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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.
The growth of Synechococcus at different intensities of white and red light caused changes in the pigment composition. The ratio of chlorophyll a to phycocyanin varied from 1:8,2 in LWLI-grown cells to 1:1,4 in cells grown at HWLI and to 1:15,7 in cultures exposed to HRLI. Acyl lipids were quantitatively determ ined and fatty acids of the individual lipid classes analysed by GLC. Phycocyanin-free photosynthetic lam ellae were obtained by fractional centrifugation. No variation was found in the acyl lipid composition of the m em brane preparations. These all contained MGDG, DGDG, SQDG and PG as components. In all the lipids investigated, palmitic, hexadecenoic and octadecenoic acids m ade up to more than 90% of total fatty acids. The pattern of these major components w ithin the lipids from the different cultures depended on the light used. No large differences were detected between zones obtained from LWLI and HRLI isolated membranes, whereas density gradient centrifugation of those from HWLI-grown cells resulted in a completely different pattern of bands. The variations in lipid and fatty acid composition are discussed with respect to changes observed in lipid composition of whole cells and the results reported on tem perature dependent shifts in lipid fluidity in cyanobacteria.
Effect of UV-B radiation on biomass production, pigmentation and protein content of marine diatoms
(1984)
Several species of marine diatoms were grown at + 18 °C and + 22 °C under normal air conditions (0.035 vol.% C02) at a light/dark alteration of 14: 8̄ h. Intensity of white light was 1 mW (~ 5000 lux). An artifical nutrient solution of 35%o salinity was used. Algae - harvested during exponential growth - were exposed to different intensities of UV-B radiation (439, 717 and 1230 J · m-2 · m-1) for 2 days. UV-B radiation depressed the growth of all tested marine diatoms. Low levels of UV-B resulted in a slight increase of the biomass production (dry weight) compared to not UV-B treated cells. Enhanced UV-B doses caused a diminution of the primary productivity in all species. Algae exposed to UV-B stress showed a marked decrease in the protein and pigment content (chlorophyll a, chlorophyll c1 + c2 and carotenoids). In + 22°C grown cells of Lauderia annulata and Thalassiosira rotula were more sensitive to UV-B radiation than those cultures grown at + 18 °C. Bellerochea yucatanensis cells grown at +22 °C were less affected after UV-B exposure than at +18°C grown algae. The UV-B sensibility and growth of the individual species varied in a mixture of several marine diatoms. Results were discussed with reference to the UV-B effect on metabolic processes.
The cyanobacterium Anabaena flos-aquae (strain 1444) grown at different intensities of white light (900, 3500 and 30000 lux) showed changes in the content and composition of the pigments. Phycocyanin was more affected by high light conditions during growth than chlorophyll a. In comparison to in low white light grown cyanobacteria number of phycobilisomes and thylakoids decreased under strong light. A diminution of 14CO2 fixation, total amino acid content, glutamic acid and glutamine pools was found in strong white light grown cells. Under these conditions the majority of 14C-labelling was measured in sugar phosphates. After pressure treatment a marked increase of 14C-incorporation into amino acids could be obtained. Results were discussed with reference to regulation of buoyancy in Anabaena flos-aquae.
The blue-green alga Anacystis nidulans (strain L 1402-1) was grown at + 37 °C in air (0.03 vol.% CO2 and in air enriched with 3.0 vol.% CO2. The effects of several inhibitors on the activity of aminotransferases, 14CO2 fixation and radioactive photosynthetic products of Anacystis were studied. No serine-pyruvate aminotransferase activity could be found in 10-2 м isonicotinyl hydrazide (INH) ; under the influence of this inhibitor aspartate and alanine aminotransferase were decreased about 49% respectively 17.6%. Serine-pyruvate and alanine aminotransferase activity decreased to more than 50% in 10-3 м glyoxalbisulfite. The obtained inhibitory effect of 10-4 м HPMS on serine-piruvate aminotransferase (35%) was stronger than on the other aminotransferases. DCMU (5 × 10-6 м) inhibition on alanine aminotransferase activity was 83.7%. Under the influence of 10-3 м glyoxalbisulfite no 14C-labelled amino acids could be detected after 5 min photosynthesis; 14C-labelling of phosphoenolpyruvate, malate, phosphoglycolate and glycolic acid increased. Isonicotinyl hydrazide (10-2 м) caused in comparison to the control experiment a lower radioactivity in aspartate, glutamate and phosphoenolpyruvate. The results are discussed with reference to the operation of the glycolate pathway and a carboxylation reaction of phosphoenolpyruvate in the blue-green alga Anacystis nidulans.
The cyanobacterium Synechococcus (Anacystis nidulans strain L 1402-1) was grown at + 37 °C in 3.0 vol.% CO2. The effect of preillumination with white light on the subsequent dark 14CO2 fixation was studied under aerobic conditions at + 30 °C. The radioactive carbon first incoiporated into 3-phosphoglyceric acid was transferred during the later periods of dark 14CO2 fixation to phosphoenolpyruvate and aspartate. No labelling or a very low label in sugar monophosphates could be observed. During the dark/light transients the initial fixation product was mainly aspartate. The pattern of 14C-incorporation into photosynthetic products under steady state conditions (10 min photosynthesis) varied with the temperature during the experiments. The radioactive carbon was firstly incorporated into 3-phosphoglyceric acid. During the later periods of photosynthetic 14CO2 fixation an increased 14C-incorporation into aspartate and glutamate could be observed. Our findings were interpreted with operating of a phosphoenolpyruvate carboxylation besides the Calvin cycle.
Mitochondria are ubiquitous organelles of eukaryotic organisms with a number of essential functions, including synthesis of iron-sulfur clusters, amino acids, lipids, and adenosine triphosphate (ATP). During aging of the fungal aging model Podospora anserina, the inner mitochondrial membrane (IMM) undergoes prominent morphological alterations, ultimately resulting in functional impairments. Since phospholipids (PLs) are key components of biological membranes, maintenance of membrane plasticity and integrity via regulation of PL biosynthesis is indispensable. Here, we report results from a lipidomic analysis of isolated mitochondria from P. anserina that revealed an age-related reorganization of the mitochondrial PL profile and the involvement of the i-AAA protease PaIAP in proteolytic regulation of PL metabolism. The absence of PaIAP enhances biosynthesis of characteristic mitochondrial PLs, leads to significant alterations in the acyl composition of the mitochondrial signature PL cardiolipin (CL), and induces mitophagy. These alterations presumably cause the lifespan increase of the PaIap deletion mutant under standard growth conditions. However, PaIAP is required at elevated temperatures and for degradation of superfluous CL synthase PaCRD1 during glycolytic growth. Overall, our study uncovers a prominent role of PaIAP in the regulation of PL homeostasis in order to adapt membrane plasticity to fluctuating environmental conditions as they occur in nature.
Animals living in human care for several generations face the risk of losing natural behaviors, which can lead to reduced animal welfare. The goal of this study is to demonstrate that meerkats (Suricata suricatta) living in zoos can assess potential danger and respond naturally based on acoustic signals only. This includes that the graded information of urgency in alarm calls as well as a response to those alarm calls is retained in captivity. To test the response to acoustic signals with different threat potential, meerkats were played calls of various animals differing in size and threat (e.g., robin, raven, buzzard, jackal) while their behavior was observed. The emitted alarm calls were recorded and examined for their graded structure on the one hand and played back to them on the other hand by means of a playback experiment to see whether the animals react to their own alarm calls even in the absence of danger. A fuzzy clustering algorithm was used to analyze and classify the alarm calls. Subsequently, the features that best described the graded structure were isolated using the LASSO algorithm and compared to features already known from wild meerkats. The results show that the graded structure is maintained in captivity and can be described by features such as noise and duration. The animals respond to new threats and can distinguish animal calls that are dangerous to them from those that are not, indicating the preservation of natural cooperative behavior. In addition, the playback experiments show that the meerkats respond to their own alarm calls with vigilance and escape behavior. The findings can be used to draw conclusions about the intensity of alertness in captive meerkats and to adapt husbandry conditions to appropriate welfare.
Three of the four species of giraffe are threatened, particularly the northern giraffe (Giraffa camelopardalis), which collectively have the smallest known wild population estimates. Among the three subspecies of the northern giraffe, the West African giraffe (Giraffa camelopardalis peralta) had declined to 49 individuals by 1996 and only recovered due to conservation efforts undertaken in the past 25 years, while the Kordofan giraffe (Giraffa camelopardalis antiquorum) remains at <2300 individuals distributed in small, isolated populations over a large geographical range in Central Africa. These combined factors could lead to genetically depauperated populations. We analyzed 119 mitochondrial sequences and 26 whole genomes of northern giraffe individuals to investigate their population structure and assess the recent demographic history and current genomic diversity of West African and Kordofan giraffe. Phylogenetic and population structure analyses separate the three subspecies of northern giraffe and suggest genetic differentiation between populations from eastern and western areas of the Kordofan giraffe’s range. Both West African and Kordofan giraffe show a gradual decline in effective population size over the last 10 ka and have moderate genome-wide heterozygosity compared to other giraffe species. Recent inbreeding levels are higher in the West African giraffe and in Kordofan giraffe from Garamba National Park, Democratic Republic of Congo. Although numbers for both West African and some populations of Kordofan giraffe have increased in recent years, the threat of habitat loss, climate change impacts, and illegal hunting persists. Thus, future conservation actions should consider close genetic monitoring of populations to detect and, where practical, counteract negative trends that might develop.
The absolute configurations of the diastereomeric 10-hydroxyaloins, which may be regarded as parent structures for other naturally occurring oxanthrone-C-glucosyls, have been established as 10R, 16 R (A) and 10 S, 16 R (B) by an X-ray structure analysis of the A-octaacetyl derivative (C 16 is the anomeric glucosyl carbon atom). The determination was confirmed by CD spectroscopic comparison with the structural analogues aloins A and B, which should prove useful for making future configurational assignments within this class of compounds. A conformational analysis by the use of a molecular modeling method based on force-field calculations reveals the presence of an extra- and an intra-form, the extra-form of which is energetically preferred.
Trypanosoma cruzi, the causative agent of Chagas disease (American trypanosomiasis), colonizes the intestinal tract of triatomines. Triatomine bugs act as vectors in the life cycle of the parasite and transmit infective parasite stages to animals and humans. Contact of the vector with T. cruzi alters its intestinal microbial composition, which may also affect the associated metabolic patterns of the insect. Earlier studies suggest that the complexity of the triatomine fecal metabolome may play a role in vector competence for different T. cruzi strains. Using high-resolution mass spectrometry and supervised machine learning, we aimed to detect differences in the intestinal metabolome of the triatomine Rhodnius prolixus and predict whether the insect had been exposed to T. cruzi or not based solely upon their metabolic profile. We were able to predict the exposure status of R. prolixus to T. cruzi with accuracies of 93.6%, 94.2% and 91.8% using logistic regression, a random forest classifier and a gradient boosting machine model, respectively. We extracted the most important features in producing the models and identified the major metabolites which assist in positive classification. This work highlights the complex interactions between triatomine vector and parasite including effects on the metabolic signature of the insect.
Different interaction modes of two cytochrome-c oxidase soluble CuA fragments with their substrates
(2003)
Cytochrome-c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria and catalyzes the formation of water by reduction of dioxygen. The first step in the cytochrome oxidase reaction is the bimolecular electron transfer from cytochrome c to the homobinuclear mixed-valence CuA center of subunit II. In Thermus thermophilus a soluble cytochrome c552 acts as the electron donor to ba3 cytochrome-c oxidase, an interaction believed to be mainly hydrophobic. In Paracoccus denitrificans, electrostatic interactions appear to play a major role in the electron transfer process from the membrane-spanning cytochrome c552. In the present study, soluble fragments of the CuA domains and their respective cytochrome c electron donors were analyzed by stopped-flow spectroscopy to further characterize the interaction modes. The forward and the reverse electron transfer reactions were studied as a function of ionic strength and temperature, in all cases yielding monoexponential time-dependent reaction profiles in either direction. From the apparent second-order rate constants, equilibrium constants were calculated, with values of 4.8 and of 0.19, for the T. thermophilus and P. denitrificans c552 and CuA couples, respectively. Ionic strength strongly affects the electron transfer reaction in P. denitrificans indicating that about five charges on the protein interfaces control the interaction, when analyzed according to the Brønsted equation, whereas in the T. thermophilus only 0.5 charges are involved. Overall the results indicate that the soluble CuA domains are excellent models for the initial electron transfer processes in cytochrome-c oxidases.
At pH 5.3 and 4.5 the half life of valyl-, threonyl-, leucyl- and seryl-tRNA from E. coli K 12 is significantly higher than at pH 6.8. While no changes were observed in the MAK elution patterns of valyl- and threonyl-tRNA, leucyl-tRNA was eluted in two peaks at pH 6.8 and 5.3 and in one broad peak at pH 4.5. Seryl-TRNA - two peaks at pH 6.8 - was separated in three peaks at pH 5.3 and 4.5. Rechromatography of these peaks at the other pH suggests the existence of at least four species of seryl-tRNA in E. coli K 12.
Lifespan Extension of Podospora anserina Mic60-Subcomplex Mutants Depends on Cardiolipin Remodeling
(2022)
Function of mitochondria largely depends on a characteristic ultrastructure with typical invaginations, namely the cristae of the inner mitochondrial membrane. The mitochondrial signature phospholipid cardiolipin (CL), the F1Fo-ATP-synthase, and the ‘mitochondrial contact site and cristae organizing system’ (MICOS) complex are involved in this process. Previous studies with Podospora anserina demonstrated that manipulation of MICOS leads to altered cristae structure and prolongs lifespan. While longevity of Mic10-subcomplex mutants is induced by mitohormesis, the underlying mechanism in the Mic60-subcomplex deletion mutants was unclear. Since several studies indicated a connection between MICOS and phospholipid composition, we now analyzed the impact of MICOS on mitochondrial phospholipid metabolism. Data from lipidomic analysis identified alterations in phospholipid profile and acyl composition of CL in Mic60-subcomplex mutants. These changes appear to have beneficial effects on membrane properties and promote longevity. Impairments of CL remodeling in a PaMIC60 ablated mutant lead to a complete abrogation of longevity. This effect is reversed by supplementation of the growth medium with linoleic acid, a fatty acid which allows the formation of tetra-octadecanoyl CL. In the PaMic60 deletion mutant, this CL species appears to lead to longevity. Overall, our data demonstrate a tight connection between MICOS, the regulation of mitochondrial phospholipid homeostasis, and aging of P. anserina.
The avian magnetic compass was analyzed by testing migratory birds, using their orientation as an indicator. These tests revealed some remarkable properties of the avian magnetic compass: (1) It is an inclination compass’, (2) it is light-dependent, with (3) receptors located in the right eye. These characteristics are in agreement with the Radical Pair model proposed by Ritz et al. (2000). Using the same experimental set-up, we tested the model by behavioral spectroscopy’, exposing migratory birds to radiofrequency fields of different frequencies and intensities. Such fields affected the orientation only when applied at an angle to the field lines. Tests with different frequencies led to an estimate of the life time of the crucial radical pair between 2-10 μs. We also could identify an extremely sensitive resonance at the Larmor frequency, which implies specific properties of the radical pair. Cryptochromes, a blue-light absorbing photopigment, has been proposed to be the receptor-molecule; it has been found to be present in the retina of birds.
In recent decades, zoos have been increasingly transformed into education centers with the goal of raising awareness about environmental issues and providing environmental education. Probably the simplest and most widespread environmental education program in the zoo is the guided tour. This study therefore aims to test whether a one hour zoo tour has an influence on the participants’ connection to nature and attitude towards species conservation. For this purpose, 269 people who had voluntarily registered for a zoo tour were surveyed before and after the tour. In addition to the regular zoo tour, special themed tours and tours with animal feedings were included. The results show a positive increase in connection to nature and a strengthening of positive attitudes towards species conservation for all tour types. For nature connectedness, in particular, people with an initial high connection to nature benefitted from the special themed tours and the tours, including animal feedings. For attitudes towards species conservation, no difference was found between the tour types. The results prove the positive influence of a very simple environmental education program, even for people with a preexisting high level of connection to nature and positive attitude towards species conservation.
Animals use the geomagnetic field and astronomical cues to obtain compass information. The magnetic compass is not a uniform mechanism, as several functional modes have been described in different animal groups. The Sun compass requires the internal clock to interpret the position of the Sun. For star compass orientation, night-migrating birds seem to use the star pattern as a whole, without involving the internal clock. Both the astronomical compass mechanisms are based on learning processes to adapt them to the geographic latitude where the animals live and, in long-living animals, to compensate for the seasonal changes. Several mechanisms are used to determine the compass course to a goal. Using information collected during the outward journey is mostly done by path integration: recording the direction with a compass and integrating its twists and turns. Migratory animals have innate programs to guide them to their still unknown goal. Highly mobile animals with large ranges develop a so-called navigational ‘map’, a mental representation of the spatial distribution of navigational factors within their home region and their migration route. The nature of the factors involved is not yet entirely clear; magnetic intensity and inclination are the ones best supported so far.
Forest species are affected by macroclimate, however, the microclimatic variability can be more extreme and change through climate change. Fungal fruiting community composition was affected by microclimatic differences. Here we ask whether differences in the fruiting community can be explained by morphological traits of the fruit body, which may help endure harsh conditions. We used a dead wood experiment and macrofungal fruit body size, color, and toughness. We exposed logs of two host tree species under closed and experimentally opened forest canopies in a random-block design for four years and identified all visible fruit bodies of two fungal lineages (Basidio- and Ascomycota). We found a consistently higher proportion of tough-fleshed species in harsher microclimates under open canopies. Although significant, responses of community fruit body size and color lightness were inconsistent across lineages. We suggest the toughness-protection hypothesis, stating that tough-fleshed fruit bodies protect from microclimatic extremes by reducing dehydration. Our study suggests that the predicted increase of microclimatic harshness with climate change will likely decrease the presence of soft-fleshed fruit bodies. Whether harsh microclimates also affect the mycelium of macrofungi with different fruit body morphology would complement our findings and increase predictability under climate change.
The tremendous body of knowledge about genetics, cell biology, and metabolism of Saccharomyces cerevisiae, as well as its long history and robustness in industrial fermentations, have made this yeast one of the most popular microbial cell factories. Novel genetic tools have enabled the rapid construction of strains producing various platform chemicals, fuels, or pharmaceuticals. The relevance of synthetic biology approaches, such as the construction of fully synthetic genomes and artificial cellular compartments are not only relevant for biotechnological applications but can also lead to new insight into basic principles of life.
Tilletia caries and T. laevis, which are the causal agents of common bunt, as well as T. controversa, which causes dwarf bunt of wheat, threaten especially organic wheat farming. The three closely related fungal species differ in their teliospore morphology and partially in their physiology and infection biology. The gene content as well as intraspecies variation in these species and the genetic basis of their separation is unknown. We sequenced the genome of four T. caries, five T. controversa, and two T. laevis and extended this dataset with five publicly available ones. The genomes of the three species displayed microsynteny with up to 94.3% pairwise aligned regions excluding repetitive regions. The majority of functionally characterized genes involved in pathogenicity, life cycle, and infection of corn smut, Ustilago maydis, were found to be absent or poorly conserved in the draft genomes and the biosynthetic pathway for trimethylamine in Tilletia spp. could be different from bacteria. Overall, 75% of the identified protein-coding genes comprising 84% of the total predicted carbohydrate utilizing enzymes, 72.5% putatively secreted proteins, and 47.4% of effector-like proteins were conserved and shared across all 16 isolates. We predicted nine highly identical secondary metabolite biosynthesis gene clusters comprising in total 62 genes in all species and none were species-specific. Less than 0.1% of the protein-coding genes were species-specific and their function remained mostly unknown. Tilletia controversa had the highest intraspecies genetic variation, followed by T. caries and the lowest in T. laevis. Although the genomes of the three species are very similar, employing 241 single copy genes T. controversa was phylogenetically distinct from T. caries and T. laevis, however these two could not be resolved as individual monophyletic groups. This was in line with the genome-wide number of single nucleotide polymorphisms and small insertions and deletions. Despite the conspicuously different teliospore ornamentation of T. caries and T. laevis, a high degree of genomic identity and scarcity of species-specific genes indicate that the two species could be conspecific.
The current outbreak of the highly infectious COVID-19 respiratory disease is caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). To fight the pandemic, the search for promising viral drug targets has become a cross-border common goal of the international biomedical research community. Within the international Covid19-NMR consortium, scientists support drug development against SARS-CoV-2 by providing publicly available NMR data on viral proteins and RNAs. The coronavirus nucleocapsid protein (N protein) is an RNA-binding protein involved in viral transcription and replication. Its primary function is the packaging of the viral RNA genome. The highly conserved architecture of the coronavirus N protein consists of an N-terminal RNA-binding domain (NTD), followed by an intrinsically disordered Serine/Arginine (SR)-rich linker and a C-terminal dimerization domain (CTD). Besides its involvement in oligomerization, the CTD of the N protein (N-CTD) is also able to bind to nucleic acids by itself, independent of the NTD. Here, we report the near-complete NMR backbone chemical shift assignments of the SARS-CoV-2 N-CTD to provide the basis for downstream applications, in particular site-resolved drug binding studies.
Flavin-based electron bifurcation is a long hidden mechanism of energetic coupling present mainly in anaerobic bacteria and archaea that suffer from energy limitations in their environment. Electron bifurcation saves precious cellular ATP and enables lithotrophic life of acetate-forming (acetogenic) bacteria that grow on H2 + CO2 by the only pathway that combines CO2 fixation with ATP synthesis, the Wood–Ljungdahl pathway. The energy barrier for the endergonic reduction of NADP+, an electron carrier in the Wood–Ljungdahl pathway, with NADH as reductant is overcome by an electron-bifurcating, ferredoxin-dependent transhydrogenase (Nfn) but many acetogens lack nfn genes. We have purified a ferredoxin-dependent NADH:NADP+ oxidoreductase from Sporomusa ovata, characterized the enzyme biochemically and identified the encoding genes. These studies led to the identification of a novel, Sporomusa type Nfn (Stn), built from existing modules of enzymes such as the soluble [Fe–Fe] hydrogenase, that is widespread in acetogens and other anaerobic bacteria.
This study was performed to identify Peronosclerospora species found in Indonesia based on sequence analysis of the cox2 gene. In addition, sequence data in total, 26 isolates of Peronosclerospora were investigated in this study. They were obtained from 7 provinces in Indonesia, namely Lampung, Jawa Timur, Jawa Barat, Sumatera Utara, Jawa Tengah, Yogyakarta, and Sulawesi Selatan. Sequence analysis of cox2 and phylogenetic inference were performed on all the 26 isolates. A set of primers developed in this study, PCOX2F and PCOX2R, was used for PCR amplification. Phylogenetic analyses showed that all the Indonesian isolates were divided into two groups. Group I contained 13 isolates; 9 isolates obtained from Lampung, 3 isolates from Sumatera Utara, and 1 isolate from Jawa Barat. Group II consisted of 13 isolates; 7 isolates from Jawa Timur, 2 isolates from Jawa Tengah, 1 isolate from Yogyakarta, and 3 isolates from Sulawesi Selatan. All the members of group I clustered with the ex-type sequence of P. australiensis. Meanwhile, all members of Group II formed the sister clade of isolates obtained from Timor-Leste and may represent P. maydis.
Carotenoids represent a class of pigmented terpenoids. They are distributed in all taxonomic groups of fungi. Most of the fungal carotenoids differ in their chemical structures to those from other organisms. The general function of carotenoids in heterotrophic organisms is protection as antioxidants against reactive oxygen species generated by photosensitized reactions. Furthermore, carotenoids are metabolized to apocarotenoids by oxidative cleavage. This review presents the current knowledge on fungal-specific carotenoids, their occurrence in different taxonomic groups, and their biosynthesis and conversion into trisporic acids. The outline of the different pathways was focused on the reactions and genes involved in not only the known pathways, but also suggested the possible mechanisms of reactions, which may occur in several non-characterized pathways in different fungi. Finally, efforts and strategies for genetic engineering to enhance or establish pathways for the production of various carotenoids in carotenogenic or non-carotenogenic yeasts were highlighted, addressing the most-advanced producers of each engineered yeast, which offered the highest biotechnological potentials as production systems.
Kálmán Vánky (15th of June 1930–18th of October 2021) was arguably the most prolific researcher of smut fungi so far. He published more than 1000 taxonomic novelties, and crowned his outstanding oeuvre with the most comprehensive monograph of the smut fungi (Smut Fungi of the World) written to date.
Establishing and maintaining protected areas (PAs) is a key action in delivering post-2020 biodiversity targets. PAs often need to meet multiple objectives, ranging from biodiversity protection to ecosystem service provision and climate change mitigation, but available land and conservation funding is limited. Therefore, optimizing resources by selecting the most beneficial PAs is vital. Here, we advocate for a flexible and transparent approach to selecting PAs based on multiple objectives, and illustrate this with a decision support tool on a global scale. The tool allows weighting and prioritization of different conservation objectives according to user-specified preferences as well as real-time comparison of the outcome. Applying the tool across 1,346 terrestrial PAs, we demonstrate that decision makers frequently face trade-offs among conflicting objectives, e.g., between species protection and ecosystem integrity. Nevertheless, we show that transparent decision support tools can reveal synergies and trade-offs associated with PA selection, thereby helping to illuminate and resolve land-use conflicts embedded in divergent societal and political demands and values.
Nature affects human well-being in multiple ways. However, the association between species diversity and human well-being at larger spatial scales remains largely unexplored. Here, we examine the relationship between species diversity and human well-being at the continental scale, while controlling for other known drivers of well-being. We related socio-economic data from more than 26,000 European citizens across 26 countries with macroecological data on species diversity and nature characteristics for Europe. Human well-being was measured as self-reported life-satisfaction and species diversity as the species richness of several taxonomic groups (e.g. birds, mammals and trees). Our results show that bird species richness is positively associated with life-satisfaction across Europe. We found a relatively strong relationship, indicating that the effect of bird species richness on life-satisfaction may be of similar magnitude to that of income. We discuss two, non-exclusive pathways for this relationship: the direct multisensory experience of birds, and beneficial landscape properties which promote both bird diversity and people's well-being. Based on these results, this study argues that management actions for the protection of birds and the landscapes that support them would benefit humans. We suggest that political and societal decision-making should consider the critical role of species diversity for human well-being.
Attitude polarization describes an increasing attitude difference between groups and is increasingly recognized as a multidimensional phenomenon. However, a unified framework to study polarization across multiple dimensions is lacking. We introduce the attitudinal space framework (ASF) to fully quantify attitudinal diversity. We highlight two key measures—attitudinal extremization and attitudinal dispersion—to quantify across- and within-group attitudinal patterns. First, we show that affective polarization in the US electorate is weaker than previously thought based on mean differences alone: in both Democrat and Republican partisans, attitudinal dispersion increased between 1988 and 2008. Second, we examined attitudes toward wolves in Germany. Despite attitude differences between regions with and without wolves, we did not find differences in attitudinal extremization or dispersion, suggesting only weak attitude polarization. These results illustrate how the ASF is applicable to a wide range of social systems and offers an important avenue to understanding societal transformations.
Na(+)/H(+) exchangers are essential for regulation of intracellular proton and sodium concentrations in all living organisms. We examined and experimentally verified a kinetic model for Na(+)/H(+) exchangers, where a single binding site is alternatively occupied by Na(+) or one or two H(+) ions. The proposed transport mechanism inherently down-regulates Na(+)/H(+) exchangers at extreme pH, preventing excessive cytoplasmic acidification or alkalinization. As an experimental test system we present the first electrophysiological investigation of an electroneutral Na(+)/H(+) exchanger, NhaP1 from Methanocaldococcus jannaschii (MjNhaP1), a close homologue of the medically important eukaryotic NHE Na(+)/H(+) exchangers. The kinetic model describes the experimentally observed substrate dependences of MjNhaP1, and the transport mechanism explains alkaline down-regulation of MjNhaP1. Because this model also accounts for acidic down-regulation of the electrogenic NhaA Na(+)/H(+) exchanger from Escherichia coli (EcNhaA, shown in a previous publication) we conclude that it applies generally to all Na(+)/H(+) exchangers, electrogenic as well as electroneutral, and elegantly explains their pH regulation. Furthermore, the electrophysiological analysis allows insight into the electrostatic structure of the translocation complex in electroneutral and electrogenic Na(+)/H(+) exchangers.
The anaerobic acetogenic bacterium Acetobacterium woodii has a novel Na(+)-translocating electron transport chain that couples electron transfer from reduced ferredoxin to NAD(+) with the generation of a primary electrochemical Na(+) potential across its cytoplasmic membrane. In previous assays in which Ti(3+) was used to reduce ferredoxin, Na(+) transport was observed, but not a Na(+) dependence of the electron transfer reaction. Here, we describe a new biological reduction system for ferredoxin in which ferredoxin is reduced with CO, catalyzed by the purified acetyl-CoA synthase/CO dehydrogenase from A. woodii. Using CO-reduced ferredoxin, NAD(+) reduction was highly specific and strictly dependent on ferredoxin and occurred at a rate of 50 milliunits/mg of protein. Most important, this assay revealed for the first time a strict Na(+) dependence of this electron transfer reaction. The Km was 0.2 mm. Na(+) could be partly substituted by Li(+). Na(+) dependence was observed at neutral and acidic pH values, indicating the exclusive use of Na(+) as a coupling ion. Electron transport from reduced ferredoxin to NAD(+) was coupled to electrogenic Na(+) transport, indicating the generation of ΔμNa(+). Vice versa, endergonic ferredoxin reduction with NADH as reductant was possible, but only in the presence of ΔμNa(+), and was accompanied by Na(+) efflux out of the vesicles. This is consistent with the hypothesis that Rnf also catalyzes ferredoxin reduction at the expense of an electrochemical Na(+) gradient. The physiological significance of this finding is discussed.
Background: Ferredoxin:NAD+-oxidoreductases (Rnf) found in many bacteria are novel ion-translocating electron transport chains.
Results: A Na+ requirement for the reaction and its reversible coupling to the transmembrane Na+ gradient are demonstrated.
Conclusion: Na+ is the coupling ion. Rnf not only generates a Na+ potential but also uses it to drive the reverse reaction.
Significance: Evidence for a function of Rnf in ferredoxin reduction is provided.
The Wood-Ljungdahl pathway of anaerobic CO(2) fixation with hydrogen as reductant is considered a candidate for the first life-sustaining pathway on earth because it combines carbon dioxide fixation with the synthesis of ATP via a chemiosmotic mechanism. The acetogenic bacterium Acetobacterium woodii uses an ancient version of the pathway that has only one site to generate the electrochemical ion potential used to drive ATP synthesis, the ferredoxin-fueled, sodium-motive Rnf complex. However, hydrogen-based ferredoxin reduction is endergonic, and how the steep energy barrier is overcome has been an enigma for a long time. We have purified a multimeric [FeFe]-hydrogenase from A. woodii containing four subunits (HydABCD) which is predicted to have one [H]-cluster, three [2Fe2S]-, and six [4Fe4S]-clusters consistent with the experimental determination of 32 mol of Fe and 30 mol of acid-labile sulfur. The enzyme indeed catalyzed hydrogen-based ferredoxin reduction, but required NAD(+) for this reaction. NAD(+) was also reduced but only in the presence of ferredoxin. NAD(+) and ferredoxin reduction both required flavin. Spectroscopic analyses revealed that NAD(+) and ferredoxin reduction are strictly coupled and that they are reduced in a 1:1 stoichiometry. Apparently, the multimeric hydrogenase of A. woodii is a soluble energy-converting hydrogenase that uses electron bifurcation to drive the endergonic ferredoxin reduction by coupling it to the exergonic NAD(+) reduction.
The transporter associated with antigen processing (TAP1/2) translocates cytosolic peptides of proteasomal degradation into the endoplasmic reticulum (ER) lumen. A peptide-loading complex of tapasin, major histocompatibility complex class I, and several auxiliary factors is assembled at the transporter to optimize antigen display to cytotoxic T-lymphocytes at the cell surface. The heterodimeric TAP complex has unique N-terminal domains in addition to a 6 + 6-transmembrane segment core common to most ABC transporters. Here we provide direct evidence that this core TAP complex is sufficient for (i) ER targeting, (ii) heterodimeric assembly within the ER membrane, (iii) peptide binding, (iv) peptide transport, and (v) specific inhibition by the herpes simplex virus protein ICP47 and the human cytomegalovirus protein US6. We show for the first time that the translocation pore of the transporter is composed of the predicted TM-(5-10) of TAP1 and TM-(4-9) of TAP2. Moreover, we demonstrate that the N-terminal domains of TAP1 and TAP2 are essential for recruitment of tapasin, consequently mediating assembly of the macromolecular peptide-loading complex.
A low potential electron carrier ferredoxin (E0′ ≈ −500 mV) is used to fuel the only bioenergetic coupling site, a sodium-motive ferredoxin:NAD+ oxidoreductase (Rnf) in the acetogenic bacterium Acetobacterium woodii. Because ferredoxin reduction with physiological electron donors is highly endergonic, it must be coupled to an exergonic reaction. One candidate is NADH-dependent caffeyl-CoA reduction. We have purified a complex from A. woodii that contains a caffeyl-CoA reductase and an electron transfer flavoprotein. The enzyme contains three subunits encoded by the carCDE genes and is predicted to have, in addition to FAD, two [4Fe-4S] clusters as cofactor, which is consistent with the experimental determination of 4 mol of FAD, 9 mol of iron, and 9 mol of acid-labile sulfur. The enzyme complex catalyzed caffeyl-CoA-dependent oxidation of reduced methyl viologen. With NADH as donor, it catalyzed caffeyl-CoA reduction, but this reaction was highly stimulated by the addition of ferredoxin. Spectroscopic analyses revealed that ferredoxin and caffeyl-CoA were reduced simultaneously, and a stoichiometry of 1.3:1 was determined. Apparently, the caffeyl-CoA reductase-Etf complex of A. woodii uses the novel mechanism of flavin-dependent electron bifurcation to drive the endergonic ferredoxin reduction with NADH as reductant by coupling it to the exergonic NADH-dependent reduction of caffeyl-CoA.
Engineering of thioesterase YciA from Haemophilus influenzae for production of carboxylic acids
(2023)
Acyl-CoA-thioesterases, which hydrolyze acyl-CoA-esters and thereby release the respective acid, have essential functions in cellular metabolism and have also been used to produce valuable compounds in biotechnological processes. Thioesterase YciA originating from Haemophilus influenzae has been previously used to produce specific dicarboxylic acids from CoA-bound intermediates of the ethylmalonyl CoA pathway (EMCP) in Methylorubrum extorquens. In order to identify variants of the YciA enzyme with the capability to hydrolyze so far inaccessible CoA-esters of the EMCP or with improved productivity, we engineered the substrate-binding region of the enzyme. Screening a small semi-rational mutant library directly in M. extorquens yielded the F35L variant which showed a drastic product level increase for mesaconic acid (6.4-fold) and 2-methylsuccinic acid (4.4-fold) compared to the unaltered YciA enzyme. Unexpectedly, in vitro enzyme assays using respective M. extorquens cell extracts or recombinantly produced thioesterases could not deliver congruent data, as the F35L variant showed strongly reduced activity in these experiments. However, applied in an Escherichia coli production strain, the protein variant again outperformed the wild-type enzyme by allowing threefold increased 3-hydroxybutyric acid product titers. Saturation mutagenesis of the codon for position 35 led to the identification of another highly efficient YciA variant and enabled structure-function interpretations. Our work describes an important module for dicarboxylic acid production with M. extorquens and can guide future thioesterase improvement approaches.
Plasmids are one of the most important genetic tools for basic research and biotechnology, as they enable rapid genetic manipulation. Here we present a novel pBBR1-based plasmid for Methylorubrum extorquens, a model methylotroph that is used for the development of C1-based microbial cell factories. To develop a vector with compatibility to the so far mainly used pCM plasmid system, we transferred the pBBR1-based plasmid pMiS1, which showed an extremely low transformation rate and caused a strong growth defect. Isolation of a suppressor mutant with improved growth led to the isolation of the variant pMis1_1B. Its higher transformation rate and less pronounced growth defect phenotype could be shown to be the result of a mutation in the promotor region of the rep gene. Moreover, cotransformation of pMis1_1B and pCM160 was possible, but the resulting transformants showed stronger growth defects in comparison with a single pMis1_1B transformant. Surprisingly, cotransformants carrying pCM160 and a pMis1_1B derivative containing a mCherry reporter construct showed higher fluorescence levels than strains containing only the pMis1_1B-based reporter plasmids or a corresponding pCM160 derivative. Relative plasmid copy number determination experiments confirmed our hypothesis of an increased copy number of pMis1_1B in the strain carrying both plasmids. Despite the slight metabolic burden caused by pMis1_1B, the plasmid strongly expands the genetic toolbox for M. extorquens.
Methylorubrum extorquens is an important model methylotroph and has enormous potential for the development of C1-based microbial cell factories. During strain construction, regulated promoters with a low background expression level are important genetic tools for expression of potentially toxic genes. Here we present an accordingly optimised promoter, which can be used for that purpose. During construction and testing of terpene production strains harbouring a recombinant mevalonate pathway, strong growth defects were observed which made strain development impossible. After isolation and characterisation of suppressor mutants, we discovered a variant of the cumate-inducible promoter PQ2148 used in this approach. Deletion of 28 nucleotides resulted in an extremely low background expression level, but also reduced the maximal expression strength to about 30% of the original promoter. This tightly repressed promoter version is a powerful module for controlled expression of potentially toxic genes in M. extorquens.
The methylotrophic bacterium Methylorubrum extorquens AM1 has the potential to become a platform organism for methanol-driven biotechnology. Its ethylmalonyl-CoA pathway (EMCP) is essential during growth on C1 compounds and harbors several CoA-activated dicarboxylic acids. Those acids could serve as precursor molecules for various polymers. In the past, two dicarboxylic acid products, namely mesaconic acid and 2-methylsuccinic acid, were successfully produced with heterologous thioesterase YciA from Escherichia coli, but the yield was reduced by product reuptake. In our study, we conducted extensive research on the uptake mechanism of those dicarboxylic acid products. By using 2,2-difluorosuccinic acid as a selection agent, we isolated a dicarboxylic acid import mutant. Analysis of the genome of this strain revealed a deletion in gene dctA2, which probably encodes an acid transporter. By testing additional single, double, and triple deletions, we were able to rule out the involvement of the two other DctA transporter homologs and the ketoglutarate transporter KgtP. Uptake of 2-methylsuccinic acid was significantly reduced in dctA2 mutants, while the uptake of mesaconic acid was completely prevented. Moreover, we demonstrated M. extorquens-based synthesis of citramalic acid and a further 1.4-fold increase in product yield using a transport-deficient strain. This work represents an important step towards the development of robust M. extorquens AM1 production strains for dicarboxylic acids.