A thylakoid biogenesis BtpA protein is required for the initial step of tetrapyrrole biosynthesis in cyanobacteria.

Biogenesis of the photosynthetic apparatus requires complicated molecular machinery, individual components of which are either poorly characterized or unknown. The BtpA protein has been described as a factor required for the stability of photosystem I (PSI) in cyanobacteria; however, how the BtpA stabilized PSI remains unexplained. To clarify the role of BtpA, we constructed and characterized the btpA-null mutant (ΔbtpA) in the cyanobacterium Synechocystis sp. PCC 6803. The mutant contained only c. 1% of chlorophyll and nearly no thylakoid membranes. However, this strain, growing only in the presence of glucose, was genetically unstable and readily generated suppressor mutations that restore the photoautotrophy. Two suppressor mutations were mapped into the hemA gene encoding glutamyl-tRNA reductase (GluTR) - the first enzyme of tetrapyrrole biosynthesis. Indeed, the GluTR was not detectable in the ΔbtpA mutant and the suppressor mutations restored biosynthesis of tetrapyrroles and photoautotrophy by increased GluTR expression or by improved GluTR stability/processivity. We further demonstrated that GluTR associates with a large BtpA oligomer and that BtpA is required for the stability of GluTR. Our results show that the BtpA protein is involved in the biogenesis of photosystems at the level of regulation of tetrapyrrole biosynthesis.

Minor pilins are involved in motility and natural competence in the cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria synthesize type IV pili, which are known to be essential for motility, adhesion and natural competence. They consist of long flexible fibers that are primarily composed of the major pilin PilA1 in Synechocystis sp. PCC 6803. In addition, Synechocystis encodes less abundant pilin-like proteins, which are known as minor pilins. In this study, we show that the minor pilin PilA5 is essential for natural transformation but is dispensable for motility and flocculation. In contrast, a set of minor pilins encoded by the pilA9-slr2019 transcriptional unit are necessary for motility but are dispensable for natural transformation. Neither pilA5-pilA6 nor pilA9-slr2019 are essential for pilus assembly as mutant strains showed type IV pili on the cell surface. Three further gene products with similarity to PilX-like minor pilins have a function in flocculation of Synechocystis. The results of our study indicate that different minor pilins facilitate distinct pilus functions. Further, our microarray analysis demonstrated that the transcription levels of the minor pilin genes change in response to surface contact. A total of 122 genes were determined to have altered transcription between planktonic and surface growth, including several plasmid genes which are involved exopolysaccharide synthesis and the formation of bloom-like aggregates.

The Ribosome-Bound Protein Pam68 Promotes Insertion of Chlorophyll into the CP47 Subunit of Photosystem II.

Photosystem II (PSII) is a large enzyme complex embedded in the thylakoid membrane of oxygenic phototrophs. The biogenesis of PSII requires the assembly of more than 30 subunits, with the assistance of a number of auxiliary proteins. In plants and cyanobacteria, the photosynthesis-affected mutant 68 (Pam68) is important for PSII assembly. However, its mechanisms of action remain unknown. Using a Synechocystis PCC 6803 strain expressing Flag-tagged Pam68, we purified a large protein complex containing ribosomes, SecY translocase, and the chlorophyll-binding PSII inner antenna CP47. Using 2D gel electrophoresis, we identified a pigmented Pam68-CP47 subcomplex and found Pam68 bound to ribosomes. Our results show that Pam68 binds to ribosomes even in the absence of CP47 translation. Furthermore, Pam68 associates with CP47 at an early phase of its biogenesis and promotes the synthesis of this chlorophyll-binding polypeptide until the attachment of the small PSII subunit PsbH. Deletion of both Pam68 and PsbH nearly abolishes the synthesis of CP47, which can be restored by enhancing chlorophyll biosynthesis. These results strongly suggest that ribosome-bound Pam68 stabilizes membrane segments of CP47 and facilitates the insertion of chlorophyll molecules into the translated CP47 polypeptide chain.

Interaction of the PsbH subunit with a chlorophyll bound to histidine 114 of CP47 is responsible for the red 77K fluorescence of Photosystem II.

A characteristic feature of the active Photosystem II (PSII) complex is a red-shifted low temperature fluorescence emission at about 693nm. The origin of this emission has been attributed to a monomeric 'red' chlorophyll molecule located in the CP47 subunit. However, the identity and function of this chlorophyll remain uncertain. In our previous work, we could not detect the red PSII emission in a mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking PsbH, a small transmembrane subunit bound to CP47. However, it has not been clear whether the PsbH is structurally essential for the red emission or the observed effect of mutation has been indirectly caused by compromised PSII stability and function. In the present work we performed a detailed spectroscopic characterization of PSII in cells of a mutant lacking PsbH and Photosystem I and we also characterized PSII core complexes isolated from this mutant. In addition, we purified and characterized the CP47 assembly modules containing and lacking PsbH. The results clearly confirm an essential role of PsbH in the origin of the PSII red emission and also demonstrate that PsbH stabilizes the binding of one ß-carotene molecule in PSII. Crystal structures of the cyanobacterial PSII show that PsbH directly interacts with a single monomeric chlorophyll ligated by the histidine 114 residue of CP47 and we conclude that this peripheral chlorophyll hydrogen-bonded to PsbH is responsible for the red fluorescence state of CP47. Given the proximity of ß-carotene this state could participate in the dissipation of excessive light energy.

Accumulation of the Type IV prepilin triggers degradation of SecY and YidC and inhibits synthesis of Photosystem II proteins in the cyanobacterium Synechocystis†PCC 6803.

Type IV pilins are bacterial proteins that are small in size but have a broad range of functions, including motility, transformation competence and secretion. Although pilins vary in sequence, they possess a characteristic signal peptide that has to be removed by the prepilin peptidase PilD during pilin maturation. We generated a pilD (slr1120) null mutant of the cyanobacterium Synechocystis 6803 that accumulates an unprocessed form of the major pilin PilA1 (pPilA1) and its non-glycosylated derivative (NpPilA1). Notably, the pilD strain had aberrant membrane ultrastructure and did not grow photoautotrophically because the synthesis of Photosystem II subunits was abolished. However, other membrane components such as Photosystem I and ATP synthase were synthesized at levels comparable to the control strain. Proliferation of the pilD strain was rescued by elimination of the pilA1 gene, demonstrating that PilA1 prepilin inhibited the synthesis of Photosystem II. Furthermore, NpPilA1 co-immunoprecipitated with the SecY translocase and the YidC insertase, and both of these essential translocon components were degraded in the mutant. We propose that unprocessed prepilins inactivate an identical pool of translocons that function in the synthesis of both pilins and the core subunits of Photosystem II.

Long-term acclimation of the cyanobacterium Synechocystis sp. PCC 6803 to high light is accompanied by an enhanced production of chlorophyll that is preferentially channeled to trimeric photosystem I.

Cyanobacteria acclimate to high-light conditions by adjusting photosystem stoichiometry through a decrease of photosystem I (PSI) abundance in thylakoid membranes. As PSI complexes bind the majority of chlorophyll (Chl) in cyanobacterial cells, it is accepted that the mechanism controlling PSI level/synthesis is tightly associated with the Chl biosynthetic pathway. However, how Chl is distributed to photosystems under different light conditions remains unknown. Using radioactive labeling by (35)S and by (14)C combined with native/two-dimensional electrophoresis, we assessed the synthesis and accumulation of photosynthetic complexes in parallel with the synthesis of Chl in Synechocystis sp. PCC 6803 cells acclimated to different light intensities. Although cells acclimated to higher irradiances (150 and 300 µE m(-2)s(-1)) exhibited markedly reduced PSI content when compared with cells grown at lower irradiances (10 and 40 µE m(-2) s(-1)), they grew much faster and synthesized significantly more Chl, as well as both photosystems. Interestingly, even under high irradiance, almost all labeled de novo Chl was localized in the trimeric PSI, whereas only a weak Chl labeling in photosystem II (PSII) was accompanied by the intensive (35)S protein labeling, which was much stronger than in PSI. These results suggest that PSII subunits are mostly synthesized using recycled Chl molecules previously released during PSII repair-driven protein degradation. In contrast, most of the fresh Chl is utilized for synthesis of PSI complexes likely to maintain a constant level of PSI during cell proliferation.

Evolutionary Patterns of Thylakoid Architecture in Cyanobacteria.

While photosynthetic processes have become increasingly understood in cyanobacterial model strains, differences in the spatial distribution of thylakoid membranes among various lineages have been largely unexplored. Cyanobacterial cells exhibit an intriguing diversity in thylakoid arrangements, ranging from simple parietal to radial, coiled, parallel, and special types. Although metabolic background of their variability remains unknown, it has been suggested that thylakoid patterns are stable in certain phylogenetic clades. For decades, thylakoid arrangements have been used in cyanobacterial classification as one of the crucial characters for definition of taxa. The last comprehensive study addressing their evolutionary history in cyanobacteria was published 15 years ago. Since then both DNA sequence and electron microscopy data have grown rapidly. In the current study, we map ultrastructural data of >200 strains onto the SSU rRNA gene tree, and the resulting phylogeny is compared to a phylogenomic tree. Changes in thylakoid architecture in general follow the phylogeny of housekeeping loci. Parietal arrangement is resolved as the original thylakoid organization, evolving into complex arrangement in the most derived group of heterocytous cyanobacteria. Cyanobacteria occupying intermediate phylogenetic positions (greater filamentous, coccoid, and baeocytous types) exhibit fascicular, radial, and parallel arrangements, partly tracing the reconstructed course of phylogenetic branching. Contrary to previous studies, taxonomic value of thylakoid morphology seems very limited. Only special cases such as thylakoid absence or the parallel arrangement could be used as taxonomically informative apomorphies. The phylogenetic trees provide evidence of both paraphyly and reversion from more derived architectures in the simple parietal thylakoid pattern. Repeated convergent evolution is suggested for the radial and fascicular architectures. Moreover, thylakoid arrangement is constrained by cell size, excluding the occurrence of complex architectures in cyanobacteria smaller than 2 µm in width. It may further be dependent on unknown (eco)physiological factors as suggested by recurrence of the radial type in unrelated but morphologically similar cyanobacteria, and occurrence of special features throughout the phylogeny. No straightforward phylogenetic congruences have been found between proteins involved in photosynthesis and thylakoid formation, and the thylakoid patterns. Remarkably, several postulated thylakoid biogenesis factors are partly or completely missing in cyanobacteria, challenging their proposed essential roles.

Synthesis of Chlorophyll-Binding Proteins in a Fully Segregated Δycf54 Strain of the Cyanobacterium Synechocystis PCC 6803.

In the chlorophyll (Chl) biosynthesis pathway the formation of protochlorophyllide is catalyzed by Mg-protoporphyrin IX methyl ester (MgPME) cyclase. The Ycf54 protein was recently shown to form a complex with another component of the oxidative cyclase, Sll1214 (CycI), and partial inactivation of the ycf54 gene leads to Chl deficiency in cyanobacteria and plants. The exact function of the Ycf54 is not known, however, and further progress depends on construction and characterization of a mutant cyanobacterial strain with a fully inactivated ycf54 gene. Here, we report the complete deletion of the ycf54 gene in the cyanobacterium Synechocystis 6803; the resulting Δycf54 strain accumulates huge concentrations of the cyclase substrate MgPME together with another pigment, which we identified using nuclear magnetic resonance as 3-formyl MgPME. The detection of a small amount (~13%) of Chl in the Δycf54 mutant provides clear evidence that the Ycf54 protein is important, but not essential, for activity of the oxidative cyclase. The greatly reduced formation of protochlorophyllide in the Δycf54 strain provided an opportunity to use (35)S protein labeling combined with 2D electrophoresis to examine the synthesis of all known Chl-binding protein complexes under drastically restricted de novo Chl biosynthesis. We show that although the Δycf54 strain synthesizes very limited amounts of photosystem I and the CP47 and CP43 subunits of photosystem II (PSII), the synthesis of PSII D1 and D2 subunits and their assembly into the reaction centre (RCII) assembly intermediate were not affected. Furthermore, the levels of other Chl complexes such as cytochrome b 6 f and the HliD- Chl synthase remained comparable to wild-type. These data demonstrate that the requirement for de novo Chl molecules differs completely for each Chl-binding protein. Chl traffic and recycling in the cyanobacterial cell as well as the function of Ycf54 are discussed.

Awakening of a Dormant Cyanobacterium from Nitrogen Chlorosis Reveals a Genetically Determined Program.

The molecular and physiological mechanisms involved in the transition of microbial cells from a resting state to the active vegetative state are critically relevant for solving problems in fields ranging from microbial ecology to infection microbiology. Cyanobacteria that cannot fix nitrogen are able to survive prolonged periods of nitrogen starvation as chlorotic cells in a dormant state. When provided with a usable nitrogen source, these cells re-green within 48 hr and return to vegetative growth. Here we investigated the resuscitation of chlorotic Synechocystis sp. PCC 6803 cells at the physiological and molecular levels with the aim of understanding the awakening process of a dormant bacterium. Almost immediately upon nitrate addition, the cells initiated a highly organized resuscitation program. In the first phase, they suppressed any residual photosynthetic activity and activated respiration to gain energy from glycogen catabolism. Concomitantly, they restored the entire translational apparatus, ATP synthesis, and nitrate assimilation. After only 12-16 hr, the cells re-activated the synthesis of the photosynthetic apparatus and prepared for metabolic re-wiring toward photosynthesis. When the cells reached full photosynthetic capacity after ∼48 hr, they resumed cell division and entered the vegetative cell cycle. An analysis of the transcriptional dynamics during the resuscitation process revealed a perfect match to the observed physiological processes, and it suggested that non-coding RNAs play a major regulatory role during the lifestyle switch in awakening cells. This genetically encoded program ensures rapid colonization of habitats in which nitrogen starvation imposes a recurring growth limitation.