Naegleria gruberi is a free-living heterotrophic aerobic amoeba well known for its ability to transform from an amoeba to a flagellate form. The genome of N. gruberi has been recently published, and in silico predictions demonstrated that Naegleria has the capacity for both aerobic respiration and anaerobic biochemistry to produce molecular hydrogen in its mitochondria. This finding was considered to have fundamental implications on the evolution of mitochondrial metabolism and of the last eukaryotic common ancestor. However, no actual experimental data have been shown to support this hypothesis. For this reason, we have decided to investigate the anaerobic metabolism of the mitochondrion of N. gruberi. Using in vivo biochemical assays, we have demonstrated that N. gruberi has indeed a functional [FeFe]-hydrogenase, an enzyme that is attributed to anaerobic organisms. Surprisingly, in contrast to the published predictions, we have demonstrated that hydrogenase is localized exclusively in the cytosol, while no hydrogenase activity was associated with mitochondria of the organism. In addition, cytosolic localization displayed for HydE, a marker component of hydrogenase maturases. Naegleria gruberi, an obligate aerobic organism and one of the earliest eukaryotes, is producing hydrogen, a function that raises questions on the purpose of this pathway for the lifestyle of the organism and potentially on the evolution of eukaryotes.

• Klíčová slova
• Naegleria, hydrogen hypothesis, hydrogenase, maturases, mitochondrial evolution,
• MeSH
• cytosol enzymologie   MeSH
• hydrogenasa genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• Naegleria enzymologie   genetika   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• vodík metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hydrogenasa MeSH
• protozoální proteiny MeSH
• vodík MeSH

Trichomonads, represented by the highly prevalent sexually transmitted human parasite Trichomonas vaginalis, are anaerobic eukaryotes with hydrogenosomes in the place of the standard mitochondria. Hydrogenosomes form indispensable FeS-clusters, synthesize ATP, and release molecular hydrogen as a waste product. Hydrogen formation is catalyzed by [FeFe] hydrogenase, the hallmark enzyme of all hydrogenosomes found in various eukaryotic anaerobes. Eukaryotic hydrogenases were originally thought to be exclusively localized within organelles, but today few eukaryotic anaerobes are known that possess hydrogenase in their cytosol. We identified a thus-far unknown hydrogenase in T. vaginalis cytosol that cannot use ferredoxin as a redox partner but can use cytochrome b5 as an electron acceptor. Trichomonads overexpressing the cytosolic hydrogenase, while maintaining the carbon flux through hydrogenosomes, show decreased excretion of hydrogen and increased excretion of methylated alcohols, suggesting that the cytosolic hydrogenase uses the hydrogen gas as a source of reducing power for the reactions occurring in the cytoplasm and thus accounts for the overall redox balance. This is the first evidence of hydrogen uptake in a eukaryote, although further work is needed to confirm it. Assembly of the catalytic center of [FeFe] hydrogenases (H-cluster) requires the activity of three dedicated maturases, and these proteins in T. vaginalis are exclusively localized in hydrogenosomes, where they participate in the maturation of organellar hydrogenases. Despite the different subcellular localization of cytosolic hydrogenase and maturases, the H-cluster is present in the cytosolic enzyme, suggesting the existence of an alternative mechanism of H-cluster assembly.

• Klíčová slova
• H-cluster, Trichomonas, cytosolic hydrogenase, hydrogenosome,
• MeSH
• cytosol metabolismus   MeSH
• ferredoxiny metabolismus   MeSH
• hydrogenasa * metabolismus   MeSH
• Trichomonas vaginalis * enzymologie   ultrastruktura   MeSH
• vodík * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ferredoxiny MeSH
• hydrogenasa * MeSH
• vodík * MeSH

Although the mitochondria of extant eukaryotes share a single origin, functionally these organelles diversified to a great extent, reflecting lifestyles of the organisms that host them. In anaerobic protists of the group Metamonada, mitochondria are present in reduced forms (also termed hydrogenosomes or mitosomes) and a complete loss of mitochondrion in Monocercomonoides exilis (Metamonada:Preaxostyla) has also been reported. Within metamonads, retortamonads from the gastrointestinal tract of vertebrates form a sister group to parasitic diplomonads (e.g. Giardia and Spironucleus) and have also been hypothesized to completely lack mitochondria. We obtained transcriptomic data from Retortamonas dobelli and R. caviae and searched for enzymes of the core metabolism as well as mitochondrion- and parasitism-related proteins. Our results indicate that retortamonads have a streamlined metabolism lacking pathways for metabolites they are probably capable of obtaining from prey bacteria or their environment, reminiscent of the biochemical arrangement in other metamonads. Retortamonads were surprisingly found do encode homologs of components of Giardia's remarkable ventral disk, as well as homologs of regulatory NEK kinases and secreted lytic enzymes known for involvement in host colonization by Giardia. These can be considered pre-adaptations of these intestinal microorganisms to parasitism. Furthermore, we found traces of the mitochondrial metabolism represented by iron‑sulfur cluster assembly subunits, subunits of mitochondrial translocation and chaperone machinery and, importantly, [FeFe]‑hydrogenases and hydrogenase maturases (HydE, HydF and HydG). Altogether, our results strongly suggest that a remnant mitochondrion is still present.

• Klíčová slova
• Anaerobic metabolism, Diplomonads, Hydrogenosome, Mitochondrion-related organelles,
• MeSH
• anaerobióza MeSH
• biologická adaptace * MeSH
• Diplomonadida cytologie   fyziologie   MeSH
• mitochondrie fyziologie   MeSH
• morčata MeSH
• nemoci hlodavců MeSH
• protozoální infekce zvířat metabolismus   parazitologie   MeSH
• Retortamonadidae cytologie   fyziologie   MeSH
• žáby MeSH
• zvířata MeSH
• Check Tag
• morčata MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

All eukaryotic organisms contain mitochondria or organelles that evolved from the same endosymbiotic event like classical mitochondria. Organisms inhabiting low oxygen environments often contain mitochondrial derivates known as hydrogenosomes, mitosomes or neutrally as mitochondrion-like organelles. The detailed investigation has shown unexpected evolutionary plasticity in the biochemistry and protein composition of these organelles in various protists. We investigated the mitochondrion-like organelle in Trimastix pyriformis, a free-living member of one of the three lineages of anaerobic group Metamonada. Using 454 sequencing we have obtained 7 037 contigs from its transcriptome and on the basis of sequence homology and presence of N-terminal extensions we have selected contigs coding for proteins that putatively function in the organelle. Together with the results of a previous transcriptome survey, the list now consists of 23 proteins - mostly enzymes involved in amino acid metabolism, transporters and maturases of proteins and transporters of metabolites. We have no evidence of the production of ATP in the mitochondrion-like organelle of Trimastix but we have obtained experimental evidence for the presence of enzymes of the glycine cleavage system (GCS), which is part of amino acid metabolism. Using homologous antibody we have shown that H-protein of GCS localizes into vesicles in the cell of Trimastix. When overexpressed in yeast, H- and P-protein of GCS and cpn60 were transported into mitochondrion. In case of H-protein we have demonstrated that the first 16 amino acids are necessary for this transport. Glycine cleavage system is at the moment the only experimentally localized pathway in the mitochondrial derivate of Trimastix pyriformis.

• MeSH
• Eukaryota genetika   metabolismus   MeSH
• exprese genu MeSH
• H-protein systému glycinového štěpení genetika   metabolismus   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• multienzymové komplexy genetika   metabolismus   MeSH
• organely metabolismus   MeSH
• oxidoreduktasy aminokyselin genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• transferasy genetika   metabolismus   MeSH
• transportní proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glycine cleavage system MeSH Prohlížeč
• H-protein systému glycinového štěpení MeSH
• mitochondriální proteiny MeSH
• multienzymové komplexy MeSH
• oxidoreduktasy aminokyselin MeSH
• transferasy MeSH
• transportní proteiny MeSH

Phylogenetic relationships for Hieracium subgen. Pilosella were inferred from chloroplast (trnT-trnL, matK) and nuclear (ITS) sequence data. Chloroplast markers revealed the existence of two divergent haplotype groups within the subgenus that did not correspond to presumed relationships. Furthermore, chloroplast haplotypes of the genera Hispidella and Andryala nested each within one of these groups. In contrast, ITS data were generally in accord with morphology and other evidence and were therefore assumed to reflect the true phylogeny. They revealed a sister relationship between Pilosella and Hispidella and a joint clade of Hieracium subgenera Hieracium and Chionoracium (Stenotheca) while genus Andryala represented a third major lineage of the final ingroup cluster. Detailed analysis of trnT-trnL character state evolution along the ITS tree suggested two intergeneric hybridization events between ancestral lineages that resulted in cytoplasmic transfer (from Hieracium/Chionoracium to Pilosella, and from the introgressed Pilosella lineage to Andryala). These chloroplast capture events, the first of which involved a now extinct haplotype, are the most likely explanation for the observed incongruencies between plastid and nuclear DNA markers.

• MeSH
• Asteraceae klasifikace   genetika   MeSH
• buněčné jádro genetika   MeSH
• DNA chloroplastová chemie   genetika   MeSH
• endoribonukleasy genetika   MeSH
• fylogeneze * MeSH
• hybridizace genetická genetika   MeSH
• intergenová DNA genetika   MeSH
• molekulární evoluce MeSH
• molekulární sekvence - údaje MeSH
• nukleotidyltransferasy genetika   MeSH
• plastidy genetika   MeSH
• RNA transferová genetika   MeSH
• sekvenční analýza DNA MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA chloroplastová MeSH
• endoribonukleasy MeSH
• intergenová DNA MeSH
• mRNA maturase MeSH Prohlížeč
• nukleotidyltransferasy MeSH
• RNA transferová MeSH

Naegleria gruberi is a free-living amoeba, closely related to the human pathogen Naegleria fowleri, the causative agent of the deadly human disease primary amoebic meningoencephalitis. Herein, we investigated the effect of iron limitation on different aspects of N. gruberi metabolism. Iron metabolism is among the most conserved pathways found in all eukaryotes. It includes the delivery, storage and utilisation of iron in many cell processes. Nevertheless, most of the iron metabolism pathways of N. gruberi are still not characterised, even though iron balance within the cell is crucial. We found a single homolog of ferritin in the N. gruberi genome and showed its localisation in the mitochondrion. Using comparative mass spectrometry, we identified 229 upregulated and 184 down-regulated proteins under iron-limited conditions. The most down-regulated protein under iron-limited conditions was hemerythrin, and a similar effect on the expression of hemerythrin was found in N. fowleri. Among the other down-regulated proteins were [FeFe]-hydrogenase and its maturase HydG and several heme-containing proteins. The activities of [FeFe]-hydrogenase, as well as alcohol dehydrogenase, were also decreased by iron deficiency. Our results indicate that N. gruberi is able to rearrange its metabolism according to iron availability, prioritising mitochondrial pathways. We hypothesise that the mitochondrion is the center for iron homeostasis in N. gruberi, with mitochondrially localised ferritin as a potential key component of this process.

• Klíčová slova
• Ferritin, Hemerythrin, Iron, Metabolism, Naegleria,
• MeSH
• anaerobióza MeSH
• biologický transport MeSH
• chromatografie kapalinová MeSH
• hemerythrin metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• Naegleria metabolismus   MeSH
• protozoální proteiny genetika   MeSH
• regulace genové exprese enzymů účinky léků   MeSH
• spotřeba kyslíku MeSH
• železo metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hemerythrin MeSH
• protozoální proteiny MeSH
• železo MeSH