New insights into the structure and oligomeric state of the bacterial multidrug transporter EmrE: an unusual asymmetric homo-dimer

EmrE is a small multidrug transporter that contains 110 amino acid residues that form four transmembrane alpha-helices. The three-dimensional structure of EmrE has been determined from two-dimensional crystals by electron cryo-microscopy. EmrE is an asymmetric homo-dimer with one substrate molecule bound in a chamber accessible laterally from one leaflet of the lipid bilayer. Evidence from substrate binding analyses and analytical ultracentrifugation of detergent-solubilised EmrE shows that the minimum functional unit for substrate binding is a dimer. However, it is possible that EmrE exists as a tetramer in vivo and plausible models are suggested based upon analyses of two-dimensional crystals.     

Breeding system evolution in Tarasa (Malvaceae) and selection for reduced pollen grain size in the polyploid species

Polyploidy, primarily allopolyploidy, has played a major role throughout flowering plant evolution with an estimated 30-80% of all extant angiosperms carrying traces of ancient or recent polyploidy. One immediate and seemingly invariant phenotypic consequence of genome doubling is larger cell size in polyploids relative to their diploid progenitors. In plants, increases in pollen grain and guard cell sizes exemplify this rule and are often used as surrogate evidence for polyploidy. Tarasa (Malvaceae), a genus of 27 species primarily distributed in the high (>3000 m) Andes, has numerous independently generated tetraploid species, most of which have pollen grains smaller than their putative diploid parents. The tetraploids are also unusual because they are annual, rather than perennial, in habit. Data correlate these apparent anomalies to a change in the breeding system within the genus from xenogamy (outcrossing) in the diploid species to autogamy (inbreeding) in the tetraploids, leading to a convergence in reduced floral morphology. The harsh environment of the high-elevation Andean habitats in which all the tetraploid annuals are found is implicated as a critical factor in shaping the evolution of these unusual polyploids.     

Comparison of characteristics and function of translation termination signals between and within prokaryotic and eukaryotic organisms

Six diverse prokaryotic and five eukaryotic genomes were compared to deduce whether the protein synthesis termination signal has common determinants within and across both kingdoms. Four of the six prokaryotic and all of the eukaryotic genomes investigated demonstrated a similar pattern of nucleotide bias both 5′ and 3′ of the stop codon. A preferred core signal of 4 nt was evident, encompassing the stop codon and the following nucleotide. Codons decoded by hyper-modified tRNAs were over-represented in the region 5′ to the stop codon in genes from both kingdoms. The origin of the 3′ bias was more variable particularly among the prokaryotic organisms. In both kingdoms, genes with the highest expression index exhibited a strong bias but genes with the lowest expression showed none. Absence of bias in parasitic prokaryotes may reflect an absence of pressure to evolve more efficient translation. Experiments were undertaken to determine if a correlation existed between bias in signal abundance and termination efficiency. In Escherichia coli signal abundance correlated with termination efficiency for UAA and UGA stop codons, but not in mammalian cells. Termination signals that were highly inefficient could be made more efficient by increasing the concentration of the cognate decoding release factor.     

Effects of Plant Species Diversity and Composition on Nitrogen Cycling and the Trace Gas Balance of Soils

Experiments addressing the role of plant species diversity for ecosystem functioning have recently proliferated. Most studies have focused on plant biomass responses. However, microbial processes involved in the production of N₂O and the oxidation of atmospheric CH₄ could potentially be affected via effects on N cycling, on soil diffusive properties (due to changes in water relations and root architecture) and by more direct interactions of plants with soil microbes. We studied ecosystem-level CH₄ and N₂O fluxes in experimental communities assembled from two pasture soils and from combinations of 1, 3, 6, 8 or 9 species typical for these pastures. The soils contrasted with respect to texture and fertility. N₂O emissions decreased with diversity and increased in the presence of legumes. Soils were sinks for CH₄ at all times; legume monocultures were a smaller sink for atmospheric CH₄ than non-legume monocultures, but no effect of species richness per se was detected. However, both the exchange of CH₄ and N₂O strongly depended on plant community composition, and on the interaction of composition with soil type, indicating that the functional role of species and their interactions differed between soils. N₂O fluxes were mainly driven by effects on soil nitrate and on nitrification while soil moisture had less of an effect. Soil microbial C and N and N mineralisation rates were not altered. The driver of the interactive soil type×plant community composition-effects was less clear. Because soil methanotrophs may take longer to respond to alterations of N cycling than the 1/2 year treatment in this study, we also tested species richness-effects in two separate 5-year field studies, but results were ambiguous, indicating complex interactions with soil disturbance. In conclusion, our study demonstrates that plant community composition can affect the soil trace gas balance, whereas plant species richness per se was less important; it also indicates a potential link between the botanical composition of plant communities and global warming.     

Distinct DNA methylation activity of Dnmt3a and Dnmt3b towards naked and nucleosomal DNA

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. De novo type DNA methyltransferases Dnmt3a and Dnmt3b are responsible for creating DNA methylation patterns during embryogenesis and in germ cells. Although their in vitro DNA methylation properties are similar, Dnmt3a and Dnmt3b methylate different genomic DNA regions in vivo. In the present study, we have examined the DNA methylation activity of Dnmt3a and Dnmt3b towards nucleosomes reconstituted from recombinant histones and DNAs, and compared it to that of the corresponding naked DNAs. Dnmt3a showed higher DNA methylation activity than Dnmt3b towards naked DNA and the naked part of nucleosomal DNA. On the other hand, Dnmt3a scarcely methylated the DNA within the nucleosome core region, while Dnmt3b significantly did, although the activity was low. We propose that the preferential DNA methylation activity of Dnmt3a towards the naked part of nucleosomal DNA and the significant methylation activity of Dnmt3b towards the nucleosome core region contribute to their distinct methylation of genomic DNA in vivo.     

Oxygen limitation modulates pH regulation of catabolism and hydrogenases, multidrug transporters, and envelope composition inEscherichia coli K-12

Background  

InEscherichia coli, pH regulates genes for amino-acid and sugar catabolism, electron transport, oxidative stress, periplasmic and envelope proteins. Many pH-dependent genes are co-regulated by anaerobiosis, but the overall intersection of pH stress and oxygen limitation has not been investigated.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.     

A role for the vesicle-associated tubulin binding protein ARL6 (BBS3) in flagellum extension in Trypanosoma brucei

The small GTPase Arl6 is implicated in the ciliopathic human genetic disorder Bardet-Biedl syndrome, acting at primary cilia in recruitment of the octomeric BBSome complex, which is required for specific trafficking events to and from the cilium in eukaryotes. Here we describe functional characterisation of Arl6 in the flagellated model eukaryote Trypanosoma brucei, which requires motility for viability. Unlike human Arl6 which has a ciliary localisation, TbARL6 is associated with electron-dense vesicles throughout the cell body following co-translational modification by N-myristoylation. Similar to the related protein ARL-3A in T. brucei, modulation of expression of ARL6 by RNA interference does not prevent motility but causes a significant reduction in flagellum length. Tubulin is identified as an ARL6 interacting partner, suggesting that ARL6 may act as an anchor between vesicles and cytoplasmic microtubules. We provide evidence that the interaction between ARL6 and the BBSome is conserved in unicellular eukaryotes. Overexpression of BBS1 leads to translocation of endogenous ARL6 to the site of exogenous BBS1 at the flagellar pocket. Furthermore, a combination of BBS1 overexpression and ARL6 RNAi has a synergistic inhibitory effect on cell growth. Our findings indicate that ARL6 in trypanosomes contributes to flagellum biogenesis, most likely through an interaction with the BBSome.     

Rapid, repeated, and clustered loss of duplicate genes in allopolyploid plant populations of independent origin

The predictability of evolution is debatable, with recent evidence suggesting that outcomes may be constrained by gene interaction networks [1]. Whole-genome duplication (WGD; polyploidization-ubiquitous in plant evolution [2]) provides the opportunity to evaluate the predictability of genome reduction, a pervasive feature of evolution [3, 4]. Repeated patterns of genome reduction appear to have occurred via duplicated gene (homeolog) loss in divergent species following ancient WGD [5-9], with evidence for preferential retention of duplicates in certain gene classes [8-10]. The speed at which these patterns arise is unknown. We examined presence/absence of 70 homeologous loci in 59 Tragopogon miscellus plants from five natural populations of independent origin; this allotetraploid arose ~80 years ago via hybridization between diploid parents and WGD [11]. Genes were repeatedly retained or lost in clusters, and the gene ontology categories of the missing genes correspond to those lost after ancient WGD in the same family (Asteraceae; sunflower family) [6] and with gene dosage sensitivity [8]. These results provide evidence that the outcomes of WGD are predictable, even in 40 generations, perhaps due to the connectivity of gene products [8, 10, 12]. The high frequency of single-allele losses detected and low frequency of changes fixed within populations provide evidence for ongoing evolution.