The plastid ancestor originated among one of the major cyanobacterial lineages.

The primary endosymbiotic origin of chloroplasts is now well established but the identification of the present cyanobacteria most closely related to the plastid ancestor remains debated. We analyse the evolutionary trajectory of a subset of highly conserved cyanobacterial proteins (core) along the plastid lineage, those which were not lost after the endosymbiosis. We concatenate the sequences of 33 cyanobacterial core proteins that share a congruent evolutionary history, with their eukaryotic counterparts to reconstruct their phylogeny using sophisticated evolutionary models. We perform an independent reconstruction using concatenated 16S and 23S rRNA sequences. These complementary approaches converge to a plastid origin occurring during the divergence of one of the major cyanobacterial lineages that include N2-fixing filamentous cyanobacteria and species able to differentiate heterocysts.

Cyanobacterial two-component proteins: structure, diversity, distribution, and evolution.

A survey of the already characterized and potential two-component protein sequences that exist in the nine complete and seven partially annotated cyanobacterial genome sequences available (as of May 2005) showed that the cyanobacteria possess a much larger repertoire of such proteins than most other bacteria. By analysis of the domain structure of the 1,171 potential histidine kinases, response regulators, and hybrid kinases, many various arrangements of about thirty different modules could be distinguished. The number of two-component proteins is related in part to genome size but also to the variety of physiological properties and ecophysiologies of the different strains. Groups of orthologues were defined, only a few of which have representatives with known physiological functions. Based on comparisons with the proposed phylogenetic relationships between the strains, the orthology groups show that (i) a few genes, some of them clustered on the genome, have been conserved by all species, suggesting their very ancient origin and an essential role for the corresponding proteins, and (ii) duplications, fusions, gene losses, insertions, and deletions, as well as domain shuffling, occurred during evolution, leading to the extant repertoire. These mechanisms are put in perspective with the different genetic properties that cyanobacteria have to achieve genome plasticity. This review is designed to serve as a basis for orienting further research aimed at defining the most ancient regulatory mechanisms and understanding how evolution worked to select and keep the most appropriate systems for cyanobacteria to develop in the quite different environments that they have successfully colonized.

Regulated expression of glutamyl-tRNA synthetase is directed by a mobile genetic element in the cyanobacterium Tolypothrix sp. PCC 7601.

The genome of Tolypothrix sp. PCC 7601 carries two copies of a novel insertion sequence, ISTosp1. One of the two copies is located upstream of the gene encoding glutamyl-tRNA synthetase, an enzyme playing a key role in protein and pigment synthesis. The tnpA gene of the IS element and gltX were co-transcribed and their expression was transiently upregulated upon retrieval of the ammonium source irrespective of whether nitrate or no nitrogen source were available. The second copy is also transcribed and shows a similar regulatory pattern. Structural elements of the promoter (-10 and -35 sequences) directing the expression of the tnpA-gltX operon have been localized within the IS. Regulatory sequences involving the NtcA transcription factor in the control of tnpA-gltX expression were found both within and in sequences upstream of the insertion element. The expression of gltX in a closely related cyanobacterium, Nostoc sp. PCC 7120, which lacks the insertion upstream of gltX, decreased upon ammonium retrieval, a regulatory pattern that markedly differs from that observed in Tolypothrix sp. PCC 7601. ISTosp1 constitutes a good example of how cells can make use of a transposable element to evolve an original regulatory mechanism.

The light-harvesting antenna of the diatom Phaeodactylum tricornutum. Evidence for a diadinoxanthin-binding subcomplex.

Diatoms differ from higher plants by their antenna system, in terms of both polypeptide and pigment contents. A rapid isolation procedure was designed for the membrane-intrinsic light harvesting complexes (LHC) of the diatom Phaeodactylum tricornutum to establish whether different LHC subcomplexes exist, as well to determine an uneven distribution between them of pigments and polypeptides. Two distinct fractions were separated that contain functional oligomeric complexes. The major and more stable complex ( approximately 75% of total polypeptides) carries most of the chlorophyll a, and almost only one type of carotenoid, fucoxanthin. The minor complex, carrying approximately 10-15% of the total antenna chlorophyll and only a little chlorophyll c, is highly enriched in diadinoxanthin, the main xanthophyll cycle carotenoid. The two complexes also differ in their polypeptide composition, suggesting specialized functions within the antenna. The diadinoxanthin-enriched complex could be where the de-epoxidation of diadinoxanthin into diatoxanthin mostly occurs.

Cyclic nucleotides, the photosynthetic apparatus and response to a UV-B stress in the Cyanobacterium Synechocystis sp. PCC 6803.

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.

Dissipation of excess energy triggered by blue light in cyanobacteria with CP43' (isiA).

The chlorophyll-protein CP43' (isiA gene) induced by stress conditions in cyanobacteria is shown to serve as an antenna for Photosystem II (PSII), in addition to its known role as an antenna for Photosystem I (PSI). At high light intensity, this antenna is converted to an efficient trap for chlorophyll excitations that protects system II from photo-inhibition. In contrast to the 'energy-dependent non-photochemical quenching' (NPQ) in chloroplasts, this photoprotective energy dissipation in cyanobacteria is triggered by blue light. The induction is proportional to light intensity. Induction and decay of the quenching exhibit the same large temperature-dependence.

Immunolocalization of NblA, a protein involved in phycobilisome turnover, during heterocyst differentiation in cyanobacteria.

In unicellular non-diazotrophic cyanobacteria, NblA is a small polypeptide required for phycobilisome degradation during macronutrient limitation. In the filamentous N(2)-fixing Tolypothrix sp., a nblA gene (nblAI) lies upstream of the cpeBA operon that encodes phycoerythrin apoproteins. Using a specific anti-NblAI antibody it was found that in strains of Tolypothrix sp. NblAI abundance increases under nitrogen-limiting conditions but the protein is also present in cells grown in nitrogen-replete medium. Gold immunolabelling experiments showed that, upon a nitrogen shift-down, NblAI is preferentially located in the differentiated heterocysts, where O(2) evolution has to be shut off for nitrogenase to operate. The results lead to the proposal that NblAI is a necessary 'cofactor' but not the triggering factor that governs phycobilisome degradation in Tolypothrix sp.

The NblAI protein from the filamentous cyanobacterium Tolypothrix PCC 7601: regulation of its expression and interactions with phycobilisome components.

Cyanobacteria respond to changes in light or nutrient availability by modifications in their photosynthetic light harvesting antenna. In unicellular cyanobacteria a small polypeptide (NblA) is required for phycobilisome degradation following environmental stresses. In the filamentous strain Tolypothrix sp. PCC 7601 the nblAI gene, encoding a NblA homologue, is located upstream of the operon coding for phycoerythrin (cpeBA). The nblAI transcripts all originate from a single transcription start point; their intracellular levels vary according to nitrogen regimes but not with light spectral quality. Using recombinant His-tagged NblAI protein, we found that in vitro NblAI has affinity for both phycocyanin and phycoerythrin subunits from Tolypothrix sp. PCC 7601, but not for allophycocyanin from this cyanobacterium or for phycobiliproteins from other cyanobacterial species. We also observed that although nblAI is mainly expressed under nitrogen starvation, NblAI polypeptides are always present in the cell; a significant portion of them co-purify with phycobilisome preparations but only if cells were grown under red light. Our data indicate that NblAI attaches to the phycobilisomes even under non-inducing conditions and suggest a preferential affinity of NblAI for phycocyanin.

In the unicellular red alga Rhodella violacea iron deficiency induces an accumulation of uncoupled LHC.

Iron plays a key role in the synthesis and functioning of the photosynthetic apparatus. Conditions of partial iron deficiency that lead to a relatively stable phenotype were established and the effects of starvation studied in the unicellular red alga, Rhodella violacea. Synthesis of the photosynthetic pigments were found to decrease, with phycobiliproteins being affected to a lesser extent than chlorophyll a. Biophysical, biochemical and immunological approaches were used to show that the PSI content is highly diminished and the PSII/PSI stoichiometry increased by a factor of 5 compared to standard conditions. Meanwhile light-harvesting complex (LHC) was still assembled in the thylakoid membranes at unchanged levels. The use of translation inhibitors for either nuclear- or plastid-encoded polypeptides revealed that uncoupled LHC may be responsible for the high wavelength-fluorescence contribution observed around 700-710 nm. There is no evidence for the synthesis of new chlorophyll-protein complexes.

Expression of the glutamyl-tRNA synthetase gene from the cyanobacterium Synechococcus sp PCC 7942 depends on nitrogen availability and the global regulator NtcA.

We report here transcriptional analyses of a cyanobacterial gene encoding an aminoacyl-tRNA synthetase (aaRS), the gltX gene from Synechoccocus sp. PCC 7942, coding for the glutamyl-tRNA synthetase. We show that the transcript levels of gltX in Synechococcus depend on nitrogen availability and do not increase with the growth rate, which is at odds with observations from other bacteria. We also demonstrate the involvement of the cyanobacterial global regulator NtcA in transcriptional control of gltX according to nitrogen status. Our results support a regulatory model in which the gltX transcript level is finely tuned by a dynamic equilibrium between activation and repression relying upon the cellular concentration of NtcA.

The ycf27 genes from cyanobacteria and eukaryotic algae: distribution and implications for chloroplast evolution.

The two ycf27 genes from the filamentous cyanobacterium Tolypothrix PCC 7601 have been cloned and sequenced. These two genes, previously designated rpaA and rpaB, encode putative transcriptional regulators of the 'OmpR' family. In Synechocystis PCC 6803, homologous genes have been linked to the regulation of transfer of excitation energy from the phycobilisome to photosystem (PS) I and PSII respectively. Partial clones from Spirulina platensis, Dactylococcopsis salina and Synechococcus PCC 7002 have also been sequenced. A table of identity between the proteins confirms that RpaB belongs in the same family as the algal ycf27 proteins. However, RpaA is a rather different protein and should lose the designation ycf27. The loss of rpaB from the plastid genomes of eukaryotic algae is associated with the loss of phycobiliproteins, so it is likely that this gene performs a similar role in algae to that in cyanobacteria. The implications for chloroplast evolution are discussed along with the possible identity of the cognate histidine kinase gene in the plastid genomes.

Co-ordinated expression of phycobiliprotein operons in the chromatically adapting cyanobacterium Calothrix PCC 7601: a role for RcaD and RcaG.

In the cyanobacterium Calothrix sp. PCC 7601 the cpc2 operon encoding phycocyanin 2 (PC2) is expressed if red radiations are available. RcaD was previously identified in extracts from red-light-grown cells as an alkaline phosphatase-sensitive protein that binds upstream of the transcription start point (TSP) of the cpc2 operon. In this work, RcaD was purified, and the corresponding gene cloned with a PCR probe obtained using degenerated primers based on RcaD peptide sequences (accession no. AJ319541). Purified RcaD binds to the cpc2 promoter region and also to those of the constitutive cpc1 and apc1 operons that encode phycocyanin 1 and allophycocyanin. Escherichia coli-overexpressed RcaD can bind to the cpc2 promoter region. The rcaD gene is upstream of an open reading frame (ORF) termed rcaG. Co-transcription of both genes was demonstrated by reverse transcription (RT)-PCR experiments, and found to be independent of the light wavelengths. A single TSP was mapped. Sequence features of RcaD and RcaG led us to propose a functional relationship between these two proteins. A rcaD mutant generated by allelic exchange exhibited altered expression of the cpc2, cpeBA, apc1 and cpc1 operons upon green to red-light shifts. RcaD seems to be a co-activator co-ordinating the transcription of the phycobiliprotein operons upon changes in light spectral quality.

Genomic survey of cAMP and cGMP signalling components in the cyanobacterium Synechocystis PCC 6803.

Cyanobacteria modulate intracellular levels of cAMP and cGMP in response to environmental conditions (light, nutrients and pH). In an attempt to identify components of the cAMP and cGMP signalling pathways in Synechocystis PCC 6803, the authors screened its complete genome sequence by using bioinformatic tools and data from sequence-function studies performed on both eukaryotic and prokaryotic cAMP/cGMP-dependent proteins. Sll1624 and Slr2100 were tentatively assigned as being two putative cyclic nucleotide phosphodiesterases. Five proteins were identified as having all the determinants required to be cyclic nucleotide receptors, two of them being probably more specific for cGMP (an element of two-component regulatory systems - Slr2104 - and a putative cyclic-nucleotide-gated cation channel - Slr1575), the three others being probably more specific for cAMP: (i) a protein of unidentified function (Slr0842); (ii) a putative cyclic-nucleotide-modulated permease (Slr0593), previously annotated as a kinase A regulatory subunit; and (iii) a putative transcription factor (CRP-SYN: =Sll1371), which possesses cAMP- and DNA-binding determinants homologous to those of the cAMP receptor protein of Escherichia coli (CRP-EC:). This homology, together with the presence in Synechocystis of CRP-EC:-like binding sites upstream of crp, cya1, slr1575, and several genes encoding enzymes involved in transport and metabolism, strongly suggests that CRP-SYN: is a global regulator.

Effect of the nitrogen source on phycobiliprotein synthesis and cell reserves in a chromatically adapting filamentous cyanobacterium.

Cyanobacteria can utilize nitrate or ammonium as a source of fixed nitrogen for cell growth. In the filamentous Calothrix sp. strain PCC 7601, these two sources of nitrogen differently influenced the phycobiliprotein composition of the phycobilisomes, the major light-harvesting antennae. When compared to nitrate, growth in the presence of ammonium resulted in intracellular steady-state levels 35% lower for phycoerythrin and 46% higher for phycocyanin. Besides these differences in cell pigmentation, a rapid but transient accumulation of cyanophycin granule polypeptide occurred in ammonium-grown cells, while these macromolecules were not detected in cells grown with nitrate. In contrast, glycogen reserves displayed a dynamic pattern of accumulation and disappearance during cell growth which varied only slightly with the nitrogen source. The observed changes in cell pigmentation are reminiscent of the phenomenon of complementary chromatic adaptation, in which green and red wavelengths promote the syntheses of phycoerythrin and phycocyanin-2, respectively. As in complementary chromatic adaptation, the regulation of synthesis of phycoerythrin and phycocyanin-2 by the nitrogen source occurred mainly at the mRNA level. Moreover, the transcriptional start sites for the expression of the cpeBA and the cpc2 operons, which respectively encode the two subunits of phycoerythrin and phycocyanin-2, were the same in cells grown in nitrate or ammonium, and identical to those in green- and red-light-grown cells. The results of this study suggest that acclimation to the spectral light quality and to the nitrogen source share some common regulatory elements.

A multigene family in Calothrix sp. PCC 7601 encodes phycocyanin, the major component of the cyanobacterial light-harvesting antenna

In cyanobacteria, light is harvested by phycobilisomes which are essentially made up of chromophoric proteins called phycobiliproteins. We have characterized two gene clusters (cpcB1, cpcA1 and cpcB3, cpcA3) each encoding the two subunits of phycocyanin (ßPC and αPC, respectively), one of the major phycobiliproteins in Calothrix 7601. Downstream from the gene encoding the PCα subunit in cluster 1, an open reading frame was found, cpcE1. These genes are organized in two transcriptional units, namely: cpcB3 A3 and cpcB1 A1 E1. All these genes are transcribed whatever the chromatic light received during cell growth. Consequently, although only one type of "constitutive" PC has been biochemically characterized, we have demonstrated that there are two cpc operons "constitutively" transcribed in this strain. With the previously described red light "inducible" cpcB2 A2 operon, there are three copies of the PC encoding genes in Calothrix 7601. The significance of this newly described multigene family in cyanobacteria is discussed. We have also mapped the 5' and 3' termini of the major transcript from the cpc1 operon. Analysis of the 5' untranslated region of this transcript has revealed alternative secondary structures which are proposed to play a role in the regulation of the expression of this operon.