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Iron-Sulfur cluster assembly in Monocercomonoides exilis
Vacek, Vojtěch ; Hampl, Vladimír (advisor) ; Balk, Janneke (referee) ; Tsaousis, Anastasios (referee)
In the search for the mitochondrion of oxymonads, DNA of Monocercomonoides exilis - an oxymonad isolated from the gut of Chinchilla, was isolated and its genome was sequenced. Sequencing resulted in a fairly complete genome which was extensively searched or genes for mitochondrion related proteins, but no reliable candidate for such gene was identified. Even genes for the ISC pathway, which is responsible for Fe-S cluster assembly and considered to be the only essential function of reduced mitochondrion-like organelles (MROs), were absent. Instead, we were able to detect the presence of a SUF pathway which functionally replaced the ISC pathway. Closer examination of the SUF pathway based on heterologous localisation revealed that this pathway localised in the cytosol. In silico analysis showed that SUF genes are highly conserved at the level of secondary and tertiary structure and most catalytic residues and motifs are present in their sequences. The functionality of these proteins was further indirectly confirmed by complementation experiments in Escherichia coli where SUF proteins of M. exilis were able to restore at least partially Fe-S cluster assembly of strains deficient in the SUF and ISC pathways. We also proved by bacterial adenylate cyclase two-hybrid system that SufB and SufC can form...
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Iron-Sulfur cluster assembly in Monocercomonoides exilis
Vacek, Vojtěch ; Hampl, Vladimír (advisor) ; Balk, Janneke (referee) ; Tsaousis, Anastasios (referee)
In the search for the mitochondrion of oxymonads, DNA of Monocercomonoides exilis - an oxymonad isolated from the gut of Chinchilla, was isolated and its genome was sequenced. Sequencing resulted in a fairly complete genome which was extensively searched or genes for mitochondrion related proteins, but no reliable candidate for such gene was identified. Even genes for the ISC pathway, which is responsible for Fe-S cluster assembly and considered to be the only essential function of reduced mitochondrion-like organelles (MROs), were absent. Instead, we were able to detect the presence of a SUF pathway which functionally replaced the ISC pathway. Closer examination of the SUF pathway based on heterologous localisation revealed that this pathway localised in the cytosol. In silico analysis showed that SUF genes are highly conserved at the level of secondary and tertiary structure and most catalytic residues and motifs are present in their sequences. The functionality of these proteins was further indirectly confirmed by complementation experiments in Escherichia coli where SUF proteins of M. exilis were able to restore at least partially Fe-S cluster assembly of strains deficient in the SUF and ISC pathways. We also proved by bacterial adenylate cyclase two-hybrid system that SufB and SufC can form...
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Genomics and cell biology of oxymonads
Treitli, Sebastian Cristian ; Hampl, Vladimír (advisor) ; Brune, Andreas (referee) ; Beneš, Vladimír (referee)
Oxymonads are a group of poorly studied protists living as intestinal endosymbionts in the gut of insects and vertebrates. In this thesis I focused on the study of phylogeny, genomics and cell biology of oxymonads. Using culture-based approaches, we uncovered the hidden diversity of small oxymonads and described one new genus and six new species. In Monocercomonoides exilis, the only oxymonad with a published genome, we investigated the genome organization using fluorescence in situ hybdridization (FISH) against the telomeric regions and single-copy genes. Our results show that the genome is most probably haploid being organized in 6-7 chromosomes. Annotation of the genome revealed that the DNA replication and repair mechanisms in M. exilis are canonical and they seem more complete than those of other metamonads whose genomes are available. Although M. exilis lacks in any traces of mitochondria, its genome annotation revealed that other cellular systems do not markedly differ from other eukaryotes. Our taxon-rich phylogenetic analyses suggested that the genus Monocercomonoides is closely related to the oxymonad Streblomastix strix, which is found exclusively in the gut of the termites. Streblomastix strix, as opposed to M. exilis, is highly adapted to harbour bacterial ectosymbionts. Since S. strix...
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Genomics of Preaxostyla flagellates
Novák, Lukáš ; Hampl, Vladimír (advisor) ; Hirt, Robert (referee) ; Van der Giezen, Mark (referee)
Protists inhabiting oxygen-depleted environments have evolved various adaptation to thrive in their niches, including modified mitochondria to various degrees adapted to anaerobiosis. The most radically altered forms of these organelles (Mitochondria-Related Organelles, MROs) have completely lost their genomes and other defining features of canonical aerobic mitochondria. Anaerobic protists are often found as endobionts (parasites, mutualists, etc.) of larger organisms. The endobiotic lifestyle combined with anaerobiosis poses another source of evolutionary pressure forcing unique adaptations in the endobionts. Here we present new insights into the adaptations of an anaerobic protistan phylum Preaxostyla, especially with regard to the reductive evolution of mitochondria, which, uniquely among all known eukaryotes, led to a complete loss of the organelle in the oxymonad Monocercomonoides exilis. We have obtained M. exilis genomic assembly of good quality and completeness, as well as genomic and transcriptomic data of varying quality and completeness from 9 other Preaxostyla species. Based on extensive, thorough gene searches and functional gene annotation on these datasets, as well as phylogenetic analyses and protein localization experiments, we conclude: 1) M. exilis has completely lost the...
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