Manfred G. Bayer

Sequence analysis of pre-ferredoxin-NADP+-reductase cDNA from Cyanophora paradoxa specifying a precursor for a nucleus-encoded cyanelle polypeptide

A cDNA clone for pre-ferredoxin-NADP+ reductase (FNR) was obtained by screening a Cyanophora paradoxa expression library with antibodies specific for cyanelle FNR. The 1.4 kb transcript was derived from a single-copy gene. The precursor (41 kDa) and mature forms (34 kDa) of FNR were identified by western blotting of in vitro translation products and cyanelle extracts, respectively. The derived amino acid sequence of the mature form was corroborated by data from N-terminal protein sequencing and yielded identity scores from 58% to 62% upon comparison with cyanobacterial FNRs. Sequence conservation seemed to be even more pronounced in comparison with enzymes from higher plants, but using the neighbor joining method the C. paradoxa sequence was clearly positioned between the prokaryotic and eukaryotic sequences. The transit peptide of 65 or 66 amino acids appeared to be totally unrelated to those from spinach, pea and ice plant but showed overall characteristics of stroma-targeting peptides.

Cyanophora paradoxa: nucleotide sequence and phylogeny of the nucleus encoded muroplast fructose-1,6-bisphosphate aldolase.

Immunoscreening of a C. paradoxa expression library against water soluble muroplast (“cyanelle”) proteins resulted in isolation of a clone encoding the nucleus-encoded muroplast class-II fructose-1,6-bisphosphate aldolase (class-II FBA§). Its nucleotide sequence was determined. The 1432 bp insert, derived from a single-copy gene transcript, bears a reading frame of 1206 bp in length, representing 402 amino acids with 346 amino acids of mature protein. The leading amino acids match structural features necessary for precursor import across chloroplast envelope membranes. In phylogenetic tree topology, the investigated mature FBA clusters within type B FBAs with Synechocystis sp. as nearest neighbor. This is the first report of a Type B class-II FBA sequence of plastids.

Ferredoxin of Cyanophora paradoxa Korsch. is encoded on cyanellar DNA

SummaryThe ferredoxin present in the chloroplasts of higher plants and algae is encoded by the nuclear genome. We found that this is not the case for the cyanellar ferredoxin of Cyanophora paradoxa. The coding and synthesizing site of this polypeptide was determined by selective inhibition of transcription and translation in the host and the cyanellar compartment, respectively. Water-soluble cyanellar proteins were isolated, extracted from in vivo 14C-labelled cells, separated by NEPHGE and analyzed by autoradiography. The results clearly showed that the ferredoxin must be encoded on the cyanellar genome. This finding is discussed in the context of the evolutionary position of C. paradoxa cyanelles as a model system with which to study intermediate stages between cyanobacteria and plastids.

Cyanellar Ferredoxin-NADP+-oxidoreductase of Cyanophora paradoxa is encoded by the nuclear genome and synthesized on cytoplasmatic 80S ribosomes

SummaryCyanophora paradoxa is an important model organism for the study of the transition from endocytobiontic cyanobacteria to factual eukaryotic cell organelles. The cyanelles of these organisms possess cyanobacterial, as well as plastidic, characteristics. Although the transfer of cyanellar proteins from cytosolic into cyanellar space has been shown, the process of translocation of a known protein across the peptidoglycan layer and the envelope membranes has not been characterized. In this study we demonstrate that a specific and obligate cyanelle protein —Ferredoxin-NADP+-oxidoreductase (FNR) — is coded on the nuclear genome, synthesized on 80S ribosomes and transported from the eukaryotic cell compartment into the cyanelles of Cyanophora paradoxa, an original intracellular host-guest relation. These results indicate a gene transfer from guest to host genome and support the view that, in spite of their cyanobacterial origin, cyanelles have been evolved to cell organelles comparable to plastids.

Ferredoxin: NADP oxidoreductase of Cyanophora paradoxa: Purification, partial characterization, and N-terminal amino acid sequence

The ferredoxin:NADP+ oxidoreductase of the protist Cyanophora paradoxa, as a descendant of a former symbiotic consortium, an important model organism in view of the Endosymbiosis Theory, is the first enzyme purified from a formerly original endocytobiont (cyanelle) that is found to be encoded in the nucleus of the host. This cyanoplast enzyme was isolated by FPLC (19% yield) and characterized with respect to the uv-vis spectrum, pH optimum (pH 9), molecular mass of 34 kDa, and an N-terminal amino acid sequence (24 residues). The enzyme shows, as known from other organisms, molecular heterogeneity. The N-terminus of a further ferredoxin:NADP+ oxidoreductase polypeptide represents a shorter sequence missing the first four amino acids of the mature enzyme.

Ferredoxin-NADP+ oxidoreductase of C. paradoxa nucleus encoded, but cyanobacterial gene transfer from symbiont to host, an evolutionary mechanism originating new species

The nucleus encoded cyanoplast (“cyanellar”) ferredoxin-NADP+ -oxidoreductase (FNR ) of Cyanophora paradoxa, characterized by an N-terminal amino acid sequence, is compared with homologous sequences of other photoautotrophic organisms. The high degree of similarity to the cyanobacterial sequences indicates a cyanobacterial origin. This could be a first direct demonstration of an intertaxonic combination: a gene transfer from an original endocytobiont (cyanobacterium) to the nucleus of its host, one of the most important demands of the Endosymbiosis Theory, an evolutionary mechanism leading to the origin of a new species.

Two-step purification of Cyanophora ferredoxin and its identification in soluble protein preparations by isoelectric focusing

Cyanophora paradoxa ferredoxin is encoded by (cyano-)plastidic DNA, in contrast to those of all other photosynthetic eukaryotes investigated so far. In the present study we report (i) the rapid purification of a chloroplast-type [2Fe-2S] ferredoxin in a two-step procedure by DEAE-Sephadex and Mono Q ion-exchange chromatography; (ii) the biochemical characterization of the purified ferredoxin by electrophoretic separation methods on a microscale; and (iii) a qualitative and quantitative ferredoxin detection method in the femtomole range that allows densitometry, semidry immunoblotting, identification of ferredoxin in soluble cell protein preparations, and analysis of protein biosynthesis from cyanoplast poly(A)- RNA in vivo and in vitro. These fast micromethods should be useful for screening phototrophic species containing ferredoxins encoded by nonnuclear DNA.