing BG11 plates. Cells were repeatedly subcultured until full segregation of the mutation was obtained. Total segregation was ascertained by PCRs performed using the flanking region oligonucleotides.

cNMP Determination—Cells (100 ml) between 6 and 6.5 µg of Chla·ml-1 were harvested by filtration under vacuum on glass fiber prefilters (Millipore: APF B04700 [GenBank] ). Filters were immediately transferred to a tube containing 10 ml of 1 mM NaH2PO4 buffer at pH 6.5 preheated at 100 °C. After 5 min at 100 °C, a known volume of the filtrate was filtered through a 0.45-µm polyvinylidene difluoride membrane (Millipore) before transfer to a Vivaspin Mr 50,000 column (Vivascience) and centrifugation for 30 min at 5000 x g. An aliquot of the eluate was freeze-dried, and the lyophilisate was resuspended in 600 µl of 30 mM, pH 6.5, of NaH2PO4 buffer. After centrifugation for 5 min at 12,000 x g, 100 µl were injected onto a high performance liquid chromatography XTerra rp18 (3.5-µm 4.6 x 150 column, Waters) equipped with a 20-mm-long precolumn. The column was developed at a flow rate of 0.8 ml·min-1 with solvent A (30 nM sodium phosphate buffer at pH 6.5) and solvent B (acetonitrile/H2O (99:1, v/v). Quantification was achieved by calibrating the system with precisely known quantities of the different cyclic nucleotides.

Thylakoid Preparation and Protein Analyses—Thylakoid membranes were prepared by breakage of the cells with glass beads (150–200 µm in diameter) at 4 °C followed by differential centrifugations according to Komenda et al. (41). Protein composition was assessed by electrophoresis in a denaturing 12–20% linear gradient polyacrylamide gel containing 6 M urea. After solubilization (41), thylakoid extracts adjusted at 0.7 µg of Chla per lane were loaded, and the gel was run overnight at 18 °C. Proteins were transferred onto nitrocellulose membranes (0.45 µm, Schleicher and Schuell) by wet blotting. The membrane was incubated with an antibody raised against the C terminus of pea D1 protein (a kind gift of P. Nixon) and then with secondary antibody-alkaline phosphatase conjugate. The antigen-antibody complexes were visualized by colorimetric reaction using a BCPIP-NBT system. Membranes were scanned, and the bands were quantified using the ImageJ program (a public domain image processing and analysis program provided by the National Institutes of Health).

Macroarray and Quantitative PCR—Total RNAs were isolated by the hot-phenol method adapted from Mohamed and Jansson (42) and treated with DNase I (Invitrogen, 1 unit/µg of RNA) according to the manufacturer's instructions before use. The absence of DNA products from these RNA preparations in standard PCR assays was checked to ascertain the removal of any DNA contaminants.

Probes for the macroarray analyses were prepared as follows. RNA was reverse-transcribed with a set of hexanucleotides (CGATCG, GGCGAT, CAAAAT, CAATGG, GGCAAT, AAATCC, CTTTTT, ACCAAT, GGCCAC, AAAACC, CCAGCA) kindly designed by Anne Morgat (INRIA Rhône-Alpes, France) so as to prime all of the 400 genes loaded on the nylon membrane while minimizing transcription from the rRNAs. The labeling reaction was performed in a total volume of 20 µl containing 1 µg of RNA, 5 pmol of each primer, 0.5 mM dCTP, dGTP, and dTTP, 5 µM dATP, 40 µCi of {alpha}-[32P]dATP, 200 units of SuperScript II reverse transcriptase, and 20 units of RNasOUT in 1x manufacturer's reaction buffer (Invitrogen). RNA and primers in RNase-free H2O were first incubated at 55 °C for 15 min and cooled down. The reaction mix containing [{alpha}-32P]dATP was then added, and incubation was done for 1 h at 37 °C before the addition of 1 mM dATP (final concentration) followed by a further 15 min of incubation at 37 °C. RNase (2 µg) was added, and after 30 min at 37 °C probes were purified using the QIA-quick nucleotide removal kit (Qiagen). Hybridizations were performed overnight at 65 °C. Membranes were then washed as follows: 2x SSC (1x SSC = 0.15 M NaCl and 0.015 M sodium citrate) for 5 min at room temperature, 1% SDS in 2x SSC for 30 min at 60 °C, and 1% SDS in 0.1x SSC for 30 min at room temperature. Quantifications of the RNA levels were performed with a Fuji Fla3000 reader.

For quantitative PCRs, primers were designed using the LightCycler software (Roche Applied Science) so as to get products with size about 150-bp long with a Tm of 60 °C. The cDNA synthesis was achieved in a final volume of 20 µl containing 1 µg of RNA, 2 pmol of the primers for each of the genes to be further amplified, 0.5 mM concentrations of each dNTP, 200 units of SuperScript II reverse transcriptase, and 20 units of RNasOUT in 1x manufacturer's reaction buffer (Invitrogen). Real-time PCRs were performed according to the manufacturer's instructions using the QuantiTect SYBR Green kit (Qiagen) and a Roche LightCycler system on 0.4 µl of the cDNA solution prepared above. Amplifications were done by incubating the reaction mixtures at 95 °C for 30 s before 45 cycles of 30 s at 95 °C (melting), 30 s at 60 °C (annealing), and 30 s at 72 °C (extension). At the end of the runs, a melting curve was generated and analyzed to ascertain that the recorded data correspond to only one PCR product of expected Tm. The relative abundance of each transcript was determined by comparison of the threshold cycle values (CT) recorded by the apparatus for the different genes to that for the rnpB product. The ratio of expression (R) of the gene of interest (goi) to rnpB was calculated using the simple equation: Rgoi/rnpB = 2(CTrnpB - CTgoi).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation and Growth Properties of the Mutants
Targeted substitution of the sll1624 and slr2100 genes by the antibiotic resistance cassettes was achieved by transforming wild type Synechocystis PCC 6803 cells with the non-replicative plasmid p{Delta}Sll1624 and p{Delta}Slr2100, respectively. Full segregation was easily obtained for the two mutations and ascertained by Southern blot experiments using slr2100, sll1624, and antibiotic resistance genes as hybridizing probes (data not shown). Under standard conditions, no significant differences in terms of growth rate could be observed between the null mutants (gene deletions) and wild type strain, with doubling times ~14 h at 30 °C and 30 µmol of photon·m-2·s-1.

Characterization of the Mutants
Pigment Composition—Because of an apparent color difference between cultures, whole cell absorption spectra were recorded (Fig. 1A). The phycocyanin over chlorophyll ratio (PC/Chl) was consistently 10% lower for the slr2100 mutant (1.01 ± 0.004) compared with the sll1624 and wild type strains (1.12 ± 0.013 and 1.09 ± 0.007, respectively). No significant difference was, however, observed in the carotenoid absorption bands under standard growth conditions, and the three strains exhibit similar rates of oxygen evolution.

Effect of the Light Regime on Oxygen Evolution—Transfer of wild type Synechocystis PCC 6803 cells from a moderate photon flux density (30 µmol of photon·m-2·s-1) to >300 µmol of photon·m-2·s-1 produces photoinhibition. Oxygen evolution is then reduced as a consequence of partial PSII inactivation. The two mutants behave similarly to the wild type upon a shift to 1500 µmol of photon·m-2·s-1 (only slr2100 is shown, Fig. 1B). In contrast, differences were observed in the response to a UV-B stress. After a 2-h exposure to UV-B (at an intensity in the range found in natural environments), oxygen evolution decreased by ~29% for the wild type and ~40% for slr2100 (Fig. 2A). The sll1624 mutant, however, does not differ from the wild type (Fig. 2B).

To determine the origin of this increased sensitivity of the slr2100 mutant to UV-B radiation, the same experiment was repeated in the presence of lincomycin, a translation inhibitor. For the wild type the relative decrease in O2 evolution was much larger in the presence of lincomycin (50 versus 29%) than for the slr2100 mutant (55 versus 40%). The impairment of O2 evolution for the wild type in the presence of lincomycin resembles that observed for slr2100 without the inhibitor. The simplest explanation is that the mutation has an effect on the cascade of events required for the repair of the damaged PSII centers, which is known to require de novo protein synthesis (43). Upon transfer back to medium intensity visible light (50 µmol·m-2·s-1) after the 2-h UV exposure, both wild type and mutant strains recover the same level of O2 evolution (90–100% of the initial value within 1 h). PSII inactivation is, thus, fully reversible for the two strains (Fig. 2A).



View larger version (7K):
[in this window]
[in a new window]
  		FIGURE 1.
A, whole cell absorption spectra of Synechocystis PCC 6803. Cells were grown under 30 µmol·m-2·s-1 in a gyratory shaker (120 rpm) under air atmosphere enriched to 1% CO2. Solid line, wild type strain; dashed line, slr2100 mutant. B, kinetics of PSII inactivation after a shift to 1500 µmol·m-2·s-1. After 2 h cells were transferred back to 50 µmol·m-2·s-1 white light. Oxygen evolution is expressed as % of the value measured at time 0. filled symbols and solid line, wild type; open symbols and dashed line, slr2100.

 
Effect of UV-B on Fluorescence Relaxation—To ascertain the previous results, the relaxation of the fluorescence induced by a saturating flash delivered to dark-adapted cells in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea was monitored for both the wild type and slr2100 strains. Recordings were made with cells sampled before the treatment, 2 h after the UV-B exposure, and after 1 h of recovery under white light conditions (Fig. 3A). As previously described (19), sigmoidal type curves are obtained when the fluorescence intensity was plotted against the log of the time. This decay was assigned to the back recombination of QA- with the S2 state of the water oxidation complex. Such experiments, thus, give information on the functional integrity of the redox components on the donor side of PSII.



View larger version (9K):
[in this window]
[in a new window]
  		FIGURE 2.
PSII inactivation during a UV-B stress. A, Synechocystis PCC 6803 wild type (filled symbols) and slr2100 (open symbols) cells were exposed to UV-B (6 µmol m-2 s-1) at time 0 for 2 h and then transferred back to white light (50 µmol·m-2·s-1). Experiments were performed in the absence (squares and circles) or presence (triangles) of lincomycin (translation inhibitor). Plotted values represent the mean of three independent measurements performed with cell suspensions at 6.5 µg of Chla·ml-1. B, Synechocystis PCC 6803 wild type (filled symbols) and sll1624 (open symbols, dashed line) cells were exposed to UV-B (6 µmol m-2 s-1) at time 0 for 2 h and then transferred back to white light (50 µmol·m-2·s-1). Plotted values represent the mean of 3 independent measurements performed with cell suspensions at 6.5 µg of Chla·ml-1. C, wild type Synechocystis PCC 6803 cells were incubated in the absence (squares, solid line) or presence (circles, dashed line) of 100 µM dipyridamole, an inhibitor of cGMP phosphodiesterase. Plotted values are the means of 4 independent measurements performed with cell suspensions adjusted to 6.3 µg of Chla·ml-1.

 
The half-life for relaxation is 0.6 s for the two strains when grown under standard conditions (Fig. 3A). After 2 h of a UV-B treatment, the decay was faster for the wild type (half-life, 0.4 s), indicating that QA- cannot recombine with the S2 state in part of the centers and exchanges with redox components closer to QA- like the redox-active tyrosine (Tyr-Z) (19, 44). The difference was even more pronounced for the slr2100 mutant (half-life, 0.3 s). This result indicates that the manganese cluster of the PSII centers was more damaged by the UV-B treatment in the mutant than in the wild type. After the recovery phase, the fluorescence relaxation profiles are similar for both strains (Fig. 3C), confirming that the mutation likely affects the PSII repair process that takes place during the UV-B exposure.

D1 Contents of the Thylakoid Membrane—The similarity in the O2 evolution profile found between the slr2100 mutant and the lincomycin-treated wild type strain (Fig. 2A) pointed to a defect in the PSII repair process. The content in mature D1 protein of the thylakoid membrane was, thus, determined by immuno-reaction with a specific antiserum. As shown in Fig. 4, there is a significant difference between the two strains, showing faster loss of the D1 protein in the mutant, which confirms an impairment of D1 turnover in slr2100.



View larger version (7K):
[in this window]
[in a new window]
  		FIGURE 3.
Relaxation of the variable chlorophyll fluorescence induced by a single saturating flash applied to Synechocystis PCC 6803 cells; squares, wild type strain; triangles, slr2100 mutant. A, cells before the UV-B treatment. B, after 2 h of UV-B irradiation. C, after a 1-h recovery under 50 µmol·m-2·s-1 white light. Cells at 6.5 µg of Chla·ml-1 were dark-adapted for 10 min before exposure to the saturating flash and recording of the variable fluorescence. Experiments were performed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and repeated three times. The S.E. of the measurements is smaller than the size of the symbols in the figure. The curves were normalized to the same amplitude. rel.u., relative units.

 
Cyclic Nucleotide Contents and Effect of a Specific Phosphodiesterase Inhibitor—The intracellular levels of cyclic nucleotides were determined for the two strains after various periods of UV-B exposure. No significant difference was detected in the cAMP concentration in the cells kept under standard conditions (T0) as well as during the first 10 min of the UV-B treatment (Fig. 5A). In contrast, under standard conditions the steady-state level of cGMP is significantly higher in slr2100 than in the wild type strain, and it almost does not vary after the UV-B exposure (Fig. 5B). For the wild type, the cGMP concentration drops rapidly by about 40% within the first 10 min of the UV-B treatment. The UV-B stress, thus, specifically affects the intracellular cGMP concentration, and slr2100 is impaired in its ability to regulate it.

To confirm that the observed mutant phenotype could be linked to its high and not regulated level of cGMP, which may result from the lack of a phosphodiesterase activity, inhibitors were tested. Dipyridamole is known to inhibit cGMP phosphodiesterases (45). O2 evolution was monitored in wild type cells after a UV-B exposure in the presence of dipyridamole. Under these conditions cells were more sensitive than without the inhibitor (Fig. 2C). The extent of inhibition of O2 evolution observed under the UV-B stress in the presence of dipyridamole (47%) is similar to that recorded for the slr2100 mutant without inhibitor (42%). In both instances cells fully recover after white light exposure, as does the mutant.



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 4.
D1 content of thylakoid membranes. Membranes were prepared at the given time points, and the D1 protein content was determined by immunodetection using an anti-D1 antiserum. Data are the means of three experiments. A, quantified D1 content. Circles, wild type; squares, slr2100. B, a representative immunoblot. T0–120, time points corresponding to the UV-B treatment; rT30–60, time points corresponding to the recovery period under white light.

 
Characterization of the slr2100 Gene
Because the slr2100 gene product can modify the response to a UV-B stress and the intracellular level of cGMP, we looked for putative regulatory sequences in front of slr2100. In Cyanobase (www.hazusa.or.ip) (46, 47), the Synechocystis PCC 6803 slr2100 sequence was annotated as an open reading frame composed of 368 amino acids, with a calculated molecular mass of 40,673 Da. No obvious ribosome binding site lies in front of the proposed ATG. The latter is nevertheless likely to be the start codon because: (i) the CheY-like phospho-acceptor domain (see below) begins immediately downstream of this ATG, and (ii) there is no other potential initiation codon preceded by a recognizable Shine-Dalgarno sequence in front of this domain. A lack of consensus ribosome binding site has already been observed in cyanobacteria, even in front of the psbA genes, which encode the very high turnover D1 protein of photosystem II and phycobiliprotein genes (48).



View larger version (6K):
[in this window]
[in a new window]
  		FIGURE 5.
Evolution of the cyclic nucleotide concentrations during a UV-B stress. A, cAMP. B, cGMP. Cells were exposed to 6 µmol·m-2·s-1 UV-B at time 0. Data are presented as the means of two duplicates performed on two independent cultures. Solid lines and filled symbols correspond to the wild type; dashed lines and empty symbols correspond to the mutant.

 



View larger version (9K):
[in this window]
[in a new window]
  		FIGURE 6.
Mapping of the stable slr2100 transcripts. A, sequencing gel with the primer extension products run along the DNA sequence performed with the oligonucleotide used to produce the extended DNA by retrotranscription. B, promoter region with relevant features (-35 and -10 boxes and translation initiation codon) in bold. The bent arrow indicates the 5'-end of the stable slr2100 mRNAs, and the dashed arrow indicates the nucleotide sequence of the oligonucleotide used for the mapping. tsp, transcriptional start site

 
Primer extension analysis was performed using a synthetic 32P end-labeled primer (nucleotides 6–26 from the slr2100 ATG) that allowed to map a stable 5' end for the slr2100 transcripts (Fig. 6A). This potential transcriptional start site is located 22 bp upstream from the slr2100 ATG and is preceded by the sequence 5'-TTGCCC-N19-TATACA-N7-T-3' (Fig. 6B). Given its sequence and the spacing between the putative -10 and -35 boxes, this promoter could be either a poor SigA-dependent or a {sigma} class II-dependent promoter. No consensus DNA sequence known as the target for transcriptional regulators (SYCRP1 or NtcA; see for example Refs. 49 and 50) could be detected in the slr2099-2100 intergenic region. Only an imperfect match (GTAATTTTTCTCAC) to the consensus E. coli CRP binding site (TGTGAT-N4-ATCACA) can be found 280 bp downstream from the slr2100 transcriptional start site.

Transcriptome Analysis
Preliminary experiments were performed using macroarrays that contain PCR products for about 400 genes (~20% of the transcription units). Most of the genes chosen encode products for photosynthetic related processes and stress responses or known regulatory proteins. Representatives of the main cellular functions have also been included (see Supplemental Table 1s). Transcription profiles of slr2100 and wild type strains grown under standard conditions and after a 30-min UV-B exposure have been compared. Transcript levels were determined by measuring counts from the nylon membrane after hybridization with 32P-labeled retrotranscription products directly obtained from total RNAs without any amplification step. As could be expected, mRNA levels vary more than 4 orders of magnitude, photosynthesis-related (psa, psb, cpc, glgA, and ftrC) and ribosomal protein (rps2) genes being the most expressed. Comparing slr2100 and wild type strains adapted under standard growth conditions, changes by a factor of 2 or more were observed for 30 genes. In particular, the mRNA level for slr0093 (dnaJ), slr2076 (groEL1), sll1932 (dnaK3), and sll0430 (htpG), which code for molecular chaperones, is 2–3 times lower in slr2100. This means that the mutant is likely affected in its abilities to properly fold or unfold polypeptides. After a 30-min UV-B exposure, no very significant differences were noticed for the wild type compared with the global analysis reported during the course of this work by Huang et al. (9). The observed changes likely result from the use in the present study of a lower and more "natural" UV-B irradiance. Changes by a factor of 2 or more between slr2100 and the wild type were observed for 17 genes (data not shown).

These results were used to select genes for a more precise analysis. We more specifically looked at genes linked to cNMPs, related to PSII and its turnover, as well as at a few genes that have been proposed to participate in light-sensing and adaptation (TABLE TWO). Transcript levels were measured using quantitative reverse transcription-PCR for 32 genes, with transcript levels normalized to that of rnpB, the gene coding for the RNA component of the RNase P (Fig. 7). This gene is classically used as an internal standard, its transcription assumed to be the less affected by the growth conditions.


View this table:
[in this window]
[in a new window]
  		TABLE TWO
Name and function of the genes used in the quantitative reverse transcription-PCR experiments (www.kazusa.or.jp/cyano)

 
As expected, because the gene was deleted, no slr2100 transcript was found in the mutant. For 23 genes we did not detect significant differences between the wild type and slr2100 strains grown under standard conditions (Fig. 7A). Thus, when the cells are adapted to their environment, the higher cGMP steady-state concentration present in the slr2100 cells does not affect the transcript level for these genes. A decrease by a factor of about 2 was observed for: (i) two photosynthesis-related genes (psbA3 cpcB), (ii) two genes involved in PSII turnover, phb1 (slr1106) and ftsH (slr0228), and (iii) a gene encoding a two-component system histidine kinase (hik33) proposed as a global regulator that helps coordinate cellular metabolism with growth limitations imposed by environmental conditions (51).

After 15 min of UV-B treatment, significant changes in transcript amounts were observed for 13 of the 32 genes with the wild type strain (Fig. 7B). Eight genes showed an ~2-fold or more increased mRNA level: psbA2, psbA3, ftsH, nblA1 (small polypeptide required for phycobilisome degradation), sigD, hliD (a high light inducible polypeptide), and gifB (a glutamine synthetase inhibitor) as well as, notably, slr2100. Transcript levels also increased for cya2 (guanylate cyclase), slr2098 (hik21), and slr1759 (hik14), which encode a two-component hybrid (sensor plus regulator) kinases. The largest decreases concerned cpcB (phycocyanin apoprotein), cph1 (hik35, cyanobacterial phytochrome 1), sodB (O2-scavenging superoxide dismutase), sll1330 (rre37, a two-component response regulator of the OmpR subfamily), and gifA (the second glutamine synthetase inhibitor). Significantly lower levels were also observed for phb1 (prohibitin), cpcF (phycocyanin {alpha}-subunit phycocyanobilin lyase), cph2 (phytochrome-like protein), and sll0396 (rre28, a two-component response regulator of the OmpR subfamily).

After the UV-B exposure, transcript levels are affected for 22 of the 32 genes in the slr2100 mutant (Fig. 7C). An increase similar to or higher than that of the wild type was monitored for cya2, psbA2, psbA3, ftsH, nblA1, sigD, hliD, hik14, and gifB. For psbA3 and ftsH, which were less expressed in slr2100 than in the wild type under standard conditions, the extent of increase is more pronounced, with levels similar in the two strains after 15 min of UV-B. Enhanced levels were also observed for 10 genes that were not affected in the wild type: phb2 (second prohibitin), clpC (ATP-dependent Clp protease ATPase subunit), sigA, sigB, plpA (hik3, phytochrome-like protein), hik33, katG (catalase peroxidase), and interestingly, three genes related to cAMP, cya1 (adenylate cyclase), sycrp1 (cAMP receptor protein), and sll1624 (the second putative cNMP phosphodiesterase). Only two of the genes for which decreased levels were monitored in the wild type after UV-B showed the same pattern; they are cpcF and cph1. The amount of stable mRNAs that were about two times lower for cpcB and higher for slr2098 in slr2100 than in the wild type before the UV-B treatment, were not modified by it. In contrast, an opposite regulation was observed in slr2100 compared with wild type cells for five genes: phb1, cph2, rre28, and sodB showed higher levels, whereas that of slr2098 was lower.

The transcription pattern of slr2100 cells after the UV-B treatment clearly differs from that of the wild type for 20 of the 32 genes (Fig. 7D). Because both cAMP synthesis (achieved by Cya1) and that of its receptor protein (SYCRP1) increase, the expression of the genes transcribed under the control of the cAMP-SYCRP1 complex likely is specifically modified in the slr2100 mutant. The later also tends to increase protective mechanisms against damaging reactive oxygen species by producing more superoxide dismutase (SodB) and catalase peroxidase (KatG). Other important differences concern genes related to: (i) PSII repair (phb1 and phb2) and PCB chromophore attachment (cpcF), (ii) transcription, sigA (house keeping) and sigB, which code for {sigma} factors, (iii) light sensing (cph1, cph2, and plpA), and (iv) two component regulatory pathways, with hik14, hik33, rre37, and rre28 more expressed and slr2099 (hik40) less expressed.



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 7.
Histograms comparing the transcript levels determined by quantitative reverse transcription-PCR for the wild type and slr2100 strains before and after 15 min UV-B as well as the ratios for slr2100 versus wild type (wt) under standard conditions and after a 15-min UV-B exposure. The correspondence between figures and gene names is given below the figure.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Because of the presence of a HD domain and of their similarity with the cAMP-dependent phosphodiesterase RegA from Dictyostelium discoideum, the two open reading frames, Sll1624 and Slr2100, have been proposed as the putative phosphodiesterases needed to ensure cyclic nucleotide homeostasis in Synechocystis PCC 6803 cells (36). This hypothesis is supported for Slr2100 by the data obtained in this study: (i) the steady-state cGMP level under standard growth conditions is two times higher in the slr2100 null mutant than in the wild type cells, (ii) after an exposure to UV-B, the decrease in intracellular cGMP that occurs in wild type does not happen in slr2100 cells, and (iii) the increased level of slr2100 mRNAs monitored in wild type cells after the UV-B treatment correlates with the decreased concentration of cGMP observed. We have observed that the mutant cells contain more cAMP after 30 min of UV-B than the wild type (data not shown), which can be correlated with the 2-fold increase in the adenylate cyclase cya1 mRNAs detected in the mutant compared with the wild type (Fig. 7). Cross-talks between cAMP and cGMP regulatory networks could, thus, exist.

When adapted to standard growth conditions, both the slr2100 and the wild type strains exhibit similar generation time, although the mutant contains less phycocyanin, meaning that light-harvesting is not rate-limiting. Accordingly, we detected a 2-fold lower steady-state level of cpcB transcripts in the mutant (Fig. 7). The expression of 3 of the five group 2 {sigma} factor genes, sigA (house-keeping), sigB, and sigD, was monitored. An accumulation of sigB transcripts has already been reported to occur under stress conditions like heat shock and high salt concentration (52), and SigD has been shown to contribute to the light-induced transcription of psbA2/A3, the two genes that produce the PSII reaction center D1 polypeptide (53). A UV-B treatment leads to increased expression of sigD in wild type cells and of the three genes in slr2100. To achieve a similar growth rate, the mutant must compensate the lack of Slr2100 protein by establishing new equilibria and likely has different enzyme and regulatory protein complements. Accordingly, not all of the genes are similarly affected by the UV-B exposure.

One such difference between the wild type and mutant strain concerns the level of expression of slr2098 (hik21) and slr2099 (hik40), two genes that encode two-component hybrid histidine kinases located immediately upstream from slr2100. It was proposed that these three genes together with slr2104 (the fourth open reading frame downstream of slr2100) could form a phosphorelay cascade (36). Slr2098 is a 1261-amino acid-long polypeptide with three MHYT domains at its N-terminal end followed by PAS-PAC, histidine kinase, two response regulators, and a histidine phospho-transfer domain. The newly identified MHYT conserved domain consists of six transmembrane segments, three of which contain conserved methionine, histidine, and tyrosine residues that are projected to lie near the outer face of the cytoplasmic membrane (54). A model of the membrane topology of the MHYT domain indicates that its conserved residues could coordinate one or two copper ions, suggesting a role in sensing oxygen, CO, or NO. Slr2099 is a 366-amino acid-long response regulator preceding the histidine kinase. Slr2104 is another more complex hybrid kinase (950 amino acids long) with the succession of GAF, PAC, histidine kinase, response regulator, and histidine phospho-transfer domains. Interestingly, as the acronym refers to (cGMP phosphodiesterase-adenylyl cyclases-bacterial transcription factors FhlA), GAF domains could bind cGMP. Based on two-hybrid experiment data, direct interactions and possible phospho-transfers have been proposed between Slr2098 (Hik21) and Slr2104 (Hik22) as well as from Slr2099 (Hik40) to Slr2100 (Rre20) (www.genome.jp/dbget-bin/show_pathway?syn02020). The lack of Slr2100 in the mutant clearly has consequences on the expression of slr2098 and slr2099 (Fig. 7).

Excess light energy as well as UV-B radiation provokes cell damages, in particular at the level of the photosynthetic apparatus. For PSII repair, photodamaged D1 polypeptides must be degraded and replaced by newly synthesized polypeptides. Compared with the wild type, neither slr2100 nor sll1624 exhibits a different behavior toward photoinhibition, but the former mutant clearly shows an increased sensitivity to UV-B radiation. The experiments of O2 evolution and relaxation of variable fluorescence demonstrate that PSII in slr2100 is damaged to an extent similar to that found for wild type cells in which translation is inhibited during the UV-B exposure. The decreased amount of D1 protein found in the thylakoids of the mutant strain exposed to UV-B compared with that of the wild type (Fig. 4) demonstrates that the degradation part of the repair cycle is not affected by the lack of the slr2100 gene. This idea is also consistent with the response of the slr0228 gene, which codes for FtsH, a key protease for PSII repair after high light (55) and UV-B light (56) damages. The level of slr0228 and psbA3 is 2-fold lower in slr2100 than in the wild type under standard conditions. The mutant cells are, thus, partly deprived of essential components for the PSII repair when exposed to UV-B radiation. However, the transcription of ftsH increases more in the mutant than in the wild type so that the ftsH mRNA level in slr2100 reaches that of the wild type after 15 min of UV-B treatment, ensuring efficient D1 degradation. The replacement of UV-damaged and degraded D1 by new copies requires an enhanced transcription of psbA genes, especially of psbA3 (21), followed by translation and incorporation of new D1 copies into the PSII reaction center complex. Although the psbA mRNA levels are lower in the mutant than in the wild type under standard conditions, the higher extent of UV-B induction compensates for this effect in the slr2100 mutant under UV-B exposure. The similar and higher mRNA amounts in UV-B-stressed wild type and mutant cells show that it is not the abundance of psbA transcripts that limits D1 synthesis. Therefore, the unregulated cGMP concentration observed in the mutant under the conditions of UV-B exposure should affect either the translation of psbA mRNA or the incorporation of newly synthesized D1 into the PSII reaction center. Altogether, our data fit with the observation that the repair of damaged PSII differs depending on whether the inhibition was induced by UV-B or high light (17), since the difference in the repair efficiency between the slr2100 and wild type strains only exists when cells are exposed to UV-B and not during photoinhibition by visible light.

This work points to a role for the cyclic nucleotides, more specifically cGMP, in the regulation and adaptation of the Synechocystis PCC 6803 photosynthetic apparatus to a UV-B stress. Even though functions such as UV-photo-protective mechanisms are common to many species, the regulatory networks by which different cyanobacteria respond to such stress seem to be achieved by different sets of proteins, or at least the domains that interact may belong to proteins that do not have the same multimodular arrangements. These differences highlight the plasticity of the ancestral cyanobacterial genome and the success of the cyanobacteria in colonizing quite different ecosystems throughout the ~3 billion years of evolution.


   FOOTNOTES
 
* This work was supported by grants from the CNRS and IMPB012 BacAttract to the FRE 2433, by Hungarian Granting Agency OTKA Grant T034321, and by European Union Grant STREP-SOLAR-H-516510. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1s. Back

1 This research is part of the Ph.D. work performed at Ecole Doctorale B2M and was supported by a bilateral French-Hungarian fellowship. Back

2 To whom correspondence should be addressed. Tel.: 33-1-44-32-3519; Fax: 33-1-44-32-3941; E-mail: jhoumard{at}biologie.ens.fr.

3 The abbreviations used are: PSII, Photosystem II; cNMP, cyclic nucleotide; Chl, chlorophyll; PC, phycocyanin. Back

4 J. Ochoa de Alda, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank A.-L. Etienne for continuing support and helpful comments. We are grateful to P. Nixon (London) for the D1 antibody and Dr. Yanjun Zhang who performed some macroarray analyses.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Tandeau de Marsac, N., and Houmard, J. (1993) FEMS Microbiol. Rev. 104, 119-189[CrossRef]
  2. Grossman, A. R., Bhaya, D., and He, Q. (2001) J. Biol. Chem. 276, 11449-11452[Free Full Text]
  3. Unsal-Kacmaz, K. A., Makhov, M., Griffith, J. D., and Sancar, A. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 6673-6678[Abstract/Free Full Text]
  4. Vladimirov, Y. A., Roshchupkin, D. I., and Fesenko, E. E. (1970) Photochem. Photobiol. 11, 227-246[Medline] [Order article via Infotrieve]
  5. Dohler, G., and Biermann, T. (1994) Z. Naturforsch. C 49, 607-614
  6. Teramura, A. H., Biggs, R. H., and Kossuth, S. (1980) Plant Physiol. 65, 483-488[Abstract/Free Full Text]
  7. Kulandaivelu, G., and Noorudeen, A. M. (1983) Physiol. Plant. 58, 389-394[CrossRef]
  8. He, Y. Y., and Hader, D. P. (2002) Photochem. Photobiol. Sci. 1, 729-736[CrossRef][Medline] [Order article via Infotrieve]
  9. Huang, L., McCluskey, M. P., Ni, H, and LaRossa, R. A. (2002) J. Bacteriol. 184, 6845-6858[Abstract/Free Full Text]
  10. Lao, K., and Glazer, A. N. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5258-5263[Abstract/Free Full Text]
  11. Trebst, A., and Depka, B. (1990) Z. Naturforsch. C 45, 765-771
  12. Greenberg, B.M., Gaba, V., Canaani, O., Malkin, S., Mattoo, A. K, and Edelman, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6617-6620[Abstract/Free Full Text]
  13. Friso, G., Spetea, C., Giacometti, G. M., Vass, I., and Barbato, R. (1994) Biochim. Biophys. Acta 1184, 78-84[CrossRef]
  14. Renger, G., Völker, M., Eckert, H. J., Fromme, R., Hohm-Veit, S., and Graber, P. (1989) Photochem. Photobiol. 49, 97-105
  15. Post, A., Lukins, P. B, Walker, P. J., and Larkum, A. W. D. (1996) Photosynth. Res. 49, 21-27[CrossRef]
  16. Spetea, C., Hideg, É., and Vass, I. (1996) Plant Sci. 115, 207-215[CrossRef]
  17. Giacometti, G. M., Barbato, R., Chiaramonte, S., Friso, G., and Rigoni, F. (1996) Eur. J. Biochem. 242, 799-806[Medline] [Order article via Infotrieve]
  18. Nedunchezhian, N., and Kulandaivelu, G. (1996) Acta Physiol. Plant. 18, 39-45
  19. Vass, I., Kirilovsky, D., and Etienne, A. L. (1999) Biochemistry 38, 12786-12794[CrossRef][Medline] [Order article via Infotrieve]
  20. Sass, L., Spetea, C., Maté, Z., Nagy, F., and Vass, I. (1997) Photosynth. Res. 54, 55-62[CrossRef]
  21. Mate, Z., Sass, L., Szekeres, M., Vass, I., and Nagy, F. (1998) J. Biol. Chem. 273, 17439-17444[Abstract/Free Full Text]
  22. Trewavas, A. J., Rodrigues, C., Rato, C., and Malho, R. (2002) Curr. Opin. Plant Biol. 5, 425-429[CrossRef][Medline] [Order article via Infotrieve]
  23. Cann, M. J. (2003) New Phytol. 161, 23-34[CrossRef]
  24. Ohmori, M., and Okamoto, S. (2004) Photochem. Photobiol. Sci. 3, 503-511[CrossRef][Medline] [Order article via Infotrieve]
  25. Francko, D., and Wetzel, R. G. (1981) J. Phycol. 17, 129-134[CrossRef]
  26. Ohmori, M., Terauchi, K., Okamoto, S., and Watanabe, M. (2002) Photochem. Photobiol. 75, 675-679[CrossRef][Medline] [Order article via Infotrieve]
  27. Okamoto, S., Kasahara, M., Kamiya, A., Nakahira, Y., and Ohmori, M. (2004) Photochem. Photobiol. 80, 429-433[CrossRef][Medline] [Order article via Infotrieve]
  28. Terauchi, K., and Ohmori, M. (2004) Mol. Microbiol. 52, 303-309[CrossRef][Medline] [Order article via Infotrieve]
  29. Terauchi, K., and Ohmori, M. (1999) Plant Cell Physiol. 40, 248-251[Abstract/Free Full Text]
  30. Yoshimura, H., Yoshihara, S., Okamoto, S., Ikeuchi, M., and Ohmori, M. (2002) Plant Cell Physiol. 43, 460-463[Abstract/Free Full Text]
  31. Elmorjani, K, and Herdman, M. (1988) J. Gen. Microbiol. 133, 1685-1694
  32. Katayama, M., and Ohmori, M. (1997) J. Bacteriol. 179, 3588-3593[Abstract/Free Full Text]
  33. Ohmori, M., Ikeuchi, M., Sato, N., Wolk, P., Kaneko, T., Ogawa, T., Kanehisa, M., Goto, S., Kawashima, S., Okamoto, S., Yoshimura, H., Katoh, H., Fujisawa, T., Ehira, S., Kamei, A., Yoshihara, S., Narikawa, R., and Tabata, S. (2001) DNA Res. 8, 271-284[Abstract]
  34. Ochoa de Alda, J. A. G., Ajlani, G., and Houmard, J. (2000) J. Bacteriol. 182, 3839-3842[Abstract/Free Full Text]
  35. Sakamoto, T., Murata, N., and Ohmori, M. (1991) Plant Cell Physiol. 32, 581-584[Abstract/Free Full Text]
  36. Ochoa de Alda, J. A. G., and Houmard, J. (2000) Microbiology 146, 3183-3194[Abstract/Free Full Text]
  37. Rippka, R. (1988) Methods Enzymol. 167, 3-27[Medline] [Order article via Infotrieve]
  38. Porra, R. J., and Grimme, L. H. (1974) Anal. Biochem. 57, 255-267[CrossRef][Medline] [Order article via Infotrieve]
  39. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, NY
  40. Golden, S., Brusslan, J., and Haselkorn, R. (1987) Methods Enzymol. 153, 215-231[Medline] [Order article via Infotrieve]
  41. Komenda, J., Lupinkova, L., and Kopecky, J. (2002) Eur. J. Biochem. 269, 610-619[Medline] [Order article via Infotrieve]
  42. Mohamed, A., and Jansson, C. (1989) Plant Mol. Biol. 13, 693-700[CrossRef][Medline] [Order article via Infotrieve]
  43. Aro, E. M., Virgin, I., and Andersson, B. (1993) Biochim. Biophys. Acta 1143, 113-134[Medline] [Order article via Infotrieve]
  44. Tichy, M., Lupinkova, L., Sicora, C., Vass, I., Kuvikova, S., Prasil, O., and Komenda, J. (2003) Biochim. Biophys. Acta 1605, 55-66[Medline] [Order article via Infotrieve]
  45. Abdollahi, M., Chan, T. S., Subrahmanyam, V., and O'Brien, P. J. (2003) Mol. Cell. Biochem. 252, 205-211[CrossRef][Medline] [Order article via Infotrieve]
  46. Kaneko, T., Sato, S., Kotani, H., Tanaka, A., Asamizu, E., Nakamura, Y., Miyajima, N., Hirosawa, M., Sugiura, M., Sasamoto, S., Kimura, T., Hosouchi, T., Matsuno, A., Muraki, A., Nakazaki, N., Naruo, K., Okumura, S., Shimpo, S., Takeuchi, C., Wada, T., Watanabe, A., Yamada, M., Yasuda, M., and Tabata, S. (1996) DNA Res. 3, (suppl.) 185-209[CrossRef][Medline] [Order article via Infotrieve]
  47. Nakamura, Y., Kaneko, T., and, Tabata, S. (2000) Nucleic Acids Res. 28, 72[Abstract/Free Full Text]
  48. Houmard, J., Capuano, V., Colombano, M. V., Coursin, T., and Tandeau de Marsac, N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2152-2156[Abstract/Free Full Text]
  49. Omagari, K., Yoshimura, H., Takano, M., Hao, D., Ohmori, M., Sarai, A., and Suyama, A. (2004) FEBS Lett. 563, 55-58[CrossRef][Medline] [Order article via Infotrieve]
  50. Vazquez-Bermudez, M. F., Flores, E., and Herrero, A. (2002) Biochim. Biophys. Acta 1578, 95-98[Medline] [Order article via Infotrieve]
  51. Tu, C. J., Shrager, J., Burnap, R. L., Postier, B. L., and Grossman, A. R. (2004) J. Bacteriol. 186, 3889-3902[Abstract/Free Full Text]
  52. Tuominen, I., Tyystjarvi, E., and Tyystjarvi, T. (2003) J. Bacteriol. 185, 1116-1119[Abstract/Free Full Text]
  53. Imamura, S., Asayama, M., Takahashi, H., Tanaka, K., Takahashi, H., and Shirai, M. (2003) FEBS Lett. 554, 357-362[CrossRef][Medline] [Order article via Infotrieve]
  54. Galperin, M. Y., Gaidenko, T. A., Mulkidjanian, A. Y., Nakano, M., and Price, C. W. (2001) FEMS Microbiol. Lett. 205, 17-23[CrossRef][Medline] [Order article via Infotrieve]
  55. Silva, P., Thompson, E., Bailey, S., Kruse, O., Mullineaux, C. W., Robinson, C., Mann, N. H., and Nixon, P. J. (2003) Plant Cell 15, 2152-2164[Abstract/Free Full Text]
  56. Sicora, C., Cheregi, O., Kos, P. B., Nixon, P. J., and Vass, I. (2005) Photosynthesis: Fundamental Aspects to Global Perspectives (Van der Est, A., and Bruce, D., eds) in press

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Bacteriol.Home page
D. Bhaya, K. Nakasugi, F. Fazeli, and M. S. Burriesci
Phototaxis and Impaired Motility in Adenylyl Cyclase and Cyclase Receptor Protein Mutants of Synechocystis sp. Strain PCC 6803.
J. Bacteriol., October 1, 2006; 188(20): 7306 - 7310.
[Abstract] [Full Text] [PDF]

Home page
 Microbiol. Mol. Biol. Rev.Home page
M. K. Ashby and J. Houmard
Cyanobacterial Two-Component Proteins: Structure, Diversity, Distribution, and Evolution
Microbiol. Mol. Biol. Rev., June 1, 2006; 70(2): 472 - 509.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/40/33935    most recent
M503153200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cadoret, J.-C.
Right arrow Articles by Houmard, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cadoret, J.-C.
Right arrow Articles by Houmard, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement