Effect of the endoplasmic reticulum stressor tunicamycin in Angomonas deanei heat-shock protein expression and on the association with the endosymbiotic bacterium.

The endoplasmic reticulum (ER) presents unique properties to establishing bacterium symbiosis in eukaryotic cells since it synthesizes and glycosylates essential molecules like proteins and lipids. Tunicamycin (TM) is an antibiotic that inhibits the first step in the N-linked glycosylation in eukaryotes and has been used as an ER stress inducer to activate the Unfolded Protein Response (UPR). Mutualistic symbiosis in trypanosomatids is characterized by structural adaptations and intense metabolic exchanges, thus we investigated the effects of TM in the association between Angomonas deanei and its symbiotic bacterium, through ultrastructural and proteomic approaches. Cells treated with the inhibitor showed a decrease in proliferation, enlargement of the ER and Golgi cisternae and an increased distance between the symbiont and the ER. TM proved to be an important tool to better understand ER stress in trypanosomatids, since changes in protein composition were observed in the host protozoan, especially the expression of the Hsp90 chaperone. Furthermore, data obtained indicates the importance of the ER for the adaptation and maintenance of symbiotic associations between prokaryotes and eukaryotes, considering that this organelle has recognized importance in the biogenesis and division of cell structures.

Contrasting the microbiomes from forest rhizosphere and deeper bulk soil from an Amazon rainforest reserve.

Pristine forest ecosystems provide a unique perspective for the study of plant-associated microbiota since they host a great microbial diversity. Although the Amazon forest is one of the hotspots of biodiversity around the world, few metagenomic studies described its microbial community diversity thus far. Understanding the environmental factors that can cause shifts in microbial profiles is key to improving soil health and biogeochemical cycles. Here we report a taxonomic and functional characterization of the microbiome from the rhizosphere of Brosimum guianense (Snakewood), a native tree, and bulk soil samples from a pristine Brazilian Amazon forest reserve (Cuniã), for the first time by the shotgun approach. We identified several fungi and bacteria taxon significantly enriched in forest rhizosphere compared to bulk soil samples. For archaea, the trend was the opposite, with many archaeal phylum and families being considerably more enriched in bulk soil compared to forest rhizosphere. Several fungal and bacterial decomposers like Postia placenta and Catenulispora acidiphila which help maintain healthy forest ecosystems were found enriched in our samples. Other bacterial species involved in nitrogen (Nitrobacter hamburgensis and Rhodopseudomonas palustris) and carbon cycling (Oligotropha carboxidovorans) were overrepresented in our samples indicating the importance of these metabolic pathways for the Amazon rainforest reserve soil health. Hierarchical clustering based on taxonomic similar microbial profiles grouped the forest rhizosphere samples in a distinct clade separated from bulk soil samples. Principal coordinate analysis of our samples with publicly available metagenomes from the Amazon region showed grouping into specific rhizosphere and bulk soil clusters, further indicating distinct microbial community profiles. In this work, we reported significant shifts in microbial community structure between forest rhizosphere and bulk soil samples from an Amazon forest reserve that are probably caused by more than one environmental factors such as rhizosphere and soil depth.

CrocoBLAST: Running BLAST efficiently in the age of next-generation sequencing.

SUMMARY: CrocoBLAST is a tool for dramatically speeding up BLAST+ execution on any computer. Alignments that would take days or weeks with NCBI BLAST+ can be run overnight with CrocoBLAST. Additionally, CrocoBLAST provides features critical for NGS data analysis, including: results identical to those of BLAST+; compatibility with any BLAST+ version; real-time information regarding calculation progress and remaining run time; access to partial alignment results; queueing, pausing, and resuming BLAST+ calculations without information loss. AVAILABILITY AND IMPLEMENTATION: CrocoBLAST is freely available online, with ample documentation (webchem.ncbr.muni.cz/Platform/App/CrocoBLAST). No installation or user registration is required. CrocoBLAST is implemented in C, while the graphical user interface is implemented in Java. CrocoBLAST is supported under Linux and Windows, and can be run under Mac OS X in a Linux virtual machine. CONTACT: jkoca@ceitec.cz. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The bacterium endosymbiont of Crithidia deanei undergoes coordinated division with the host cell nucleus.

In trypanosomatids, cell division involves morphological changes and requires coordinated replication and segregation of the nucleus, kinetoplast and flagellum. In endosymbiont-containing trypanosomatids, like Crithidia deanei, this process is more complex, as each daughter cell contains only a single symbiotic bacterium, indicating that the prokaryote must replicate synchronically with the host protozoan. In this study, we used light and electron microscopy combined with three-dimensional reconstruction approaches to observe the endosymbiont shape and division during C. deanei cell cycle. We found that the bacterium replicates before the basal body and kinetoplast segregations and that the nucleus is the last organelle to divide, before cytokinesis. In addition, the endosymbiont is usually found close to the host cell nucleus, presenting different shapes during the protozoan cell cycle. Considering that the endosymbiosis in trypanosomatids is a mutualistic relationship, which resembles organelle acquisition during evolution, these findings establish an excellent model for the understanding of mechanisms related with the establishment of organelles in eukaryotic cells.

Phosphatidylcholine synthesis in Crithidia deanei: the influence of the endosymbiont.

In this study, the role of phospholipid biosynthetic pathways was investigated in the establishment of the mutualistic relationship between the trypanosomatid protozoan Crithidia deanei and its symbiotic bacterium. Although the endosymbiont displays two unit membranes, it lacks a typical Gram-negative cell wall. As in other intracellular bacteria, phosphatidylcholine is a major component of the symbiont envelope. Here, it was shown that symbiont-bearing C. deanei incorporates more than two-fold (32)Pi into phospholipids as compared with the aposymbiotic strain. The major phospholipid synthesized by both strains was phosphatidylcholine, followed by phosphatidylethanolamine and phosphatidylinositol. Cellular fractioning indicated that (32)Pi-phosphatidylcholine is the major phospholipid component of the isolated symbionts, as well as of mitochondria. Although the data indicated that isolated symbionts synthesized phospholipids independently of the trypanosomatid host, a key finding was that the isolated bacteria synthesized mostly phosphatidylethanolamine, rather than phosphatidylcholine. These results indicate that phosphatidylcholine production by the symbiont depends on metabolic exchanges with the host protozoan. Insight about the mechanisms underlying lipid biosynthesis in symbiont-bearing C. deanei might help to understand how the prokaryote/trypanosomatid relation has evolved in the establishment of symbiosis.