Activity and stability of different immobilized preparations of recombinant E. coli cells containing ω-transaminase

Production of chiral amines using ω-transaminases has been thoroughly studied in recent years. Immobilized ω-transaminases, however, have been used on relatively few occasions despite potential benefits such as reuse of enzyme and ease of product purification. In this study principally different methods including surface immobilization, entrapment and sweep flocculation using titanium oxide, Ca-alginate and chitosan respectively were evaluated for the immobilization of recombinant Escherichia coli cells. The enzyme expressed was a modified Arthrobacter citreus ω-transaminase with improved thermostability. The preparations were compared in terms of cell loading capacity, operational stability in repeated batches and storage stability using the conversion of methylbenzylamine to acetophenone.The use of chitosan for cell immobilization proved to be the method of choice since it was both very simple and effective. At a very high cell loading of 3.2 g cells/g chitosan >60% activity was observed. The preparation was reused in eight successive 1-h batches with >90% remaining activity. To further demonstrate its usability the preparation was used for asymmetric synthesis of (S)-4′-cyano-(α)-methylbenzylamine in three repeated bathes (cycle time >20 h), using isopropylamine as the amine donor. Storage stability was comparable with that of non-immobilized cells. It was concluded that the chitosan method due to its properties and simplicity would be advantageous for use also on a larger scale.     

Why are population growth rate estimates of past and present hunter–gatherers so different?

Hunter–gatherer population growth rate estimates extracted from archaeological proxies and ethnographic data show remarkable differences, as archaeological estimates are orders of magnitude smaller than ethnographic and historical estimates. This could imply that prehistoric hunter–gatherers were demographically different from recent hunter–gatherers. However, we show that the resolution of archaeological human population proxies is not sufficiently high to detect actual population dynamics and growth rates that can be observed in the historical and ethnographic data. We argue that archaeological and ethnographic population growth rates measure different things; therefore, they are not directly comparable. While ethnographic growth rate estimates of hunter–gatherer populations are directly linked to underlying demographic parameters, archaeological estimates track changes in the long-term mean population size, which reflects changes in the environmental productivity that provide the ultimate constraint for forager population growth. We further argue that because of this constraining effect, hunter–gatherer populations cannot exhibit long-term growth independently of increasing environmental productivity.This article is part of the theme issue ‘Cross-disciplinary approaches to prehistoric demography’.     

E. coli biofilm formation and its susceptibility towards T4 bacteriophages studied in a continuously operating mixing – tubular bioreactor system

A system consisting of a connected mixed and tubular bioreactor was designed to study bacterial biofilm formation and the effect of its exposure to bacteriophages under different experimental conditions. The bacterial biofilm inside silicone tubular bioreactor was formed during the continuous pumping of bacterial cells at a constant physiological state for 2 h and subsequent washing with a buffer for 24 h. Monitoring bacterial and bacteriophage concentration along the tubular bioreactor was performed via a piercing method. The presence of biofilm and planktonic cells was demonstrated by combining the piercing method, measurement of planktonic cell concentration at the tubular bioreactor outlet, and optical microscopy. The planktonic cell formation rate was found to be 8.95 × 10⁻³ h⁻¹ and increased approximately four-fold (4×) after biofilm exposure to an LB medium. Exposure of bacterial biofilm to bacteriophages in the LB medium resulted in a rapid decrease of biofilm and planktonic cell concentration, to below the detection limit within < 2 h. When bacteriophages were supplied in the buffer, only a moderate decrease in the concentration of both bacterial cell types was observed. After biofilm washing with buffer to remove unadsorbed bacteriophages, its exposure to the LB medium (without bacteriophages) resulted in a rapid decrease in bacterial concentration: again below the detection limit in < 2 h.     

Why do mountains support so many species of birds?

Although topographic complexity is often associated with high bird diversity at broad geographic scales, little is known about the relative contributions of geomorphologic heterogeneity and altitudinal climatic gradients found in mountains. We analysed the birds in the western mountains of the New World to examine the two‐fold effect of topography on species richness patterns, using two grains at the intercontinental extent and within temperate and tropical latitudes. Birds were also classified as montane or lowland, based on their overall distributions in the hemisphere. We estimated range in temperature within each cell and the standard deviation in elevation (topographic roughness) based on all pixels within each cell. We used path analysis to test for the independent effects of topographic roughness and temperature range on species richness while controlling for the collinearity between topographic variables. At the intercontinental extent, actual evapotranspiration (AET) was the primary driver of species richness patterns of all species taken together and of lowland species considered separately. In contrast, within‐cell temperature gradients strongly influenced the richness of montane species. Regional partitioning of the data also suggested that range in temperature either by itself or acting in combination with AET had the strongest “effect” on montane bird species richness everywhere. Topographic roughness had weaker “effects” on richness variation throughout, although its positive relationship with richness increased slightly in the tropics. We conclude that bird diversity gradients in mountains primarily reflect local climatic gradients. Widespread (lowland) species and narrow‐ranged (montane) species respond similarly to changes in the environment, differing only in that the richness of lowland species correlates better with broad‐scale climatic effects (AET), whereas mesoscale climatic variation accounts for richness patterns of montane species. Thus, latitudinal and altitudinal gradients in species richness can be explained through similar climatic‐based processes, as has long been argued.     

Predicting the strength of UP-elements and full-length E. coli σE promoters

Predicting the location and strength of promoters from genomic sequence requires accurate sequenced-based promoter models. We present the first model of a full-length bacterial promoter, encompassing both upstream sequences (UP-elements) and core promoter modules, based on a set of 60 promoters dependent on σ(E), an alternative ECF-type σ factor. UP-element contribution, best described by the length and frequency of A- and T-tracts, in combination with a PWM-based core promoter model, accurately predicted promoter strength both in vivo and in vitro. This model also distinguished active from weak/inactive promoters. Systematic examination of promoter strength as a function of RNA polymerase (RNAP) concentration revealed that UP-element contribution varied with RNAP availability and that the σ(E) regulon is comprised of two promoter types, one of which is active only at high concentrations of RNAP. Distinct promoter types may be a general mechanism for increasing the regulatory capacity of the ECF group of alternative σ's. Our findings provide important insights into the sequence requirements for the strength and function of full-length promoters and establish guidelines for promoter prediction and for forward engineering promoters of specific strengths.     

Why do Plasmodium falciparumm-infected erythrocytes form spontaneous erythrocyte rosettes?

Plasmodium falciparum malaria is one o f the most widespread o f human parasitic diseases and is responsible for the deaths of several million people in subtropical and tropical regions o f the world. The interaction o f malarial merozoites with erythrocytes and the adherence o f infected erythrocytes to the endothelium are among the cellular interactions extensively studied to define candidate antigens for a blood stage vaccine. However, the exact mechanisms underlying the invasion o f erythrocytes by P. falciparum merozoites and their subsequent binding to endothelium are not yet understood. Here Mats Wahlgren, Johan Carlson, Rachonee Udomsangpetch and Peter Perlmonn discuss a novel cytoodherence phenomenon which may be o f great importance in this context, that is, the spontaneous binding o f uninfected erythrocytes to those infected with late-stage parasites (trophozoites/schizonts).     

Codon Bias Patterns of E. coli’s Interacting Proteins

Synonymous codons, i.e., DNA nucleotide triplets coding for the same amino acid, are used differently across the variety of living organisms. The biological meaning of this phenomenon, known as codon usage bias, is still controversial. In order to shed light on this point, we propose a new codon bias index, CompAI, that is based on the competition between cognate and near-cognate tRNAs during translation, without being tuned to the usage bias of highly expressed genes. We perform a genome-wide evaluation of codon bias for E.coli, comparing CompAI with other widely used indices: tAI, CAI, and Nc. We show that CompAI and tAI capture similar information by being positively correlated with gene conservation, measured by the Evolutionary Retention Index (ERI), and essentiality, whereas, CAI and Nc appear to be less sensitive to evolutionary-functional parameters. Notably, the rate of variation of tAI and CompAI with ERI allows to obtain sets of genes that consistently belong to specific clusters of orthologous genes (COGs). We also investigate the correlation of codon bias at the genomic level with the network features of protein-protein interactions in E.coli. We find that the most densely connected communities of the network share a similar level of codon bias (as measured by CompAI and tAI). Conversely, a small difference in codon bias between two genes is, statistically, a prerequisite for the corresponding proteins to interact. Importantly, among all codon bias indices, CompAI turns out to have the most coherent distribution over the communities of the interactome, pointing to the significance of competition among cognate and near-cognate tRNAs for explaining codon usage adaptation. Notably, CompAI may potentially correlate with translation speed measurements, by accounting for the specific delay induced by wobble-pairing between codons and anticodons.     

Are faces of different species perceived categorically by human observers?

What are the species boundaries of face processing? Using a face-feature morphing algorithm, image series intermediate between human, monkey (macaque), and bovine faces were constructed. Forced-choice judgement of these images showed sharply bounded categories for upright face images of each species. These predicted the perceptual discrimination boundaries for upright monkey-cow and cow-human images, but not human-monkey images. Species categories were also well-judged for inverted face images, but these did not give sharpened discrimination (categorical perception) at the category boundaries. While categorical species judgements are made reliably, only the distinction between primate faces and cow faces appears to be categorically perceived, and only in upright faces. One inference is that humans may judge monkey faces in terms of human characteristics, albeit distinctive ones.     

Optimization of conditions for expression of recombinant interferon-γ in E.coli

Interferon gamma (IFN-γ) is an important immunoregulatory cytokine that has a central role against viral and bacterial infections. In this study, the cDNA encoding 141 amino acids of mature IFN-γ from mice splenocytes was cloned in a prokaryotic expression vector pQE 30. Optimization of expression conditions resulted in high IFN-γ protein. Western blot showed that recombinant IFN-γ was specifically recognized by its counterpart anti-mouse IFN-γ antibodies. In vitro dose-dependent studies, with A549 and HeLa cell lines, showed that cloned IFN-γ was safe and had no effect on cell proliferation. The protein prediction and analysis using SOPMA program, revealed that IFN-γ had 80 α-helices, 8 β-turns jointed by 9 extended strands and 44 random coils. A total of four major clusters were observed with murine IFN-γ sharing 39 % homology with human IFN-γ. Pair-wise alignment studies with human revealed 26 % identity and 43.3 % similarity. The recovery of bioactive proteins from inclusion bodies (IBs) is a complex process and various protocols have been developed. We report here a simple, robust and inexpensive purification approach for obtaining recombinant IFN-γ protein expressed as IBs in E.coli.     

Why does phenology drive species distribution?

Despite the numerous studies which have been conducted during the past decade on species ranges and their relationship to the environment, our understanding of how environmental conditions shape species distribution is still far from complete. Yet, some process-based species distribution models have been able to simulate plants and insects distribution at a global scale. These models strongly rely on the completion of the annual cycle of the species and therefore on their accomplished phenology. In particular, they have shown that the northern limit of species'' ranges appears to be caused mainly by the inability to undergo full fruit maturation, while the southern limit appears to be caused by the inability to flower or unfold leaves owing to a lack of chilling temperatures that are necessary to break bud dormancy. I discuss here why phenology is a key adaptive trait in shaping species distribution using mostly examples from plant species, which have been the most documented. After discussing how phenology is involved in fitness and why it is an adaptive trait susceptible to evolve quickly in changing climate conditions, I describe how phenology is related to fitness in species distribution process-based models and discuss the fate of species under climate change scenarios using model projections and experimental or field studies from the literature.     

Why are estimates of the terrestrial carbon balance so different?

The carbon balance of the world's terrestrial ecosystems is uncertain. Both top‐down (atmospheric) and bottom‐up (forest inventory and land‐use change) approaches have been used to calculate the sign and magnitude of a net terrestrial flux. Different methods often include different processes, however, and comparisons can be misleading. Differences are not necessarily the result of uncertainties or errors, but often result from incomplete accounting inherent in some of the methods. Recent estimates are reviewed here. Overall, a northern mid‐latitude carbon sink of approximately 2 Pg C yr^?1 appears robust, although the mechanisms responsible are uncertain. Several lines of evidence point to environmentally enhanced rates of carbon accumulation. Other lines suggest that recovery from past disturbances is largely responsible for the sink. The tropics appear to be a small net source of carbon or nearly neutral, and the same uncertainties of mechanism exist. In addition, studies in the tropics do not permit an unequivocal choice between two alternatives: large emissions of carbon from deforestation offset by large sinks in undisturbed forests, or moderate emissions from land‐use change with essentially no change in the carbon balance in undisturbed forests. Resolution of these uncertainties is most likely to result from spatially detailed historical reconstructions of land‐use change and disturbance in selected northern mid‐latitude regions where such data are available, and from systematic monitoring of changes in the area of tropical forests with satellite data of high spatial resolution collected over the last decades and into the future.