Evolutionary conservation and in vitro reconstitution of microsporidian iron–sulfur cluster biosynthesis

This work was supported by Marie Curie Postdoctoral Fellowships to T.A.W., E. H. and S. L., a European Research Council Advanced Investigator Grant (ERC-2010-AdG-268701) to T.M.E., and a Wellcome Trust Programme Grant (number 045404) to T.M.E. and J.M.L. R.L. acknowledges generous financial support from Deutsche Forschungsgemeinschaft (SFB 593, SFB 987, GRK 1216, LI 415/5), LOEWE program of state Hessen, Max-Planck Gesellschaft, von Behring-Röntgen StiftungMicrosporidians are a diverse group of obligate intracellular parasites that have minimized their genome content and simplified their sub-cellular structures by reductive evolution. Functional studies are limited because we lack reliable genetic tools for their manipulation. Here, we demonstrate that the cristae-deficient mitochondrion (mitosome) of the microsporidian Trachipleistophora hominis is the functional site of iron-sulphur cluster (ISC) assembly, which we suggest is the essential task of this organelle. Cell fractionation, fluorescence imaging and fine-scale immunoelectron microscopy demonstrate that mitosomes contain a complete pathway for [2Fe-2S] cluster biosynthesis that we biochemically reconstituted using purified recombinant mitosomal ISC proteins. Reconstitution proceeded as rapidly and efficiently as observed for yeast or fungal mitochondrial ISC components. Core components of the T. hominis cytosolic iron-sulphur protein assembly (CIA) pathway were also identified including the essential Cfd1-Nbp35 scaffold complex that assembles a [4Fe-4S] cluster as shown by spectroscopic methods in vitro. Phylogenetic analyses reveal that both the ISC and CIA biosynthetic pathways are predominantly bacterial, but their cytosolic and nuclear target Fe/S proteins are mainly archaeal. This mixed evolutionary history of the Fe/S-related proteins and pathways, and their strong conservation among highly reduced parasites, provides additional compelling evidence for the ancient chimeric ancestry of eukaryotes.Publisher PDFPeer reviewe

The ArfGEF GBF-1 Is Required for ER Structure, Secretion and Endocytic Transport in C. elegans

Small GTPases of the Sar/Arf family are essential to generate transport containers that mediate communication between organelles of the secretory pathway. Guanine nucleotide exchange factor (GEFs) activate the small GTPases and help their anchorage in the membrane. Thus, GEFs in a way temporally and spatially control Sar1/Arf1 GTPase activation. We investigated the role of the ArfGEF GBF-1 in C. elegans oocytes and intestinal epithelial cells. GBF-1 localizes to the cis-Golgi and is part of the t-ER-Golgi elements. GBF-1 is required for secretion and Golgi integrity. In addition, gbf-1(RNAi) causes the ER reticular structure to become dispersed, without destroying ER exit sites (ERES) because the ERES protein SEC-16 was still localized in distinct punctae at t-ER-Golgi units. Moreover, GBF-1 plays a role in receptor-mediated endocytosis in oocytes, without affecting recycling pathways. We find that both the yolk receptor RME-2 and the recycling endosome-associated RAB-11 localize similarly in control and gbf-1(RNAi) oocytes. While RAB5-positive early endosomes appear to be less prominent and the RAB-5 levels are reduced by gbf-1(RNAi) in the intestine, RAB-7-positive late endosomes were more abundant and formed aggregates and tubular structures. Our data suggest a role for GBF-1 in ER structure and endosomal traffic

Microbial Ecology of Lake Kivu

peer reviewedWe review available data on archaea, bacteria and small eukaryotes in an attempt to provide a general picture of microbial diversity, abundances and microbe-driven processes in Lake Kivu surface and intermediate waters (ca. 0–100 m). The various water layers present contrasting physical and chemical properties and harbour very different microbial communities supported by the vertical redox structure. For instance, we found a clear vertical segregation of archaeal and bacterial assemblages between the oxic and the anoxic zone of the surface waters. The presence of specific bacterial (e.g. Green Sulfur Bacteria) and archaeal (e.g. ammonia-oxidising archaea) communities and the prevailing physico-chemical conditions point towards the redoxcline as the most active and metabolically diverse water layer. The archaeal assemblage in the surface and intermediate water column layers was mainly composed by the phylum Crenarchaeota , by the recently defined phylum Thaumarchaeota and by the phylum Euryarchaeota . In turn, the bacterial assemblage comprised mainly ubiquitous members of planktonic assemblages of freshwater environments (Actinobacteria, Bacteroidetes and Betaproteobacteria among others) and other less commonly retrieved phyla (e.g. Chlorobi, Clostridium and Deltaproteobacteria). The community of small eukaryotes (<5 µm) mainly comprised Stramenopiles , Alveolata , Cryptophyta , Chytridiomycota , Kinetoplastea and Choanoflagellida, by decreasing order of richness. The total prokaryotic abundance ranged between 0.5 × 10^6 and 2.0 × 10^6 cells mL−1 , with maxima located in the 0–20 m layer, while phycoerythrin-rich Synechococcus-like picocyanobacteria populations were comprised between 0.5 × 10^5 and 2.0 × 10^5 cells mL−1 in the same surface layer. Brown-coloured species of Green Sulfur Bacteria permanently developed at 11m depth in Kabuno Bay and sporadically in the anoxic waters of the lower mixolimnion of the main basin. The mean bacterial production was estimated to 336 mg C m−2 day−1 . First estimates of the re-assimilation by bacterioplankton of dissolved organic matter excreted by phytoplankton showed high values of dissolved primary production (ca. 50% of total production). The bacterial carbon demand can totally be fuelled by phytoplankton production. Overall, recent studies have revealed a high microbial diversity in Lake Kivu, and point towards a central role of microbes in the biogeochemical and ecological functioning of the surface layers, comprising the mixolimnion and the upper chemocline

Down under the tunic: bacterial biodiversity hotspots and widespread ammonia-oxidizing archaea in coral reef ascidians

14 páginas, 3 tablas, 3 figuras.Ascidians are ecologically important components of marine ecosystems yet the ascidian microbiota remains largely unexplored beyond a few model species. We used 16S rRNA gene tag pyrosequencing to provide a comprehensive characterization of microbial symbionts in the tunic of 42 Great Barrier Reef ascidian samples representing 25 species. Results revealed high bacterial biodiversity (3 217 unique operational taxonomic units (OTU0.03) from 19 described and 14 candidate phyla) and the widespread occurrence of ammonia-oxidizing Thaumarchaeota in coral reef ascidians (24 of 25 host species). The ascidian microbiota was clearly differentiated from seawater microbial communities and included symbiont lineages shared with other invertebrate hosts as well as unique, ascidian-specific phylotypes. Several rare seawater microbes were markedly enriched (200–700 fold) in the ascidian tunic, suggesting that the rare biosphere of seawater may act as a conduit for horizontal symbiont transfer. However, most OTUs (71%) were rare and specific to single hosts and a significant correlation between host relatedness and symbiont community similarity was detected, indicating a high degree of host-specificity and potential role of vertical transmission in structuring these communities. We hypothesize that the complex ascidian microbiota revealed herein is maintained by the dynamic microenvironments within the ascidian tunic, offering optimal conditions for different metabolic pathways such as ample chemical substrate (ammonia-rich host waste) and physical habitat (high oxygen, low irradiance) for nitrification. Thus, ascidian hosts provide unique and fertile niches for diverse microorganisms and may represent an important and previously unrecognized habitat for nitrite/nitrate regeneration in coral reef ecosystems.This research was funded by the Marie Curie International Reintegration Grant FP7-PEOPLE-2010-RG 277038 (within the 7th European Community Framework Program), the Spanish Government projects CTM2010-17755 and CTM2010-22218 and the Catalan Government grant 2009 SGR-484 for Consolidated Research Groups. NSW was funded through an Australian Research Council Future Fellowship (FT1200100480).Peer reviewe