Supercomplexes of IsiA and photosystem I in a mutant lacking subunit PsaL

The cyanobacterium Synechocystis PCC 6803 grown under short-term iron-deficient conditions assembles a supercomplex consisting of a trimeric Photosystem I (PSI) complex encircled by a ring of 18 IsiA complexes. Furthermore, it has been shown that single or double rings of IsiA with up to 35 copies in total can surround monomeric PSI. Here we present an analysis by electron microscopy and image analysis of the various PSI-IsiA supercomplexes from a Synechocystis PCC 6803 mutant lacking the PsaL subunit after short- and long-term iron-deficient growth. In the absence of PsaL, the tendency to form complexes with IsiA is still strong, but the average number of complete rings is lower than in the wild type. The majority of IsiA copies binds into partial double rings at the side of PsaF/J subunits rather than in complete single or double rings, which also cover the PsaL side of the PSI monomer. This indicates that PsaL facilitates the formation of IsiA rings around PSI monomers but is not an obligatory structural component in the formation of PSI-IsiA complexes.     

Comparison of chitin-induced natural transformation in pandemic Vibrio cholerae O1 El Tor strains

The human pathogen Vibrio cholerae serves as a model organism for many important processes ranging from pathogenesis to natural transformation, which has been extensively studied in this bacterium. Previous work has deciphered important regulatory circuits involved in natural competence induction as well as mechanistic details related to its DNA acquisition and uptake potential. However, since competence was first reported for V. cholerae in 2005, many researchers have struggled with reproducibility in certain strains. In this study, we therefore compare prominent seventh pandemic V. cholerae isolates, namely strains A1552, N16961, C6706, C6709, E7946, P27459, and the close relative MO10, for their natural transformability and decipher underlying defects that mask the high degree of competence conservation. Through a combination of experimental approaches and comparative genomics based on new whole-genome sequences and de novo assemblies, we identify several strain-specific defects, mostly in genes that encode key players in quorum sensing. Moreover, we provide evidence that most of these deficiencies might have recently occurred through laboratory domestication events or through the acquisition of mobile genetic elements. Lastly, we highlight that differing experimental approaches between research groups might explain more of the variations than strain-specific alterations.     

Predicting loss of evolutionary history: Where are we?

The Earth's evolutionary history is threatened by species loss in the current sixth mass extinction event in Earth's history. Such extinction events not only eliminate species but also their unique evolutionary histories. Here we review the expected loss of Earth's evolutionary history quantified by phylogenetic diversity (PD) and evolutionary distinctiveness (ED) at risk. Due to the general paucity of data, global evolutionary history losses have been predicted for only a few groups, such as mammals, birds, amphibians, plants, corals and fishes. Among these groups, there is now empirical support that extinction threats are clustered on the phylogeny; however this is not always a sufficient condition to cause higher loss of phylogenetic diversity in comparison to a scenario of random extinctions. Extinctions of the most evolutionarily distinct species and the shape of phylogenetic trees are additional factors that can elevate losses of evolutionary history. Consequently, impacts of species extinctions differ among groups and regions, and even if global losses are low within large groups, losses can be high among subgroups or within some regions. Further, we show that PD and ED are poorly protected by current conservation practices. While evolutionary history can be indirectly protected by current conservation schemes, optimizing its preservation requires integrating phylogenetic indices with those that capture rarity and extinction risk. Measures based on PD and ED could bring solutions to conservation issues, however they are still rarely used in practice, probably because the reasons to protect evolutionary history are not clear for practitioners or due to a lack of data. However, important advances have been made in the availability of phylogenetic trees and methods for their construction, as well as assessments of extinction risk. Some challenges remain, and looking forward, research should prioritize the assessment of expected PD and ED loss for more taxonomic groups and test the assumption that preserving ED and PD also protects rare species and ecosystem services. Such research will be useful to inform and guide the conservation of Earth's biodiversity and the services it provides.     

Functional Reconstitution into Liposomes of Purified Human RhCG Ammonia Channel

Background

Rh glycoproteins (RhAG, RhBG, RhCG) are members of the Amt/Mep/Rh family which facilitate movement of ammonium across plasma membranes. Changes in ammonium transport activity following expression of Rh glycoproteins have been described in different heterologous systems such as yeasts, oocytes and eukaryotic cell lines. However, in these complex systems, a potential contribution of endogenous proteins to this function cannot be excluded. To demonstrate that Rh glycoproteins by themselves transport NH₃, human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties.

Methodology/Principal Findings

An HA-tag introduced in the second extracellular loop of RhCG was used to purify to homogeneity the HA-tagged RhCG glycoprotein from detergent-solubilized recombinant HEK293E cells. Electron microscopy analysis of negatively stained purified RhCG-HA revealed, after image processing, homogeneous particles of 9 nm diameter with a trimeric protein structure. Reconstitution was performed with sphingomyelin, phosphatidylcholine and phosphatidic acid lipids in the presence of the C₁₂E₈ detergent which was subsequently removed by Biobeads. Control of protein incorporation was carried out by freeze-fracture electron microscopy. Particle density in liposomes was a function of the Lipid/Protein ratio. When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively). This strong NH₃ transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy.

Conclusions/Significance

This study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent Punit_NH3 (around 1×10⁻³ µm³.s⁻¹) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60×10⁻³ µm³.s⁻¹), and in human red blood cells endogenously expressing RhAG (2.18×10⁻³ µm³.s⁻¹). The major finding of this study is that RhCG protein is active as an NH₃ channel and that this function does not require any protein partner.     

APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment

Signals generated in response to extracellular stimuli at the plasma membrane are transmitted through cytoplasmic transduction cascades to the nucleus. We report the identification of a pathway directly linking the small GTPase Rab5, a key regulator of endocytosis, to signal transduction and mitogenesis. This pathway operates via APPL1 and APPL2, two Rab5 effectors, which reside on a subpopulation of endosomes. In response to extracellular stimuli such as EGF and oxidative stress, APPL1 translocates from the membranes to the nucleus where it interacts with the nucleosome remodeling and histone deacetylase multiprotein complex NuRD/MeCP1, an established regulator of chromatin structure and gene expression. Both APPL1 and APPL2 are essential for cell proliferation and their function requires Rab5 binding. Our findings identify an endosomal compartment bearing Rab5 and APPL proteins as an intermediate in signaling between the plasma membrane and the nucleus.     

Neural correlates of experience-induced deficits in learned vocal communication

Songbirds are one of the few vertebrate groups (including humans) that evolved the ability to learn vocalizations. During song learning, social interactions with adult models are crucial and young songbirds raised without direct contacts with adults typically produce abnormal songs showing phonological and syntactical deficits. This raises the question of what functional representation of their vocalizations such deprived animals develop. Here we show that young starlings that we raised without any direct contact with adults not only failed to differentiate starlings' typical song classes in their vocalizations but also failed to develop differential neural responses to these songs. These deficits appear to be linked to a failure to acquire songs' functions and may provide a model for abnormal development of communicative skills, including speech.     

Using SD-AFLP and MSAP to assess CCGG methylation in the banana genome

Two amplified fragment length polymorphism (AFLP)-derived techniques were used to assess methylation at CCGG sites in the banana genome. Assessment of these techniques revealed that, while amplification steps are very reproducible, the ligation step is more subject to variability. Overall, these techniques produced an error rate of 0.2% per analysed band. Statistical approach highlights the fact that sample duplication is necessary to produce reliable results. This study involved 18 primer pairs and found that in the banana genome, roughly 80% of CCGG sites are unmethylated, 5% are methylated at the internal cytosine, and 15% are methylated at the external or both cytosines.     

Targeted genome enrichment for efficient purification of endosymbiont DNA from host DNA

Wolbachia endosymbionts are widespread in arthropods and are generally considered reproductive parasites, inducing various phenotypes including cytoplasmic incompatibility, parthenogenesis, feminization and male killing, which serve to promote their spread through populations. In contrast, Wolbachia infecting filarial nematodes that cause human diseases, including elephantiasis and river blindness, are obligate mutualists. DNA purification methods for efficient genomic sequencing of these unculturable bacteria have proven difficult using a variety of techniques. To efficiently capture endosymbiont DNA for studies that examine the biology of symbiosis, we devised a parallel strategy to an earlier array-based method by creating a set of SureSelect? (Agilent) 120-mer target enrichment RNA oligonucleotides (“baits”) for solution hybrid selection. These were designed from Wolbachia complete and partial genome sequences in GenBank and were tiled across each genomic sequence with 60 bp overlap. Baits were filtered for homology against host genomes containing Wolbachia using BLAT and sequences with significant host homology were removed from the bait pool. Filarial parasite Brugia malayi DNA was used as a test case, as the complete sequence of both Wolbachia and its host are known. DNA eluted from capture was size selected and sequencing samples were prepared using the NEBNext® Sample Preparation Kit. One-third of a 50 nt paired-end sequencing lane on the HiSeq? 2000 (Illumina) yielded 53 million reads and the entirety of the Wolbachia genome was captured. We then used the baits to isolate more than 97.1 % of the genome of a distantly related Wolbachia strain from the crustacean Armadillidium vulgare, demonstrating that the method can be used to enrich target DNA from unculturable microbes over large evolutionary distances.     

The Characterization of Novel Tissue Microbiota Using an Optimized 16S Metagenomic Sequencing Pipeline

Background

Substantial progress in high-throughput metagenomic sequencing methodologies has enabled the characterisation of bacteria from various origins (for example gut and skin). However, the recently-discovered bacterial microbiota present within animal internal tissues has remained unexplored due to technical difficulties associated with these challenging samples.

Results

We have optimized a specific 16S rDNA-targeted metagenomics sequencing (16S metabarcoding) pipeline based on the Illumina MiSeq technology for the analysis of bacterial DNA in human and animal tissues. This was successfully achieved in various mouse tissues despite the high abundance of eukaryotic DNA and PCR inhibitors in these samples. We extensively tested this pipeline on mock communities, negative controls, positive controls and tissues and demonstrated the presence of novel tissue specific bacterial DNA profiles in a variety of organs (including brain, muscle, adipose tissue, liver and heart).

Conclusion

The high throughput and excellent reproducibility of the method ensured exhaustive and precise coverage of the 16S rDNA bacterial variants present in mouse tissues. This optimized 16S metagenomic sequencing pipeline will allow the scientific community to catalogue the bacterial DNA profiles of different tissues and will provide a database to analyse host/bacterial interactions in relation to homeostasis and disease.     

Impact of Selection for Digestive Efficiency on Microbiota Composition in the Chicken

Objectives

Feed efficiency and its digestive component, digestive efficiency, are key factors in the environmental impact and economic output of poultry production. The interaction between the host and intestinal microbiota has a crucial role in the determination of the ability of the bird to digest its food and to the birds’ feed efficiency. We therefore investigated the phenotypic and genetic relationships between birds’ efficiency and the composition of the cecal microbiota in a F2 cross between broiler lines divergently selected for their high or low digestive efficiency.

Methods

Analyses were performed on 144 birds with extreme feed efficiency values at 3 weeks, with feed conversion values of 1.41±0.05 and 2.02±0.04 in the efficient and non-efficient groups, respectively. The total numbers of Lactobacillus, L. salivarius, L. crispatus, C. coccoides, C. leptum and E. coli per gram of cecal content were measured.

Results

The two groups mainly differed in larger counts of Lactobacillus, L. salivarius and E. coli in less efficient birds. The equilibrium between bacterial groups was also affected, efficient birds showing higher C. leptum, C. coccoides and L. salivarius to E. coli ratios. The heritability of the composition of microbiota was also estimated and L. crispatus, C. leptum, and C. coccoides to E. coli ratios were moderately but significantly heritable (0.16 to 0.24). The coefficient of fecal digestive use of dry matter was genetically and positively correlated with L. crispatus, C. leptum, C. coccoides (0.50 to 0.76) and negatively with E. coli (-0.66). Lipid digestibility was negatively correlated with E. coli (-0.64), and AMEn positively correlated with C. coccoides and with the C. coccoides to Lactobacillus ratio (0.48 to 0.64). We also detected 14 Quantitative Trait Loci (QTL) for microbiota on the host genome, mostly on C. leptum and Lactobacillus. The QTL for C. leptum on GGA6 was close to genome-wide significance. This region mainly includes genes involved in anti-inflammatory responses and in the motility of the gastrointestinal tract.