Pericentrin-mediated SAS-6 recruitment promotes centriole assembly.

The centrosome is composed of two centrioles surrounded by a microtubule-nucleating pericentriolar material (PCM). Although centrioles are known to regulate PCM assembly, it is less known whether and how the PCM contributes to centriole assembly. Here we investigate the interaction between centriole components and the PCM by taking advantage of fission yeast, which has a centriole-free, PCM-containing centrosome, the SPB. Surprisingly, we observed that several ectopically-expressed animal centriole components such as SAS-6 are recruited to the SPB. We revealed that a conserved PCM component, Pcp1/pericentrin, interacts with and recruits SAS-6. This interaction is conserved and important for centriole assembly, particularly its elongation. We further explored how yeasts kept this interaction even after centriole loss and showed that the conserved calmodulin-binding region of Pcp1/pericentrin is critical for SAS-6 interaction. Our work suggests that the PCM not only recruits and concentrates microtubule-nucleators, but also the centriole assembly machinery, promoting biogenesis close by.

Disulfiram, an alcohol dependence therapy, can inhibit the in vitro growth of Francisella tularensis.

Disulfiram (DSF) can help treat alcohol dependency by inhibiting aldehyde dehydrogenase (ALDH). Genomic analysis revealed that Francisella tularensis, the causative agent of tularemia, has lost all but one ALDH-like domain and that this domain retains the target of DSF. In this study, minimum inhibitory concentration (MIC) assays demonstrated that both DSF and its primary metabolite diethyldithiocarbamate (DDC) have strong antimicrobial activity against F. tularensis strain SCHU S4, with the MIC of DSF determined as 2 µg/mL in comparison with 8 µg/mL for DDC. The activity of DSF was further confirmed using an in vitro human macrophage infection assay. Francisella tularensis bacteria in DSF-treated cells were reduced in comparison with untreated and DDC-treated cells, comparable with that observed in doxycycline-treated cells. This suggests that DSF may be suitable for further investigation as an in vivo therapy for tularemia.

Diversity and Composition of Pelagic Prokaryotic and Protist Communities in a Thin Arctic Sea-Ice Regime.

One of the most prominent manifestations of climate change is the changing Arctic sea-ice regime with a reduction in the summer sea-ice extent and a shift from thicker, perennial multiyear ice towards thinner, first-year ice. These changes in the physical environment are likely to impact microbial communities, a key component of Arctic marine food webs and biogeochemical cycles. During the Norwegian young sea ICE expedition (N-ICE2015) north of Svalbard, seawater samples were collected at the surface (5 m), subsurface (20 or 50 m), and mesopelagic (250 m) depths on 9 March, 27 April, and 16 June 2015. In addition, several physical and biogeochemical data were recorded to contextualize the collected microbial communities. Through the massively parallel sequencing of the small subunit ribosomal RNA amplicon and metagenomic data, this work allows studying the Arctic's microbial community structure during the late winter to early summer transition. Results showed that, at compositional level, Alpha- (30.7%) and Gammaproteobacteria (28.6%) are the most frequent taxa across the prokaryotic N-ICE2015 collection, and also the most phylogenetically diverse. Winter to early summer trends were quite evident since there was a high relative abundance of thaumarchaeotes in the under-ice water column in late winter while this group was nearly absent during early summer. Moreover, the emergence of Flavobacteria and the SAR92 clade in early summer might be associated with the degradation of a spring bloom of Phaeocystis. High relative abundance of hydrocarbonoclastic bacteria, particularly Alcanivorax (54.3%) and Marinobacter (6.3%), was also found. Richness showed different patterns along the depth gradient for prokaryotic (highest at mesopelagic depth) and protistan communities (higher at subsurface depths). The microbial N-ICE2015 collection analyzed in the present study provides comprehensive new knowledge about the pelagic microbiota below drifting Arctic sea-ice. The higher microbial diversity found in late winter/early spring communities reinforces the need to continue with further studies to properly characterize the winter microbial communities under the pack-ice.

Rabifier2: an improved bioinformatic classifier of Rab GTPases.

Summary: The Rab family of small GTPases regulates and provides specificity to the endomembrane trafficking system; each Rab subfamily is associated with specific pathways. Thus, characterization of Rab repertoires provides functional information about organisms and evolution of the eukaryotic cell. Yet, the complex structure of the Rab family limits the application of existing methods for protein classification. Here, we present a major redesign of the Rabifier, a bioinformatic pipeline for detection and classification of Rab GTPases. It is more accurate, significantly faster than the original version and is now open source, both the code and the data, allowing for community participation. Availability and Implementation: Rabifier and RabDB are freely available through the web at http://rabdb.org . The Rabifier package can be downloaded from the Python Package Index at https://pypi.python.org/pypi/rabifier , the source code is available at Github https://github.com/evocell/rabifier . Contact: jsurkont@igc.gulbenkian.pt or jleal@igc.gulbenkian.pt. Supplementary information: Supplementary data are available at Bioinformatics online.

Are There Rab GTPases in Archaea?

A complex endomembrane system is one of the hallmarks of Eukaryotes. Vesicle trafficking between compartments is controlled by a diverse protein repertoire, including Rab GTPases. These small GTP-binding proteins contribute identity and specificity to the system, and by working as molecular switches, trigger multiple events in vesicle budding, transport, and fusion. A diverse collection of Rab GTPases already existed in the ancestral Eukaryote, yet, it is unclear how such elaborate repertoire emerged. A novel archaeal phylum, the Lokiarchaeota, revealed that several eukaryotic-like protein systems, including small GTPases, are present in Archaea. Here, we test the hypothesis that the Rab family of small GTPases predates the origin of Eukaryotes. Our bioinformatic pipeline detected multiple putative Rab-like proteins in several archaeal species. Our analyses revealed the presence and strict conservation of sequence features that distinguish eukaryotic Rabs from other small GTPases (Rab family motifs), mapping to the same regions in the structure as in eukaryotic Rabs. These mediate Rab-specific interactions with regulators of the REP/GDI (Rab Escort Protein/GDP dissociation Inhibitor) family. Sensitive structure-based methods further revealed the existence of REP/GDI-like genes in Archaea, involved in isoprenyl metabolism. Our analysis supports a scenario where Rabs differentiated into an independent family in Archaea, interacting with proteins involved in membrane biogenesis. These results further support the archaeal nature of the eukaryotic ancestor and provide a new insight into the intermediate stages and the evolutionary path toward the complex membrane-associated signaling circuits that characterize the Ras superfamily of small GTPases, and specifically Rab proteins.

Coiled-coil length: Size does matter.

Protein evolution is governed by processes that alter primary sequence but also the length of proteins. Protein length may change in different ways, but insertions, deletions and duplications are the most common. An optimal protein size is a trade-off between sequence extension, which may change protein stability or lead to acquisition of a new function, and shrinkage that decreases metabolic cost of protein synthesis. Despite the general tendency for length conservation across orthologous proteins, the propensity to accept insertions and deletions is heterogeneous along the sequence. For example, protein regions rich in repetitive peptide motifs are well known to extensively vary their length across species. Here, we analyze length conservation of coiled-coils, domains formed by an ubiquitous, repetitive peptide motif present in all domains of life, that frequently plays a structural role in the cell. We observed that, despite the repetitive nature, the length of coiled-coil domains is generally highly conserved throughout the tree of life, even when the remaining parts of the protein change, including globular domains. Length conservation is independent of primary amino acid sequence variation, and represents a conservation of domain physical size. This suggests that the conservation of domain size is due to functional constraints.

Evolutionary patterns in coiled-coils.

Models of protein evolution are used to describe evolutionary processes, for phylogenetic analyses and homology detection. Widely used general models of protein evolution are biased toward globular domains and lack resolution to describe evolutionary processes for other protein types. As three-dimensional structure is a major constraint to protein evolution, specific models have been proposed for other types of proteins. Here, we consider evolutionary patterns in coiled-coil forming proteins. Coiled-coils are widespread structural domains, formed by a repeated motif of seven amino acids (heptad repeat). Coiled-coil forming proteins are frequently rods and spacers, structuring both the intracellular and the extracellular spaces that often form protein interaction interfaces. We tested the hypothesis that due to their specific structure the associated evolutionary constraints differ from those of globular proteins. We showed that substitution patterns in coiled-coil regions are different than those observed in globular regions, beyond the simple heptad repeat. Based on these substitution patterns we developed a coiled-coil specific (CC) model that in the context of phylogenetic reconstruction outperforms general models in tree likelihood, often leading to different topologies. For multidomain proteins containing both a coiled-coil region and a globular domain, we showed that a combination of the CC model and a general one gives higher likelihoods than a single model. Finally, we showed that the model can be used for homology detection to increase search sensitivity for coiled-coil proteins. The CC model, software, and other supplementary materials are available at http://www.evocell.org/cgl/resources (last accessed January 29, 2015).