Arsenic efflux governed by the arsenic resistance determinant of Staphylococcus aureus plasmid pI258.

The arsenic resistance operon of Staphylococcus aureus plasmid pI258 determined lowered net cellular uptake of 73As by an active efflux mechanism. Arsenite was exported from the cells; intracellular arsenate was first reduced to arsenite and then transported out of the cells. Resistant cells showed lower accumulation of 73As originating from both arsenate and arsenite. Active efflux from cells loaded with arsenite required the presence of the plasmid-determined arsB gene. Efflux of arsenic originating as arsenate required the presence of the arsC gene and occurred more rapidly with the addition of arsB. Inhibitor studies with S. aureus loaded with arsenite showed that arsenite efflux was energy dependent and appeared to be driven by the membrane potential. With cells loaded with 73AsO4(3-), a requirement for ATP for energy was observed, leading to the conclusion that ATP was required for arsenate reduction. When the staphylococcal arsenic resistance determinant was cloned into Escherichia coli, lowered accumulation of arsenate and arsenite and 73As efflux from cells loaded with arsenate were also found. Cloning of the E. coli plasmid R773 arsA gene (the determinant of the arsenite-dependent ATPase) in trans to the S. aureus gene arsB resulted in increased resistance to arsenite.     

Ultracytochemical localization of dihydroorotate dehydrogenase in mitochondria and vacuoles ofSaccharomyces cerevisiae

The coenzyme-independent dihydroorotate dehydrogenase (EC 1.3.3.1) linking the pyrimidine biosynthetic pathway to the respiratory chain, was ultracytochemically localized by the tetrazolium method in derepressed exponential-phase cultures ofSaccharomyces cerevisiae. Biochemical analysis showed a considerable variation of this enzyme activity in inverse proportion to the aeration of the yeast cultures. The assay also showed that after prefixation of yeast cells with 1% glutaraldehyde at 0°C for 20 min, approximately one-half of the enzyme activity was preserved. The cytochemical reaction mixture contained dihydroorotate (2 mmol/L), thiocarbamyl nitroblue tetrazolium (0.44 mmol/L), phenazine methosulfate (0.16 mmol/L) and KCN (1.7 mmol/L) in Tris-HCl buffer (100 mmol/L) of pH 8.0. The osmicated formazan deposits featured envelopes of mitochondria and of nuclei and were prominent in the mitochondrial inclusions and in the vacuolar membranes. The latter sites of dihydroorotate dehydrogenase activity represent biosynthetic activity in yeast vacuoles, still generally assumed to function as yeast lysosomes and storage organelles. In the light of the generally observed invasions of juvenile yeast vacuoles into mitochondria, the enzymic sites observed in mitochondrial inclusion were considered as evidence of the interactions of yeast vacuoles and mitochondria. Transfer of vacuolar membranes with dihydroorotate dehydrogenase activity into mitochondrial matrix is suggested.     

Seasonal Variations in Rubber Biosynthesis, 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase,and Rubber Transferase Activities in Parthenium argentatum in the Chihuahuan Desert

The rubber content and the activities of enzymes in the polyisoprenoid pathway in Parthenium argentatum (guayule) were examined throughout the growing season in field plots in the Chihuahuan Desert. The rubber content of the plants was low in July and August and slowly increased until October. From October to December there was a rapid increase in rubber formation (per plant) from 589.0 mg to 4438.0 mg. The percentage of rubber in the plants increased from 0.7% (mg/g dry weight) in August and 1.27% in October to 5.5% in December. The rapid increase in rubber formation may result from exposing the plants to low temperatures of 5 to 7[deg]C. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) was 21.1 nmol mevalonic acid (MVA) h-1 g-1 fresh weight in the bark of the lower stems in June during seedling growth and decreased to 5.1 nmol MVA h-1g-1 fresh weight in July and 2.9 nmol MVA h-1 g-1 fresh weight in September. From October to December, the activity increased from 5.0 to 29.9 nmol MVA h-1 g-1 fresh weight. The activity of rubber transferase was 65.5 nmol isopentenyl pyrophosphate (IPP) h-1 g-1fresh weight in the bark in September and increased to 357.5 nmol IPP h-1 g-1 fresh weight in December. The rapid increase in the activities of HMGR and rubber transferase coincided with the rapid increase in rubber formation. The activities of MVA kinase and IPP isomerase did not significantly increase in the fall and winter. A tomato HMGR-1 cDNA probe containing a highly conserved C-terminal region of HMGR genes hybridized at low stringency with several bands on blots of HindIII-digested genomic DNA from guayule. In northern blots with the HMGR-1 cDNA probe at low stringency, HMGR mRNA was high in June and November, corresponding to periods of high HMGR activity during seedling growth and rapid increase in rubber formation. The seasonal variations in rubber formation and HMGR mRNA, HMGR activity, and rubber transferase activity may be due to low temperature stimulation in the fall and winter months.     

湖羊瘤胃微生物GH9家族葡聚糖酶基因IDSGLUC9-25的表达与功能表征

【目的】葡聚糖酶是饲用添加剂的重要成分,本研究旨在从湖羊消化道微生物中挖掘性质优良的GH9家族葡聚糖酶基因,用于研发新型饲用酶制剂。【方法】从湖羊瘤胃微生物cDNA中扩增IDSGLUC9-25基因,在大肠杆菌中进行异源表达,对重组蛋白进行诱导表达和纯化,研究重组蛋白的酶学性质和底物水解模式。【结果】IDSGLUC9-25基因编码527个氨基酸,包含一个CelD_N结构和一个GH9家族催化结构域;重组蛋白rIDSGLUC9-25分子量约为62.7 kDa,最适反应温度和pH分别为40℃和6.0,在30-50℃下活性较高,在pH 4.0-8.0范围内能够保持较高的稳定性,经pH 4.0-8.0缓冲液处理1 h后残余活性均大于90%;底物谱分析表明,rIDSGLUC9-25能催化大麦β-葡聚糖、苔藓地衣多糖、魔芋胶和木葡聚糖,比活性分别为(443.55±24.48)、(65.56±5.98)、(122.37±2.85)和(159.16±7.73) U/mg;利用薄层色谱法(thin layer chromatography, TLC)和高效液相色谱法(high performance liquid chromatography, HPLC)分析水解产物发现,rIDSGLUC9-25降解大麦葡聚糖主要生成纤维三糖(占总还原糖64.19%±1.19%)和纤维四糖(占总还原糖26.24%±0.12%),催化地衣多糖主要生成纤维三糖(占总还原糖78.46%±0.89%)。【结论】本研究报道了一种来自密螺旋体属细菌的内切β-1,4-葡聚糖酶IDSGLUC9-25 (EC 3.2.1.4),能高效催化多糖底物生成纤维三糖和纤维四糖,为研发饲用酶制剂和制备低聚寡糖建立基础。     

Ecological and methodological drivers of non-stationarity in tree growth response to climate

Radial tree growth is sensitive to environmental conditions, making observed growth increments an important indicator of climate change effects on forest growth. However, unprecedented climate variability could lead to non-stationarity, that is, a decoupling of tree growth responses from climate over time, potentially inducing biases in climate reconstructions and forest growth projections. Little is known about whether and to what extent environmental conditions, species, and model type and resolution affect the occurrence and magnitude of non-stationarity. To systematically assess potential drivers of non-stationarity, we compiled tree-ring width chronologies of two conifer species, Picea abies and Pinus sylvestris, distributed across cold, dry, and mixed climates. We analyzed 147 sites across the Europe including the distribution margins of these species as well as moderate sites. We calibrated four numerical models (linear vs. non-linear, daily vs. monthly resolution) to simulate growth chronologies based on temperature and soil moisture data. Climate–growth models were tested in independent verification periods to quantify their non-stationarity, which was assessed based on bootstrapped transfer function stability tests. The degree of non-stationarity varied between species, site climatic conditions, and models. Chronologies of P. sylvestris showed stronger non-stationarity compared with Picea abies stands with a high degree of stationarity. Sites with mixed climatic signals were most affected by non-stationarity compared with sites sampled at cold and dry species distribution margins. Moreover, linear models with daily resolution exhibited greater non-stationarity compared with monthly-resolved non-linear models. We conclude that non-stationarity in climate–growth responses is a multifactorial phenomenon driven by the interaction of site climatic conditions, tree species, and methodological features of the modeling approach. Given the existence of multiple drivers and the frequent occurrence of non-stationarity, we recommend that temporal non-stationarity rather than stationarity should be considered as the baseline model of climate–growth response for temperate forests.     

艾滋病病毒蛋白酶高效表达载体及体外活性检测系统的构建

艾滋病是本世纪80年代初发现的一种烈性传染病,5年病死率为100％,致病因子为人免疫缺陷病毒,该病毒的蛋白酶在病毒复制和成熟中具有决定性的意义。由于目前国内外尚未获得艾滋病病毒蛋白酶的高效表达的重组子及活性检测系统,限制了它的研究与应用。本文利用PCR技术修饰了艾滋病病毒蛋白酶的基因,使其具有便于克隆及表达用的限制酶切位点及转录终止码,井在其C末端设置了一个可用于检验该酶活性的特殊序列。DNA序列分析揭示上述突变策略成功,将修饰后的艾滋病病毒蛋白酶基因克隆入大肠杆菌表达系统,并获得高效表达(>30％),Western-Bolt鉴定结果表明所表达的蛋白为艾滋病病毒所特有,并具有较好的生物活性。     

Ontogenetic change in effectiveness of chemical defence against different predators in Oxycarenus true bugs

Many prey species change their antipredator defence during ontogeny, which may be connected to different potential predators over the life cycle of the prey. To test this hypothesis, we compared reactions of two predator taxa – spiders and birds – to larvae and adults of two invasive true bug species, Oxycarenus hyalinipennis and Oxycarenus lavaterae (Heteroptera: Oxycarenidae) with life-stage-specific chemical defence mechanisms. The reactions to larvae and adults of both true bug species strikingly differed between the two predator taxa. The spiders were deterred by the defences of adult bugs, but the larval defences were ineffective against them. By contrast, birds attacked the larvae considerably less often than the adult bugs. The results indicate a predator-specific ontogenetic change in defence effectiveness of both Oxycarenus species. The change in defence is likely linked to the life-stage-specific composition of secretions in both species: whereas secretions of larvae are dominated by unsaturated aldehydes, secretions of adults are rich in terpenoids, which probably serve dual function of defensive chemicals and pheromones. Our results highlight the variation in defence between different life stages and the importance of testing responses of different types of predators.     

Decomposing trends in bird populations: Climate,life histories and habitat affect different aspects of population change

Aim

Despite the complexity of population dynamics, most studies concerning current changes in bird populations reduce the trajectory of population change to a linear trend. This may hide more complex patterns reflecting responses of bird populations to changing anthropogenic pressures. Here, we address this complexity by means of multivariate analysis and attribute different components of bird population dynamics to different potential drivers.

Location

Czech Republic.

Methods

We used data on population trajectories (1982–2019) of 111 common breeding bird species, decomposed them into independent components by means of the principal component analysis (PCA), and related these components to multiple potential drivers comprising climate, land use change and species' life histories.

Results

The first two ordination axes explained substantial proportion of variability of population dynamics (42.0 and 12.5% of variation in PC1 and PC2 respectively). The first axis captured linear population trend. Species with increasing populations were characterized mostly by long lifespan and warmer climatic niches. The effect of habitat was less pronounced but still significant, with negative trends being typical for farmland birds, while positive trends characterized birds of deciduous forests. The second axis captured the contrast between hump-shaped and U-shaped population trajectories and was even more strongly associated with species traits. Species migrating longer distances and species with narrower temperature niches revealed hump-shaped population trends, so that their populations mostly increased before 2000 and then declined. These patterns are supported by the trends of total abundances of respective ecological groups.

Main Conclusion

Although habitat transformation apparently drives population trajectories in some species groups, climate change and associated species traits represent crucial drivers of complex population dynamics of central European birds. Decomposing population dynamics into separate components brings unique insights into non-trivial patterns of population change and their drivers, and may potentially indicate changes in the regime of anthropogenic effects on biodiversity.     

Metabarcoding reveals seasonal and spatial patterns of arthropod community assemblages in two contrasting habitats: Desert and oasis of the Baja California Peninsula,Mexico

Aim

Desert springs or oases are the only permanent mesic environments in highly water-limited arid regions. Oases have immense cultural, evolutionary and ecological importance for people and a high number of endemic and relic species. Nevertheless, they are also highly vulnerable ecosystems, with invasive species, overexploitation and climate change being the primary threats. We used the arthropod communities' spatiotemporal diversity and distribution patterns as a proxy to understand biodiversity dynamics in two geographically close but ecologically contrasting and highly threatened ecosystems: deserts and oases.

Location

Baja California Peninsula, Mexico.

Methods

Arthropod communities at five oases and surrounding desert scrub areas were sampled in two seasons. Using DNA metabarcoding and traditional taxonomic surveys, we tried to identify what biotic and abiotic characteristics of the habitat are important drivers of arthropod diversity and how these characteristics can change across spatial and temporal scales.

Results

Over 6200 individuals representing 23 orders were collected. In oasis samples, the community composition fluctuated more in space (i.e. among sites) than in time (i.e. seasons). Thus, seasonal changes did not affect oasis community diversity and composition, but the dissimilarity among sites increased with geographic distance. Moreover, anthropic activities negatively correlated with arthropod diversity in oases. On the other hand, the season, geography (e.g. latitude) and biotic characteristics of the habitat (e.g. sampled scrub species) significantly affected the diversity and composition of the desert arthropod communities.

Main Conclusions

Neutral dynamics (e.g. historical climatic events, dispersal limitation and spatial component) and human impact significantly influenced the biodiversity patterns of each oasis. In contrast, the habitat's seasonal variation and biotic characteristics were the most important variables influencing the diversity of the desert communities. Baja California oases harbour distinct invertebrate communities; therefore, each oasis should be conserved individually to preserve these unique assemblages.