Oligomeric behavior of the RND transporters CusA and AcrB in micellar solution of detergent

We have used analytical ultracentrifugation to explore the oligomeric states of AcrB and CusA in micellar solution of detergent. These two proteins belong to the resistance, nodulation and cell division (RND) family of efflux proteins that are involved in multiple drug and heavy metal resistance. Only the structure of AcrB has been determined so far. Although functional RND proteins should assemble as trimers as AcrB does, both AcrB and CusA form a mixture of quaternary structures (from monomer to heavy oligomer) in detergent solution. The distribution of the oligomeric states was studied as a function of different parameters: nature and concentration of the detergent, ionic strength, pH, protein concentration. This pseudo-heterogeneity does not hamper the crystallization of AcrB as a homotrimer.     

Biophysical analysis of the EPEC translocated intimin receptor-binding domain

Enteropathogenic Escherichia coli (EPEC) are Gram (-) bacteria responsible for widespread illness in the form of diarrhea. EPEC cells attach to the intestinal epithelium using a Type III secretion system common to many Gram (-) bacteria. The translocated intimin receptor (TIR) is the first protein secreted through the EPEC secretion complex, and is absolutely required for pathogenesis. It inserts into the intestinal epithelium, serving as an anchor responsible for the attachment of EPEC to the host epithelial cell. Intimin is a transmembrane protein displayed on the EPEC cell surface with an extracellular domain that binds TIR. Observation of a TIR-TIR dimer in the X-ray co-crystal structure of the extracellular domains of intimin and TIR raised the question of how these protein domains interact and function in solution. Herein we report that the extracellular domain of TIR exists in a folded and active monomeric state in solution, as confirmed by analytical ultracentrifugation, analytical size-exclusion HPLC, isothermal titration calorimetry, and surface plasmon resonance.     

Coding sequence polymorphism in avian mitochondrial genomes reflects population histories

Nucleotide sequence diversity at mitochondrial protein-coding loci from 72 species of birds from different geographical regions was analysed in order to test the hypothesis that temperate zone species show population genetic effects of past glaciation. Temperate zone species showed reduced nucleotide diversity in comparison to tropical mainland species, suggesting that the latter have long-term effective population sizes due to population bottleneck effects during the most recent glaciation. This hypothesis was further supported by evidence of an unusually high estimated rate of population growth in species breeding in North America and wintering in the New World tropics (Nearctic migrants), consistent with population expansion after a bottleneck. Nearctic migrants also showed evidence of an abundance of rare nonsynonymous (amino acid-altering) polymorphisms, a pattern suggesting that slightly deleterious polymorphisms drifted to high frequencies during a bottleneck and are now being eliminated by selection. Because the shape of the North American land mass limited the area available for refugia during glaciation, the bottleneck effects are predicted to have been particularly strong in Nearctic migrants, and this prediction was supported. The reduced genetic diversity of Nearctic migrants provides an additional basis for concern for the survival of these species, which are threatened by loss of habitat in the winter range and by introduced disease.     

Recent demographic bottlenecks are not accompanied by a genetic signature in banner-tailed kangaroo rats (Dipodomys spectabilis)

Single-sample methods of bottleneck detection are now routine analyses in studies of wild populations and conservation genetics. Three common approaches to bottleneck detection are the heterozygosity excess, mode-shift, and M-ratio tests. Empirical groundtruthing of these methods is difficult, but their performances are critical for the accurate reconstruction of population demography. We use two banner-tailed kangaroo rat (Dipodomys spectabilis) populations from southeastern Arizona (USA) that are known to have experienced recent demographic reductions to search for genetic bottleneck signals with eight microsatellite loci. Over eight total sample-years, neither population showed a genetic bottleneck signature. M-ratios in both populations were large, stable, and never fell below a critical significance value (Mc). The mode shift test did not detect any distortion of allele frequencies, and tests of heterozygosity excess were not significant in postbottleneck samples when we used standard microsatellite mutation models. The genetic effects of bottlenecks like those experienced by our study populations should be strongly influenced by rates of mutation and migration. We used genetic parentage data to estimate a relatively high mutation rate in D. spectabilis (0.0081 mutants/generation/locus), but mutation alone is unlikely to explain the temporal distribution of rare alleles that we observed. Migration (gene flow) is a more likely explanation, despite prior mark-recapture analysis that estimated very low rates of interpopulation dispersal. We interpret our kangaroo rat data in light of the broader literature and conclude that in natural populations connected by dispersal, demographic bottlenecks may prove difficult to detect using molecular genetic data.     

Genetic variation and differentiation in captive and wild zebra finches (Taeniopygia guttata)

The zebra finch (Taeniopygia guttata) is a small Australian grassland songbird that has been domesticated over the past two centuries. Because it is easy to breed in captivity, it has become a widely used study organism, especially in behavioural research. Most work has been conducted on domesticated populations maintained at numerous laboratories in Europe and North America. However, little is known about the extent to which, during the process of domestication, captive populations have gone through bottlenecks in population size, leading to inbred and potentially genetically differentiated study populations. This is an important issue, because (i) behavioural studies on captive populations might suffer from artefacts arising from high levels of inbreeding or lack of genetic variation in such populations, and (ii) it may hamper the comparability of research findings. To address this issue, we genotyped 1000 zebra finches from 18 captive and two wild populations at 10 highly variable microsatellite loci. We found that all captive populations have lost some of the genetic variability present in the wild, but there is no evidence that they have gone through a severe bottleneck, as the average captive population still showed a mean of 11.7 alleles per locus, compared to a mean of 19.3 alleles/locus for wild zebra finches. We found significant differentiation between the captive populations (F(ST) = 0.062). Patterns of genetic similarity closely match geographical relationships, so the most pronounced differences occur between the three continents: Australia, North America, and Europe. By providing a tree of the genetic similarity of the different captive populations, we hope to contribute to a better understanding of variation in research findings obtained by different laboratories.     

Growth inhibitory factors in bovine faeces impairs detection of Salmonella Dublin by conventional culture procedure

AIMS: To analyse the relative importance of different biological and technical factors on the analytical sensitivity of conventional culture methods for detection of Salmonella Dublin in cattle faeces. METHODS AND RESULTS: Faeces samples collected from six adult bovines from different salmonella-negative herds were split into subpools and spiked with three strains of S. Dublin at a concentration level of c. 10 CFU g(-1) faeces. Each of the 18 strain-pools was divided into two sets of triplicates of four volumes of faecal matter (1, 5, 10 and 25 g). The two sets were pre-enriched with and without novobiocin, followed by combinations of culture media (three types) and selective media (two types). The sensitivity of each combination and sources of variation in detection were determined by a generalized linear mixed model using a split-plot design. CONCLUSIONS: Biological factors, such as faecal origin and S. Dublin strain influenced the sensitivity more than technical factors. Overall, the modified semi-solid Rappaport Vassiliadis (MSRV)-culture medium had the most reliable detection capability, whereas detection with selenite cystine broth and Mueller Kauffman tetrathionate broth combinations varied more in sensitivity and rarely reached the same level of detection as MSRV in this experiment. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed that for MSRV-culture medium and xylose lysine decarboxylase agar as the indicative medium, the sensitivity of the faecal culture method may be improved by focusing on the strain variations and the ecology of the faecal sample. Detailed investigation of the faecal flora (pathogens and normal flora) and the interaction with chemical factors may result in developing an improved method for detection of S. Dublin.     

Solution Structural Studies of Pre-amyloid Oligomer States of the Biofilm Protein Aap

Staphylococcus epidermidis is a commensal bacterium on human skin that is also the leading cause of medical device-related infections. The accumulation-associated protein (Aap) from S. epidermidis is a critical factor for infection via its ability to mediate biofilm formation. The B-repeat superdomain of Aap is composed of 5 to 17 Zn²⁺-binding B-repeats, which undergo rapid, reversible assembly to form dimer and tetramer species. The tetramer can then undergo a conformational change and nucleate highly stable functional amyloid fibrils. In this study, multiple techniques including analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS) are used to probe a panel of B-repeat mutant constructs that assemble to distinct oligomeric states to define the structural characteristics of B-repeat dimer and tetramer species. The B-repeat region from Aap forms an extremely elongated conformation that presents several challenges for standard SAXS analyses. Specialized approaches, such as cross-sectional analyses, allowed for in-depth interpretation of data, while explicit-solvent calculations via WAXSiS allowed for accurate evaluation of atomistic models. The resulting models suggest mechanisms by which Aap functional amyloid fibrils form, illuminating an important contributing factor to recurrent staphylococcal infections.     

Direct and indirect causal effects of heterozygosity on fitness-related traits in Alpine ibex

Heterozygosity–fitness correlations (HFCs) are a useful tool to investigate the effects of inbreeding in wild populations, but are not informative in distinguishing between direct and indirect effects of heterozygosity on fitness-related traits. We tested HFCs in male Alpine ibex (Capra ibex) in a free-ranging population (which suffered a severe bottleneck at the end of the eighteenth century) and used confirmatory path analysis to disentangle the causal relationships between heterozygosity and fitness-related traits. We tested HFCs in 149 male individuals born between 1985 and 2009. We found that standardized multi-locus heterozygosity (MLH), calculated from 37 microsatellite loci, was related to body mass and horn growth, which are known to be important fitness-related traits, and to faecal egg counts (FECs) of nematode eggs, a proxy of parasite resistance. Then, using confirmatory path analysis, we were able to show that the effect of MLH on horn growth was not direct but mediated by body mass and FEC. HFCs do not necessarily imply direct genetic effects on fitness-related traits, which instead can be mediated by other traits in complex and unexpected ways.     

Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85α Homodimer

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide. We have demonstrated in vivo concentration-dependent dimerization of p85α using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.     

Structural Characterization of Bardet-Biedl Syndrome 9 Protein (BBS9)

The Bardet-Biedl syndrome protein complex (BBSome) is an octameric complex that transports membrane proteins into the primary cilium signaling organelle in eukaryotes and is implicated in human disease. Here we have analyzed the 99-kDa human BBS9 protein, one of the eight BBSome components. The protein is composed of four structured domains, including a β-stranded N-terminal domain. The 1.8 Å crystal structure of the 46-kDa N-terminal domain reveals a seven-bladed β-propeller. A structure-based homology search suggests that it functions in protein-protein interactions. We show that the Bardet-Biedl syndrome-causing G141R mutation in BBS9 likely results in misfolding of the β-propeller. Although the C-terminal half of BBS9 dimerizes in solution, the N-terminal domain only does so in the crystal lattice. This C-terminal dimerization interface might be important for the assembly of the BBSome.