Eucarotic carboxysomes in Cyanophora paradoxa

Photosynthesis without doubt is basic to advanced forms of life on our planet. About 50% of the enormous amount of more than 2 x 1011 tons CO 2 that are converted into biomass per year is contributed by aquatic microorganisms. This is made possible through the operation of inorganic carbon-concentrating mechanisms (CCM). The evolution of CCM will be studied using the "living fossil" Cyanophora paradoxa, Glaucocystophyta. This primordial phototrophic eukaryote (phylogenetic analyses place glaucocystophytes on the first branch after the single primary endosymbiotic event that gave rise to the kingdom "Plantae") holds a unique bridge position between cyanobacteria and algae. Only glaucocystophytes harbour plastids (cyanelles) that are surrounded by a peptidoglycan wall and - possibly -contain the key enzyme Rubisco compartmentalized in the form of a carboxysome. Both these features are normally restricted to prokaryotes. In algae, "solid" Rubisco often is organized in a functionally similar microcompartment, the pyrenoid. Two theories describe the origin of the CCM: one puts it early on the time scale, prior to the primary endosymbiotic event, i.e. more than 1.2 billion years ago. In this scenario, the pyrenoids of algae would have been derived from the carboxysomes of the cyanobacterial endosymbionts and C. paradoxa could still possess a carboxysomal CCM. Thus the cyanelles accumulate bicarbonate to such an extent that they would burst without the stabilizing wall. The second hypothesis posits that CCMs in cyanobacteria and algae arose independently and much later, about 400 million years ago. The localization of carbonic anhydrase (CA) catalyzing the interconversion of CO 2 and bicarbonate is crucial. In carboxysomes, this enzyme is co-packaged with Rubisco, in pyrenoids it localizes to the lumen of thylakoid membranes traversing the microcompartment. In the literature, the electron-dense central body of cyanelles often is named carboxysome. This view has its points but important data are still lacking. We want to investigate cyanelle CA with various methods: information about the corresponding gene and protein should be obtained via EST-sequencing, PCR using degenerate primers, mass spectrometry of protein bands from isolated central bodies, heterologous westerns, heterologous immuno-EM, etc. Once the gene is available, the labelled precursor will be imported in vitro into cyanelles and the incorporation of the mature protein into the putative carboxysome will be determined. Only then a decision can be made, including the possibility that we deal with a type of microcompartment in between carboxysomes and pyrenoids. The microarrays established in the previous project should show enhanced effects of low CO 2 concentrations on gene expression when the conditions are changed (high light, nitrogen limitation). The enrichment factor for bicarbonate in cyanelles will be determined, to clarify if osmoprotection is the "raison d`etre" for the unique eukaryotic peptidoglycan. Other components of the CCM known from cyanobacteria and/or algae, as shell proteins, established or putative Ci transporters, etc. should also be demonstrated in cyanelles or in the genome of C. paradoxa.

The unicellular alga Cyanophora paradoxa (Glaucocystophyta) is a key organism for understanding plastid evolution from endosymbiotic cyanobacteria. The unique features of the photosynthetic organelles (cyanelles) of this `living fossil` are a peptidoglycan wall (otherwise found in bacteria only) and the presence of carboxysomes: our hypothesis. Carboxysomes are cyanobacterial microcompartments that contain the bulk of ribulose-1, 5- bisphosphate carboxylase (Rubisco) in quasi-crystalline form and play an important role in the inorganic carbon concentrating mechanism (CCM). Bicarbonate is strongly enriched within the cell, diffuses into in the carboxysomes and is converted by their co-packaged carbonic anhydrase (CA) into CO 2 which is efficiently fixed through the high concentration of surrounding Rubisco. This enables photosynthetic microorganisms tho thrive despite the even lower CO 2 concentrations in aqueous solution and to contribute about 50% (i. e. in the same range as the rain forest) to the global CO 2 balance. Most eukaryotic algae also possess a CCM which is based on a similar Rubisco-microcompartment, the pyrenoid. This is considerably larger than carboxysomes (in general one per chloroplast) and lacks their polyhedric structure and their confining electron-dense layer consisting of `shell` proteins. The pyrenoid is traversed by thylakoid membranes containing a lumenal CA, at least in Chlamydomonas reinhardtii. When its size and its presence in an alga are considered, the Rubisco-microcompartment of C. paradoxa should be classified as a pyrenoid. However, an interesting theory links this putative `eukaryotic carboxysome` with the peptidoglycan wall and gives a plausible explanation for the retainment of the latter: upon primary endosymbiosis, the carboxysomal CCM was transferred to the glaucocystophytes. The concentration of bicarbonate by a factor of >1000 in the organelles is possible provided the osmotic stress-bearing peptidoglycan layer is retained. In the course of evolution, all other algae converted their CCM into a pyrenoidal one (factor only 70) and abandoned the organelle wall. The goals of our work were: i) to demonstrate the operation of a CCM in C. paradoxa, ii) to perform electronmicroscopic investigations, and iii) to identify CA and shell proteins in `carboxysome` preparations using (genomic and) proteomic methods. Outcome: i) The operation of a CCM in C. paradoxa was unequivocally demonstrated, enabling us to publish our data on CO 2 -responsive genes; ii) EM revealed interesting morphological differences of the microcompartments at high and low [CO2 ], respectively, but no confirmation of a proteinaceous shell; iii) a number of unknown proteins were obtained which at present could not yet be identified as CA or shell proteins. In summary, despite considerable progress, the crucial question - carboxysome or pyrenoid - still remains open for the cyanelle microcompartment of C. paradoxa.