Functional characterization of putative Fe-hydrogenase maturases from the hydrogenosomes of Trichomonas vaginalis

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As descendants of endosymbiotic eubacteria hydrogenosomes - anaerobic forms of mitochondria - represent a highly reduced form of intracellular life in eukaryotic cells. By comparing the protein complement of hydrogenosomes that evolved independently in chytrid fungi, ciliate, and trichomonad lineages, we want to study the extent of functional similarity between these organelles and determine whether they were all shaped by parallel selective forces (adaptation to anaerobic habitat). We propose to generate 5000 ESTs from the chytrid fungus Neocallimastix frontalis as the basis for the identification of hydrogenosomal proteins isolated by 2D-electrophoresis and sequenced by mass spectrometry. Together with data from a similar project on trichomonads and EST data from the anaerobic ciliate Plagiopyla frontata we expect to obtain insight into the relationships between hydrogenosomes from different eukaryotic groups. In addition, we propose to explore the unusual association of hydrogenosomes in anaerobic ciliates with endosymbiotic methanogenic archaebacteria to determine the influence of the endosymbionts on gene expression in the host cells. For that purpose we will establish ciliate cultures without endosymbionts and use messenger-RNA from cultures with and without endosymbiotic methanogens to assay expression levels on arrays of Plagiopyla cDNAs selected from the EST data. Together, these experiments should provide a substantial advance in the understanding of intracellular life in anaerobic eukaryotic cells.