Complementation of a phycocyanin-bilin lyase from Synechocystis sp. PCC 6803 with a nucleomorph-encoded open reading frame from the cryptophyte Guillardia theta.

BACKGROUND: Cryptophytes are highly compartmentalized organisms, expressing a secondary minimized eukaryotic genome in the nucleomorph and its surrounding remnant cytoplasm, in addition to the cell nucleus, the mitochondrion and the plastid. Because the members of the nucleomorph-encoded proteome may contribute to essential cellular pathways, elucidating nucleomorph-encoded functions is of utmost interest. Unfortunately, cryptophytes are inaccessible for genetic transformations thus far. Therefore the functions of nucleomorph-encoded proteins must be elucidated indirectly by application of methods in genetically accessible organisms. RESULTS: Orf222, one of the uncharacterized nucleomorph-specific open reading frames of the cryptophyte Guillardia theta, shows homology to slr1649 of Synechocystis sp. PCC 6803. Recently a further homolog from Synechococcus sp. PCC 7002 was characterized to encode a phycocyanin-beta155-bilin lyase. Here we show by insertion mutagenesis that the Synechocystis sp. PCC 6803 slr1649-encoded protein also acts as a bilin lyase, and additionally contributes to linker attachment and/or stability of phycobilisomes. Finally, our results indicate that the phycocyanin-beta155-bilin lyase of Synechocystis sp. PCC 6803 can be complemented in vivo by the nucleomorph-encoded open reading frame orf222. CONCLUSION: Our data show that the loss of phycocyanin-lyase function causes pleiotropic effects in Synechocystis sp. PCC 6803 and indicate that after separating from a common ancestor protein, the phycoerythrin lyase from Guillardia theta has retained its capacity to couple a bilin group to other phycobiliproteins. This is a further, unexpected example of the universality of phycobiliprotein lyases.

Unique tRNA introns of an enslaved algal cell.

Nucleomorphs are remnant nuclei of eukaryotic, secondary endosymbionts exclusively found in cryptophytes and chlorarachniophytes. The nucleomorph of the cryptophyte Guillardia theta codes for 36 transfer RNA (tRNA) genes, 15 of them predicted to contain introns and 1 pseudo-tRNA. Some of the predicted intervening sequences are manifested at positions not known in Eukarya, even tRNAs with more than one intron were suggested. By isolating reverse transcriptase-polymerase chain reaction products of the spliced tRNAs we verify the processing of all predicted intron-harboring tRNAs and demonstrate the splicing of the smallest introns (3 nt) investigated so far. However, the spliced intervening sequences are in some cases shifted in respect to the predicted ones. Moreover, we show that introns, if inserted into the B-box of tRNA genes in the nucleomorph of cryptophytes, mimic promoter regions and do not abolish transcription by RNA polymerase III. Consequently, internal nucleomorph-encoded tRNA promoter regions are in some cases dissected from the sequence of the mature tRNAs. By reanalyzing tRNA introns of a recently sequenced red algae we furthermore show that splicing of introns at unusual positions may be introduced in cryptophytes by its secondary endosymbiont. However, in contrast to the rest of the symbiont genome, introns are not minimized in quantity but are instead scattered along the tRNA genes.