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Multiple independent losses of photosynthesis have occurred among several unrelated eukaryotic lineages including red and green algae, land plants, alveolates, stramenopiles, cryptophytes and euglenophytes. Most plastid genomes of non-photosynthetic eukaryotes do not contain genes associated with photosynthesis, but they usually encode at least one gene product involved in an essential non-photosynthetic metabolic pathway. Complete loss of plastid genome and simultaneous retention of plastid compartment is rare, and complete plastid loss is documented only for few alveolate species. Non-photosynthetic plastids are often involved in essential fatty acid, isoprenoid, Fe-S cluster, and heme synthesis.

Calpains are cysteine proteases involved in many cellular processes. They are an ancient and large superfamily of enzymes responsible for the cleavage and irreversible modification of a large variety of substrates. They have been intensively studied in humans and other mammals, but information about calpains in bacteria is scarce. Calpains have not been found among Archaea to date. In this study, we have investigated the presence of calpains in selected cyanobacterial species using in silico analyses. We show that calpains defined by possessing CysPC core domain are present in cyanobacterial genera Anabaena, Aphanizomenon, Calothrix, Chamaesiphon, Fischerella, Microcystis, Scytonema and Trichormus. Based on in silico protein interaction analysis, we have predicted putative interaction partners for identified cyanobacterial calpains. The phylogenetic analysis including cyanobacterial, other bacterial and eukaryotic calpains divided bacterial and eukaryotic calpains into two separate monophyletic clusters. We propose two possible evolutionary scenarios to explain this tree topology: (1) the eukaryotic ancestor or an archaeal ancestor of eukaryotes obtained calpain gene from an unknown bacterial donor, or alternatively (2) calpain gene had been already present in the last common universal ancestor and subsequently lost by the ancestor of Archaea, but retained by the ancestor of Bacteria and by the ancestor of Eukarya. Both scenarios would require multiple independent losses of calpain genes in various bacteria and eukaryotes.

Aims: Euglena gracilis is used as model organism for various microbiological, molecular biological and biotechnological studies. Its most studied wild-type strains are Z and bacillaris, but their discrimination by standard molecular methods is difficult.Therefore, we decided to test the suitability of MALDI-TOF MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) for identification of E. gracilis and for discrimination of these two strains possessing functional chloroplasts. MALDI-TOF MS profiling was also tested for two white (non-photosynthetic) stable E. gracilis mutant strains W gm ZOflL and W 10 BSmL. Methods and results:We have successfully obtained main spectrum profiles (MSPs) of E. gracilis strains Z, SAG 1224-5/25 and bacillaris, SAG 1224-5/15 using protein extraction procedure. Subsequent MALDI-TOF MS profiling of a number of tested samples and the comparison of the obtained protein profiles with our in-house database including MSPs of both strains have revealed that these two strains can be easily distinguished by MALDI-TOF MS based on score values over two in most cases.This method has also confirmed the ancestry of white mutant strains W gm ZOflL and W 10 BSmL, originally derived from strains Z and bacillaris, respectively.Conclusions: MALDI-TOF MS is suitable, accurate and rapid method for discrimination of E. gracilis strains. Significance and Impact of the Study: These results can have broad practical implications for laboratories cultivating various strains of euglenids, and they can be applied for their discrimination by MALDI-TOF MS.