570 Biowissenschaften; Biologie
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We have developed a genetic circuit in Escherichia coli that can be used to select for protein-protein interactions of different strengths by changing antibiotic concentrations in the media. The genetic circuit links protein-protein interaction strength to beta-lactamase activity while simultaneously imposing tuneable positive and negative selection pressure for beta-lactamase activity. Cells only survive if they express interacting proteins with affinities that fall within set high- and low-pass thresholds; i.e. the circuit therefore acts as a bandpass filter for protein-protein interactions. We show that the circuit can be used to recover protein-protein interactions of desired affinity from a mixed population with a range of affinities. The circuit can also be used to select for inhibitors of protein-protein interactions of defined strength. (C) 2018 Elsevier Ltd. All rights reserved.
The organohalide-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B-12, which serves as cofactor of the tetrachloroethene (PCE) reductive dehalogenase (PceA). As previously reported, a replacement of the adeninyl moiety, the lower base of the cofactor, by exogenously applied 5,6-dimethylbenzimidazole led to inactive PceA. To explore the general effect of benzimidazoles on the PCE metabolism, the susceptibility of the organism for guided biosynthesis of various singly substituted benzimidazolyl-norcobamides was investigated, and their use as cofactor by PceA was analyzed. Exogenously applied 5-methylbenzimidazole (5-MeBza), 5-hydroxybenzimidazole (5-OHBza), and 5-methoxybenzimidazole (5-OMeBza) were found to be efficiently incorporated as lower bases into norcobamides (NCbas). Structural analysis of the NCbas by nuclear magnetic resonance spectroscopy uncovered a regioselectivity in the utilization of these precursors for NCba biosynthesis. When 5-MeBza was added, a mixture of 5-MeBza-norcobamide and 6-MeBza-norcobamide was formed, and the PceA enzyme activity was affected. In the presence of 5-OHBza, almost exclusively 6-OHBza-norcobamide was produced, while in the presence of 5-OMeBza, predominantly 5-OMeBza-norcobamide was detected. Both NCbas were incorporated into PceA, and no negative effect on the PceA activity was observed. In crystal structures of PceA, both NCbas were bound in the base-off mode with the 6-OHBza and 5-OMeBza lower bases accommodated by the same solvent-exposed hydrophilic pocket that harbors the adenine as the lower base of authentic norpseudo-B-12. In this study, a selective production of different norcobamide isomers containing singly substituted benzimidazoles as lower bases is shown, and unique structural insights into their utilization as co-factors by a cobamide-containing enzyme are provided. IMPORTANCE Guided biosynthesis of norcobamides containing singly substituted benzimidazoles as lower bases by the organohalide-respiring epsilonproteobacterium Sulfurospirillum multivorans is reported. An unprecedented specificity in the formation of norcobamide isomers containing hydroxylated or methoxylated benzimidazoles was observed that implicated a strict regioselectivity of the norcobamide biosynthesis in the organism. In contrast to 5,6-dimethylbenzimidazolyl-norcobamide, the incorporation of singly substituted benzimidazolyl-norcobamides as a cofactor into the tetrachloroethene reductive dehalogenase was not impaired. The enzyme was found to be functional with different isomers and not limited to the use of adeninyl-norcobamide. Structural analysis of the enzyme equipped with either adeninyl-or benzimidazolyl-norcobamide cofactors visualized for the first time structurally different cobamides bound in base-off conformation to the cofactor-binding site of a cobamide-containing enzyme.
Intensive rice-wheat cultivation cycle in Northern belt of India in general and in the State of Punjab in particular results in large volumes of straw and other post-harvest residue annually. The agricultural area, bordering the districts of Nawanshahr and Hoshiarpur, is popularly known as the seleniferous belt of India. The agri-residues, generated in seleniferous region of this state, are observed to contain significantly high concentration of selenium (Se). The present study was aimed to evaluate the Se uptake by different mushroom species: Pleurotus sajorcaju, Pleurotus ostreatus, Pleurotus citrinopileatus, Agaricus bisporus, and Volvariella volvacea, cultivated on Se-rich wheat and paddy straw from the seleniferous region. Wheat (Pleurotus species and A. bisporus) and paddy straw (V. volvacea) was inoculated with the mycelium spawn and left for 7-20 days, depending on the species, to grow. Control mushrooms were grown analogously using the agricultural residues from non-seleniferous area of the State of Punjab. All fruiting bodies were collected and analyzed in triplicate. Se was quantified using inductively coupled plasma sector field mass spectrometry. The Se accumulation was high in all species under study, being the highest in A. bisporus (1396 mu g/g vs. 46.8 mu g/g in controls - dry weight) and V. volvacea (231 mu g/g vs. 3.77 mu g/g - dry weight). The observed biological efficiency and total yield for all mushroom species showed good and unaltered productivity in Se-rich conditions, if compared to the controls. The Se-rich mushrooms can be prospective Se-supplements sourcing and biofortified foods, providing readily bioavailable and accessible Se for the diets deficient of this biologically essential element.
While click chemistry reactions for biopolymer network formation are attractive as the defined reactions may allow good control of the network formation and enable subsequent functionalization, tailoring of gelatin network properties over a wide range of mechanical properties has yet to be shown. Here, it is demonstrated that copper-catalyzed alkyne-azide cycloaddition of alkyne functionalized gelatin with diazides gave hydrogel networks with properties tailorable by the ratio of diazide to gelatin and diazide rigidity. 4,4′-diazido-2,2′-stilbenedisulfonic acid, which has been used as rigid crosslinker, yielded hydrogels with Young’s moduli E of 50–390 kPa and swelling degrees Q of 150–250 vol.%, while the more flexible 1,8-diazidooctane resulted in hydrogels with E = 125–280 kPa and Q = 225–470 vol.%. Storage moduli could be varied by two orders of magnitude (G′ = 100–20,000 Pa). An indirect cytotoxicity test did not show cytotoxic properties. Even when employing 1:1 ratios of alkyne and azide moieties, the hydrogels were shown to contain both, unreacted alkyne groups on the gelatin backbone as well as dangling chains carrying azide groups as shown by reaction with functionalized fluorescein. The free groups, which can be tailored by the employed ratio of the reactants, are accessible for covalent attachment of drugs, as was demonstrated by functionalization with dexamethasone. The sequential network formation and functionalization with click chemistry allows access to multifunctional materials relevant for medical applications.
Sex-specific differences in nutritional requirements may crucially influence the performances of the sexes, which may have implications for sexual reproduction and thus is of great ecological and evolutionary interest. In the freshwater model species Daphnia magna, essential lipid requirements have been extensively studied. Dietary deficiencies in sterols and polyunsaturated fatty acids (PUFA) have been shown to constrain somatic growth and parthenogenetic reproduction of female Daphnia. In contrast, nutrient requirements of male Daphnia have not been studied yet. Supplementation experiments were conducted to investigate differences in sterol (cholesterol) and PUFA (eicosapentaenoic acid, EPA) requirements between female and male D. magna. Thresholds for sterol-limited juvenile growth were higher in females than in males, suggesting that females are more susceptible to dietary sterol deficiencies than males. Sex-specific differences in maximum somatic growth rates were evident primarily in the presence of dietary EPA; females could not exploit their generally higher growth potential in the absence of dietary PUFA. However, the thresholds for EPA-limited growth did not differ between sexes, suggesting that both sexes have similar dietary EPA requirements during juvenile growth. During a life history experiment, the gain in body dry mass was higher in females than in males, irrespective of food treatment. In both sexes, the gain in body dry mass increased significantly upon EPA supplementation, indicating that both sexes benefited from dietary EPA supply also later in life. However, the positive effects of EPA supplementation were most pronounced for female reproduction-related traits (i.e., clutch sizes, egg dry masses, and total dry mass investment in reproduction). The high maternal investment in reproduction resulted in a depletion of nutrients in female somata. In contrast, the comparatively low paternal investment in reproduction allowed for the accumulation of nutrients in male somata. We conclude that males are generally less susceptible to dietary nutrient deficiencies than females, because they can rely more on internal body stores. Our data suggest that the performances of the sexes are differentially influenced by lipid-mediated food quality, which may have consequences for sexual reproduction and thus the production of resting eggs and the maintenance of Daphnia populations.
Non-vascular vegetation has been shown to capture considerable quantities of rainfall, which may affect the hydrological cycle and climate at continental scales. However, direct measurements of rainfall interception by non-vascular vegetation are confined to the local scale, which makes extrapolation to the global effects difficult. Here we use a process-based numerical simulation model to show that non-vascular vegetation contributes substantially to global rainfall interception. Inferred average global water storage capacity including non-vascular vegetation was 2.7 mm, which is consistent with field observations and markedly exceeds the values used in land surface models, which average around 0.4 mm. Consequently, we find that the total evaporation of free water from the forest canopy and soil surface increases by 61% when non-vascular vegetation is included, resulting in a global rainfall interception flux that is 22% of the terrestrial evaporative flux (compared with only 12% for simulations where interception excludes non-vascular vegetation). We thus conclude that non-vascular vegetation is likely to significantly influence global rainfall interception and evaporation with consequences for regional-to continental-scale hydrologic cycling and climate.
Background
Semi-natural plant communities such as field boundaries play an important ecological role in agricultural landscapes, e.g., provision of refuge for plant and other species, food web support or habitat connectivity. To prevent undesired effects of herbicide applications on these communities and their structure, the registration and application are regulated by risk assessment schemes in many industrialized countries. Standardized individual-level greenhouse experiments are conducted on a selection of crop and wild plant species to characterize the effects of herbicide loads potentially reaching off-field areas on non-target plants. Uncertainties regarding the protectiveness of such approaches to risk assessment might be addressed by assessment factors that are often under discussion. As an alternative approach, plant community models can be used to predict potential effects on plant communities of interest based on extrapolation of the individual-level effects measured in the standardized greenhouse experiments. In this study, we analyzed the reliability and adequacy of the plant community model IBC-grass (individual-based plant community model for grasslands) by comparing model predictions with empirically measured effects at the plant community level.
Results
We showed that the effects predicted by the model IBC-grass were in accordance with the empirical data. Based on the species-specific dose responses (calculated from empirical effects in monocultures measured 4 weeks after application), the model was able to realistically predict short-term herbicide impacts on communities when compared to empirical data.
Conclusion
The results presented in this study demonstrate an approach how the current standard greenhouse experiments—measuring herbicide impacts on individual-level—can be coupled with the model IBC-grass to estimate effects on plant community level. In this way, it can be used as a tool in ecological risk assessment.
Artificial illumination increases around the globe and this has been found to affect many groups of organisms and ecosystems. By manipulating nocturnal illumination using one large experimental field site with 24 streetlights and one dark control, we assessed the impact of artificial illumination on slugs over a period of 4 years. The number of slugs, primarily Arionidae, increased strongly in the illuminated site but not on the dark site. There are several nonexclusive explanations for this effect, including reduced predation and increased food quality in the form of carcasses of insects attracted by the light. As slugs play an important role in ecosystems and are also important pest species, the increase of slugs under artificial illumination cannot only affect ecosystem functioning but also have important economic consequences.
The principles of protein-glycan binding are still not well understood on a molecular level. Attempts to link affinity and specificity of glycan recognition to structure suffer from the general lack of model systems for experimental studies and the difficulty to describe the influence of solvent. We have experimentally and computationally addressed energetic contributions of solvent in protein-glycan complex formation in the tailspike protein (TSP) of E. coli bacteriophage HK620. HK620TSP is a 230 kDa native trimer of right-handed, parallel beta-helices that provide extended, rigid binding sites for bacterial cell surface O-antigen polysaccharides. A set of high affinity mutants bound hexa- or pentasaccharide O-antigen fragments with very similar affinities even though hexasaccharides introduce an additional glucose branch into an occluded protein surface cavity. Remarkably different thermodynamic binding signatures were found for different mutants; however, crystal structure analyses indicated that no major oligosaccharide or protein topology changes had occurred upon complex formation. This pointed to a solvent effect. Molecular dynamics simulations using a mobility-based approach revealed an extended network of solvent positions distributed over the entire oligosaccharide binding site. However, free energy calculations showed that a small water network inside the glucose-binding cavity had the most notable influence on the thermodynamic signature. The energy needed to displace water from the glucose binding pocket depended on the amino acid at the entrance, in agreement with the different amounts of enthalpy-entropy compensation found for introducing glucose into the pocket in the different mutants. Studies with small molecule drugs have shown before that a few active water molecules can control protein complex formation. HK620TSP oligosaccharide binding shows that similar fundamental principles also apply for glycans, where a small number of water molecules can dominate the thermodynamic signature in an extended binding site.