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Diurnal and nocturnal behaviour of cheetahs (Acinonyx jubatus) and lions (Panthera leo) in zoos
(2022)
Mammals are constantly exposed to exogenous and endogenous influences that affect their behaviour and daily activity. Light and temperature, as well as anthropogenic factors such as husbandry routines, visitors, and feeding schedules are potential influences on animals in zoological gardens. In order to investigate the effects of some of these factors on animal behaviour, observational studies based on the analyses of activity budgets can be used. In this study, the daily and nightly activity budgets of six lions (Panthera leo) and five cheetahs (Acinonyx jubatus) from four EAZA institutions were investigated. Focused on the influencing factor light and feeding, we analysed these activity budgets descriptively. Behaviour was recorded and analysed during the winter months over an observation period of 14 days and 14 nights using infrared-sensitive cameras. Our results show that lions and cheetahs exhibit activity peaks at crepuscular and feeding times, regardless of husbandry. Thus, lions in captivity shift nocturnal behaviour familiar from the wild to crepuscular and diurnal times. In cheetahs, in contrast, captive and wild individuals show similar 24 h behavioural rhythms. The resting behaviour of both species is more pronounced at night, with cheetahs having a shorter overall sleep duration than lions. This study describes the results of the examined animals and is not predictive. Nevertheless, the results of this study make an important contribution to gaining knowledge about possible factors influencing the behaviour of lions and cheetahs in zoos and offer implications that could be useful for improving husbandry and management.
The success of the increasing use of technology in education is highly dependent on learner acceptance. Although the Technology Acceptance Model (TAM) is dominant in research for surveying acceptance of technology, it does not allow the prediction of a successful first time use of technology. The successful first time use can be determined with the survey of technology affinity, as it corresponds to the expression of certain personality traits of users and is thus detached from the specific technology. Since there are no measurement instruments for the educational sector so far and existing instruments for measuring technology affinity do not meet the specific requirements for use in the educational context (e.g., limited time for questioning), we present the single item Inclusion of Technology Affinity in Self-Scale (ITAS). In study 1 we provide evidence of convergent and discriminant validity within the general population so that a generalization of its applicability is possible. In study 2 we subsequently tested ITAS in the actual target group, the educational sector. The high correlations of the ITAS with the ATI and the control instrument TA-EG (ranging from rs = 0.679 to rs = 0.440) show that ITAS is suitable for use in research. Furthermore, the newly developed instrument convinces with its low complexity, the graphical component, which requires little text understanding and the high time saving. This research thus can contribute to the investigation of technology affinity in the educational sector helping educators to conduct technical activities with their learning group, to predict possible difficulties and adjust their planning accordingly.
Microplastics are small plastic fragments that are widely distributed in marine and terrestrial environments. While the soil ecosystem represents a large reservoir for plastic, research so far has focused mainly on the impact on aquatic ecosystems and there is a lack of information on the potentially adverse effects of microplastics on soil biota. Earthworms are key organisms of the soil ecosystem and are due to their crucial role in soil quality and fertility a suitable and popular model organism in soil ecotoxicology.
Therefore, the aim of this study was to gain insight into the effects of environmentally relevant concentrations of microplastics on the earthworm Eisenia andrei on multiple levels of biological organization after different exposure periods. Earthworms were exposed to two types of microplastics: (1) polystyrene-HBCD and (2) car tire abrasion in natural soil for 2, 7, 14 and 28 d. Acute and chronic toxicity and all subcellular investigations were conducted for all exposure times, avoidance behavior assessed after 48 h and reproduction after 28 d. Subcellular endpoints included enzymatic biomarker responses, namely, carboxylesterase, glutathione peroxidase, acetylcholinesterase, glutathione reductase, glutathione S-transferase and catalase activities, as well as fluorescence-based measurements of oxidative stress-related markers and multixenobiotic resistance activity. Multiple biomarkers showed significant changes in activity, but a recovery of most enzymatic activities could be observed after 28 d. Overall, only minor effects could be observed on a subcellular level, showing that in this exposure scenario with environmentally relevant concentrations based on German pollution levels the threat to soil biota is minimal. However, in areas with higher concentrations of microplastics in the environment, these results can be interpreted as an early warning signal for more adverse effects. In conclusion, these findings provide new insights regarding the ecotoxicological effects of environmentally relevant concentrations of microplastics on soil organisms.
Holocarpic oomycetes infecting freshwater diatoms are obligate endobiotic parasites reported from a wide range of habitats. So far, the taxonomy of and phylogeny of most species remains unresolved, since most have not been reported throughout the past decades and sequence data are available for only the four species, Aphanomycopsis bacillariacearum, Diatomophthora gillii, Ectrogella bacillariacearum, and the recently-discovered species Miracula moenusica. In the current study, a new freshwater diatom parasite resembling Ectrogella bacillariacearum in the sense of Scherffel was discovered from pennate diatoms (Ulnaria acus, Ulnaria ulna) collected from the small stream Einbúalækur on Víkurskarð, North Iceland and investigated for its life cycle and phylogenetic placement. In contrast to the original description, Scherffel reports an achlya-like spore discharge for Ectrogella bacillariacearum. The phylogenetic reconstruction and morphological characterisation in this study revealed that Scherffel’s E. bacillariacearum is largely unrelated to the epitype of the species and is a member of the early-diverging genus Miracula. Consequently, the new species is described as M. einbuarlaekurica in the present study. This adds a second freshwater member to the genus, demonstrating the high ecological adaptability of the genus, which thrives in both freshwater and marine ecosystems.
Despite islands contributing only 6.7% of land surface area, they harbor ~20% of the Earth's biodiversity, but unfortunately also ~50% of the threatened species and 75% of the known extinctions since the European expansion around the globe. Due to their geological and geographic history and characteristics, islands act simultaneously as cradles of evolutionary diversity and museums of formerly widespread lineages—elements that permit islands to achieve an outstanding endemicity. Nevertheless, the majority of these endemic species are inherently vulnerable due to genetic and demographic factors linked with the way islands are colonized. Here, we stress the great variation of islands in their physical geography (area, isolation, altitude, latitude) and history (age, human colonization, human density). We provide examples of some of the most species rich and iconic insular radiations. Next, we analyze the natural vulnerability of the insular biota, linked to genetic and demographic factors as a result of founder events as well as the typically small population sizes of many island species. We note that, whereas evolution toward island syndromes (including size shifts, derived insular woodiness, altered dispersal ability, loss of defense traits, reduction in clutch size) might have improved the ability of species to thrive under natural conditions on islands, it has simultaneously made island biota disproportionately vulnerable to anthropogenic pressures such as habitat loss, overexploitation, invasive species, and climate change. This has led to the documented extinction of at least 800 insular species in the past 500 years, in addition to the many that had already gone extinct following the arrival of first human colonists on islands in prehistoric times. Finally, we summarize current scientific knowledge on the ongoing biodiversity loss on islands worldwide and express our serious concern that the current trajectory will continue to decimate the unique and irreplaceable natural heritage of the world's islands. We conclude that drastic actions are urgently needed to bend the curve of the alarming rates of island biodiversity loss.
Spatial genome organization is tightly controlled by several regulatory mechanisms and is essential for gene expression control. Nuclear receptors are ligand-activated transcription factors that modulate physiological and pathophysiological processes and are primary pharmacological targets. DNA binding of the important loop-forming insulator protein CCCTC-binding factor (CTCF) was modulated by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). We performed CTCF HiChIP assays to produce the first genome-wide dataset of CTCF long-range interactions in 1,25(OH)2D3-treated cells, and to determine whether dynamic changes of spatial chromatin interactions are essential for fine-tuning of nuclear receptor signaling. We detected changes in 3D chromatin organization upon vitamin D receptor (VDR) activation at 3.1% of all observed CTCF interactions. VDR binding was enriched at both differential loop anchors and within differential loops. Differential loops were observed in several putative functional roles including TAD border formation, promoter-enhancer looping, and establishment of VDR-responsive insulated neighborhoods. Vitamin D target genes were enriched in differential loops and at their anchors. Secondary vitamin D effects related to dynamic chromatin domain changes were linked to location of downstream transcription factors in differential loops. CRISPR interference and loop anchor deletion experiments confirmed the functional relevance of nuclear receptor ligand-induced adjustments of the chromatin 3D structure for gene expression regulation.
The archaeal ATP synthase is a multisubunit complex that consists of a catalytic A(1) part and a transmembrane, ion translocation domain A(0). The A(1)A(0) complex from the hyperthermophile Pyrococcus furiosus was isolated. Mass analysis of the complex by laser-induced liquid bead ion desorption (LILBID) indicated a size of 730 +/- 10 kDa. A three-dimensional map was generated by electron microscopy from negatively stained images. The map at a resolution of 2.3 nm shows the A(1) and A(0) domain, connected by a central stalk and two peripheral stalks, one of which is connected to A(0), and both connected to A(1) via prominent knobs. X-ray structures of subunits from related proteins were fitted to the map. On the basis of the fitting and the LILBID analysis, a structural model is presented with the stoichiometry A(3)B(3)CDE(2)FH(2)ac(10).
Climate forecasts show that in many regions the temporal distribution of precipitation events will become less predictable. Root traits may play key roles in dealing with changes in precipitation predictability, but their functional plastic responses, including transgenerational processes, are scarcely known. We investigated root trait plasticity of Papaver rhoeas with respect to higher versus lower intra-seasonal and inter-seasonal precipitation predictability (i.e., the degree of temporal autocorrelation among precipitation events) during a four-year outdoor multi-generation experiment. We first tested how the simulated predictability regimes affected intra-generational plasticity of root traits and allocation strategies of the ancestors, and investigated the selective forces acting on them. Second, we exposed three descendant generations to the same predictability regime experienced by their mothers or to a different one. We then investigated whether high inter-generational predictability causes root trait differentiation, whether transgenerational root plasticity existed and whether it was affected by the different predictability treatments. We found that the number of secondary roots, root biomass and root allocation strategies of ancestors were affected by changes in precipitation predictability, in line with intra-generational plasticity. Lower predictability induced a root response, possibly reflecting a fast-acquisitive strategy that increases water absorbance from shallow soil layers. Ancestors’ root traits were generally under selection, and the predictability treatments did neither affect the strength nor the direction of selection. Transgenerational effects were detected in root biomass and root weight ratio (RWR). In presence of lower predictability, descendants significantly reduced RWR compared to ancestors, leading to an increase in performance. This points to a change in root allocation in order to maintain or increase the descendants’ fitness. Moreover, transgenerational plasticity existed in maximum rooting depth and root biomass, and the less predictable treatment promoted the lowest coefficient of variation among descendants’ treatments in five out of six root traits. This shows that the level of maternal predictability determines the variation in the descendants’ responses, and suggests that lower phenotypic plasticity evolves in less predictable environments. Overall, our findings show that roots are functional plastic traits that rapidly respond to differences in precipitation predictability, and that the plasticity and adaptation of root traits may crucially determine how climate change will affect plants.
Biodiversity patterns of marine crustaceans are still unknown in many locations or might have been overlooked due to our knowledge gaps, despite increasing sampling and data sharing efforts during the last decades. By analysing big data extracted from open portals such as Ocean Biodiversity Information System (OBIS) and Global Biodiversity Information System (GBIF), we aim to revisit the distribution and biodiversity patterns of the highly speciose and abundant Crustacea in the Northwest Pacific (NWP) from shallowest depths to the deep sea. This study focussed on selected benthic and pelagic crustacean (sub) classes and their species richness, sampling effort, and expected species richness (ES50) using equal/sized hexagonal cells, 5° latitudinal bands, 500 m depth intervals were analyzed. Crustacean species richness was highest in the tropical Philippines as well as around the Japanese islands. Pelagic crustacean species richness peaked at 30° latitude and declined beyond that. Benthic taxa; however, depicted high levels of species richness across most of the latitudinal gradient, reaching its highest point at 45° latitude. Due to the prevalence of certain crustacean orders in the deep sea, benthic species richness showed a distribution pattern with two distinct peaks across bathymetric gradients; with highest species richness recorded at shallow-water depths and also at abyssal depths. The most important environmental drivers of benthic and pelagic crustacean species richness were primary productivity (positive correlation) and salinity (negative correlation). Our study provides first insights into biodiversity patterns of the highly diverse Crustacea in the NWP and highlights strong differences between benthic and pelagic taxa. The results presented here could help us to better understand whether benthic or pelagic taxa might respond differently to climate changes in the NWP based on their distinct physiological and biological characteristics. This information is crucial in establishing species management strategies and ecosystem restorations in both shallow water and deep-sea environments.
Mining is one of the major pollution sources worldwide, causing huge disturbances to the environment. Industrial and artisanal mining activities are widespread in Mexico, a major global producer of various metals. This study aimed to assess the ecological impairments resulting from mining activities using aquatic macroinvertebrates assemblages (MA). A multiple co-inertia analysis was applied to determine the relationships between environmental factors, habitat quality, heavy metals, and aquatic macroinvertebrates in 15 study sites in two different seasons (dry and wet) along two rivers running across the Central Plateau of Mexico. The results revealed three contrasting environmental conditions associated with different MAs. High concentrations of heavy metals, nutrients, and salinity limit the presence of several families of seemingly sensitive macroinvertebrates. These factors were found to influence structural changes in MAs, showing that not only mining activities, but also agriculture and presence of villages in the basin, exert adverse effects on macroinvertebrate assemblages. Diversity indices showed that the lowest diversity matched both the most polluted and the most saline rivers. The rivers studied displayed high alkalinity and hardness levels, which can reduce the availability of metals and cause adverse effects on periphyton by inhibiting photosynthesis and damaging MAs. Aquatic biomonitoring in rivers, impacted by mining and other human activities, is critical for detecting the effect of metals and other pollutants to improve management and conservation strategies. This study supports the design of cost-effective and accurate water quality biomonitoring protocols in developing countries.
The ability of wild animals to navigate and survive in complex and dynamic environments depends on their ability to store relevant information and place it in a spatial context. Despite the centrality of spatial memory, and given our increasing ability to observe animal movements in the wild, it is perhaps surprising how difficult it is to demonstrate spatial memory empirically. We present a cognitive analysis of movements of several wolves (Canis lupus) in Finland during a summer period of intensive hunting and den-centered pup-rearing. We tracked several wolves in the field by visiting nearly all GPS locations outside the den, allowing us to identify the species, location and timing of nearly all prey killed. We then developed a model that assigns a spatially explicit value based on memory of predation success and territorial marking. The framework allows for estimation of multiple cognitive parameters, including temporal and spatial scales of memory. For most wolves, fitted memory-based models outperformed null models by 20 to 50% at predicting locations where wolves chose to forage. However, there was a high amount of individual variability among wolves in strength and even direction of responses to experiences. Some wolves tended to return to locations with recent predation success—following a strategy of foraging site fidelity—while others appeared to prefer a site switching strategy. These differences are possibly explained by variability in pack sizes, numbers of pups, and features of the territories. Our analysis points toward concrete strategies for incorporating spatial memory in the study of animal movements while providing nuanced insights into the behavioral strategies of individual predators.
Forest wildflowers bloom earlier as Europe warms: lessons from herbaria and spatial modelling
(2022)
Today plants often flower earlier due to climate warming. Herbarium specimens are excellent witnesses of such long-term changes. However, the magnitude of phenological shifts may vary geographically, and the data are often clustered. Therefore, large-scale analyses of herbarium data are prone to pseudoreplication and geographical biases.
We studied over 6000 herbarium specimens of 20 spring-flowering forest understory herbs from Europe to understand how their phenology had changed during the last century. We estimated phenology trends with or without taking spatial autocorrelation into account.
On average plants now flowered over 6 d earlier than at the beginning of the last century. These changes were strongly associated with warmer spring temperatures. Flowering time advanced 3.6 d per 1°C warming. Spatial modelling showed that, in some parts of Europe, plants flowered earlier or later than expected. Without accounting for this, the estimates of phenological shifts were biased and model fits were poor.
Our study indicates that forest wildflowers in Europe strongly advanced their phenology in response to climate change. However, these phenological shifts differ geographically. This shows that it is crucial to combine the analysis of herbarium data with spatial modelling when testing for long-term phenology trends across large spatial scales.
An important goal is to identify the direct activation domain (AD)-interacting components of the transcriptional machinery within the context of native complexes. Toward this end, we first demonstrate that the multisubunit TFIID, SAGA, mediator, and Swi/Snf coactivator complexes from transcriptionally competent whole-cell yeast extracts were all capable of specifically interacting with the prototypic acidic ADs of Gal4 and VP16. We then used hexahistidine tags as genetically introduced activation domain-localized cross-linking receptors. In combination with immunological reagents against all subunits of TFIID and SAGA, we systematically identified the direct AD-interacting subunits within the AD-TFIID and AD-SAGA coactivator complexes enriched from whole-cell extracts and confirmed these results using purified TFIID and partially purified SAGA. Both ADs directly cross-linked to TBP and to a subset of TFIID and SAGA subunits that carry histone-fold motifs.
In (eco-)toxicological studies the light/dark transition (LDT) test is one of the most frequently used behaviour assays with zebrafish eleutheroembryos. However, study results vary regarding data presentation and analysis and mostly focus on a limited amount of the recorded data. In this study, we investigated whether monitoring two behavioural outcomes (time and distance moved) together with analysing multiple parameters can improve test sensitivity and data interpretation. As a proof of principle 5-day old zebrafish (Danio rerio) eleutheroembryos exposed to either endocrine disruptors (EDs) or acetylcholine esterase (AChE) inhibitors were investigated. We analysed conventional parameters such as mean and sum and implemented additional endpoints such as minimum or maximum distance moved and new parameters assessing the bursting response of eleutheroembryos. Furthermore, changes in eleutheroembryonic behaviour during the moment of the light to dark transition were added. To improve data presentation control-normalised results were displayed in radar charts, enabling the simultaneous presentation of different parameters in relation to each other. This enabled us to identify parameters most relevant to a certain behavioural response. A cut off threshold using control data was applied to identify parameters that were altered in a biological relevant manner. Our approach was able to detect effects on different parameters that remained undetected when analysis was done using conventional bar graphs on - in most cases analysed - averaged, mean distance moved values. By combining the radar charts with additional parameters and by using control-based thresholds, we were able to increase the test sensitivity and promote a deeper understanding of the behaviour response of zebrafish eleutheroembryos in the LDT test and thereby increased its usability for behavioural toxicity studies.
In the past two decades, an increasing body of studies has been published on the intersex phenomenon in separate-sexed crustaceans from marine and freshwater ecosystems. Various causes are being considered that could have an influence on the occurrence of intersex. Besides genetic factors, environmental conditions such as photoperiodicity, temperature, salinity and parasitism, but also environmental pollution with endocrine disrupting chemicals (EDCs) are discussed. As part of a long-term monitoring (2012 – 2020) in north-west Brittany, we recorded the occurrence of intersex in the marine amphipod Echinogammarus marinus. We quantified the intersex incidence at marine and estuarine sites and analyzed the incidence in relation to the endocrine potential of the sediments. Intersex occurred with mean frequencies between 0.87% and 12%. It was striking that the incidence of intersex increased with increasing distance from the sea. Since the highest incidence was observed at the range boundary of this stenohaline species, we assume that intersex is triggered by endocrine potential and increasing stress due to increasing freshwater content − and thus an interplay of different environmental factors.
Nanoplastics affect the inflammatory cytokine release by primary human monocytes and dendritic cells
(2022)
So far, the human health impacts of nano- and microplastics are poorly understood. Thus, we investigated whether nanoplastics exposure induces inflammatory processes in primary human monocytes and monocyte-derived dendritic cells. We exposed these cells in vitro to nanoplastics of different shapes (irregular vs. spherical), sizes (50–310 nm and polydisperse mixtures) and polymer types (polystyrene; polymethyl methacrylate; polyvinyl chloride, PVC) using concentrations of 30–300 particles cell−1. Our results show that irregular PVC particles induce the strongest cytokine release of these nanoplastics. Irregular polystyrene triggered a significantly higher pro-inflammatory response compared to spherical nanoplastics. The contribution of chemicals leaching from the particles was minor. The effects were concentration-dependent but varied markedly between cell donors. We conclude that nanoplastics exposure can provoke human immune cells to secrete cytokines as key initiators of inflammation. This response is specific to certain polymers (PVC) and particle shapes (fragments). Accordingly, nanoplastics cannot be considered one homogenous entity when assessing their health implications and the use of spherical polystyrene nanoplastics may underestimate their inflammatory effects.
Dealing with potential stress in species that have high husbandry requirements, such as elephants, is a challenge for zoos. The objective of the present study was to determine whether positive reinforcement training (PRT) and exposure to a novel object (NOV) for enrichment induced a salivary cortisol response indicative of activation of the hypothalamic–pituitary–adrenal (HPA) axis and which factors determine individual variation in this regard in captive African elephants. We repeatedly sampled the saliva of ten animals (three zoos) for the analysis of cortisol (SACort) before and up to 60 min (in 10–15 min intervals) after the onset of PRT (three repeats) or NOV (nine repeats), which lasted 10 min. There was considerable individual variation in SACort in response to PRT or NOV. Using mixed models, we were able to control these and to reveal that PRT was associated with high SACort before and relatively low SACort after PRT, while NOV induced a moderate SACort increase. The individual differences in SACort were related to age and sex (NOV), while the effects of zoo, handling method (free vs. protected contact) and reproductive and social status were variable. We conclude that positive affective states, such as anticipation or arousal, should be taken into account when interpreting the differences in the SACort responses between PRT and NOV. In addition, understanding the individuality of stress will support management decisions aimed at promoting captive elephant welfare.
Biallelic pathogenic variants in CLPP, encoding mitochondrial matrix peptidase ClpP, cause a rare autosomal recessive condition, Perrault syndrome type 3 (PRLTS3). It is characterized by primary ovarian insufficiency and early sensorineural hearing loss, often associated with progressive neurological deficits. Mouse models showed that accumulations of (i) its main protein interactor, the substrate-selecting AAA+ ATPase ClpX, (ii) mitoribosomes, and (iii) mtDNA nucleoids are the main cellular consequences of ClpP absence. However, the sequence of these events and their validity in human remain unclear. Here, we studied global proteome profiles to define ClpP substrates among mitochondrial ClpX interactors, which accumulated consistently in ClpP-null mouse embryonal fibroblasts and brains. Validation work included novel ClpP-mutant patient fibroblast proteomics. ClpX co-accumulated in mitochondria with the nucleoid component POLDIP2, the mitochondrial poly(A) mRNA granule element LRPPRC, and tRNA processing factor GFM1 (in mouse, also GRSF1). Only in mouse did accumulated ClpX, GFM1, and GRSF1 appear in nuclear fractions. Mitoribosomal accumulation was minor. Consistent accumulations in murine and human fibroblasts also affected multimerizing factors not known as ClpX interactors, namely, OAT, ASS1, ACADVL, STOM, PRDX3, PC, MUT, ALDH2, PMPCB, UQCRC2, and ACADSB, but the impact on downstream metabolites was marginal. Our data demonstrate the primary impact of ClpXP on the assembly of proteins with nucleic acids and show nucleoid enlargement in human as a key consequence.
The maintenance of cellular homeostasis over time is essential to avoid the degeneration of biological systems leading to aging and disease. Several interconnected pathways are active in this kind of quality control. One of them is autophagy, the vacuolar degradation of cellular components. The absence of the sorting nexin PaATG24 (SNX4 in other organisms) has been demonstrated to result in impairments in different types of autophagy and lead to a shortened lifespan. In addition, the growth rate and the size of vacuoles are strongly reduced. Here, we report how an oleic acid diet leads to longevity of the wild type and a PaAtg24 deletion mutant (ΔPaAtg24). The lifespan extension is linked to altered membrane trafficking, which abrogates the observed autophagy defects in ΔPaAtg24 by restoring vacuole size and the proper localization of SNARE protein PaSNC1. In addition, an oleic acid diet leads to an altered use of the mitochondrial respiratory chain: complex I and II are bypassed, leading to reduced reactive oxygen species (ROS) production. Overall, our study uncovers multiple effects of an oleic acid diet, which extends the lifespan of P. anserina and provides perspectives to explain the positive nutritional effects on human aging.
The accumulation of functionally impaired mitochondria is a key event in aging. Previous works with the fungal aging model Podospora anserina demonstrated pronounced age-dependent changes of mitochondrial morphology and ultrastructure, as well as alterations of transcript and protein levels, including individual proteins of the oxidative phosphorylation (OXPHOS). The identified protein changes do not reflect the level of the whole protein complexes as they function in-vivo. In the present study, we investigated in detail the age-dependent changes of assembled mitochondrial protein complexes, using complexome profiling. We observed pronounced age-depen-dent alterations of the OXPHOS complexes, including the loss of mitochondrial respiratory supercomplexes (mtRSCs) and a reduction in the abundance of complex I and complex IV. Additionally, we identified a switch from the standard complex IV-dependent respiration to an alternative respiration during the aging of the P. anserina wild type. Interestingly, we identified proteasome components, as well as endoplasmic reticulum (ER) proteins, for which the recruitment to mitochondria appeared to be increased in the mitochondria of older cultures. Overall, our data demonstrate pronounced age-dependent alterations of the protein complexes involved in energy transduction and suggest the induction of different non-mitochondrial salvage pathways, to counteract the age-dependent mitochondrial impairments which occur during aging.
Mutations in the clk-1 gene result in slower development and increased life span in Caenorhabditis elegans. The Saccharomyces cerevisiae homologue COQ7/CAT5 is essential for several metabolic pathways including ubiquinone biosynthesis, respiration, and gluconeogenic gene activation. We show here that Coq7p/Cat5p is a mitochondrial inner membrane protein directly involved in ubiquinone biosynthesis, and that the defect in gluconeogenic gene activation in coq7/cat5 null mutants is a general consequence of a defect in respiration. These results obtained in the yeast model suggest that the effects on development and life span in C. elegans clk-1 mutants may relate to changes in the amount of ubiquinone, an essential electron transport component and a lipid soluble antioxidant.
Interest is an important factor for successful learning that has been the subject of intensive research for decades. Although interest in nature is of great importance for environmental education, to date there is no valid and reliable measurement tool. Therefore, the purpose of this study was to develop and test a scale for interest in nature, the Nature Interest Scale (NIS). In study 1, nine items were selected based on the three dimensions of the psychological interest construct to represent interest in nature. The factor structure of this new measurement instrument, was tested using confirmatory factor analyses. The results show that the instrument represents the three dimensions of the interest construct well. In study 2 the validity (discriminant and convergent validity) as well as the reliability (internal consistency, composite reliability, test-retest reliability) of the NIS were demonstrated. In study 3, the applicability of the NIS was tested with a different target group, students with learning disabilities. The results of this factor analysis also confirm the factor structure of the scale. Thus, this study provides a valid and reliable measurement tool for individual interest in nature that can be used for future research.
In plants, a family of more than 20 heat stress transcription factors (Hsf) controls the expression of heat stress (hs) genes. There is increasing evidence for the functional diversification between individual members of the Hsf family fulfilling distinct roles in response to various environmental stress conditions and developmental signals. In response to hs, accumulation of both heat stress proteins (Hsp) and Hsfs is induced. In tomato, the physical interaction between the constitutively expressed HsfA1 and the hs-inducible HsfA2 results in synergistic transcriptional activation (superactivation) of hs gene expression. Here, we show that the interaction is strikingly specific and not observed with other class A Hsfs. Hetero-oligomerization of the two-component Hsfs is preferred to homo-oligomerization, and each Hsf in the HsfA1/HsfA2 hetero-oligomeric complex has its characteristic contribution to its function as superactivator. Distinct regions of the oligomerization domain are responsible for specific homo- and hetero-oligomeric interactions leading to the formation of hexameric complexes. The results are summarized in a model of assembly and function of HsfA1/A2 superactivator complexes in hs gene regulation.
Establishing management programs to preserve the benthic communities along the NW Pacific and the Arctic Ocean (AO) requires a deep understanding of the composition of communities and their responses to environmental stressors. In this study, we thus examine patterns of benthic community composition and patterns of species richness along the NW Pacific and Arctic Seas and investigate the most important environmental drivers of those patterns. Overall we found a trend of decreasing species richness toward higher latitudes and deeper waters, peaking in coastal waters of the eastern Philippines. The most dominant taxa along the entire study area were Arthropoda, Mollusca, Cnidaria, Echinodermata, and Annelida. We found that depth, not temperature, was the main driver of community composition along the NW Pacific and neighboring Arctic Seas. Depth has been previously suggested as a factor driving species distribution in benthic fauna. Following depth, the most influential environmental drivers of community composition along the NW Pacific and the Arctic Ocean were silicate, light, and currents. For example, silicate in Hexactinellida, Holothuroidea, and Ophiuroidea; and light in Cephalopoda and Gymnolaemata had the highest correlations with community composition. In this study, based on a combination of new samples and open-access data, we show that different benthic communities might respond differently to future climatic changes based on their taxon-specific biological, physiological, and ecological characteristics. International conservation efforts and habitat preservation should take an adaptive approach and apply measures that take the differences among benthic communities in responding to future climate change into account. This facilitates implementing appropriate conservation management strategies and sustainable utilization of the NW Pacific and Arctic marine ecosystems.
Insects with aquatic life stages can transfer sediment and water pollutants to terrestrial ecosystems, which has been described for metals, polyaromatic hydrocarbons, and polychlorinated chemicals. However, knowledge of the transfer of aquatic micropollutants released by wastewater treatment plants is scarce despite some preliminary studies on their occurrence in riparian spiders. In our study, we address a major analytical gap focusing on the transfer of the micropollutant carbamazepine from the larvae to the adult midges of Chironomus riparius using an optimized QuEChERS extraction method and HPLC–MS/MS applicable to both life stages down to the level of about three individuals. We show that the uptake of carbamazepine by larvae is concentration-dependent and reduces the emergence rate. Importantly, the body burden remained constant in adult midges. Using this information, we estimated the daily exposure of insectivorous tree swallows as terrestrial predators to carbamazepine using the energy demand of the predator and the energy content of the prey. Assuming environmentally relevant water concentrations of about 1 μg/L, the daily dose per kilogram of body weight for tree swallows was estimated to be 0.5 μg/kg/day. At places of high water contamination of 10 μg/L, the exposure may reach 5 μg/kg/day for this micropollutant of medium polarity. Considering body burden changes upon metamorphosis, this study fills the missing link between aquatic contamination and exposure in terrestrial habitats showing that wastewater pollutants can impact birds’ life. Clearly, further analytical methods for biota analysis in both habitats are urgently required to improve risk assessment.
Bird-mediated seed dispersal is crucial for the regeneration and viability of ecosystems, often resulting in complex mutualistic species networks. Yet, how this mutualism drives the evolution of seed dispersing birds is still poorly understood. In the present study we combine whole genome re-sequencing analyses and morphometric data to assess the evolutionary processes that shaped the diversification of the Eurasian nutcracker (Nucifraga), a seed disperser known for its mutualism with pines (Pinus). Our results show that the divergence and phylogeographic patterns of nutcrackers resemble those of other non-mutualistic passerine birds and suggest that their early diversification was shaped by similar biogeographic and climatic processes. The limited variation in foraging traits indicates that local adaptation to pines likely played a minor role. Our study shows that close mutualistic relationships between bird and plant species might not necessarily act as a primary driver of evolution and diversification in resource-specialized birds.
The majority of bacterial membrane-bound NiFe-hydrogenases and formate dehydrogenases have homologous membrane-integral cytochrome b subunits. The prototypic NiFe-hydrogenase of Wolinella succinogenes (HydABC complex) catalyzes H2 oxidation by menaquinone during anaerobic respiration and contains a membrane-integral cytochrome b subunit (HydC) that carries the menaquinone reduction site. Using the crystal structure of the homologous FdnI subunit of Escherichia coli formate dehydrogenase-N as a model, the HydC protein was modified to examine residues thought to be involved in menaquinone binding. Variant HydABC complexes were produced in W. succinogenes, and several conserved HydC residues were identified that are essential for growth with H2 as electron donor and for quinone reduction by H2. Modification of HydC with a C-terminal Strep-tag II enabled one-step purification of the HydABC complex by Strep-Tactin affinity chromatography. The tagged HydC, separated from HydAB by isoelectric focusing, was shown to contain 1.9 mol of heme b/mol of HydC demonstrating that HydC ligates both heme b groups. The four histidine residues predicted as axial heme b ligands were individually replaced by alanine in Strep-tagged HydC. Replacement of either histidine ligand of the heme b group proximal to HydAB led to HydABC preparations that contained only one heme b group. This remaining heme b could be completely reduced by quinone supporting the view that the menaquinone reduction site is located near the distal heme b group. The results indicate that both heme b groups are involved in electron transport and that the architecture of the menaquinone reduction site near the cytoplasmic side of the membrane is similar to that proposed for E. coli FdnI.
In Archaea, bacteria, and eukarya, ATP provides metabolic energy for energy-dependent processes. It is synthesized by enzymes known as A-type or F-type ATP synthase, which are the smallest rotatory engines in nature (Yoshida, M., Muneyuki, E., and Hisabori, T. (2001) Nat. Rev. Mol. Cell. Biol. 2, 669-677; Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312-2315). Here, we report the first projected structure of an intact A(1)A(0) ATP synthase from Methanococcus jannaschii as determined by electron microscopy and single particle analysis at a resolution of 1.8 nm. The enzyme with an overall length of 25.9 nm is organized in an A(1) headpiece (9.4 x 11.5 nm) and a membrane domain, A(0) (6.4 x 10.6 nm), which are linked by a central stalk with a length of approximately 8 nm. A part of the central stalk is surrounded by a horizontal-situated rodlike structure ("collar"), which interacts with a peripheral stalk extending from the A(0) domain up to the top of the A(1) portion, and a second structure connecting the collar structure with A(1). Superposition of the three-dimensional reconstruction and the solution structure of the A(1) complex from Methanosarcina mazei Gö1 have allowed the projections to be interpreted as the A(1) headpiece, a central and the peripheral stalk, and the integral A(0) domain. Finally, the structural organization of the A(1)A(0) complex is discussed in terms of the structural relationship to the related motors, F(1)F(0) ATP synthase and V(1)V(0) ATPases.
Unlike other eukaryotes, plants possess a complex family of heat stress transcription factors (Hsfs) with usually more than 20 members. Among them, Hsfs A4 and A5 form a group distinguished from other Hsfs by structural features of their oligomerization domains and by a number of conserved signature sequences. We show that A4 Hsfs are potent activators of heat stress gene expression, whereas A5 Hsfs act as specific repressors of HsfA4 activity. The oligomerization domain of HsfA5 alone is necessary and sufficient to exert this effect. Due to the high specificity of the oligomerization domains, other class A Hsfs are not affected. Pull-down assay and yeast two-hybrid interaction tests demonstrate that the tendency to form HsfA4/A5 heterooligomers is stronger than the formation of homooligomers. The specificity of interaction between Hsfs A4 and A5 was confirmed by bimolecular fluorescence complementation experiments. The major role of the representatives of the HsfA4/A5 group, which are not involved in the conventional heat stress response, may reside in cell type-specific functions connected with the control of cell death triggered by pathogen infection and/or reactive oxygen species.
Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis.
Background: Although Tic22 is involved in protein import into chloroplasts, the function in cyanobacteria is unknown.
Results: Cyanobacterial Tic22 is required for OM biogenesis, shares structural features with chaperones, and can be substituted by plant Tic22.
Conclusion: Tic22, involved in outer membrane biogenesis, is functionally conserved in cyanobacteria and plants.
Significance: The findings are important for the understanding of periplasmic protein transport.
Proteins of the Omp85 family are conserved in all kingdoms of life. They mediate protein transport across or protein insertion into membranes and reside in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Omp85 proteins contain a C-terminal transmembrane β-barrel and a soluble N terminus with a varying number of polypeptide-transport-associated or POTRA domains. Here we investigate Omp85 from the cyanobacterium Anabaena sp. PCC 7120. The crystallographic three-dimensional structure of the N-terminal region shows three POTRA domains, here named P1 to P3 from the N terminus. Molecular dynamics simulations revealed a hinge between P1 and P2 but in contrast show that P2 and P3 are fixed in orientation. The P2-P3 arrangement is identical as seen for the POTRA domains from proteobacterial FhaC, suggesting this orientation is a conserved feature. Furthermore, we define interfaces for protein-protein interaction in P1 and P2. P3 possesses an extended loop unique to cyanobacteria and plantae, which influences pore properties as shown by deletion. It now becomes clear how variations in structure of individual POTRA domains, as well as the different number of POTRA domains with both rigid and flexible connections make the N termini of Omp85 proteins versatile adaptors for a plentitude of functions.
Precursor protein translocation across the outer chloroplast membrane depends on the action of the Toc complex, containing GTPases as recognizing receptor components. The G domains of the GTPases are known to dimerize. In the dimeric conformation an arginine contacts the phosphate moieties of bound nucleotide in trans. Kinetic studies suggested that the arginine in itself does not act as an arginine finger of a reciprocal GTPase-activating protein (GAP). Here we investigate the specific function of the residue in two GTPase homologues. Arginine to alanine replacement variants have significantly reduced affinities for dimerization compared with wild-type GTPases. The amino acid exchange does not impact on the overall fold and nucleotide binding, as seen in the monomeric x-ray crystallographic structure of the Arabidopsis Toc33 arginine-alanine replacement variant at 2.0A. We probed the catalytic center with the transition state analogue GDP/AlF(x) using NMR and analytical ultracentrifugation. AlF(x) binding depends on the arginine, suggesting the residue can play a role in catalysis despite the non-GAP nature of the homodimer. Two non-exclusive functional models are discussed: 1) the coGAP hypothesis, in which an additional factor activates the GTPase in homodimeric form; and 2) the switch hypothesis, in which a protein, presumably the large Toc159 GTPase, exchanges with one of the homodimeric subunits, leading to activation.
Abstract
Natural plant populations often harbour substantial heritable variation in DNA methylation. However, a thorough understanding of the genetic and environmental drivers of this epigenetic variation requires large-scale and high-resolution data, which currently exist only for a few model species. Here, we studied 207 lines of the annual weed Thlaspi arvense (field pennycress), collected across a large latitudinal gradient in Europe and propagated in a common environment. By screening for variation in DNA sequence and DNA methylation using whole-genome (bisulfite) sequencing, we found significant epigenetic population structure across Europe. Average levels of DNA methylation were strongly context-dependent, with highest DNA methylation in CG context, particularly in transposable elements and in intergenic regions. Residual DNA methylation variation within all contexts was associated with genetic variants, which often co-localized with annotated methylation machinery genes but also with new candidates. Variation in DNA methylation was also significantly associated with climate of origin, with methylation levels being lower in colder regions and in more variable climates. Finally, we used variance decomposition to assess genetic versus environmental associations with differentially methylated regions (DMRs). We found that while genetic variation was generally the strongest predictor of DMRs, the strength of environmental associations increased from CG to CHG and CHH, with climate-of-origin as the strongest predictor in about one third of the CHH DMRs. In summary, our data show that natural epigenetic variation in Thlaspi arvense is significantly associated with both DNA sequence and environment of origin, and that the relative importance of the two factors strongly depends on the sequence context of DNA methylation. T. arvense is an emerging biofuel and winter cover crop; our results may hence be relevant for breeding efforts and agricultural practices in the context of rapidly changing environmental conditions.
Author summary
Variation within species is an important level of biodiversity, and it is key for future adaptation. Besides variation in DNA sequence, plants also harbour heritable variation in DNA methylation, and we want to understand the evolutionary significance of this epigenetic variation, in particular how much of it is under genetic control, and how much is associated with the environment. We addressed these questions in a high-resolution molecular analysis of 207 lines of the common plant field pennycress (Thlaspi arvense), which we collected across Europe, propagated under standardized conditions, and sequenced for their genetic and epigenetic variation. We found large geographic variation in DNA methylation, associated with both DNA sequence and climate of origin. Genetic variation was generally the stronger predictor of DNA methylation variation, but the strength of environmental association varied between different sequence contexts. Climate-of-origin was the strongest predictor in about one third of the differentially methylated regions in the CHH context, which suggests that epigenetic variation may play a role in the short-term climate adaptation of pennycress. As pennycress is currently being domesticated as a new biofuel and winter cover crop, our results may be relevant also for agriculture, particularly in changing environments.
Background: Genome sequencing of all known eukaryotes on Earth promises unprecedented advances in biological sciences and in biodiversity-related applied fields such as environmental management and natural product research. Advances in long-read DNA sequencing make it feasible to generate high-quality genomes for many non–genetic model species. However, long-read sequencing today relies on sizable quantities of high-quality, high molecular weight DNA, which is mostly obtained from fresh tissues. This is a challenge for biodiversity genomics of most metazoan species, which are tiny and need to be preserved immediately after collection. Here we present de novo genomes of 2 species of submillimeter Collembola. For each, we prepared the sequencing library from high molecular weight DNA extracted from a single specimen and using a novel ultra-low input protocol from Pacific Biosciences. This protocol requires a DNA input of only 5 ng, permitted by a whole-genome amplification step.
Results: The 2 assembled genomes have N50 values >5.5 and 8.5 Mb, respectively, and both contain ∼96% of BUSCO genes. Thus, they are highly contiguous and complete. The genomes are supported by an integrative taxonomy approach including placement in a genome-based phylogeny of Collembola and designation of a neotype for 1 of the species. Higher heterozygosity values are recorded in the more mobile species. Both species are devoid of the biosynthetic pathway for β-lactam antibiotics known in several Collembola, confirming the tight correlation of antibiotic synthesis with the species way of life.
Conclusions: It is now possible to generate high-quality genomes from single specimens of minute, field-preserved metazoans, exceeding the minimum contig N50 (1 Mb) required by the Earth BioGenome Project.
Earliella scabrosa is a pantropical species of Polyporales (Basidiomycota) and well-studied concerning its morphology and taxonomy. However, its pantropical intraspecific genetic diversity and population differentiation is unknown. We initiated this study to better understand the genetic variation within E. scabrosa and to test if cryptic species are present. Sequences of three DNA regions, the nuclear ribosomal internal transcribed spacer (ITS), the large subunit ribosomal DNA (LSU), and the translation elongation factor (EF1α) were analysed for 66 samples from 15 geographical locations. We found a high level of genetic diversity (haplotype diversity, Hd = 0.88) and low nucleotide diversity (π = 0.006) across the known geographical range of E. scabrosa based on ITS sequences. The analysis of molecular variance (AMOVA) indicates that the genetic variability is mainly found among geographical populations. The results of Mantel tests confirmed that the genetic distance among populations of E. scabrosa is positively correlated with the geographical distance, which indicates that geographical isolation is an important factor for the observed genetic differentiation. Based on phylogenetic analyses of combined dataset ITS-LSU-EF1α, the low intraspecific divergences (0–0.3%), and the Automated Barcode Gap Discovery (ABGD) analysis, E. scabrosa can be considered as a single species with five different geographical populations. Each population might be in the process of allopatric divergence and in the long-term they may evolve and become distinct species.
RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA-protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process.
Highlights
• Protocol for extracting and analyzing pollen grains from fossil insects
• Individual fossil grains can be analyzed using a combined approach
• Simple and fast TEM embedding and sectioning protocol
• Protocol enables a taxonomic assignment of pollen
Summary
This protocol explains how to extract pollen from fossil insects with subsequent descriptions of pollen treatment. We also describe how to document morphological and ultrastructural features with light-microscopy and electron microscopy. It enables a taxonomic assignment of pollen that can be used to interpret flower-insect interactions, foraging and feeding behavior of insects, and the paleoenvironment. The protocol is limited by the state of the fossil, the presence/absence of pollen on fossil specimens, and the availability of extant pollen for comparison.
Highlights
• Seed size mediates seedling recruitment in tropical forests and pastures.
• Large-seeded species recruited better than small-seeded species in the forest.
• Recruitment of large-seeded species in pastures was limited by surface temperature.
• Large-seeded species should be protected against drought in regenerating pastures.
Abstract
Seedling recruitment is a key process of plant regeneration that often depends on plant functional traits, such as seed size. To optimize forest restoration efforts, we need to better understand how seedling recruitment of different seed sizes varies along environmental gradients with strong variation in abiotic and biotic factors. To understand these interacting effects, we conducted a sowing experiment with different-sized seeds in forests and pastures in the tropical mountains of southern Ecuador. We quantified seedling recruitment in relation to temperature, soil moisture and biotic pressures. We sowed seeds of five tree species of varying seed size at three elevations (1000, 2000 and 3000 m a.s.l.) in primary forest and pastures. We tested (1) how habitat type influences the recruitment of seedlings belonging to three small- and two large-seeded species, and (2) how abiotic and biotic factors limit seedling recruitment of species with different seed sizes. We found that seedlings of the two large-seeded species recruited better than seedlings of the three small-seeded species, but only in the forest habitat. Seedling recruitment of large seeds was primarily limited by high surface temperature, which explains lower recruitment of large seeds in pastures compared to forests. Our study shows that seed size can be a key trait mediating variability in seedling recruitment in tropical ecosystems. We conclude that restoration measures should aim to mitigate extreme temperatures in tropical pastures to aid the natural regeneration of large-seeded tree species.
Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D. M. (1990) Biochemistry 29, 4031–4037) is postulated to proceed in 2 steps: partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two, and the first three transmembrane (TM) segments, as well as a construct comprising TM1–TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin, A., Cohen, L. S., Arshava, B., Tantry, S., Becker, J. M., Zerbe, O., and Naider, F. (2009) Biophys. J. 96, 3187–3196), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123, and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior.
Lantibiotics are peptide-derived antibiotics that inhibit the growth of Gram-positive bacteria via interactions with lipid II and lipid II-dependent pore formation in the bacterial membrane. Due to their general mode of action the Gram-positive producer strains need to express immunity proteins (LanI proteins) for protection against their own lantibiotics. Little is known about the immunity mechanism protecting the producer strain against its own lantibiotic on the molecular level. So far, no structures have been reported for any LanI protein. We solved the structure of SpaI, a LanI protein from the subtilin producing strain Bacillus subtilis ATCC 6633. SpaI is a 16.8-kDa lipoprotein that is attached to the outside of the cytoplasmic membrane via a covalent diacylglycerol anchor. SpaI together with the ABC transporter SpaFEG protects the B. subtilis membrane from subtilin insertion. The solution-NMR structure of a 15-kDa biologically active C-terminal fragment reveals a novel fold. We also demonstrate that the first 20 N-terminal amino acids not present in this C-terminal fragment are unstructured in solution and are required for interactions with lipid membranes. Additionally, growth tests reveal that these 20 N-terminal residues are important for the immunity mediated by SpaI but most likely are not part of a possible subtilin binding site. Our findings are the first step on the way of understanding the immunity mechanism of B. subtilis in particular and of other lantibiotic producing strains in general.
The solution structure of the lantibiotic immunity protein NisI and its interactions with nisin
(2015)
Many Gram-positive bacteria produce lantibiotics, genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. To protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from Bacillus subtilis. To understand the structural basis for LanI-mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI-mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity.
Previous studies towards reduced oxygen availability have mostly focused on changes in total mRNA expression, neglecting underlying transcriptional and post-transcriptional events. Therefore, we generated a comprehensive overview of hypoxia-induced changes in total mRNA expression, global de novo transcription, and mRNA stability in monocytic THP-1 cells. Since hypoxic episodes often persist for prolonged periods, we further compared the adaptation to acute and chronic hypoxia. While total mRNA changes correlated well with enhanced transcription during short-term hypoxia, mRNA destabilization gained importance under chronic conditions. Reduced mRNA stability not only added to a compensatory attenuation of immune responses, but also, most notably, to the reduction in nuclear-encoded mRNAs associated with various mitochondrial functions. These changes may prevent the futile production of new mitochondria under conditions where mitochondria cannot exert their full metabolic function and are indeed actively removed by mitophagy. The post-transcriptional mode of regulation might further allow for the rapid recovery of mitochondrial capacities upon reoxygenation. Our results provide a comprehensive resource of functional mRNA expression dynamics and underlying transcriptional and post-transcriptional regulatory principles during the adaptation to hypoxia. Furthermore, we uncover that RNA stability regulation controls mitochondrial functions in the context of hypoxia.
Background: The causative agent of Chagas disease, Trypanosoma cruzi, and its nonpathogenic relative, Trypanosoma rangeli, are transmitted by haematophagous triatomines and undergo a crucial ontogenetic phase in the insect’s intestine. In the process, the parasites interfere with the host immune system as well as the microbiome present in the digestive tract potentially establishing an environment advantageous for development. However, the coherent interactions between host, pathogen and microbiota have not yet been elucidated in detail. We applied a metagenome shotgun sequencing approach to study the alterations in the microbiota of Rhodnius prolixus, a major vector of Chagas disease, after exposure to T. cruzi and T. rangeli focusing also on the functional capacities present in the intestinal microbiome of the insect.
Results: The intestinal microbiota of R. prolixus was dominated by the bacterial orders Enterobacterales, Corynebacteriales, Lactobacillales, Clostridiales and Chlamydiales, whereas the latter conceivably originated from the blood used for pathogen exposure. The anterior and posterior midgut samples of the exposed insects showed a reduced overall number of organisms compared to the control group. However, we also found enriched bacterial groups after exposure to T. cruzi as well as T rangeli. While the relative abundance of Enterobacterales and Corynebacteriales decreased considerably, the Lactobacillales, mainly composed of the genus Enterococcus, developed as the most abundant taxonomic group. This applies in particular to vectors challenged with T. rangeli and at early timepoints after exposure to vectors challenged with T. cruzi. Furthermore, we were able to reconstruct four metagenome-assembled genomes from the intestinal samples and elucidate their unique metabolic functionalities within the triatomine microbiome, including the genome of a recently described insect symbiont, Candidatus Symbiopectobacterium, and the secondary metabolites producing bacteria Kocuria spp.
Conclusions: Our results facilitate a deeper understanding of the processes that take place in the intestinal tract of triatomine vectors during colonisation by trypanosomal parasites and highlight the influential aspects of pathogen-microbiota interactions. In particular, the mostly unexplored metabolic capacities of the insect vector’s microbiome are clearer, underlining its role in the transmission of Chagas disease.
Understanding how species relate mechanistically to their environment via traits is a central goal in ecology. Many macroecological rules were found for macroorganisms, however, whether they can explain microorganismal macroecological patterns still requires investigation. Further, whether macroecological rules are also applicable in microclimates is largely unexplored. Here we use fruit body-forming fungi to understand both aspects better. A recent study showed first evidence for the thermal-melanism hypothesis (Bogert’s rule) in fruit body-forming fungi and relied on a continental spatial scale with large grid size. At large spatial extent and grid sizes, other factors like dispersal limitation or local microclimatic variability might influence observed patterns besides the rule of interest. Therefore, we test fungal assemblage fruit body color lightness along a local elevational gradient (mean annual temperature gradient of 7°C) while considering the vegetation cover as a proxy for local variability in microclimate. Using multivariate linear modeling, we found that fungal fruiting assemblages are significantly darker at lower mean annual temperatures supporting the thermal-melanism hypothesis. Further, we found a non-significant trend of assemblage color lightness with vegetation cover. Our results support Bogert’s rule for microorganisms with macroclimate, which was also found for macroorganisms.
Out-of-school laboratories, also called student labs, are an advantageous opportunity to teach biological subjects. Particularly in the case of complex fields such as neurobiology, student labs offer the opportunity to learn about difficult topics in a practical way. Due to numerous advantages, digital student labs are becoming increasingly popular nowadays. In this study, we investigated the effect of an electrophysiological setup for a virtual experiment with and without hands-on elements on participant motivation and technology acceptance. For this purpose, 235 students were questioned during a student laboratory day. The surveyed students showed high motivation and technology acceptance for the virtual experiment. In the comparison, the electrophysiological setup with hands-on elements performs better in the intrinsic components than the setup without hands-on elements: Thus, the hands-on approach is rated as more interesting and the perceived enjoyment scores higher. Nevertheless, both experimental groups show high values, so that the results of the study support the positive influence of digital laboratory as well as a positive influence of hands-on elements.
Highlights
• PUR, PVC and PLA microplastics affect life-history parameters of Daphnia magna.
• Natural kaolin particles are less toxic than microplastics.
• Microplastic toxicity is material-specific, e.g. PVC is most toxic on reproduction.
• In case of PVC, plastic chemicals are the main driver of microplastic toxicity.
• PLA bioplastics are similarly toxic as conventional plastics.
Abstract
Given the ubiquitous presence of microplastics in aquatic environments, an evaluation of their toxicity is essential. Microplastics are a heterogeneous set of materials that differ not only in particle properties, like size and shape, but also in chemical composition, including polymers, additives and side products. Thus far, it remains unknown whether the plastic chemicals or the particle itself are the driving factor for microplastic toxicity. To address this question, we exposed Daphnia magna for 21 days to irregular polyvinyl chloride (PVC), polyurethane (PUR) and polylactic acid (PLA) microplastics as well as to natural kaolin particles in high concentrations (10, 50, 100, 500 mg/L, ≤ 59 μm) and different exposure scenarios, including microplastics and microplastics without extractable chemicals as well as the extracted and migrating chemicals alone. All three microplastic types negatively affected the life-history of D. magna. However, this toxicity depended on the endpoint and the material. While PVC had the largest effect on reproduction, PLA reduced survival most effectively. The latter indicates that bio-based and biodegradable plastics can be as toxic as their conventional counterparts. The natural particle kaolin was less toxic than microplastics when comparing numerical concentrations. Importantly, the contribution of plastic chemicals to the toxicity was also plastic type-specific. While we can attribute effects of PVC to the chemicals used in the material, effects of PUR and PLA plastics were induced by the mere particle. Our study demonstrates that plastic chemicals can drive microplastic toxicity. This highlights the importance of considering the individual chemical composition of plastics when assessing their environmental risks. Our results suggest that less studied polymer types, like PVC and PUR, as well as bioplastics are of particular toxicological relevance and should get a higher priority in ecotoxicological studies.
Highlights
• Three ecological groups were identified based on distributional patterns.
• Old assessments were confirmed with the latest occurrence data.
• For each group, we derived different population trends in times of global change.
• Global change elevates importance of vector-borne diseases.
• Our results serve as base for effective Simuliidae monitoring.
Abstract
The black fly genus Simulium includes medically and ecologically important species, characterized by a wide variation of ecological niches largely determining their distributional patterns. In a rapidly changing environment, species-specific niche characteristics determine whether a species benefits or not. With aquatic egg, larval and pupal stages followed by a terrestrial adult phase, their spatial arrangements depend upon the interplay of aquatic conditions and climatic-landscape parameters in the terrestrial realm. The aim of this study was to enhance the understanding of the distributional patterns among Simulium species and their ecological drivers. In an ecological niche modelling approach, we focused on 12 common black fly species with different ecological requirements. Our modelling was based on available distribution data along with five stream variables describing the climatic, land-cover, and topographic conditions of river catchments. The modelled freshwater habitat suitability was spatially interpolated to derive an estimate of the adult black flies' probability of occurrence. Based on similarities in the spatial patterns of modelled habitat suitability we were able to identify three biogeographical groups, which allows us to confirm old assessments with current occurrence data: (A) montane species, (B) broad range species and (C) lowland species. The five veterinary and human medical relevant species Simulium equinum, S. erythrocephalum, S. lineatum, S. ornatum and S. reptans are mainly classified in the lowland species group. In the course of climatic changes, it is expected that biocoenosis will slightly shift towards upstream regions, so that the lowland group will presumably emerge as the winner. This is mainly explained by wider ecological niches, including a higher temperature tolerance and tolerance to various pollutants. In conclusion, these findings have significant implications for human and animal health. As exposure to relevant Simulium species increases, it becomes imperative to remain vigilant, particularly in investigating the potential transmission of pathogens.
Calcium (Ca2+) elevation is an essential secondary messenger in many cellular processes, including disease progression and adaptation to external stimuli, e.g., gravitational load. Therefore, mapping and quantifying Ca2+ signaling with a high spatiotemporal resolution is a key challenge. However, particularly on microgravity platforms, experiment time is limited, allowing only a small number of replicates. Furthermore, experiment hardware is exposed to changes in gravity levels, causing experimental artifacts unless appropriately controlled. We introduce a new experimental setup based on the fluorescent Ca2+ reporter CaMPARI2, onboard LED arrays, and subsequent microscopic analysis on the ground. This setup allows for higher throughput and accuracy due to its retrograde nature. The excellent performance of CaMPARI2 was demonstrated with human chondrocytes during the 75th ESA parabolic flight campaign. CaMPARI2 revealed a strong Ca2+ response triggered by histamine but was not affected by the alternating gravitational load of a parabolic flight.
In Saccharomyces cerevisiae, the NDI1 gene encodes a mitochondrial NADH dehydrogenase, the catalytic side of which projects to the matrix side of the inner mitochondrial membrane. In addition to this NADH dehydrogenase, S. cerevisiae exhibits another mitochondrial NADH-dehydrogenase activity, which oxidizes NADH at the cytosolic side of the inner membrane. To investigate whether open reading frames YMR145c/NDE1 and YDL 085w/NDE2, which exhibit sequence similarity with NDI1, encode the latter enzyme, NADH-dependent mitochondrial respiration was assayed in wild-type S. cerevisiae and nde deletion mutants. Mitochondria were isolated from aerobic, glucose-limited chemostat cultures grown at a dilution rate (D) of 0. 10 h-1, in which reoxidation of cytosolic NADH by wild-type cells occurred exclusively by respiration. Compared with the wild type, rates of mitochondrial NADH oxidation were about 3-fold reduced in an nde1Delta mutant and unaffected in an nde2Delta mutant. NADH-dependent mitochondrial respiration was completely abolished in an nde1Delta nde2Delta double mutant. Mitochondrial respiration of substrates other than NADH was not affected in nde mutants. In shake flasks, an nde1Delta nde2Delta mutant exhibited reduced specific growth rates on ethanol and galactose but not on glucose. Glucose metabolism in aerobic, glucose-limited chemostat cultures (D = 0.10 h-1) of an nde1Delta nde2Delta mutant was essentially respiratory. Apparently, under these conditions alternative systems for reoxidation of cytosolic NADH could replace the role of Nde1p and Nde2p in S. cerevisiae.
In the diazotroph Klebsiella pneumoniae the flavoprotein NifL inhibits the activity of the nif-specific transcriptional activator NifA in response to molecular oxygen and combined nitrogen. Sequestration of reduced NifL to the cytoplasmic membrane under anaerobic and nitrogen-limited conditions impairs inhibition of cytoplasmic NifA by NifL. To analyze whether NifL is reduced by electrons directly derived from the reduced menaquinone pool, we studied NifL reduction using artificial membrane systems containing purified components of the anaerobic respiratory chain of Wolinella succinogenes. In this in vitro assay using proteoliposomes containing purified formate dehydrogenase and purified menaquinone (MK6) or 8-methylmenaquinone (MMK6) from W. succinogenes, reduction of purified NifL was achieved by formate oxidation. Furthermore, the respective reduction rates, which were determined using equal amounts of NifL, have been shown to be directly dependent on the concentration of both formate dehydrogenase and menaquinones incorporated into the proteoliposomes, demonstrating a direct electron transfer from menaquinone to NifL. When purified hydrogenase and MK6 from W. succinogenes were inserted into the proteoliposomes, NifL was reduced with nearly the same rate by hydrogen oxidation. In both cases reduced NifL was found to be highly associated to the proteoliposomes, which is in accordance with our previous findings in vivo. On the bases of these experiments, we propose that the redox state of the menaquinone pool is the redox signal for nif regulation in K. pneumoniae by directly transferring electrons onto NifL under anaerobic conditions.
Arsenic trioxide is a toxic metalloid and carcinogen that is also used as an anticancer drug, and for this reason it is important to identify the routes of arsenite uptake by cells. In this study the ability of hexose transporters to facilitate arsenic trioxide uptake in Saccharomyces cerevisiae was examined. In the absence of glucose, strains with disruption of the arsenite efflux gene ACR3 accumulated high levels of (73)As(OH)(3). The addition of glucose inhibited uptake by approximately 80%. Disruption of FPS1, the aquaglyceroporin gene, reduced glucose-independent uptake by only about 25%, and the residual uptake was nearly completely inhibited by hexoses, including glucose, galactose, mannose, and fructose but not pentoses or disaccharides. A strain lacking FPS1, ACR3, and all genes for hexose permeases except for HXT3, HXT6, HXT7, and GAL2 exhibited hexose-inhibitable (73)As(OH)(3) uptake, whereas a strain lacking all 18 hexose transport-related genes (HXT1 to HXT17 and GAL2), FPS1 and ACR3, exhibited <10% of wild type (73)As(OH)(3) transport. When HXT1, HXT3, HXT4, HXT5, HXT7, or HXT9 was individually expressed in that strain, hexose-inhibitable (73)As(OH)(3) uptake was restored. In addition, the transport of [(14)C]glucose was inhibited by As(OH)(3). These results clearly demonstrate that hexose permeases catalyze the majority of the transport of the trivalent metalloid arsenic trioxide.
Secretins are a family of large bacterial outer membrane protein complexes mediating the transport of complex structures, such as type IV pili, DNA and filamentous phage, or various proteins, such as extracellular enzymes and pathogenicity determinants. PilQ of the thermophilic bacterium Thermus thermophilus HB27 is a member of the secretin family required for natural transformation. Here we report the isolation, structural, and functional analyses of a unique PilQ from T. thermophilus. Native PAGE, gel filtration chromatography, and electrophoretic mobility shift analyses indicated that PilQ forms a macromolecular homopolymeric complex that binds dsDNA. Electron microscopy showed that the PilQ complex is 15 nm wide and 34 nm long and consists of an extraordinary stable "cone" and "cup" structure and five ring structures with a large central channel. Moreover, the electron microscopic images together with secondary structure analyses combined with structural data of type II protein secretion system and type III protein secretion system secretins suggest that the individual rings are formed by conserved domains of alternating α-helices and β-sheets. The unprecedented length of the PilQ complex correlated well with the distance between the inner and outer membrane of T. thermophilus. Indeed, PilQ was found immunologically in both membranes, indicating that the PilQ complex spans the entire cell periphery of T. thermophilus. This is consistent with the hypothesis that PilQ accommodates a PilA4 comprising pseudopilus mediating DNA transport across the outer membrane and periplasmic space in a single-step process.
DNA translocators of natural transformation systems are complex systems critical for the uptake of free DNA and provide a powerful mechanism for adaptation to changing environmental conditions. In natural transformation machineries, outer membrane secretins are suggested to form a multimeric pore for the uptake of external DNA. Recently, we reported on a novel structure of the DNA translocator secretin complex, PilQ, in Thermus thermophilus HB27 comprising a stable cone and cup structure and six ring structures with a large central channel. Here, we report on structural and functional analyses of a set of N-terminal PilQ deletion derivatives in T. thermophilus HB27. We identified 136 N-terminal residues exhibiting an unusual ααβαββα fold as a ring-building domain. Deletion of this domain had a dramatic effect on twitching motility, adhesion, and piliation but did not abolish natural transformation. These findings provide clear evidence that the pilus structures of T. thermophilus are not essential for natural transformation. The truncated complex was not affected in inner and outer membrane association, indicating that the 136 N-terminal residues are not essential for membrane targeting. Analyses of complex formation of the truncated PilQ monomers revealed that the region downstream of residue 136 is required for multimerization, and the region downstream of residue 207 is essential for monomer stability. Possible implications of our findings for the mechanism of DNA uptake are discussed.
Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic beta-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (Kd = 45 nm, DeltaH = 15.6 kcal/mol, TDeltaS = 25.7 kcal/mol, DeltaCp = -354 cal mol(-1) K(-1)). With k(off) = 1.3 x 10(-2) s(-1), the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs.
The traffic AAA-ATPase PilF is essential for pilus biogenesis and natural transformation of Thermus thermophilus HB27. Recently, we showed that PilF forms hexameric complexes containing six zinc atoms coordinated by conserved tetracysteine motifs. Here we report that zinc binding is essential for complex stability. However, zinc binding is neither required for pilus biogenesis nor natural transformation. A number of the mutants did not exhibit any pili during growth at 64 °C but still were transformable. This leads to the conclusion that type 4 pili and the DNA translocator are distinct systems. At lower growth temperatures (55 °C) the zinc-depleted multiple cysteine mutants were hyperpiliated but defective in pilus-mediated twitching motility. This provides evidence that zinc binding is essential for the role of PilF in pilus dynamics. Moreover, we found that zinc binding is essential for complex stability but dispensable for ATPase activity. In contrast to many polymerization ATPases from mesophilic bacteria, ATP binding is not required for PilF complex formation; however, it significantly increases complex stability. These data suggest that zinc and ATP binding increase complex stability that is important for functionality of PilF under extreme environmental conditions.
Background: In the face of ongoing climate warming, vector-borne diseases are expected to increase in Europe, including tick-borne diseases (TBD). The most abundant tick-borne diseases in Germany are Tick-Borne Encephalitis (TBE) and Lyme Borreliosis (LB), with Ixodes ricinus as the main vector.
Methods: In this study, we display and compare the spatial and temporal patterns of reported cases of human TBE and LB in relation to some associated factors. The comparison may help with the interpretation of observed spatial and temporal patterns.
Results: The spatial patterns of reported TBE cases show a clear and consistent pattern over the years, with many cases in the south and only few and isolated cases in the north of Germany. The identification of spatial patterns of LB disease cases is more difficult due to the different reporting practices in the individual federal states. Temporal patterns strongly fluctuate between years, and are relatively synchronized between both diseases, suggesting common driving factors. Based on our results we found no evidence that weather conditions affect the prevalence of both diseases. Both diseases show a gender bias with LB bing more commonly diagnosed in females, contrary to TBE being more commonly diagnosed in males.
Conclusion: For a further investigation of of the underlying driving factors and their interrelations, longer time series as well as standardised reporting and surveillance system would be required.
The hydrophobic thickness of membranes, which is manly defined by fatty acids, influences the packing of transmembrane domains of proteins and thus can modulate the activity of these proteins. We analyzed the dynamics of the dimerization of Glycophorin A (GpA) by molecular dynamics simulations to describe the fatty acid dependence of the transmembrane region assembly. GpA represents a well-established model for dimerization of single transmembrane helices containing a GxxxG motif in vitro and in silico. We performed simulations of the dynamics of the NMR-derived dimer as well as self-assembly simulations of monomers in membranes composed of different fatty acid chains and monitored the formed interfaces and their transitions. The observed dimeric interfaces, which also include the one known from NMR, are highly dynamic and converted into each other. The frequency of interface formation and the preferred transitions between interfaces similar to the interface observed by NMR analysis strongly depend on the fatty acid used to build the membrane. Molecular dynamic simulations after adaptation of the helix topology parameters to better represent NMR derived structures of single transmembrane helices yielded an enhanced occurrence of the interface determined by NMR in molecular dynamics simulations. Taken together we give insights into the influence of fatty acids and helix conformation on the dynamics of the transmembrane domain of GpA.
ω-Azido fatty acids as probes to detect fatty acid biosynthesis, degradation, and modification
(2014)
FAs play a central role in the metabolism of almost all known cellular life forms. Although GC-MS is regarded as a standard method for FA analysis, other methods, such as HPLC/MS, are nowadays widespread but are rarely applied to FA analysis. Here we present azido-FAs as probes that can be used to study FA biosynthesis (elongation, desaturation) or degradation (β-oxidation) upon their uptake, activation, and metabolic conversion. These azido-FAs are readily accessible by chemical synthesis and their matization with high sensitivity by HPLC/MS, contributing a powerful tool to FA analysis, and hence, lipid analysis in general.
A first model of the three-dimensional structure of the photosynthetic reaction center of the mutant T1 (SerL 223 → Ala, ArgL 217 → His) from Rhodopseudomonas viridis, resistant toward the triazine herbicide terbutryn (2-methylthio-4-ethylamino-6-f-butylamino-5-triazine), has been developed from X-ray data measured to a resolution of 2.5 Å. The secondary quinone, QB, which in T1 binds better than in the wild type, is present in the crystals. Both substituted residues are clearly visible in the difference fourier map. The replacement of these two residues in the QB site causes only minor changes in the overall structure of the protein.
Young poplar cuttings (Populus nigra L. cv. Loenen and P. maximowiczii Henry x P. nigra L. cv. Rochester) were exposed for six weeks in open-top chambers to realistic concentrations of pollutant mixtures: 1) control; 2) SO2/NOx; 3)O3/ NOx and 4)SO2/O3/NOx. In this sequence of fumigation variants, the degree of influence of the various parameters of the nitrogen metabolism and of premature leaf drop increased very frequently compared to the control plants, P. nigra L. proving to be the more sensitive species.
The elevated Kjeldahl nitrogen content of the fumigated leaves was accompanied by either an increase in free amino acids or in total protein or, in the case of particularly large rises (SO2/O3/NOx variants), by increases in both substance groups. Proteolytic processes as a cause of the elevated content of free amino acids could be excluded to a large extent. A diminished de novo synthesis of proteins obviously led to a shift in the amino acid/protein relationship. In the younger fumigated leaves, the total concentration of free amino acids exceeded the values of the older leaves. The elevated amino acid content of the fumigated leaves was produced to a high degree by the glycolate pathway and the Krebs cycle. The increased turnover of the carbon skeletons was connected with a drastic starch degradation, especially in the older leaves.
The interaction of the amino acid and carbohydrate metabolisms is probably an important regulator in the promotion of rapid growth of young leaves in order to compensate premature leaf loss.
Membrane-embedded β-barrel proteins are found in the outer membranes (OM) of Gram-negative bacteria, mitochondria and chloroplasts. In eukaryotic cells, precursors of these proteins are synthesized in the cytosol and have to be sorted to their corresponding organelle. Currently, the signal that ensures their specific targeting to either mitochondria or chloroplasts is ill-defined. To address this issue, we studied targeting of the chloroplast β-barrel proteins Oep37 and Oep24. We found that both proteins can be integrated in vitro into isolated plant mitochondria. Furthermore, upon their expression in yeast cells Oep37 and Oep24 were exclusively located in the mitochondrial OM. Oep37 partially complemented the growth phenotype of yeast cells lacking Porin, the general metabolite transporter of this membrane. Similarly to mitochondrial β-barrel proteins, Oep37 and Oep24 expressed in yeast cells were assembled into the mitochondrial OM in a pathway dependent on the TOM and TOB complexes. Taken together, this study demonstrates that the central mitochondrial components that mediate the import of yeast β-barrel proteins can deal with precursors of chloroplast β-barrel proteins. This implies that the mitochondrial import machinery does not recognize signals that are unique to mitochondrial β-barrel proteins. Our results further suggest that dedicated targeting factors had to evolve in plant cells to prevent mis-sorting of chloroplast β-barrel proteins to mitochondria.
A non-radioactive cell-free assay was developed to quantitatively determine inhibition of plant-type phytoene desaturase by bleaching herbicides. An active desaturase was prepared from an appropriately cloned E. coli transformant. Another E. coli transformant was used to produce the required phytoene. Phytofluene and t-carotene, the products of the desaturase reaction, were either determined by HPLC or optical absorption spectra. Enzyme kinetics and inhibition data for the bleaching tetrazole herbicide WL110547 are presented as an example.
The accumulation and distribution of characteristic secondary products in the different organs of an Aloe plant (A. succotrina Lam.) were studied by high performance liquid chromatography for the first time. In the leaves of the Aloe plant, only anthrone-C-glycosyls of the 7-hydroxyaloin type and, for the first time in plant material, the free anthraquinone 7-hydroxyaloeemodin were found. In contrast to previous reports on the distribution of secondary products in Aloe plants, anthrone-C-glycosyls were also detected in flowers, bracts and the inflorescence axis of the species examined. Aloesaponol I, a tetrahydroanthracene aglycone, was only present in the underground organs and in the stem. The 2-alkylchromone-C-glucosyl aloeresin B showed no specific occurrence as it was found in every type of organ. Based on these results and the findings of recent studies on Aloe roots and flowers, a distribution scheme of polyketide types in the Aloe plant was established. It suggests a separate and independent anthranoid metabolism for underground Aloe organs and stem on the one hand, and for leaves and inflorescence organs on the other hand. In the latter structures anthranoid metabolism seems to be additionally compartmentalized as the anthranoid pro files of inflorescence organs and leaves differ in two points relevant to anthranoid biosynthe sis: firstly, the occurrence of anthrone aglycones and secondly, the individual content of corresponding anthrone-C-glucosyl diastereomers.
The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent nonaethylene glycol lauryl ether and then reconstituted in spherical egg phosphatidylcholine bilayers as described earlier (U. Scheuring, K. Kollewe, W. Haase, and D. Schubert, J. Membrane Biol. 90, 123-135 (1986)). The resulting paucilamellar proteoliposom es of average diameter 70 nm were transformed into smaller vesicles by French press treatment and fractionated according to size by gel filtration. The smallest protein-containing liposomes obtained had diameters around 32 nm; still smaller vesicles were free of protein. All proteoliposome samples studied showed a rapid sulfate efflux which was sensitive to specific inhibitors of band 3-mediated anion exchange. In addition, the orientation of the transport protein in the vesicle membranes was found to be “right-side-out” in all samples. This suggests that the orientation of the protein in the vesicle membranes is dictated by the shape of the protein’s intramembrane domain and that this domain has the form of a truncated cone or pyramid.
Physiological conditions which lead to changes in total carotenoid content in tomato plantlets were identified. Carotenoid levels were found to increase after the onset of a dark period during a normal 24h cycle. This rapid initial increase is followed by a steady decrease in carotenoid content throughout the night. A decrease in the expression of several carotenogenic genes, namely pds, zds (carotenoid desaturases) and ptox (plastid terminal oxidase), was observed following the removal of the light (when carotenoid content is at its highest). An increase in gene expression was observed before the return to light for pds and zds (when carotenoid levels were at their lowest), or following the return to light for ptox. The phytoene desaturation inhibitor norflurazon leads to a decrease coloured carotenoid content and, in the light, this correlated with pds and zds gene induction. In the dark, norflurazon treatment led to only a weak decrease in carotenoid content and only a small increase in pds and zds gene expression. The striking absence of phytoene accumulation under norflurazon treatment in the dark suggests a down-regulation of carotenoid formation in darkness. However, prolonged dark conditions, or treatment with photosynthetic inhibitors, surprisingly led to higher carotenoid levels, which correlated with decreased expression of most examined genes. In addition to light, which acts in a complex way on carotenoid accumulation and gene expression, our results are best explained by a regulatory effect of carotenoid levels on the expression of several biosynthetic genes. In addition, monitoring of protein amounts for phytoene desaturase and plastid terminal oxidase (which sometimes do not correlate with gene expression) indicate an even more complex regulatory pattern.
Pheromonal synergism and inhibition in P. flammea was further studied through electrophysiological and field trapping tests. Z11-tetradecenyl acetate and Z11-hexa - decenyl acetate, each acting upon a separate type of male sensory cell, were equally effective in synergizing attraction responses to the major pheromone component, Z9-tetradecenyl acetate. Addition of Z7-dodecenyl acetate to these lures reduced captures. Male attraction specificity markedly varied with local moth density.
The role of TolC has largely been explored in proteobacteria, where it functions as a metabolite and protein exporter. In contrast, little research has been carried out on the function of cyanobacterial homologues, and as a consequence, not much is known about the mechanism of cyanobacterial antibiotic uptake and metabolite secretion in general. It has been suggested that the TolC-like homologue of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, termed heterocyst glycolipid deposition protein D (HgdD), is involved in both protein and lipid secretion. To describe its function in secondary metabolite secretion, we established a system to measure the uptake of antibiotics based on the fluorescent molecule ethidium bromide. We analyzed the rate of porin-dependent metabolite uptake and confirmed the functional relation between detoxification and the action of HgdD. Moreover, we identified two major facilitator superfamily proteins that are involved in this process. It appears that anaOmp85 (Alr2269) is not required for insertion or assembly of HgdD, because an alr2269 mutant does not exhibit a phenotype similar to the hgdD mutant. Thus, we could assign components of the metabolite efflux system and describe parameters of detoxification by Anabaena sp. PCC 7120.
By a comparative thin layer chromatographic screening of the methanol-soluble leaf exudates from more than 400 Aloe plants (183 species), 5-hydroxyaloin A was identified in 20 species. Whilst 13 of the 20 species revealed interindividual variations concerning to the occurrence of 5-hydroxyaloin A, this anthrone-C-glucosyl was unambiguously detected in each individual of 6 Aloe species. In the leaf exudates from A. marlothii Berger 5-hydroxyaloin A was only traceable in the aloin-containing chemivars. The complete anthrone-C-glucosyl pattern of these 7 clearly characterized species has been determined additionally by qualitative and quantitative high performance liquid chromatography: The results obtained demonstrate that 5-hydroxyaloin only occurs in the more stable A-configuration (10 R, 1′S), thus being till now the only anthrone-C-glycosyl which has not been found as diastereomeric pair genuinely in plants. As well, 5-hydroxyaloin A characterizes a quantitatively significant hydroxylating pathway in biosynthesis of anthranoids. It is discussed as a chemotaxonomic marker of the genus Aloe, especially of the sections Pachydendron and Eualoe.
In haploid and diploid S. cerevisiae the dimer yield ratio TT̂/CT̂ is found to be 1.2/1 and 1.3/1, resp., at the UV (254 nm) unit dose 1 erg/mm2, the share of TT̂ and CT̂ in a UV (254 nm) lethal hit being 0.7 TT̂ and 0.6 CT̂. A general formulation of the UV lethal hit is given and discussed. The TT̂ + CT̂ yields obtained for S. cerevisiae are compared to those reported for other organisms. It is found that there obviously exists a directly proportional linear correlation between genome size and TT̂ + CT̂ yield for the UV dose range well below the stationary levels of the TT̂ and CT̂ formation kinetics.
A screening procedure is presented which allows the isolation of yeast mutants (typ tlr) with highly efficient utilization of exogenous deoxythymidine-5′-monophosphate (5′-dTMP) (>50% ). Data are given concerning the phenomenon of 5′-dTMP utilization in general: (i) The ability of S. cerevisiae to incorporate exogenous 5′-dTMP was found to already be a wild type feature of this yeast, i. e. apparently not to be due to any mutation such as typ , tup, tmp per or tum. Consequently these mutations are interpreted as amplifiers of a pre-given wild type potency. So far eight stages of 5′-dTMP utilization were detected as classified by the optimal 5′-dTMP requirement, with 5′-dTMP biosynthesis blocked, of the corresponding mutant strains isolated. All of them fit well into a mathematical series of the type “2n × 1.5” (n = 0, 1, 2, … , 11), where the product term for n = 11 represents the 5′-dTMP requirement (μg/ml) of the best 5′-dTMP utilizing wild type strain found, (ii) Amplification of the 5′-dTMP utilizing potency obviously is due to any genetically determined alteration of the yeast 5′-dTMP uptaking principle itself or of physiological processes accompanying the monophosphate’s uptake, (iii) The functioning of 5′-dTMP uptake requires acidic (≦ pH 6) conditions in the yeast cell’s outer environment, (iv) Some yeast typ and typ tlr mutants were found to exhibit a more or less pronounced sensitivity towards exogenously offered 5′dTM P. The response of a sensitive strain towards inhibitory concentrations of the nucleotide apparently is co-conditioned by the presence or absence of thymidylate biosynthesis. With 5′-dTMP biosynthesis blocked the 5′-dTMP mediated inhibition is a permanent one and finally leads to the death of a cell. With a functioning thymidylate biosynthesis, in contrast, the inhibition is only temporary, (v) Yeast typ or typ tlr strains were observed to dephosphorylate exogenous 5′-dTMP to thymidine due to a phosphatase activity which cannot be eliminated at pH 7 + 70 mм inorganic phosphate conditions in the growth medium. This 5′-dTMP cleavage obviously occurs outside the cell and does not seem to be correlated both to the monophosphate’s uptake and to the phenomenon of 5′-dTMP sensitivity. The destruction of 5′-dTMP does not disturb (5′-dTMP) DNA-specific labelling.
An improved method for isolation of yeast m utants auxotrophic for 5′-dTM P is presented. The procedure employs the two folic acid antagonists am inopterin and sulfanilam ide (SAA). Selectiveness of the procedure depends on concentration of SAA and time of incubation.
44 mutants auxotrophic and 3 conditionally auxotrophic for 5′-dTMP were isolated. All belong to one complementation group. The corresponding gene was designated TMP1. Tetrad dissection revealed its chromosomal nature. TMP1 is not closely linked to the genes ADE2,, LEU1, ARG 4, ILV2, HIS5, LYS1 and the mating type locus. With the centromere-linked genes ARG4 and LEU1 I gene TMP1 exhibited second division segregation frequencies of 0.42 and 0.53 respectively, indicative of centromere-linkage.
Strains auxotrophic and conditionally auxotrophic for 5′-dTM P were all respiratory deficient (petite). Genetical analysis indicates that the petite phenotype is due to loss of the rho factor in cells harbouring either tmp1 or tmp1ts alleles.
A quantitative determination method of gallic and protocatechuic acid in cultures and liquid nutrient of Phycomyces blakesleeanus was described. Both phenolic acids were separated by TLC and the colour reaction with Folin reagent was used for a colorimetric test. This procedure was employed for investigating the formation of gallic and protocatechuic acid in cultures with optimal (10-4 m) and reduced (1.3 × 10-6 ᴍ) zinc supply showing that their production is stimulated by zinc ions.
In addition, the inhibiting effect of light on the accumulation of gallic acid was manifested, however, its excretion into the medium was uneffected by light and protocatechuic acid was not excreted at all. During the development of Phycomyces gallic and protocatechuic acid could be detected in two days old mycelium . With the sporangiophore production both acids are accumulated more rapidly in the sporangiophores. After the end of sporangiophore formation the gallic acid content increases only slightly. In contrast the total content of protocatechuic acid decreases sharply. As no excretion occurs a degradation of at least protocatechuic acid must be taken into consideration.
Phosphoenolpyruvate carboxykinase (PEPCK) from Phycomyces blakesleeanus was partially purified by protamine sulfate precipitation, ammoniumsulfate precipitation, and diethylamino ethyl cellulose (DEAE) treatment. This preparation was employed for the characterization of the enzyme. The Km values for phosphoenolpyruvate (PEP) and ADP were determined as 1.6 and 0.42 mᴍ. The nucleotid specifity was demonstrated for ADP exclusively. The use of sulfuryl reagents showed the presence of thiol groups sensitive against p-hydroxymercuribenzoate but not effected by N-ethylmaleimide.
The function of gene sll0033 from Synechocystis 6803 which is homologous to the bacterial crtI-type phytoene desaturase genes was elucidated as a novel carotene isomerase. Escherichia coli transformed with all genes necessary for the formation of ζ-carotene and expressing a ζ-carotene desaturase synthesized the positional isomer prolycopene (7,9,7′,9′Z lycopene) which cannot be cyclized in the subsequent reactions to a- and β-carotene. Upon cotransformation with sll0033, the formation of all-E lycopene is mediated instead.
A role of the Qв binding protein in the mechanism of cyanobacterial adaptation to light intensity?
(1986)
Growth of the unicellular blue-green alga Anacystis nidulans in media containing sublethal concentrations of DCMU-type inhibitors of photosynthetic electron transport in strong white light gave rise to shade type appearance in this organism, as characterized by an increased ratio of phycocyanin to chlorophyll and reduced ratios, both, of carotenoids to chlorophyll and of total chlorophyll to P700. Shade type in Anacystis was caused neither by phenolic inhibitors tested nor by those known to bind to the cytochrome b6/f-complex. Surprisingly enough, the molar ratio of phycocyanin to chlorophyll in artificially shade adapted Anacystis1 grown in strong white light in the presence of 10-6 м atrazine, was found to increase with temperature for a given light intensity and with light intensity for a given temperature.
Mutants of Anaeystis with a reduced binding capacity for DCMU-type herbicides due to an amino acid exchange in the 32 kDa Qв-binding polypeptide, also called D-1 protein, were ob- served to show shade type appearance in strong light, to respond very little to changes in light intensity and to show a reduced capability to further change their appearance to shade type by binding of competitors of Ob to the 32 kDa polypeptide.
In Anaeystis a concentration of atrazine (10-7 м), ten times lower than the one causing the highest rate of shade adaptation (10-6 м), was shown to induce an optimum in cell density, which in turn resulted in an optimum in light-dependent O2 evolution. Both factors together might be responsible for the so-called greening effect observed in higher plants treated with sublethal concentrations of DCMU-type inhibitors of photosynthetic electron transport.
A thylakoid membrane preparation isolated from the blue-green alga Anacystis nidulans was freed from carboxysomes, soluble enzymes and the pigment P750 by floating in a discontinuous sucrose density gradient. In a buffer containing sucrose and the zwitterionic detergent Miranol S2M-SF the thylakoids were loaded on a linear 10-18% sucrose density gradient which also contained Miranol. The sedimentation yielded three bands, the lower two of which were green and the upper one was orange. The light green band in the middle of the gradient was the only one to show any photosystem II activity. This was measured as light-induced electron transport from diphenylcarbazide (DPC) to dichlorophenol-indophenol (DCPIP). The activity was sensitive to dichlorophenyl-dimethylurea (DCMU).
The red absorption maximum of the particles in this middle band - henceforth called photosystem II particles - was found at 672 nm and the maximum of their low temperature fluorescence emission spectrum at 685 nm upon excitation with blue light. Cytochrome b559 was the only cytochrome found in these particles; it was present at an average ratio of one molecule cytochrome per 40 -50 molecules chlorophyll a. C550 photoreduction with accompanying photooxidation of cytochrome b559 was also observed in the photosystem II particles. Good photosystem II preparations did not contain any detectable amounts of P 700.
By means of sodium dodecylsulfate polyacrylamide gel electrophoresis the polypeptide composition of the photosystem II particles was studied. Dissolution of the chlorophyll protein complexes was done under strongly denaturing conditions; consequently, no green bands were observed on the gels. The polypeptide pattern of the photosystem II particles showed two strong predominant bands of protein components with apparent molecular weights (app. mol. wts.) of about 50 000 and 48 000. These two bands are unique for photosystem II. Two other weaker bands were also found characteristic for photosystem II, the band of a polypeptide with an app. mol. wt. of 38 000 and that of a polypeptide with an app. mol. wt. of 31 000. Sometimes in addition the weak band of a polypeptide with the app. mol. wt. 27 000 was observed on the gel. The polypeptide 38 000 aggregated upon boiling of the sample in the presence of the denaturing agents prior to the electrophoresis, yielding an aggregate with an app. mol. wt. of 50 000. Additional polypeptides which were often found in the photosystem II particle preparation could be identified as subunits of the coupling factor of photophosphorylation CF1. None of the polypeptides described as characteristic for photosystem II are due to proteolytic activity.
As the observed photosystem II activity was found to be DCMU-sensitive it appears that the DCMU-binding protein is among the here described photosystem II polypeptides. Moreover, the authors have reason to believe that one of the major protein components found characteristic for photosystem II is cytochrome b559.
Mutants of Anacystis R2 with different amino acid exchanges in positions 255 and/or 264 in copy I of the psbA gene, leading to different tolerances to DCMU-type herbicides, are com- pared with the respective wild type concerning pigmentation and incorporation of 35S into the D1 protein upon growth in the presence of [35S]methionine. All mutants have shade-type appearance compared to the wild type, although to different extents depending on site and mode of the amino acid exchange in the D1 protein. Except for 3 mutants, there is no correlation between shade-type appearance on one hand and resistance towards a certain inhibitor on the other hand.
Not only the molar ratio of phycocyanin (PC) to chlorophyll (Chi) is higher in all mutants compared to the respective wild type, but also the rate of synthesis of the D1 protein. On the background of different levels of total 35S incorporation within 18 min, D1 synthesis can be related to shade adaptation. Degradation of the D1 protein remains to be thoroughly studied in this context.
No reproducible differences in whole chain electron transport were observed between mutants and wild type.
In addition to the importance of many Dioscorea species (yams) as starchy staple food, some representatives are known and still used as a source for the steroidal sapogenin diosgenin, which, besides phytosterols derived from tall-oil, is an important precursor for partial synthesis of steroids for pharmaceutical research and applications. While in edible yams the diosgenin content should be as low as possible, a high yield of the compound is preferable for cultivars which are grown for the extraction of sterols. In the past, miscalculations and insufficiently precise techniques for quantification of diosgenin prevailed. Therefore we set out to re-evaluate the steroid content of a world collection of Dioscorea species, using leaves as sample material. We optimized diosgenin quantification techniques and fingerprinted the whole collection with the DNA amplification fingerprinting (DAF) technique. Total diosgenin contents ranged from 0.04 to 0.93% of dry weight within the collection. Several Dioscorea cultivars can be characterized via their DAF fingerprint patterns.
Chromatin, RNA Polymerase, Potato Tuber Tissue, Aging Phenomenon The synthesis of RNA by chromatin-bound RNA polymerase (E.C. 2.7.7.6.) from white potato tubers proceeds at a low rate, which is enhanced after slicing the tissue, however. Concomitantly DNA template availability as measured with saturating amounts of Escherichia coli polymerase is diminished drastically. Nearest neighbor frequency analysis proved that the RNA synthesized on chromatin of intact tubers is different from that synthesized on chromatin of sliced tissue.
The RNA polymerase of white potato tubers is dependent on all four ribonucleoside triphos phates and a divalent metal ion such as Mg2+ or Mn2+ and totally inhibited by the presence of pyrophosphate. Actinomycin D blocks the formation of the RNA product, which could be shown to be a heteropolymer by nearest neighbour frequency technique. The Km of the chromatin-bound enzyme with regard to ATP, GTP, CTP and UTP was 5.1 X10-5 M, 1.6X10-5 M, 0.9X10-5 M and 0.45 X 10-5M/1 respectively, α-amanitin inhibits the overall activity to about 50%, which indicates the presence of equal amounts of polymerase I and polymerase If.
Testosterone, Androst-4-en-3,17-dione, Enzyme Induction, S trep to m yces hydrogenans After cultivation of S trep to m yces hydrogenan s in the presence of 3H-labelled testosterone, radio active steroids were extracted separately from the cytosolic, ribosomal and cell wall-membrane fraction of the cells and from the culture medium, respectively.. The separation of the steroids was performed by one-and two-dimensional thin layer chromatography (TLC). The identification of the main metabolites was achieved by crystallization to constant specific radioactivity, specific staining procedures and acetylation. The oxidation of testosterone to androst-4-en-3,17-dione is by far the predominating reaction, which is almost finished after 3 h cultivation. Androst-4-en-3,17-dione is mainly transferred into the culture medium and partly accumulated within the cell wall-membrane fraction. High polar steroid metabolites and androstane derivatives are present in very small amounts only.
Resting potato tuber tissue possesses only faint activity of the two dehydrogenases of the oxidative pentose phosphate cycle, glucose-6-phosphate- and 6-phosphogluconate dehydrogenase. Slicing of the tissue, however, greatly enhances the action of both enzymes. The slicing-induced increase in activity is a consequence of intensified action of at least 5 glucose-6-phosphate dehydrogenase isozymes and a more differentiated activation/inactivation of seven 6-phosphogluconate dehydrogenase isozymes.
Using density labelling and isopycnic equilibrium centrifugation it could be demonstrated, that the bulk of both enzymes appearing after slicing the tissue is the result of de novo synthesis rather than activation of pre-existing proenzymes.
In the course of the odontogenesis of bovine incisors several clearly distinguishable phosphohydrolase activities are observed in the pulp and in dental hard tissues. Using various substrates and inhibitors, unspecific alkaline phosphatase, two isoenzymes of acid phosphatase, Ca2+-activated ATPase and inorganic pyrophosphatase are characterized. The enzymatic activity of alkaline phosphatase in pulp and hard tissues is significantly high at the beginning of dentine and enamel mineralization. The specific activity of this enzyme decreases quite fast with the beginning of root formation, then more slowly, until it reaches a constant final value. Histochemical studies show that during mineralization the maximum of alkaline phosphatase activity is in the subodontoblasts. Lower enzyme concentrations are found in the stratum intermedium and in the outer enamel epithelium during that process.
The specific activities of ATPase, acid phosphatases and pyrophosphatase show little temporal variation during tooth development, but they also appear in a characteristic spatial pattern in the dental tissues.
The genus Ebolavirus comprises some of the deadliest viruses for primates and humans and associated disease outbreaks are increasing in Africa. Different evidence suggests that bats are putative reservoir hosts and play a major role in the transmission cycle of these filoviruses. Thus, detailed knowledge about their distribution might improve risk estimations of where future disease outbreaks might occur. A MaxEnt niche modelling approach based on climatic variables and land cover was used to investigate the potential distribution of 9 bat species associated to the Zaire ebolavirus. This viral species has led to major Ebola outbreaks in Africa and is known for causing high mortalities. Modelling results suggest suitable areas mainly in the areas near the coasts of West Africa with extensions into Central Africa, where almost all of the 9 species studied find suitable habitat conditions. Previous spillover events and outbreak sites of the virus are covered by the modelled distribution of 3 bat species that have been tested positive for the virus not only using serology tests but also PCR methods. Modelling the habitat suitability of the bats is an important step that can benefit public information campaigns and may ultimately help control future outbreaks of the disease.
Bleaching of chlorophyll was studied in the leaves of rye seedlings (Secale cereale L.) treated with four chlorosis-inducing herbicides of different potency (weak photodestructions, group 1: aminotriazole, haloxidine; strong photodestructions, group 2: San 6706, difunone). Chlorophyll deficiency and particularly the inactivation of a chloroplast marker enzyme, NADP-dependent glyceraldehyde-3-P dehydrogenase, that occurred in the presence of group 2 herbicides were stronger in red, than in blue, light.
When grown in white light of low intensity (10 lx) herbicide-treated leaves contained chloro phyll, 70 S ribosomes and unimpaired activities of NADP-dependent glyceraldehyde-3-P de hydrogenase. At 10 lx only the leaves treated with SAN 6706 and difunone were strongly carotenoid-deficient but not those treated with group 1 herbicides. After all herbicide treatments 10 lx-grown leaf tissue was, however, not capable of photosynthetic O2-evolution indicating some disorder of photosynthetic electron transport. Leaf segments grown at 10 lx were exposed to a high light intensity of 30000 lx at either 0 ° C or 30 °C. In treatments with group 1 herbicides chlorophyll accumulation was stopped in bright light at 30 °C but breakdown was not apparent. Only at 0 °C and in the presence of high, growth-reducing, herbicide concentrations chlorophyll was slightly degraded. The RNAs o f the 70S ribosomes were, however, clearly destroyed at 30000 lx and 30 °C in aminotriazole-treated leaves. In leaves treated with group 2 herbicides chlorophyll was rapidly degraded at 30000 lx both at 0 ° C and 30 °C, however, only in the presence of O2, indicating a true photooxidative and mainly photochemical nature o f the reactions involved. This chlorophyll breakdown was accompanied by the photodestruction of 70S ribosomes and the inactivation of NADP-glyceraldehyde-3-P dehydrogenase.In treatments with group 1 herbicides photoinactivation of the latter enzyme did not occur, although it was clearly localized in the bleached plastids, as demonstrated by gradient separation of organelles.
In the presence of group 2 herbicides the chlorosis was originating from a direct photo oxidation of chlorophyll, accompanied by a massive destruction of other plastid constituents and functions. In treatments with group 1 herbicides photodestructions appeared to be much weaker and insufficient to affect chlorophyll directly. Mediated through some photodestructive inter ference with obviously more sensitive plastid components, such as their ribosomes, further chlorophyll accumulation was, however, prevented.
Cucumber plants (Cucumis sativus L.) were grown under controlled conditions and fumigated with either O3, diluted automobile exhaust or a combination of both. The ratio of variable to maximum chlorophyll fluorescence (Fv/Fm) was estimated as a measure of PSII activity Activities of the enzymes catalase, glutathione reductase and guaiacol-dependent peroxidase and contents of the antioxidants ascorbate and glutathione were assayed as potential indicators of oxidative stress. The behavior of catalase and of PSII are of particular diagnostic interest because they require continuous repair in light. Exposures of up to 13 days to moderate concentrations of the pollutant gases alone did not induce striking changes in any of the activities that were assayed. A lso when the plants were subjected to an additional stress treatment by exposing them to 4 short cold treatments (2h each at 0 - 4 °C in light on days 12-15 after sowing) which induced marked declines of the Fv/Fm ratio, the chlorophyll content and the catalase activity, these cold-induced symptoms of photodamage were not significantly enhanced by the fumigation treatments. However, increases of the activities of glutathione reductase and peroxidase observed during a period of recovery following the cold-exposures were markedly higher in O3-fumigated plants, as compared to plants grown in filtered air or fumigated with car exhaust alone. The results emphasize that effects of moderate pollutant exposures may be latent or delayed over long time periods and that defence responses can be enhanced when plants are exposed to additional, naturally occurring stress situations.
Glycolate oxidase that was partially purified from pea leaves was inactivated in vitro by blue light in the presence of FMN. Inactivation was greatly retarded in the absence of O2. Under aerobic conditions H2O2 was formed. The presence of catalase, GSH or dithiothreitol protected glycolate oxidase against photoinactivation. Less efficient protection was provided by ascorbate, histidine, tryptophan or EDTA. The presence of superoxide dismutase or of hydroxyl radical scavengers had no, or only minor, effects. Glutathione suppressed H2O2 accumulation and was oxidized in the presence of glycolate oxidase in blue light. Glycolate oxidase was also inactivated in the presence of a superoxide-generating system or by H2O2 in darkness. In intact leaves photoinactivation of glycolate oxidase was not observed. However, when catalase was inactivated by the application of 3-amino-1,2,4-triazole or depleted by prolonged exposure to cycloheximide a strong photoinactivation of glycolate oxidase was also seen in leaves. In vivo blue and red light were similarly effective. Furthermore, glycolate oxidase was photoinactivated in leaves when the endogenous GSH was depleted by the application of buthionine sulfoximine. Both catalase and antioxidants, in particular GSH, appear to be essential for the protection of glycolate oxidase in the peroxisomes in vivo.
Formation of major prenylquinones and carotenoids was investigated by comparing the incorporation of [14C]mevalonate into segments of different age from green and etiolated leaves of 22 C-grown rye seedlings (Secale cereale L.) and from 32 C-grown rye leaves which contained bleached and proplastid-like ribosome-deficient plastids, due to a heat-sensitivity of 70S ribosome formation. The contents of plastidic isoprenoids were much lower (between 2 - 30%) in the achlorophyllous than in green leaves. In green leaves [14C]mevalonate incorporation into non-polar lipids and into plastoquinone was partially inhibited in the presence of gabaculin, an inhibitor of chlorophyll synthesis. However, except for β-carotene, [14C]mevalonate incorporation into isoprenoids continuously increased with age also in achlorophyllous etiolated or 32 °C-grown, as in green, leaves and was, except for P-carotene and plastoquinone, higher in etiolated than in green leaves. In bleached °32 C-grown leaves [14C]mevalonate incorporation into all plastidic isoprenoids was strikingly (up to 45-fold) higher than in green control leaves. While degradation of P-carotene was greatly enhanced in bleached 32 °C-grown leaves, relative to green control leaves, and could thus compensate for a higher apparent synthesis, chase experiments did not reveal any marked differences of the turnover of other isoprenoids. The half times of plastoquinone. phylloquinone and lutein were in the order of 2-3 days. Within a 24 h chase period a-tocopherol degradation did not become apparent. Uptake of [14C]mevalonate and [14C]isopentenyl pyrophosphate by isolated bleached plastids from 32 °C-grown leaves was much more rapid than by chloroplasts and resulted in higher precursor accumulation within the organelle. While mevalonate incorporation into isoprenoid lipids was not detected, isopentenyl pyrophosphate was incorporated into isoprenoid lipids, including plastoquinone. Rates of incorporation by isolated chloroplasts or bleached plastids were of similar order. The results illustrate that divergent types of plastid differentiation are associated with fundamental developmental changes of the metabolic flow of isoprenoid precursors between different products and compartments and, in particular, with changes of import into the plastid compartment.
Among chlorosis-inducing herbicides that interfere with carotenoid synthesis two groups of different potency can be discriminated (group 1: aminotriazole amd haloxidine; group 2 with more extensive photodestructions: pyridazinone herbicides and difunon). After application of herbicides of group 2 colored carotenoids were completely absent and preexisting chlorophyll was degraded by photochemical reactions requiring high light intensity and O2, that occurred also at 0°C. In treatments with group 1 herbicides direct photodegradation of chlorophyll was not sufficient to generate the chlorosis. Light-induced interference with constituents of the chloroplast protein synthesis apparatus being more sensitive to photooxidative damage than chlorophyll, appeared to indirectly mediate the chlorosis. In the absence of chloroplast protein synthesis further chlorophyll accumulation is prevented. Photodegradation of chlorophyll in the presence of group 2 herbicides involved the participation of O2- radicals and was accompanied by lipid peroxidation. In all herbicide treatments the catalase activity of the leaves was very low. Only in the presence of group 2 herbicides chloroplast enzymes of cytoplasmic origin (e.g. NADP-glyceraldehyde-3-phosphate dehydrogenase) were also inactivated. Rapid inactivation of catalase as well as of NADP-glyceraldehyde-3-phosphate dehydrogenase was induced by exposure of dim-light-grown herbicide-treated leaves to bright light, also at 0°C. In treatments with herbicides of group 2 also other peroxisomal enzymes (e.g. glycolate oxidate, hydroxy-pyruvate reductase) were affected. The elimination of these peroxisomal enzymes also appeared to depend on photooxidative processes of the chloroplast.
Synechococcus (Anacystis nidulans, strain L 1402-1) were grown at + 37 °C in an atmosphere of 0.04 vol.% CO2 using different light conditions. Changing the culture conditions caused alterations in pigment ratios and ultrastructure of Synechococcus. In comparison to the low white and red light grown cells under strong white light the number of thylakoids decreased and an accumulation of storage carbohydrates could be observed. The number of the polyhedral bodies also varied with culture conditions. The results are discussed with reference to the pigment composition and the function of the polyhedral bodies.
The cyanobacteria Anabaena cylindrica and Synechococcus leopoliensis (= Anacystis nidulans) were grown at different levels of UV-B radiation (439. 717, 1230 and 1405 J m -2d-1 weighted according Caldwell, 1971) for 2 days. Dry weight was hardly affected but phycocyanin content of both species decreased linearly to the level of UV-B radiation. Contents of protein, carotenoids and chlorophyll a were reduced only after exposure to high doses (1230 J m-2d-1) of UV-B radiation. Photosynthetic 14CO2 fixation of Anabaena cells was reduced linearly with increasing UV-B dose whereas no effect could be observed in Synechococcus. A depression of photosynthetic 15N-nitrate uptake was found after UV-B stress in both species. UV-B irradiance caused an increase of 15N-incorporation into glutamine, but no effect was noted for incorporation into alanine or aspartic acid. An increase of 15N-excess in glutamic acid linear with the UV-B dose was observed in Synechococcus, only. Patterns of 14C-labelled photosynthetic products were either less affected by UV-B radiation (Anabaena) or an enhancement of 14C-label in total amino acids was detected (Synechococcus). The amount of total free amino acids increased parallel to the level of UV-B radiation. Only, the high dose of UV-B (1405 J m-2d-1, weighted) results in a decrease of the glutamine pool. Our results indicate an inhibition of glutamate synthase by UV-B irradiation in Anabaena, only. Results were discussed with reference to the damage of the photosynthetic apparatus.
The marine diatoms Bellerochea yucatanensis and Thalassiosira rotula were grown at different salinities (20/25, 35, and 40/45‰ salinity (S), respectively) under normal air (0.035 vol.% CO2). No significant variations in the percentage of gross photosynthetic products (e.g. total amino acids, sugar phosphates) were found as a function of salinity during growth. The bulk of the soluble 14C-radioactivity was detected in amino acids. 14C-labelling of glutamine increased markedly with salinity. Low salt - grown algae are characterized by enhanced amino acid pools, mainly of aspartic acid, asparagine and glutamine. It was found that the tested amino acids are not involved in osmoregulation.
The cyanobacterium Synechococcus (Anacystis nidulans, strain L 1401-1) grown under different light conditions showed variations in pigmentation. Ratios of photosynthetic pigments and the effect on quantum requirement and oxygen evolution were studied. An increase in the ratio of chlorophyll a forms with absorption maxima in the far red regime to total chlorophyll a forms was observed in cells grown in strong white light. The quantum efficiency of orange light (637 nm) - absorbed by phycocyanin - was higher after growth of Synechococcus in white than in red light. The quantum efficiency at 677 nm increased when cells were grown in red light and decreased strongly after transfering red light grown cells to conditions of strong white light. The results show an adaptation of pigment composition to light regimes during growth and its effect on photosynthesis.
14C-and 15N-Assimilation, 15N-Labelled Amino Acids, M arine D iatom s The marine diatoms Bellerochea yucatanensis and Skeletonema costatum were grown at +20 °C in 0.03 vol.% CO2 with nitrate or ammonia. The 15N -am m onia and 15N -nitrate assim ila tion and 15N -incorporation into various amino acids were studied of both diatom s during exponential growth phase in dependence of different nitrogen conditions. In all experiments the 15N -am m onia uptake was lower than the 15N -nitrate assim ilation rate up to 20-40 min photo synthesis. N itrate lim itation -cells grown in nitrate followed by growth in nitrogen-free m edium for 24 h — caused a strong 15N-label into aspartate after adding 15NH 4C1 (1 m M). In cells grown in nitrate highest enrichment of 15N was found in glutamine. Results were discussed with reference to the operating of the GS/GOGAT system and glutam ic acid dehydrogenase pathway. Photosynthetic 14CO2 fixation experiments showed a very high labelling of aspartate which was interpreted with a phosphoenolpyruvate carboxylation catalysed by phosphoenolpyruvate carb-oxykinase.
The cyanobacterium Synechococcus (Anacystis nidulans strain L 1402-1) was grown at +35 °C in air and in air enriched with 2.2 vol.% CO2. The effect of different oxygen concentrations (0, 2, 20, 50, 75 and 99.97 or 97.8 vol.%) was studied in low (0.03 vol.%) and high (2.2 vol.%) CO2 concentrations at + 35 °C. After exposure to a nitrogen atmosphere and low CO2 content I4C-bicarbonate was mainly incorporated into aspartate and glycine/serine. During oxygenic photosynthetic CO2 fixation label in aspartate decreased and a high degree of radioactivity could be found in 3-phosphoglyceric acid and sugar monophosphates. The Calvin cycle was the main fixing pathway in 2.2 vol.% CO2 during anoxygenic and oxygenic conditions independent on the O2 concentrations during the experiments. No oxygen enhancement of photosynthetic CO2 fixation could be found. Possible mechanism involved in CO2 fixation pathways and glycolate metabolism underlying the effect of oxygen was discussed.
The marine diatom Ditylum brightwellii (West) Grunow isolated from the Baltic Sea could be synchronized by a light/dark rhythm of 6.5:17.5 h (white light intensity 8 W m-2) at 18 °C and 0.035 vol.% CO2. Content of protein, DNA and RNA increased linearly up to the end of the cell cycle. Pigments (chlorophyll a, chlorophyll c1 + c2, carotenoids) and galactolipids were synthesized in the light period only. A lag phase of 2 h was observed in the biosynthesis of sulphoquinovosyl diacylglycerol and phosphatidylglycerol. Formation of phosphatidylglycerol and phosphatidylcholin continued in the dark period (30% and 28%, respectively). The pattern of major fatty acids (C14:0, C16:1, C16:0, C18:1 and C20:5) varied during the cell cycle of Ditylum.
Biosynthesis of acyl lipids was reduced in dependence on the UV-B dose. The most sensitive lipid was digalactosyl diacylglycerol (total inhibition at 585 J m-2), whereas phosphatidylcholin was less affected (20% reduction). UV-B radiation during the dark period had no effect on the lipid and pigment content. Strongest inhibitory effect of UV-B on cell division, synthesis of protein, pigments, sulphoquinovosyl diacylglycerol and phosphatidylglycerol was found after UV-B radiation at the beginning of the cell cycle (0.-2. h). An exposure time at the end of the light period (4.-6. h) led to a marked damage on the synthesis of monogalactosyl diacylglycerol and phosphatidylglycerol. These findings indicate a stage-dependent response of Ditylum to UV-B irradiance. The impact of UV-B resulted in an increase of unsaturated long chained fatty acids (C18, C20) and in a diminution of short chained fatty acids (C14, C16). Content of ATP was not affected by UV-B radiation under the used conditions. The inhibitory effect of UV-B on synthesis of DNA, RNA, protein and acyl lipids was mainly reversible. Results were discussed with reference to UV-B damage on the enzymes involved in the biosynthesis of acyl lipids and by a reduction of available metabolites.
The cyanobacterium Synechococcus leopoliensis (Anacystis nidulans, strain L 1402-1) grown at 39 °C and 2 vol. % CO : could be synchronized by a light/dark regime of 3:5 h (white light intensity 1.5 × 104 erg cm-2 sec-1). Content of pigments (chlorophyll a. phycocyanin and carotenoids), R N A and proteins increased linearly up to 100% at the end of the light period while DNA synthesis was lower. Chlorophyll a synthesis was correlated to the photosystem I activity of the isolated thylakoids and to the formation of MGD G . Galacto lipids were synthesized in the light period, only. A lag phase of 2h was observed in the biosynthesis of SQDG and PG. No significant differences were found between the cell and thylakoid fractions. Palmitic (C16:0), hexadecenoic (C16:1) and octadecenoic (C18:1) acid as major com ponents accounted for more than 90% of total fatty acids in MGD G , DGDG and SQDG . PG contains a small amount of stearic (C18:0) and heptadecenoic (C17:1) acid. No significant variations in the fatty acid distribution of all lipids could be detected in the cell fraction during the division cycle. Changes in the ratio of saturated to unsaturated fatty acids were found in isolated thylakoids. only. In experiments with [14C]bicarbonate main radioactivity was measured in galacto lipids while using [14C]acetate SQDG and PG were markedly [14C]labelled. Results were discussed with reference to the findings of eucaryotic algae and the formation of photosynthetic membranes.