Three old and one new: protein import into red algal-derived plastids surrounded by four membranes

Engulfment of a red or green alga by another eukaryote and subsequent reduction of the symbiont to an organelle, termed a complex plastid, is a process known as secondary endosymbiosis and is shown in a diverse group of eukaryotic organisms. Important members are heterokontophytes, haptophytes, cryptophytes, and apicomplexan parasites, all of them with complex plastids of red algal origin surrounded by four membranes. Although the evolutionary relationship between these organisms is still debated, they share common mechanisms for plastid protein import. In this review, we describe recent findings and current models on preprotein import into complex plastids with a special focus on the second outermost plastid membrane. Derived from the plasma membrane of the former endosymbiont, the evolution of protein transport across this so-called periplastidal membrane most likely represented the challenge in the transition from an endosymbiont to a host-dependent organelle. Here, remodeling and relocation of the symbiont endoplasmic reticulum-associated degradation (ERAD) machinery gave rise to a translocon complex termed symbiont-specific ERAD-like machinery and provides a fascinating insight into complex cellular evolution.     

Camphor Pathway Redux: Functional Recombinant Expression of 2,5- and 3,6-Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453 with Their Cognate Flavin Reductase Catalyzing Baeyer-Villiger Reactions

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (−) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (K_m = 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (K_m = 3.6 μM; k_cat = 283 s⁻¹) in preference to flavin adenine dinucleotide (FAD) (K_m = 19 μM; k_cat = 128 s⁻¹). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE_25–1 for 2,5-DKCMO-1 and camE_25–2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE₃₆). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.     

Synthesis and acetylcholinesterase inhibitory activity of polyhydroxylated sulfated steroids: Structure/activity studies

Disulfated and trisulfated steroids have been synthesized from cholesterol and their acetylcholinesterase inhibitory activity has been evaluated. In our studies we have found that the activity was not only dependent on the location of the sulfate groups but on their configurations. 2β,3α,6α-trihydroxy-5α-cholestan-6-one trisulfate (18) was the most active steroid with an IC₅₀ value of 15.48 μM comparable to that of 2β,3α-dihydroxy-5α-cholestan-6-one disulfate (1). Both compounds were found to be less active than the reference compound eserine. The butyrylcholinesterase activity of 1 and 18 was one magnitude lower than that against acetylcholinesterase revealing a selective inhibitor profile.     

Invasion genetics of American cherry fruit fly in Europe and signals of hybridization with the European cherry fruit fly

The American cherry fruit fly is an invasive pest species in Europe, of serious concern in tart cherry production as well as for the potential to hybridize with the European cherry fruit fly, Rhagoletis cerasi L. (Diptera: Tephritidae), which might induce new pest dynamics. In the first European reports, the question arose whether only the eastern American cherry fruit fly, Rhagoletis cingulata (Loew) (Diptera: Tephritidae), is present, or also the closely related western American cherry fruit fly, Rhagoletis indifferens Curran. In this study, we investigate the species status of European populations by comparing these with populations of both American species from their native ranges, the invasion dynamics in German (first report in 1993) and Hungarian (first report in 2006) populations, and we test for signals of hybridization with the European cherry fruit fly. Although mtDNA sequence genealogy could not separate the two American species, cross‐species amplification of 14 microsatellite loci separated them with high probabilities (0.99–1.0) and provided evidence for R. cingulata in Europe. German and Hungarian R. cingulata populations differed significantly in microsatellite allele frequencies, mtDNA haplotype and wing pattern distributions, and both were genetically depauperate relative to North American populations. The diversity suggests independent founding events in Germany and Hungary. Within each country, R. cingulata displayed little or no structure in any trait, which agrees with rapid local range expansions. In cross‐species amplifications, signals of hybridization between R. cerasi and R. cingulata were found in 2% of R. cingulata individuals and in 3% of R. cerasi. All putative hybrids had R. cerasi mtDNA indicating that the original between‐species mating involved R. cerasi females and R. cingulata males.     

GENETIC DIFFERENTIATION AND SELECTION AGAINST MIGRANTS IN EVOLUTIONARILY REPLICATED EXTREME ENVIRONMENTS

We investigated mechanisms of reproductive isolation in livebearing fishes (genus Poecilia) inhabiting sulfidic and nonsulfidic habitats in three replicate river drainages. Although sulfide spring fish convergently evolved divergent phenotypes, it was unclear if mechanisms of reproductive isolation also evolved convergently. Using microsatellites, we found strongly reduced gene flow between adjacent populations from different habitat types, suggesting that local adaptation to sulfidic habitats repeatedly caused the emergence of reproductive isolation. Reciprocal translocation experiments indicate strong selection against immigrants into sulfidic waters, but also variation among drainages in the strength of selection against immigrants into nonsulfidic waters. Mate choice experiments revealed the evolution of assortative mating preferences in females from nonsulfidic but not from sulfidic habitats. The inferred strength of sexual selection against immigrants (RI_s) was negatively correlated with the strength of natural selection (RI_m), a pattern that could be attributed to reinforcement, whereby natural selection strengthens behavioral isolation due to reduced hybrid fitness. Overall, reproductive isolation and genetic differentiation appear to be replicated and direct consequences of local adaptation to sulfide spring environments, but the relative contributions of different mechanisms of reproductive isolation vary across these evolutionarily independent replicates, highlighting both convergent and nonconvergent evolutionary trajectories of populations in each drainage.     

Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries

Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062–147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217–73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples.     

Pleiotropy in the melanocortin system: expression levels of this system are associated with melanogenesis and pigmentation in the tawny owl (Strix aluco)

The adaptive function of melanin‐based coloration is a long‐standing debate. A recent genetic model suggested that pleiotropy could account for covariations between pigmentation, behaviour, morphology, physiology and life history traits. We explored whether the expression levels of genes belonging to the melanocortin system (MC1R, POMC, PC1/3, PC2 and the antagonist ASIP), which have many pleiotropic effects, are associated with melanogenesis (through variation in the expression of the genes MITF, SLC7A11, TYR, TYRP1) and in turn melanin‐based coloration. We considered the tawny owl (Strix aluco) because individuals vary continuously from light to dark reddish, and thus, colour variation is likely to stem from differences in the levels of gene expression. We measured gene expression in feather bases collected in nestlings at the time of melanin production. As expected, the melanocortin system was associated with the expression of melanogenic genes and pigmentation. Offspring of darker reddish fathers expressed PC1/3 to lower levels but tended to express PC2 to higher levels. The convertase enzyme PC1/3 cleaves the POMC prohormone to obtain ACTH, while the convertase enzyme PC2 cleaves ACTH to produce α‐melanin‐stimulating hormone (α‐MSH). ACTH regulates glucocorticoids, hormones that modulate stress responses, while α‐MSH induces eumelanogenesis. We therefore conclude that the melanocortin system, through the convertase enzymes PC1/3 and PC2, may account for part of the interindividual variation in melanin‐based coloration in nestling tawny owls. Pleiotropy may thus account for the covariation between phenotypic traits involved in social interactions (here pigmentation) and life history, morphology, behaviour and physiology.     

Dissecting the energy metabolism in Mycoplasma pneumoniae through genome‐scale metabolic modeling

Mycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint‐based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time‐dependent changes, albeit using a static model. By performing an in silico knock‐out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms.     

Transfer of Natural Micro Structures to Bionic Lightweight Design Proposals

The abstraction of complex biological lightweight structure features into a producible technical component is a funda- mental step within the transfer of design principles from nature to technical lightweight solutions. A major obstacle for the transfer of natural lightweight structures to technical solutions is their peculiar geometry. Since natural lightweight structures possess irregularities and often have extremely complex forms due to elaborate growth processes, it is usually necessary to simplify their design principles. This step of simplification/abstraction has been used in different biomimetic methods, but so far, it has an arbitrary component, i.e. it crucially depends on the competence of the person who executes the abstraction. This paper describes a new method for abstraction and specialization of natural micro structures for technical lightweight compo- nents. The new method generates stable lightweight design principles by using topology optimization within a design space of preselected biological archetypes such as diatoms or radiolarian. The resulting solutions are adapted to the technical load cases and production processes, can be created in a large variety, and may be further optimized e.g. by using parametric optimization.     

Measuring Information-Transfer Delays

In complex networks such as gene networks, traffic systems or brain circuits it is important to understand how long it takes for the different parts of the network to effectively influence one another. In the brain, for example, axonal delays between brain areas can amount to several tens of milliseconds, adding an intrinsic component to any timing-based processing of information. Inferring neural interaction delays is thus needed to interpret the information transfer revealed by any analysis of directed interactions across brain structures. However, a robust estimation of interaction delays from neural activity faces several challenges if modeling assumptions on interaction mechanisms are wrong or cannot be made. Here, we propose a robust estimator for neuronal interaction delays rooted in an information-theoretic framework, which allows a model-free exploration of interactions. In particular, we extend transfer entropy to account for delayed source-target interactions, while crucially retaining the conditioning on the embedded target state at the immediately previous time step. We prove that this particular extension is indeed guaranteed to identify interaction delays between two coupled systems and is the only relevant option in keeping with Wiener’s principle of causality. We demonstrate the performance of our approach in detecting interaction delays on finite data by numerical simulations of stochastic and deterministic processes, as well as on local field potential recordings. We also show the ability of the extended transfer entropy to detect the presence of multiple delays, as well as feedback loops. While evaluated on neuroscience data, we expect the estimator to be useful in other fields dealing with network dynamics.