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Protistology ■ 59
the natural fluorescence of cellular photosynthetic pigments demonstrated that the observed increase in transcription was not directly linked to their concentration in cells. Moreover, this analysis revealed heterogeneity in the pigment fluorescence among distinct cells within the model culture. Funded in part by RFBR, project 15-29-02706.
THE HIGH COMPLEXITY AND DYNAMIC EVOLUTION OF THE RAS SUPERFAMILY OF GTPASES IN NAEGLERIA Petrzelkova R.1, Herman E.K.2, Dacks J.B.2, Elias M.1
1 - University of Ostrava, Faculty ofScience, Department ofBiology and Ecology, Ostrava, Czech Republic
2 - Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada [email protected]
Ras superfamily GTPases constitute a vast group of proteins involved in many eukaryote-specific processes. The last eukaryotic common ancestor appears to have possessed at least several tens of Ras superfamily genes, but gene duplications and losses in different eukaryotic lineages have modified this ancestral set such that substantially different gene complements may be present in different eukaryote groups. One extreme are taxa harbouring an extensively expanded Ras superfamily complement, as is the case of a free-living amoeboflagellate Nae-gleria gruberi (Heterolobosea). Recently, genome sequences of three strains of Naegleria fowleri, a causative agent of primary amebic meningoencephalitis (PAM), became available for analysis. In order to assess the differences between the two species and the three strains, we identified and annotated the Ras superfamily genes in the newly sequenced N. fowleri genomes and reannotated the respective gene complement in the previously published N. gruberi genome. The sets of Ras superfamily genes turned out to differ substantially between the two species, as N. gruberi harbours over 350 genes, whereas N. fowleri exhibits a much less expanded set with "only" over 200 genes. In contrast, little, if any, differences were found for the three N. fowleri strains. Phylogenetic analyses revealed both species-specific duplications and losses as the factors responsible for the different gene numbers in the two species. The evolution of the Ras superfamily in the genus Naegleria is thus surprisingly dynamic and points to a hidden level of differentiation in cellular physiology of different Naegleria species.
EXPRESSION AND PURIFICATION OF A PNEUMOCYSTIS JIROVECII SYNTHETIC RECOMBINANT ANTIGEN AND APPLICATION IN THE DEVELOPMENT OF A SEROLOGICAL RAPID DIAGNOSTIC TEST Pinto Mafalda12, Cardoso Fernando2, Gomes Ines1,3, Pereira Eulália4, Peixoto Miguel4, Franco Ricardo1, Matos Olga2
1 - UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa (UNL), Portugal
2 - Medical Parasitology Unit, Group ofOpportunistic Protozoa/HIV and Other Protozoa, GHTM, Instituto de Higiene e Medicina Tropical, UNL, Portugal
3 - Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal
4 - UCIBIO, REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciencias, Universidade do Porto, Portugal
Pneumocystis pneumonia (PcP) is an infectious disease caused by Pneumocystis jirovecii, an atypical fungus. PcP remains a major cause of respiratory illness among immunosuppressed patients. Current PcP diagnosis is based on the detection of P. jirovecii in respiratory specimens, obtained by invasive methods such as bronchoalveolar lavage, followed by cytochemical staining, immunofluorescent staining with monoclonal antibodies (IF/Mab) or PCR. Therefore, the possibility of an early diagnostic method allowing the use of biological specimens obtained non-invasively, is highly desirable. Rapid diagnostic tests (RDTs) using gold nanoparticles (AuNPs) allow a more sensitive, fast and cheap diagnosis, to be used in developing countries. The goal of this work is to develop an immunochromatographic RDT for the detection of P. jirovecii in non-invasive specimens like serum. In this test, spherical AuNPs are conjugated with a multi-epitope synthetic recombinant antigen (msr) which will allow the detection of circulating anti-P. jirovecii antibodies in sera. In order to obtain the highest amount of pure antigen, the expression vector pLATE 31, which contains the coding sequence for the MSG g antigen, was isolated and cloned in E. coli XJb (DE3). Extraction and purification through affinity chromatography with immobilized metallic ions, ELISA, SDS-PAGE and Western-Blot, were performed with the objective to obtain the maximum quantity of antigen and determine its purity. The antigen was then used to form bionanoconjugates with AuNPs, previously
60 • "PROTIST—2016
functionalized with several types of ligands, which will be the core of the immunochromatographic RDT with potential for point-of-care diagnostics. Acknowledgments: Partially supported by Gilead GENESE-PGG/001/2014.
DIVERSITY OF PROTISTS IN SALINE AND BRACKISH CONTINENTAL WATER BODIES REVEALED BY HIGH-THROUGHPUT SEQUENCING
Plotnikov Andrey12, Gerasimova Elena1, Poshvina Daria1, Gogoleva Natalya3, Khlopko Yuri1
1 - Institute for Cellular and Intracellular Symbiosis UB RAS, Orenburg, Russia
2 - Orenburg State Medical University, Orenburg, Russia
3 - Kazan Institute ofBiochemistry and Biophysics KSC RAS, Kazan, Russia
Modern methods of high-throughput sequencing (NGS) are widely used for characterization of protists biodiversity in fresh and marine waters and often result in new data changing our knowledge about natural microbial communities. At present only protistian communities from marine biotops have been studied with NGS, whereas the data on continental saline water bodies are rare (Heidelberg et al., 2013; Triado-Margarit and Casamayor, 2013). The aim ofthis investigation was characterization of structure and biodiversity ofprotistian communities in saline and brackish water bodies of the South Urals (Russia) with 18S metagenomic sequencing. For this purpose water samples from saline and brackish lakes and a brackish river were filtered through membranes with diameter ofpores 0.45 ^m. Total DNA was isolated from the filters and DNAlibraries were made by PCR with universal primers for V4 region of the gene 18S. High-throughput sequencing was conducted with MiSeq (Illumina). The obtained reads were treated with complex of bioinformatic tools. In the report the first data on the biodiversity of eukaryotes in the deeply continental saline and brackish water bodies of the South Urals (Russia) will be presented.
The research was performed in the Center of Shared Scientific Equipment «Persistence of microorganisms» of ICIS UB RAS and was supported by RFBR (16-44-560234, 15-29-02749, 15-2902518).
AN EARLY-BRANCHING CYANOBACTE-RIUM AT THE ORIGIN OF PRIMARY PHO-TOSYNTHETIC EUKARYOTES Ponce-Toledo R.I.1, Deschamps P.1, Lopez-Garcia P.1, Zivanovic Y.2, Benzerara K.3, Moreira D.1
1 - Unité d'Ecologie, Systématique et Evolution, Centre National de la Recherche Scientifique UMR 8079, Université Paris-Sud, 91405 Orsay, France
2 - Institut de Génétique et Microbiologie, Centre National de la Recherche Scientifique UMR 8621, Université Paris-Sud, 91405 Orsay, France
3 - IMPMC, Sorbonne Universités, Paris 75005, France
Primary plastid-bearing eukaryotes evolved by the endosymbiosis of a cyanobacterium within a heterotrophic host. This gave rise to the supergroup called Archaeplastida, comprising Viridiplantae (green algae and land plants), Rhodophyta (red algae) and Glaucophyta. Although the monophyly of primary plastids has been extensively recovered, the present-day closest cyanobacterial lineage to the chloroplast ancestor is still debated. We performed phylogenetic analyses using two concatenated datasets containing 97 plastid-encoded proteins and the plastid 16S+23S rRNA cluster, and found in both phylogenetic reconstructions that the ancestor of primary plastids was an early-branching cyanobacterium related to Gloeomargarita lithopho-ra, the first cultured member of a recently discovered freshwater cyanobacterial lineage widely present in stromatolites and thermophilic microbial mats. This discovery has implications for the environmental conditions in which the endosymbiosis took place.
THE SPECIAL CASE OF HOLOSPORA CARYO-PHILA, BACTERIAL SYMBIONT OF CILIATES PARAMECIUM
Potekhin A.1, Schrallhammer M.2, Schweikert M.3, Nekrasova I.1, Lebedeva N.4, Kaltz O.5, Petroni G.6
1 - Faculty of Biology, St Petersburg State University, Saint Petersburg, Russia
2 - Institute ofBiology, University ofFreiburg, Freiburg, Germany
3 - Institute of Biology, University of Stuttgart, Stuttgart, Germany
4 - Centre of Core Facilities "Culture Collections of Microorganisms", St Petersburg State University, Saint Petersburg, Russia
5 - Institut of Evolutionary Science, Montpellier University 2, Montpellier, France
6 - Department of Biology, University of Pisa, Pisa, Italy
Infectious bacterium Holospora caryophila, described as symbiont of the macronucleus of Paramecium biaurelia, is an unconventional Holospora. While other Holospora species are highly selective for the host, H. caryophila has been isolated from nature in several species of the P. aurelia complex, and in
